Jundt F, etal., Blood. 2004 May 1;103(9):3511-5. Epub 2004 Jan 15.
Notch receptors expressed on hematopoietic stem cells interact with their ligands on bone marrow stromal cells and thereby control cell fate decisions and survival. We recently demonstrated that Notch signaling is involved in proliferation and survival of B cell-derived tumor cells of classic Hodgki
n disease and described a novel mechanism for the oncogenic capacity of Notch. In this study we investigated whether Notch signaling is involved in the tight interactions between neoplastic plasma cells and their bone marrow microenvironment, which are essential for tumor cell growth in multiple myeloma (MM). Here we demonstrate that Notch receptors and their ligand Jagged1 are highly expressed in cultured and primary MM cells, whereas nonneoplastic counterparts show low to undetectable levels of Notch. Functional data indicate that ligand-induced Notch signaling is a growth factor for MM cells and suggest that these interactions contribute to myelomagenesis in vivo.
Jundi K and Greene CM, Biomolecules. 2015 Jul 1;5(3):1386-98. doi: 10.3390/biom5031386.
Interleukin-8 (IL-8) is a neutrophil chemokine that is encoded on the CXCL8 gene. Normally CXCL8 expression is repressed due to histone deacetylation, octamer-1 binding to the promoter and the inhibitory effect of nuclear factor-kappaB repressing factor (NRF). However, in response to a suitable stim
ulus, the human CXCL8 gene undergoes transcription due to its inducible promoter that is regulated by the transcription factors nuclear factor-kappaB (NF-kappaB), activating protein (AP-1), CAAT/enhancer-binding protein beta (C/EBPbeta, also known as NF-IL-6), C/EBP homologous protein (CHOP) and cAMP response element binding protein (CREB). CXCL8 mRNA is then stabilised by the activity of p38 mitogen-activated protein kinase (p38 MAPK). Cystic fibrosis (CF) lung disease is characterised by a neutrophil-dominated airway inflammatory response. A major factor contributing to the large number of neutrophils is the higher than normal levels of IL-8 that are present within the CF lung. Infection and inflammation, together with intrinsic alterations in CF airway cells are responsible for the abnormally high intrapulmonary levels of IL-8. Strategies to inhibit aberrantly high CXCL8 expression hold therapeutic potential for CF lung disease.
Mathas S, etal., EMBO J 2002 Aug 1;21(15):4104-13.
AP-1 family transcription factors have been implicated in the control of proliferation, apoptosis and malignant transformation. However, their role in oncogenesis is unclear and no recurrent alterations of AP-1 activities have been described in human cancers. Here, we show that constitutively activa
ted AP-1 with robust c-Jun and JunB overexpression is found in all tumor cells of patients with classical Hodgkin's disease. A similar AP-1 activation is present in anaplastic large cell lymphoma (ALCL), but is absent in other lymphoma types. Whereas c-Jun is up-regulated by an autoregulatory process, JunB is under control of NF-kappa B. Activated AP-1 supports proliferation of Hodgkin cells, while it suppresses apoptosis of ALCL cells. Furthermore, AP-1 cooperates with NF-kappa B and stimulates expression of the cell-cycle regulator cyclin D2, proto-oncogene c-met and the lymphocyte homing receptor CCR7, which are all strongly expressed in primary HRS cells. Together, these data suggest an important role of AP-1 in lymphoma pathogenesis.
Li R, etal., J Immunol. 2015 Oct 15;195(8):3901-11. doi: 10.4049/jimmunol.1500967. Epub 2015 Sep 14.
Earlier studies reported that a cell membrane protein, Annexin A2 (AnxA2), plays multiple roles in the development, invasion, and metastasis of cancer. Recent studies demonstrated that AnxA2 also functions in immunity against infection, but the underlying mechanism remains largely elusive. Using a m
ouse infection model, we reveal a crucial role for AnxA2 in host defense against Pseudomonas aeruginosa, as anxa2(-/-) mice manifested severe lung injury, systemic dissemination, and increased mortality compared with wild-type littermates. In addition, anxa2(-/-) mice exhibited elevated inflammatory cytokines (TNF-α, IL-6, IL-1β, and IFN-γ), decreased bacterial clearance by macrophages, and increased superoxide release in the lung. We further identified an unexpected molecular interaction between AnxA2 and Fam13A, which activated Rho GTPase. P. aeruginosa infection induced autophagosome formation by inhibiting Akt1 and mTOR. Our results indicate that AnxA2 regulates autophagy, thereby contributing to host immunity against bacteria through the Akt1-mTOR-ULK1/2 signaling pathway.
Nadiri A, etal., Int Immunol. 2015 Nov;27(11):555-65. doi: 10.1093/intimm/dxv030. Epub 2015 May 14.
CD40, a member of the tumor necrosis factor receptor superfamily, plays a key role in both adaptive and innate immunity. Engagement of CD40 with its natural trimeric ligand or with cross-linked antibodies results in disulfide-linked CD40 (dl-CD40) homodimer formation, a process mediated by the cyst
eine-238 residues of the cytoplasmic tail of CD40. The present study was designed to elucidate the biological relevance of cysteine-238-mediated dl-CD40 homodimers to the expression of CD23 on B cells and to investigate its possible involvement in the innate response. Our results indicate that cysteine-238-mediated dl-CD40 homodimerization is required for CD40-induced activation of PI3-kinase/Akt signaling and the subsequent CD23 expression, as inhibition of dl-CD40 homodimer formation through a point mutation-approach specifically impairs these responses. Interestingly, cysteine-238-mediated dl-CD40 homodimers are also shown to play a crucial role in Toll-like receptor 4-induced CD23 expression, further validating the importance of this system in bridging innate and adaptive immune responses. This process also necessitates the activation of the PI3-kinase/Akt cascade. Thus, our results highlight new roles for CD40 and cysteine-238-mediated CD40 homodimers in cell biology and identify a potential new target for therapeutic strategies against CD40-associated chronic inflammatory diseases.
