Enhancement of p53 expression in keratinocytes by the bioflavonoid apigenin is associated with RNA-binding protein HuR.
ABSTRACT: We have reported previously that apigenin, a naturally occurring nonmutagenic flavonoid, increased wild-type p53 protein expression in the mouse keratinocyte 308 cell line by a mechanism involving p53 protein stabilization. Here we further demonstrated that the increase in p53 protein level induced by apigenin treatment of 308 keratinoyctes was not the result of enhanced transcription, mRNA stabilization or cytoplasmic export of p53 mRNA. Instead, biosynthetic labeling showed that apigenin increased nascent p53 protein synthesis by enhancing p53 translation. The AU-rich element (ARE) within the 3'-untranslated region (UTR) of p53 mRNA was found to be responsible for apigenin's ability to increase p53 translation, as demonstrated in studies wherein the 3'-UTR of p53 mRNA containing the ARE was fused downstream of a luciferase reporter gene. Furthermore, apigenin treatment increased the level of association of the RNA binding protein HuR with endogenous p53 mRNA. Apigenin treatment also augmented HuR translocation into the cytoplasm. Overexpression of HuR enhanced apigenin-induced p53 protein expression in 308 keratinocytes, whereas siRNA-mediated HuR reduction suppressed apigenin-induced p53 protein expression and de novo translation of p53. Moreover, apigenin treatment of cells induced p16 protein expression, which in turn was correlated with cytoplasmic localization of HuR induced by apigenin. Overall, these findings indicate that, in addition to modulating p53 protein stability, one of the mechanisms by which apigenin induces p53 protein expression is enhancement of translation through the RNA binding protein HuR.
Project description:Plant flavonoid apigenin prevents and inhibits UVB-induced carcinogenesis in the skin and has strong anti-proliferative and anti-angiogenic properties. Here we identify mechanisms, by which apigenin controls these oncogenic events. We show that apigenin acts, at least in part, via endogenous angiogenesis inhibitor, thrombospondin-1 (TSP1). TSP1 expression by the epidermal keratinocytes is potently inhibited by UVB. It inhibits cutaneous angiogenesis and UVB-induced carcinogenesis. We show that apigenin restores TSP1 in epidermal keratinocytes subjected to UVB and normalizes proliferation and angiogenesis in UVB-exposed skin. Importantly, reconstituting TSP1 anti-angiogenic function in UVB-irradiated skin with a short bioactive peptide mimetic representing exclusively its anti-angiogenic domain reproduced the anti-proliferative and anti-angiogenic effects of apigenin. Cox-2 and HIF-1? are important mediators of angiogenesis. Both apigenin and TSP1 peptide mimetic attenuated their induction by UVB. Finally we identified the molecular mechanism, whereby apigenin did not affect TSP1 mRNA, but increased de novo protein synthesis. Knockdown studies implicated the RNA-binding protein HuR, which controls mRNA stability and translation. Apigenin increased HuR cytoplasmic localization and physical association with TSP1 mRNA causing de novo TSP1 synthesis. HuR cytoplasmic localization was, in turn, dependent on CHK2 kinase. Together, our data provide a new mechanism, by which apigenin controls UVB-induced carcinogenesis.
Project description:Tumor suppressor protein p53 plays a crucial role in maintaining genomic integrity in response to DNA damage. Regulation of translation of p53 mRNA is a major mode of regulation of p53 expression under genotoxic stress. The AU/U-rich element-binding protein HuR has been shown to bind to p53 mRNA 3'UTR and enhance translation in response to DNA-damaging UVC radiation. On the other hand, the microRNA miR-125b is reported to repress p53 expression and stress-induced apoptosis. Here, we show that UVC radiation causes an increase in miR-125b level in a biphasic manner, as well as nuclear cytoplasmic translocation of HuR. Binding of HuR to the p53 mRNA 3'UTR, especially at a site adjacent to the miR-125b target site, causes dissociation of the p53 mRNA from the RNA-induced silencing complex (RISC) and inhibits the miR-125b-mediated translation repression of p53. HuR prevents the oncogenic effect of miR-125b by reversing the decrease in apoptosis and increase in cell proliferation caused by the overexpression of miR-125b. The antagonistic interplay between miR-125b and HuR might play an important role in fine-tuning p53 gene expression at the post-transcriptional level, and thereby regulate the cellular response to genotoxic stress.
