Department of Molecular Biology, Division of Cancer Cell Biology, Showa University School of Pharmacy, Tokyo 142-8555, Japan.
J Biol Chem. 2012 Nov 9;287(46):38854-65. doi: 10.1074/jbc.M112.377721. Epub 2012 Sep 24.
Anchorage loss elicits a set of responses in cells, such as transcriptional changes, in order to prevent inappropriate cell growth in ectopic environments. However, the mechanisms underlying these responses are poorly understood. In this study, we investigated the transcriptional up-regulation of cyclin-dependent kinase inhibitor p21(Cip1) during anchorage loss, which is important for cell cycle arrest of nonadherent cells in the G1 phase. Up-regulation was mediated by an upstream element, designated as the detachment-responsive element (DRE), that contained Kruppel-like factor 4 (KLF4) and runt-related transcription factor 1 (RUNX1) recognition sites; both of these together were necessary for transactivation, as individually they were insufficient. RNAi experiments revealed that KLF4 and a multidomain adaptor protein, hydrogen peroxide-inducible clone 5 (HIC-5), were critically involved in DRE transactivation. The role of HIC-5 in this mechanism was to tether KLF4 to DNA sites in response to cellular detachment. In addition, further analysis suggested that oligomerization and subsequent nuclear matrix localization of HIC-5, which was accelerated spontaneously in cells during anchorage loss, was assumed to potentiate the scaffolding function of HIC-5 in the nucleus and consequently regulate p21(Cip1) transcription in a manner responding to anchorage loss. At the RUNX1 site, a LIM-only protein, CRP2, imposed negative regulation on transcription, which appeared to be removed by anchorage loss and contributed to increased transcriptional activity of DRE together with regulation at the KLF4 sites. In conclusion, this study revealed a novel transcriptional mechanism that regulated gene expression in a detachment-dependent manner, thereby contributing to anchorage-dependent cell growth.
锚定丢失会引发细胞一系列反应，如转录变化，以防止异位环境中细胞的不当生长。然而，这些反应的机制还知之甚少。在这项研究中，我们研究了锚定丢失过程中细胞周期蛋白依赖性激酶抑制剂 p21(Cip1)的转录上调，这对于非贴壁细胞在 G1 期的细胞周期阻滞很重要。上调是由一个上游元件介导的，称为脱离反应元件(DRE)，它包含 Kruppel 样因子 4(KLF4)和 runt 相关转录因子 1(RUNX1)识别位点;这两者一起是转录激活所必需的，因为单独它们是不够的。RNAi 实验表明，KLF4 和一种多结构域衔接蛋白，过氧化氢诱导克隆 5(HIC-5)，在 DRE 转录激活中起着关键作用。HIC-5 在这种机制中的作用是在细胞脱离时将 KLF4 连接到 DNA 位点。此外，进一步的分析表明，HIC-5 的寡聚化和随后的核基质定位，在锚定丢失过程中细胞会自发加速，这被认为增强了 HIC-5 在核中的支架功能，并因此以响应锚定丢失的方式调节 p21(Cip1)转录。在 RUNX1 位点，LIM 仅蛋白 CRP2 对转录施加负调节，这种调节似乎通过锚定丢失而被去除，并与 KLF4 位点的调节一起增加 DRE 的转录活性。总之，这项研究揭示了一种新的转录机制，以依赖于脱离的方式调节基因表达，从而促进依赖于锚定的细胞生长。