Li X, etal., PLoS Pathog. 2016 Jan 6;12(1):e1005363. doi: 10.1371/journal.ppat.1005363. eCollection 2016 Jan.
Extracellular bacteria, such as Pseudomonas aeruginosa and Klebsiella pneumoniae, have been reported to induce autophagy; however, the role and machinery of infection-induced autophagy remain elusive. We show that the pleiotropic Src kinase Lyn mediates phagocytosis and autophagosome maturation in a
lveolar macrophages (AM), which facilitates eventual bacterial eradication. We report that Lyn is required for bacterial infection-induced recruitment of autophagic components to pathogen-containing phagosomes. When we blocked autophagy with 3-methyladenine (3-MA) or by depleting Lyn, we observed less phagocytosis and subsequent bacterial clearance by AM. Both morphological and biological evidence demonstrated that Lyn delivered bacteria to lysosomes through xenophagy. TLR2 initiated the phagocytic process and activated Lyn following infection. Cytoskeletal trafficking proteins, such as Rab5 and Rab7, critically facilitated early phagosome formation, autophagosome maturation, and eventual autophagy-mediated bacterial degradation. These findings reveal that Lyn, TLR2 and Rab modulate autophagy related phagocytosis and augment bactericidal activity, which may offer insight into novel therapeutic strategies to control lung infection.
Emmerich F, etal., Blood. 1999 Nov 1;94(9):3129-34.
The transcription factor NF kappa B (NF-kappaB) mediates the expression of numerous genes involved in diverse functions such as inflammation, immune response, apoptosis, and cell proliferation. We recently identified constitutive activation of NF-kappaB (p50/p65) as a common feature of Hodgkin/Reed-
Sternberg (HRS) cells preventing these cells from undergoing apoptosis and triggering proliferation. To examine possible alterations in the NF-kappaB/IkappaB system, which might be responsible for constitutive NF-kappaB activity, we have analyzed the inhibitor I kappa B alpha (IkappaBalpha) in primary and cultured HRS cells on protein, mRNA, and genomic levels. In lymph node biopsy samples from Hodgkin's disease patients, IkappaBalpha mRNA proved to be strongly overexpressed in the HRS cells. In 2 cell lines (L428 and KM-H2), we detected mutations in the IkappaBalpha gene, resulting in C-terminally truncated proteins, which are presumably not able to inhibit NF-kappaB-DNA binding activity. Furthermore, an analysis of the IkappaBalpha gene in single HRS cells micromanipulated from frozen tissue sections showed a monoallelic mutation in 1 of 10 patients coding for a comparable C-terminally truncated IkappaBalpha protein. We suggest that the observed IkappaBalpha mutations contribute to constitutive NF-kappaB activity in cultured and primary HRS cells and are therefore involved in the pathogenesis of these Hodgkin's disease (HD) patients. The demonstrated constitutive overexpression of IkappaBalpha in HRS cells evidences a deregulation of the NF-kappaB/IkappaB system also in the remaining cases, probably due to defects in other members of the IkappaB family.
Somatic mutations of TP53 are among the most common in cancer and germline mutations of TP53 (usually missense) can cause Li-Fraumeni syndrome (LFS). Recently, recurrent genomic rearrangements in intron 1 of TP53 have been described in osteosarcoma (OS), a highly malignant neoplasm of bone belonging
to the spectrum of LFS tumors. Using whole-genome sequencing of OS, we found features of TP53 intron 1 rearrangements suggesting a unique mechanism correlated with transcription. Screening of 288 OS and 1,090 tumors of other types revealed evidence for TP53 rearrangements in 46 (16%) OS, while none were detected in other tumor types, indicating this rearrangement to be highly specific to OS. We revisited a four-generation LFS family where no TP53 mutation had been identified and found a 445 kb inversion spanning from the TP53 intron 1 towards the centromere. The inversion segregated with tumors in the LFS family. Cancers in this family had loss of heterozygosity, retaining the rearranged allele and resulting in TP53 expression loss. In conclusion, intron 1 rearrangements cause p53-driven malignancies by both germline and somatic mechanisms and provide an important mechanism of TP53 inactivation in LFS, which might in part explain the diagnostic gap of formerly classified "TP53 wild-type" LFS.
A better understanding of events triggering chronic myeloid leukemia progression is critical for optimized clinical management of chronic myeloid leukemia (CML). We sought to validate that increased expression of Musashi 2 (MSI2), a post-transcription regulator, is associated with progression and
prognosis. Screening of 152 patients with CML showed that MSI2 was significantly decreased among patients with CML in chronic phase (CP) at diagnosis (p < 0.0001), but found no significant difference between the normal control group and treated patients with CML in CP. Moreover MSI2 was significantly increased (p < 0.0001) in patients with advance disease (AD) CML. Furthermore, our human hematopoietic cell line data imply that MSI2 and BCR-ABL1 mRNA expression are correlated. However, these data cast a doubt on earlier reports that MSI2 effects HES1 expression via NUMB-NOTCH signaling.
Pokrywczynska M, etal., Stem Cell Res Ther. 2019 Jun 13;10(1):176. doi: 10.1186/s13287-019-1266-1.
