Springhorn JP, etal., J Biol Chem 1994 Feb 18;269(7):5132-6.
The transcriptional regulatory protein Id, a negatively trans-acting protein with a helix-loop-helix motif that is expressed in many proliferating tissues early in development, continues to be expressed in postmitotic adult cardiac myocytes and vascular smooth muscle. Following the observation of a
"doublet" band of 1.1 and 1.25 kilobases on Northern hybridizations of Id1 cDNA with mRNA isolated from both cardiac muscle and vascular smooth muscle cells, we identified and sequenced an alternatively spliced Id1 gene product containing an insert of 214 base pairs within the coding domain of the original Id1 cDNA. A protein with a molecular mass corresponding to that predicted by the Id1.25-kilobase mRNA sequence could be identified on immunoblots of cell lysates from neonatal and adult rat ventricular myocytes. The insert appears to be a "coding intron," based on the presence of intron-exon consensus sequences at the insert boundaries and the presence of the originally described Id1 carboxyl-terminal coding sequence immediately downstream from, and out of frame with, this insert. In contrast to Id1 and Id2, which do not form homodimers, the carboxyl-terminal sequence of this alternatively spliced Id1 transcript, termed Id1.25, permits homodimerization. Thus, alternative splicing of Id1 may allow for tissue-specific expression of Id1, while formation of homodimers could provide a post-translational mechanism to regulate the ability of Id1.25 to bind and inactivate E2A gene products.
Tzeng SF and de Vellis J, Glia. 1998 Dec;24(4):372-81.
Id1, Id2, and Id3 mRNA are expressed mainly in the proliferating ependymal cell zone of the mouse brain during embryogenesis. In this study, the expression pattern and cell phenotypes of the Id family mRNA were examined in postnatal and adult rat brain. The exp
ression of Idl and Id3 mRNA in rat brain was observed in the cortex layer 1, corpus callosum, ventricular/subventricular zone (VZ/ SVZ), and the CA1-4 layers of the hippocampus at postnatal day 1 (P1) through P14, whereby it declined at 2 months. In general, the developmental pattern of Idl mRNA coincided with the pattern observed for Id3 mRNA. Similar to Id1 and Id3, Id2 mRNA was highly expressed in the corpus callosum, VZ/SVZ, and the hippocampus. Examination of Id2 mRNA revealed high levels in the cortex and caudate putamen at P1 through P14, whereas a decline was observed in its expression in the adult cortex. In P5 rat cerebellum, all Id mRNA examined were found in the internal granular cell layers; however, at this time point, only Id2 mRNA expression was detected in the differentiating zone of the external granular cell layers, preferentially localizing to adult Purkinje cells. Furthermore, only Id2 mRNA expression in brain was observed in NF+ neurons at P5. Examination of S100alpha+ and GFAP+ astrocytes, revealed the presence of all three mRNAs, whereas the expression of Id2 and Id3 mRNA was absent in 04+ immature oligodendrocytes. These data suggest that the spatial and temporal kinetic patterns during development, as well as cellular specificity, of the Id gene family may play a critical role in neural precursor cell proliferation and cell divergence.
Man N, etal., Blood. 2016 May 12;127(19):2322-6. doi: 10.1182/blood-2015-11-677708. Epub 2016 Mar 4.
Inhibitor of DNA binding 1 (Id1) functions as an E protein inhibitor, and overexpression of Id1 is seen in acute myeloid leukemia (AML) patients. To define the effects of Id1 on leukemog
enesis, we expressed MLL-AF9 in fetal liver (FL) cells or bone marrow (BM) cells isolated from wild-type, Id1(-/-), p21(-/-), or Id1(-/-)p21(-/-) mice, and transplanted them into syngeneic recipient mice. We found that although mice receiving MLL-AF9-transduced FL or BM cells develop AML, loss of Id1 significantly prolonged the median survival of mice receiving FL cells but accelerated leukemogenesis in recipients of BM cells. Deletion of Cdkn1a (p21), an Id1 target gene, can rescue the effect of Id1 loss in both models, suggesting that Cdkn1a is a critical target of Id1 in leukemogenesis. It has been suggested that the FL transplant model mimics human fetal-origin (infant) MLL fusion protein (FP)-driven leukemia, whereas the BM transplantation model resembles postnatal MLL leukemia; in fact, the analysis of clinical samples from patients with MLL-FP(+) leukemia showed that Id1 expression is elevated in the former and reduced in the latter type of MLL-FP(+) AML. Our findings suggest that Id1 could be a potential therapeutic target for infant MLL-AF9-driven leukemia.
