Sheng George, Bernabe Kathryn Q, Guo Jun, Warner Brad W
Department of Surgery, University of Cincinnati College of Medicine, Ohio, USA.
Gastroenterology. 2006 Jul;131(1):153-64. doi: 10.1053/j.gastro.2006.05.007.
BACKGROUND & AIMS: Epidermal growth factor receptor (EGFR)-mediated increase in enterocyte proliferation following massive resection is a major mechanism by which the small intestine adapts to the loss of its mucosal surface area. In addition, expression of the cyclin-dependent kinase inhibitor p21(waf1/cip1) is required for resection-induced enterocyte proliferation. This study sought to establish a mechanistic link between EGFR-mediated intestinal epithelial cell proliferation and p21(waf1/cip1) expression.
EGF was used to stimulate IEC-6 and HCA-7 cells. P21(waf1/cip1) messenger RNA (mRNA) and protein expression were measured by real-time polymerase chain reaction and Western blot, respectively. P21(waf1/cip1) promoter studies were performed using p21(waf1/cip1) promoter-driven luciferase assay. Pharmacologic inhibitors of PI3-kinase and mitogen activated protein kinase (MAPK) were used to block these pathways downstream of the activated EGFR. Constitutively active Ras, Raf, or MEK-1 constructs were transfected into cells for overexpression studies. Cell proliferation was measured by bromodeoxyuridine incorporation following p21(waf1/cip1) silencing with RNAi. Finally, Cyclin D(1)/Cdk interaction was evaluated by immunoprecipitation.
EGFR activation in intestinal epithelial cells induced the expression of p21(waf1/cip1) mRNA and protein This event was transcriptionally regulated via a 50-bp segment of the p21(waf1/cip1) promoter as a result of MAPK activation. Exogenous EGF failed to induce proliferation in p21(waf1/cip1)-silenced cells and adaptive proliferation after intestinal resection in p21(waf1/cip1)-null mice. Functionally, p21(waf1/cip1) up-regulation was required for stabilizing Cyclin D/Cdk 4 complexes and intestinal cell proliferation.
EGFR-mediated induction of enterocyte proliferation requires MAPK-dependent increase in p21(waf1/cip1) expression in intestinal epithelial cells. These studies elucidate an important mechanism for resection-induced enterocyte proliferation during intestinal adaptation.
表皮生长因子受体（EGFR）介导的大量肠切除术后肠上皮细胞增殖增加是小肠适应其黏膜表面积减少的主要机制。此外，细胞周期蛋白依赖性激酶抑制剂p21（waf1/cip1）的表达是切除诱导的肠上皮细胞增殖所必需的。本研究旨在建立EGFR介导的肠上皮细胞增殖与p21（waf1/cip1）表达之间的机制联系。
使用表皮生长因子（EGF）刺激IEC-6和HCA-7细胞。分别通过实时聚合酶链反应和蛋白质印迹法检测p21（waf1/cip1）信使核糖核酸（mRNA）和蛋白质表达。使用p21（waf1/cip1）启动子驱动的荧光素酶测定法进行p21（waf1/cip1）启动子研究。使用磷脂酰肌醇-3激酶（PI3-激酶）和丝裂原活化蛋白激酶（MAPK）的药理抑制剂来阻断活化的EGFR下游的这些信号通路。将组成型活性Ras、Raf或MEK-1构建体转染到细胞中进行过表达研究。在用RNA干扰使p21（waf1/cip1）沉默后，通过溴脱氧尿苷掺入法测量细胞增殖。最后，通过免疫沉淀评估细胞周期蛋白D（1）/细胞周期蛋白依赖性激酶（Cdk）相互作用。
肠上皮细胞中的EGFR激活诱导了p21（waf1/cip1）mRNA和蛋白质的表达。由于MAPK激活，该事件通过p21（waf1/cip1）启动子的一段50个碱基对的片段进行转录调控。外源性EGF未能在p21（waf1/cip1）沉默的细胞中诱导增殖，也未能在p21（waf1/cip1）基因敲除小鼠的肠切除术后诱导适应性增殖。在功能上，p21（waf1/cip1）的上调是稳定细胞周期蛋白D/Cdk 4复合物和肠细胞增殖所必需的。
EGFR介导的肠上皮细胞增殖诱导需要MAPK依赖性地增加肠上皮细胞中p21（waf1/cip1）的表达。这些研究阐明了肠适应过程中切除诱导的肠上皮细胞增殖的重要机制。
