Cui Wei, De Jesus Kristine, Zhao Hong, Takasawa Shin, Shi Bingyin, Srikant Coimbatore B, Liu Jun-Li
School of Medicine, The First Affiliated Hospital, Xi'an Jiao Tong University, Xi'an, Shaanxi, PR China.
Growth Factors. 2009 Jun;27(3):195-202. doi: 10.1080/08977190902863548.
Regenerating gene (Reg) family protein Reg3alpha is normally expressed in pancreatic acinar and endocrine cells. In order to explore its effect on islet beta-cell replication, insulinoma MIN6 cells were stably transfected with murine Reg3alpha cDNA. Determined using real-time PCR and Western blots, the levels of Reg3alpha mRNA and protein in Reg3alpha-transfected clones were increased 10- and 6-fold, respectively. Western blots also revealed that the protein was released into the culture medium, consistent with an endocrine effect. In MTT cell proliferation assay, Reg3alpha-overexpressing cells exhibited a 2-fold increase in the rate of cell growth. In order to investigate the intracellular mechanism, we studied cell cycle regulatory proteins. In Reg3alpha-expressing cells, we detected 2.2- and 2.5-fold increased levels of cyclin D1 and CDK4, respectively, which paralleled a 1.8-fold increase in the rate of Akt phosphorylation. It is established that beta-cell replication is associated with increased cyclin D1 and CDK4 levels; deficiency in CDK4 or cyclin D2 results in reduced beta-cell mass and diabetes. Our results suggest that Reg3alpha stimulates beta-cell replication, by activating Akt kinase and increasing the levels of cyclin D1/CDK4.
再生基因（Reg）家族蛋白Reg3α通常在胰腺腺泡细胞和内分泌细胞中表达。为了探究其对胰岛β细胞复制的影响，用小鼠Reg3α cDNA稳定转染胰岛素瘤MIN6细胞。通过实时PCR和蛋白质印迹法测定，Reg3α转染克隆中Reg3α mRNA和蛋白水平分别增加了10倍和6倍。蛋白质印迹法还显示该蛋白被释放到培养基中，这与内分泌作用一致。在MTT细胞增殖试验中，过表达Reg3α的细胞生长速率增加了2倍。为了研究细胞内机制，我们研究了细胞周期调节蛋白。在表达Reg3α的细胞中，我们分别检测到细胞周期蛋白D1和细胞周期蛋白依赖性激酶4（CDK4）水平增加了2.2倍和2.5倍，这与Akt磷酸化速率增加1.8倍相平行。已知β细胞复制与细胞周期蛋白D1和CDK4水平增加有关；CDK4或细胞周期蛋白D2缺乏会导致β细胞数量减少和糖尿病。我们的结果表明，Reg3α通过激活Akt激酶并增加细胞周期蛋白D1/CDK4水平来刺激β细胞复制。
