ID1 and ID3 are Negative Regulators of TGFß2-Induced Ocular Hypertension and Compromised Aqueous Humor Outflow Facility in Mice.

Purpose: In POAG, elevated IOP remains the major risk factor in irreversible vision loss. Increased TGFß2 expression in POAG aqueous humor and in the trabecular meshwork (TM) amplifies extracellular matrix (ECM) deposition and reduces ECM turnover in the TM, leading to a decreased aqueous humor (AH) outflow facility and increased IOP. Inhibitor of DNA binding proteins (ID1 and ID3) inhibit TGFß2-induced fibronectin and PAI-1 production in TM cells. We examined the effects of ID1 and ID3 gene expression on TGFß2-induced ocular hypertension and decreased AH outflow facility in living mouse eyes. Methods: IOP and AH outflow facility changes were determined using a mouse model of Ad5-hTGFß2C226S/C288S-induced ocular hypertension. The physiological function of ID1 and ID3 genes were evaluated using Ad5 viral vectors to enhance or knockdown ID1/ID3 gene expression in the TM of BALB/cJ mice. IOP was measured in conscious mice using a Tonolab impact tonometer. AH outflow facilities were determined by constant flow infusion in live mice. Results: Over-expressing ID1 and ID3 significantly blocked TGFß2-induced ocular hypertension (P < 0.0001). Although AH outflow facility was significantly decreased in TGFß2-transduced eyes (P < 0.04), normal outflow facility was preserved in eyes injected concurrently with ID1 or ID3 along with TGFß2. Knockdown of ID1 or ID3 expression exacerbated TGFß2-induced ocular hypertension. Conclusions: Increased expression of ID1 and ID3 suppressed both TGFß2-elevated IOP and decreased AH outflow facility. ID1 and/or ID3 proteins thus may show promise as future candidates as IOP-lowering targets in POAG.

JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).

Osteoblasts are versatile cells involved in multiple whole-body processes, including bone formation and immune response. Secretory amounts and patterns of osteoblast-derived proteins such as osteopontin (OPN) and osteocalcin (OCN) modulate osteoblast function. However, the regulatory mechanism of OPN and OCN expression remains unknown. Here, we demonstrate that p54/p46 c-jun N-terminal kinase (JNK) inhibition suppresses matrix mineralization and OCN expression but increases OPN expression in MC3T3-E1 cells and primary osteoblasts treated with differentiation inducers, including ascorbic acid, bone morphogenic protein-2, or fibroblast growth factor 2. Preinhibition of JNK before the onset of differentiation increased the number of osteoblasts that highly express OPN but not OCN (OPN-OBs), indicating that JNK affects OPN secretory phenotype at the early stage of osteogenic differentiation. Additionally, we identified JNK2 isoform as being critically involved in OPN-OB differentiation. Microarray analysis revealed that OPN-OBs express characteristic transcription factors, cell surface markers, and cytokines, including glycoprotein hormone α2 and endothelial cell-specific molecule 1. Moreover, we found that inhibitor of DNA binding 4 is an important regulator of OPN-OB differentiation and that dual-specificity phosphatase 16, a JNK-specific phosphatase, functions as an endogenous regulator of OPN-OB induction. OPN-OB phenotype was also observed following LPS from Porphyromonas gingivalis stimulation during osteogenic differentiation. Collectively, these results suggest that the JNK-Id4 signaling axis is crucial in the control of OPN and OCN expression during osteoblastic differentiation.-Kusuyama, J., Amir, M. S., Albertson, B. G., Bandow, K., Ohnishi, T., Nakamura, T., Noguchi, K., Shima, K., Semba, I., Matsuguchi, T. JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).

Id Proteins Suppress E2A-Driven Invariant Natural Killer T Cell Development prior to TCR Selection.

A family of transcription factors known as E proteins, and their antagonists, Id proteins, regulate T cell differentiation at critical developmental checkpoints. Id proteins promote the differentiation of conventional αß T cells and suppress the expansion of innate-like αß T cells known as invariant natural killer T (iNKT) cells. However, it remains to be determined whether Id proteins differentially regulate these distinct lineage choices in early stages of T cell development. In this manuscript, we report that in Id-deficient mice, uninhibited activity of the E protein family member E2A mediates activation of genes that support iNKT cell development and function. There is also biased rearrangement in Id-deficient DP cells that promotes selection into the iNKT lineage in these mice. The observed expansion of iNKT cells is not abrogated by blocking pre-TCR signaling, which is required for conventional αß T cell development. Finally, E2A is found to be a key transcriptional regulator of both iNKT and γδNKT lineages, which appear to have shared lineage history. Therefore, our study reveals a previously unappreciated role of E2A in coordinating the development of the iNKT lineage at an early stage, prior to their TCR-mediated selection alongside conventional αß T cells.