BACKGROUND: Molecular mechanisms underlying the regenerative process induced by stem cells in tissue-engineered urinary bladder are poorly explained. The study was performed to explore the pathways associated with regeneration process in the urinary bladder reconstructed with adipose tiss
ue-derived mesenchymal stromal cells (ASCs). METHODS: Rat urinary bladders were reconstructed with bladder acellular matrix (BAM) (n = 52) or BAM seeded with adipose tissue-derived mesenchymal stromal cells (ASCs) (n = 52). The process of bladder healing was analyzed at 7, 30, 90, and 180 days postoperatively using macroscopic histologic and molecular techniques. Gene expression was analyzed by microarrays and confirmed by real-time PCR. RESULTS: Numerous differentially expressed genes (DEGs) were identified between the bladders augmented with BAM seeded with ASCs or BAM only. Pathway analysis of DEGs allows to discover numerous pathways among them Hedgehog, TGF-β, Jak-STAT, PI3-Akt, and Hippo modulated by ASCs during the healing process of tissue-engineered urinary bladder. Real-time PCR analysis confirmed upregulation of genes involved in the Hedgehog signaling pathway including Shh, Gli1, Smo, Bmp2, Bmp4, Wnt2, Wnt2b, Wnt4, Wnt5a, and Wnt10 in urinary bladders reconstructed with ASC-seeded grafts. CONCLUSION: The study provided the unequivocal evidence that ASCs change the molecular pattern of healing in tissue-engineered urinary bladder and indicated which signaling pathways triggered by ASCs can be associated with the regenerative process. These pathways can be used as targets in the future studies on induced urinary bladder regeneration. Of particular interest is the Hedgehog signaling pathway that has been upregulated by ASCs during healing of tissue-engineered urinary bladder.
Pokrywczynska M, etal., Arch Immunol Ther Exp (Warsz). 2015 Oct;63(5):377-84. doi: 10.1007/s00005-015-0340-3. Epub 2015 May 10.
Pancreatic islet implantation has been recently shown to be an efficient method of treatment for type 1 diabetes. However, limited availability of donor islets reduces its use. Bone morrow would provide potentially unlimited source of stem cells for generation of insulin-producing cells. This study
was performed to evaluate the influence of extracellular matrix proteins like collagen, laminin, and vitronectin on bone marrow mesenchymal stem cells (BM-MSCs) transdifferentiation into islet-like cells (ILCs) in vitro. To our knowledge, this is the first report evaluating the importance of vitronectin in transdifferentiation of BM-MSCs into ILCs. Rat BM-MSCs were induced to ILCs using four-step protocol on plates coated with collagen type IV, laminin type I and vitronectin type I. Quantitative real-time PCR was performed to detect gene expression related to pancreatic beta cell development. The induced cells expressed islet-related genes including: neurogenin 3, neurogenic differentiation 1, paired box 4, NK homeobox factor 6.1, glucagon, insulin 1 and insulin 2. Laminin but not collagen type IV or vitronectin enhanced expression of insulin and promoted formation of islet-like structures in monolayer culture. Laminin triggered transdifferentiation of BM-MSCs into ILCs.
Short JD and Pfarr CM, J Biol Chem 2002 Sep 6;277(36):32697-705.
JunD, a member of the Jun family of nuclear transcription proteins, dimerizes with Fos family members or other Jun proteins (c-Jun or JunB) to form the activator protein 1 (AP-1) transcription factor. The junD gene contains no introns and generates a single mRNA. Here we show that two predominant Ju
nD isoforms are generated by alternative initiation of translation, a 39-kDa full-length JunD protein (JunD-FL) by initiation at the first AUG codon downstream of the mRNA 5' cap and a shorter, 34-kDa JunD protein (DeltaJunD) by initiation at a second in-frame AUG codon. The JunD mRNA contains a long, G/C-rich 5'-untranslated region that is predicted to be highly structured and is important for regulating the ratio of JunD-FL and DeltaJunD protein expression. A third functional out-of-frame AUG directs translation from a short open reading frame positioned between the JunD-FL and DeltaJunD start sites. In addition, three non-AUG codons also support translation, an ACG codon (in-frame with JunD) and a CUG are positioned in the 5'-untranslated region, and a CUG codon (also in-frame with JunD) is located downstream of the short open reading frame. Mutation of these start sites individually had no affect on DeltaJunD protein levels, but mutation of multiple upstream start sites led to an increase in DeltaJunD protein levels, indicating that these codons can function cumulatively to suppress DeltaJunD translation. Finally, we show that the JunD mRNA does not possess an internal ribosome entry site and is translated in a cap-dependent manner.
The AP-1 transcription factor c-Jun is required for Ras-driven tumorigenesis in many tissues and is considered as a classical proto-oncogene. To determine the requirement for c-Jun in a mouse model of K-RasG12D-induced lung adenocarcinoma, we inducibly deleted c-Jun in the adult lung. Surprisingly,
we found that inactivation of c-Jun, or mutation of its JNK phosphorylation sites, actually increased lung tumor burden. Mechanistically, we found that protein levels of the Jun family member JunD were increased in the absence of c-Jun. In c-Jun-deficient cells, JunD phosphorylation was increased, and expression of a dominant-active JNKK2-JNK1 transgene further increased lung tumor formation. Strikingly, deletion of JunD completely abolished Ras-driven lung tumorigenesis. This work identifies JunD, not c-Jun, as the crucial substrate of JNK signaling and oncogene required for Ras-induced lung cancer.
Kamme F and Wieloch T, Brain Res Mol Brain Res. 1996 Dec 31;43(1-2):51-6. doi: 10.1016/s0169-328x(96)00151-9.
The expression of junD was studied in the rat hippocampus by in situ hybridization after 15 min of normothermic (37 degrees C) and hypothermic (33 degrees C) transient forebrain ischemia. Ischemia was induced by common carotid artery occlusion combined with hypotension leading to damage in the CA1 r
egion of the hippocampus which was prevented by hypothermia. junD mRNA was induced in the hippocampus within 2 h of reperfusion and was strong in the dentate gyrus but weak in the CA3 and CA1 subregions. Intraischemic hypothermia significantly augmented the junD induction in the dentate gyrus. During late reperfusion (between 12 and 36 h after ischemia) a transient increase in junD mRNA was seen in the normothermic CA3 which was abolished in the hypothermic brains. In contrast, in the normothermic CA1 a continuous increase of junD was seen. This was significantly reduced by intraischemic hypothermia. We suggest that the early induction in junD expression in the dentate gyrus and in the hypothermic CA3 region is a protective reaction to the ischemic stress. The marked increase in resistant brain areas could be due to the preserved intracellular signaling pathways and a subsequent maintenance of protein synthesis. The late continuous increase, unique to the vulnerable normothermic CA1 region, suggests that junD participates in a transcriptional process that may be important for delayed neuronal death in the hippocampus following transient forebrain ischemia.
