Influence of the RNA-binding protein HuR in pVHL-regulated p53 expression in renal carcinoma cells
Influence of the RNA-binding protein HuR in pVHL-regulated p53 expression in renal carcinoma cells
复制标题
DOI：
10.1128/mcb.23.20.7083-7095.2003
复制
发表时间：
2003-10-01
影响因子：
5.3
通讯作者：
Gorospe, M
中科院分区：
文献类型：
--
作者：
Galbán, S;Martindale, JL;Gorospe, M
最近对肾细胞癌细胞中基因表达的分析导致鉴定了其翻译依赖于von Hippel-Lindau（VHL）肿瘤抑制基因产物pVHL的存在的mRNA。在这里，我们调查的发现，pVHL表达RCC细胞（VHL+）表现出升高的多核糖体相关的p53 mRNA水平和增加p53蛋白水平相比，VHL缺陷（VHL-）细胞。我们的研究结果表明，p53翻译是特别提高VHL+细胞，鉴于（i）p53 mRNA丰度在VHL+和VHL-细胞是可比的，（ii）p53降解并没有显着影响p53的表达，（iii）p53合成显着诱导VHL+细胞。电泳迁移率变化和免疫沉淀试验检测内源性和放射性标记的p53转录本显示，RNA结合蛋白HuR，以前显示调节mRNA的周转和翻译，能够结合到3'非翻译区的p53 mRNA的VHL依赖性的方式。有趣的是，虽然VHL+和VHL-细胞中HuR的全细胞水平相当，但HuR在VHL+细胞的细胞质和多核糖体相关组分中明显更丰富。与早期报道一致，VHL+细胞中升高的胞质HuR可能是由于这些细胞中AMP激活的激酶活性降低。通过使用RNA干扰获得了HuR确实有助于VHL+细胞中p53表达增加的证明，RNA干扰有效地降低了HuR表达，进而导致p53翻译和p53丰度的显著降低。总之，我们的研究结果支持pVHL在升高p53表达中的作用，暗示HuR在增强VHL介导的p53翻译中的作用，并表明VHL介导的p53上调可能有助于pVHL在肾细胞癌中的肿瘤抑制功能。
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.