ID4 levels dictate the stem cell state in mouse spermatogonia.

Spermatogenesis is a classic model of cycling cell lineages that depend on a balance between stem cell self-renewal for continuity and the formation of progenitors as the initial step in the production of differentiated cells. The mechanisms that guide the continuum of spermatogonial stem cell (SSC) to progenitor spermatogonial transition and precise identifiers of subtypes in the process are undefined. Here we used an Id4-eGfp reporter mouse to discover that EGFP intensity is predictive of the subsets, with the ID4-EGFPBright population being mostly, if not purely, SSCs, whereas the ID4-EGFPDim population is in transition to the progenitor state. These subsets are also distinguishable by transcriptome signatures. Moreover, using a conditional overexpression mouse model, we found that transition from the stem cell to the immediate progenitor state requires downregulation of Id4 coincident with a major change in the transcriptome. Collectively, our results demonstrate that the level of ID4 is predictive of stem cell or progenitor capacity in spermatogonia and dictates the interface of transition between the different functional states.

Id4 functions downstream of Bmp signaling to restrict TCF function in endocardial cells during atrioventricular valve development.

The atrioventricular canal (AVC) connects the atrial and ventricular chambers of the heart and its formation is critical for the development of the cardiac valves, chamber septation and formation of the cardiac conduction system. Consequently, problems in AVC formation can lead to congenital defects ranging from cardiac arrhythmia to incomplete cardiac septation. While our knowledge about early heart tube formation is relatively comprehensive, much remains to be investigated about the genes that regulate AVC formation. Here we identify a new role for the basic helix-loop-helix factor Id4 in zebrafish AVC valve development and function. id4 is first expressed in the AVC endocardium and later becomes more highly expressed in the atrial chamber. TALEN induced inactivation of id4 causes retrograde blood flow at the AV canal under heat induced stress conditions, indicating defects in AV valve function. At the molecular level, we found that id4 inactivation causes misexpression of several genes important for AVC and AV valve formation including bmp4 and spp1. We further show that id4 appears to control the number of endocardial cells that contribute to the AV valves by regulating Wnt signaling in the developing AVC endocardium.

Effects of upregulation of Id3 in human lung adenocarcinoma cells on proliferation, apoptosis, mobility and tumorigenicity.

The inhibitor of DNA-binding/differentiation 3 (Id3) protein is a helix-loop-helix transcription factor and may have an important role in cell proliferation and differentiation. This study was to evaluate the effects of upregulation of Id3 in human lung adenocarcinoma cells on proliferation, apoptosis, mobility and tumorigenicity. Short interference RNA suppression of Id3 (miRId3) in A549 cells was used to investigate the functional role(s) of Id3. Next, we used in vitro wound-healing assay and trans-well assay to study the effects of overexpressed Id3 on migration and invasion of A549 cells. Furthermore, to explore the influence of overexpressed Id3 on in vivo tumorigenesis, adenoviruses containing Id3 gene (Ad-Id3) and empty vector (Ad-LacZ) were generated. Co-transfection of pcDNA/miRId3 and pEGFP/Id3 into A549 cells reversed the Id3-induced cell proliferation inhibition and apoptosis. Upon Id3 transfection, A549 cells displayed decreased migratory and invasive capabilities, however, co-transfection of miRId3 and Id3 into A549 cells reversed the Id3-induced inhibitions of migratory and invasive capabilities. Three groups of nude mice were inoculated with Ad-LacZ, Ad-Id3 transfectants and untransfected A549 cells, respectively. Twenty-eight days after inoculation, tumors induced by Ad-Id3 transfectants grew much more slowly compared with Ad-LacZ transfectants and control group. This study provides for the first time both in vitro and in vivo proofs that forced expression of Id3 in lung adenocarcinoma cells reduces tumor growth rate and may be a potential target for tumor suppression.

B-1b Cells Secrete Atheroprotective IgM and Attenuate Atherosclerosis.

