Niehof M, etal., Mol Cell Biol. 1997 Jul;17(7):3600-13.
LAP/C/EBP beta is a member of the C/EBP family of transcription factors and is involved in hepatocyte-specific gene expression. Recently we showed that, besides its posttranscriptional regulation, LAP/C/EBP
ght:700;'>EBP beta mRNA is modulated during liver regeneration. Therefore, in this study we investigated mechanisms which control LAP/C/EBP beta gene transcription. Deletion analysis of the 5'-flanking region, located upstream of the start site of transcription in the LAP/C/EBP beta gene, demonstrated that a small region in close proximity to the TATA box is important in maintaining a high level of transcription of the luciferase reporter gene constructs. In gel shift experiments two sites were identified which are important for specific complex formation within this region. Further analysis by cross-linking, super shift, and competition experiments was performed with liver cell nuclear extracts, hepatoma cell nuclear extracts, or recombinant CREB protein. These experiments conclusively demonstrated that CREB binds to both sites in the LAP/C/EBP beta promoter with an affinity similar to that with the CREB consensus sequence. Transfection experiments with promoter constructs where the CREB sites were mutated showed that these sites are important to maintain both basal promoter activity and LAP/C/EBP beta inducibility through CREB. Northern blot analysis and runoff transcription assays demonstrated that the protein kinase A pathway not only stimulated the activity of the luciferase reporter construct but also the transcription of the endogenous LAP/C/EBP beta gene in different cell types. Western blot analysis of rat liver cell nuclear extracts and runoff transcription assays of rat liver cell nuclei after two-thirds hepatectomy showed a functional link between the induction of CREB phosphorylation and LAP/C/EBP beta mRNA transcription during liver regeneration. These results demonstrate that the two CREB sites are important to control LAP/C/EBP beta transcription in vivo. As several pathways control CREB phosphorylation, our results provide evidence for the transcriptional regulation of LAP/C/EBP beta via CREB under different physiological conditions.
Landschulz WH, etal., Genes Dev 1988 Jul;2(7):786-800.
In two previous studies we described the properties of a heat-stable DNA-binding protein present in rat liver nuclei. This protein, hereafter termed C/EBP, is capable of selective binding to the CCAAT homology of several viral promoters (Graves et al. 1986), as
well as the core homology common to many viral enhancers (Johnson et al. 1987). We now report the isolation of a recombinant clone of the gene that encodes C/EBP. Expression of the clone in bacterial cells yields a protein that binds in vitro to both the CCAAT homology and the enhancer core homology, providing conclusive evidence that a single gene product accounts for both binding activities. By examining the properties of protease-derived fragments of C/EBP, we have localized its DNA-binding domain to a 14-kD fragment. A 60-amino-acid segment located within the DNA-binding domain of C/EBP bears sequence similarity to the products of the myc and fos oncogenes.
Bek MF, etal., Am J Pathol. 2006 Jan;168(1):20-32.
Podocytes are crucial for the permeability of the glomerular filtration barrier. In glomerular disease, however, reactive oxygen species (ROS) may be involved in podocyte injury and subsequent proteinuria. Here, we describe ROS-dependent gene induction in differentiated podocytes stimulated with H(2
)O(2) or xanthine/xanthine-oxidase. Superoxide anions and H(2)O(2) increased mRNA and protein expression of GAS5 (growth arrest-specific protein 5) and CHOP (C/EBP homology protein). Cultured podocytes overexpressing CHOP showed increased generation of superoxide anions compared to controls. In addition, the expression of alpha(3)/beta(1) integrins, crucial for cell-matrix interaction of podocytes, was down-regulated, leading to increased cell-matrix adhesion and cell displacement. The altered cell-matrix adhesion was antagonized by the ROS scavenger 1,3-dimethyl-2-thiourea, and the increase in cell displacement could be mimicked by stimulating untransfected podocytes with puromycin, an inductor of ROS. We next performed immunohistochemical staining of human kidney tissue (normal, membranous nephropathy, focal segmental glomerulosclerosis, and minimal change nephropathy) as well as sections from rats with puromycin nephrosis, a model of minimal change nephropathy. CHOP was weakly expressed in podocytes of control kidneys but up-regulated in most proteinuric human kidneys and in rat puromycin nephrosis. Our data suggest that CHOP-via increased ROS generation-regulates cell-matrix adhesion of podocytes in glomerular disease.
Bellizzi A, etal., Cell Cycle. 2015;14(13):2075-9. doi: 10.1080/15384101.2015.1042631. Epub 2015 May 27.
Endoplasmic reticulum (ER) stress is caused by the accumulation of misfolded or unfolded proteins in the lumen of the endoplasmic reticulum. CCAAT/enhancer binding proteins are one of the cellular proteins whose expression is upregulated during ER stress. Previously, we have identified C/EBP
='font-weight:700;'>EBPbeta isoforms, especially LIP, as a negative regulator of polyomavirus JC (JCV), the causative agent of the demyelinating disease progressive multifocal leukoencephalopathy (PML). Here, we show that the induction of ER stress by thapsigargin increase the expression of endogenous LIP and the degradation of JCV T-antigen in a JCV-transgenic mouse tumor cell line. Our results also revealed that overexpression of LIP significantly reduced the level of T-Ag and this effect is reversed upon siRNA-mediated silencing of LIP. Immunoprecipitation/Western blot experiments indicated that LIP interacts with T-antigen directly. Treatment of cells that overexpress LIP with MG115, a proteasome inhibitor, partially rescued LIP-mediated degradation of T-antigen. Our observations point to a role of LIP in ER stress regulation of T-antigen stability and may open a new avenue to study host-virus interaction during ER stress.
Furtado LV, etal., Am J Med Genet A. 2010 Nov;152A(11):2838-44. doi: 10.1002/ajmg.a.33674.
Mutations of the gene coding for emopamil binding protein (EBP) can lead to deficient activity of 3-beta-hydroxysteroid Delta(8), Delta(7) isomerase and are most commonly identified in. association with the X-linked dominant (male lethal) chondrodysplasia punct
ata (CDPX2), also known as Conradi-Hunermann syndrome. Our group has identified a hemizygous EBP mutation in males with a phenotype remarkable for Dandy-Walker malformation, cataracts, collodion skin and cryptorchidism. Additional findings of hydrocephalus, dysplasia of the corpus callosum, cardiovascular, craniofacial and skeletal anomalies were regularly seen in affected males and the family histories were supportive of an X-linked -recessive condition. The regularly reproducible constellation of cardinal features aligns very nicely with other disorders of sterol biosynthesis and is further distinguished by an absence of arty clinical manifestations in obligate carrier females. Biochemical analysis of blood from cases demonstrated markedly increased levels of 8(9)-cholestenol, and 8-dehydroeholesterol and a mildly increased level of 7-dehydrocholesterol; a similar pattern to what is seen in CDPX2. Sequence analysis of EJJP revealed a novel hemizygous missense mutation at position 141, predictive of a tryptophan to cysteine substitution (c.141G>T, p.W47C). The unaffected mothers were heterozygous for the c.141G>T mutation arid showed random X-inactivation pattern upon.
Pope RM, etal., Clin Immunol. 1999 Jun;91(3):271-82.
Rheumatoid arthritis synovial tissue was examined and compared with osteoarthritis tissue for the presence of the nuclear transcription factor C/EBP beta (NF-IL-6). The region (lining or sublining), cell type, and subcellular distribution (cytoplasmic or nuclea
r) of the expression of C/EBP beta was characterized. Rheumatoid arthritis synovial fluid and blood and normal peripheral blood were also examined. C/EBP beta was detected in the synovial lining and in sublining cells of synovial tissue from patients with both rheumatoid and osteoarthritis. A significant (P < 0.001 and < 0.05, respectively) increase in the percentage of cells with nuclear staining was seen in the lining layer, compared to cells in the sublining region, in rheumatoid and osteoarthritis. In both diseases a strong correlation (r = 0.79, P < 0.001) was observed between the percentage of cells in the synovial lining that were positive for nuclear C/EBP beta and lining cell depth. Two-color immunohistochemistry demonstrated that both macrophages and fibroblast-like synoviocytes were positive for nuclear C/EBP beta. The presence of C/EBP beta was confirmed by immunohistochemistry and Western blot analysis with isolated synovial fibroblasts. Nuclear C/EBP beta was also detected in rheumatoid synovial fluid monocytes/macrophages, but not in lymphocytes or neutrophils. Western blot analysis confirmed the presence of C/EBP beta in these cells. The intensity of C/EBP beta staining was greater (P < 0.001) in synovial fluid monocytes than in those from normal or rheumatoid peripheral blood. In conclusion, the enhanced nuclear staining for C/EBP beta in the synovial lining, compared to the sublining, suggesting activation in the lining, and the positive correlation of lining layer depth with the percentage of cells in the lining positive for nuclear C/EBP beta, suggest a potential role for C/EBP beta in chronic inflammation. The regulation of the production or activity of C/EBP beta, to inhibit inflammatory mediator expression by synovial macrophages and fibroblasts, offers a novel approach to therapeutic intervention.
Kolyada AY, etal., Am J Physiol. 1995 Dec;269(6 Pt 1):C1408-16.
We have recently shown that regulatory element D (nucleotides -239 to -215) of the 0.25-kb promoter of the human growth factor-activatable Na+/H+ exchanger (NHE1) is important for gene transcription in cells of hepatic origin (Hep G2) and vascular smooth muscle origin (VSM A7r5). This element contai
ns a sequence (nucleotides -230 to -222) with complete homology to the C/EBP binding site. We now demonstrate that nucleotide substitution mutations disrupting this C/EBP site suppressed transcription in Hep G2 cells, VSM A7r5 cells, and Sprague-Dawley VSM cells in primary culture. These mutations abolished the binding of rat liver nuclear activities as well as transcription factors C/EBP alpha, C/EBP beta, and C/EBP delta expressed in COS-1 cell lysates to element D. Anti-C/EBP antibodies supershifted DNA-protein complexes formed between hepatic nuclear activities or C/EBP proteins expressed in COS-1 cell lysates and regulatory element D. Finally, cotransfection experiments of NHE1 0.25-kb promoter-chloramphenicol acetyltransferase (CAT) construct and C/EBP expression vectors showed that C/EBP alpha and C/EBP delta are transactivators of the NHE1 proximal promoter in Hep G2 and VSM A7r5 cells. These results indicate that members of the C/EBP family of transcription factors are involved in the regulation of hepatic and vascular smooth muscle transcription of the human NHE1 gene.
Sepsis-induced muscle cachexia is associated with increased expression of several genes in the ubiquitin-proteasome proteolytic pathway, but little is known about the activation of transcription factors in skeletal muscle during sepsis. We tested the hypothesis that sepsis upregulates the expression
and activity of the transcription factors CCAAT/enhancer binding protein (C/EBP)-beta and -delta in skeletal muscle. Sepsis was induced in rats by cecal ligation and puncture, and control rats were sham operated. C/EBP-beta and -delta DNA-binding activity was determined by electrophoretic mobility shift assay and supershift analysis. In addition, C/EBP-beta and -delta nuclear protein levels were determined by Western blot analysis. Sepsis resulted in increased DNA-binding activity of C/EBP, and supershift analysis suggested that this reflected activation of the beta- and delta-isoforms of C/EBP. Concomitantly, C/EBP-beta and -delta protein levels were increased in the nuclear fraction of skeletal muscle. In additional experiments, we tested the role of glucocorticoids in sepsis-induced activation of C/EBP-beta and -delta by treating rats with the glucocorticoid receptor antagonist RU-38486. This treatment inhibited the sepsis-induced activation of C/EBP-beta and -delta, suggesting that glucocorticoids participate in the upregulation of C/EBP in skeletal muscle during sepsis. The present results suggest that C/EBP-beta and -delta are activated in skeletal muscle during sepsis and that this response is, at least in part, regulated by glucocorticoids.
Hernandez-Encinas E, etal., J Neuroinflammation. 2016 Oct 21;13(1):276. doi: 10.1186/s12974-016-0742-0.
BACKGROUND: The CCAAT/enhancer-binding protein β (C/EBPβ) is a transcription factor implicated in the control of proliferation, differentiation, and inflammatory processes mainly in adipose tissue and liver; although more recent results have revealed
an important role for this transcription factor in the brain. Previous studies from our laboratory indicated that CCAAT/enhancer-binding protein β is implicated in inflammatory process and brain injury, since mice lacking this gene were less susceptible to kainic acid-induced injury. More recently, we have shown that the complement component 3 gene (C3) is a downstream target of CCAAT/enhancer-binding protein β and it could be a mediator of the proinflammatory effects of this transcription factor in neural cells. METHODS: Adult male Wistar rats (8-12 weeks old) were used throughout the study. C/EBPβ+/+ and C/EBPβ-/- mice were generated from heterozygous breeding pairs. Animals were injected or not with kainic acid, brains removed, and brain slices containing the hippocampus analyzed for the expression of both CCAAT/enhancer-binding protein β and C3. RESULTS: In the present work, we have further extended these studies and show that CCAAT/enhancer-binding protein β and C3 co-express in the CA1 and CA3 regions of the hippocampus after an excitotoxic injury. Studies using CCAAT/enhancer-binding protein β knockout mice demonstrate a marked reduction in C3 expression after kainic acid injection in these animals, suggesting that indeed this protein is regulated by C/EBPβ in the hippocampus in vivo. CONCLUSIONS: Altogether these results suggest that CCAAT/enhancer-binding protein β could regulate brain disorders, in which excitotoxic and inflammatory processes are involved, at least in part through the direct regulation of C3.
Yang F, etal., Zhen Ci Yan Jiu. 2014 Aug;39(4):267-71.
OBJECTIVE: To observe the effect of acupuncture intervention on expression of glucose-regulated protein 78 (Grp 78) and C/EBP homologous protein (CHOP) in the hippocampus in epilepsy rats so as to explore its mechanism underlying improvement of hyperspasmia-ind
uced brain injury. METHODS: Forty-two SD rats were randomly divided into normal control group (n = 6), model group (n = 18), and acupuncture group (n = 18). The epileptic seizure model was established by intraperitonel injection of Pentylenetetrazol (50 mg/kg, 2 mL). Manual acupuncture stimulation of "Baihui" (GV 20) and "Dazhui" (GV 14) was conducted for rats of the acupuncture group for 30 min. Two hours (h), 12 h and 48 h after acupuncture intervention, the hippocampal tissue was sampled (6 rats at each time-point). The expression levels of Grp 78 and CHOP proteins in the hippocampal CA 1 region were detected by immunohistochemistry. RESULTS: Compared with the normal group, the expression levels of Grp 78 protein at time-points of 2 h and 12 h, and those of CHOP protein at 2 h, 12 h and 48 h after epilpeptic seizure were significantly increased in the model group (P < 0.01). After acupuncture treatment, the expression levels of Grp 78 at 12 and 48 h were significantly increased, and those of CHOP protein at 2 h, 12 h and 24 h in the acupuncture group were considerably downregulated (P < 0.05, P < 0.01). CONCLUSION: Acupuncture treatment can up-regulate Grp 78 protein expression and down-regulate CHOP protein expression level in epilepsy rats , which may contribute to its protective effect on seizure-induced brain injury.
Williams SC, etal., Genes Dev 1991 Sep;5(9):1553-67.
Mouse and rat genomic DNA libraries were screened by reduced stringency hybridization with the DNA-binding domain of the c/ebp gene as a probe. Three genes were isolated that encode bZIP DNA-binding proteins (designated CRP1, CRP2, and CRP3) with strong amino ac
id sequence similarities to the C/EBP-binding domain. CRP2 is identical to the protein described recently by other groups as NF-IL6, LAP, IL-6DBP, and AGP/EBP, whereas CRP1 and CRP3 represent novel proteins. Several lines of evidence indicate that these three proteins, along with C/EBP, comprise a functional family. Each bacterially expressed polypeptide binds to DNA as a dimer with recognition properties that are virtually identical to those of C/EBP. Every member also bears a conserved cysteine residue at or near the carboxyl terminus, immediately following the leucine zipper, that at least in vitro allows efficient disulfide cross-linking between paired zipper helices. We developed a gel assay for covalent dimers to assess leucine zipper specificities among the family members. The results demonstrate that all pairwise combinations of dimer interactions are possible. To the extent that we have examined them, the same heterodimeric complexes can be detected intracellularly following cotransfection of the appropriate pair of genes into recipient cells. All members are also capable of activating in vivo transcription from promoters that contain a C/EBP-binding site. Our findings indicate that a set of potentially interacting C/EBP-like proteins exists, whose complexity is comparable to that of other bZIP protein subfamilies such as Jun, Fos, and ATF/CREB.
Liu Z, etal., Chem Res Toxicol. 2016 Sep 19;29(9):1510-8. doi: 10.1021/acs.chemrestox.6b00181. Epub 2016 Aug 16.
Tetrachlorobenzoquinone (TCBQ) is a downstream metabolite of pentachlorophenol (PCP). Previously, we demonstrated that TCBQ caused cytotoxicity due to mitochondrial-related apoptosis. Here, we confirmed the upregulation of death receptor 5 (DR5) followed by the construction of the death-inducing sig
naling complex (DISC). We also detected the activation of the caspase cascade, which was correlated with TCBQ-induced apoptotic cell death in PC12 cells. The upregulation of DR5 included transcriptional activation and de novo protein synthesis in response to TCBQ. We also identified the endoplasmic reticulum (ER) as a new target for the TCBQ challenge in PC12 cells. The protein kinase R-like ER kinase/eukaryotic translation initiation factor 2α (PERK/eIF2α)-mediated activating transcription factor 4 (ATF4)-ATF3-C/EBP homologous protein (CHOP) signaling pathway contributed to the process of TCBQ-induced ER stress. Blocking ATF4, ATF3, or CHOP signaling by gene silencing technology resulted in decreased cell apoptosis after exposure to TCBQ. Finally, NAC ameliorated TCBQ-induced apoptosis and ER stress, which illustrated that TCBQ-induced apoptosis is somehow ROS-dependent. In summary, this study provided important mechanistic insight into how TCBQ utilizes ER stress-related signaling to exhibit pro-apoptotic activity in PC12 cells.
The participation of C/EBP alpha and C/EBP beta in the transcriptional regulation of the haptoglobin (Hp) gene throughout liver development and the acute-phase (AP) response was examined. Western immunoblot analysis revealed
that the relative concentrations of C/EBP alpha and C/EBP beta increased during differentiation in two nuclear protein fractions - the nuclear extract and nuclear matrix. The AP reaction was accompanied by a decrease of the relative concentration of C/EBP alpha and an increase of C/EBP beta during development in both protein fractions. Using Western analysis after DNA-affinity chromatography it was observed that a 45 kDa C/EBP alpha isoform displayed a binding affinity towards the Hp gene hormone responsive element (HRE) in both pre- and postnatal livers. In the course of the AP response DNA binding of the 45 kDa isoform was detected only in the adult, when its binding affinity decreased. The 35 kDa C/EBP beta isoform exhibited a binding affinity towards the Hp HRE after the second week from birth, whereas the AP response promoted an enhanced binding of 35 kDa isoform after the first postnatal week. These results indicate that Hp gene transcription is regulated by C/EBP alpha during normal liver development, whereas C/EBP beta is involved in the AP regulation during the later phase of differentiation and in the adult.
C/EBP homologous protein (CHOP), a transcription factor for the expression of apoptosis-related genes, plays an important role in endoplasmic reticulum (ER) stress-related organ diseases, including diseases of the kidney. Here, we investigated the role of CHOP i
n ischemia/reperfusion (I/R)-induced acute kidney injury using CHOP-knockout (CHOP(-/-)) and wild type (CHOP(+/+)) mice. Fifteen or thirty minutes of bilateral renal ischemia (I/R) insult resulted in necrotic and apoptotic tubular epithelial cell death, together with increases in plasma creatinine (PCr) and blood urea nitrogen (BUN) concentrations. After I/R, BiP/GRP78 and CHOP expressions in the kidney gradually increased over time. CHOP expression was greater in the outer medulla than that in the cortex and localized intensely in the nucleus. I/R caused apoptosis of tubular epithelial cells in both CHOP(-/-) and CHOP(+/+) mice. The number of apoptotic cells after I/R was lower in CHOP(-/-) mice than that in CHOP(+/+) mice. Consistent with the degree of apoptosis, I/R-induced kidney morphological and functional damages were milder in CHOP(-/-) than that in CHOP(+/+) mice. The cleavage of procaspase-3 and the induction of Bax protein after I/R were lower in CHOP(-/-) than that in CHOP(+/+) mice. In contrast, the expression levels of Bcl-2, Bcl-xL, cIAP2, Mcl-1, and XIAP were higher in CHOP(-/-) than that in CHOP(+/+) mice. These results indicate that I/R induces ER stress, leading to the activation of CHOP-associated apoptosis signals, resulting in renal functional and histological damages.
RATIONALE: Myeloid-derived C/EBP-homologous protein (CHOP), an effector of the endoplasmic reticulum stress-induced unfolded protein response, promotes macrophage apoptosis in advanced atherosclerosis, but the role of CHOP in vascular smooth muscle cells (VSMCs)
in atherosclerosis is not known. OBJECTIVE: To investigate the role of CHOP in SM22alpha(+) VSMCs in atherosclerosis. METHODS AND RESULTS: Chop(fl/fl) mice were generated and crossed into the Apoe(-/-) and SM22alpha-CreKI(+) backgrounds. SM22alpha-CreKI causes deletion of floxed genes in adult SMCs. After 12 weeks of Western-type diet feeding, the content of alpha-actin-positive cells in aortic root lesions was decreased in Chop(fl/fl)SM22alpha-CreKI(+)Apoe(-/-) versus control Chop(fl/fl)Apoe(-/-) mice, and aortic explant-derived VSMCs from the VSMC-CHOP-deficient mice displayed reduced proliferation. Kruppel-like factor 4 (KLF4), a key suppressor of VSMC proliferation, was increased in lesions and aortic VSMCs from Chop(fl/fl)SM22alpha-CreKI(+)Apoe(-/-) mice, and silencing Klf4 in CHOP-deficient VSMCs restored proliferation. CHOP deficiency in aortic VSMCs increased KLF4 through 2 mechanisms mediated by the endoplasmic reticulum stress effector activating transcription factor 4: transcriptional induction of Klf4 mRNA and decreased proteasomal degradation of KLF4 protein. CONCLUSIONS: These findings in SM22alpha-CHOP-deficient mice imply that CHOP expression in SM22alpha(+) VSMCs promotes cell proliferation by downregulating KLF4. The mechanisms involve newly discovered roles of CHOP in the transcriptional and post-translational regulation of KLF4.
Courselaud B, etal., J Biol Chem 2002 Oct 25;277(43):41163-70.
Originally identified as a gene up-regulated by iron overload in mouse liver, the HEPC gene encodes hepcidin, the first mammalian liver-specific antimicrobial peptide and potential key regulator of iron metabolism. Here we demonstrate that during rat liver development, amounts of HEPC transcripts we
re very low in fetal liver, strongly and transiently increased shortly after birth, and reappeared in adult liver. To gain insight into mechanisms that regulate hepatic expression of hepcidin, 5'-flanking regions of human and mouse HEPC genes were isolated and analyzed by functional and DNA binding assays. Human and mouse HEPC promoter-luciferase reporter vectors exhibited strong basal activity in hepatoma HuH-7 and mouse hepatocytes, respectively, but not in non-hepatic U-2OS cells. We found that CCAAT/enhancer-binding protein alpha (C/EBPalpha) and C/EBPbeta were respectively very potent and weak activators of both human and mouse promoters. In contrast, co-expression of hepatocyte nuclear factor 4alpha (HNF4alpha) failed to induce HEPC promoter activity. By electrophoretic mobility shift assay we demonstrated that one putative C/EBP element found in the human HEPC promoter (-250/-230) predominantly bound C/EBPalpha from rat liver nuclear extracts. Hepatic deletion of the C/EBPalpha gene resulted in reduced expression of HEPC transcripts in mouse liver. In contrast, amounts of HEPC transcripts increased in liver-specific HNF4alpha-null mice. Decrease of hepcidin mRNA in mice lacking hepatic C/EBPalpha was accompanied by iron accumulation in periportal hepatocytes. Finally, iron overload led to a significant increase of C/EBPalpha protein and HEPC transcripts in mouse liver. Taken together, these data demonstrate that C/EBPalpha is likely to be a key regulator of HEPC gene transcription and provide a novel mechanism for cross-talk between the C/EBP pathway and iron metabolism.
Taniguchi M, etal., FEBS Lett. 2005 Oct 24;579(25):5785-90. Epub 2005 Oct 6.
The rat catalase gene carries a TATA-less promoter and its transcriptional mechanism is interesting because of downregulation in liver injury. We characterized the core element in the promoter and found that C/EBP-beta binding downstream of the transcription ini
tiation site plays a crucial role for transcription. The multiple complexes binding to the promoter were composed of homodimers and heterodimers of C/EBP-beta isoforms. Transduction of the C/EBP-beta gene showed complete reconstitution of multiple binding complexes in HeLa cells, similar to normal liver. Furthermore, C/EBP-beta was observed to bind to the endogenous catalase promoter. These data suggest that multiple complex formation of C/EBP-beta regulates transcription in the TATA-less catalase promoter.