TWIST1 plays a crucial role in dentinogenesis, and its activity depends on both a dimerization partner selection and phosphorylation. Other factors, like Id proteins, can affect the availability of dimerization partners for TWIST1, subsequently leading to diverse biological outcomes. The purpose of
this study was to evaluate an impact of Id1 expression on differentiation of dental pulp stem cells (DPSCs). The altered expression of Id1 was achieved by transfection of human DPSCs with lentiviral vectors either driving an entire sequence of Id1, hence leading to Id1 overexpression, or carrying the Id1 silencing sequence. We observed that both overexpression and silencing of Id1 modulated human DPSC differentiation. Id1 overexpression resulted in a prevailing formation of TWIST1 homodimer and increased expression of genes encoding dentin sialophosphoprotein and dentin matrix protein 1, which confirm an enhanced odontogenic differentiation of DPSCs. Concurrently, Id1 silencing produced an opposite effect, slowing DPSC differentiation. These results highlight Id1 as an important modulator of molecular events during DPSC commitment and differentiation, which should be considered in dental research on tissue engineering. Moreover, we assume that the balance between TWIST1 dimerization forms in DPSCs might function in a cell-type-specific manner.
Nagata Y and Todokoro K, Biochem Biophys Res Commun 1994 Mar 30;199(3):1355-62.
Id is a nuclear factor containing a helix-loop-helix (HLH) motif. It has been reported that Id functions as an inhibitor of cell differentiation and its gene expression is down-regulated during cell differentiation. We have characterized three Id-related cDNAs, Id1
an>, Id2 and Id3, isolated from nerve growth factor (NGF)-stimulated PC12 cells. Structural analysis revealed that all three Id's contain putative phosphorylation sites for cyclic AMP-dependent kinase, casein kinase II and protein kinase C near and inside the HLH motifs. In contradiction to the previous reports, Northern blot analysis revealed that NGF induces rapid and transient increase of all three Id gene transcriptions. Furthermore, in situ hybridization of rat embryo showed that all three Id genes are highly expressed in neural precursors rather than differentiated neural cells. These results indicate that Id family members may function as immediate-early gene products, and that the expression of the Id family may play an important role in the early stage of neural differentiation.
Patil M, etal., Diabetes. 2017 Jun;66(6):1611-1625. doi: 10.2337/db16-1079. Epub 2017 Mar 7.
Obesity results from increased energy intake or defects in energy expenditure. Brown adipose tissue (BAT) is specialized for energy expenditure, a process called adaptive thermogenesis. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) controls BAT-mediated thermo
genesis by regulating the expression of Ucp1 Inhibitor of differentiation 1 (Id1) is a helix-loop-helix transcription factor that plays an important role in cell proliferation and differentiation. We demonstrate a novel function of Id1 in BAT thermogenesis and programming of beige adipocytes in white adipose tissue (WAT). We found that adipose tissue-specific overexpression of Id1 causes age-associated and high-fat diet-induced obesity in mice. Id1 suppresses BAT thermogenesis by binding to and suppressing PGC1α transcriptional activity. In WAT, Id1 is mainly localized in the stromal vascular fraction, where the adipose progenitor/precursors reside. Lack of Id1 increases beige gene and Ucp1 expression in the WAT in response to cold exposure. Furthermore, brown-like differentiation is increased in Id1-deficient mouse embryonic fibroblasts. At the molecular level, Id1 directly interacts with and suppresses Ebf2 transcriptional activity, leading to reduced expression of Prdm16, which determines beige/brown adipocyte cell fate. Overall, the study highlights the existence of novel regulatory mechanisms between Id1/PGC1α and Id1/Ebf2 in controlling brown fat metabolism, which has significant implications in the treatment of obesity and its associated diseases, such as diabetes.
Zhou JD, etal., Int J Clin Exp Pathol. 2015 May 1;8(5):5336-44. eCollection 2015.