RATIONALE: B cells contribute to atherosclerosis through subset-specific mechanisms. Whereas some controversy exists about the role of B-2 cells, B-1a cells are atheroprotective because of secretion of atheroprotective IgM antibodies independent of antigen. B-1b cells, a unique subset of B-1 cells that respond specifically to T-cell-independent antigens, have not been studied within the context of atherosclerosis. OBJECTIVE: To determine whether B-1b cells produce atheroprotective IgM antibodies and function to protect against diet-induced atherosclerosis. METHODS AND RESULTS: We demonstrate that B-1b cells are sufficient to produce IgM antibodies against oxidation-specific epitopes on low-density lipoprotein both in vitro and in vivo. In addition, we demonstrate that B-1b cells provide atheroprotection after adoptive transfer into B- and T-cell deficient (Rag1(-/-)Apoe(-/-)) hosts. We implicate inhibitor of differentiation 3 (Id3) in the regulation of B-1b cells as B-cell-specific Id3 knockout mice (Id3(BKO)Apoe(-/-)) have increased numbers of B-1b cells systemically, increased titers of oxidation-specific epitope-reactive IgM antibodies, and significantly reduced diet-induced atherosclerosis when compared with Id3(WT)Apoe(-/-) controls. Finally, we report that the presence of a homozygous single nucleotide polymorphism in ID3 in humans that attenuates Id3 function is associated with an increased percentage of circulating B-1 cells and anti-malondialdehyde-low-density lipoprotein IgM suggesting clinical relevance. CONCLUSIONS: These results provide novel evidence that B-1b cells produce atheroprotective oxidation-specific epitope-reactive IgM antibodies and protect against atherosclerosis in mice and suggest that similar mechanisms may occur in humans.

Id3 induces an Elk-1-caspase-8-dependent apoptotic pathway in squamous carcinoma cells.

Inhibitor of differentiation/DNA-binding (Id) proteins are helix-loop-helix (HLH) transcription factors. The Id protein family (Id1-Id4) mediates tissue homeostasis by regulating cellular processes including differentiation, proliferation, and apoptosis. Ids typically function as dominant negative HLH proteins, which bind other HLH proteins and sequester them away from DNA promoter regions. Previously, we have found that Id3 induced apoptosis in immortalized human keratinocytes upon UVB exposure, consistent with its role as a tumor suppressor. To investigate the role of Id3 in malignant squamous cell carcinoma (SCC) cells (A431), a tetracycline-regulated inducible system was used to induce Id3 in cell culture and mouse xenograft models. We found that upon Id3 induction, there was a decrease in cell number under low serum conditions, as well as in soft agar. Microarray, RT-PCR, immunoblot, siRNA, and inhibitor studies revealed that Id3 induced expression of Elk-1, an E-twenty-six (ETS)-domain transcription factor, inducing procaspase-8 expression and activation. Id3 deletion mutants revealed that 80 C-terminal amino acids, including the HLH, are important for Id3-induced apoptosis. In a mouse xenograft model, Id3 induction decreased tumor size by 30%. Using immunofluorescent analysis, we determined that the tumor size decrease was also mediated through apoptosis. Furthermore, we show that Id3 synergizes with 5-FU and cisplatin therapies for nonmelanoma skin cancer cells. Our studies have shown a molecular mechanism by which Id3 induces apoptosis in SCC, and this information can potentially be used to develop new treatments for SCC patients.

Inhibitor of differentiation 4 (ID4): From development to cancer.

Highly conserved Inhibitors of DNA-Binding (ID1-ID4) genes encode multi-functional proteins whose transcriptional activity is based on dominant negative inhibition of basic helix-loop-helix (bHLH) transcription factors. Initial animal models indicated a degree of compensatory overlap between ID genes such that deletion of multiple ID genes was required to generate easily recognizable phenotypes. More recently, new model systems have revealed alterations in mice harboring deletions in single ID genes suggesting complex gene and tissue specific functions for members of the ID gene family. Because ID genes are highly expressed during development and their function is associated with a primitive, proliferative cellular phenotype there has been significant interest in understanding their potential roles in neoplasia. Indeed, numerous studies indicate an oncogenic function for ID1, ID2 and ID3. In contrast, the inhibitor of differentiation 4 (ID4) presents a paradigm shift in context of well-established role of ID1, ID2 and ID3 in development and cancer. Apart from some degree of functional redundancy such as HLH dependent interactions with bHLH protein E2A, many of the functions of ID4 are distinct from ID1, ID2 and ID3: ID4 proteins a) regulate distinct developmental processes and tissue expression in the adult, b) promote stem cell survival, differentiation and/or timing of differentiation, c) epigenetic inactivation/loss of expression in several advanced stage cancers and d) increased expression in some cancers such as those arising in the breast and ovary. Thus, in spite of sharing the conserved HLH domain, ID4 defies the established model of ID protein function and expression. The underlying molecular mechanism responsible for the unique role of ID4 as compared to other ID proteins still remains largely un-explored. This review will focus on the current understanding of ID4 in context of development and cancer.

