Fosbrink Matthew, Cudrici Cornelia, Tegla Cosmin A, Soloviova Kateryna, Ito Takahiro, Vlaicu Sonia, Rus Violeta, Niculescu Florin, Rus Horea
Department of Neurology, University of Maryland, School of Medicine, Baltimore, MD 21201, USA.
Exp Mol Pathol. 2009 Apr;86(2):87-94. doi: 10.1016/j.yexmp.2008.12.005. Epub 2009 Jan 7.
Proliferation of vascular endothelial cells (EC) and smooth muscle cells (SMC) is a critical event in angiogenesis and atherosclerosis. We previously showed that the C5b-9 assembly during complement activation induces cell cycle in human aortic EC (AEC) and SMC. C5b-9 can induce the expression of Response Gene to Complement (RGC)-32 and over expression of this gene leads to cell cycle activation. Therefore, the present study was carried out to test the requirement of endogenous RGC-32 for the cell cycle activation induced by C5b-9 by knocking-down its expression using siRNA. We identified two RGC-32 siRNAs that can markedly reduce the expression of RGC-32 mRNA in AEC. RGC-32 silencing in these cells abolished DNA synthesis induced by C5b-9 and serum growth factors, indicating the requirement of RGC-32 activity for S-phase entry. RGC-32 siRNA knockdown also significantly reduced the C5b-9 induced CDC2 activation and Akt phosphorylation. CDC2 does not play a role in G1/S transition in HeLa cells stably overexpressing RGC-32. RGC-32 was found to physically associate with Akt and was phosphorylated by Akt in vitro. Mutation of RGC-32 protein at Ser 45 and Ser 47 prevented Akt mediated phosphorylation. In addition, RGC-32 was found to regulate the release of growth factors from AEC. All these data together suggest that cell cycle induction by C5b-9 in AEC is RGC-32 dependent and this is in part through regulation of Akt and growth factor release.
血管内皮细胞（EC）和平滑肌细胞（SMC）的增殖是血管生成和动脉粥样硬化中的关键事件。我们之前表明，补体激活过程中形成的C5b-9复合物可诱导人主动脉内皮细胞（AEC）和平滑肌细胞的细胞周期。C5b-9可诱导补体反应基因（RGC）-32的表达，该基因的过表达会导致细胞周期激活。因此，本研究通过使用小干扰RNA（siRNA）敲低其表达来测试内源性RGC-32对C5b-9诱导的细胞周期激活的必要性。我们鉴定出两种RGC-32 siRNA，它们可显著降低AEC中RGC-32 mRNA的表达。这些细胞中RGC-32的沉默消除了C5b-9和血清生长因子诱导的DNA合成，表明进入S期需要RGC-32的活性。RGC-32 siRNA敲低还显著降低了C5b-9诱导的细胞周期蛋白依赖性激酶2（CDC2）激活和蛋白激酶B（Akt）磷酸化。在稳定过表达RGC-32的HeLa细胞中，CDC在G1/S期转换中不起作用。发现RGC-32与Akt存在物理结合，并在体外被Akt磷酸化。RGC-32蛋白第45位丝氨酸和第47位丝氨酸的突变阻止了Akt介导的磷酸化。此外，发现RGC-32可调节AEC中生长因子的释放。所有这些数据共同表明，C5b-9在AEC中诱导的细胞周期是RGC-32依赖性的，这部分是通过调节Akt和生长因子释放来实现的。