To investigate the clinical significance of ID1 expression in Chinese de novo AML patients. Real-time quantitative PCR was carried out to detect the status of ID1 expression in 102 de novo AML patients and 28 controls. ... (more)
n style='font-weight:700;'>ID1 transcript level was significantly increased in AML compared to normal controls (p=0.029). The age in the patients with high ID1 expression is significantly older than in those with low ID1 expression (p=0.044). ID1 overexpression occurred with the highest frequency in the patients with poor karyotype (7/7, 100%), lower frequency in the patients with intermediate karyotype (28/60, 47%), and the lowest frequency in the patients with favorable karyotype (12/31, 39%). Both whole AML and non-M3 patients with high ID1 expression had significantly lower rate of complete remission than those with low ID1 expression (p=0.007 and 0.038). ID1 high-expressed patients showed significantly shorter overall survival (OS) than ID1 low-expressed patients in both whole AML and non-M3 according to Kaplan-Meier analysis (p=0.007 and 0.040). However, multivariate analysis indicated that ID1 overexpression was not an independent risk factor in both whole AML and non-M3 patients. However, the adverse impact of ID1 overexpression on outcome was revealed by both Kaplan-Meier analysis and multivariate analysis in the non-M3 patients less than 60 years old. Our study reveals that ID1 overexpression may be associated with higher risk karyotype classification and act as an independent risk factor in young non-M3 patients.
Wiercinska E, etal., Hepatology. 2006 May;43(5):1032-41.
Transforming growth factor (TGF)-beta is critically involved in the activation of hepatic stellate cells (HSCs) that occurs during the process of liver damage, for example, by alcohol, hepatotoxic viruses, or aflatoxins. Overexpression of the TGF-beta antagonist Smad7 inhibits transdifferentiation a
nd arrests HSCs in a quiescent stage. Additionally, bile duct ligation (BDL)-induced fibrosis is ameliorated by introducing adenoviruses expressing Smad7 with down-regulated collagen and alpha-smooth muscle actin (alpha-SMA) expression. The aim of this study was to further characterize the molecular details of TGF-beta pathways that control the transdifferentiation process. In an attempt to elucidate TGF-beta target genes responsible for fibrogenesis, an analysis of Smad7-dependent mRNA expression profiles in HSCs was performed, resulting in the identification of the inhibitor of differentiation 1 (Id1) gene. Ectopic Smad7 expression in HSCs strongly reduced Id1 mRNA and protein expression. Conversely, Id1 overexpression in HSCs enhanced cell activation and circumvented Smad7-dependent inhibition of transdifferentiation. Moreover, knock-down of Id1 in HSCs interfered with alpha-SMA fiber formation, indicating a pivotal role of Id1 for fibrogenesis. Treatment of HSCs with TGF-beta1 led to increased Id1 protein expression, which was not directly mediated by the ALK5/Smad2/3, but the ALK1/Smad1 pathway. In vivo, Id1 expression and Smad1 phosphorylation were co-induced during fibrogenesis. In conclusion, Id1 is identified as TGF-beta/ALK1/Smad1 target gene in HSCs and represents a critical mediator of transdifferentiation that might be involved in hepatic fibrogenesis. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).
Li B, etal., Clin Cancer Res. 2016 Mar 1;22(5):1243-55. doi: 10.1158/1078-0432.CCR-15-1196. Epub 2015 Oct 16.
PURPOSE: Chemoresistance is a major obstacle in cancer therapy. We found that fluorouracil (5-FU)-resistant esophageal squamous cell carcinoma cell lines, established through exposure to increasing concentrations of 5-FU, showed upregulation of Id1, I
GF2, and E2F1. We hypothesized that these genes may play an important role in cancer chemoresistance. EXPERIMENTAL DESIGN: In vitro and in vivo functional assays were performed to study the effects of Id1-E2F1-IGF2 signaling in chemoresistance. Quantitative real-time PCR, Western blotting, immunoprecipitation, chromatin immunoprecipitation, and dual-luciferase reporter assays were used to investigate the molecular mechanisms by which Id1 regulates E2F1 and by which E2F1 regulates IGF2. Clinical specimens, tumor tissue microarray, and Gene Expression Omnibus datasets were used to analyze the correlations between gene expressions and the relationships between expression profiles and patient survival outcomes. RESULTS: Id1 conferred 5-FU chemoresistance through E2F1-dependent induction of thymidylate synthase expression in esophageal cancer cells and tumor xenografts. Mechanistically, Id1 protects E2F1 protein from degradation and increases its expression by binding competitively to Cdc20, whereas E2F1 mediates Id1-induced upregulation of IGF2 by binding directly to the IGF2 promoter and activating its transcription. The expression level of E2F1 was positively correlated with that of Id1 and IGF2 in human cancers. More importantly, concurrent high expression of Id1 and IGF2 was associated with unfavorable patient survival in multiple cancer types. CONCLUSIONS: Our findings define an intricate E2F1-dependent mechanism by which Id1 increases thymidylate synthase and IGF2 expressions to promote cancer chemoresistance. The Id1-E2F1-IGF2 regulatory axis has important implications for cancer prognosis and treatment.
