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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 the 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., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The bone morphogenetic protein (BMP) signaling pathway regulates a wide range of cellular responses in metazoans. A key step in the canonical BMP signaling is the phosphorylation and activation of transcription factors Smad1, Smad5, and Smad8 (collectively Smad1/5/8) by the type I BMP receptors. We previously identified PPM1A as a phosphatase toward dephosphorylation of all receptor-regulated Smads (R-Smads), including Smad1/5/8. Here we report another nuclear phosphatase named SCP4/CTDSPL2, belonging to the FCP/SCP family, as a novel Smad phosphatase in the nucleus. SCP4 physically interacts with and specifically dephosphorylates Smad1/5/8, and as a result attenuates BMP-induced transcriptional responses. Knockdown of SCP4 in multipotent mesenchymal C2C12 cells leads to increased expression of BMP target genes and consequently promotes BMP-induced osteogenic differentiation. Collectively, our results demonstrate that SCP4, as a Smad phosphatase, plays a critical role in BMP-induced signaling and cellular functions., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Activation of mesangial cells (MCs), which is characterized by induction of smooth muscle α-actin (SMA) expression, contributes to a key event in various renal diseases; however, the mechanisms controlling MC differentiation are still largely undefined. Activated Smad1 induced SMA in a dose-dependent manner in MCs. As a direct regulating molecule for SMA, we identified and characterized scleraxis (Scx) as a new phenotype modulator in advanced glycation end product (AGE)-exposed MCs. Scx physically associated with E12 and bound the E-box in the promoter of SMA and negatively regulated the AGE-induced SMA expression. Scx induced expression and secretion of bone morphogenetic protein 4 (BMP4), thereby controlling the Smad1 activation in AGE-treated MCs. In diabetic mice, Scx was concomitantly expressed with SMA in the glomeruli. Inhibitor of differentiation 1 (Id1) was further induced by extended treatment with AGE, thereby dislodging Scx from the SMA promoter. These data suggest that Scx and Id1 are involved in the BMP4-Smad1-SMA signal transduction pathway besides the TGFβ1-Smad1-SMA signaling pathway and modulate phenotypic changes in MCs in diabetic nephropathy.

Increasing evidence supports a role for PKCα in growth arrest and tumor suppression in the intestinal epithelium. In contrast, the Id1 transcriptional repressor has pro-proliferative and tumorigenic properties in this tissue. Here, we identify Id1 as a novel target of PKCα signaling. Using a highly specific antibody and a combined morphological/biochemical approach, we establish that Id1 is a nuclear protein restricted to proliferating intestinal crypt cells. A relationship between PKCα and Id1 was supported by the demonstration that (a) down-regulation of Id1 at the crypt/villus junction coincides with PKCα activation, and (b) loss of PKCα in intestinal tumors is associated with increased levels of nuclear Id1. Manipulation of PKCα activity in IEC-18 nontransformed intestinal crypt cells determined that PKCα suppresses Id1 mRNA and protein via an Erk-dependent mechanism. PKCα, but not PKCδ, also inhibited Id1 expression in colon cancer cells. Id1 was found to regulate cyclin D1 levels in IEC-18 and colon cancer cells, pointing to a role for Id1 suppression in the antiproliferative/tumor suppressive activities of PKCα. Notably, Id1 expression was elevated in the intestinal epithelium of PKCα-knock-out mice, confirming that PKCα regulates Id1 in vivo. A wider role for PKCα in control of inhibitor of DNA binding factors is supported by its ability to down-regulate Id2 and Id3 in IEC-18 cells, although their suppression is more modest than that of Id1. This study provides the first demonstrated link between a specific PKC isozyme and inhibitor of DNA binding factors, and it points to a role for a PKCα → Erk ⊣ Id1 → cyclin D1 signaling axis in the maintenance of intestinal homeostasis., (© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.)