The concept of macrophage polarization toward different phenotypes after CNS injury has been increasingly discussed. Here, we propose that CD200 treatment may help shift pro-inflammatory macrophages to an arginase 1 (Arg1)-, transglutaminase 2 (TGM2)-, and transforming growth factor beta 1 (TGF-beta
)-positive phenotype. Rat macrophages were stimulated by interferon gamma and lipopolysaccharide (LPS) to induce pro-inflammatory phenotypes. Treatment with human CD200-Fc up-regulated expression levels of alternatively activated M2-like markers such as Arg1 and TGM2 but suppressed pro-inflammatory M1-like markers such as toll-like receptor 4, interleukin 1 beta (IL-1beta), IL-6, and GM-CSF. Concomitantly, CD200-Fc enhanced (CCAAT/enhancer-binding protein) C/EBP-beta promoter activity, whereas NF-kappaB activity was suppressed. Treatment with CD200-Fc also up-regulated potentially beneficial TGF-beta expression in macrophages. When C/EBP-beta signaling was suppressed with siRNA, the effect of CD200-Fc on Arg1, TGM2 and TGF-beta up-regulation was canceled. Taken together, these data provide proof-of-principle that targeting CD200 signaling may be a novel therapeutic approach to shift macrophages toward M2-like polarization via modulating cAMP-response element binding protein-C/EBP-beta transcriptional activity. We showed that CD200 treatment decreased pro-inflammatory cytokines (IL-1beta, IL-6, and GM-CSF) along with suppressed inflammatory NF-kappaB activity in pro-inflammatory Mphi. On the other hand, CD200 increased Arg1, TGM2, and TGF-beta production through CREB-C/EBPbeta signaling. We think that these findings provide proof-of-concept that CD200 signaling may play a key role in regulating macrophage polarization toward anti-inflammatory phenotypes.
Ron D and Habener JF, Genes Dev. 1992 Mar;6(3):439-53.
We report on the identification of a nuclear protein that serves as a dominant-negative inhibitor of the transcription factors C/EBP and LAP. A 32P-labeled LAP DNA-binding and dimerization domain "zipper probe" was used to isolate a clone that encodes a new C/... (more)
pan style='font-weight:700;'>EBP-homologous protein: CHOP-10. CHOP-10 has strong sequence similarity to C/EBP-like proteins within the bZIP region corresponding to the DNA-binding domain consisting of a leucine zipper and a basic region. Notably, however, CHOP-10 contains 2 prolines substituting for 2 residues in the basic region, critical for binding to DNA. Thus, heterodimers of CHOP-10 and C/EBP-like proteins are unable to bind their cognate DNA enhancer element. CHOP-10 mRNA is expressed in many different rat tissues. Antisera raised against CHOP-10 recognize a nuclear protein with an apparent molecular mass of 29 kD. CHOP-10 is induced upon differentiation of 3T3-L1 fibroblasts to adipocytes, and cytokine-induced dedifferentiation of adipocytes is preceded by the loss of nuclear CHOP-10. Coimmunoprecipitation of CHOP-10 and LAP from transfected COS-1 cells demonstrated a direct interaction between the two proteins, in vivo. Consistent with the structure of its defective basic region, bacterially expressed CHOP-10 inhibits the DNA-binding activity of C/EBP and LAP by forming heterodimers that cannot bind DNA. In transfected HepG2 cells, expression of CHOP-10 attenuates activation of C/EBP- and LAP-driven promoters. We suggest that CHOP-10 is a negative modulator of the activity of C/EBP-like proteins in certain terminally differentiated cells, similar to the regulatory function of Id on the activity of MyoD and MyoD-related proteins important in the development of muscle cells.
Xi S, etal., PLoS One. 2010 Oct 29;5(10):e13764. doi: 10.1371/journal.pone.0013764.
BACKGROUND: Limited information is available regarding mechanisms by which miRNAs contribute to pulmonary carcinogenesis. The present study was undertaken to examine expression and function of miRNAs induced by cigarette smoke condensate (CSC) in normal human respiratory epithelia and lun
g cancer cells. METHODOLOGY: Micro-array and quantitative RT-PCR (qRT-PCR) techniques were used to assess miRNA and host gene expression in cultured cells, and surgical specimens. Software-guided analysis, RNA cross-link immunoprecipitation (CLIP), 3' UTR luciferase reporter assays, qRT-PCR, focused super-arrays and western blot techniques were used to identify and confirm targets of miR-31. Chromatin immunoprecipitation (ChIP) techniques were used to evaluate histone marks and transcription factors within the LOC554202 promoter. Cell count and xenograft experiments were used to assess effects of miR-31 on proliferation and tumorigenicity of lung cancer cells. RESULTS: CSC significantly increased miR-31 expression and activated LOC554202 in normal respiratory epithelia and lung cancer cells; miR-31 and LOC554202 expression persisted following discontinuation of CSC exposure. miR-31 and LOC554202 expression levels were significantly elevated in lung cancer specimens relative to adjacent normal lung tissues. CLIP and reporter assays demonstrated direct interaction of miR-31 with Dickkopf-1 (Dkk-1) and DACT-3. Over-expression of miR-31 markedly diminished Dkk-1 and DACT3 expression levels in normal respiratory epithelia and lung cancer cells. Knock-down of miR-31 increased Dkk-1 and DACT3 levels, and abrogated CSC-mediated decreases in Dkk-1 and DACT-3 expression. Furthermore, over-expression of miR-31 diminished SFRP1, SFRP4, and WIF-1, and increased Wnt-5a expression. CSC increased H3K4Me3, H3K9/14Ac and C/EBP-β levels within the LOC554202 promoter. Knock-down of C/EBP-β abrogated CSC-mediated activation of LOC554202. Over-expression of miR-31 significantly enhanced proliferation and tumorigenicity of lung cancer cells; knock-down of miR-31 inhibited growth of these cells. CONCLUSIONS: Cigarette smoke induces expression of miR-31 targeting several antagonists of cancer stem cell signaling in normal respiratory epithelia and lung cancer cells. miR-31 functions as an oncomir during human pulmonary carcinogenesis.
T/EBP/NKX2.1, a member of the NKX family of homeodomain-containing transcription factors, regulates the expression of a number of genes in lung and thyroid. Here we describe the isolation and characterization of a novel target gene, termed claudin-18, that is do
wn-regulated in the lungs of T/ebp/Nkx2.1-null mouse embryos. The gene product exhibits an amino acid sequence similar to those of the claudin multigene family of proteins that constitute tight junction strands in epithelial cells. The gene was localized by fluorescence in situ hybridization to mouse chromosome 9 at region 9E3-F1 and to human chromosome 3 at region 3q21-23. The claudin-18 gene has two promoters, each with its own unique exon 1 that is spliced to common exons 2 through 5. Alternative usage of these promoters leads to production of lung and stomach-specific transcripts. The downstream lung-specific promoter contains two T/EBP/NKX2.1 binding sites responsible for trans activation of the gene by T/EBP/NKX2.1 in lung cells. Only claudin-18 was down-regulated in T/ebp/Nkx2.1-null embryo lungs among 11 claudin transcripts examined. Furthermore, the claudin-18 transcript has an alternative 12-bp insertion derived from the 5' end of intron 4, which produces a C-terminally truncated isoform in lung and stomach. Immunohistochemistry demonstrated complete membrane localization of claudin-18 with small focal dots in the lung and stomach epithelial cells. Immunogold electron microscopy analysis revealed that claudin-18 is concentrated at the cell-cell borders of epithelial cells. These unique features suggest a potentially important role for claudin-18 in the structure and function of tight junctions in lung and stomach.
Davydov IV, etal., Gene. 1995 Aug 19;161(2):271-5.
The positive regulatory element-I (PRE-I) is a strong enhancer element essential for expression of the human interleukin-4 (IL-4)-encoding gene. In order to identify transcription factors binding to PRE-I, we screened a cDNA expression library from human Jurkat T-cells. A cDNA encoding the human CC
AAT/enhancer binding protein-gamma (hC/EBP gamma) was cloned. The deduced amino acid (aa) sequence of HC/EBP gamma contains 150 aa with high homology to mouse Ig/EBP-1 and rat C/EBP gamma. The mRNA of hC/EBP gamma is expressed at a high level in Jurkat T-cells in three forms generated via differential polyadenylation. DNA-binding experiments with recombinant protein produced in bacteria demonstrate that hC/EBP gamma binds to PRE-I, but not to unrelated DNA fragments. Our data also show that hC/EBP gamma may cooperate with Fos to bind PRE-I.
Jiang L, etal., Biochemistry. 1996 Oct 8;35(40):13136-46.
Primer extension and RNase protection analyses of the rat beta 2-adrenergic receptor (beta 2AR) gene identify two transcription start points at -64 and -220 nt, respectively. Transient transfections of putative promoter/pCAT constructs into DDT1 MF-2 cells indicate that fragments -36 to -100 (PI) a
nd -186 to -312 (P2) are sufficient to promote transcription, whereas -911 to -1122 contains a negative regulatory element(s). RNase protection analysis of the 3' untranslated region (3'-UTR) indicates the presence of two transcripts with 3'-UTR of 111 and 604 nt exclusive of the poly(A+) tails. Northern blots of beta 2AR mRNA using full-length and partial cDNA probes indicate that a major 2.2 kb and a minor 1.6 kb species arise from the use of alternative promoters as well as different polyadenylation signals. DNase I footprinting and DNA mobility shift assays (DMSA) using rat liver nuclear extracts identify a number of transcription factors binding to sequence elements within or upstream from P1 and P2, including Spl, CRE, CPl, AP-2, NF-1, NF-kappa B, and C/EBP. Supershift assays using antibodies against C/EBP alpha and C/EBP beta and mutational analyses indicate that the protein binding to the C/EBP consensus recognition site at -925 to -933 is C/EBP alpha. The activity of promoter/CAT constructs containing the C/EBP recognition site is significantly decreased by cotransfection of C/EBP alpha but not C/EBP alpha but not C/EBP beta into either DDT1 MF-2 cells or primary rat hepatocytes. Partial hepatectomy causes a transient decrease in C/EBP alpha, as measured by DMSA, and an increase in beta 2 AR mRNA levels and rate of transcription in the remnant liver. Thus, derepression via C/EBP alpha is likely involved in the up-regulation of beta 2AR in the regenerating rat liver.
BACKGROUND/AIMS: Several cytochromes P450 (CYPs) are expressed in differentiated hepatocytes, but downregulated in growth-stimulated cells. We determined the signals involved in CYP downregulation by epidermal growth factor (EGF). METHODS: Rat hepatocytes were cultured with or without diverse subst
ances for 72 h and EGF for the last 48 h. RESULTS: EGF increased c-myc mRNA and protein, and decreased CYP mRNAs and proteins; both effects were prevented by two agents blocking c-myc transcription (retinoic acid and DMSO) and two antisense c-myc oligomers. Despite unchanged CCAAT-enhancer binding protein alpha (C/EBPalpha) and increased C/EBPbeta levels, nuclear proteins of EGF-treated cells did not bind to a C/EBP DNA probe in a gel mobility shift assay. This binding was restored when cells were co-treated with both EGF and c-myc antisense oligomers (preventing c-Myc induction). The N-terminal c-Myc domain added to control nuclear extracts prevented C/EBP DNA binding. A monoclonal anti-c-Myc antibody co-immunoprecipitated c-Myc, C/EBPalpha and C/EBPbeta from nuclear extracts. In cells not treated with EGF, an antisense C/EBPalpha oligomer decreased CYP expression. CONCLUSIONS: EGF overexpresses c-Myc, decreases C/EBP binding to DNA and downregulates CYPs. We suggest that c-Myc may form inactive complexes with C/EBPs, thus decreasing C/EBP-mediated CYP transactivation.
Fujinami Y, etal., Neurochem Int. 2010 Feb;56(3):487-94. doi: 10.1016/j.neuint.2009.12.008. Epub 2009 Dec 21.
We previously reported that treatment of the rat cochlea with a mitochondrial toxin, 3-nitropropionic acid (3-NP), causes temporary to permanent hearing loss depending on the amount of the drug. Furthermore, apoptosis of cochlear lateral wall fibrocytes, which are important for maintaining the endol
ymph, is a predominant pathological feature in this animal model. 3-NP is known to induce oxidative stress as well as neuronal apoptosis. C/EBP homologous protein gene (chop) is one of the marker genes induced during endoplasmic reticulum (ER) stress, and is also considered to be involved in apoptosis. To elucidate the molecular mechanism of cochlear fibrocyte apoptosis induced by 3-NP, we studied spatiotemporal expression of C/EBP homologous protein (CHOP) and other signaling molecules related to ER stress as well as the appearance of apoptotic cells in the cochlear lateral wall after 3-NP treatment. Quantitative real-time PCR revealed that chop and activating transcription factor 4 gene (atf-4) showed marked increase within 6h, whereas expression of other ER stress-responsive genes such as grp78 and grp94 did not change. Immunohistochemistry showed that 3-NP treatment caused up-regulation of CHOP, especially in type II and type IV fibrocytes, followed by the appearance of terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling (TUNEL)-positive apoptotic cells in the same confined area. Thus, apoptosis of lateral wall fibrocytes induced by 3-NP is likely to be mediated by induction of CHOP. These results contribute clarification of pathological mechanism of cochlear fibrocytes and may lead to development of novel therapeutic strategy for hearing loss.
Sarkar N, etal., J Viral Hepat. 2015 Oct;22(10):817-27. doi: 10.1111/jvh.12390. Epub 2015 Feb 26.
Effective recognition of viral infection and successive activation of antiviral innate immune responses are vital for host antiviral defence, which largely depends on multiple regulators, including Toll-like receptors (TLRs) and microRNAs. Several early reports suggest that specific TLR-mediated imm
une responses can control hepatitis B virus (HBV) replication and express differentially with disease outcome. Considering the versatile function of miR-155 in the TLR-mediated innate immune response, we aimed to study the association between miR-155 and TLRs and their subsequent impact on HBV replication using both a HBV-replicating stable cell line (HepG2.2.15) and HBV-infected liver biopsy and serum samples. Our results showed that miR-155 was suppressed during HBV infection and a subsequent positive correlation of miR-155 with TLR7 activation was noted. Further, ectopic expression of miR-155 in vitro reduced HBV load as evidenced from reduced viral DNA, mRNA and subsequently reduced level of secreted viral antigens (HBsAg and HBeAg). Our results further suggested that CCAAT/enhancer-binding protein-beta (C/EBP-beta), a positive regulator of HBV transcription, was inhibited by miR-155. Taken together, our study established a correlation between miR-155 and TLR7 during HBV infection and also demonstrated in vitro that increased miR-155 level could help to reduce HBV viral load by targeting C/EBP-beta.
NFIL3 (nuclear factor IL-3 regulated) is a multifunctional transcription factor implicated in a wide range of physiological processes, including cellular survival, circadian gene expression and natural killer cell development. We recently demonstrated that NFIL3 acts as a repressor of CREB-induced g
ene expression underlying the regeneration of axotomized DRG sensory neurons. In this study we performed chromatin immunoprecipitation assays combined with microarray technology (ChIP-chip) to reveal direct NFIL3 and CREB target genes in an in vitro cell model for regenerating DRG neurons. We identified 505 promoter regions bound by NFIL3 and 924 promoter regions bound by CREB. Based on promoter analysis of NFIL3-bound genes, we were able to redefine the NFIL3 consensus-binding motif. Histone H3 acetylation profiling and gene expression microarray analysis subsequently indicated that a large fraction (>60%) of NFIL3 target genes were transcriptionally silent, whereas CREB target genes in general were transcriptionally active. Only a small subset of NFIL3 target genes also bound CREB. Computational analysis indicated that a substantial number of NFIL3 target genes share a C/EBP (CCAAT/Enhancer Binding Protein) DNA binding motif. ChIP analysis confirmed binding of C/EBPs to NFIL3 target genes, and knockdown of C/EBPα, C/EBPβ and C/EBPδ, but not C/EBPγ, significantly reduced neurite outgrowth in vitro. Together, our findings show that NFIL3 is a general feed-forward repressor of basic leucine zipper transcription factors that control neurite outgrowth.
Ren YA, etal., Biol Reprod. 2016 Feb;94(2):44. doi: 10.1095/biolreprod.115.133058. Epub 2016 Jan 6.
Ovulation and luteinization are initiated in preovulatory follicles by the luteinizing hormone (LH) surge; however, the signaling events that mediate LH actions in these follicles remain incompletely defined. Two key transcription factors that are targets of LH surge are C/EBP
00;'>EBPalpha and C/EBPbeta, and their depletion in granulosa cells results in complete infertility. Microarray analyses of these mutant mice revealed altered expression of a number of genes, including growth arrest specific-1 (Gas1). To investigate functions of Gas1 in ovulation- and luteinization-related processes, we crossed Cyp19a1-Cre and Gas1(flox/flox) mice to conditionally delete Gas1 in granulosa and cumulus cells. While expression of Gas1 is dramatically increased in granulosa and cumulus cells around 12-16 h post-human chorionic gonadotropin (hCG) stimulation in wild-type mice, this increase is abolished in Cebpa/b double mutant and in Gas1 mutant mice. GAS1 is also dynamically expressed in stromal cells of the ovary independent of C/EBPalpha/beta. Female Gas1 mutant mice are fertile, exhibit enhanced rates of ovulation, increased fertility, and higher levels of Areg and Lhcgr mRNA in granulosa cells. The morphological appearance and vascularization of corpora lutea appeared normal in these mutant females. Interestingly, levels of mRNA for a number of genes (Cyp11a1, Star, Wnt4, Prlr, Cd52, and Sema3a) associated with luteinization are decreased in corpora lutea of Gas1 mutant mice as compared with controls at 24 h post-hCG; these differences were no longer detectable by 48 h post-hCG. The C/EBP target Gas1 is induced in granulosa cells and is associated with ovulation and luteinization.
To study the role of nuclear regulatory proteins in mediating dietary effects, hepatic CCAAT/enhancer binding protein (C/EBP), mRNA and transcription rate were measured for C/EBP-alpha and C/EBP
P-beta in nutritional states that profoundly alter energy metabolism and growth. Weanling male Sprague-Dawley rats were fed riboflavin-sufficient (R+) or deficient (R-) diets for 4 wk. A diet-restricted, pair-fed (RP) group was maintained concurrently, because riboflavin-deficient rats voluntarily decrease food consumption by approximately 50% compared with controls. Half of each group was deprived of food for 48 h. The 4-wk treatment altered hepatic levels of both proteins (P < 0.05). C/EBP-alpha protein levels were increased -twofold by diet restriction. C/ EBP-beta protein levels were increased nearly threefold by riboflavin deficiency. Starvation had no significant effect on the expression of either protein. We investigated the mechanism responsible for increased protein by measuring steady-state mRNA levels and transcription rates for C/EBP-alpha and C/EBP-beta. In both isoforms, increases in mRNA were parallel to increases in transcription rates. The nutrient-induced changes in protein, mRNA and transcription rates could not be attributed only to alterations in serum glucagon or insulin concentrations. We conclude that 1) C/EBP-alpha and C/EBP-beta expression responds to diet but may involve different dietary signals for diet restriction vs. riboflavin deficiency; 2) the dietary regulation of C/EBP-alpha and C/EBP-beta expression seems to be controlled in part at the level of gene transcription; and 3) C/EBP-alpha and C/EBP-beta nuclear proteins, by virtue of their increased quantities, may participate in regulating altered energy metabolism and growth by influencing hepatic transcription of key metabolic enzymes.
Zhang J, etal., J Surg Res. 2015 May 15;195(2):588-95. doi: 10.1016/j.jss.2015.01.039. Epub 2015 Jan 28.
BACKGROUND: Endoplasmic reticulum (ER) stress and autophagy each play important roles in hepatocyte cell injury. We hypothesized that gene expression of C/EBP-homologous protein (CHOP) and the BH3 proteins Bcl2-interacting mediator of cell death (BIM)
and BH3-interacting domain death agonist (BID) are involved in a complex interplay that regulates ER stress-induced autophagy and cell death. MATERIALS AND METHODS: Hepatocytes were cultured from lean Zucker rats. Confluent hepatocytes were incubated with single or combined small interfering RNA for CHOP, BIM, and/or BID for 24 h providing gene inhibition. Incubation with tunicamycin (TM) for another 24 h stimulated ER stress. Quantitative real-time polymerase chain reaction determined the expression levels of CHOP, BIM, and BID. Immunostaining with microtubule-associated protein 1 light chain 3 measured autophagy activity. Trypan blue exclusion determined the cell viability. RESULTS: TM treatment increased the messenger RNA levels of CHOP and BIM but decreased the messenger RNA levels of BID. TM increased autophagy and decreased cell viability. Individual inhibition of CHOP, BIM, or BID protected against autophagy and cell death. However, simultaneous treatment with any combination of CHOP, BIM, and BID small interfering RNAs reduced autophagy activity but increased cell death independent of ER stress induction. CONCLUSIONS: Autophagy in hepatocytes results from acute ER stress and involves interplay, at the gene expression level, of CHOP, BIM, and BID. Inhibition of any one of these individual genes during acute ER stress is protective against cell death. Conversely, inhibition of any two of the three genes results in increased nonautophagic cell death independent of ER stress induction. This study suggests interplay between CHOP, BIM, and BID expression that can be leveraged for protection against ER stress-related cell death. However, disruption of the CHOP/BH3 gene expression homeostasis is detrimental to cell survival independent of other cellular stress.
Macrophages and dendritic cells (DC) are distributed throughout the body and play important roles in pathogen detection and tissue homeostasis. In tissues, resident macrophages exhibit distinct phenotypes and activities, yet the transcriptional pathways that specify tissue-specific macrophages are l
argely unknown. We investigated the functions and origins of two peritoneal macrophage populations in mice: small and large peritoneal macrophages (SPM and LPM, respectively). SPM and LPM differ in their ability to phagocytose apoptotic cells, as well as in the production of cytokines in response to LPS. In steady-state conditions, SPM are sustained by circulating precursors, whereas LPM are maintained independently of hematopoiesis; however, both populations are replenished by bone marrow precursors following radiation injury. Transcription factor analysis revealed that SPM and LPM express abundant CCAAT/enhancer binding protein (C/EBP)-β. Cebpb(-/-) mice exhibit elevated numbers of SPM-like cells but lack functional LPM. Alveolar macrophages are also missing in Cebpb(-/-) mice, although macrophage populations in the spleen, kidney, skin, mesenteric lymph nodes, and liver are normal. Adoptive transfer of SPM into Cebpb(-/-) mice results in SPM differentiation into LPM, yet donor SPM do not generate LPM after transfer into C/EBPβ-sufficient mice, suggesting that endogenous LPM inhibit differentiation by SPM. We conclude that C/EBPβ plays an intrinsic, tissue-restricted role in the generation of resident macrophages.
Cooper C, etal., Nucleic Acids Res. 1995 Nov 11;23(21):4371-7.
Analysis of cDNA and genomic clones shows that the murine Ig/EBP (C/EBP gamma) gene encodes a small protein with a predicted molecular weight of 16.4 kDa which contains C/EBP family bas
ic and leucine zipper domains but lacks the transcriptional activation domains present in C/EBP (C/EBP alpha) and NF-IL6 (C/EBP beta). In transfection assays Ig/EBP is neither an activator nor a repressor of transcription; however, Ig/EBP inhibits the transcriptional ability of NF-IL6 (C/EBP beta) and C/EBP (C/EBP alpha), acting as a transdominant negative regulator. Thus Ig/EBP resembles LIP, another negative regulator of the C/EBP family, in both structure and transcriptional activity. Of the three known C/EBP family inhibitors, Ig/EBP, LIP and CHOP-10, only Ig/EBP is ubiquitously expressed. Therefore, Ig/EBP may act as a general buffer for C/EBP activators in many cell types.
We analyzed a family of proteins from hepatoma cell nuclei that bind to interleukin-6 responsive elements (IL-6REs) of several acute-phase genes. This family is characterized by leucine zipper domains compatible with that of the CCAAT/enhancer binding protein (C/EBP
pan>). A cDNA clone coding for a member of the family, IL-6DBP, was isolated; it is strongly homologous to C/EBP in the region of the basic domain and in the leucine zipper sequence. IL-6DBP and C/EBP can interact in vitro to form heterodimers that bind to DNA with the same specificity as the respective homodimers, and they can interact functionally in vivo. Both the DNA binding activity and the trans-activating capacity of IL-6DBP are induced in hepatoma cells by treatment with IL-6 through a posttranslational mechanism, implicating it as a nuclear target of IL-6 and as a mediator of the IL-6-dependent transcriptional activation of liver genes during the acute-phase response.