Project description:Expression of tumor suppressor p53 is regulated at multiple levels, disruption of which often leads to cancer. We have adopted an approach combining computational systems modeling with experimental validation to elucidate the translation regulatory network that controls p53 expression post DNA damage. The RNA-binding protein HuR activates p53 mRNA translation in response to UVC-induced DNA damage in breast carcinoma cells. p53 and HuR levels show pulsatile change post UV irradiation. The computed model fitted with the observed pulse of p53 and HuR only when hypothetical regulators of synthesis and degradation of HuR were incorporated. miR-125b, a UV-responsive microRNA, was found to represses the translation of HuR mRNA. Furthermore, UV irradiation triggered proteasomal degradation of HuR mediated by an E3-ubiquitin ligase tripartite motif-containing 21 (TRIM21). The integrated action of miR-125b and TRIM21 constitutes an intricate control system that regulates pulsatile expression of HuR and p53 and determines cell viability in response to DNA damage.
Project description:A recent analysis of gene expression in renal cell carcinoma cells led to the identification of mRNAs whose translation was dependent on the presence of the von Hippel-Lindau (VHL) tumor suppressor gene product, pVHL. Here, we investigate the finding that pVHL-expressing RCC cells (VHL(+)) exhibited elevated levels of polysome-associated p53 mRNA and increased p53 protein levels compared with VHL-defective (VHL(-)) cells. Our findings indicate that p53 translation is specifically heightened in VHL(+) cells, given that (i) p53 mRNA abundance in VHL(+) and VHL(-) cells was comparable, (ii) p53 degradation did not significantly influence p53 expression, and (iii) p53 synthesis was markedly induced in VHL(+) cells. Electrophoretic mobility shift and immunoprecipitation assays to detect endogenous and radiolabeled p53 transcripts revealed that the RNA-binding protein HuR, previously shown to regulate mRNA turnover and translation, was capable of binding to the 3' untranslated region of the p53 mRNA in a VHL-dependent fashion. Interestingly, while whole-cell levels of HuR in VHL(+) and VHL(-) cells were comparable, HuR was markedly more abundant in the cytoplasmic and polysome-associated fractions of VHL(+) cells. In keeping with earlier reports, the elevated cytoplasmic HuR in VHL(+) cells was likely due to the reduced AMP-activated kinase activity in these cells. Demonstration that HuR indeed contributed to the increased expression of p53 in VHL(+) cells was obtained through use of RNA interference, which effectively reduced HuR expression and in turn caused marked decreases in p53 translation and p53 abundance. Taken together, our findings support a role for pVHL in elevating p53 expression, implicate HuR in enhancing VHL-mediated p53 translation, and suggest that VHL-mediated p53 upregulation may contribute to pVHL's tumor suppressive functions in renal cell carcinoma.
Project description:Exposure to short-wavelength UV light (UVC) strongly induces p53 expression. In human RKO colorectal carcinoma cells, this increase was not due to elevated p53 mRNA abundance, cytoplasmic export of p53 mRNA, or UVC-triggered stabilization of the p53 protein. Instead, p53 translation was potently enhanced after UVC irradiation. The 3' UTR of p53 was found to be a target of the RNA-binding protein HuR in a UVC-dependent manner in vitro and in vivo. HuR-overexpressing RKO cells displayed elevated p53 levels, whereas cells expressing reduced HuR showed markedly diminished p53 abundance and p53 translation. Our results demonstrate a role for HuR in binding to the p53 mRNA and enhancing its translation.
Project description:HuR, a RNA binding protein, is known to function as a tumor maintenance gene in breast cancer and associated with tumor growth and poor prognosis. However, the cellular function of this protein remains largely unknown in normal mammary epithelial cells. Here, we showed that in immortalized MCF10A mammary epithelial cells, HuR knockdown inhibits cell proliferation and enhances premature senescence. We also showed that in three-dimensional culture, MCF10A cells with HuR knockdown form abnormal acini with filled lumen and an aberrant expression pattern of the extracellular matrix protein laminin V. In addition, we showed that HuR knockdown increases ?Np63, but decreases wild-type p53, expression in MCF10A cells. Moreover, we showed that ?Np63 knockdown partially rescues the proliferative defect induced by HuR knockdown in MCF10A cells. Consistent with this, we identified two U-rich elements in the 3'-untranslated region of p63 mRNA, to which HuR specifically binds. Finally, we showed that HuR knockdown enhances ?Np63 mRNA translation but has no effect on p63 mRNA turnover. Together, our data suggest that HuR maintains cell proliferation and polarity of mammary epithelial cells at least in part via ?Np63.