The highly expressed Id1 (inhibitor of DNA binding/differentiation) protein promotes angiogenesis in HCC and is a well established target for anti-angiogenesis therapeutic strategies. Heparan sulfate (HS) mimetics such as PI-88 can abrogate HS-protein interactions to inhibit angiogenesis. Id1 is the direct downstream effector of bone morphogenetic proteins (BMPs), which are angiogenic and HS-binding proteins. Thus, targeting BMPs by HS mimetics may inhibit angiogenesis via attenuating Id1 expression. We report here that a HS mimetic WSS25 potently inhibited the tube formation of HMEC-1 cells on Matrigel and their migration. Meanwhile, WSS25 (25 μg/ml) nearly completely blocked Id1 expression in the HMEC-1 cells as demonstrated by oligo-angiogenesis microarray analysis and further confirmed by RT-PCR and Western blotting. BMP/Smad/Id1 signaling also was blocked by WSS25 treatment in HMEC-1 cells. Importantly, Id1 knockdown in HMEC-1 cells caused the disruption of their tube formation on Matrigel. By employing quartz crystal microbalance analysis, we found that WSS25 strongly bound to BMP2. Moreover, WSS25 impaired BMP2-induced tube formation of HMEC-1 cells on Matrigel and angiogenesis in Matrigel transplanted into C57BL6 mice. Furthermore, WSS25 (100 mg/kg) abrogated the growth of HCC cells xenografted in male nude mice. Immunohistochemical analysis showed that both the expression of Id1 and the endothelial cell marker CD31 were lower in the WSS25-treated tumor tissue than in the control. Therefore, WSS25 is a potential drug candidate for HCC therapy as a tumor angiogenesis inhibitor.

Cyclooxygenase-2 (COX-2) and Id-1 are overexpressed in a variety of human malignancies. Recently, each of these genes was found to play a role in mediating breast cancer metastasis to the lungs, but their potential interdependence was not evaluated. Hence, the main objective of the current study was to determine whether COX-2-derived prostaglandin (PGE(2)) activated Id-1 transcription, leading in turn to increased invasiveness of mammary epithelial cells. In MDA-MB-231 cells, treatment with PGE(2) induced Id-1, an effect that was mimicked by an EP(4) agonist. PGE(2) via EP(4) activated the epidermal growth factor receptor (EGFR) --> ERK1/2 pathway, which led to increased expression of Egr-1. PGE(2) stimulated EGFR signaling by inducing the release of amphiregulin, an EGFR ligand. The ability of PGE(2) to activate Id-1 transcription was mediated by enhanced binding of Egr-1 to the Id-1 promoter. Silencing of COX-2 or pharmacological inhibition of COX-2 led to reduced PGE(2) production, decreased Id-1 expression, and reduced migration of cells through extracellular matrix. A similar decrease in cell migration was found when Id-1 was silenced. The interrelationship between COX-2, PGE(2), Id-1, and cell invasiveness was also compared in nontumorigenic SCp2 and tumorigenic SCg6 mammary epithelial cells. Consistent with the findings in MDA-MB-231 cells, COX-2-derived PGE(2) induced Id-1, leading in turn to increased cell invasiveness. Taken together, these results suggest that PGE(2) via EP(4) activated the EGFR --> ERK1/2 --> Egr-1 pathway, leading to increased Id-1 transcription and cell invasion. These findings provide new insights into the relationship between COX-2 and Id-1 and their potential role in metastasis.

Recently, ID-1 (inhibitor of differentiation/DNA binding) is suggested as an oncogene and is reported to promote cell proliferation, invasion, and survival in several types of human cancer cells through multiple signaling pathways. However, how Id-1 interacts with these pathways and the immediate downstream effectors of the Id-1 protein are not known. In this study, using a yeast two-hybrid screening technique, we identified a novel Id-1-interacting protein, caveolin-1 (Cav-1), a cell membrane protein, and a positive regulator of cell survival and metastasis in prostate cancer. Using an immunoprecipitation method, we found that the helix-loop-helix domain of the Id-1 protein was essential for the physical interaction between Id-1 and Cav-1. In addition, we also demonstrated that the physical interaction between Id-1 and Cav-1 played a key role in the epithelial-mesenchymal transition and increased cell migration rate as well as resistance to taxol-induced apoptosis in prostate cancer cells. Furthermore, our results revealed that this effect was regulated by Id-1-induced Akt activation through promoting the binding activity between Cav-1 and protein phosphatase 2A. Our study demonstrates a novel Id-1 binding partner and suggests a molecular mechanism that mediates the function of Id-1 in promoting prostate cancer progression through activation of the Akt pathway leading to cancer cell invasion and resistance to anticancer drug-induced apoptosis.