Sylvester SL, etal., J Biol Chem. 1994 Aug 5;269(31):20119-25.
CCAAT/enhancer-binding proteins (C/EBPs) comprise a homologous group of transcriptional regulators that control liver and fat differentiation and are involved in regulating the expression of acute phase reactants during the host response to inflammation. GADD153
, a unique member of the C/EBP family, has been proposed to act as a dominant negative inhibitor of other C/EBPs, but little is known about its expression in liver or its role in the processes described above. We have examined its expression during the acute phase response (APR) and have shown that like C/EBP beta and C/EBP delta, GADD153 mRNA is markedly induced in livers of rats treated with lipopolysaccharide to initiate the APR. Interestingly, its induction is temporally delayed relative to that of C/EBP beta and C/EBP delta but is similar to that of acute phase reactants shown to be regulated by C/EBPs. Footprint analysis of the GADD153 promoter showed binding of proteins in liver extracts of both untreated and lipopolysaccharide-injected rats to a putative C/EBP regulatory site. Gel shift analysis showed that although present constitutively, binding activity was increased in extracts from lipopolysaccharide-treated animals. Both C/EBP alpha and C/EBP beta were shown to contribute to the binding activity with the contribution by C/EBP beta increasing during the APR. Support for the functional role of this C/EBP-binding site and its interaction with C/EBPs in regulating GADD153 expression was obtained with cultured HepG2 hepatoma cells in which overexpression of C/EBP beta was found to transactivate expression of a plasmid containing the GADD153 promoter linked to a reporter gene. These findings suggest that the GADD153 gene is itself regulated by C/EBPs during the host response to inflammation and that GADD153 is likely to contribute to the regulation of other genes whose expression is altered during the APR.
Gong B, etal., Mol Immunol. 2013 Dec;56(4):619-29. doi: 10.1016/j.molimm.2013.06.016. Epub 2013 Aug 1.
BACKGROUND: Complement component C5-derived C5a locally generated in the brain has been shown to protect against glutamate-induced neuronal apoptosis and beta-amyloid (Abeta) toxicity, but the mechanism is not clear. In this study, we tested the hypothesis that C5a influences upstream signal transd
uction pathways associated with cAMP-response element-binding protein (CREB) activation, in which alterations of CREB levels are associated with cognitive deterioration in AD. METHODS: CREB signaling pathway, synaptic plasticity and cognitive function were studied in C5a receptor knockout mice (C5aR(-/-)), C5a over expressing mice (C5a/GFAP) and in Tg2576 mice, an AD mouse model. RESULTS: (1) Cognitive function is severely impaired in C5aR(-/-) mice, coincident with the down-regulated CREB/CEBP pathway in brain. (2) Either the application of recombinant-human-C5a (hrC5a) or exogenous expression of C5a in the brain of a mouse model (C5a/GFAP) enhances this pathway. (3) Application of hrC5a in brain slices from Tg2576 mice significantly improves deficits in long-term potentiation (LTP), while this effect is blocked by a specific AMPA receptor antagonist. (4) Searching for a pharmacological approach to locally mediate C5a responses in the brain, we found that low-dose human intravenous immunoglobulin (IVIG) treatment improves synaptic plasticity and cognitive function through C5a-mediated induction of the CREB/CEBP pathway, while the levels of Abeta in the brain are not significantly affected. CONCLUSION: This study for the first time provides novel evidence suggesting that C5a may beneficially influence cognitive function in AD through an up-regulation of AMPA-CREB signaling pathway. IVIG may systematically improve cognitive function in AD brain by passing Abeta toxicity.
Strategies to stimulate endogenous surfactant production require a detailed understanding of the regulation of lipogenesis in alveolar type II cells. We developed culture conditions in which keratinocyte growth factor (KGF) stimulates fatty acid and phospholipid synthesis. KGF stimulated acetate inc
orporation into phosphatidylcholine, disaturated phosphatidylcholine, and phosphatidylglycerol more than 5% rat serum alone. To determine the mRNA levels of lipogenic enzymes and transport proteins, we analyzed gene expression by oligonucleotide microarrays. KGF increased the mRNA levels for fatty acid synthase, stearoyl-CoA desaturase-1 (SCD-1), and epidermal fatty acid-binding protein more than rat serum alone. In addition, KGF increased the mRNA levels of the transcription factors CCAAT/enhancer-binding protein alpha (C/EBPalpha) and C/EBPdelta as well as SREBP-1c (ADD-1), but not PPARgamma. These changes in C/EBPalpha and C/EBPdelta were confirmed by in situ hybridization. SCD-1 was also found to be highly expressed in alveolar type II cells in vivo. Furthermore, KGF increased protein levels of fatty acid synthase, C/EBPalpha, C/EBPdelta, SREBP-1, epidermal fatty acid-binding protein, and SCD. Finally, the liver X receptor agonist T0901317 increased acetate incorporation and SREBP-1 but not SREBP-2 protein levels. In summary, KGF stimulates lipogenesis in type II cells by a coordinated expression of lipogenic enzymes and transport proteins regulated by C/EBP isoforms and SREBP-1c.
Descombes P, etal., Genes Dev 1990 Sep;4(9):1541-51.
A gene, encoding a liver-enriched transcriptional activator protein (LAP) has been isolated. LAP is a 32-kD protein that stimulates the transcription of chimeric genes containing albumin D-promoter elements both in vivo and in vitro. LAP shares extensive sequence homology (71%) in its DNA-binding an
d leucine zipper domains with C/EBP. As a consequence, these two proteins show an indistinguishable DNA-binding specificity and readily heterodimerize. In addition, both genes, lap and cebp, are devoid of intervening sequences. Although correctly initiated transcripts from the LAP gene accumulate in the six examined tissues--liver, lung, spleen, kidney, brain, and testis--LAP protein is highly enriched in liver nuclei. Thus, the preferential accumulation of LAP protein in liver appears to be regulated post-transcriptionally.
Rosenberg E, etal., Biochim Biophys Acta 2002 May 3;1575(1-3):82-90.
Members of the CCAAT enhancer binding protein (C/EBP) transcription factor family were detected in fetal lung of both human and rat. In rat lung, the level of C/EBPs increased with time of gestation, peaking around birth. In
adult rat lung, C/EBPs were localized to the alveolar type II cells. The effect of C/EBPs on pulmonary surfactant protein A (SP-A), which is also expressed late in gestation, was investigated. In contrast to control plasmids, C/EBP delta expressing plasmids reversed the action of a transcriptional silencer just upstream of the rat SP-A promoter. In order to test the effect of C/EBPs on endogenous SP-A gene expression, cells that express SP-A were exposed to a phosphorothioate-substituted, double-stranded oligonucleotide matching the consensus C/EBP binding site (decoy oligonucleotide) at concentrations from 0.5 to 10 microM for 72 h. A mutant oligonucleotide with an 8-base pair (bp) substitution served as a control. The decoy oligonucleotide reduced SP-A mRNA as much as 75% compared to a mutant oligonucleotide both in the human lung cell line, NCI-H441, and in primary human fetal alveolar type II cells. The data indicate that C/EBPs facilitate SP-A gene expression, possibly by overcoming transcriptional silencing.
For efficient clearance of Mycobacterium tuberculosis (Mtb), macrophages tilt towards M1 polarization leading to the activation of transcription factors associated with the production of antibacterial effector molecules such as nitric oxide (NO) and proinflammatory cytokines such as interleukin 1 β
(IL-1β) and tumor necrosis factor α (TNF-α). At the same time, resolution of inflammation is associated with M2 polarization with increased production of arginase and cytokines such as IL-10. The transcriptional and post-transcriptional mechanisms that govern the balance between M1 and M2 polarization, and bacteria-containing processes such as autophagy and trafficking of Mtb to lysosomes, are incompletely understood. Here we report for the first time, that the transcription factor KLF4 is targeted by microRNA-26a (miR-26a). During Mtb infection, downregulation of miR-26a (observed both ex vivo and in vivo) facilitates upregulation of KLF4 which in turn favors increased arginase and decreased iNOS activity. We further demonstrate that KLF4 prevents trafficking of Mtb to lysosomes. The CREB-C/EBPβ signaling axis also favors M2 polarization. Downregulation of miR-26a and upregulation of C/ebpbeta were observed both in infected macrophages as well as in infected mice. Knockdown of C/ebpbeta repressed the expression of selected M2 markers such as Il10 and Irf4 in infected macrophages. The importance of these pathways is substantiated by observations that expression of miR-26a mimic or knockdown of Klf4 or Creb or C/ebpbeta, attenuated the survival of Mtb in macrophages. Taken together, our results attribute crucial roles for the miR-26a/KLF4 and CREB-C/EBPβsignaling pathways in regulating the survival of Mtb in macrophages. These studies expand our understanding of how Mtb hijacks host signaling pathways to survive in macrophages, and open up new exploratory avenues for host-targeted interventions.
Kageyama R, etal., J Biol Chem 1991 Aug 15;266(23):15525-31.
A cAMP response element (CRE) plays an important role in the cAMP-mediated gene regulation. Several factors that recognize a CRE have been characterized, and it has been shown that they need either covalent modification by protein kinase A or a cofactor such as the adenovirus Ela to function as an a
ctivator. In this study we show that the substance P precursor gene expression is regulated by protein kinase A and identify the CRE sequence in its promoter region. We find that a novel factor and ATF2 bind to the region containing the CRE of the substance P precursor gene. The sequence analysis indicates that the novel protein, designated CELF, has a significant homology to C/EBP gene family proteins in the carboxyl-terminal part containing the basic region and the leucine zipper motif. Ubiquitous expression of CELF suggests that this factor is utilized by various genes. Cell-free transcription analyses indicate that CELF is a constitutive transcriptional activator without apparent phosphorylation by protein kinase A. These results demonstrate that multiple factors are responsible for transcriptional control of the substance P precursor gene through the CRE region.
Thomassin H, etal., Nucleic Acids Res 1992 Jun 25;20(12):3091-8.
In an attempt to identify proteins that may regulate alpha 1-fetoprotein (AFP) gene expression, we screened a cDNA expression library from neonatal rat liver with two essential cis-elements of the AFP promoter and enhancer. We isolated two cDNAs which were found to correspond to leucine zipper prote
ins of the CC-AAT/enhancer binding protein (C/EBP) family: C/EBP beta and C/EBP gamma. The three related proteins C/EBP alpha, beta and gamma bind with indistinguishable specificity to multiple DNA sites in the promoter and the enhancer of the AFP gene. In addition, C/EBP beta and C/EBP gamma readily heterodimerize with each other as well as with C/EBP alpha. The mRNAs coding for C/EBP beta and C/EBP gamma are expressed in a wider variety of rat tissues than C/EBP alpha mRNA, including yolk sac and fetal liver. The steady-state levels of C/EBP alpha, beta and gamma mRNAs increase during liver development, in parallel with their respective gene transcriptional rates. The high levels of C/EBP beta and gamma mRNAs in rat yolk sac and fetal liver, where C/EBP alpha is poorly expressed, suggest that C/EBP beta and/or gamma could be preponderant or early regulators of the AFP gene in these tissues.
Milunsky JM, etal., Am J Med Genet A. 2003 Jan 30;116A(3):249-54. doi: 10.1002/ajmg.a.10849.
X-linked dominant Conradi-Hunermann-Happle syndrome (CDPX2; MIM 302960) is a rare chondrodysplasia punctata primarily affecting females. CDPX2 is presumed lethal in males, although a few affected males have been reported. CDPX2 is a cholesterol biosynthetic disorder due to 3-beta-hydroxysteroid-delt
a8,delta7-isomerase deficiency caused by mutations in the emopamil binding protein (EBP) gene. A 2.5-year-old Caucasian male was followed from the age of 6 weeks and noted to have significant developmental delay, hypotonia, seizures, and patchy hypopigmentation. Multiple congenital anomalies included a unilateral cataract, esotropia, crossed renal ectopia, stenotic ear canals, and failure to thrive, requiring G-tube placement. Multiple minor anomalies and ptosis were noted. No skeletal asymmetry or chondrodysplasia punctata were noted on skeletal survey at 6 weeks and 13 months. An extensive genetic work-up including cholesterol (126-176 mg/dl) and 7-dehydrocholesterol was unrevealing. However, the levels of 8(9)-cholestenol and 8-dehydrocholesterol were mildly increased in plasma, which was confirmed in cultured fibroblasts. This prompted molecular analysis of the EBP gene, which revealed a novel hemizygous (nonmosaic) mutation in exon 2 (L18P). Two restriction digests were developed that confirmed this mutation in skin fibroblasts, blood, and buccal cells (all nonmosaic). We determined that the patient's mother (adopted) also has the L18P mutation enabling prenatal diagnosis of a normal male fetus. She has normal stature, no asymmetry, no cataracts at this time, and has a patch of hyperpigmentation on her chest best visualized on Woods lamp examination, characteristic of CDPX2. The mild maternal phenotype has been described previously. However, this nonmosaic missense mutation has resulted in a severe phenotype in her surviving son.
Gangwani MR and Kumar A, PLoS One. 2015 Aug 13;10(8):e0135633. doi: 10.1371/journal.pone.0135633. eCollection 2015.
Neurocognitive impairments affect a substantial population of HIV-1 infected individuals despite the success of anti-retroviral therapy in controlling viral replication. Astrocytes are emerging as a crucial cell type that might be playing a very important role in the persistence of neuroinflammation
seen in patients suffering from HIV-1 associated neurocognitive disorders. HIV-1 viral proteins including Vpr exert neurotoxicity through direct and indirect mechanisms. Induction of IL-8 in microglial cells has been shown as one of the indirect mechanism through which Vpr reduces neuronal survival. We show that HIV-1 Vpr induces IL-6 and IL-8 in astrocytes in a time-dependent manner. Additional experiments utilizing chemical inhibitors and siRNA revealed that HIV-1 Vpr activates transcription factors NF-kappaB, AP-1 and C/EBP-delta via upstream protein kinases PI3K/Akt, p38-MAPK and Jnk-MAPK leading to the induction of IL-6 and IL-8 in astrocytes. We demonstrate that one of the mechanism for neuroinflammation seen in HIV-1 infected individuals involves induction of IL-6 and IL-8 by Vpr in astrocytes. Understanding the molecular pathways involved in the HIV-1 neuroinflammation would be helpful in the design of adjunct therapy to ameliorate some of the symptoms associated with HIV-1 neuropathogenesis.
Persistent exposure of rats to 6-propyl-2-thiouracil (PTU) from birth resulted in decreases in plasma thyroid hormone (TH) levels and hepatic expression of catalase and CCAAT enhancer binding protein beta (C/EBP-beta). Catalase promoter region (-185 to +52) tha
t contains binding sites for C/EBP-beta showed an augmentation in the methylation level along with a change in methylation pattern of CpG islands in response to PTU treatment. PTU withdrawal on 30 days of birth restored TH levels and C/EBP-beta to control rats in adulthood. Although catalase expression was restored to some extent in adult rats in response to PTU withdrawal, a permanent change in its promoter CpG methylation pattern was recorded. The results suggest that downregulation of adult hepatic catalase gene in response to persistent neonatal PTU exposure may not solely be attributed to thyroid-disrupting properties of PTU. It is possible that besides thyroid-disrupting behavior, PTU may impair expression of hepatic catalase by altering methylation pattern of its promoter.
We report that TGF alpha induces activation of the p90 ribosomal S kinase (RSK), which results in the phosphorylation of rat C/EBP beta on Ser-105 and of mouse C/EBP beta on Thr-217 and concomitantly stimulates proliferatio
n in differentiated hepatocytes. Moreover, C/EBP beta-/- mouse hepatocytes respond to TGF alpha when wild-type C/EBP beta is reexpressed, whereas they remain refractory to the growth effect of TGF alpha when expressing phosphoacceptor mutants rat C/EBP beta Ala-105 or mouse C/EBP beta Ala-217. In contrast, C/EBP beta-/- hepatocytes expressing the phosphorylation mimic mutants, rat C/EBP beta Asp-105 or mouse C/EBP beta Glu-217, exhibited marked proliferation in the absence of TGF alpha. Thus, a site-specific phosphorylation of the transcription factor C/EBP beta is critical for hepatocyte proliferation induced by TGF alpha and other stimuli that activate RSK.
Jimenez-Preitner M, etal., Cell Metab. 2011 Nov 2;14(5):658-70. doi: 10.1016/j.cmet.2011.08.008. Epub 2011 Oct 6.
Brown adipocytes oxidize fatty acids to produce heat in response to cold or to excessive energy intake; stimulation of brown fat development and function may thus counteract obesity. Brown adipogenesis requires activation of the transcription factor C/EBPß and r
ecruitment of the zinc finger protein Prdm16, but upstream inducers of these proteins are incompletely defined. Here, we show that genetic inactivation of Plac8, a gene encoding an evolutionarily conserved protein, induces cold intolerance, and late-onset obesity, as well as abnormal morphology and impaired function of brown adipocytes. Using brown preadipocyte lines we show that Plac8 is required for brown fat differentiation, that its overexpression induces C/EBPß and Prdm16, and that upon induction of differentiation Plac8 associates with C/EBPß and binds to the C/EBPß promoter to induce its transcription. Thus, Plac8 is a critical upstream regulator of brown fat differentiation and function that acts, at least in part, by inducing C/EBPß expression.
Hong Z, etal., Neurol Res. 2012 Jan;34(1):85-90. doi: 10.1179/1743132811Y.0000000026.
OBJECTIVES: Erythropoietin (EPO) is a variety of tissue-protective functions, including spinal cord. This study aimed to determine the neuron protective effect of erythropoietin on spinal cord injury (SCI) by assessing C/EBP-homologous protein (CHOP) in the deve
lopment of a rat model of SCI. METHODS: Sixty Sprague-Dawley rats were randomly assigned to three groups: sham-operation control group, SCI group, and EPO treatment group. By using a weight-drop contusion SCI model, the rats in the SCI group and EPO treatment group were killed at 1 and 7 days subsequently. The Basso, Beattie, and Bresnahan (BBB) scores were examined for locomotor function. Pathological changes were observed after hematoxylin-eosin (H&E) staining. The expression of CHOP was determined by immunohistochemical staining and RT-PCR analysis. RESULTS: BBB scores showed more quick recovery in the erythropoietin treatment group than that in the SCI group (P < 0.01). Pathological changes also revealed a reduction in the volume of cavitations and more neurons regeneration in the EPO treatment rats than that of the SCI rats. The number of CHOP positive cells in the SCI group on day 1 and 7 days after SCI increased compared with the erythropoietin treatment group and sham-operation control group (P < 0.01). CHOP mRNA folds in sham-operation control rat from 1 to 7 days showed the same trend. CONCLUSIONS: Endoplasmic reticulum (ER) stress was triggered at the early stage of SCI. Increased expression of CHOP can be found in the injured segment of the spinal cord after injury. EPO treatment could prevent pathological alterations from severe spinal cord injury by reducing expression of CHOP.
Iwama A, etal., J Exp Med 2002 Mar 4;195(5):547-58.
Myeloid progenitor cells give rise to a variety of progenies including dendritic cells. However, the mechanism controlling the diversification of myeloid progenitors into each progeny is largely unknown. PU.1 and CCAAT/enhancing binding protein (C/EBP) family tr
anscription factors have been characterized as key regulators for the development and function of the myeloid system. However, the roles of C/EBP transcription factors have not been fully identified because of functional redundancy among family members. Using high titer--retroviral infection, we demonstrate that a dominant-negative C/EBP completely blocked the granulocyte--macrophage commitment of human myeloid progenitors. Alternatively, Langerhans cell (LC) commitment was markedly facilitated in the absence of tumor necrosis factor (TNF)alpha, a strong inducer of LC development, whereas expression of wild-type C/EBP in myeloid progenitors promoted granulocytic differentiation, and completely inhibited TNFalpha-dependent LC development. On the other hand, expression of wild-type PU.1 in myeloid progenitors triggered LC development in the absence of TNFalpha, and its instructive effect was canceled by coexpressed C/EBP. Our findings establish reciprocal roles for C/EBP and PU.1 in LC development, and provide new insight into the molecular mechanism of LC development, which has not yet been well characterized.
Adipogenesis comprises a complex network of signaling pathways and transcriptional cascades; the GSK3ß-C/EBPß-srebf1a axis is a critical signaling pathway at early stages leading to the expression of PPAR¿2, the master regulator of adipose differentiation. Previ
ous work has demonstrated that retinoic acid inhibits adipogenesis affecting different signaling pathways. Here, we evaluated the anti-adipogenic effect of retinoic acid on the adipogenic transcriptional cascade, and the expression of adipogenic genes cebpb, srebf1a, srebf1c, pparg2, and cebpa. Our results demonstrate that retinoic acid blocks adipose differentiation during commitment, returning cells to an apparent non-committed state, since they have to be newly induced to adipose conversion after the retinoid is removed from the culture medium. Retinoic acid down regulates the expression of the adipogenic genes, srebf1a, srebf1c, pparg2, and cebpa; however, it did not down regulate the expression of cebpb, but it inhibited C/EBPß phosphorylation at Thr188, a critical step for the progression of the adipogenic program. We also found that RA inhibition of adipogenesis did not increase the expression of dlk1, the gene encoding for Pref1, a well-known anti-adipogenic factor.
Alam S, etal., Biosci Rep. 2017 Jul 16;37(4):BSR20170898. doi: 10.1042/BSR20170898. Print 2017 Aug 31.
C/EBP-homologous protein (CHOP) is a ubiquitously expressed stress-inducible transcription factor robustly induced by maladaptive endoplasmic reticulum (ER) stresses in a wide variety of cells. Here, we examined a novel function of Sigma 1 receptor (Sigmar1) in
regulating CHOP expression under ER stress in cardiomyocytes. We also defined Sigmar1-dependent activation of the adaptive ER-stress pathway in regulating CHOP expression. We used adenovirus-mediated Sigmar1 overexpression as well as Sigmar1 knockdown by siRNA in neonatal rat ventricular cardiomyocytes (NRCs); to induce ER stress, cardiomyocytes were treated with tunicamycin. Sigmar1-siRNA knockdown significantly increased the expression of CHOP and significantly induced cellular toxicity by sustained activation of ER stress in cardiomyocytes. Sigmar1 overexpression decreased the expression of CHOP and significantly decreased cellular toxicity in cells. Using biochemical and immunocytochemical experiments, we also defined the specific ER-stress pathway associated with Sigmar1-dependent regulation of CHOP expression and cellular toxicity. We found that Sigmar1 overexpression significantly increased inositol requiring kinase 1α (IRE1α) phosphorylation and increased spliced X-box-binding proteins (XBP1s) expression as well as nuclear localization. In contrast, Sigmar1 knockdown significantly decreased IRE1α phosphorylation and decreased XBP1s expression as well as nuclear transport. Taken together, these results indicate that Sigmar1-dependent activation of IRE1α-XBP1s ER-stress response pathways are associated with inhibition of CHOP expression and suppression of cellular toxicity. Hence, Sigmar1 is an essential component of the adaptive ER-stress response pathways eliciting cellular protection in cardiomyocytes.
Uematsu S, etal., J Immunol. 2007 Oct 15;179(8):5378-86. doi: 10.4049/jimmunol.179.8.5378.
The C/ebpb gene is translated into three different protein isoforms, two transcriptional activating proteins (38-kDa Full and 34-kDa liver-enriched transcriptional activation protein (LAP)) and one transcriptional inhibitory protein, by alternative use of differ
ent AUG initiation codons within the same open reading frame. The isoform 34-kDa LAP is thought to be the most transcriptionally active form of C/EBPbeta in macrophages. To assess the function of the 34-kDa LAP in vivo, we generated knock-in mice, in which methionine 20 of C/EBPbeta, the start site for the 34-kDa LAP is replaced with an alanine. The expression of the 34-kDa LAP was abolished in C/ebpb(M20A/M20A) mice. The induction of C/EBPbeta target genes, such as inflammatory cytokines, chemokines, prostanoid synthetase, and antimicrobial peptides, was abolished in C/ebpb(M20A/M20A) macrophages, and C/ebpb(M20A/M20A) mice were susceptible to Listeria monocytogenes infection. Furthermore, the heat-killed Propionibacterium acnes-induced Th1 response, granuloma formation, and LPS shock were severely impaired. Nevertheless, impairment of intracellular bacteria killing, which is the most prominent phenotype in C/EBPbeta-deficient mice, was not observed in C/ebpb(M20A/M20A) mice. Collectively, we demonstrated that 34-kDa LAP is responsible for NF-IL6-mediated gene induction, but not essential for intracellular bacteria killing in activated macrophages.
Narayanan K, etal., J Biol Chem. 2004 Oct 29;279(44):45423-32. Epub 2004 Aug 12.