The testicular Sertoli cells support spermatogenesis by providing a microenvironment and structural support for the developing germ cells. Sertoli cell functions are regulated by the gonadotropin FSH. Sertoli cells become a terminally differentiated nongrowing cell population in the adult. In respon
se to FSH, the Sertoli cells express a large number of differentiated gene products, such as transferrin, which transports iron to the developing germ cells. Previously, members of the basic helix-loop-helix (bHLH) family of transcription factors have been shown to influence FSH-mediated gene expression in Sertoli cells. The functions of the bHLH proteins are modulated by Id (inhibitor of differentiation) proteins, which lack the DNA-binding basic domain. The Id proteins form transcriptionally inactive dimers with bHLH proteins and thus regulate cell proliferation and differentiation. The current study investigated the expression and function of Id proteins in the postmitotic Sertoli cell. Freshly isolated and cultured Sertoli cells coexpress all four isoforms of Id (Id1, Id2, Id3, and Id4), as determined by immunoprecipitation with isoform-specific anti-Id antibodies, RT-PCR, and Northern blot analysis. Id2 and Id3 expression levels seem higher than Id1. Interestingly, the expression of Id4 in Sertoli cells is only detectable after stimulation with FSH or cAMP. The Id1 expression is down-regulated by FSH and cAMP, whereas Id2 and Id3 levels remain unchanged in response to FSH. In contrast, serum induces the expression of Id1, Id2, and Id3. Treatment of Sertoli cells with serum significantly reduces the expression of the larger 4-kb Id4 transcript and promotes the presence of a novel 1.3-kb transcript of Id4. The regulatory role of FSH in the expression of all four isoforms of Id is mimicked by a cAMP analog, suggesting that the actions of FSH are mediated through the protein kinase A pathway. An antisense approach was used to study the functional significance of Id proteins in Sertoli cells. Antisense to Id1 stimulated transferrin promoter activity in a transient transfection assay. Interestingly, an antisense to Id2 down-regulated transferrin promoter activity. Id3 and Id4 antisense oligonucleotides had no effect on FSH-mediated transferrin promoter activation. Contrary to the hypothesis that Id proteins have redundant functions, the results of the current study suggest that Id1, Id2, Id3, and Id4 are differentially regulated and may have distinct functions. Id1 may act to maintain Sertoli cell growth potential, whereas Id2 and Id4 may be involved in the differentiation and hormone regulation of Sertoli cells.
Yu Y, etal., Mol Cell Biochem. 2016 Jan;411(1-2):289-98. doi: 10.1007/s11010-015-2591-z. Epub 2015 Oct 17.
The migration and proliferation of EPCs are crucial for re-endothelialization in vascular repair and development. Id1 has a regulatory role in the regulation of EPCs migration and proliferation. Based on these findings, we hypothesized that Id1
ight:700;'>Id1 plays a regulatory role in modulating the migration and proliferation of EPCs by interaction with other factors. Herein, we report that the Id1 protein and E-box protein E2-2 regulate EPCs function with completely opposite effects. Id1 plays a positive role in the regulation of EPC proliferation and migration, while endogenous E2-2 appears to be a negative regulator. Immunoprecipitation and immunofluorescence assay revealed that the Id1 protein interacts and co-localizes with the E2-2 protein in EPCs. Further, endogenous E2-2 protein was found to block EPCs function via the inhibition of FGFR1 and VEGFR2 expression. The overexpression and silencing of Id1 have no direct regulatory role on VEGFR2 and FGFR1 expression. On the other hand, Id1 relieves the E2-2-mediated repression of FGFR1 and VEGFR2 expression to modulate EPCs proliferation, migration, and tube formation in vitro. In summary, we demonstrated that Id1 and E2-2 are critical regulators of EPCs function in vitro. Id1 interacts with E2-2 and relieves the E2-2-mediated repression of FGFR1 and VEGFR2 expression to modulate EPCs functions. Id1 and E2-2 may represent novel therapeutic targets for re-endothelialization in vascular damage and repair.