Guida N, etal., Toxicol Sci. 2018 Jun 1;163(2):569-578. doi: 10.1093/toxsci/kfy051.
Methylmercury (MeHg) causes neuronal death through different pathways. Particularly, we found that in cortical neurons it increased the expression of Repressor Element-1 Silencing Transcription Factor (REST), histone deacetylase (HDAC)4, Specificity Protein (Sp)1, Sp4, and reduced the levels of brai
n-derived neurotrophic factor (BDNF). Herein, in rat cortical neurons we investigated whether microRNA (miR)206 can modulate MeHg-induced cell death by regulating REST/HDAC4/Sp1/Sp4/BDNF axis. MeHg (1 µM) reduced miR206 expression after both 12 and 24 h and miR206 transfection prevented MeHg-induced neuronal death. Furthermore, miR206 reverted MeHg-induced REST and Sp4 increase and BDNF reduction at gene and protein level, and reverted HDAC4 protein increase, but not HDAC4 mRNA upregulation. Moreover, since no miR206 seed sequences were identified in the 3'-untranslated regions (3'-UTRs) of REST and SP4, we investigated the role of JunD, that presents a consensus motif on REST, Sp4, and BDNF promoters. Indeed, MeHg increased JunD mRNA and protein levels, and JunD knockdown counteracted MeHg-induced REST, Sp4 increase, but not BDNF reduction. Furthermore, we identified a miR206 binding site in the 3'-UTR of JunD mRNA (miR206/JunD) and mutagenesis of miR206/JunD site reverted JunD luciferase activity reduction induced by miR206. Finally, miR206 prevented MeHg-increased JunD binding to REST and Sp4 promoters, and MeHg-reduced BDNF expression was determined by the increase of HDAC4 binding on BDNF promoter IV. Collectively, these results suggest that miR206 downregulation induced by MeHg exposure determines an upregulation of HDAC4, that in turn downregulated BDNF, and the activation of JunD that, by binding REST and Sp4 gene promoters, increased their expression.
Valastro B, etal., Neuroscience. 2007 Jan 5;144(1):198-207. doi: 10.1016/j.neuroscience.2006.09.001. Epub 2006 Oct 19.
In this study, we have used 6-hydroxydopamine-lesioned rats to examine changes in striatal junD and fosB/deltafosB expression induced by acute and chronic treatment with L-DOPA (5 and 15 days). Changes at the protein levels were studied using Western immunoblotting while mRNA changes were compared u
sing in situ hybridization histochemistry. We observed a significant increase in the level of deltaFosB proteins after chronic treatment with L-DOPA, an effect that was not observed for JunD proteins. In addition, the upregulation of deltaFosB was already present after an acute treatment but increased upon chronic treatment. By contrast, junD and deltafosB mRNA were both upregulated significantly above control levels after an acute injection of L-DOPA. In conclusion, this study suggests a differential expression pattern of junD and deltafosB in a rat model of L-DOPA-induced dyskinesia. The upregulation of deltaFosB protein, but not JunD, is likely to reflect an increased stability of the deltaFosB proteins without ongoing enhanced transcription of the encoding genes.
The aryl hydrocarbon receptor (AhR) is a transcription factor involved in physiological processes, but also mediates most, if not all, toxic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Activation of the AhR by TCDD leads to its dimerization with aryl hydrocarbon nuclear translocator (AR
NT) and transcriptional activation of several phase I and II metabolizing enzymes. However, this classical signalling pathway so far failed to explain the pleiotropic hazardous effects of TCDD, such as developmental toxicity and tumour promotion. Thus, there is an urgent need to define genetic programmes orchestrated by AhR to unravel its role in physiology and toxicology. Here we show that TCDD treatment of rat liver oval cells leads to induction of the transcription factor JunD, resulting in transcriptional upregulation of the proto-oncogene cyclin A which finally triggers a release from contact inhibition. Ectopic expression of cyclin A in confluent cultures overcomes G(1) arrest, indicating that increased cyclin A levels are indeed sufficient to bypass contact inhibition. Functional interference with AhR-, but not with ARNT, abolished TCDD-induced increase in JunD and cyclin A and prevented loss of contact inhibition. In summary, we have discovered a novel AhR-dependent and probably ARNT-independent signalling pathway involving JunD and cyclin A, which mediates TCDD-induced deregulation of cell cycle control.
De León M, etal., J Neurosci Res. 1995 Oct 15;42(3):391-401. doi: 10.1002/jnr.490420314.