Terminal differentiation of odontoblasts, the principal cells in dentin formation, proceeds by synthesis of type I collagen and noncollagenous proteins. DSP and DPP are specific markers for terminally differentiated odontoblasts and are encoded by a single gene DSPP (dentin sialophosphoprotein). In
an attempt to understand the molecular mechanisms required for tissue-specific expression of the DSPP gene, we have identified a novel interaction between two bZIP transcription factors, Nrf1 and the CCAAT enhancer-binding protein (C/EBP)beta. This interaction was confirmed by both immunoprecipitation and chromatin immunoprecipitation assays. In undifferentiated odontoblasts, Nrf1 and C/EBPbeta repress DSPP promoter activity individually and synergistically by cooperatively interacting with each other. This mutual interaction is facilitated by the bZIP domains in both the proteins. The repression domain in both Nrf1 and C/EBPbeta was determined, and deletion of this domain abolished transcriptional repression. In fully differentiated odontoblasts, the loss of interaction between Nrf1 and C/EBPbeta results in an increased DSPP transcription. Further, this interaction was found to be dependent on phosphorylation at Ser(599) of Nrf1. Thus, the physical interaction between Nrf1 and C/EBPbeta provide a novel mechanism for the transcriptional regulation of DSPP in odontoblasts.
BACKGROUND: The transcription factors CCAAT/enhancer binding proteins (C/EBP) alpha, beta and delta have been shown to be expressed in brain and to be involved in regulation of inflammatory genes in concert with nuclear factor kappaB (NF-kappaB). In general, C/<
span style='font-weight:700;'>EBPalpha is down-regulated, whereas both C/EBPbeta and delta are up-regulated in response to inflammatory stimuli. In Alzheimer's disease (AD) one of the hallmarks is chronic neuroinflammation mediated by astrocytes and microglial cells, most likely induced by the formation of amyloid-beta (Abeta) deposits. The inflammatory response in AD has been ascribed both beneficial and detrimental roles. It is therefore important to delineate the inflammatory mediators and signaling pathways affected by Abeta deposits with the aim of defining new therapeutic targets. METHODS: Here we have investigated the effects of Abeta on expression of C/EBP family members with a focus on C/EBPdelta in rat primary astro-microglial cultures and in a transgenic mouse model with high levels of fibrillar Abeta deposits (tg-ArcSwe) by western blot analysis. Effects on DNA binding activity were analyzed by electrophoretic mobility shift assay. Cross-talk between C/EBPdelta and NF-kappaB was investigated by analyzing binding to a kappaB site using a biotin streptavidin-agarose pull-down assay. RESULTS: We show that exposure to fibril-enriched, but not oligomer-enriched, preparations of Abeta inhibit up-regulation of C/EBPdelta expression in interleukin-1beta-activated glial cultures. Furthermore, we observed that, in aged transgenic mice, C/EBPalpha was significantly down-regulated and C/EBPbeta was significantly up-regulated. C/EBPdelta, on the other hand, was selectively down-regulated in the forebrain, a part of the brain showing high levels of fibrillar Abeta deposits. In contrast, no difference in expression levels of C/EBPdelta between wild type and transgenic mice was detected in the relatively spared hindbrain. Finally, we show that interleukin-1beta-induced C/EBPdelta DNA binding activity to both C/EBP and kappaB sites is abolished after exposure to Abeta. CONCLUSIONS: These data suggest that both expression and function of C/EBPdelta are dysregulated in Alzheimer's disease. C/EBPdelta seems to be differently regulated in response to different conformations of Abeta. We propose that Abeta induces an imbalance between NF-kappaB and C/EBP transcription factors that may result in abnormal responses to inflammatory stimuli.
Adzic M, etal., Behav Brain Res. 2015 Sep 15;291:130-9. doi: 10.1016/j.bbr.2015.05.029. Epub 2015 May 27.
Peripheral inflammation induced by lipopolysaccharide (LPS) causes behavioural changes indicative for depression. The possible mechanisms involve the interference with neuroinflammatory, neuroendocrine, and neurotrophic processes. Apart from heterogeneity in the molecular background, sexual context
may be another factor relevant to the manifestation of mood disturbances upon an immune challenge. We investigated sex-dependent effects of a 7-day LPS treatment of adult Wistar rats on depressive-like behaviour and their relation with hypothalamic neuroendocrine factor, corticotrophin-releasing hormone (CRH), proplastic brain-derived neurotropic factor (BDNF), pro-inflammatory cyclooxygenase-2 (COX-2) and nuclear factor kappa beta (NFkB). Also, their regulators, the glucocorticoid receptor (GR) and CCAAT enhancer-binding protein (C/EBP) beta were followed. LPS induced depressive-like behaviour in females was associated with the increased hypothalamic CRH and decreased BDNF, but not with COX-2. These changes were paralleled by an increase in nuclear GR, NFkB and 20 kDa C/EBPbeta. LPS also altered behaviour in males and increased CRH expression, but in contrast to females, this was accompanied with the elevated COX-2, accumulation of cytosolic GR and elevated nuclear 38 kDa C/EBPbeta and NFkB. In conclusion, depressive-like phenotype induced by LPS in both sexes emerges from similar HPA axis activation and sex-specific alterations of hypothalamic molecular signalling: in males it is related to compromised control of neuroinflamation connected with cytoplasmic GR retention, while in females it is related to diminished proplastic capacity of BDNF. Sex-dependent mechanisms by which inflammation alters hypothalamic processes and cause pathological behaviour in animals, could be operative in the treatment of depression-related brain inflammation.
Metz R and Ziff E, Oncogene 1991 Dec;6(12):2165-78.
We show that members of two major families of transcription factors, the helix-loop-helix and C/EBP families, interact with the c-fos serum response element (SRE). Two cDNA clones encoding SRE binding factors (clones 9 and 21) were isolated by the direct screeni
ng of a PC12 lambda gt11 cDNA library using SRE oligonucleotide sequences as probes. Clone 9 encodes the rat homolog of the human HLH transcription factor, E12 (called here rE12). Clone 21 encodes a b-zip domain polypeptide that is related to the liver transcription factor C/EBP, and is homologous to the human NFIL-6 transcription factor (called here rNFIL-6). Using in vitro-translated products we show that rNFIL-6 recognizes a 'CCAATT' motif which overlaps the c-fos dyad symmetry element (DSE), the binding site for serum regulatory factor (SRF). Factor rE12 binds to an E-box enhancer sequence, 'CATCTG', immediately adjacent to the rNFIL-6 site, within the SRE. Antibodies specific to rE12 and rNFIL-6 disrupt nucleoprotein complexes with these DNA-binding sites, confirming the interaction of native in vivo factors. We present evidence that rNFIL-6 and SRF binding are mutually exclusive, consistent with the overlap of their binding sites. The demonstration that rE12 and rNFIL-6 bind to the SRE at sites adjacent to the major c-fos regulatory element, the DSE, raises the possibility that helix-loop-helix and C/EBP families regulate the SRE and provide a new basis for the multifunctional properties of the SRE, including possible tissue specificity of expression.
Tao LL, etal., Int J Clin Exp Pathol. 2015 Oct 1;8(10):13102-7. eCollection 2015.
AIM: To investigate the expression of CCAAT enhancer binding protein-alpha (C/EBP-alpha) in normal human liver and liver fibrosis and its probable association with autophagy. METHODS: Double label immunohistochemistry was used to detect the location of C/EBP
tyle='font-weight:700;'>EBP-alpha in hepatocytes and hepatic stellate cells (HSCs). The expression of C/EBP-alpha, Atg5, and Atg6 was also evaluated by immunohistochemistry in paraffin sections of human liver. HSC-T6 cells were treated with rapamycin and 3-methyladenine (3MA) to induce or inhibit autophagy, and the expression of C/EBP-alpha protein was detected by Western blotting. RESULTS: Double label immunohistochemistry showed that C/EBP-alpha was predominantly located in hepatocytes and that its expression was significantly decreased in fibrosis compared with normal liver. Atg5 expression was increased in fibrosis but was located primarily in liver septa and peri-vascular areas, which was consistent with the distribution of HSCs. In contrast, Atg6 was not expressed in normal or fibrotic liver. Treatment of HSC-T6 cells in culture with rapamycin or 3MA decreased or increased C/EBP-alpha expression, respectively, as shown by Western blotting. CONCLUSION: C/EBP-alpha was primarily expressed in hepatocytes in normal liver, but its expression decreased significantly in liver fibrosis. Autophagy might play a role in liver fibrosis through its association with C/EBP-alpha, but this hypothesis warrants further investigation.
Kordula T and Travis J, Biochem J. 1996 Feb 1;313 ( Pt 3):1019-27.
The rat serine proteinase inhibitor 3 gene is activated by interleukin 6 (IL-6) and glucocorticoids in hepatic cells. We report here that a 147 bp promoter is sufficient for both IL-6 stimulation and glucocorticoid enhancement of IL-6 induced transcription. Within this region we identified two funct
ional elements binding transcription factors from the C/EBP (CCAAT/enhancer binding proteins) and Stat (signal transducers and activators of transcription) families. Mutations introduced into the Stat binding site resulted in a loss of responsiveness, showing that this element is indispensable for activation. In contrast, the promoter containing the mutated C/EBP binding site was still responsive to IL-6 and glucocorticoids; however, the magnitude of the induction was decreased by 50%. The Stat binding element is an enhancer capable of conferring both responsiveness to IL-6 and partial enhancement of glucocorticoids on to a heterologous promoter. In response to IL-6 this element rapidly binds acute-phase response factor (APRF/Stat3) and, later, the protein(s) that require ongoing protein synthesis and is recognized by anti-Stat3 antibodies. In addition, long-term treatment with IL-6 results in sustained phosphorylation of APRF /Stat3.
The thyroid-specific enhancer-binding protein (T/ebp) gene was disrupted by homologous recombination in embryonic stem cells to generate mice lacking T/EBP expression. Heterozygous animals developed normally, whereas mice ho
mozygous for the disrupted gene were born dead and lacked the lung parenchyma. Instead, they had a rudimentary bronchial tree associated with an abnormal epithelium in their pleural cavities. Furthermore, the homozygous mice had no thyroid gland but had a normal parathyroid. In addition, extensive defects were found in the brain of the homozygous mice, especially in the ventral region of the forebrain. The entire pituitary, including the anterior, intermediate, and posterior pituitary, was also missing. In situ hybridization showed that the T/ebp gene is expressed in the normal thyroid, lung bronchial epithelium, and specific areas of the forebrain during early embryogenesis. These results establish that the expression of T/EBP, a transcription factor known to control thyroid-specific gene transcription, is also essential for organogenesis of the thyroid, lung, ventral forebrain, and pituitary.
Antonio V, etal., Biochem J 2002 Dec 1;368(Pt 2):415-24.
The abundant secretion of type IIA secreted phospholipase A(2) (sPLA(2)) is a major feature of the inflammatory process of atherosclerosis. sPLA(2) is crucial for the development of inflammation, as it catalyses the production of lipid mediators and induces the proliferation of smooth muscle cells.
We have analysed the activation of sPLA(2) transcription by cAMP and interleukin-1beta (IL-1beta), and shown that the 500 bp region upstream of the transcription start site of the rat sPLA(2) gene is implicated in activation by synergistically acting cAMP and IL-1beta. We transiently transfected and stimulated rat smooth muscle cells in primary culture and measured the promoter activities of serial and site-directed deletion mutants of sPLA(2)-luciferase constructs. A distal region, between -488 and -157 bp, bearing a CAAT/enhancer binding protein (C/EBP)-responsive element (-242 to -223) was sufficient for cAMP/protein kinase A-mediated sPLA(2) promoter activation. We find evidence for the first time that activation of the sPLA(2) promoter by IL-1beta requires activation of an Ets-responsive element in the -184 to -180 region of the distal promoter via the Ras pathway and a nuclear factor-kappaB site at positions -141 to -131 of the proximal promoter. We also used electrophoretic mobility shift assays to identify five binding sites for the Sp1 factor; a specific inhibitor of Sp1, mithramycin A, showed that this factor is crucial for the basal activity of the sPLA(2) promoter.
Stephens JM and Pekala PH, J Biol Chem. 1992 Jul 5;267(19):13580-4.
We have previously demonstrated the ability of tumor necrosis factor-alpha (TNF) to down-regulate the expression of GLUT4 (insulin-responsive glucose transporter) and C/EBP-alpha (CCAAT/enhancer-binding protein) (Stephens J. M., and Pekala, P. H. (1991) J. Bio
l. Chem. 266, 21839-21845). As C/EBP-alpha has been suggested to control GLUT4 expression, we have examined the time course for attenuation of transcription of these genes. Run-on transcription assays indicate a coordinate transcriptional repression of both GLUT4 and C/EBP-alpha genes (as well as the 422/aP2 gene, the adipocyte lipid-binding protein, whose expression has also been proposed to be controlled by C/EBP-alpha). Inhibition of transcription was observed within 1 h of TNF addition, with maximal suppression observed after 4 h. The inhibition was not blocked by cycloheximide. Okadaic acid treatment (1 h, 0.5 microM) also resulted in the coordinate transcriptional repression of the C/EBP-alpha, GLUT4, and 422/aP2 genes, consistent with involvement of a kinase-phosphatase system in the regulation of these genes. The decrease in C/EBP-alpha protein content was detectable 4 h after TNF addition and declined to 25% of controls within 24 h. A minor decrease in the protein content of GLUT4 was observed during the first 24 h of exposure to TNF; however, after 72 h of exposure GLUT4 protein was not detectable. The rapid coordinate transcriptional regulation of C/EBP-alpha, GLUT4, and 422/aP2 by TNF in the presence of cycloheximide suggests that the TNF-induced loss of GLUT4 protein may be mediated by a post-translational modification of an existing transcription factor. However, the rapid loss of C/EBP-alpha protein may be a contributing factor to further transcriptional suppression of the GLUT4 gene at the later time points. In addition to the transcriptional effect, we report that TNF-induced destabilization of these mRNAs contributes to decreased expression of all three genes.
Sun R, etal., Am J Physiol Cell Physiol. 2016 Jul 1;311(1):C101-15. doi: 10.1152/ajpcell.00344.2015. Epub 2016 Apr 27.
Muscle wasting is the hallmark of cancer cachexia and is associated with poor quality of life and increased mortality. Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, has important biological effects in the treatment of muscular dystrophy. To verify whether VPA could ameliorate muscle w
asting induced by cancer cachexia, we explored the role of VPA in two cancer cachectic mouse models [induced by colon-26 (C26) adenocarcinoma or Lewis lung carcinoma (LLC)] and atrophied C2C12 myotubes [induced by C26 cell conditioned medium (CCM) or LLC cell conditioned medium (LCM)]. Our data demonstrated that treatment with VPA increased the mass and cross-sectional area of skeletal muscles in tumor-bearing mice. Furthermore, treatment with VPA also increased the diameter of myotubes cultured in conditioned medium. The skeletal muscles in cachectic mice or atrophied myotubes treated with VPA exhibited reduced levels of CCAAT/enhancer binding protein beta (C/EBPβ), resulting in atrogin1 downregulation and the eventual alleviation of muscle wasting and myotube atrophy. Moreover, atrogin1 promoter activity in myotubes was stimulated by CCM via activating the C/EBPβ-responsive cis-element and subsequently inhibited by VPA. In contrast to the effect of VPA on the levels of C/EBPβ, the levels of inactivating forkhead box O3 (FoxO3a) were unaffected. In summary, VPA attenuated muscle wasting and myotube atrophy and reduced C/EBPβ binding to atrogin1 promoter locus in the myotubes. Our discoveries indicate that HDAC inhibition by VPA might be a promising new approach for the preservation of skeletal muscle in cancer cachexia.
Schmid metaphyseal chondrodysplasia (MCDS) involves dwarfism and growth plate cartilage hypertrophic zone expansion resulting from dominant mutations in the hypertrophic zone collagen, Col10a1. Mouse models phenocopying MCDS through the expression of an exogenous misfolding protein in the endoplasmi
c reticulum (ER) in hypertrophic chondrocytes have demonstrated the central importance of ER stress in the pathology of MCDS. The resultant unfolded protein response (UPR) in affected chondrocytes involved activation of canonical ER stress sensors, IRE1, ATF6, and PERK with the downstream effect of disrupted chondrocyte differentiation. Here, we investigated the role of the highly conserved IRE1/XBP1 pathway in the pathology of MCDS. Mice with a MCDS collagen X p.N617K knock-in mutation (ColXN617K) were crossed with mice in which Xbp1 was inactivated specifically in cartilage (Xbp1CartDeltaEx2), generating the compound mutant, C/X. The severity of dwarfism and hypertrophic zone expansion in C/X did not differ significantly from ColXN617K, revealing surprising redundancy for the IRE1/XBP1 UPR pathway in the pathology of MCDS. Transcriptomic analyses of hypertrophic zone cartilage identified differentially expressed gene cohorts in MCDS that are pathologically relevant (XBP1-independent) or pathologically redundant (XBP1-dependent). XBP1-independent gene expression changes included large-scale transcriptional attenuation of genes encoding secreted proteins and disrupted differentiation from proliferative to hypertrophic chondrocytes. Moreover, these changes were consistent with disruption of C/EBP-beta, a master regulator of chondrocyte differentiation, by CHOP, a transcription factor downstream of PERK that inhibits C/EBP proteins, and down-regulation of C/EBP-beta transcriptional co-factors, GADD45-beta and RUNX2. Thus we propose that the pathology of MCDS is underpinned by XBP1 independent UPR-induced dysregulation of C/EBP-beta-mediated chondrocyte differentiation. Our data suggest that modulation of C/EBP-beta activity in MCDS chondrocytes may offer therapeutic opportunities.
Chen W, etal., Proc Natl Acad Sci U S A. 2013 Apr 30;110(18):7294-9. doi: 10.1073/pnas.1211383110. Epub 2013 Apr 11.
Despite recent insights gained from the effects of targeted deletion of the Finkel-Biskis-Jinkins osteosarcoma oncogene (c-fos), Spleen focus-forming virus (SFFV) proviral integration 1 (PU.1), microphthalmia-associated transcription factor, NF-kappaB, and nuclear factor of activated cells cytoplasm
ic 1 (NFATc1) transcription factor genes, the mechanism underlying transcription factors specifying osteoclast (OC) lineage commitment from monocyte/macrophage remains unclear. To characterize the mechanism by which transcription factors regulate OC lineage commitment, we mapped the critical cis-regulatory element in the promoter of cathepsin K (Ctsk), which is expressed specifically in OCs, and found that CCAAT/enhancer binding protein alpha (C/EBPalpha) is the critical cis-regulatory element binding protein. Our results indicate that C/EBPalpha is highly expressed in pre- OCs and OCs. The combined presence of macrophage colony-stimulating factor and receptor activator of NF-kappaB ligand significantly induces high C/EBPalpha expression. Furthermore, C/EBPalpha(-/-) newborn mice exhibited impaired osteoclastogenesis, and a severe osteopetrotic phenotype, but unaffected monocyte/macrophage development. Impaired osteoclastogenesis of C/EBPalpha(-/-) mouse bone marrow cells can be rescued by c-fos overexpression. Ectopic expression of C/EBPalpha in mouse bone marrow cells and monocyte/macrophage cells, in the absence of receptor activator of NF-kappaB ligand, induces expression of receptor activator of NF-kappaB, c-fos, Nfatc1, and Ctsk, and it reprograms monocyte/macrophage cells to OC-like cells. Our results demonstrate that C/EBPalpha directly up-regulates c-fos expression. C/EBPalpha(+/-) mice exhibit an increase in bone density compared with C/EBPalpha(+/+) controls. These discoveries establish C/EBPalpha as the key transcriptional regulator of OC lineage commitment, providing a unique therapeutic target for diseases of excessive bone resorption, such as osteoporosis and arthritis.
Bararia D, etal., Nat Commun. 2016 Mar 23;7:10968. doi: 10.1038/ncomms10968.
CCAAT/enhancer-binding protein alpha (C/EBPalpha) is an essential transcription factor for myeloid lineage commitment. Here we demonstrate that acetylation of C/EBPalpha at lysine residues K298 and K302, mediated at least in
part by general control non-derepressible 5 (GCN5), impairs C/EBPalpha DNA-binding ability and modulates C/EBPalpha transcriptional activity. Acetylated C/EBPalpha is enriched in human myeloid leukaemia cell lines and acute myeloid leukaemia (AML) samples, and downregulated upon granulocyte-colony stimulating factor (G-CSF)- mediated granulocytic differentiation of 32Dcl3 cells. C/EBPalpha mutants that mimic acetylation failed to induce granulocytic differentiation in C/EBPalpha-dependent assays, in both cell lines and in primary hematopoietic cells. Our data uncover GCN5 as a negative regulator of C/EBPalpha and demonstrate the importance of C/EBPalpha acetylation in myeloid differentiation.
Redondo-Angulo I, etal., Int J Cardiol. 2016 Jan 1;202:819-28. doi: 10.1016/j.ijcard.2015.10.005. Epub 2015 Oct 9.
AIM: Pregnancy is a physiological model of adaptive and reversible heart enlargement, but the molecular mechanisms determining this kind of physiologic cardiac hypertrophy are poorly known. Here, we analyzed the role of the transcription factor C/EBPbeta in the
development of pregnancy-induced cardiac hypertrophy. RESULTS: C/EBPbeta+/- mice at day 18 of gestation were used as happloinsufficiency model of late pregnancy. We found that C/EBPbeta expression was specifically increased in hearts from Wt pregnant mice whereas expression of other C/EBP subtypes (alpha and delta) was not affected by gestation. Pregnancy-induced changes in systemic metabolic and hormonal profiles were not essentially different in Wt versus C/EBPbeta+/- mice. However, C/EBPbeta+/- mice developed pregnancy-induced heart hypertrophy to a lower extent relative to Wt mice. Furthermore, hearts from C/EBPbeta+/- mice have alterations in fatty acid oxidation genes and reductions in the expression levels of glucose transporters that may compromise metabolic cardiac function during pregnancy. Among marker genes of inflammation, interleukin-6 (Il-6) showed a marked differential behavior in C/EBPbeta+/- pregnant mice: pregnancy strongly induced cardiac Il-6 expression in wt, a phenomenon that did not occur in C/EBPbeta+/- mice. Moreover, marker genes for M2 macrophages were decreased in C/EBPbeta+/- pregnant mice and in C/EBPbeta-/- mice subjected to LPS stimulus. CONCLUSIONS: Here we found that normal levels of C/EBPbeta are required for hypertrophy development during pregnancy. Events such as the increase in IL-6 in the heart of pregnant mice are prevented in C/EBPbeta+/- animals. Moreover, C/EBPbeta controls M2-macrophage gene expression in the heart. Thus, C/EBPbeta appears as a transcription factor required for cardiac hypertrophy response to gestation.
Neuroinflammation contributes to the pathogenesis of several neurological disorders and pericytes are implicated in brain inflammatory processes. Cellular inflammatory responses are orchestrated by transcription factors but information on transcriptional control in pericytes is lacking. Because th
e transcription factor CCAAT/enhancer binding protein delta (C/EBPdelta) is induced in a number of inflammatory brain disorders, we sought to investigate its role in regulating pericyte immune responses. Our results reveal that C/EBPdelta is induced in a concentration- and time-dependent fashion in human brain pericytes by interleukin-1beta (IL-1beta). To investigate the function of the induced C/EBPdelta in pericytes we used siRNA to knockdown IL-1beta-induced C/EBPdelta expression. C/EBPdelta knockdown enhanced IL-1beta-induced production of intracellular adhesion molecule-1 (ICAM-1), interleukin-8, monocyte chemoattractant protein-1 (MCP-1) and IL-1beta, whilst attenuating cyclooxygenase-2 and superoxide dismutase-2 gene expression. Altered ICAM-1 and MCP-1 protein expression were confirmed by cytometric bead array and immunocytochemistry. Our results show that knock-down of C/EBPdelta expression in pericytes following immune stimulation increased chemokine and adhesion molecule expression, thus modifying the human brain pericyte inflammatory response. The induction of C/EBPdelta following immune stimulation may act to limit infiltration of peripheral immune cells, thereby preventing further inflammatory responses in the brain.
Liu L, etal., Genet Mol Res. 2015 Oct 2;14(4):11860-6. doi: 10.4238/2015.October.2.19.
In this study, the ErbB3-binding protein (Ebp1) and p53 protein expression in cervical cancer tissues, and its significance in the prognosis of the disease was investigated. Ebp1 and p53 protein expression was detected by im
munohistochemical analysis in cervical cancer tissues (N = 60) and normal tissues adjacent to the cancer tissues (N = 60). The rates of positive Ebp1 and p53 protein expression were 35.0 and 60.0%, respectively. Ebp1 and p53 were overexpressed in cervical cancer tissues, compared to normal tissues (P < 0.05). Ebp1 and p53 protein expression was not correlated with age, tumor size, or family tumor history (P > 0.05). However, high levels of expression of Ebp1 and p53 were positively correlated with the TNM stage and lymphatic metastasis in cervical cancer patients (P < 0.05). The combined determination of Ebp1 and p53 expression levels in cervical cancer patients could support the effective prediction of metastatic potential and patient prognosis.
Liu Z, etal., Proc Natl Acad Sci U S A. 2006 Jul 18;103(29):10917-22. Epub 2006 Jul 10.
