Han Ya-Ling, Xu Hong-Mei, Deng Jie, Hu Ye, Kang Jian, Liu Hai-Wei, Yan Cheng-Hui
Department of Cardiology, Shenyang General Hospital, Cardiovascular Research Institute of PLA, Shenyang, China.
Sheng Li Xue Bao. 2006 Aug 25;58(4):324-30.
To investigate the effects and molecular mechanisms of the cellular repressor of E1A-stimulated genes (CREG) on the apoptosis of vascular smooth muscle cells (VSMCs), the human internal thoracic artery-Shenyang (HITASY) cells were infected with sense-CREG [pLNCX(2)(+)/CREG] and antisense-CREG [pLXSN(-)/CREG] retrovirus respectively. The stably infected cells were obtained by screening the G418-resistant clones. DAPI nuclei staining and Annexin V/PI FASC assay indicated that over-expression of CREG in HITASY cells infected with pLNCX(2) (+)/CREG inhibited VSMC apoptosis induced by serum deprivation, accompanied with decreased expression of caspase-9 mRNA detected by RT-PCR. Furthermore, Western blot analysis showed that p38 mitogen activated protein kinase (p38 MAPK) expression and activation were significantly enhanced in HITASY cells infected with pLNCX(2) (+)/CREG. The inhibition of CREG protein expression in cells infected with pLXSN(-)/CREG promoted the VSMC spontaneous apoptosis, as well as down-regulated p38 MAPK expression and activation, when cells were cultured with 10% fetal bovine serum (FBS) mediums. These results implicate that the CREG protein has the ability to regulate VSMC apoptosis in which the activation of p38 MAPK is possibly involved. To further identify the role of p38 MAPK in VSMC apoptosis, SB203580, a specific inhibitor of p38 MAPK, was used to inhibit p38 MAPK activity. When p38 MAPK signaling pathway was blocked, the effects that over-expression of CREG protein inhibited VSMC apoptosis disappeared. Taken together, the present work indicates that over-expression of CREG protein inhibits VSMC apoptosis, and this inhibitory effect is partly mediated by p38 MAPK signaling pathway.
为了研究E1A刺激基因的细胞抑制因子（CREG）对血管平滑肌细胞（VSMC）凋亡的影响及其分子机制，分别用正义CREG[ pLNCX(2)(+)/CREG]和反义CREG[ pLXSN(-)/CREG]逆转录病毒感染人胸廓内动脉-沈阳（HITASY）细胞。通过筛选G418抗性克隆获得稳定感染的细胞。DAPI细胞核染色和Annexin V/PI FASC分析表明，感染pLNCX(2)(+)/CREG的HITASY细胞中CREG的过表达抑制了血清剥夺诱导的VSMC凋亡，同时RT-PCR检测到caspase-9 mRNA表达降低。此外，蛋白质印迹分析表明，感染pLNCX(2)(+)/CREG的HITASY细胞中p38丝裂原活化蛋白激酶（p38 MAPK）的表达和活化显著增强。当用10%胎牛血清（FBS）培养基培养时，感染pLXSN(-)/CREG的细胞中CREG蛋白表达的抑制促进了VSMC的自发凋亡，同时下调了p38 MAPK的表达和活化。这些结果表明，CREG蛋白具有调节VSMC凋亡的能力，其中p38 MAPK的活化可能参与其中。为了进一步确定p38 MAPK在VSMC凋亡中的作用，使用p38 MAPK的特异性抑制剂SB203580抑制p38 MAPK活性。当p38 MAPK信号通路被阻断时，CREG蛋白过表达抑制VSMC凋亡的作用消失。综上所述，目前的研究表明，CREG蛋白的过表达抑制VSMC凋亡，且这种抑制作用部分由p38 MAPK信号通路介导。