Papaspyridonos M, etal., Nat Commun. 2015 Apr 29;6:6840. doi: 10.1038/ncomms7840.
A central mechanism of tumour progression and metastasis involves the generation of an immunosuppressive 'macroenvironment' mediated in part through tumour-secreted factors. Here we demonstrate that upregulation of the Inhibitor of Differentiation 1 (Id1), in r
esponse to tumour-derived factors, such as TGFbeta, is responsible for the switch from dendritic cell (DC) differentiation to myeloid-derived suppressor cell expansion during tumour progression. Genetic inactivation of Id1 largely corrects the myeloid imbalance, whereas Id1 overexpression in the absence of tumour-derived factors re-creates it. Id1 overexpression leads to systemic immunosuppression by downregulation of key molecules involved in DC differentiation and suppression of CD8 T-cell proliferation, thus promoting primary tumour growth and metastatic progression. Furthermore, advanced melanoma patients have increased plasma TGFbeta levels and express higher levels of ID1 in myeloid peripheral blood cells. This study reveals a critical role for Id1 in suppressing the anti-tumour immune response during tumour progression and metastasis.
In rats under continuous stress (CS) there is decreased hypothalamic dopaminergic innervation to the intermediate lobe (IL) of the pituitary gland, which causes hyperactivation and subsequent degeneration of melanotrophs in the IL. In this study, we investigated the molecular basis for the changes t
hat occur in melanotrophs during CS. Using microarray analysis, we identified several genes differentially expressed in the IL under CS conditions. Among the genes up-regulated under CS conditions, we focused on the inhibitor of DNA binding/differentiation (Id) family of dominant negative basic helix-loop-helix (bHLH) transcription factors. RT-PCR, Western blotting and in situ hybridization confirmed the significant inductions of Id1, Id2 and Id3 in the IL of CS rats. Administration of the dopamine D2 receptor agonist bromocriptine prevented the inductions of Id1-3 in the IL of CS rats, whereas application of the dopamine D2 antagonist sulpiride induced significant expressions of Id1-3 in the IL of normal rats. Moreover, an in vitro study using primary cultured melanotrophs demonstrated a direct effect on Id1-3 inductions by dopamine suppression. These results suggest that the decreased dopamine levels in the IL during CS induce Id1-3 expressions in melanotrophs. Because Id family members inhibit various bHLH transcription factors, it is conceivable that the induced Id1-3 would cooperatively modulate gene expressions in melanotrophs under CS conditions to induce hormone secretion.
Zhao Y, etal., J Biol Chem. 2016 Mar 25;291(13):6831-42. doi: 10.1074/jbc.M115.704361. Epub 2016 Feb 8.
ID1 (inhibitor of differentiation/DNA binding 1) acts an important role in metastasis, tumorigenesis, and maintenance of cell viability. It has been shown that the up-regulation of ID1 is correlated with poor prognosis and t
he resistance to chemotherapy of human cancers. However, the underlying molecular mechanism remains elusive. Here, we determined for the first time that up-regulating ID1 upon etoposide activation was mediated through AP-1 binding sites within theID1promoter and confirmed that ID1 enhanced cell resistance to DNA damage-induced apoptosis in esophageal squamous cell carcinoma cells. Ablation of c-Jun/c-Fos or ID1 expression enhanced etoposide-mediated apoptosis through increasing activity of caspase 3 and PARP cleavage. Moreover, c-Jun/c-Fos and ID1 were positively correlated in human cancers. More importantly, simultaneous high expression of ID1 and c-Jun or c-Fos was correlated with poor survival in cancer patients. Collectively, we demonstrate the importance of c-Jun/c-Fos-ID1 signaling pathway in chemoresistance of esophageal cancer cells and provide considerable insight into understanding the underlying molecular mechanisms in esophageal squamous cell carcinoma cell biology.