Ebp1, an ErbB3 receptor-binding protein, inhibits the proliferation and induces the differentiation of human cancer cells. Ebp1 binds nuclear Akt and prevents DNA fragmentation by inhibiting caspase-activated DNase. Here, we
show that Ebp1 possesses two different isoforms, p48 and p42, which differentially mediate PC12 cell survival and differentiation. The longer-form p48 localizes in both the cytoplasm and the nucleus and suppresses apoptosis, whereas the shorter-form p42 predominantly resides in the cytoplasm and promotes cell differentiation. EGF strongly stimulates p42 to bind ErbB3, and the association depends on PKC-mediated phosphorylation of Ebp1. By contrast, p48 does not bind to ErbB3 regardless of EGF treatment. Overexpression of p48 provokes cell proliferation, which is inhibited by p42. Moreover, nerve growth factor elicits extensive sprouting in p42 stably transfected PC12 cells, whereas p48 cells reveal modest neurite outgrowth. Although mitogen-activated protein kinase cascade remains similar in both cells, Akt is more active in p48 cells than in p42 cells. Thus, Ebp1 might regulate cell survival and differentiation through two distinctive p48 and p42 isoforms.
Liu L, etal., Genet Mol Res. 2015 May 22;14(2):5496-500. doi: 10.4238/2015.May.22.20.
The objectives of this study were to observe the changes in expression of ErbB-3 binding protein (Ebp1) in cervical cancer and to investigate their clinic significance. We detected the expression level of Ebp1 in cancerous
and adjacent tissues from 56 patients with cervical cancer. We identified 21 Ebp1 positive samples (37.5%) from among the 56 cervical cancer tissue samples and 5 Ebp1 positive samples (8.9%) in the corresponding adjacent tissues; the difference was statistically significant (P < 0.05). No statistically significant (P > 0.05) differences in the rates of positive Ebp1 expression were found between patients under 60 years of age and those equal to or over this age. No statistically significant differences (P > 0.05) were found between patients whose tumor diameters were under 5 cm and those with tumor diameters over 5 cm. No statistically significant differences (P > 0.05) in the Ebp1 positive rates were found among the cervical cancer samples when stratified by grade (I, II, or III). Together, these results demonstrate that Ebp1 protein expression is upregulated in cervical cancer tissues but is not related to clinical pathologic factors such as patient age or tumor size or differentiation level, suggesting that Ebp1 plays an important role in the genesis and growth process of cervical cancer.
Mori M, etal., Respir Res. 2015 Oct 29;16:133. doi: 10.1186/s12931-015-0297-0.
The expression of CCAAT/enhancer-binding protein (C/EBP)beta in the small airway epithelium of COPD is unknown. C/EBPbeta was assessed in peripheral lung tissue of non-smoking/smoking controls and patients with GOLD I-IV CO
PD by quantitative immunohistochemistry. The expression of C/EBPbeta was decreased in smokers compared to never smokers. Furthermore, C/EBPbeta was significantly elevated in advanced COPD vs. asymptomatic smokers, and the expression correlated to lung function decline. As C/EBPbeta exerts pro-inflammatory effects in the context of cigarette smoke, the elevated C/EBPbeta in advanced COPD may be an indication of a breakdown of regulatory mechanisms and excessive inflammation.
Ming J, etal., Biosci Rep. 2015 Oct 8;35(6). pii: e00266. doi: 10.1042/BSR20150167.
Cx43 (connexin43) is an enhancer of the metastasis of breast cancer cells. Our previous study identified miR-381 as an indirect suppressor of Cx43 gene expression, with the precise mechanism being not understood. In the present study, using a reporter gene assay, we found that miR-381 suppressed Cx4
3 gene expression via the promoter region -500/-250. With site-directed gene mutation, we demonstrated that miR-381 could directly bind with the sequences CACUUGUAU in the 3'-UTR so as to inhibit C/EBPalpha (CCAAT/enhancer-binding protein alpha) expression. C/EBPalpha was further identified as a novel transcription factor by binding to a canonic element (AATTGTC) locating at -459/-453 in the promoter region of the Cx43 gene. Functionally, we demonstrated that miR-381 suppressed C/EBPalpha- and Cx43-dependent migration and invasion of breast cancer cells. Finally, we revealed that decreased levels of miR-381 as well as increased expression of C/EBPalpha and Cx43 in the metastatic breast cancer cells and tissues. Therefore we are the first to identify that miR-381 suppresses C/EBPalpha-dependent Cx43 expression in breast cancer cells. The miR-381-C/EBPalpha-Cx43 axis might be a useful diagnostic and therapeutic target of metastatic breast cancer.
In the past decade, miRNA emerges as a vital player in orchestrating gene regulation and maintaining cellular homeostasis. It is well documented that miRNA influences a variety of biological events, including embryogenesis, cell fate decision, and cellular differentiation. Adipogenesis is an organiz
ed process of cellular differentiation by which pre-adipocytes differentiate towards mature adipocytes. It has been shown that adipogenesis is tightly modulated by a number of transcription factors such as PPARgamma, KLF4, and C/EBPalpha. However, the molecular mechanisms underlying the missing link between miRNA and adipogenesis-related transcription factors remain elusive. In this study, we unveiled that miR-25, a member of miR-106b-25 cluster, was remarkably downregulated during 3T3-L1 adipogenesis. Restored expression of miR-25 significantly impaired 3T3-L1 adipogenesis and downregulated the expression of serial adipogenesis-related genes. Further experiments presented that ectopic expression of miR-25 did not affect cell proliferation and cell cycle progression. Finally, KLF4 and C/EBPalpha, two key regulators of adipocyte differentiation, were experimentally identified as bona fide targets for miR-25. These data indicate that miR-25 is a novel negative regulator of adipocyte differentiation and it suppressed 3T3-L1 adipogenesis by targeting KLF4 and C/EBPalpha, which provides novel insights into the molecular mechanism of miRNA-mediated cellular differentiation.
Tian J, etal., Proc Natl Acad Sci U S A. 2016 Jul 19;113(29):8182-7. doi: 10.1073/pnas.1608987113. Epub 2016 Jul 5.
Insulin increases lipid synthesis in liver by activating transcription of the gene encoding sterol regulatory element-binding protein-1c (SREBP-1c). SREBP-1c activates the transcription of all genes necessary for fatty acid
synthesis. Insulin induction of SREBP-1c requires LXRalpha, a nuclear receptor. Transcription of SREBP-1c also requires transcription factor C/EBPbeta, but a connection between LXRalpha and C/EBPbeta has not been made. Here we show that LXRalpha and C/EBPbeta form a complex that can be immunoprecipitated from rat liver nuclei. Chromatin immunoprecipitation assays showed that the LXRalpha-C/EBPbeta complex binds to the SREBP-1c promoter in a region that contains two binding sites for LXRalpha and is known to be required for insulin induction. Knockdown of C/EBPbeta in fresh rat hepatocytes or mouse livers in vivo reduces the ability of insulin to increase SREBP-1c mRNA. The LXRalpha-C/EBPbeta complex is bound to the SREBP-1c promoter in the absence or presence of insulin, indicating that insulin acts not by increasing the formation of this complex, but rather by activating it.
Humans or mice subjected to immunosuppression, such as corticosteroids or anti-cytokine biologic therapies, are susceptible to mucosal infections by the commensal fungus Candida albicans. Recently it has become evident that the Th17/IL-17 axis is essential for immunity to candidiasis, but the downst
ream events that control immunity to this fungus are poorly understood. The CCAAT/Enhancer Binding Protein-beta (C/EBPbeta) transcription factor is important for signaling by multiple inflammatory stimuli, including IL-17. C/EBPbeta is regulated in a variety of ways by IL-17, and controls several downstream IL-17 target genes. However, the role of C/EBPbeta in vivo is poorly understood, in part because C/EBPbeta-deficient mice are challenging to breed and work with. In this study, we sought to understand the role of C/EBPbeta in the context of an IL-17-dependent immune response, using C. albicans infection as a model system. Confirming prior findings, we found that C/EBPbeta is required for immunity to systemic candidiasis. In contrast, C/EBPbeta(-/-) mice were resistant to oropharyngeal candidiasis (OPC), in a manner indistinguishable from immunocompetent WT mice. However, C/EBPbeta(-/-) mice experienced more severe OPC than WT mice in the context of cortisone-induced immunosuppression. Expression of the antimicrobial peptide beta-defensin (BD)-3 correlated strongly with susceptibility in C/EBPbeta(-/-) mice, but no other IL-17-dependent genes were associated with susceptibility. Therefore, C/EBPbeta contributes to immunity to mucosal candidiasis during cortisone immunosuppression in a manner linked to beta-defensin 3 expression, but is apparently dispensable for the IL-17-dependent response.
The C/EBPalpha transcription factor is required for differentiation of adipocytes and neutrophil granulocytes, and controls cellular proliferation in vivo. To address the molecular mechanisms of C/EBPalpha action, we have id
entified C/EBPalpha mutants defective in repression of E2F-dependent transcription and found them to be impaired in their ability to suppress cellular proliferation, and to induce adipocyte differentiation in vitro. Using targeted mutagenesis of the mouse germline, we show that E2F repression-deficient C/EBPalpha alleles failed to support adipocyte and granulocyte differentiation in vivo. These results indicate that E2F repression by C/EBPalpha is critical for its ability to induce terminal differentiation, and thus provide genetic evidence that direct cell cycle control by a mammalian lineage-instructive transcription factor couples cellular growth arrest and differentiation.
Xi Y, etal., Biochem Biophys Res Commun. 2016 Apr 15;472(4):617-23. doi: 10.1016/j.bbrc.2016.03.015. Epub 2016 Mar 8.
Adipogenesis is orchestrated by a highly ordered network of transcription factors including peroxisome-proliferator activated receptor-gamma (PPARgamma) and CCAAT-enhancer binding protein (C/EBP) family proteins. High mobility group protein AT-hook 2 (HMGA2), an
architectural transcription factor, has been reported to play an essential role in preadipocyte proliferation, and its overexpression has been implicated in obesity in mice and humans. However, the direct role of HMGA2 in regulating the gene expression program during adipogenesis is not known. Here, we demonstrate that HMGA2 is required for C/EBPbeta-mediated expression of PPARgamma, and thus promotes adipogenic differentiation. We observed a transient but marked increase of Hmga2 transcript at an early phase of differentiation of mouse 3T3-L1 preadipocytes. Importantly, Hmga2 knockdown greatly impaired adipocyte formation, while its overexpression promoted the formation of mature adipocytes. We found that HMGA2 colocalized with C/EBPbeta in the nucleus and was required for the recruitment of C/EBPbeta to its binding element at the Ppargamma2 promoter. Accordingly, HMGA2 and C/EBPbeta cooperatively enhanced the Ppargamma2 promoter activity. Our results indicate that HMGA2 is an essential constituent of the adipogenic transcription factor network, and thus its function may be affected during the course of obesity.
Recent evidence indicates that transcription factor Runx1 modulates the expression of several phenotypic markers in dorsal root ganglia (DRGs) neurons, including the pain-related P2X3 receptor. In several cell lineages C/EBP transcription factors interact with t
he Runx factor family members to jointly bind and activate transcription of target genes. Here, we examine whether these two transcription factors directly regulate P2X3 gene expression. Through in silico analyses of the first 2 kb of the P2X3 gene promoter we identified putative consensus-binding sites for both Runx1 and C/EBPbeta transcription factors. Transient over-expression in PC12 cells of either Runx1 or C/EBPbeta increases P2X3 gene promoter activity and co-expression of both factors results in an additive stimulatory effect on the promoter function. Accordingly, chromatin immunoprecipitation assays demonstrate that both Runx1 and C/EBPbeta bind to the P2X3 promoter in PC12 cells expressing this gene. Site-directed mutagenesis of the proximal Runx1 and C/EBPbeta consensus elements in the P2X3 promoter decrease Runx1- and C/EBPbeta-mediated transcriptional activity. Moreover, C/EBPbeta-mediated enhancement of the P2X3 promoter requires a functional Runx1 binding site. Altogether our results support a functional and coordinated role for Runx1 and C/EBPbeta transcription factors during activation of P2X3 gene transcription.
The CEBPA gene is mutated in 9% of patients with acute myeloid leukemia (AML). Selective expression of a short (30-kDa) CCAAT-enhancer binding protein-alpha (C/EBPalpha) translational isoform, termed p30, represents the most
common type of CEBPA mutation in AML. The molecular mechanisms underlying p30-mediated transformation remain incompletely understood. We show that C/EBPalpha p30, but not the normal p42 isoform, preferentially interacts with Wdr5, a key component of SET/MLL (SET-domain/mixed-lineage leukemia) histone-methyltransferase complexes. Accordingly, p30-bound genomic regions were enriched for MLL-dependent H3K4me3 marks. The p30-dependent increase in self-renewal and inhibition of myeloid differentiation required Wdr5, as downregulation of the latter inhibited proliferation and restored differentiation in p30-dependent AML models. OICR-9429 is a new small-molecule antagonist of the Wdr5-MLL interaction. This compound selectively inhibited proliferation and induced differentiation in p30-expressing human AML cells. Our data reveal the mechanism of p30-dependent transformation and establish the essential p30 cofactor Wdr5 as a therapeutic target in CEBPA-mutant AML.
Cohen DM, etal., Elife. 2015 Jun 25;4:e06821. doi: 10.7554/eLife.06821.
A well-established cascade of transcription factor (TF) activity orchestrates adipogenesis in response to chemical cues, yet how cell-intrinsic determinants of differentiation such as cell shape and/or seeding density inform this transcriptional program remain enigmatic. Here, we uncover a novel mec
hanism licensing transcription in human mesenchymal stem cells (hMSCs) adipogenically primed by confluence. Prior to adipogenesis, confluency promotes heterodimer recruitment of the bZip TFs C/EBPbeta and ATF4 to a non-canonical C/EBP DNA sequence. ATF4 depletion decreases both cell-density-dependent transcription and adipocyte differentiation. Global profiling in hMSCs and a novel cell-free assay reveals that ATF4 requires C/EBPbeta for genomic binding at a motif distinct from that bound by the C/EBPbeta homodimer. Our observations demonstrate that C/EBPbeta bridges the transcriptional programs in naive, confluent cells and early differentiating pre-adipocytes. Moreover, they suggest that homo- and heterodimer formation poise C/EBPbeta to execute diverse and stage-specific transcriptional programs by exploiting an expanded motif repertoire.
Georgescu MM, etal., Acta Neuropathol Commun. 2015 Mar 8;3:11. doi: 10.1186/s40478-015-0197-z.
NHERF1/EBP50, an adaptor protein required for epithelial morphogenesis, has been implicated in the progression of various human malignancies. NHERF1-deficient mice have intestinal brush border structural defects and we report here that they also have disorganiz
ed ependymal cilia with development of non-obstructive hydrocephalus. Examination of mouse and human brain tissues revealed highest NHERF1 expression at the apical plasma membrane of ependymal cells. In ependymal tumors, NHERF1 expression was retained in polarized membrane structures, such as microlumens, rosettes and canals, where it co-localized with some of its ligands, such as moesin and PTEN. Analysis of a comprehensive panel of 113 tumors showed robust NHERF1 labeling of microlumens in 100% of ependymomas, subependymomas, and pediatric anaplastic ependymomas, and in 67% of adult anaplastic ependymomas. NHERF1 staining was present in 35% of ependymoma cases that lacked reactivity for EMA, the routine immunohistochemical marker used for ependymoma diagnosis. NHERF1 labeling of microlumens was either absent or rarely seen in other types of brain tumors analyzed, denoting NHERF1 as a reliable diagnostic marker of ependymal tumors. Anaplastic foci and a subset of adult anaplastic ependymomas showed complete absence of NHERF1-labeled polarity structures, consistent with a loss of differentiation in these aggressive tumors. These data highlight a role for NHERF1 in ependymal morphogenesis with direct application to the diagnosis of ependymal tumors.
The transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) coordinates proliferation arrest and the differentiation of myeloid progenitors, adipocytes, hepatocytes, keratinocytes, and cells of the lung and placenta. C/EBP
0;'>EBPalpha transactivates lineage-specific differentiation genes and inhibits proliferation by repressing E2F-regulated genes. The myeloproliferative C/EBPalpha BRM2 mutant serves as a paradigm for recurrent human C-terminal bZIP C/EBPalpha mutations that are involved in acute myeloid leukemogenesis. BRM2 fails to repress E2F and to induce adipogenesis and granulopoiesis. The data presented here show that, independently of pocket proteins, C/EBPalpha interacts with the dimerization partner (DP) of E2F and that C/EBPalpha-E2F/DP interaction prevents both binding of C/EBPalpha to its cognate sites on DNA and transactivation of C/EBP target genes. The BRM2 mutant, in addition, exhibits enhanced interaction with E2F-DP and reduced affinity toward DNA and yet retains transactivation potential and differentiation competence that becomes exposed when E2F/DP levels are low. Our data suggest a tripartite balance between C/EBPalpha, E2F/DP, and pocket proteins in the control of proliferation, differentiation, and tumorigenesis.
Zhang J, etal., Int J Clin Exp Pathol. 2015 Nov 1;8(11):14472-8. eCollection 2015.
C/EBPalpha plays an important role in the modulation of cell proliferation, differentiation or apoptosis in various tissues. Most recently, reduced expression of C/EBPalpha and growth inhibitory effect was found in primary m
ammary carcinomas. However, the underlying mechanism is still not fully aware. Here, we firstly identified miR-134 as a target of C/EBPalpha in MCF7 breast cancer cell lines. C/EBPalpha overexpression promoted miR-134 expression, causing suppression of apoptosis- protective genes CREB and Bcl-2, and resulted in the proliferation inhibition of MCF7 cells. Moreover, anti-miR-134 rescued the proliferation inhibition of MCF7 cells and the suppression of anti-apoptotic genes CREB and Bcl-2 caused by C/EBPalpha overexpression. Collectively, C/EBPalpha inhibited cell growth in breast cancer cells via a novel pathway miR-134/CREB.
Baeyens N, etal., J Cell Biochem. 2011 Sep;112(9):2574-84. doi: 10.1002/jcb.23183.
Ezrin, Radixin, Moesin binding phosphoprotein 50 (EBP50) is a scaffold protein that possesses two PDZ interacting domains. We have shown that, in isolated artery stimulated with noradrenaline, EBP50 interacts with several el
ements of the cytoskeleton. However, the contribution of EBP50 to the organization of the cytoskeleton is unknown. We have used primary cultured vascular smooth muscle cells to investigate the involvement of EBP50 in the regulation of cell architecture, motility and cell cycle, and to identify its target proteins and subsequent action mechanism. The results showed that depletion of EBP50 by siRNA transfection induced changes in cell architecture and increased cell migration. The same phenotype was induced by inhibition of myosin IIa and this effect was not additive in cells depleted for EBP50. Moreover, a larger proportion of binucleated cells was observed after EBP50 depletion, indicating a defect in cytokinesis. The identification, after co-immunoprecipitation, of a direct interaction of EBP50 with both tubulin and myosin IIa suggested that EBP50 could regulate cell migration and cytokinesis by linking myosin IIa fibers and microtubule network. Indeed, depletion of EBP50 also dismantled myosin IIa fibers and induced the formation of stable microtubules in lamellae expansions and Rac1 activation. This signaling cascade leads to the formation of lamellipodia, trailing tails and decrease of focal adhesion formation, triggering cell migration.
Zhang F, etal., Am J Respir Cell Mol Biol 2004 Feb;30(2):174-83. Epub 2003 Aug 01.
Alveolar type II cells increase lipogenesis and convert glycogen into the phospholipids of surfactant in the late term fetal lung. Recent studies suggest that CCAAT/enhancing-binding protein (C/EBP) isoforms and sterol regulatory element binding protein (SREBP
style='font-weight:700;'>EBP)-1c regulate fatty acid synthesis in adult type II cells in vitro. To define the temporal relationships and enzymes involved in lipogenesis in fetal rat lung, the mRNA levels of selected transcription factors and enzymes were determined. There was an increase in the mRNA levels of C/EBPalpha, C/EBPbeta, C/EBPdelta, peroxisomal proliferator-activated receptor gamma (PPARgamma), and SREBP-1c, but not SREBP-1a or SREBP-2 from fetal Days 19-21. There was also an increase in the mRNA levels of fatty acid synthase, stearoyl-CoA desaturase 1 (SCD-1), fatty acid translocase, glycerol-3-P acyl transferase, and phosphatidate cytidylyltransferase. By in situ hybridization, there was detectible expression of fatty acid synthase, SCD-1, and C/EBPalpha along the alveolar septae with the same distribution pattern as surfactant protein-C, whereas PPARgamma expression appeared to be restricted to macrophages. Regulation of lipogenesis at the mRNA level is predominately on enzymes of fatty acid synthesis and appears to be regulated by C/EBPalpha and SREBP-1c. SCD-1 and phosphatidate cytidylyltransferase are important components of the lipogenic response in the fetal lung that have not been recognized previously.
Samuelsson M, etal., Biochem Biophys Res Commun. 2008 Jun 13;370(4):619-22. doi: 10.1016/j.bbrc.2008.03.150. Epub 2008 Apr 9.
Members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors have been reported to be up-regulated in Alzheimer's disease. In the present study, we have investigated the effects of amyloid-beta (Abeta) peptides on C/EBP
ight:700;'>EBPbeta and C/EBPdelta, previously shown to be induced by inflammatory stimuli in glial cells. Surprisingly, electrophoretic mobility shift assay showed that both Abeta(1-42) and Abeta(25-35) blocked C/EBP activation induced by the inflammatory cytokine interleukin-1beta (IL-1beta) or lipopolysaccharide (LPS) in mixed primary glial cell cultures from rat. Abeta also blocked IL-1beta- or LPS-induced C/EBP protein levels. The most prominent effects were observed on DNA binding activity and protein levels of C/EBPdelta. Our results demonstrate a dysregulation of C/EBP when glial cells are activated in the presence of Alzheimer Abeta peptides.
B cells have been shown to be refractory to reprogramming and B-cell-derived induced pluripotent stem cells (iPSC) have only been generated from murine B cells engineered to carry doxycycline-inducible Oct4, Sox2, Klf4 and Myc (OSKM) cassette in every tissue and from EBV/SV40LT-immortalized lymphobl
astoid cell lines. Here, we show for the first time that freshly isolated non-cultured human cord blood (CB)- and peripheral blood (PB)-derived CD19+CD20+ B cells can be reprogrammed to iPSCs carrying complete VDJH immunoglobulin (Ig) gene monoclonal rearrangements using non-integrative tetracistronic, but not monocistronic, OSKM-expressing Sendai Virus. Co-expression of C/EBPalpha with OSKM facilitates iPSC generation from both CB- and PB-derived B cells. We also demonstrate that myeloid cells are much easier to reprogram than B and T lymphocytes. Differentiation potential back into the cell type of their origin of B-cell-, T-cell-, myeloid- and fibroblast-iPSCs is not skewed, suggesting that their differentiation does not seem influenced by 'epigenetic memory'. Our data reflect the actual cell-autonomous reprogramming capacity of human primary B cells because biased reprogramming was avoided by using freshly isolated primary cells, not exposed to cytokine cocktails favoring proliferation, differentiation or survival. The ability to reprogram CB/PB-derived primary human B cells offers an unprecedented opportunity for studying developmental B lymphopoiesis and modeling B-cell malignancies.
Anxa2 is dysregulated in many types of carcinomas and implicated in several pivotal biological functions, such as angiogenesis, cell proliferation, invasion, and metastasis. We previously demonstrated that upregulation of Anxa2 enhances the proliferation and invasion of breast cancer cells. However,
the detailed mechanism remains unclear. In this study, co-immunoprecipitation and LC-MS/MS-based interactome approach were employed to screen potential Anxa2 binding proteins. A total of 312 proteins were identified as candidate Anxa2 interacting partners. Using Gene Ontology, pathway annotation, and protein-protein interaction analyses, we constructed a connected network for Anxa2 interacting proteins, and Ebp1 may function as a "hub" in the Anxa2 interaction network. Moreover, Ebp1 knockdown resulted in enhanced cell proliferation and invasion, as well as increased expression of Anxa2. Furthermore, the abundance of cyclin D1 and the phosphorylation of Erk1/2 were increased in Ebp1 inhibited cells. This finding is consistent with a previous study, in which upregulation of Anxa2 results in an increased cyclin D1 expression and Erk1/2 activation. Our results suggest a novel function of Ebp1 as a binding protein and negative regulator of Anxa2. The functional association between Anxa2 and EBP1 may also participate in regulating cancer cell proliferation and invasion, thereby contributing to cancer progression.
Venot Q, etal., PLoS One. 2016 Jan 20;11(1):e0146962. doi: 10.1371/journal.pone.0146962. eCollection 2016.