The potential role of inhibitor of differentiation-3 in human adipose tissue remodeling and metabolic health.

Metabolic health in obesity is known to differ among individuals, and the distribution of visceral (VAT) and subcutaneous adipose tissue (SAT) plays an important role in this regard. Adipose tissue expansion is dependent on new blood vessel formation in order to prevent hypoxia and inflammation in the tissue. Regulation of angiogenesis in SAT and VAT in response to diet is therefore crucial for the metabolic outcome in obesity. Knowledge about the underlying genetic mechanisms determining metabolic health in obesity is very limited. We aimed to review the literature of the inhibitor of differentiation-3 (ID3) gene in relation to adipose tissue and angiogenesis in humans in order to determine whether ID3 could be involved in the regulation of adipose tissue expansion and metabolic health in human obesity. We find evidence that ID3 is involved in regulatory mechanisms in adipose tissue and regulates angiogenesis in many tissues including adipose tissue. We discuss how this might influence obesity and metabolic health in obesity and further discuss some potential mechanisms by which ID3 might regulate visceral and subcutaneous adipose tissue expansion. The combined results from the reviewed literature suggest ID3 to play a potential role in the underlying regulatory mechanisms of metabolic health in human obesity. The literature is still sparse and further studies focusing on human ID3 in relation to the nature of obesity are warranted.

Dual roles for Id4 in the regulation of estrogen signaling in the mammary gland and ovary.

The HLH transcriptional regulator Id4 exerts important roles in different organs, including the neural compartment, where Id4 loss usually results in early lethality. To explore the role of this basally restricted transcription factor in the mammary gland, we generated a cre-inducible mouse model. MMTV- or K14-cre-mediated deletion of Id4 led to a delay in ductal morphogenesis, consistent with previous findings using a germ-line knockout mouse model. A striking increase in the expression of ERα (Esr1), PR and FoxA1 was observed in both the basal and luminal cellular subsets of Id4-deficient mammary glands. Together with chromatin immunoprecipitation of Id4 on the Esr1 and Foxa1 promoter regions, these data imply that Id4 is a negative regulator of the ERα signaling axis. Unexpectedly, examination of the ovaries of targeted mice revealed significantly increased numbers of secondary and antral follicles, and reduced Id4 expression in the granulosa cells. Moreover, expression of the cascade of enzymes that are crucial for estrogen biosynthesis in the ovary was decreased in Id4-deficient females and uterine weights were considerably lower, indicating impaired estrogen production. Thus, compromised ovarian function and decreased circulating estrogen likely contribute to the mammary ductal defects evident in Id4-deficient mice. Collectively, these data identify Id4 as a novel regulator of estrogen signaling, where Id4 restrains ERα expression in the basal and luminal cellular compartments of the mammary gland and regulates estrogen biosynthesis in the ovary.

Inhibitor of differentiation 4 (ID4) inactivation promotes de novo steroidogenesis and castration-resistant prostate cancer.

Prostate cancer (PCa) is the most commonly diagnosed cancer in men in the Western world. The transition of androgen-dependent PCa to castration-resistant (CRPC) is a major clinical manifestation during disease progression and presents a therapeutic challenge. Our studies have shown that genetic ablation of inhibitor of differentiation 4 (Id4), a dominant-negative helix loop helix protein, in mice results in prostatic intraepithelial neoplasia lesions and decreased Nkx3.1 expression without the loss of androgen receptor (Ar) expression. ID4 is also epigenetically silenced in the majority of PCa. However, the clinical relevance and molecular pathways altered by ID4 inactivation in PCa are not known. This study investigates the effect of loss of ID4 in PCa cell lines on tumorigenicity and addresses the underlying mechanism. Stable silencing of ID4 in LNCaP cells (L-ID4) resulted in increased proliferation, migration, invasion, and anchorage-independent growth. An increase in the rate of tumor growth, weight, and volume was observed in L-ID4 xenografts compared with that in the LNCaP cells transfected with nonspecific short hairpin RNA (L+ns) in noncastrated mice. Interestingly, tumors were also observed in castrated mice, suggesting that loss of ID4 promotes CRPC. RNA sequence analysis revealed a gene signature mimicking that of constitutively active AR in L-ID4, which was consistent with gain of de novo steroidogenesis. Prostate-specific antigen expression as a result of persistent AR activation was observed in L-ID4 cells but not in L+ns cells. The results demonstrate that ID4 acts as a tumor suppressor in PCa, and its loss, frequently observed in PCa, promotes CRPC through constitutive AR activation.