Project description:The RNA-binding protein HuR (also known as ELAV1) binds to the 3'-untranslated region of mRNAs and regulates transcript stability and translation. However, the in vivo functions of HuR are not well understood. Here, we report that murine HuR is essential for life; postnatal global deletion of Elavl1 induced atrophy of hematopoietic organs, extensive loss of intestinal villi, obstructive enterocolitis, and lethality within 10 days. Upon Elavl1 deletion, progenitor cells in the BM, thymus, and intestine underwent apoptosis, whereas quiescent stem cells and differentiated cells were unaffected. The survival defect of hematopoietic progenitor cells was cell intrinsic, as transplant of Elavl1-/- BM led to compromised hematopoietic reconstitution but did not cause lethality. Expression of p53 and its downstream effectors critical for cell death were induced in progenitor cells as HuR levels declined. In mouse embryonic fibroblasts, HuR bound to and stabilized the mRNA for Mdm2, a critical negative regulator of p53. Furthermore, cell survival was restored by expression of Mdm2 in Elavl1-/- cells, suggesting that HuR keeps p53 levels in check in progenitor cells and thereby promotes cell survival. This regulation of cell stress response by HuR in progenitor cells, which we believe to be novel, could potentially be exploited in cytotoxic anticancer therapies as well as stem cell transplant therapy.
Project description:Noncoding RNAs (ncRNAs) and RNA-binding proteins are potent post-transcriptional regulators of gene expression. The ncRNA 7SL is upregulated in cancer cells, but its impact upon the phenotype of cancer cells is unknown. Here, we present evidence that 7SL forms a partial hybrid with the 3'-untranslated region (UTR) of TP53 mRNA, which encodes the tumor suppressor p53. The interaction of 7SL with TP53 mRNA reduced p53 translation, as determined by analyzing p53 expression levels, nascent p53 translation and TP53 mRNA association with polysomes. Silencing 7SL led to increased binding of HuR to TP53 mRNA, an interaction that led to the promotion of p53 translation and increased p53 abundance. We propose that the competition between 7SL and HuR for binding to TP53 3'UTR contributes to determining the magnitude of p53 translation, in turn affecting p53 levels and the growth-suppressive function of p53. Our findings suggest that targeting 7SL may be effective in the treatment of cancers with reduced p53 levels.
Project description:IL-17, a major inflammatory cytokine plays a critical role in the pathogenesis of many autoimmune inflammatory diseases. In this study, we report a new function of RNA-binding protein HuR in IL-17-induced Act1-mediated chemokine mRNA stabilization. HuR deficiency markedly reduced IL-17-induced chemokine expression due to increased mRNA decay. Act1-mediated HuR polyubiquitination was required for the binding of HuR to CXCL1 mRNA, leading to mRNA stabilization. Although IL-17 induced the coshift of Act1 and HuR to the polysomal fractions in a sucrose gradient, HuR deficiency reduced the ratio of translation-active/translation-inactive IL-17-induced chemokine mRNAs. Furthermore, HuR deletion in distal lung epithelium attenuated IL-17-induced neutrophilia. In summary, HuR functions to couple receptor-proximal signaling to posttranscriptional machinery, contributing to IL-17-induced inflammation.
Project description:Occludin is a transmembrane tight junction (TJ) protein that plays an important role in TJ assembly and regulation of the epithelial barrier function, but the mechanisms underlying its post-transcriptional regulation are unknown. The RNA-binding protein HuR modulates the stability and translation of many target mRNAs. Here, we investigated the role of HuR in the regulation of occludin expression and therefore in the intestinal epithelial barrier function. HuR bound the 3'-untranslated region of the occludin mRNA and enhanced occludin translation. HuR association with the occludin mRNA depended on Chk2-dependent HuR phosphorylation. Reduced HuR phosphorylation by Chk2 silencing or by reduction of Chk2 through polyamine depletion decreased HuR-binding to the occludin mRNA and repressed occludin translation, whereas Chk2 overexpression enhanced (HuR/occludin mRNA) association and stimulated occludin expression. In mice exposed to septic stress induced by cecal ligation and puncture, Chk2 levels in the intestinal mucosa decreased, associated with an inhibition of occludin expression and gut barrier dysfunction. These results indicate that HuR regulates occludin mRNA translation through Chk2-dependent HuR phosphorylation and that this influence is crucial for maintenance of the epithelial barrier integrity in the intestinal tract.