The present study was designed to compare the expression of the Jun family of protooncogenes following nerve injury. Adult rats were anesthetized and the sciatic nerve transected. Dorsal root ganglia (DRG) at 1, 2, 3, and 7 days after nerve transection were collected, their total RNA extracted, and
Northern blots performed using 32P-labeled oligonucleotide probes. The constitutive expression of c-jun mRNA was very low in DRG. Induction of c-jun mRNA was observed by day 1 after nerve transection, with a sixfold peak at 3 days and a twofold induction still present by day 7. The constitutive expression of junB mRNA was also low in the DRG, and sciatic nerve transection produced only a modest induction (1.7-fold by day 3) in the DRG ipsilateral to the nerve cut. junD mRNA was constitutively expressed at high levels in the DRG, and its level of expression did not in the DRG, and its level of expression did not change after sciatic nerve transection. Immunocytochemistry studies demonstrated a pattern of c-Jun, JunB, and JunD immunoreactivity (IR) associated with the cell nuclei of DRG neurons. c-Jun IR was found at very low levels in the undamaged contralateral DRG neurons, but sciatic nerve transection dramatically increased the number of c-Jun-immunoreactive neurons. Dot blot immunoblotting assay confirmed that the DRG ipsilateral to the sciatic nerve cut contained a higher level of c-Jun protein than the contralateral control DRG. Similar to c-Jun IR, JunB IR was minimal in the undamaged contralateral DRG. However, the DRG ipsilateral to the nerve transection did not show an increase in the number of immunoreactive neurons. JunD protein was expressed at high levels in the contralateral DRG, and this level of expression persisted after sciatic nerve transection in the ipsilateral DRG. DNA gel retardation assay experiments with an AP-1 consensus sequence showed a single DNA-protein complex. This complex was increased in ipsilateral as compared with contralateral DRG extracts. The amount of DNA-protein complex was reduced by c-Jun protein antiserum but was not altered when treated with a Fos antibody. We conclude that c-jun, junB and junD mRNAs and proteins are differentially regulated in the DRG after sciatic nerve transection.
Gillardon F, etal., Neurosci Lett. 1992 Feb 17;136(1):87-90. doi: 10.1016/0304-3940(92)90654-p.
Noxious peripheral stimulation induces the expression of various proto-oncogenes in rat spinal neurons. However, proto-oncogene expression seems to differ depending on the mode of the stimulus. Here, we report that noxious cutaneous ultraviolet (UV) irradiation results in a nearly 8-fold increase in
junD mRNA levels in the rat lumbar spinal cord. RNA slot-blotting and hybridization techniques revealed a transcriptional activation of the junD proto-oncogene after 6 h, but not 1 h following UV exposure. These results suggest that low-frequency ongoing afferent impulse discharge is reflected by an accumulation in junD transcripts.
Borowiak M, etal., Carcinogenesis. 2013 Nov;34(11):2664-72. doi: 10.1093/carcin/bgt221. Epub 2013 Jun 19.
Telomerase activity in cancer cells is dependent on the transcriptional regulation of the human telomerase reverse transcriptase (hTERT) gene, encoding the catalytic subunit of human telomerase. We have shown previously that HTLV-1 basic leucine zipper (HBZ), a viral regulatory protein encoded by th
e human retrovirus, human T-cell leukemia virus, type 1 (HTLV-1) cooperates with JunD to enhance hTERT transcription in adult T-cell leukemia (ATL) cells. Menin, the product of the tumor-suppressor MEN-1 gene, also interacts with JunD, represses its transcriptional activity and downregulates telomerase expression. The main objective of this study was to examine how menin and HBZ get involved in the regulation of hTERT transcription. In this study, we report that JunD and menin form a repressor complex of hTERT transcription in HBZ-negative cells. Conversely, in HBZ-positive cells, the formation of a JunD/HBZ/menin ternary complex and the recruitment of p300 histone acetyl transferase activity by HBZ lead to a decreased activity of the JunD-menin suppressor unit that correlates with the activation of hTERT transcription. Silencing HBZ or menin expression in ATL cells confirms that these proteins are differentially involved in telomerase regulation. These results propose that HBZ, by impeding the tumor-suppressor activity of menin, functions as a leukemogenic cofactor to upregulate gene transcription and promote JunD-mediated leukemogenesis.
Xiao L, etal., Am J Physiol Cell Physiol. 2010 May;298(5):C1226-34. doi: 10.1152/ajpcell.00021.2010. Epub 2010 Feb 24.
Intestinal epithelium is a rapidly self-renewing tissue in the body, and its homeostasis is tightly regulated by numerous factors including polyamines. Decreased levels of cellular polyamines increase activating transcription factor (ATF)-2, but the exact role and mechanism of induced ATF-2 in the
regulation of intestinal epithelial cell (IEC) growth remain elusive. Cyclin-dependent kinase (CDK) 4 is necessary for the G1-to-S phase transition during the cell cycle, and its expression is predominantly controlled at the transcription level. Here, we reported that induced ATF-2 following polyamine depletion repressed CDK4 gene transcription in IECs by increasing formation of the ATF-2/JunD heterodimers. ATF-2 formed complexes with JunD as measured by immunoprecipitation using the ATF-2 and JunD antibodies and by glutathione S-transferase (GST) pull-down assays using GST-ATF-2 fusion proteins. Studies using various mutants of GST-ATF-2 revealed that formation of the ATF-2/JunD dimers depended on the COOH-terminal basic region-leucine zipper domain of ATF-2. Polyamine depletion increased ATF-2/JunD complex and inhibited CDK4 transcription as indicated by a decrease in the levels of CDK4-promoter activity and its mRNA. ATF-2 silencing not only prevented inhibition of CDK4 transcription in polyamine-deficient cells but also abolished repression of CDK4 expression induced by ectopic JunD overexpression. ATF-2 silencing also promoted IEC growth in polyamine-depleted cells. These results indicate that induced ATF-2/JunD association following polyamine depletion represses CDK4 transcription, thus contributing to the inhibition of IEC growth.