ABCB4/MDR3, a member of the ABC superfamily, is an ATP-dependent phosphatidylcholine translocator expressed at the canalicular membrane of hepatocytes. Defects in the ABCB4 gene are associated with rare biliary diseases. It is essential to understand the mechanisms of its canalicular membrane expres
sion in particular for the development of new therapies. The stability of several ABC transporters is regulated through their binding to PDZ (PSD95/DglA/ZO-1) domain-containing proteins. ABCB4 protein ends by the sequence glutamine-asparagine-leucine (QNL), which shows some similarity to PDZ-binding motifs. The aim of our study was to assess the potential role of the QNL motif on the surface expression of ABCB4 and to determine if PDZ domain-containing proteins are involved. We found that truncation of the QNL motif decreased the stability of ABCB4 in HepG2-transfected cells. The deleted mutant ABCB4-DeltaQNL also displayed accelerated endocytosis. EBP50, a PDZ protein highly expressed in the liver, strongly colocalized and coimmunoprecipitated with ABCB4, and this interaction required the QNL motif. Down-regulation of EBP50 by siRNA or by expression of an EBP50 dominant-negative mutant caused a significant decrease in the level of ABCB4 protein expression, and in the amount of ABCB4 localized at the canalicular membrane. Interaction of ABCB4 with EBP50 through its PDZ-like motif plays a critical role in the regulation of ABCB4 expression and stability at the canalicular plasma membrane.
Bogojevic D, etal., Gen Physiol Biophys. 2003 Jun;22(2):279-85.
Interactions of nuclear extract and nuclear matrix proteins from rat hepatocytes with the hormone response element of the alpha2-macroglobulin gene were studied. By Western and South-Western blot analysis we have shown the presence of C/EBPbeta in the examined n
uclear fractions as well as its increased binding affinity to the examined gene sequence during the acute-phase response. The results suggest that both nuclear protein fractions could participate in the transcriptional regulation of the alpha2-macroglobulin gene in the rat hepatocytes.
Del-1 is an endothelial cell-secreted anti-inflammatory protein. In humans and mice, Del-1 expression is inversely related to that of IL-17, which inhibits Del-1 through hitherto unidentified mechanism(s). Here we show that IL-17 downregulates human endothelial cell expression of Del-1 by targeting
a critical transcription factor, C/EBPbeta. Specifically, IL-17 causes GSK-3beta-dependent phosphorylation of C/EBPbeta, which is associated with diminished C/EBPbeta binding to the Del-1 promoter and suppressed Del-1 expression. This inhibitory action of IL-17 can be reversed at the GSK-3beta level by PI3K/Akt signalling induced by D-resolvins. The biological relevance of this regulatory network is confirmed in a mouse model of inflammatory periodontitis. Intriguingly, resolvin-D1 (RvD1) confers protection against IL-17-driven periodontal bone loss in a Del-1-dependent manner, indicating an RvD1-Del-1 axis against IL-17-induced pathological inflammation. The dissection of signalling pathways regulating Del-1 expression provides potential targets to treat inflammatory diseases associated with diminished Del-1 expression, such as periodontitis and multiple sclerosis.
Arambasic J, etal., IUBMB Life. 2010 Mar;62(3):227-36.
Upregulation of haptoglobin (Hp) expression in the rat during the acute phase (AP) response is the result of synergistic effects of IL-6-, IL-1beta-, and corticosterone-activated signaling pathways. IL-6 signaling terminates in cis-trans interactions of the Hp gene hormone-responsive element (HRE) w
ith transcription factors STAT3 and C/EBPbeta. The aim of this study was to examine the unresolved molecular mechanism of glucocorticoid action. A 3-fold rise in serum corticosterone at 2 and 4 h of the AP response induced by turpentine administration preceded a 2.3-fold increase in the rate of Hp gene transcription at 12 h that was accompanied by a 4.8-fold increase in glucocorticoid receptor (GR), the appearance of an 86-kDa STAT3 isoform and 3.9-, 1.9-, and 1.7-fold increased amounts of 91-kDa STAT3, 35- and 42-kDa C/EBPbeta isoforms in the nucleus. These events resulted in 4.6- and 2.5-fold increased Hp levels in the liver and serum at 24 h. HRE affinity chromatography and immunoblot analysis revealed that maximal occupancy of the HRE with GR, STAT3, and C/EBPbeta at 12 h correlated with increased transcriptional activity of the Hp gene. Coimmunoprecipitation experiments showed that activated GR established de novo interaction with STAT3 isoforms while GR-C/EBPbeta interactions observed during basal transcription increased during the AP response. Computer analysis of the HRE disclosed two potential GR-binding sites: one overlapping STAT3, another adjacent to a C/EBPbeta-binding site. This finding and the experimental results suggest that activated GR through direct interactions with STAT3 and C/EBPbeta, participates in Hp gene upregulation as a transcriptional coactivator.
Al Ghouleh I, etal., Proc Natl Acad Sci U S A. 2016 Sep 6;113(36):E5308-17. doi: 10.1073/pnas.1514161113. Epub 2016 Aug 18.
Despite numerous reports implicating NADPH oxidases (Nox) in the pathogenesis of many diseases, precise regulation of this family of professional reactive oxygen species (ROS) producers remains unclear. A unique member of this family, Nox1 oxidase, functions as either a canonical or hybrid system
using Nox organizing subunit 1 (NoxO1) or p47(phox), respectively, the latter of which is functional in vascular smooth muscle cells (VSMC). In this manuscript, we identify critical requirement of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50; aka NHERF1) for Nox1 activation and downstream responses. Superoxide (O2 (*-)) production induced by angiotensin II (AngII) was absent in mouse EBP50 KO VSMC vs. WT. Moreover, ex vivo incubation of aortas with AngII showed a significant increase in O2 (*-) in WT but not EBP50 or Nox1 nulls. Similarly, lipopolysaccharide (LPS)-induced oxidative stress was attenuated in femoral arteries from EBP50 KO vs. WT. In silico analyses confirmed by confocal microscopy, immunoprecipitation, proximity ligation assay, FRET, and gain-/loss-of-function mutagenesis revealed binding of EBP50, via its PDZ domains, to a specific motif in p47(phox) Functional studies revealed AngII-induced hypertrophy was absent in EBP50 KOs, and in VSMC overexpressing EBP50, Nox1 gene silencing abolished VSMC hypertrophy. Finally, ex vivo measurement of lumen diameter in mouse resistance arteries exhibited attenuated AngII-induced vasoconstriction in EBP50 KO vs. WT. Taken together, our data identify EBP50 as a previously unidentified regulator of Nox1 and support that it promotes Nox1 activity by binding p47(phox) This interaction is pivotal for agonist-induced smooth muscle ROS, hypertrophy, and vasoconstriction and has implications for ROS-mediated physiological and pathophysiological processes.
Di Stefano B, etal., Nat Cell Biol. 2016 Apr;18(4):371-81. doi: 10.1038/ncb3326. Epub 2016 Mar 14.
Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) is typically inefficient and has been explained by elite-cell and stochastic models. We recently reported that B cells exposed to a pulse of C/EBPalpha (Balpha' cells) behave as elite cells,
in that they can be rapidly and efficiently reprogrammed into iPSCs by the Yamanaka factors OSKM. Here we show that C/EBPalpha post-transcriptionally increases the abundance of several hundred proteins, including Lsd1, Hdac1, Brd4, Med1 and Cdk9, components of chromatin-modifying complexes present at super-enhancers. Lsd1 was found to be required for B cell gene silencing and Brd4 for the activation of the pluripotency program. C/EBPalpha also promotes chromatin accessibility in pluripotent cells and upregulates Klf4 by binding to two haematopoietic enhancers. Balpha' cells share many properties with granulocyte/macrophage progenitors, naturally occurring elite cells that are obligate targets for leukaemic transformation, whose formation strictly requires C/EBPalpha.
Liu GF, etal., Biochim Biophys Acta. 2016 Feb;1859(2):348-54. doi: 10.1016/j.bbagrm.2015.12.004. Epub 2015 Dec 15.
Mammalian Sirtuin proteins (SIRTs) are homologs of yeast Sir2, and characterized as class III histone deacetylases of NAD(+) dependence. Unlike their lower counterparts that are directly involved in the extending of lifespan, mammalian SIRTs mainly function in metabolism and cellular homeostasis, a
mong them, SIRT7 is the least understood. SIRT7 is localized in the nucleus and rich in nucleoli associated with RNA polymerase I, and correlated with cell proliferation. In contrast, SIRT7 has recently been demonstrated to specifically deacetylate H3K18ac in the chromatin, and in most cases represses proliferation. Although MicroRNA as miR-125b has been reported to down-regulate SIRT7 by binding to its 3'UTR, however, how SIRT7 gene is regulated remains unclear. Here, we identified the transcription initiation site of human SIRT7 gene at the upstream 23rd A nucleotide respective to the translational codon, and the SIRT7 is a TATA-less and initiator-less gene. The sequences in the upstream region between -256 and -129 bp are identical with important functions in the three species detected. A C/EBPalpha responding element is found that binds both C/EBPalpha and C/EBPbeta in vitro. We showed TSA induced SIRT7 gene transcription and only the HDAC3, but not its catalytic domain depleted mutant, interacted with C/EBPalpha to occupy the C/EBPalpha element and repressed SIRT7 gene in the hepatocellular carcinoma cells. To our knowledge, this is the first report on the regulation mechanism of SIRT7 gene, in which, HDAC3 collaborated with C/EBPalpha to occupy its responding element in the upstream region of SIRT7 gene and repressed its expression in human cells.
Hepatocellular carcinoma is associated with high mortality, and tumor metastasis is an important reason for poor prognosis. However, metastasis has not been effectively prevented in clinical therapy and the mechanisms underlying metastasis have not been fully characterized. CCAAT/enhancer-binding p
rotein-alpha (C/EBPalpha) is a transcriptional regulator with an essential role in tumor metastasis. We used short-activating RNAs (saRNA) to enhance expression of C/EBPalpha. Intravenous injection of C/EBPalpha-saRNA in a nude mouse liver orthotopic xenograft tumor model inhibited intrahepatic and distant metastasis. C/EBPalpha-saRNA-treated mice showed increased serum levels of albumin and decreased alanine aminotransferase (ALT), glutamic-oxalacetic transaminase (AST), indicating a role of C/EBPalpha in improving liver function. Migration and invasion were inhibited in hepatoma cell lines transfected with C/EBPalpha-saRNA. We also observed an inhibition of epithelial-mesenchymal transition (EMT) and suppression of epidermal growth factor receptor (EGFR), EGFR phosphorylation, and beta-catenin in C/EBPa-saRNA-transfected cells. Our results suggested that C/EBPalpha-saRNA successfully inhibited HCC metastasis by inhibiting EGFR/beta-catenin signaling pathway mediated EMT in vitro and in vivo.
Accumulating evidences indicate that different long non-coding RNAs (lncRNAs) might play a relevant role in tumorigenesis, with their expression and function already associated to cancer development and progression. CCAAT/enhancer-binding protein-alpha (CEBPA)
is a critical regulator of myeloid differentiation whose inactivation contributes to the development of acute myeloid leukemia (AML). Mutations in C/EBPalpha occur in around 10% of AML cases, leading to the expression of a 30-kDa dominant negative isoform (C/EBPalpha-p30). In this study, we identified the oncogenic urothelial carcinoma associated 1 (UCA1) lncRNA as a novel target of the C/EBPalpha-p30. We show that wild-type C/EBPalpha and C/EBPalpha-p30 isoform can bind the UCA1 promoter but have opposite effects on UCA1 expression. While wild-type C/EBPalpha represses, C/EBPalpha-p30 can induce UCA1 transcription. Notably, we also show that UCA1 expression increases in cytogenetically normal AML cases carrying biallelic CEBPA mutations. Furthermore, we demonstrate that UCA1 sustains proliferation of AML cells by repressing the expression of the cell cycle regulator p27kip1. Thus, we identified, for the first time, an oncogenic lncRNA functioning in concert with the dominant negative isoform of C/EBPalpha in AML.
Hirata M, etal., Hum Mol Genet. 2012 Mar 1;21(5):1111-23. doi: 10.1093/hmg/ddr540. Epub 2011 Nov 17.
To elucidate the molecular mechanism underlying the endochondral ossification process during the skeletal growth and osteoarthritis (OA) development, we examined the signal network around CCAAT/enhancer-binding protein-beta (C/EBPbeta, encoded by CEBP
ont-weight:700;'>EBPB), a potent regulator of this process. Computational predictions and a C/EBP motif-reporter assay identified RUNX2 as the most potent transcriptional partner of C/EBPbeta in chondrocytes. C/EBPbeta and RUNX2 were induced and co-localized in highly differentiated chondrocytes during the skeletal growth and OA development of mice and humans. The compound knockout of Cebpb and Runx2 in mice caused growth retardation and resistance to OA with decreases in cartilage degradation and matrix metalloproteinase-13 (Mmp-13) expression. C/EBPbeta and RUNX2 cooperatively enhanced promoter activity of MMP13 through specific binding to a C/EBP-binding motif and an osteoblast-specific cis-acting element 2 motif as a protein complex. Human genetic studies failed to show the association of human CEBPB gene polymorphisms with knee OA, nor was there a genetic variation around the identified responsive region in the human MMP13 promoter. However, hypoxia-inducible factor-2alpha (HIF-2alpha), a functional and genetic regulator of knee OA through promoting endochondral ossification, was identified as a potent and functional inducer of C/EBPbeta expression in chondrocytes by the CEBPB promoter assay. Hence, C/EBPbeta and RUNX2, with MMP-13 as the target and HIF-2alpha as the inducer, control cartilage degradation. This molecular network in chondrocytes may represent a therapeutic target for OA.
Tumor necrosis factor (TNF)-alpha is a pleiotropic cytokine that triggers cell proliferation, cell death, or inflammation. Besides its cytotoxic effect on cancer cells, TNF-alpha exerts tumor promoting activity. Aberrant TNF-alpha signaling promotes cancer cell motility, invasiveness, and enhances c
ancer metastasis. Exaggerated tumor cell migration, invasion, and metastasis by TNF-alpha has been attributed to the activation of NF-kappaB signaling. It is yet to be elucidated if other signaling pathways and effector molecules are involved in TNF-alpha-induced cancer cell migration and metastasis. Expression of C/EBPbeta, a transcription factor involved in metabolism, inflammation, and cancer, is increased upon TNF-alpha treatment. TNF-alpha induces C/EBPbeta expression by enhancing its transcription and protein stability. Activation of p38 MAPK, but not NF-kappaB or JNK, is responsible for TNF-alpha-induced stabilization of C/EBPbeta protein. C/EBPbeta is involved in TNF-alpha-induced cancer cell migration. Knockdown of C/EBPbeta inhibits TNF-alpha-induced cell migration, while overexpression of C/EBPbeta increases migration of cancer cells. C/EBPbeta is translated into transcriptional activator LAP1 and LAP2 and transcriptional repressor LIP utilizing alternative in-frame translation start sites. Despite TNF-alpha induces expression of all three isoforms, LAP1/2, but not LIP, promote cancer cell migration. TNF-alpha induced MMP1/3 expression, which was abrogated by C/EBPbeta knockdown or p38 MAPK inhibition. MMP inhibitor or knockdown of MMP1/3 diminished TNF-alpha- and C/EBPbeta-induced cell migration. Thus, C/EBPbeta mediates TNF-alpha-induced cancer cell migration by inducing MMP1/3 expression, and may participate in the regulation of inflammation-associated cancer metastasis.
Barakat DJ, etal., Cancer Lett. 2016 May 28;375(1):152-61. doi: 10.1016/j.canlet.2016.03.005. Epub 2016 Mar 8.
The purpose of this study was to ascertain the mechanisms by which advanced prostate cancer cells resist bortezomib therapy. Several independent studies have shown that cells are protected from proteasome inhibition by increased autophagic activity. We investigated whether C/EBP
:700;'>EBPbeta, a transcription factor involved in the control of autophagic gene expression, regulates resistance to proteasome inhibition. In PC3 cells over-expressing C/EBPbeta, turnover of autophagic substrates and expression of core autophagy genes were increased. Conversely, C/EBPbeta knockdown suppressed autophagosome-lysosome fusion. We also found that C/EBPbeta knockdown suppressed REDD1 expression to delay early autophagy, an effect rescued by exogenous REDD1. Cells with suppressed C/EBPbeta levels showed delayed autophagy activation upon bortezomib treatment. Knockdown of C/EBPbeta sensitized PC3 cells to bortezomib, and blockade of autophagy by chloroquine did not further increase cell death in cells expressing shRNA targeting C/EBPbeta. Lastly, we observed a decreased growth of PC3 cells and xenografts with C/EBPbeta knockdown and such xenografts were sensitized to bortezomib treatment. Our results demonstrate that C/EBPbeta is a critical effector of autophagy via regulation of autolysosome formation and promotes resistance to proteasome inhibitor treatment by increasing autophagy.
The transcription factor C/EBPbeta plays a key role in monocytic differentiation and inflammation. Its small isoform LIP is associated with proliferation at early premonocytic developmental stages and regulated via mTOR-dependent signalling. During later stages
of (pre)monocytic differentiation there is a considerable increase in the large C/EBPbeta isoforms LAP*/LAP which inhibit proliferation thus supporting terminal differentiation. Here, we showed in different models of monocytic differentiation that this dramatic increase in the LAP*/LAP protein and LAP/LIP ratio was accompanied by an only modest/retarded mRNA increase suggesting an important role for (post)translational mechanisms. We found that LAP*/LAP formation was induced via MEK/RSK-dependent cascades, whereas mTOR/S6K1 were not involved. Remarkably, LAP*/LAP expression was dependent on phosphorylated eIF4B, an acceleratory protein of RNA helicase eIF4A. PKR inhibition reduced the expression of eIF4B and C/EBPbeta in an eIF2alpha-independent manner. Furthermore, under our conditions a marked stabilisation of LAP*/LAP protein occurred, accompanied by reduced chymotrypsin-like proteasome/calpain activities and increased calpastatin levels. Our study elucidates new signalling pathways inducing LAP*/LAP expression and indicates new alternative PKR functions in monocytes. The switch from mTOR- to RSK-mediated signalling to orchestrate eIF4B-dependent LAP*/LAP translation, accompanied by increased protein stability but only small mRNA changes, may be a prototypical example for the regulation of protein expression during selected processes of differentiation/proliferation.
Williams SC, etal., J Biol Chem 1998 May 29;273(22):13493-501.
C/EBPepsilon is a member of the CCAAT/enhancer binding protein family of basic region/leucine zipper transcriptional activators. The C/EBPepsilon protein is highly conserved between rodents and humans, and its domain structu
re is very similar to C/EBPalpha. In mice C/EBPepsilon mRNA is only detected in hematopoietic tissues, including embryonic liver and adult bone marrow and spleen. Within the hematopoietic system, C/EBPepsilon is expressed primarily in myeloid cells, including promyelocytes, myelomonocytes, and their differentiated progeny. To identify potential functions of C/EBPepsilon, cell lines over-expressing the C/EBPepsilon protein were generated in the P388 lymphoblastic cell line. In contrast to the parental cell line, C/EBPepsilon-expressing cell lines displayed lipopolysaccharide-inducible expression of the interleukin-6 and monocyte chemoattractant protein 1 (MCP-1) genes as well as elevated basal expression of the MIP-1alpha and MIP-1beta chemokine genes. In the EML-C1 hematopoietic stem cell line, C/EBPepsilon mRNA levels increased as the cells progressed along the myeloid lineage, just preceding activation of the gene encoding the receptor for macrophage-colony-stimulating factor (M-CSFR). M-CSFR expression was stimulated in C/EBPepsilon-expressing P388 cell lines, when compared with either the parental P388 cells or P388 cell lines expressing either C/EBPalpha or C/EBPbeta. These results suggest that C/EBPepsilon may be an important regulator of differentiation of a subset of myeloid cell types and may also participate in the regulation of cytokine gene expression in mature cells.
The integrated stress response (ISR) controls cellular adaptations to nutrient deprivation, redox imbalances, and endoplasmic reticulum (ER) stress. ISR genes are upregulated in stressed cells, primarily by the bZIP transcription factor ATF4 through its recruitment to cis-regulatory C/EBP
ont-weight:700;'>EBP:ATF response elements (CAREs) together with a dimeric partner of uncertain identity. Here, we show that C/EBPgamma:ATF4 heterodimers, but not C/EBPbeta:ATF4 dimers, are the predominant CARE-binding species in stressed cells. C/EBPgamma and ATF4 associate with genomic CAREs in a mutually dependent manner and coregulate many ISR genes. In contrast, the C/EBP family members C/EBPbeta and C/EBP homologous protein (CHOP) were largely dispensable for induction of stress genes. Cebpg(-/-) mouse embryonic fibroblasts (MEFs) proliferate poorly and exhibit oxidative stress due to reduced glutathione levels and impaired expression of several glutathione biosynthesis pathway genes. Cebpg(-/-) mice (C57BL/6 background) display reduced body size and microphthalmia, similar to ATF4-null animals. In addition, C/EBPgamma-deficient newborns die from atelectasis and respiratory failure, which can be mitigated by in utero exposure to the antioxidant, N-acetyl-cysteine. Cebpg(-/-) mice on a mixed strain background showed improved viability but, upon aging, developed significantly fewer malignant solid tumors than WT animals. Our findings identify C/EBPgamma as a novel antioxidant regulator and an obligatory ATF4 partner that controls redox homeostasis in normal and cancerous cells.
Valls Seron M, etal., J Neuroinflammation. 2015 May 10;12:88. doi: 10.1186/s12974-015-0309-5.
BACKGROUND: The prognosis of bacterial meningitis largely depends on the severity of the inflammatory response. The transcription factor CAAT/enhancer-binding protein delta (C/EBPdelta) plays a key role in the regulation of the inflammatory response during bacte
rial infections. Consequently, we assessed the role of C/EBPdelta during experimental meningitis. METHODS: Wild-type and C/EBPdelta-deficient mice (C/EBPdelta(-/-)) were intracisternally infected with Streptococcus pneumoniae and sacrificed after 6 or 30 h, or followed in a survival study. RESULTS: In comparison to wild-type mice, C/EBPdelta(-/-) mice showed decreased bacterial loads at the primary site of infection and decreased bacterial dissemination to lung and spleen 30 h after inoculation. Expression levels of the inflammatory mediators IL-10 and KC were lower in C/EBPdelta(-/-) brain homogenates, whereas IL-6, TNF-alpha, IL-1beta, and MIP-2 levels were not significantly different between the two genotypes. Moreover, C/EBPdelta(-/-) mice demonstrated an attenuated systemic response as reflected by lower IL-10, IL-6, KC, and MIP-2 plasma levels. No differences in clinical symptoms or in survival were observed between wild-type and C/EBPdelta(-/-) mice. CONCLUSION: C/EBPdelta in the brain drives the inflammatory response and contributes to bacterial dissemination during pneumococcal meningitis. C/EBPdelta does, however, not affect clinical parameters of the disease and does not confer a survival benefit.
Neuropeptide Y (NPY), a peptide released by nervous cells, appears to contribute to adiposity regulation by increasing food intake and inhibiting lipolysis. New NPY receptor related antagonists such as S.A.0204 are being developed as potential anti-obesity drugs affecting adipocyte lipid metabolism
and thermogenesis. In this sense, those animals fed on a high-energy yielding (cafeteria) diet decreased body fat weight as compared to overweight controls, when they were administered with S.A.0204, and increased body temperature, which statistically correlated with high UCP2 mRNA expression levels in white adipose tissue. In addition, the in vivo NPY-antagonist administration was able to prevent white adipose tissue growth in animals fed the cafeteria (high-fat) diet by impairing PPARy and CIEBPalpha mRNA expression in white fat cells. In summary, this novel NPY related-antagonist S.A.0204 may regulate body fat deposition by affecting both energy dissipation and white adipose tissue deposition, representing a potential new pharmacological strategy for obesity management.
The transcription of the alpha1-acid glycoprotein gene is induced by inflammatory cytokines and glucocorticoids. C/EBPbeta is a major transcription factor involved in the induction of the agp gene by some cytokines. In this report, we have identified a novel tra
nscriptional intermediary factor, TIF1beta, which could enhance the transcription of the agp gene by the glucocorticoid receptor (GR) and C/EBPbeta. TIF1beta belongs to a subgroup of RING (really interesting new gene) finger proteins that contain a RING finger preceding two B box-type fingers and a putative coiled-coil domain (RBCC domain). Immunoprecipitation experiments showed that the interaction between GR and TIF1beta is ligand independent. The overexpression of the TIF1beta gene enhances GR-regulated expression in a ligand- and glucocorticoid-responsive element (GRE)-dependent manner. TIF1beta can also augment C/EBPbeta-mediated activity on wild-type and GRE-mutated agp genes, but this augmentation is diminished when all three C/EBPbeta-binding elements are mutated. Functional and biochemical characterizations indicated that the bZIP domain of C/EBPbeta and the RBCC domain, plant homeodomain finger, and bromodomain of TIF1beta are crucial for the interactions of these proteins. Taken together, these results suggest that TIF1beta serves as a converging mediator of signal transduction pathways of glucocorticoids and some inflammatory cytokines.
