Hydrogen peroxide-inducible clone-5 (Hic-5, or androgen receptor-associated protein 55) is a transforming growth factor-β-inducible LIM protein whose deregulation is implicated in the progression of ...prostate cancer. Here, we report that Hic-5 binds to Smads 1, 5 and 8, and represses bone morphogenetic protein (BMP) signaling responses. Myc-Hic-5 but not Myc-paxillin was specifically immunoprecipitated with anti-FLAG IgG1 from lysates of HEK293 co-transfected with either Myc-Hic-5 or Myc-paxillin and FLAG-tagged Smads 1, 5 or 8. We showed that such interactions require the LIM3 domain of Hic-5 and the MH2 domain of those Smads. Anti-Hic-5 antibody specifically pulled down endogenous Smad1 in both the PC3 human prostate cell line and primary cultures of rat prostate fibroblasts, supporting that Hic-5 binds to Smad1 at the endogenous level. Bacterially expressed glutathione S-transferase (GST)-Smads 1, 5 or 8, but not GST alone, pulled down in vitro transcribed and translated Hic-5, implicating that Hic-5 binds directly to Smads 1, 5 and 8. Significantly, using Hic-5 small hairpin RNA silencing and overexpression systems, we show that Hic-5 (at both the endogenous and exogenous levels) represses the ability of BMP4 to induce expression of the inhibitor of differentiation-1 (Id1; a downstream target gene of BMP), activate the Id1 gene promoter and induce apoptosis in human and rat prostate epithelial cells. Moreover, silencing of Hic-5 in PC3 cells as well as in the WPMY-1 human prostate stroma cell line greatly enhances the levels of endogenous phospho-Smad1/5/8. Finally, we provide fluorescent microscopic imaging to support that Smad1 and Hic-5 mutually interact also at the level of their nuclear export mechanisms. Collectively, these results provide the first evidence for a physical and mutual functional interaction between Hic-5 and the BMP signaling pathway.
We recently reported that hydrogen peroxide-inducible clone-5 (Hic-5, also named androgen receptor-associated protein 55) can bind to the transforming growth factor-beta (TGF-beta)-signaling ...regulator Smad3, thereby inhibiting certain Smad3-dependent TGF-beta responses. We now show that Hic-5 can also control TGF-beta responses through an alternative mechanism involving Smad7, a key negative regulator of TGF-beta signaling. Hic-5 binds directly to Smad7. This interaction requires the LIM3 domain of Hic-5, and enhances TGF-beta signaling through causing loss of Smad7 protein but not mRNA. Enforced expression of Hic-5 reverses the ability of Smad7 to suppress TGF-beta-induced phosphorylation of Smads 2 and 3 and activation of the plasminogen activator inhibitor-1 promoter (in NRP-154 and PC3 prostate carcinoma and WPMY-1 prostate myofibroblast cell lines). Lentiviral-mediated small-hairpin RNA silencing of endogenous Hic-5 reduced TGF-beta responses in PC3 and WPMY-1 cells. Further work suggests that the level of Smad7 is modulated by its physical interaction with Hic-5 and targeted to a degradation pathway not likely to be proteasomal. Our findings support that Hic-5 functions as a cell-type-specific activator of TGF-beta signaling through its ability to physically interact with and neutralize Smad7.
Survivin is a prosurvival protein overexpressed in many cancers through mechanisms that remain poorly explored, and is implicated in control of tumor progression and resistance to cancer ...chemotherapeutics. Here, we report a critical role for survivin in the induction of apoptosis by transforming growth factor-beta (TGF-beta). We show that TGF-beta rapidly downregulates survivin expression in prostate epithelial cells, through a unique mechanism of transcriptional suppression involving Smads 2 and 3, Rb/E2F4, and the cell-cycle repressor elements CDE and CHR. This TGF-beta response is triggered through a Smad2/3-dependent hypophosphorylation of Rb and the subsequent association of the Rb/E2F4 repressive complex to CDE/CHR elements in the proximal region of the survivin promoter. Viral-mediated gene delivery experiments, involving overexpressing or silencing survivin, reveal critical roles of survivin in apoptosis induced by TGF-beta alone or in cooperation with cancer therapeutic agents. We propose a novel TGF-beta/Rb/survivin axis with a putative role in the functional switch of TGF-beta from tumor suppressor to tumor promoter.
αA-crystallin is a molecular chaperone and an antiapoptotic protein. This study investigated the mechanism of inhibition of apoptosis by human αA-crystallin and determined if the chaperone activity ...of αA-crystallin is required for the antiapoptotic function. αA-crystallin inhibited chemical-induced apoptosis in Chinese hamster ovary (CHO) cells and HeLa cells by inhibiting activation of caspase-3 and -9. In CHO cells, it inhibited apoptosis induced by the overexpression of human proapoptotic proteins, Bim and Bax. αA-crystallin inhibited doxorubicin-mediated activation of human procaspase-3 in CHO cells and it activated the PI3K/Akt cell survival pathway by promoting the phosphorylation of PDK1, Akt and phosphatase tensin homologue in HeLa cells. The phosphoinositide 3 kinase (PI3K) activity was increased by αA-crystallin overexpression but the protein content was unaltered. Downregulation of PI3K by the expression of a dominant-negative mutant or inhibition by LY294002 abrogated the ability of αA-crystallin to phosphorylate Akt. These antiapoptotic functions of αA-crystallin were enhanced in a mutant protein (R21A) that shows increased chaperone activity than the wild-type (Wt) protein. Interestingly, a mutant protein (R49A) that shows decreased chaperone activity was far weaker than the Wt protein in its antiapoptotic functions. Together, our study results show that αA-crystallin inhibits apoptosis by enhancing PI3K activity and inactivating phosphatase tensin homologue and that the antiapoptotic function is directly related to its chaperone activity.