OBJECTIVE: Mice deficient for the AP-1 transcription factor JunD, the only Jun protein constitutively expressed and clearly detectable in the mammalian heart, develop enhanced cardiac hypertrophy in response to chronic pressure overload. Catecholamines inducing alpha-adrenergic receptor-m
ediated signaling have been implicated in the neurohumoral response to pressure overload and the development of left ventricular hypertrophy. In the present study we analyzed the mechanistic role of JunD in cardiomyocyte hypertrophy in vitro in response to alpha-adrenergic agonist phenylephrine (PE). METHODS: Cardiomyocytes were isolated from 1- to 3-day-old rats and transfected with adenoviruses expressing LacZ or wild-type JunD, or with expression vectors encoding LacZ, wild-type JunD, mutated JunD forming only JunD homodimers (JunDeb1), mutated JunD lacking the JNK site (JunD-Delta 162), or c-Jun. After stimulation with PE (10(-5) mol/L), hypertrophic growth of cardiomyocytes (cross-sectional area and [3H]-leucine incorporation) and mRNA expression of JunD, c-Jun, c-Fos, and atrial natriuretic peptide (ANP) were analyzed. Transcriptional activation was determined by luciferase activity in cardiomyocytes transfected with AP-1 or ANP luciferase reporter plasmids. Gel shift assays with an AP-1 consensus oligonucleotide were performed to analyze AP-1 DNA binding activities. RESULTS: PE augmented mRNA levels of c-Jun and c-Fos, but decreased JunD transcript levels. Adenoviral over-expression of wild-type JunD blunted PE-induced hypertrophic growth and expression of ANP mRNA. Over-expression of JunD in cardiomyocytes caused enhanced AP-1 protein-DNA binding, without increasing the transcriptional response from AP-1 or ANP luciferase reporter plasmids at baseline or upon PE stimulation. Moreover, over-expression of JunDeb1 attenuated transcription from AP-1 or ANP luciferase reporter plasmids and blunted c-Jun-mediated acceleration of AP-1 transcriptional activity at baseline and in response to PE. CONCLUSIONS: Our observations establish a novel role for JunD as a negative regulator of cardiomyocyte hypertrophy in response to hypertrophic stimuli by inhibiting AP-1 transcriptional activity.
Crescentic glomerulonephritis is an important cause of human kidney failure for which the underlying molecular basis is largely unknown. In previous studies, we mapped several susceptibility loci, Crgn1-Crgn7, for crescentic glomerulonephritis in the Wistar Kyoto (WKY) rat. Here we show by combined
congenic, linkage and microarray studies that the activator protein-1 (AP-1) transcription factor JunD is a major determinant of macrophage activity and is associated with glomerulonephritis susceptibility. Introgression of Crgn2 from the nonsusceptible Lewis strain onto the WKY background leads to significant reductions in crescent formation, macrophage infiltration, Fc receptor-mediated macrophage activation and cytokine production. Haplotype analysis restricted the Crgn2 linkage interval to a 430-kb interval containing Jund, which is markedly overexpressed in WKY macrophages and glomeruli. Jund knockdown in rat and human primary macrophages led to significantly reduced macrophage activity and cytokine secretion, indicating conservation of JunD function in macrophage activation in rats and humans and suggesting in vivo inhibition of Jund as a possible new therapeutic strategy for diseases characterized by inflammation and macrophage activation.
Smart DE, etal., J Biol Chem. 2001 Jun 29;276(26):24414-21. doi: 10.1074/jbc.M101840200. Epub 2001 May 3.
Activation of hepatic stellate cells (HSCs) to a myofibroblast-like phenotype is the pivotal event in hepatic wound healing and fibrosis. Rat HSCs activated in vitro express JunD, Fra2, and FosB as the predominant AP-1 DNA-binding proteins, and all three associate with an AP-1 sequence that is essen
tial for activity of the tissue inhibitor of metalloproteinases-1 (TIMP-1) promoter. In this study, we used expression vectors for wild-type, dominant-negative, and forced homodimeric (Jun/eb1 chimeric factors) forms of JunD and other Fos and Jun proteins to determine the requirement for JunD in the transcriptional regulation of the TIMP-1 and interleukin-6 (IL-6) genes. JunD activity was required for TIMP-1 gene promoter activity, whereas overexpression of Fra2 or FosB caused a repression of promoter activity. The ability of homodimeric JunD/eb1 to elevate TIMP-1 promoter activity supports a role for JunD homodimers as the major AP-1-dependent transactivators of the TIMP-1 gene. IL-6 promoter activity was induced upon activation of HSCs and also required JunD activity; however, expression of JunD/eb1 homodimers resulted in transcriptional repression. Mutagenesis of the IL-6 promoter showed that an AP-1 DNA-binding site previously reported to be an activator of transcription in fibroblasts functions as a suppressor of promoter activity in HSCs. We conclude that JunD activates IL-6 gene transcription as a heterodimer and operates at an alternative DNA-binding site in the promoter. The relevance of these findings to events occurring in the injured liver was addressed by showing that AP-1 DNA-binding complexes are induced during HSC activation and contain JunD as the predominant Jun family protein. JunD is therefore an important transcriptional regulator of genes responsive to Jun homo- and heterodimers in activated HSCs.
Li L, etal., Gastroenterology. 2002 Sep;123(3):764-79. doi: 10.1053/gast.2002.35386.
BACKGROUND & AIMS: Normal intestinal mucosal growth requires cellular polyamines that regulate expression of various genes involved in cell proliferation, growth arrest, and apoptosis. We have recently shown that growth inhibition after polyamine depletion is associated with an increase i
n JunD/AP-1 activity in normal intestinal epithelial cells (IEC-6 line). The current study tests the hypothesis that polyamine depletion-induced JunD/activator protein 1 (AP-1) activity results from the activation of junD gene expression and plays a critical role in regulation of intestinal epithelial cell growth. METHODS: The junD gene transcription was examined by nuclear run-on assays, and messenger RNA (mRNA) stability was measured by determination of JunD mRNA half-life. Functions of JunD were investigated by using JunD antisense oligodeoxyribonucleotides and transient transfection with the junD-expressing vector. RESULTS: Depletion of cellular polyamines by DL-alpha-difluoromethylornithine (DFMO) induced levels of JunD mRNA and protein, which was associated with an increase in G(1) phase growth arrest. Polyamine depletion did not increase the rate of junD gene transcription but significantly increased the stability of JunD mRNA. Decreasing JunD protein by using JunD antisense oligomers promoted cell growth in polyamine-deficient cells. Growth arrest following polyamine depletion also was accompanied by increases in both p21 expression and its promoter activity. Treatment with JunD antisense oligomers inhibited the p21 promoter and prevented the increase in p21 expression in the presence of DFMO. Ectopic expression of the wild-type junD increased p21-promoter activity and inhibited epithelial cell growth. CONCLUSIONS: Polyamines negatively regulate junD gene expression posttranscriptionally, and increased JunD/AP-1 inhibits intestinal epithelial cell proliferation at least partially through the activation of p21 promoter.