Inhibitor of differentiation 3, a transcription factor, regulates hyperlipidemia-associated kidney disease.

BACKGROUND: Lipoprotein abnormalities are associated with a rapid decline in renal function in patients of chronic kidney disease. In addition, hyperlipidemia is associated with an increased risk of developing renal insufficiency. The underlying molecular mechanisms for these clinical findings are unclear. We have previously reported a role for inhibitor of differentiation 3 (ID3), a transcription factor, in regulating kidney disease in hyperlipidemia. Introducing a genetic deficiency of Id3 in spontaneously hyperlipidemic apolipoprotein E knockout (Apoe(-/-)) mice led to accelerated mesangioproliferative glomerulonephritis. The present study was carried out to further investigate the contribution of ID3 in hyperlipidemia-associated kidney disease. METHODS: Female C57BL/6 mice that were ID3-sufficient wild-type (WT) or ID3-deficient (Id3(-/-)) were fed a Western diet and evaluated for proteinuria, glomerular pathology, and immune infiltrating cells. Primary mesangial cell lines were generated from both mouse strains and stimulated with oxidized phospholipids. Cytokines and chemokines produced were measured by multiplex assays, ELISA, and QPCR. Glomerular isolates were studied for CXCL1 expression by QPCR. RESULTS: Id3(-/-) mice on a Western diet developed accelerated proteinuria and mesangioproliferative glomerulonephritis compared to WT controls. In vitro, Id3(-/-) glomerular mesangial cell lines produced higher levels of the monocyte chemoattractant CXCL1 in response to oxidized phospholipids. This was consistent with the rapid increase in glomerular CXCL1 expression followed by macrophage infiltration in Id3(-/-) mice fed a Western diet. CONCLUSIONS: A functional ID3 influences susceptibility to kidney disease and prevents glomerular injury by regulating local chemokine production and inflammatory cell recruitment.

Id3 and Id2 act as a dual safety mechanism in regulating the development and population size of innate-like γδ T cells.

The innate-like T cells expressing Vγ1.1 and Vδ6.3 represent a unique T cell lineage sharing features with both the γδ T and the invariant NKT cells. The population size of Vγ1.1(+)Vδ6.3(+) T cells is tightly controlled and usually contributes to a very small proportion of thymic output, but the underlying mechanism remains enigmatic. Deletion of Id3, an inhibitor of E protein transcription factors, can induce an expansion of the Vγ1.1(+)Vδ6.3(+) T cell population. This phenotype is much stronger on the C57BL/6 background than on the 129/sv background. Using quantitative trait linkage analysis, we identified Id2, a homolog of Id3, to be the major modifier of Id3 in limiting Vγ1.1(+)Vδ6.3(+) T cell expansion. The Vγ1.1(+)Vδ6.3(+) phenotype is attributed to an intrinsic weakness of Id2 transcription from Id2 C57BL/6 allele, leading to an overall reduced dosage of Id proteins. However, complete removal of both Id2 and Id3 genes in developing T cells suppressed the expansion of Vγ1.1(+)Vδ6.3(+) T cells because of decreased proliferation and increased cell death. We showed that conditional knockout of Id2 alone is sufficient to promote a moderate expansion of γδ T cells. These regulatory effects of Id2 and Id3 on Vγ1.1(+)Vδ6.3(+) T cells are mediated by titration of E protein activity, because removing one or more copies of E protein genes can restore Vγ1.1(+)Vδ6.3(+) T cell expansion in Id2 and Id3 double conditional knockout mice. Our data indicated that Id2 and Id3 collaboratively control survival and expansion of the γδ lineage through modulating a proper threshold of E proteins.