Id1, an inhibitory partner of basic-helix-loop-helix transcriptional factors, has recently been recognized as a potent contributor to angiogenesis. However, the molecular mechanism underlying its role in angiogenesis remains essentially unknown. Herein we demonstrate the subcellular localization of Id1 to be altered depending on the cellular context of vascular endothelial cells. Id1 was localized in the nuclei of human umbilical vein endothelial cells (HUVECs) cultured on uncoated plates, whereas it was translocated to the cytoplasm in HUVECs on Matrigel along with the formation of capillary-like structures. Treatment with the nuclear export inhibitor leptomycin B and mutagenesis analysis using green fluorescent protein-fused Id1 revealed CRM1/exportin-dependent nuclear export of Id1 in HUVECs on Matrigel. This nuclear export of Id1 was inhibited by protein kinase A (PKA) activation by dibutyryl cyclic AMP and forskolin but was promoted by PKA inactivation by H-89 and MDL-12,330A. Mutagenesis analysis of Id1 showed the phosphorylation of Ser-5 to possibly mediate the effect of PKA. These results suggest the function of Id1 as a transcriptional factor to be controlled by nucleocytoplasmic shuttling during angiogenesis and that PKA might be involved in this process. This may serve as a novel mechanism regulating angiogenesis and as a possible target for therapeutic vascular regeneration.

Transforming growth factor-beta1 (TGF-beta1) and BMP-7 (bone morphogenetic protein-7; OP-1) play central, antagonistic roles in kidney fibrosis, a setting in which the expression of endoglin (CD105), an accessory TGF-beta type III receptor, is increased. So far, endoglin is known as a negative regulator of TGF-beta/ALK-5 signaling. Here we analyzed the effect of BMP-7 on TGF-beta1 signaling and the role of endoglin for both pathways in endoglin-deficient L(6)E(9) cells. In this myoblastic cell line, TGF-beta1 and BMPs are opposing cytokines, interfering with myogenic differentiation. Both induce specific target genes of which Id1 (for BMPs) and collagen I (for TGF-beta1) are two examples. TGF-beta1 activated two distinct type I receptors, ALK-5 and ALK-1, in these cells. Although the ALK-5/Smad3 signaling pathway mediated collagen I expression, ALK-1/Smad1/Smad5 signaling mediated a transient Id1 up-regulation. In contrast, BMP-7 exclusively activated Smad1/Smad5 resulting in a more prolonged Id1 expression. Although BMP-7 had no impact on collagen I abundance, it antagonized TGF-beta1-induced collagen I expression and (CAGA)(12)-MLP-Luc activity, effects that are mediated by the ALK-5/Smad3 pathway. Finally, we found that the transient overexpression of endoglin, previously shown to inhibit TGF-beta1-induced ALK-5/Smad3 signaling, enhanced the BMP-7/Smad1/Smad5 pathway.

The yeast reverse two-hybrid method was developed to identify mutations disrupting protein-protein interactions. Adoption of the method has been slow, in large part, due to the high frequency of truncation and frameshift mutants typically observed with current protocols. We have developed a new strategy, based on in vitro recombinational cloning and full-length selection in Escherichia coli, to eliminate this background and dramatically increase the efficiency of the reverse two-hybrid protocol. The method was tested by generating an allele library of MyoD1 and selecting for alleles with defective interaction with Id1. Our results confirm that most of the interaction-defective alleles contain a single point mutation in the known interaction domain, the basic helix-loop-helix region. Moreover analysis of the crystal structure of MyoD reveals that the majority of these mutations occurred at the interaction interface. The results obtained using this novel approach for allele library generation demonstrate a significant advancement in the application of yeast reverse two-hybrid screens. Furthermore this method is applicable to any loss-of-function mutant screen where truncated proteins are a source of high background.

Leukemic transformation often requires activation of protein kinase B (PKB/c-Akt) and is characterized by increased proliferation, decreased apoptosis, and a differentiation block. PKB phosphorylates and inactivates members of the FOXO subfamily of Forkhead transcription factors. It has been suggested that hyperactivation of PKB maintains the leukemic phenotype through actively repressing FOXO-mediated regulation of specific genes. We have found expression of the transcriptional repressor Id1 (inhibitor of DNA binding 1) to be abrogated by FOXO3a activation. Inhibition of PKB activation or growth factor deprivation also resulted in strong down-regulation of Id1 promoter activity, Id1 mRNA, and protein expression. Id1 is highly expressed in Bcr-Abl-transformed K562 cells, correlating with high PKB activation and FOXO3a phosphorylation. Inhibition of Bcr-Abl by the chemical inhibitor STI571 resulted in activation of FOXO3a and down-regulation of Id1 expression. By performing chromatin immunoprecipitation assays and promoter-mutation analysis, we demonstrate that FOXO3a acts as a transcriptional repressor by directly binding to the Id1 promoter. STI571 treatment, or expression of constitutively active FOXO3a, resulted in erythroid differentiation of K562 cells, which was inhibited by ectopic expression of Id1. Taken together our data strongly suggest that high expression of Id1, through PKB-mediated inhibition of FOXO3a, is critical for maintenance of the leukemic phenotype.