BACKGROUND: Human nucleus pulposus cell (HNPC) apoptosis plays an important role in the development of intervertebral disc degeneration (IVDD). Our previous research revealed that among all of the dysregulated microRNAs in the degenerated nucleus pulposus tissues of patient with IVDD, miRNA-494 (mi
R-494) is the most significantly increased. However, the influence of miR-494 HNPC apoptosis has not been confirmed. OBJECTIVE: This study was designed to evaluate the effect of miR-494 on the HNPC apoptosis induced by TNF-alpha and to explore the possible mechanism of this process. METHODS: First, HNPCs were stimulated with TNF-alpha at different concentrations (0 ng/ml, 10 ng/ml, 50 ng/ml, or 100 ng/ml) for 0 h, 8 h, 16 h, or 24 h. Annexin V-PE/7-AAD assays and real-time quantitative PCR were used to detect the cell apoptosis rates and miR-494 expression. Second, we successfully knocked down endogenous miR-494 in HNPCs via lentiviral antigomiR-494 vector infection and then stimulated with TNF-alpha (100 ng/ml, 16 h). The rates of apoptosis and miR-494 expression were then detected again. Additionally, a dual-luciferase reporter assay and western blotting were used to determine whether JunD is a target of miR-494. Finally, western blotting was used to analyze the expression of cytochrome C. RESULTS: We found that the rate of apoptosis increased with concentration, time (p < 0.05) and miR-494 expression (p < 0.05). The rate of apoptosis in the 100 ng/ml, 16 h group appeared to be suitable. After transfection, the apoptosis rate and miR-494 expression were significantly decreased in the antigomiR-494+TNF-alpha group compared to the controls (p < 0.05). We also revealed that JunD is a target of miR-494. Western blotting analysis demonstrated that treatment with the lentiviral antigomiR-494 vector resulted in increased expression of JunD (p < 0.05) and decreased expression of cytochrome C (p < 0.05). CONCLUSION: These results indicated that miR-494 is a novel regulator of HNPC apoptosis induced by TNF-alpha. The knock-out of miR-494 expression protected the HNPCs from apoptosis via the up-regulation of JunD, which was possibly mediated via cytochrome C apoptotic signaling. These findings suggest that the miR-494/JunD signaling pathway might represent a novel therapeutic target for the prevention of IVDD.
Costantino S, etal., Eur Heart J. 2019 Mar 21;40(12):997-1008. doi: 10.1093/eurheartj/ehy903.
AIMS: Metabolic cardiomyopathy (MC)-characterized by intra-myocardial triglyceride (TG) accumulation and lipotoxic damage-is an emerging cause of heart failure in obese patients. Yet, its mechanisms remain poorly understood. The Activator Protein 1 (AP-1) member JunD was recently identifi
ed as a key modulator of hepatic lipid metabolism in obese mice. The present study investigates the role of JunD in obesity-induced MC. METHODS AND RESULTS: JunD transcriptional activity was increased in hearts from diet-induced obese (DIO) mice and was associated with myocardial TG accumulation and left ventricular (LV) dysfunction. Obese mice lacking JunD were protected against MC. In DIO hearts, JunD directly binds PPARγ promoter thus enabling transcription of genes involved in TG synthesis, uptake, hydrolysis, and storage (i.e. Fas, Cd36, Lpl, Plin5). Cardiac-specific overexpression of JunD in lean mice led to PPARγ activation, cardiac steatosis, and dysfunction, thereby mimicking the MC phenotype. In DIO hearts as well as in neonatal rat ventricular myocytes exposed to palmitic acid, Ago2 immunoprecipitation, and luciferase assays revealed JunD as a direct target of miR-494-3p. Indeed, miR-494-3p was down-regulated in hearts from obese mice, while its overexpression prevented lipotoxic damage by suppressing JunD/PPARγ signalling. JunD and miR-494-3p were also dysregulated in myocardial specimens from obese patients as compared with non-obese controls, and correlated with myocardial TG content, expression of PPARγ-dependent genes, and echocardiographic indices of LV dysfunction. CONCLUSION: miR-494-3p/JunD is a novel molecular axis involved in obesity-related MC. These results pave the way for approaches to prevent or treat LV dysfunction in obese patients.
Zou T, etal., Mol Cell Biol. 2010 Nov;30(21):5021-32. doi: 10.1128/MCB.00807-10. Epub 2010 Aug 30.
Polyamines critically regulate all mammalian cell growth and proliferation by mechanisms such as the repression of growth-inhibitory proteins, including JunD. Decreasing the levels of cellular polyamines stabilizes JunD mRNA without affecting its transcription, but the exact mechanism whereby polya
mines regulate JunD mRNA degradation has not been elucidated. RNA-binding proteins HuR and AUF1 associate with labile mRNAs bearing AU-rich elements located in the 3' untranslated regions (3'-UTRs) and modulate their stability. Here, we show that JunD mRNA is a target of HuR and AUF1 and that polyamines modulate JunD mRNA degradation by altering the competitive binding of HuR and AUF1 to the JunD 3'-UTR. The depletion of cellular polyamines enhanced HuR binding to JunD mRNA and decreased the levels of JunD transcript associated with AUF1, thus stabilizing JunD mRNA. The silencing of HuR increased AUF1 binding to the JunD mRNA, decreased the abundance of HuR-JunD mRNA complexes, rendered the JunD mRNA unstable, and prevented increases in JunD mRNA and protein in polyamine-deficient cells. Conversely, increasing the cellular polyamines repressed JunD mRNA interaction with HuR and enhanced its association with AUF1, resulting in an inhibition of JunD expression. These results indicate that polyamines modulate the stability of JunD mRNA in intestinal epithelial cells through HuR and AUF1 and provide new insight into the molecular functions of cellular polyamines.