Chemoresistance to anticancer drugs substantially reduces survival in epithelial ovarian carcinoma (EOC). Here, microarray analysis showed that collagen type XI alpha 1 (COL11A1) is a chemotherapy response-associated gene. Chemoresistant cells expressed higher COL11A1 and c/EBP
:700;'>EBPbeta than chemosensitive cells. COL11A1 or c/EBPbeta downregulation suppressed chemoresistance, whereas COL11A1 overexpression attenuated sensitivity to cisplatin and paclitaxel.The c/EBPbeta binding site in the COL11A1 promoter was identified as the major determinant of cisplatin- and paclitaxel-induced COL11A1 expression. Immunoprecipitation and immunofluorescence showed that in resistant cells, Akt and PDK1 were highly expressed and that anticancer drugs enhanced binding activity between COL11A1 and PDK1 binding and attenuated PDK1 ubiquitination and degradation. Conversely, chemosensitive cells showed decreased activity of COL11A1 binding to PDK1 and increased PDK1 ubiquitination, which were reversed by COL11A1 overexpression. Analysis of 104 EOC patients showed that high COL11A1 mRNA levels are significantly associated with poor chemoresponse and clinical outcome.
Pedersen TA, etal., Genes Dev. 2001 Dec 1;15(23):3208-16.
Chromatin remodeling is an important step in promoter activation during cellular lineage commitment and differentiation. We show that the ability of the C/EBPalpha transcription factor to direct adipocyte differentiation of uncommitted fibroblast precursors and
to activate SWI/SNF-dependent myeloid-specific genes depends on a domain, C/EBPalpha transactivation element III (TE-III), that binds the SWI/SNF chromatin remodeling complex. TE-III collaborates with C/EBPalpha TBP/TFIIB interaction motifs during induction of adipogenesis and adipocyte-specific gene expression. These results indicate that C/EBPalpha acts as a lineage-instructive transcription factor through SWI/SNF-dependent modification of the chromatin structure of lineage-specific genes, followed by direct promoter activation via recruitment of the basal transcription-initiation complex, and provide a mechanism by which C/EBPalpha can mediate differentiation along multiple cellular lineages.
Lu H, etal., Med Oncol. 2015 Mar;32(3):82. doi: 10.1007/s12032-015-0544-8. Epub 2015 Feb 21.
CUGBP1, which is involved in posttranscriptional regulatory networks, may control cell growth, activation and differentiation. Meanwhile, CCAAT/enhancer-binding protein alpha (C/EBPalpha) acts as a basic leucine zipper transcription factor which controls differe
ntiation-dependent gene expression and inhibits cell proliferation. To date, very little is known about the association between CUGBP 1 and C/EBPalpha in regulating cell proliferation and apoptosis in non-small cell lung cancer (NSCLC). CUGBP1 and C/EBPalpha mRNA expressions were analyzed in NSCLC tumor and adjacent normal tissues, and the relationship in clinicopathological parameters was evaluated. Knockdown of CUGBP1 and C/EBPalpha regulated by CUGBP1 in NSCLC cell line was identified by real-time PCR and Western blot. The effect of depletion of CUGBP1 was evaluated by MTT assay and Annexin/Propidium Iodide Apoptosis assay. CUGBP1 is highly expressed and expression of C/EBPalpha is low in NSCLC tissues. The correlation analysis revealed that there was negative correlation between the expression of CUGBP 1 and C/EBPalpha. Knockdown of CUGBP1 effectively silenced the expression of CUGBP1 and up-regulated C/EBPalpha. Also, suppression of CUGBP1 inhibits proliferation and induces apoptosis in A549 cells. These observations suggest that the first proof the overexpression of CUGBP1 in NSCLC contributes to tumorigenesis through down-regulation of C/EBPalpha. Knockdown of CUGBP1 or up-regulation C/EBPalpha might be a potential therapeutic approach for human non-small cell lung cancers.
C/EBPbeta (CCAAT/enhancer-binding protein beta) is a transcriptional regulator of the UCP1 (uncoupling protein-1) gene, the specific marker gene of brown adipocytes that is responsible for their thermogenic capacity. To investigate the role of C/EBP
t-weight:700;'>EBPbeta in brown fat, we studied the C/EBPbeta-null mice. When placed in the cold, C/EBPbeta(-/-) mice did not maintain body temperature. This cold-sensitive phenotype occurred, although UCP1 and PGC-1alpha (peroxisome-proliferator-activated receptor gamma co-activator-1alpha) gene expression was unaltered in brown fat of C/EBPbeta(-/-) mice. The UCP1 gene promoter was repressed by the truncated inhibitory C/EBPbeta isoform LIP (liver-enriched transcriptional inhibitory protein, the truncated inhibitory C/EBPbeta isoform). Since C/EBPbeta-null mice lack both C/EBPbeta isoforms, active LAP (liver-enriched transcriptional activatory protein, the active C/EBPbeta isoform) and LIP, the absence of LIP may have a stronger effect than the absence of LAP upon UCP1 gene expression. Gene expression for UCP2 and UCP3 was not impaired in all tissues analysed. In primary brown adipocytes from C/EBPbeta(-/-) mice, induction of gene expression by noradrenaline was preserved. In contrast, the expression of genes related to lipid storage was impaired, as was the amount of triacylglycerol mobilized after acute cold exposure in brown fat from C/EBPbeta(-/-) mice. LPL (lipoprotein lipase) activity was also impaired in brown fat, but not in other tissues of C/EBPbeta(-/-) mice. LPL protein levels were also diminished, but this effect was independent of changes in LPL mRNA, suggesting that C/EBPbeta is involved in the post-transcriptional regulation of LPL gene expression in brown fat. In summary, defective thermoregulation owing to the lack of C/EBPbeta is associated with the reduced capacity to supply fatty acids as fuels to sustain brown fat thermogenesis.
Park JY, etal., Biochem Cell Biol. 2015 Aug;93(4):290-7. doi: 10.1139/bcb-2014-0145. Epub 2015 Feb 26.
Ezrin-radixin-moesin-binding protein 50 (EBP50) is a scaffolding protein expressed in polarized epithelial cells in various organs, including the liver, kidney, and small intestine, in which it regulates the trafficking and targeting cellular proteins. EBP
yle='font-weight:700;'>EBP50 contains two postsynaptic density-95/disk-large/ZO-1 homology (PDZ) domains (e.g., PDZ1 and PDZ2) and an ezrin/radixin/moesin-binding (EB) domain. PDZ domains are one of the major scaffolding domains regulating protein-protein interactions with critical biological roles in cell polarity, migration, proliferation, recognition, and cell-cell interaction. PDZ1 and PDZ2 in EBP50 have different ligand selectivity, although several high-resolution structural studies of isolated PDZ1 and PDZ2 showed similar structures. We studied the conformations of full-length EBP50 and isolated PDZ1 and PDZ2 using hydrogen/deuterium exchange mass spectrometry (HDX-MS). The deuterium uptake profiles of isolated PDZ1 and PDZ2 were similar to those of full-length EBP50. Interestingly, PDZ1 was more dynamic than PDZ2, and these PDZ domains underwent different conformational changes upon ligand binding. These results might explain the differences in ligand-selectivity between PDZ1 and PDZ2.
Roth M, etal., N Engl J Med. 2004 Aug 5;351(6):560-74.
BACKGROUND: Increased proliferation of bronchial smooth-muscle cells may lead to increased muscle mass in the airways of patients with asthma. The antiproliferative effect of glucocorticoids in bronchial smooth-muscle cells in subjects without asthma is mediated by a complex of the glucocorticoid re
ceptor and the CCAAT/enhancer binding protein alpha (C/EBPalpha). We examined the signaling pathway controlling the inhibitory effect of glucocorticoids on cell proliferation and interleukin-6 synthesis in bronchial smooth-muscle cells of subjects with asthma and those without asthma. METHODS: Lines of bronchial smooth-muscle cells were established from cells from 20 subjects with asthma, 8 subjects with emphysema, and 26 control subjects. Cell proliferation was determined by means of cell counts and [3H]thymidine incorporation. Signal transduction was studied by means of an electrophoretic DNA mobility-shift assay, a supershift electrophoretic-mobility assay, immunoblotting, use of C/EBPalpha antisense oligonucleotides, and use of a human C/EBPalpha expression vector. Interleukin-6 release was determined by means of an enzyme-linked immunosorbent assay. RESULTS: Glucocorticoids activated the glucocorticoid receptor and inhibited serum-induced secretion of interleukin-6 in bronchial smooth-muscle cells from both subjects with asthma and those without asthma; however, glucocorticoids inhibited proliferation only in bronchial smooth-muscle cells from subjects without asthma. C/EBPalpha protein was detected by immunoblotting in all bronchial smooth-muscle cells from subjects without asthma but not in those with asthma, whereas the protein was expressed in lymphocytes from both groups of subjects. C/EBPalpha antisense oligonucleotides or the glucocorticoid-receptor inhibitor mifepristone reversed the antiproliferative effect of glucocorticoids in bronchial smooth-muscle cells from subjects without asthma. When bronchial smooth-muscle cells from subjects with asthma were transiently transfected with an expression vector for human C/EBPalpha, two forms of the protein were expressed, and subsequent administration of glucocorticoids inhibited cell proliferation. CONCLUSIONS: We hypothesize that a cell-type-specific absence of C/EBPalpha is responsible for the enhanced proliferation of bronchial smooth-muscle cells derived from subjects with asthma and that it explains the failure of glucocorticoids to inhibit proliferation in vitro.
Chiang MC, etal., Hum Mol Genet. 2007 Mar 1;16(5):483-98. Epub 2007 Jan 9.
Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion in the Huntingtin (Htt) gene. Using two mouse models of HD, we demonstrate that the urea cycle deficiency characterized by hyperammonemia, high blood citrulline and suppression of ure
a cycle enzymes is a prominent feature of HD. The resultant ammonia toxicity might exacerbate the neurological deficits of HD. Suppression of C/EBPalpha, a crucial transcription factor for the transcription of urea cycle enzymes, appears to mediate the urea cycle deficiency in HD. We found that in the presence of mutant Htt, C/EBPalpha loses its ability to interact with an important cofactor (CREB-binding protein). Moreover, mutant Htt recruited C/EBPalpha into aggregates, as well as suppressed expression of the C/EBPalpha gene. Consumption of protein-restricted diets not only led to the restoration of C/EBPalpha's activity, and repair of the urea cycle deficiency and hyperammonemia, but also ameliorated the formation of Htt aggregates, the motor deterioration, the suppression of striatal brain-derived neurotrophic factor and the normalization of three protein chaperones (Hsp27, Hsp70 and Hsp90). Treatments aimed at repairing the urea cycle deficiency may provide a new strategy for dealing with HD.
Squatrito M, etal., Oncogene 2004 May 27;23(25):4454-65.
EBP1 was identified as a protein that interacts with the ErbB-3 receptor and possibly contributes to transducing growth regulatory signals. The existence of EBP1 homologs across species from simple eukaryotes to humans and i
ts wide tissue expression pattern suggest that EBP1 acts as a general signaling molecule. We provide evidence that EBP1 is localized to the cytoplasm and to the nucleolus, and that its nucleolar localization requires amino-acid sequences present at both the amino- and carboxy-terminus of the molecule. We also show that EBP1 overexpression inhibits proliferation of human fibroblasts, and that this effect is linked to its nucleolar localization. Using mass spectrometry we demonstrate that EBP1 is part of ribonucleoprotein complexes and associates with different rRNA species. It is becoming clear that cell growth and proliferation are actively coordinated with rRNA processing and ribosome assembly. Our findings indicate that EBP1 is a nucleolar growth-regulating protein, and we propose that it could represent a new link between ribosome biosynthesis and cell proliferation.
Pisapia L, etal., Int J Oncol. 2015 Aug;47(2):481-9. doi: 10.3892/ijo.2015.3051. Epub 2015 Jun 16.
Many solid tumours including melanoma, glioblastoma, and breast carcinomas express MHC class II molecules (MHC II). The surface expression of these molecules confers to non-hematopoietic tumour cells the role of non-professional antigen presenting cells and the ability to potentially stimulate tumo
ur-specific CD4+ T cell response. We studied EBP1, an ErbB3 binding protein, and the effects of p48 and p42 isoforms on the MHC II expression in U87 glioblastoma, M14 melanoma and MCF7 mammary carcinoma cell lines. We found that overexpression of p48 increases MHC II transcription in U87 and M14, through upregulation of CIITA transactivator and STAT1 phosphorylation. In addition, p48 protein influences MHC II expression by increasing mRNA stability. In melanoma and glioblastoma cell lines, p48 isoform functions as oncogene promoting tumour growth, while p42 isoform, that does not affect MHC II expression, acts as a tumour suppressor by blocking cell growth and inducing apoptosis. In contrast, p48 seems to act as tumour suppressor in breast carcinoma inhibiting proliferation, favouring apoptosis, and inducing a slight increase of MHC II expression similar to p42. Our data highlight the tissue specificity function of EBP1 isoforms and demonstrate that only the oncogene p48 activates MHC II expression in human solid tumours, via STAT1 phosphorylation, in order to affect tumour progression by triggering specific immune response.
Liu H, etal., Tumour Biol. 2015 Nov;36(11):8325-31. doi: 10.1007/s13277-015-3523-y. Epub 2015 May 26.
Ebp1, a protein identified by its interactions with the ErbB3 receptor, has been characterized as a negative regulator of cancers. RAS GTPase-activating protein (RasGAP), RASAL1, was recently identified as a major tumor suppressor in thyroid cancer. In this st
udy, we examined EBP1 expression in papillary and follicular thyroid cancer cells. We found that compared with normal thyroid cells, TPC1, WRO, and FTC133 thyroid tumor cells exhibited lower EBP1 expression at messenger RNA (mRNA) and protein levels. We then investigated the effects of forced EBP1 expression on growth, migration, and invasiveness of thyroid tumor cells. By using MTT and Boyden chamber assays, we showed that EBP1 overexpression dramatically reduced growth rate, migration, and invasiveness of K1 and FTC133 thyroid tumor cells. Furthermore, we explored the molecular mechanism underlying the effects of EBP1 on the cells by disclosing the correlation of EBP1 and RASAL1 expression. RASAL expression was elevated in thyroid tumor cells overexpressing EBP1. Knockdown RASAL by transduction of RASAL1 shRNA lentiviral particles markedly reduced RASAL levels with restoration of EBP1, and RASAL1 knockdown abrogated the effects of forced EBP1 expression on cell growth, migration, and invasiveness of thyroid tumor cells. These findings suggest that Ebp1 suppressed thyroid cancer cell lines by upregulating RASRAL expression.
Fouassier L, etal., J Biol Chem 2000 Aug 11;275(32):25039-45.
Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) is a versatile membrane-cytoskeleton linking protein that binds to the COOH-tail of specific integral membrane proteins through its two PDZ domains. These EBP50 bi
nding interactions have been implicated in sequestering interactive sets of proteins into common microdomains, regulating the activity of interacting proteins, and modulating membrane protein trafficking. With only two PDZ domains, it is unclear how EBP50 forms multiprotein complexes. Other PDZ proteins increase their breadth and diversity of protein interactions through oligomerization. Hypothesizing that EBP50 self-associates to amplify its functional capacity, far-Western blotting of cholangiocyte epithelial cell proteins with EBP50 fusion protein revealed that EBP50 binds to a 50-kDa protein. Far-Western blotting of EBP50 isolated by two-dimensional gel electrophoresis or immunoprecipitation demonstrates that the 50-kDa binding partner is itself EBP50. Further, co-transfection/co-precipitation studies show the self-association can occur in an intracellular environment. In vitro analysis of the EBP50-EBP50 binding interaction indicates it is both saturable and of relatively high affinity. Analysis of truncated EBP50 proteins indicates EBP50 self-association is mediated through its PDZ domains. The ability to self-associate provides a mechanism for EBP50 to expand its capacity to form multiprotein complexes and regulate membrane transport events.
Uterine epithelial cells (UECs) undergo extensive morphological remodelling in preparation for an implanting blastocyst. This remodelling involves changes in the actin cytoskeleton and surface structures including microvilli. Ezrin and ezrin-radixin-moesin-binding protein-50-kDa (EBP
eight:700;'>EBP50) link actin filaments to intra-membranous adhesion molecules and are important molecules in polarised epithelia. The current study is the first to describe the colocalisation and molecular association of ezrin and EBP50 in rat UECs by using immunofluorescence microscopy and immunoprecipitation techniques. These proteins have also been localised in relation to uterine epithelial cytoskeletal rearrangement during early pregnancy in the rat and to the effect of apical surface contact between opposing epithelial cells, blastocyst contact and contact with a silicon filament. Immunofluorescence microscopy has revealed that ezrin and EBP50 respond to contact between opposing epithelial cells and increase apically on day 6 of pregnancy. This apical distribution is also observed in UECs in contact with a silicon filament. Ezrin and EBP50 are however absent within the implantation chamber itself, seemingly mimicking the events that take place in leucocyte-endothelium binding. Thus, ezrin and EBP50 occur apically in UECs at the time of implantation in the rat and in response to a substitute blastocyst (filament) suggesting a role for these proteins in the cytoskeletal rearrangements that facilitate uterine receptivity and blastocyst-epithelial adhesion. Their loss within the implantation chamber possibly allows the subsequent invasion of the embryo.
Ki SH, etal., Mol Cell Biol. 2005 May;25(10):4150-65.
The expression of the glutathione S-transferase gene (GST), whose induction accounts for cancer chemoprevention, is regulated by activation of CCAAT/enhancer binding protein beta (C/EBPbeta) and NF-E2-related factor 2 (Nrf2). The present study investigated the r
epressing effects of activating glucocorticoid receptor (GR) on C/EBPbeta- and Nrf2-mediated GSTA2 gene induction and the mechanism. Dexamethasone that activates GR inhibited constitutive and oltipraz- or tert-butylhydroquinone (t-BHQ)-inducible GSTA2 expression in H4IIE cells. Also, dexamethasone repressed GSTA2 promoter-luciferase gene activity. Dexamethasone-GR activation did not inhibit nuclear translocation of C/EBPbeta or Nrf2 nor their DNA binding activities induced by oltipraz or t-BHQ. Deletion of the glucocorticoid response element (GRE) in the GSTA2 promoter abolished dexamethasone inhibition of the gene induction. Immunoprecipitation-immunoblotting, chromatin immunoprecipitation, and GST pull-down assays revealed that silencing mediator for retinoid and thyroid hormone receptors (SMRT), a corepressor recruited to steroid-GR complex for histone deacetylation, bound to TAD domain of C/EBPbeta and Neh4/5 domain of Nrf2. The GSTA2 promoter-luciferase activities were decreased by SMRT but not by truncated SMRTs. The small interference RNA (siRNA) against SMRT abolished SMRT repression of the gene induction by C/EBPbeta or Nrf2. The plasmid transfection and siRNA experiments directly evidenced the functional role of SMRT in GSTA2 repression. In conclusion, dexamethasone antagonizes C/EBPbeta- and Nrf2-mediated GSTA2 gene induction via ligand-GR binding to the GRE, and steroid-mediated GSTA2 repression involves inactivation of C/EBPbeta and Nrf2 by SMRT recruited to steroid-GR complex.
The expression changes of liver X receptor alpha (LXRalpha), histone deacetylase 3 (HDAC3) and CCAAT/enhancer binding protein alpha (C/EBPalpha) were detected in liver tissues of our high-fat-diet E3 rat model. The aim of this study is to pinpoint the molecula
r mechanism of HDAC3 and C/EBPalpha to orchestrate LXRalpha expression in hepatocytes. We confirmed that LXRalpha and its target genes were negatively regulated by HDAC3 in stable expressed clones with pEGFP-Hdac3 or shRNA-Hdac3 vector. However, transient pEGFP-C/EBPalpha plasmid transfection showed an upregulation of LXRalpha expression and C/EBPalpha enhanced LXRalpha promoter activity in a dose-dependent manner in CBRH-7919 cells. By using 5'-serial deletion reporter analysis, we identified that fragment from -2881 to -1181bp of LXRalpha promoter was responsible for C/EBPalpha binding to the promoter, especially CBS1 and CBS4 were identified essentially by using ChIP and luciferase reporter assay. Co-IP, qRT-PCR and ChIP revealed that HDAC3 interacted with C/EBPalpha co-regulated LXRalpha expression. Sumoylation of C/EBPalpha at lysine 159 was detected in CBRH-7919 cells with transient overexpressed C/EBPalpha, and Co-IP assay detected that sumoylated C/EBPalpha interacted with more HDAC3 than C/EBPalpha K159L mutant. Luciferase reporter assay demonstrated that C/EBPalpha participated in HDAC3-repressed LXRalpha transcription, and HDAC3 was involved in sumoylated C/EBPalpha-inactivated LXRalpha activity. Luciferase reporter assay demonstrated that sumoylation of C/EBPalpha by SUMO-1 directly reversed the activation of C/EBPalpha on LXRalpha promoter. The results suggested that HDAC3 interacts with sumoylated C/EBPalpha to negatively regulate the LXRalpha expression.
Bloomer SA, etal., Arch Gerontol Geriatr. 2014 Jan-Feb;58(1):145-52. doi: 10.1016/j.archger.2013.07.012. Epub 2013 Aug 8.
Elevations in hepatic iron content occur with aging and physiological stressors, which may promote oxidative injury to the liver. Since dysregulation of the iron regulatory hormone, hepcidin, can cause iron accumulation, our goal was to characterize the regulation of hepcidin in young (6 mo) and o
ld (24 mo) Fischer 344 rats exposed to environmental heat stress. Liver and blood samples were taken in the control condition and after heating. Hepcidin expression did not differ between young and old rats in the control condition, despite higher levels of hepatic iron and IL-6 mRNA in the latter. Following heat stress, pSTAT3 increased in both groups, but C/EBPalpha and hepcidin mRNA increased only in old rats. Despite this, serum iron decreased in both age groups 2 h after heat stress, suggesting hepcidin-independent hypoferremia in the young rats. The differential regulation of hepcidin between young and old rats after hyperthermia may be due to the enhanced expression of C/EBPalpha protein in old rats. These data support the concept of "inflammaging" and suggest that repeated exposures to stressors may contribute to the development of anemia in older individuals.
Nakamori D, etal., Biochem Biophys Res Commun. 2016 Jan 15;469(3):424-9. doi: 10.1016/j.bbrc.2015.12.007. Epub 2015 Dec 9.
Hepatocyte-like cells differentiated from human iPS cells (human iPS-HLCs) are expected to be utilized in drug development and research. However, recent hepatic characterization of human iPS-HLCs showed that these cells resemble fetal hepatocytes rather than adult hepatocytes. Therefore, in this stu
dy, we aimed to develop a method to enhance the hepatic function of human iPS-HLCs. Because the gene expression levels of the hepatic transcription factors (activating transcription factor 5 (ATF5), CCAAT/enhancer-binding protein alpha (c/EBPalpha), and prospero homeobox protein 1 (PROX1)) in adult liver were significantly higher than those in human iPS-HLCs and fetal liver, we expected that the hepatic functions of human iPS-HLCs could be enhanced by adenovirus (Ad) vector-mediated ATF5, c/EBPalpha, and PROX1 transduction. The gene expression levels of cytochrome P450 (CYP) 2C9, 2E1, alpha-1 antitrypsin, transthyretin, Na+/taurocholate cotransporting polypeptide, and uridine diphosphate glucuronosyl transferase 1A1 and protein expression levels of CYP2C9 and CYP2E1 were upregulated by ATF5, c/EBPalpha, and PROX1 transduction. These results suggest that the hepatic functions of the human iPS-HLCs could be enhanced by ATF5, c/EBPalpha, and PROX1 transduction. Our findings would be useful for the hepatic maturation of human iPS-HLCs.
Gong C, etal., Tumour Biol. 2015 Dec;36(12):9189-99. doi: 10.1007/s13277-015-3625-6. Epub 2015 Jun 19.
Recent studies have identified that ErbB3 binding protein 1 (EBP1) is broadly expressed in various cancer tissues and critically involved in plenty of biological processes in this regard. However, the functional role of EBP1
in pancreatic ductal adenocarcinoma (PDAC) has never been elucidated. In this study, we found that EBP1 could serve as a prognostic biomarker of PDAC. Western blot analysis revealed that EBP1 was remarkably upregulated in PDAC tissues and cell lines. Using immunohistochemical analysis, we showed that the expression of EBP1 was correlated with tumor size (P = 0.004), histological differentiation (P = 0.041), and tumor node metastasis (TNM) stage (P = 0.000). Notably, Kaplan-Meier curve showed that high expression of EBP1 predicted significantly worsened prognosis of PDAC patients (P = 0.001). In addition, knockdown of EBP1 expression suppressed PDAC cell proliferation and retarded cell cycle progression. Furthermore, depletion of EBP1 induced the apoptosis of Panc-1 cells. Of great interest, we found that EBP1 interacted with anti-apoptotic protein, Bcl-xL, and promoted its accumulation. In summary, our results suggest that EBP1 is a novel prognostic indicator and potential therapeutic target of PDAC, shedding new insights into the important role of EBP1 in cancer development.
