Giles Keith M, Daly John M, Beveridge Dianne J, Thomson Andrew M, Voon Dominic C, Furneaux Henry M, Jazayeri Jalal A, Leedman Peter J
Laboratory for Cancer Medicine and University Department of Medicine, Western Australian Institute for Medical Research and Centre for Medical Research, the University of Western Australia, Perth, Western Australia 6001, Australia.
J Biol Chem. 2003 Jan 31;278(5):2937-46. doi: 10.1074/jbc.M208439200. Epub 2002 Nov 12.
Despite promoting growth in many cell types, epidermal growth factor (EGF) induces growth inhibition in a variety of cancer cells that overexpress its receptor. The cyclin-dependent kinase inhibitor p21(WAF1) is a central component of this pathway. We found in human MDA-468 breast cancer cells that EGF up-regulates p21(WAF1) mRNA and protein, through a combination of increased mRNA stability and transcription. The decay rate of a hybrid luciferase reporter full-length p21(WAF1) 3'-untranslated region (UTR) mRNA was significantly faster than that of a control mRNA. Transfections with a variety of p21(WAF1) 3'-UTR constructs identified multiple cis-acting elements capable of reducing basal reporter activity. Short wavelength ultraviolet light induced reporter activity in constructs containing the 5' region of the p21(WAF1) 3'-UTR, whereas EGF induced reporter activity in constructs containing sequences 3' of the UVC-responsive region. These cis-elements bound multiple proteins from MDA-468 cells, including HuR and poly(C)-binding protein 1 (CP1). Immunoprecipitation studies confirmed that HuR and CP1 associate with p21(WAF1) mRNA in MDA-468 cells. Over- and underexpression of HuR in MDA-468 cells did not affect EGF-induced p21(WAF1) protein expression or growth inhibition. However, binding of HuR to its target 3'-UTR cis-element was regulated by UVC but not by EGF, suggesting that these stimuli modulate the stability of p21(WAF1) mRNA via different mechanisms. We conclude that EGF-induced p21(WAF1) protein expression is mediated largely by stabilization of p21(WAF1) mRNA elicited via multiple 3'-UTR cis-elements. Although HuR binds at least one of these elements, it does not appear to be a major modulator of p21(WAF1) expression or growth inhibition in this system. CP1 is a novel p21(WAF1) mRNA-binding protein that may function cooperatively with other mRNA-binding proteins to regulate p21(WAF1) mRNA stability.
尽管表皮生长因子（EGF）能促进多种细胞类型的生长，但它却能诱导多种过表达其受体的癌细胞生长受抑制。细胞周期蛋白依赖性激酶抑制剂p21（WAF1）是该信号通路的核心组成部分。我们在人MDA - 468乳腺癌细胞中发现，EGF通过增加mRNA稳定性和转录的共同作用，上调p21（WAF1）的mRNA和蛋白水平。杂交荧光素酶报告基因全长p21（WAF1）3' - 非翻译区（UTR）mRNA的降解速率明显快于对照mRNA。用多种p21（WAF1）3' - UTR构建体转染后，鉴定出多个能够降低基础报告基因活性的顺式作用元件。短波长紫外线可诱导含有p21（WAF1）3' - UTR 5'区域的构建体中的报告基因活性，而EGF可诱导含有UVC反应区域3'序列的构建体中的报告基因活性。这些顺式元件结合了来自MDA - 468细胞的多种蛋白质，包括HuR和多聚（C）结合蛋白1（CP1）。免疫沉淀研究证实，HuR和CP1在MDA - 468细胞中与p21（WAF1）mRNA相关联。在MDA - 468细胞中过表达和低表达HuR均不影响EGF诱导的p21（WAF1）蛋白表达或生长抑制。然而，HuR与其靶标3' - UTR顺式元件的结合受UVC调节，而不受EGF调节，这表明这些刺激通过不同机制调节p21（WAF1）mRNA的稳定性。我们得出结论，EGF诱导的p21（WAF1）蛋白表达主要是由多个3' - UTR顺式元件引发的p21（WAF1）mRNA稳定性介导的。尽管HuR结合了这些元件中的至少一个，但在该系统中它似乎不是p21（WAF1）表达或生长抑制的主要调节因子。CP1是一种新型的p21（WAF1）mRNA结合蛋白，可能与其他mRNA结合蛋白协同作用来调节p21（WAF1）mRNA的稳定性。
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