Sharma SC and Richards JS, J Biol Chem. 2000 Oct 27;275(43):33718-28.
AP1 transcription factors control rapid responses of mammalian cells to stimuli that impact proliferation, differentiation, and transformation. To determine which AP1 factors are present in and regulated by hormones in ovarian cells during specific stages of proliferation and differentiation, we use
d both in vitro and in vivo models, Western blotting, immunohistochemistry, DNA binding assays, and transfections of AP1 promoter-reporter constructs. The expression patterns of Jun and Fos family members in response to hormones (follicle-stimulating hormone (FSH), luteinizing hormone (LH), and cAMP) were distinct. JunB, c-Jun, c-Fos, and Fra2 were rapidly but transiently induced by FSH in immature granulosa cells. JunD and Fra2 were induced by LH and maintained as granulosa cells terminally differentiated into luteal cells. Forskolin and phorbol myristate acetate acted synergistically to enhance transcription of an AP1(-73COL)-luciferase construct. JunD appears to be one mediator of this effect, since JunD was a major component of the AP1-DNA binding complex in granulosa cells, and menin, a selective inhibitor of JunD, blocked transcription of -73COL-luciferase. Thus, FSH and LH via cAMP induce specific AP1 factors, the AP1 expression patterns are distinct, and that of JunD and Fra2 correlates with the transition of proliferating granulosa cells to terminally differentiated, non-dividing luteal cells.
Yao ST, etal., J Neuroendocrinol. 2012 Dec;24(12):1542-52. doi: 10.1111/j.1365-2826.2012.02362.x.
The magnocellular neurones (MCN) of the supraoptic nucleus (SON) undergo reversible changes during dehydration. We hypothesise that alterations in steady-state transcript levels might be partially responsible for this plasticity. In turn, regulation of transcript abundance might be mediated by trans
cription factors. We have previously used microarrays to identify changes in the expression of mRNAs encoding transcription factors in response to water deprivation. We observed down-regulation of 11 and up-regulation of 31 transcription factor transcripts, including members of the activator protein-1 gene family, namely c-fos, c-jun, fosl1 and junD. Because JunD expression and regulation within the SON has not been previously described, we have used in situ hybridisation and the quantitative reverse transcriptase-polymerase chain reaction to confirm the array results, demonstrating a significant increase in JunD mRNA levels following 24 and 72 h of water deprivation. Western blot and immunohistochemistry revealed a significant increase in JunD protein expression following dehydration. Double-staining fluorescence immunohistochemistry with a neurone-specific marker (NeuN) demonstrated that JunD staining is predominantly neuronal. Additionally, JunD immunoreactivity is observed primarily in vasopressin-containing neurones with markedly less staining seen in oxytocin-containing MCNs. Furthermore, JunD is highly co-expressed with c-Fos in MCNs of the SON following dehydration. These results suggest that JunD plays a role in the regulation of gene expression within MCNs of the SON in association with other Fos and Jun family members.
Papoudou-Bai A, etal., Leuk Lymphoma. 2016;57(1):143-50. doi: 10.3109/10428194.2015.1034704. Epub 2015 May 12.
We analyzed the expression of Jun family in relation to CD30 expression, cell proliferation and B-cell differentiation immunophenotypes [Germinal Center and non-Germinal Center] in diffuse large B-cell lymphomas (DLBCL). Expression and high expression of phosphorylated-c-Jun (p-c-Jun), JunB, JunD an
d CD30 (cut-off scores 20% and 50%, respectively) was found in 18/103, 49/103, 72/101 and 26/102 cases, respectively, and in 6/103, 27/103, 60/101 and 21/102 cases, respectively. The following significant positive correlations were observed: (a) JunB with cyclin A (p = 0.046), cyclin B1 (p = 0.033), cyclin E (p = 0.003), MUM-1 (p = 0.002) and CD30 (p < 0.001), (b) JunD with Ki67 (p = 0.002) and cyclin E (p = 0.014), (c) p-c-Jun with CD30 (p = 0.015), and (d) high p-c-Jun with cyclin A (p = 0.034). The positive correlation between expression of JunB, JunD and p-c-Jun and tumor cell proliferation in DLBCL, suggests that increased JunB, JunD and p-c-Jun expression may be involved in the pathogenesis of DLBCL by increasing tumor cell proliferation.
Jalali A, etal., Jundishapur J Nat Pharm Prod. 2014 Oct 10;9(4):e17741. eCollection 2014 Nov.
BACKGROUND: High expression of p-glycoprotein (P-gp) has been associated with a poor prognosis in patients with hepatocellular carcinoma (HCC). It is likely that P-gp overexpression is responsible for multidrug resistance in HCC. OBJECTIVES: The aim of this study was to elucidate the effect of poten
t carcinogen nitrosamine with and without verapamil and rifampin drugs on P-gp expression at the mRNA level in HCC. MATERIALS AND METHODS: Four groups of rats (n = 5) were selected with different treatments and one group as control. mRNA concentration changes were monitored using quantitative PCR (QPCR). RESULTS: A significant difference was found between verapamil treated group and the control regarding the mRNA level. The mdr1a mRNA was significantly decreased in the verapamil group (P