Li DD, etal., Cell Cycle. 2015;14(21):3461-74. doi: 10.1080/15384101.2015.1093704.
Although Hmgn1 is involved in the regulation of gene expression and cellular differentiation, its physiological roles on the differentiation of uterine stromal cells during decidualization still remain unknown. Here we showed that Hmgn1 mRNA was highly expressed in the decidua on days 6-8 of pregnan
cy. Simultaneously, increased expression of Hmgn1 was also observed in the artificial and in vitro induced decidualization models. Hmgn1 induced the proliferation of uterine stromal cells and expression of Ccna1, Ccnb1, Ccnb2 and Cdk1 in the absence of estrogen and progesterone. Overexpression of Hmgn1 could enhance the expression of Prl8a2 and Prl3c1 which were 2 well-known differentiation markers for decidualization, whereas inhibition of Hmgn1 with specific siRNA could reduce their expression. Further studies found that Hmgn1 could mediate the effects of C/EBPbeta on the expression of Prl8a2 and Prl3c1 during in vitro decidualization. In the uterine stromal cells, cAMP analog 8-Br-cAMP could stimulate the expression of Hmgn1 via C/EBPbeta. Moreover, siRNA-mediated down-regulation of Hmgn1 could attenuate the effects of cAMP on the differentiation of uterine stromal cells. During in vitro decidualization, Hmgn1 might act downstream of C/EBPbeta to regulate the expression of Cox-2, mPGES-1 and Vegf. Progesterone could up-regulate the expression of Hmgn1 in the ovariectomized mouse uterus, uterine epithelial cells and stromal cells. Knockdown of C/EBPbeta with siRNA alleviated the up-regulation of progesterone on Hmgn1 expression. Collectively, Hmgn1 may play an important role during mouse decidualization.
Transcription factor C/EBPbeta has been known to regulate a wide array of genes including those involved in the acute-phase response. One of the molecular mechanisms underlying transcription activation by C/EBPbeta is throug
h protein-protein interaction with other transcription factors. Here we report the identification and characterization of physical and functional interactions between C/EBPbeta and heterogeneous nuclear ribonucleoprotein (hnRNP) K. This interaction results in the repression of C/EBPbeta-dependent trans-activation of the agp gene. Footprinting assays indicate that hnRNP K cannot bind to the promoter region of agp gene or interfere with the binding of C/EBPbeta to its cognate DNA site. Furthermore, agp gene activation by the synergistic interaction of Nopp140 and C/EBPbeta is abolished by hnRNP K. The kinetics of appearance of C/EBPbeta-hnRNP K complex in the nuclear extract after initiation of acute-phase reaction indicates that hnRNP K functions as a negative regulator of C/EBPbeta-mediated activation of agp gene.
Interleukin-6 (IL-6) has an important role in the pathogenesis of chronic viral hepatitis and related liver diseases. Although host genetics associated with the response to anti-viral treatment have been reported, little is known about the relationship between IL6 genetic polymorphisms and the outc
ome of hepatitis B virus (HBV) infection. In this study, we determined the genotype distribution of rs1800796 polymorphism in healthy controls and cases including chronic HBV (CHB), hepatitis C virus and HIV infection. The rs1800796 was found to be associated with clinical outcome of CHB in experimental and validation cohort. The rs1800796C allele has twofold higher promoter activity than G allele. Consistently, CD14(+) monocytes from subjects carrying the rs1800796C allele produced more IL-6 in response to in vitro HBV core antigen stimulation than those carrying G allele. Moreover, CHB patients carrying rs1800796C allele have significantly higher T-helper 17 (Th17) and regulatory T cell (Treg) ratio. Finally, a transcription factor C/EBPalpha binds in higher affinity to rs1800796C allele than to G allele. These results suggest that genetic predisposition to higher IL-6 production is associated with increased risk to HBV infection and hepatic inflammation, which might be due to C/EBPalpha-mediated regulatory effect on Th17 and Treg responses. Appropriate manipulation of IL-6 expression might be used to prevent and treat HBV infection.
Kelicen P and Tindberg N, J Biol Chem. 2004 Apr 16;279(16):15734-42. Epub 2003 Dec 11.
Cytochrome P450 2E1 (CYP2E1) is highly inducible in a subset of astrocytes in vivo following ischemic or mechanical injury and in vitro by lipopolysaccharide (LPS) or interleukin-1beta. We have studied the mechanism of induction, and found that transcriptional activation of CYP2E1 occurred within 3
h, and CYP2E1 dependent catalytic activity was induced more than 4-fold within 5 h. The induction was sensitive to several tyrosine kinase inhibitors, and was further modulated by inhibitors of p38 MAP kinase. MAP kinase kinase-3 (MKK3) was phosphorylated in response to LPS, and expression of constitutively active MKK3, but not the MAP kinase kinases MEKK1 or MKK1, activated CYP2E1. Transcriptional activation was mediated through a C/EBPbeta and -delta binding element situated at -486/-474, and appeared to involve activation of prebound factors as well as recruitment of newly synthesized C/EBPbeta and -delta. It is thus suggested that LPS induces MKK3 activation in astrocytes, which in turn stimulates a C/EBPbeta and -delta binding element to mediate transcriptional activation of CYP2E1.
Global ischemia after cardiac arrest, intraoperative hypoxia/hypotension, and hemorrhagic shock causes brain injury resulting in severe neurological and neurobehavioral deficits. Neurodegeneration can be prevented by local aromatase expression, and estrogen synthesis can be neuroprotective in ischem
ia/reperfusion. Therefore, aromatase, the enzyme that transforms androgens to estrogens, may be a potential target for the study of reperfusion injury after brain ischemia. We investigated the expression of aromatase and C/EBPbeta using western blotting in rat hippocampus after transient global ischemia plus hypotension. Immunohistochemical analysis was performed for aromatase. After 10min of ischemia, aromatase and C/EBPbeta expression in cytosolic extracts were observed after 10min and 24h of reperfusion. The expression of both proteins was similar in control and damaged tissues. Immunoblot analysis demonstrated that the highest aromatase expression appeared in damaged hippocampi after 1week and was gradually reduced after 2-10weeks. C/EBPbeta expression increased at 1week in nuclear extracts of damaged hippocampi. The aromatase inhibitor megestrol acetate (20mg/kg/day) suppressed aromatase and nuclear C/EBPbeta levels in ischemic hippocampi. Our findings indicate that ischemia as well as chronic neurodegenerative processes leads to an increase in cytoplasmic aromatase and nuclear C/EBPbeta. Thus, it is possible to hypothesize an interaction between this enzyme gene and transcription factor.
Peritoneal dissemination of tumor has high mortality and is associated with the loss of epithelial features, acquisition of motile mesenchymal morphology characteristics, and invasive properties by tumor cells. Melatonin is an endogenously produced molecule in all plant species that is known to exer
t antitumor activity, but to date, its underlying mechanisms and antiperitoneal metastasis efficacy is not well defined. This study determined the antiperitoneal dissemination potential of melatonin in vivo and assessed its association with the inhibition of epithelial-to-mesenchymal transition (EMT) signaling mechanism by endoplasmic reticulum (ER) stress, which may be a major molecular mechanism of melatonin against cancer. The results demonstrate that melatonin inhibited peritoneal metastasis in vivo and activated ER stress in Cignal ERSE Reporter Assay, organelle structure in transmission electron microscopy images, calpain activity, and protein biomarkers like p-elf2alpha. Moreover, the overexpression of transcription factor C/EBPbeta in gastric cancer interacted with NFkappaB and further regulates COX-2 expression. These were dissociated and downregulated by melatonin, as proven by immunofluorescence imaging, immunoprecipitation, EMSA, and ChIP assay. Melatonin or gene silencing of C/EBPbeta decreased the EMT protein markers (E-cadherin, Snail, and Slug) and Wnt/beta-catenin activity by Topflash activity, and increased ER stress markers. In an animal study, the results of melatonin therapy were consistent with those of in vitro findings and attenuated systemic proangiogenesis factor production. In conclusion, C/EBPbeta and NFkappaB inhibition by melatonin may impede both gastric tumor growth and peritoneal dissemination by inducing ER stress and inhibiting EMT.
Several miRNAs have recently been identified to regulate adipocyte or osteoblast differentiation or both. In this study, miR-223 was found to be involved in the reciprocal regulation of adipocyte and osteoblast differentiation. miR-223 was induced in primary cultured mouse marrow stromal cell, mese
nchymal line C3H10T1/2 and stromal line ST2 after adipogenic treatment. Conversely, it was reduced in preosteoblast MC3T3-E1 after osteogenic treatment. Supplementing miR-223 levels using synthetic miR-223 mimics significantly suppressed the growth of the C3H10T1/2 and ST2 cells and induced the progenitor cells to fully differentiate into adipocytes, along with induction of adipocyte-specific transcription factors peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein-alpha (C/EBPalpha), and marker genes aP2 and adipsin. By contrast, depletion of the endogenous miR-223 using synthetic miR-223 inhibitor repressed the progenitor cells to differentiate. The effects of miR-223 on adipocyte formation from ST2 cells were also demonstrated by using lentivirus that overexpresses miR-223. Conversely, supplementing miR-223 blocked ST2 to differentiate into osteoblasts. Fibroblast growth factor receptor 2 (Fgfr2), a critical regulator of osteoblast, was shown to be a direct target of miR-223 by using dual luciferase reporter assay. Knockdown of Fgfr2 in C3H10T1/2 downregulated phosphorylation of ERK1/2 and upregulated expression of C/EBPalpha and dramatically enhanced the differentiation of the cells into adipocytes. Further investigation of mechanisms that control miR-223 expression demonstrated that C/EBPs induced miR-223 expression through binding to the promoter regions of the miR-223. Taken together, our study provides evidences that miR-223 regulates adipocyte and osteoblast differentiation through a novel C/EBPs/miR-223/FGFR2 regulatory feedback loop.
Ahn JY, etal., EMBO J. 2006 May 17;25(10):2083-95. Epub 2006 Apr 27.
Akt promotes cell survival through phosphorylation. The physiological functions of cytoplasmic Akt have been well defined, but little is known about the nuclear counterpart. Employing a cell-free apoptotic assay and NGF-treated PC12 nuclear extracts, we purified Ebp
span>1 as a factor, which contributes to inhibition of DNA fragmentation by CAD. Depletion of Ebp1 from nuclear extracts or knockdown of Ebp1 in PC12 cells abolishes the protective effects of nerve growth factor, whereas overexpression of Ebp1 prevents apoptosis. Ebp1 (S360A), which cannot be phosphorylated by PKC, barely binds Akt or inhibits DNA fragmentation, whereas Ebp1 S360D, which mimics phosphorylation, strongly binds Akt and suppresses apoptosis. Further, phosphorylated nuclear but not cytoplasmic Akt interacts with Ebp1 and enhances its antiapoptotic action independent of Akt kinase activity. Moreover, knocking down of Akt diminishes the antiapoptotic effect of Ebp1 in the nucleus. Thus, nuclear Akt might contribute to suppressing apoptosis through interaction with Ebp1.
Muller C, etal., EMBO J. 2010 Mar 3;29(5):897-909. doi: 10.1038/emboj.2009.404. Epub 2010 Jan 14.
The messenger RNA of the intronless CEBPA gene is translated into distinct protein isoforms through the usage of consecutive translation initiation sites. These translational isoforms have distinct functions in the regulation of differentiation and proliferation
due to the presence of different N-terminal sequences. Here, we describe the function of an N-terminally extended protein isoform of CCAAT enhancer-binding protein alpha (C/EBPalpha) that is translated from an alternative non-AUG initiation codon. We show that a basic amino-acid motif within its N-terminus is required for nucleolar retention and for interaction with nucleophosmin (NPM). In the nucleoli, extended-C/EBPalpha occupies the ribosomal DNA (rDNA) promoter and associates with the Pol I-specific factors upstream-binding factor 1 (UBF-1) and SL1 to stimulate rRNA synthesis. Furthermore, during differentiation of HL-60 cells, endogenous expression of extended-C/EBPalpha is lost concomitantly with nucleolar C/EBPalpha immunostaining probably reflecting the reduced requirement for ribosome biogenesis in differentiated cells. Finally, overexpression of extended-C/EBPalpha induces an increase in cell size. Altogether, our results suggest that control of rRNA synthesis is a novel function of C/EBPalpha adding to its role as key regulator of cell growth and proliferation.
INTRODUCTION: Oxidative stress-induced trophoblast cell dysfunction is a major pathology in preeclampsia (PE). Recently, CCAAT/enhancer binding protein beta (C/EBPbeta) has been investigated as a tumor suppressor that participates in tumor invasion. However, the
function of C/EBPbeta in trophoblast cells remains unknown. Our study was designed to detect the expression of C/EBPbeta in the preeclamptic placenta and to identify the underlying mechanisms of oxidative stress. METHODS: Human placental tissues with PE were collected. The expression of C/EBPbeta and beta-catenin were detected. Human first trimester extravillous trophoblast cell (HTR8/SVneo) line exposed to hypoxia/reoxygenation (H/R) was employed as an oxidative stress model in vitro to investigate the effects of C/EBPbeta on invasion and the expression of beta-catenin. Moreover, first trimester-derived placental villous explants were used to verify the effects of C/EBPbeta and beta-catenin in placentation. RESULTS: In preeclamptic placentas, C/EBPbeta was overexpressed and beta-catenin was decreased. In addition, C/EBPbeta was found to have increased expression in H/R-treated HTR8/SVneo cells and villous explants. C/EBPbeta knockdown and beta-catenin activation could significantly promote the invasion of HTR8/SVneo cells, enhance the outgrowth and migration in villous explants and inhibit the excessive generation of intracellular ROS. These findings might be related to the increased activities of MMP-2/9 and the decreased expression of TIMP-1/2. Meanwhile, C/EBPbeta knockdown remarkably increased the expression of beta-catenin. DISCUSSION: We hypothesize that the oxidative stress-induced overexpression of C/EBPbeta might influence the activity of MMPs by regulating the Wnt/beta-catenin signaling pathway to affect the invasion of trophoblast cells, which then participate in the pathogenesis of preeclampsia.
Mouthiers A, etal., Mol Endocrinol. 2005 May;19(5):1135-46. doi: 10.1210/me.2004-0188. Epub 2005 Jan 20.
Recently, the role of the peroxisome proliferator-activated receptor alpha (PPARalpha) in the hepatic inflammatory response has been associated to the decrease of acute phase protein transcription, although the molecular mechanisms are still to be elucidated. Here, we were interested in the regulati
on by Wy-14643 (PPARalpha agonist) of alpha1-acid glycoprotein (AGP), a positive acute phase protein, after stimulation by Dexamethasone (Dex), a major modulator of the inflammatory response. In cultured rat hepatocytes, we demonstrate that PPARalpha inhibits at the transcriptional level the Dex-induced AGP gene expression. PPARalpha exerts this inhibitory effect by antagonizing the CCAAT/enhancer binding protein (C/EBPbeta) transcription factor that is involved in Dex-dependent up-regulation of AGP gene expression. Overexpression of C/EBPbeta alleviates the repressive effect of PPARalpha, thus restoring the Dex-stimulated AGP promoter activity. Furthermore, glutathione-S-transferase GST pull-down and coimmunoprecipitation experiments evidenced, for the first time, a physical interaction between PPARalpha and the C-terminal DNA binding region of C/EBPbeta, thus preventing it from binding to specific sequence elements of the AGP promoter. Altogether, these results provide an additional molecular mechanism of negative regulation of acute phase protein gene expression by sequestration of the C/EBPbeta transcription factor by PPARalpha and reveal the high potency of the latter in controlling inflammation.
Song GJ, etal., J Biol Chem. 2015 Jan 30;290(5):2879-87. doi: 10.1074/jbc.M114.609768. Epub 2014 Dec 9.
The regulation of the cell cycle by the ubiquitin-proteasome system is dependent on the activity of E3 ligases. Skp2 (S-phase kinase associated protein-2) is the substrate recognition subunit of the E3 ligase that ubiquitylates the cell cycle inhibitors p21(cip1) and p27(kip1) thus promoting cell
cycle progression. Increased expression of Skp2 is frequently observed in diseases characterized by excessive cell proliferation, such as cancer and neointima hyperplasia. The stability and cellular localization of Skp2 are regulated by Akt, but the molecular mechanisms underlying these effects remain only partly understood. The scaffolding protein Ezrin-Binding Phosphoprotein of 50 kDa (EBP50) contains two PDZ domains and plays a critical role in the development of neointimal hyperplasia. Here we report that EBP50 directly binds Skp2 via its first PDZ domain. Moreover, EBP50 is phosphorylated by Akt on Thr-156 within the second PDZ domain, an event that allosterically promotes binding to Skp2. The interaction with EBP50 causes cytoplasmic localization of Skp2, increases Skp2 stability and promotes proliferation of primary vascular smooth muscle cells. Collectively, these studies define a novel regulatory mechanism contributing to aberrant cell growth and highlight the importance of scaffolding function of EBP50 in Akt-dependent cell proliferation.
Jang JH, etal., J Cell Biochem. 2016 Feb;117(2):361-9. doi: 10.1002/jcb.25278.
RU486 (Mifepristone) is known as an antagonist of the progesterone receptor and glucocorticoid receptor. Here, we investigated the mechanism underlying anti-tumor activity of RU486 in renal carcinoma Caki cells. Treatment of Caki cells with RU486 was found to induce several signature ER stress marke
rs; including ER stress-specific XBP1 splicing, and the up-regulation of glucose-regulated protein (GRP)-78 and CCAAT/enhancer-binding protein homologous protein (CHOP) expression. RU486-induced expression of CHOP involves the putative C/EBPdelta site within the CHOP promoter region. Using a combination of C/EBPdelta cDNA transfection, the luciferase assay with a mutated C/EBPdelta binding site and siRNA-mediated C/EBPdelta knockdown, we found that the C/EBPdelta site is required for RU486-mediated activation of the CHOP promoter. In addition, RU486-induced CHOP expression is down-regulated by inhibition of the p38 MAPK and JNK signaling pathways at the post-translational levels. RU486 dose-dependently induced apoptotic cell death in renal carcinoma cells. Suppression of CHOP expression by CHOP siRNA attenuated RU486-induced apoptosis. Taken together, RU486 induces pro-apoptotic ER stress through the induction of CHOP expression.
Uskokovic A, etal., Cell Biol Int. 2002;26(5):451-61.
The greatest part of nuclear C/EBPbeta (a major 35 kD protein, 30 and 38 kD isoforms) was observed to partition with the nuclear matrix. Cross-linking experiments with formaldehyde suggested that the association reflected the in situ juxtapositioning of C/EBP
style='font-weight:700;'>EBPbeta to nuclear matrix proteins in isolated nuclei. The association of C/EBPbeta with the nuclear matrix resisted RNase and DNase treatment and extraction with protein sulfhydryl reducing agents combined with high ionic strength salt. C/EBPbeta displayed a proclivity to extensively reassemble with the filament-forming nuclear matrix proteins after a cycle of solubilization with urea, followed by its removal by dialysis. These findings suggest that the C/EBPbeta moieties were anchored to the nuclear matrix through hydrophobic protein-protein interactions with the lamins. Subsequent separation of nuclear matrix-associated C/EBPbeta into insoluble, reassembling, and soluble nuclear matrix protein (SNMP) fractions after a cycle of solubilization/reassembly pointed to the sub-partitioning of C/EBPbeta on the nuclear matrix. DNA affinity chromatography using the rat haptoglobin gene cis -element and SNMP revealed the binding of p35 during basal transcription, and p35 and p30 during elevated haptoglobin gene transcription in the course of the acute-phase (AP) response. It was concluded that the appearance of cis -element-binding p30 in the SNMP fraction resulted from its increased solubility (decreased hydrophobicity) and inability to reassociate with the lamins during urea removal. The observed solubility partitioning of C/EBPbeta on the nuclear matrix framework could represent a level of control of the general availability of regulatory proteins for establishing interactions with DNA.
Zhang J, etal., FASEB J. 2014 Mar;28(3):1511-25. doi: 10.1096/fj.13-242693. Epub 2013 Dec 16.
CCAAT/enhancer-binding protein (C/EBPbeta)-enhanced IL-6 and TGF-beta1 promoter activity and p300-mediated C/EBPbeta acetylation were involved in up-regulation of IL-6 and TGF-beta1 expression in GMCs attacked by sublytic C5
b-9. In detail, the elements of C/EBPbeta binding to rat IL-6 and TGF-beta1 promoter and 3 acetylated sites of rat C/EBPbeta protein were first revealed. Furthermore, silencing the p300 or C/EBPbeta gene in rat kidney significantly reduced the production of IL-6 and TGF-beta1 and renal lesions in Thy-1N rats. Together, these data indicate that the mechanism of IL-6 and TGF-beta1 production in renal tissue of Thy-1N rats is associated with sublytic C5b-9 up-regulated p300 and p300-mediated C/EBPbeta acetylation as well as C/EBPbeta-activated IL-6 and TGF-beta1 genes.
Muller C, etal., J Biol Chem 2004 Feb 20;279(8):7353-8. Epub 2003 Dec 04.
The transcription factor CCAAT enhancer-binding protein alpha (C/EBPalpha) is a tumor suppressor in myeloid cells and inhibits proliferation in all cell types examined. C/EBPalpha interacts with the SWI/SNF chromatin-remodel
ing complex during the regulation of differentiation-specific genes. Here we show that C/EBPalpha fails to suppress proliferation in SWI/SNF defective cell lines after knock-down of SWI/SNF core components or after deletion of the SWI/SNF interaction domain in C/EBPalpha, respectively. Reconstitution of SWI/SNF function restores C/EBPalpha-dependent proliferation arrest. Our results show that the anti-proliferation activity of C/EBPalpha critically depends on components of the SWI/SNF core complex and suggest that the functional interaction between SWI/SNF and C/EBPalpha is a prerequisite for proliferation arrest.
Gautier-Stein A, etal., J Biol Chem. 2006 Oct 20;281(42):31268-78. Epub 2006 Aug 7.
Gluconeogenesis is induced in both the liver and intestine by increased cAMP levels. However, hepatic and intestinal glucose production can have opposite effects on glucose homeostasis. Glucose release into the portal vein by the intestine increases glucose uptake and reduces food intake. In contras
t, glucose production by the liver contributes to hyperglycemia in type II diabetes. Glucose-6-phosphatase (Glc6Pase) is the key enzyme of gluconeogenesis in both the liver and intestine. Here we specify the cAMP/protein kinase A regulation of the Glc6Pase gene in the intestine compared with the liver. Similarly to the liver, the molecular mechanism of cAMP/protein kinase A regulation involves cAMP-response element-binding protein, HNF4alpha, CAAT/enhancer-binding protein, and HNF1. In contrast to the situation in the liver, we find that different isoforms of CAAT/enhancer-binding protein and HNF1 contribute to the specific regulation of the Glc6Pase gene in the intestine. Moreover, we show that cAMP-response element binding modulator specifically contributes to the regulation of the Glc6Pase gene in the intestine but not in the liver. These results allow us to identify intestine-specific regulators of the Glc6Pase gene and to improve the understanding of the differences in the regulation of gluconeogenesis in the intestine compared with the liver.
Garcia-Trevijano ER, etal., Hepatology. 1999 Mar;29(3):960-70.
Oxidative stress plays a key role in liver fibrosis. Both inflammatory cells and activated Kupffer cells produce H2O2, an oxidant involved in the activation of hepatic stellate cells (HSC). Increased production of reactive oxygen intermediates (ROIs) in fibrotic livers is associated in part with the
up-regulation of transforming growth factor beta (TGF-beta), and this cytokine enhances collagen production by cultured HSC. However, the possible link between oxidative stress and the molecular mechanisms by which TGF-beta induces collagen gene expression in HSC remains to be elucidated. To address this question, we investigated whether H2O2 is a mediator of TGF-beta-elicited alpha1(I) collagen gene (col1a1) up-regulation. We demonstrated that TGF-beta induces the accumulation of H2O2, and that this oxidant is, in turn, directly involved in up-regulating the expression of the col1a1 gene. While the addition of H2O2 to HSC induced the expression of alpha1(I) procollagen mRNA, catalase, an H2O2 enzyme scavenger, abrogated TGF-beta-mediated col1a1 gene up-regulation. We transfected HSC with chimeric plasmids driven by different segments of the mouse col1a1 promoter and mapped a cis-acting element (-370 to -344) essential for TGF-beta responsiveness. We further showed that TGF-beta induced the activation and binding of a C/EBPbeta-containing transcriptional complex to this sequence, an effect that was also mimicked by the addition of H2O2. Taken together, these data demonstrate a direct connection between TGF-beta-mediated accumulation of H2O2 and the up-regulation of col1a1 gene in HSC.
