Characterization of Platelet-Derived Growth Factor-Induced p38 Mitogen-Activated Protein Kinase Activation and its Biological Effects in the Diabetic Vascular Wall Cells

本实验旨在研究血小板衍生生长因子(PDGF)诱导糖尿病血管平滑肌细胞(VSMCs)p38丝裂原活化蛋白激酶(P38)活化的特性及其生物学效应。PDGF-BB呈剂量依赖性地激活p38的磷酸化，且在糖尿病细胞中的水平更为剧烈。这些磷酸化可被p38的特异性抑制剂SB-203580特异性抑制，但不能被PD-98059抑制。但PDGF-BB不影响p38的蛋白水平。血小板衍生生长因子BB还可激活蛋白激酶C-δ的易位和MKK3/MKK6的磷酸化，但对这两种应激激活的蛋白激酶均无激活作用。在上游水平，PDGF诱导的p38激活受小G蛋白成员之一Rho A的调控。SB-203580可剂量依赖性地抑制PDGFBB刺激的DNA合成量和迁移量。尽管用TUNEL法检测细胞凋亡，但无论有没有SB-203580，PDGFBB刺激的VSMCs都没有显示出凋亡的变化。此外，PDGF-BB刺激可使糖尿病大鼠VSMC花生四烯酸释放和COX-2水平升高，其中尤以糖尿病大鼠VSMC中花生四烯酸释放和COX-2水平升高更为明显。这些值也被SB-203580降低了。这些结果证实了PDGF-BB激活p38并随后调节VSMCs的细胞生长，提供了p38丝裂原蛋白激酶导致包括动脉粥样硬化在内的心血管疾病发展的分子机制。此外，这种激活在糖尿病患者中增强得更多。

The aim of this experiment was to examine the characterization of platelet-derived growth factor (PDGF)-induced p38 mitogen-activated protein kinase (p38) activation and its biological effects in the diabetic vascular smooth muscle cells (VSMCs). PDGF-BB activated p38 phosphorylation dose-dependently, and the level was more drastic in diabetic cells. These phosphorylations were specifically inhibited by SB-203580, a specific inhibitor of p38, but not by PD-98059. However, PDGF-BB did not affect the protein level of p38. PDGF-BB also activated the translocation of protein kinase C (PKC) - δ and the phosphorylation of MKK 3 / MKK 6 but not that of either stress-activated protein kinase. In the upstream level, PDGF-induced p38 activation was regulated the Rho A, one of the members of small G proteins. The amounts of DNA synthesis and migration stimulated by PDGF-BB were prevented by SB-203580 dose-dependently. Althou the detection of apoptotic cells was evaluated by the TUNEL method, PDGF-BB-stimulated VSMCs did not show apoptotic change in spite of the presence or absence of SB-203580. In addition, the stimulation of PDGF-BB enhanced the levels of arachidonic release and COX-2, and these levels were more increased in VSMCs from diabetic rats. These values were also decreased by SB-203580. These results established that PDGF-BB activated p38 and subsequently regulated cell growth in VSMCs, providing a molecular mechanism by which p38 MAP kinase can cause the development of cardiovascular diseases, including atherosclerosis. Furthermore, this activation was more enhanced in diabetes.