Inhibitor of differentiation/DNA binding (Id)1 is a crucial regulator of mammary development and breast cancer progression. However, its effect on stemness and tumorigenesis in mammary epithelial cells remains undefined. Herein, we demonstrate that Id1 induces
mammary tumorigenesis by increasing normal and malignant mammary stem cell (MaSC) activities in transgenic mice. MaSC-enriched basal cell expansion and increased self-renewal and in vivo regenerative capacity of MaSCs are observed in the mammary glands of MMTV-Id1 transgenic mice. Furthermore, MMTV-Id1 mice develop ductal hyperplasia and mammary tumors with highly expressed basal markers. Id1 also increases breast cancer stem cell (CSC) population and activity in human breast cancer lines. Moreover, the effects of Id1 on normal and malignant stem cell activities are mediated by the Wnt/c-Myc pathway. Collectively, these findings provide in vivo genetic evidence of Id1 functions as an oncogene in breast cancer and indicate that Id1 regulates mammary basal stem cells by activating the Wnt/c-Myc pathway, thereby contributing to breast tumor development.
Young VJ, etal., Sci Rep. 2015 Nov 18;5:16859. doi: 10.1038/srep16859.
VEGF-A, an angiogenic factor, is increased in the peritoneal fluid of women with endometriosis. The cytokine TGF-beta1 is thought to play a role in the establishment of endometriosis lesions. Inhibitor of DNA binding (ID) proteins are transcriptional targets of TGF-beta1 and ID1
t:700;'>ID1 has been implicated in VEGF-A regulation during tumor angiogenesis. Herein, we determined whether peritoneal expression of VEGF-A is regulated by TGF-beta1 through the ID1 pathway in women with endometriosis. VEGF-A was measured in peritoneal fluid by ELISA (n = 16). VEGF-A and ID1 expression was examined in peritoneal biopsies (n = 13), and primary peritoneal and immortalized mesothelial cells (MeT5A) by immunohistochemistry, qRT-PCR and ELISA. VEGF-A was increased in peritoneal fluid from women with endometriosis and levels correlated with TGF-beta1 concentrations (P < 0.05). VEGF-A was immunolocalized to peritoneal mesothelium and TGF-beta1 increased VEGFA mRNA (P < 0.05) and protein (P < 0.05) in mesothelial cells. ID1 was increased in peritoneum from women with endometriosis and TGF-beta1 increased concentrations of ID1 mRNA (P < 0.05) in mesothelial cells. VEGF-A regulation through ID1 was confirmed by siRNA in MeT5A cells (P < 0.05). Our data supports role for ID1 in the pathophysiology of endometriosis, as an effector of TGFbeta1 dependent upregulation of VEGF-A, and highlights a novel potential therapeutic target.
Engineering living, multilayered blood vessels to form in vivo arteries is a promising alternative to peripheral artery bypass using acellular grafts restricted by thrombosis and occlusion at long term. Bone Morphogenetic Protein 2 (BMP2) is a growth factor determining in the early vascular embryoni
c development. The aim of the present study was evaluate the collaborative effect of recombinant human--BMP2 and Bone marrow--Mesenchymal stem cells (BM-MSCs) seeded on vascular patch to regenerate a vascular arterial wall in a rat model. BM-MSCs expressing green fluorescent protein (GFP) seeded on vascular patch were cultured in presence of recombinant human-BMP2 [100 ng/mL] during 1 week before their implantation on the abdominal aorta of Wistar rats. We observed after 2 weeks under physiological arterial flow a regeneration of a three layers adult-like arterial wall with a middle layer expressing smooth muscle proteins and a border layer expressing endothelial marker. In vitro study, using Matrigel assay and co-culture of BM-MSCs with endothelial cells demonstrated that rh-BMP2 promoted tube-like formation even at long term (90 days) allowing the organization of thick rails. We demonstrated using inhibitors and siRNAs that rh-BMP2 enhanced the expression of HIF-1alpha and Id1 through, at least in part, the stimulation of JAK2/STAT3/STAT5 signaling pathways. Rh-BMP2 by mimicking embryological conditions allowed vascular BM-MSCs differentiation.
Einarson MB and Chao MV, Mol Cell Biol. 1995 Aug;15(8):4175-83.