Id2, Id3 and Id4 overcome a Smad7-mediated block in tumorigenesis, generating TGF-ß-independent melanoma.

The role for the inhibitors of differentiation (Ids) proteins in melanomagenesis has been poorly explored. In other cell types, Ids have been shown to contribute to cell proliferation, migration and angiogenesis and, along with a number of other genes, are direct downstream targets of the transforming growth factor (TGF)-ß pathway. Expression of Smad7, which suppress TGF-ß signaling, or synthetic TGF-ß inhibitors, was shown to potently suppress melanomagenesis. We found that endogenous Id2, Id3 and Id4 expression was elevated in 1205Lu versus 1205Lu cells constitutively expressing Smad7, indicating Ids may play a role in melanomagenesis. Therefore, the effects of Tet-inducible expression of Id2, Id3 or Id4 along with Smad7 in TGF-ß-dependent 1205Lu human melanoma cells were explored in vitro and in vivo. 1205Lu cells formed subcutaneous tumors in athymic mice, whereas cells expressing Smad7 failed to form tumors. However, 1205Lu cells expressing Smad7 along with doxycycline-induced Id2, Id3 or Id4 were able to overcome the potent tumorigenic block mediated by S7, to varying degrees. Conversely, Id small interfering RNA knockdown suppressed anchorage-independent growth of melanoma. Histology of tumors from 1205Lu cells expressing Smad7 + Id4 revealed an average of 31% necrosis, compared with 5.2% in tumors from 1205Lu with vector only. Downstream, Ids suppressed cyclin-dependent kinase inhibitors, and re-upregulated invasion and metastasis-related genes matrix metalloproteinase 2 (MMP2), MMP9, CXCR4 and osteopontin, shown previously to be downregulated in response to Smad7. This study shows that Id2, Id3 and Id4 are each able to overcome TGF-ß dependence, and establish a role for Ids as key mediators of TGF-ß melanomagenesis.

Essential functions for ID proteins at multiple checkpoints in invariant NKT cell development.

Invariant NKT (iNKT) cells display characteristics of both adaptive and innate lymphoid cells (ILCs). Like other ILCs, iNKT cells constitutively express ID proteins, which antagonize the E protein transcription factors that are essential for adaptive lymphocyte development. However, unlike ILCs, ID2 is not essential for thymic iNKT cell development. In this study, we demonstrated that ID2 and ID3 redundantly promoted iNKT cell lineage specification involving the induction of the signature transcription factor PLZF and that ID3 was critical for development of TBET-dependent NKT1 cells. In contrast, both ID2 and ID3 limited iNKT cell numbers by enforcing the postselection checkpoint in conventional thymocytes. Therefore, iNKT cells show both adaptive and innate-like requirements for ID proteins at distinct checkpoints during iNKT cell development.

Id4 promotes senescence and sensitivity to doxorubicin-induced apoptosis in DU145 prostate cancer cells.

UNLABELLED: Inhibitor of differentiation proteins (Id1, 2, 3 and 4) are dominant negative regulators of basic helix loop helix transcription factors and play dominant roles in cancer cells, spanning several molecular pathways including senescence, invasion, metastasis, proliferation and apoptosis. In contrast to high Id1, Id2 and Id3 expression, the expression of Id4 is epigenetically silenced in prostate cancer. In the present study we demonstrated a novel role of Id4, that of promotion of cellular senescence in prostate cancer cells. MATERIALS AND METHODS: Id4 was ectopically expressed in DU145 cells (DU145+Id4). The cells treated with Doxorubicin (0-500 nm) or vehicle control were analyzed for apoptosis, senescence (SA-beta Galactosidase), and expression of CDKN1A (p21), CDKN1B(p27), CDKN2A (p16), E2F1, vimentin and E-cadherin by immuno-histochemistry and/or Western blot. RESULTS: In the present study we demonstrated that Id4 promotes cellular senescence in prostate cancer cell line DU145. Ectopic overexpression of Id4 in androgen receptor-negative DU145 prostate cancer cells resulted in increased expression of p16, p21, p27, E-cadherin and vimentin but down-regulated E2F1 expression. Id4 also potentiated the effect of doxorubicin induced senescence and apoptosis. CONCLUSION: The absence of functional p16, pRB and p53 in DU145 suggests that Id4 could alter additional molecular pathways such as those involving E2F1 to promote senescence and increased sensitivity to doxorubicin-induced apoptosis. The results of the present study support the role of Id4 as a tumor suppressor in prostate cancer.

