Department of Surgery, University of Maryland School of Medicine, Baltimore, MD, USA.
Am J Physiol Cell Physiol. 2010 May;298(5):C1226-34. doi: 10.1152/ajpcell.00021.2010. Epub 2010 Feb 24.
Intestinal epithelium is a rapidly self-renewing tissue in the body, and its homeostasis is tightly regulated by numerous factors including polyamines. Decreased levels of cellular polyamines increase activating transcription factor (ATF)-2, but the exact role and mechanism of induced ATF-2 in the regulation of intestinal epithelial cell (IEC) growth remain elusive. Cyclin-dependent kinase (CDK) 4 is necessary for the G1-to-S phase transition during the cell cycle, and its expression is predominantly controlled at the transcription level. Here, we reported that induced ATF-2 following polyamine depletion repressed CDK4 gene transcription in IECs by increasing formation of the ATF-2/JunD heterodimers. ATF-2 formed complexes with JunD as measured by immunoprecipitation using the ATF-2 and JunD antibodies and by glutathione S-transferase (GST) pull-down assays using GST-ATF-2 fusion proteins. Studies using various mutants of GST-ATF-2 revealed that formation of the ATF-2/JunD dimers depended on the COOH-terminal basic region-leucine zipper domain of ATF-2. Polyamine depletion increased ATF-2/JunD complex and inhibited CDK4 transcription as indicated by a decrease in the levels of CDK4-promoter activity and its mRNA. ATF-2 silencing not only prevented inhibition of CDK4 transcription in polyamine-deficient cells but also abolished repression of CDK4 expression induced by ectopic JunD overexpression. ATF-2 silencing also promoted IEC growth in polyamine-depleted cells. These results indicate that induced ATF-2/JunD association following polyamine depletion represses CDK4 transcription, thus contributing to the inhibition of IEC growth.
肠道上皮细胞是体内快速自我更新的组织，其稳态受到包括多胺在内的多种因素的严格调控。细胞多胺水平降低会增加激活转录因子（ATF）-2，但诱导的 ATF-2 在调节肠道上皮细胞（IEC）生长中的确切作用和机制仍不清楚。细胞周期蛋白依赖性激酶（CDK）4 是细胞周期中从 G1 期向 S 期过渡所必需的，其表达主要受转录水平控制。在这里，我们报道了多胺耗竭后诱导的 ATF-2 通过增加 ATF-2/JunD 异二聚体的形成来抑制 IEC 中 CDK4 基因的转录。ATF-2 与 JunD 形成复合物，如通过使用 ATF-2 和 JunD 抗体进行免疫沉淀和使用 GST-ATF-2 融合蛋白进行谷胱甘肽 S-转移酶（GST）下拉测定来测量。使用各种 GST-ATF-2 突变体的研究表明，ATF-2/JunD 二聚体的形成取决于 ATF-2 的 COOH 末端碱性区域-亮氨酸拉链结构域。多胺耗竭增加了 ATF-2/JunD 复合物，并抑制了 CDK4 转录，如 CDK4 启动子活性及其 mRNA 水平降低所示。ATF-2 沉默不仅防止了在多胺缺乏的细胞中 CDK4 转录的抑制，而且还消除了由异位 JunD 过表达诱导的 CDK4 表达的抑制。ATF-2 沉默也促进了多胺耗竭细胞中 IEC 的生长。这些结果表明，多胺耗竭后诱导的 ATF-2/JunD 结合抑制 CDK4 转录，从而有助于抑制 IEC 生长。