The Id proteins play an important role in proliferation, differentiation and tumorigenesis. Many tumors are hypoxic, but it is unknown if expression of Id proteins is regulated in hypoxic cells. Here we show that Id-1 is down-regulated in multiple primary, immortalized, and neoplastic hypoxic cell lines, and the transcriptional repressor ATF-3 is both necessary and sufficient for this hypoxia-induced repression of Id-1. Hypoxic up-regulation of ATF-3 is due in part to activation of the unfolded protein response, a cellular stress response. Remarkably, we observe that the unfolded protein response is de-regulated in all neuroblastoma cell lines tested. Indeed, in the absence of ATF-3 the hypoxia-induced transcription factor HIF-1 up-regulates Id-1 in hypoxic neuroblastoma cells. Hypoxic neuroblastoma cells diminish expression of some neuronal differentiation markers, and forced expression of ATF-3 in hypoxic neuroblastoma cells represses Id-1 and prevents the loss of these markers. The divergent regulation of Id proteins in distinct hypoxic cells may explain some of the varied effects hypoxia has on cellular differentiation and proliferation.

E2A and HEB are basic helix-loop-helix transcription factors that play important roles in T cell development. Expression of Id1, one of their inhibitors, severely impairs T cell development in transgenic mice. Aberrant activation of NF-kappaB transcription factors has been shown to contribute to the developmental defects, but it is not clear whether NF-kappaB activation is directly due to Id1 expression or is secondary to an abnormal thymic environment in Id1 transgenic mice. Here, by using a T cell line model, we demonstrate that Id1 expression stimulates basal levels of NF-kappaB activity and further enhances NF-kappaB activation upon T cell receptor (TCR) signaling achieved by anti-CD3 and anti-CD28 stimulation. Activation of NF-kappaB is partially mediated by the classical pathway involving the interaction between the regulatory subunit, NF-kappaB essential modulator (NEMO), and the catalytic subunit, IkappaB kinase beta. However, a NEMO-independent pathway also appears to be at play. Id1-potentiated activation of NF-kappaB leads to overproduction of cytokines such as tumor necrosis factor alpha and interferon-gamma in a T cell line as well as in thymocytes. Among members of the NF-kappaB family, c-Rel appears to be preferentially activated by Id1, especially during TCR stimulation. Consistently, c-rel deficiency diminishes tumor necrosis factor alpha and interferon-gamma expression induced by Id1 and TCR signaling.

We reported in the accompanying article (Ding, B., Liu, C., Huang, Y., Hickey, R. P., Yu, J., Kong, W., and Lengyel, P. (2006) J. Biol. Chem. 281, 14882-14892) that (i) the p204 protein is required for the differentiation of murine P19 embryonal carcinoma stem cells to beating cardiac myocytes, and (ii) the expression of p204 in the differentiating P19 cells is synergistically transactivated by the cardiac transcription factors Gata4, Nkx2.5, and Tbx5. Here we report that endogenous or ectopic inhibitor of differentiation (Id) proteins inhibited the differentiation of P19 cells to myocytes. This was in consequence of the binding of Id1, Id2, or Id3 protein to the Gata4 and Nkx2.5 proteins and the resulting inhibitions (i) of the binding of these transcription factors to each other and to DNA and (ii) of their synergistic transactivation of the expression of various genes, including atrial natriuretic factor and Ifi204 (encoding p204). p204 overcame this inhibition by Id proteins in consequence of (i) binding and sequestering Id proteins, (ii) accelerating their ubiquitination and degradation by proteasomes, and (iii) decreasing the level of Id proteins in the nucleus by increasing their translocation from the nucleus to the cytoplasm. Points (ii) and (iii) depended on the presence of the nuclear export signal in p204. In the course of the differentiation, Gata4, Nkx2.5, and p204 were components of a positive feedback loop. This loop arose in consequence of it that p204 overcame the inhibition of the synergistic activity of Gata4 and Nkx2.5 by the Id proteins.