Cell differentiation in the nervous system is dictated by specific patterns of gene expression. We have investigated the role of helix-loop-helix (HLH) proteins during differentiation of PC12 pheochromocytoma cells in response to nerve growth factor. Gel mobility shift assays using PC12 cell nuclear
extracts demonstrated that active basic HLH complexes exist throughout differentiation. Addition of exogeneous Id1 protein, a negative regulator of basic HLH proteins, disrupted specific complexes formed by PC12 cell nuclear extracts on a CANNTG consensus oligonucleotide. To identify possible novel basic HLH proteins in these complexes, a glutathione S-transferase-Id1 fusion protein was used to screen a PC12 cell cDNA expression library. A single clone representing the rat E2-2 gene was identified. Sequential immunoprecipitations with antibodies to each HLH protein revealed an association between Id1 and E2-2 that could be detected in both untreated and nerve growth factor-treated PC12 cell lysates. These experiments define a new HLH interaction between Id1 and E2-2 in neuronal cells and suggest that neuronal differentiation may be regulated by HLH proteins in a distinctive manner.
Growth factors and peptides playing important roles during early development of the central nervous system have also been shown to maintain their regulation of cell genesis in the adult brain. We have previously described that endogenous opioids, expressed in the developing hippocampus, regulate pro
liferation and differentiation in the adult rat hippocampus. The aim of this study was to investigate the effects of the opioid beta-endorphin on gene expression and glial differentiation in cultures of adult rat hippocampal progenitors (AHPs). Changes in gene expression after stimulation of AHPs with beta-endorphin for 48 h were investigated using cDNA arrays. Confirmation experiments verified that stimulation with beta-endorphin increased the mRNA levels of myelin basic protein, glutathione S-transferase pi, c-junD and rab16 (P < 0.05), genes that are associated with oligodendrogenesis. Furthermore, beta-endorphin increased the levels of Id1, but not Id3, mRNA on the arrays. Incubation of AHPs with beta-endorphin resulted in a threefold increase in oligodendrogenesis (P < 0.01) but no significant change in astrogliogenesis. No effect on oligodendrogenesis was observed in the presence of the opioid antagonist naloxone. Coincubation of beta-endorphin with Id1 antisense oligonucleotides for 10 days also entirely blocked the induced oligodendrogenesis in our AHP cultures. Moreover, a subpopulation of AHPs (25%) showed nuclear expression of the proneural transcriptional activator Mash1 that was reduced to approximately 5% of the cells when exposed to beta-endorphin. We suggest a requirement for Id1 in opioid-induced oligodendrogenesis in cultured AHPs possibly acting on opioid-responsive AHPs expressing the proneural transcriptional activator Mash1.
Yang J, etal., Circ Res. 2010 Jul 23;107(2):252-62. doi: 10.1161/CIRCRESAHA.109.209940. Epub 2010 Jun 3.
RATIONALE: Mutations in the bone morphogenetic protein type II receptor (BMPR-II) are responsible for the majority of cases of heritable pulmonary arterial hypertension (PAH). Mutations lead to reduced Smad1/5-driven expression of inhibitor of DNA binding protein 1 (Id1
1) and loss of the growth suppressive effects of BMPs. The impact of existing PAH therapies on BMP signaling is lacking. OBJECTIVE: Because prostacyclin analogues are effective treatments for clinical PAH, we hypothesized that these agents enhance Smad1/Id1 signaling. METHODS AND RESULTS: Iloprost alone induced Id1 expression in human pulmonary artery smooth muscle cells (PASMCs), an effect that was independent of Smad1/5 activation but dependent on a cAMP-responsive element in the Id1 promoter. In addition, iloprost and treprostinil enhanced BMP-induced phosphorylation of Smad1/5 and Id1 expression in a cAMP-dependent manner. The mechanism involved suppression of inhibitory Smad, Smad6. Furthermore, iloprost rescued the deficit in Smad1/5 phosphorylation and Id gene expression in PASMCs harboring mutations in BMPR-II and restored growth suppression to BMP4 in mutant PASMCs. We confirmed a critical role for Id1 in PASMC proliferation. Reduced expression of Id1 was observed in concentric intimal lesions of heritable PAH cases. In the monocrotaline rat model of PAH, associated with reduced BMPR-II expression, we confirmed that treprostinil inhibited smooth muscle cell proliferation and prevented progression of PAH while enhancing Smad1/5 phosphorylation and Id1 gene expression. CONCLUSIONS: Prostacyclin analogues enhance Id1 expression in vitro and in vivo and restore deficient BMP signaling in BMPR-II mutant PASMCs.
