Graduate School of Third Military Medical University, Chongqing 400038, China.
Int J Mol Sci. 2013 Sep 6;14(9):18437-56. doi: 10.3390/ijms140918437.
Cellular repressor of E1A-stimulated genes (CREG) is a recently discovered secreted glycoprotein involved in homeostatic modulation. We previously reported that CREG is abundantly expressed in the adult vascular endothelium and dramatically downregulated in atherosclerotic lesions. In addition, CREG participates in the regulation of apoptosis, inflammation and wound healing of vascular endothelial cells. In the present study, we attempted to investigate the effect of CREG on the proliferation of vascular endothelial cells and to decipher the underlying molecular mechanisms. Overexpression of CREG in human umbilical vein endothelial cells (HUVEC) was obtained by infection with adenovirus carrying CREG. HUVEC proliferation was investigated by flow cytometry and 5-bromo-2'-deoxy-uridine (BrdU) incorporation assays. The expressions of cyclins, cyclin-dependent kinases and signaling molecules were also examined. In CREG-overexpressing cells, we observed a marked increase in the proportion of the S and G2 population and a decrease in the G0/G1 phase population. The number of BrdU positively-stained cells also increased, obviously. Furthermore, silencing of CREG expression by specific short hairpin RNA effectively inhibited the proliferation of human umbilical vein endothelial cells (HUVEC). CREG overexpression induced the expression of cyclin E in both protein and mRNA levels to regulate cell cycle progression. Further investigation using inhibitor blocking analysis identified that ERK activation mediated the CREG modulation of the proliferation and cyclin E expression in HUVEC. In addition, blocking vascular endothelial growth factor (VEGF) in CREG-overexpressed HUVEC and supplementation of VEGF in CREG knocked-down HUVEC identified that the pro-proliferative effect of CREG was partially mediated by VEGF-induced ERK/cyclin E activation. These results suggest a novel role of CREG to promote HUVEC proliferation through the ERK/cyclin E signaling pathway.
细胞 E1A 刺激基因的转录抑制因子（CREG）是一种新发现的分泌糖蛋白，参与体内平衡的调节。我们之前的研究表明，CREG 在成年血管内皮细胞中大量表达，并在动脉粥样硬化病变中显著下调。此外，CREG 参与血管内皮细胞凋亡、炎症和伤口愈合的调节。在本研究中，我们试图研究 CREG 对血管内皮细胞增殖的影响，并阐明其潜在的分子机制。通过感染携带 CREG 的腺病毒，使人类脐静脉内皮细胞（HUVEC）过表达 CREG。通过流式细胞术和 5-溴-2'-脱氧尿苷（BrdU）掺入实验检测 HUVEC 的增殖情况。还检测了细胞周期蛋白、细胞周期蛋白依赖性激酶和信号分子的表达。在 CREG 过表达的细胞中，我们观察到 S 和 G2 期细胞比例明显增加，G0/G1 期细胞比例减少。BrdU 阳性染色细胞数量也明显增加。此外，特异性短发夹 RNA 沉默 CREG 表达可有效抑制人脐静脉内皮细胞（HUVEC）的增殖。CREG 过表达诱导 cyclin E 在蛋白和 mRNA 水平的表达增加，从而调节细胞周期进程。使用抑制剂阻断分析的进一步研究表明，ERK 激活介导了 CREG 对 HUVEC 增殖和 cyclin E 表达的调节。此外，在 CREG 过表达的 HUVEC 中阻断血管内皮生长因子（VEGF）和在 CREG 敲低的 HUVEC 中补充 VEGF，表明 CREG 的促增殖作用部分通过 VEGF 诱导的 ERK/cyclin E 激活介导。这些结果表明 CREG 通过 ERK/cyclin E 信号通路促进 HUVEC 增殖的新作用。
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