Cited (CBP/p300-interacting transactivators with glutamic acid (E)/aspartic acid (D)-rich C-terminal domain) 2, which is a CBP/p300-binding transcription co-activator without typical DNA-binding ...domains, has been implicated in control of cell growth and malignant transformation in Rat1 cells. In this report, we provide evidence that Cited2 is an important regulator of transforming growth factor (TGF)-beta signaling. Overexpression of Cited2 enhanced TGF-beta-mediated transcription of a Smad-Binding Element-containing luciferase reporter construct, SBE4-Luc. This may occur through a direct physical association of Cited2 with Smads 2 and 3, as supported by co-immunoprecipitation, mammalian two-hybrid and glutathione S-transferase-pull down assays. The transcription factor p300, which binds to Smad3, was shown to further enhance the interaction between Cited2 and Smad3, and the transcriptional responses of Smad3 by Cited2 in reporter assays. Cited2 enhances TGF-beta-mediated upregulation of matrix metalloproteinase 9 (MMP9) in Cited2 inducible mouse embryo fibroblasts. Overexpression of Cited2 enhanced TGF-beta-mediated MMP9 promoter reporter activity. Moreover, knockdown of Cited2 in MDA-MB-231 cells attenuated TGF-beta-mediated upregulation of MMP9 and TGF-beta-mediated cell invasion. Chromatin immunoprecipitation showed that Cited2 and Smad3 were recruited to MMP9 promoter upon TGF-beta stimulation. This is the first demonstration that Cited2 functions as a Smad3/p300-interacting transcriptional co-activator in modulating the expression of MMP9, which could affect tumor cell invasion mediated by TGF-beta.
Earlier, we have reported that 70 kDa subunit of Ku protein heterodimer (Ku70) binds and inhibits Bax activity in the cytosol and that ubiquitin (Ub)-dependent proteolysis of cytosolic Ku70 ...facilitates Bax-mediated apoptosis. We found that Hdm2 (human homolog of murine double minute) has an ability to ubiquitinate Ku70 and that Hdm2 overexpression in cultured cells causes a decrease in Ku70 expression levels. An interaction between Ku70 and Hdm2 was shown by means of immunoprecipitation, whereas none could be shown between 80 kDa subunit of Ku protein heterodimer and Hdm2. Vascular endothelial growth factor (VEGF) is known to inhibit endothelial cell (EC) apoptosis through an Akt-mediated survival kinase signal; however, the mechanism underlying this inhibition of apoptosis has not been fully elucidated. We found that VEGF inhibited cytosolic Ku70 degradation induced by apoptotic stress. It is known that Akt-dependent phosphorylation of Hdm2 causes nuclear translocation of Hdm2 followed by Hdm2-mediated inactivation of p53. We found that VEGF stimulated nuclear translocation of Hdm2 in EC and efficiently inhibited Ku70 degradation. We also found that constitutively active Akt, but not kinase-dead Akt, inhibited Ku70 degradation in the cytosol. Furthermore, Ku70 knockdown diminished antiapoptotic activity of Akt. Taken together, we propose that Hdm2 is a Ku70 Ub ligase and that Akt inhibits Bax-mediated apoptosis, at least in part, by maintaining Ku70 levels through the promotion of Hdm2 nuclear translocation.
The prostate is a highly androgen-dependent tissue that in humans exhibits marked susceptibility to carcinogenesis. The malignant epithelium generated from this tissue ultimately loses dependence on ...androgens despite retention or amplification of the androgen receptor. Accumulating evidence support that transforming growth factor-β (TGF-β) plays key roles in the control of androgen dependence and acquisition of resistance to such hormonal control. Although TGF-β functions as a key tumour suppressor of the prostate, it can also promote malignant progression and metastasis of the advanced disease, through undefined mechanisms. In addition to giving an overview of the TGF-β field as related to its function in prostate cancer, this Review focuses on novel findings that support the tumour suppressor function of TGF-β is lost or altered by changes in the activity of the androgen receptor, insulin-like growth factor-I, Akt, and mTOR during malignant progression. Understanding the mechanisms of cross-talk between TGF-β and such growth modulators has important implications for the rational therapeutics of prostate cancer.
Transforming growth factor-beta (TGF-beta) elicits a potent growth inhibitory effect on many normal cells by binding to specific serine/threonine kinase receptors and activating specific Smad ...proteins, which regulate the expression of cell cycle genes, including the p21 cyclin-dependent kinase (CDK) inhibitor gene. Interestingly, cancer cells are often insensitive to the anti-mitogenic effects of TGF-beta for which the molecular mechanisms are not well understood. In this study, we found that the candidate prostate cancer susceptibility gene ELAC2 potentiates TGF-beta/Smad-induced transcriptional responses. ELAC2 associates with activated Smad2; the C-terminal MH2 domain of Smad2 interacts with the N-terminal region of ELAC2. Small interfering siRNA-mediated knock-down of ELAC2 in prostate cells suppressed TGF-beta-induced growth arrest. Moreover, ELAC2 was shown to specifically associate with the nuclear Smad2 partner, FAST-1 and to potentiate the interaction of activated Smad2 with transcription factor Sp1. Furthermore, activation of the p21 CDK inhibitor promoter by TGF-beta is potentiated by ELAC2. Taken together our data indicate an important transcriptional scaffold function for ELAC2 in TGF-beta/Smad signaling mediated growth arrest.
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