B-cell aortic homing and atheroprotection depend on Id3.

RATIONALE: B cells are abundant in the adventitia of normal and diseased vessels. Yet, the molecular and cellular mechanisms mediating homing of B cells to the vessel wall and B-cell effects on atherosclerosis are poorly understood. Inhibitor of differentiation-3 (Id3) is important for atheroprotection in mice and polymorphism in the human ID3 gene has been implicated as a potential risk marker of atherosclerosis in humans. Yet, the role of Id3 in B-cell regulation of atherosclerosis is unknown. OBJECTIVE: To determine if Id3 regulates B-cell homing to the aorta and atheroprotection and identify molecular and cellular mechanisms mediating this effect. METHODS AND RESULTS: Loss of Id3 in Apoe(-/-) mice resulted in early and increased atherosclerosis. Flow cytometry revealed a defect in Id3(-/-) Apoe(-/-) mice in the number of B cells in the aorta but not the spleen, lymph nodes, and circulation. Similarly, B cells transferred from Id3(-/-) Apoe(-/-) mice into B-cell-deficient mice reconstituted spleen, lymph node, and blood similarly to B cells from Id3(+/+) Apoe(-/-) mice, but aortic reconstitution and B-cell-mediated inhibition of diet-induced atherosclerosis was significantly impaired. In addition to retarding initiation of atherosclerosis, B cells homed to regions of existing atherosclerosis, reduced macrophage content in plaque, and attenuated progression of disease. The chemokine receptor CCR6 was identified as an important Id3 target mediating aortic homing and atheroprotection. CONCLUSIONS: Together, these results are the first to identify the Id3-CCR6 pathway in B cells and demonstrate its role in aortic B-cell homing and B-cell-mediated protection from early atherosclerosis.

EGFR-AKT-Smad signaling promotes formation of glioma stem-like cells and tumor angiogenesis by ID3-driven cytokine induction.

Aberrant activation of receptor tyrosine kinases (RTK) is causally linked to the pathobiological traits of glioblastoma and genesis of glioma stem-like cells (GSC), but the underlying mechanism is still unknown. Here, we show that epidermal growth factor receptor (EGFR) signaling regulates the proliferation, angiogenesis, and acquisition of GSC characteristics by inducing inhibitor of differentiation 3 (ID3) and ID3-regulated cytokines [GRO1 and interleukins (IL)-6 and 8] induction. We found that EGFR-mediated ID3 expression was regulated by Smad5, which was directly phosphorylated by AKT. Furthermore, ID3 alone imparted GSC features to primary astrocytes derived from Ink4a/Arf-deficient mouse, and EGFR-ID3-IL-6 signaling axis gave rise to tumor cell heterogeneity. Conversely, EGFR inhibitors suppressed EGFR-AKT-Smad5-driven induction of ID3, which led to a decrease in the tumorsphere forming ability of GSCs and U87MG cells that possess an active mutant EGFR, EGFRvIII, without obvious cytotoxic effects. However, these cells seemed to regain colonogenic ability after removal of the EGFR inhibitors. Together, the results delineate a novel integrative molecular mechanism in which the RTK-ID signaling pathway governs genesis and maintenance of GBM histopathologic features, such as GSCs-based tumor initiation, progression, and angiogenesis.

The opposing roles of the transcription factor E2A and its antagonist Id3 that orchestrate and enforce the naive fate of T cells.

It is established that the transcription factor E2A and its antagonist Id3 modulate the checkpoints consisting of the precursor to the T cell antigen receptor (pre-TCR) and the TCR. Here we demonstrate that Id3 expression was higher beyond the pre-TCR checkpoint, remained high in naive T cells and showed a bimodal pattern in the effector-memory population. We show how E2A promoted T lineage specification and how pre-TCR-mediated signaling affected E2A genome-wide occupancy. Thymi in Id3-deficient mice had aberrant development of effector-memory cells, higher expression of the chemokine receptor CXCR5 and the transcriptional repressor Bcl-6 and, unexpectedly, T cell-B cell conjugates and B cell follicles. Collectively, our data show how E2A acted globally to orchestrate development into the T lineage and that Id3 antagonized E2A activity beyond the pre-TCR checkpoint to enforce the naive fate of T cells.