Analysis of the Beta2-chimaerin signaling in phenotypiC modulation of vascular smooth muscle cells

血管平滑肌细胞(SMC)的表型改变是动脉粥样硬化形成的重要方面。损伤血管中激活的细胞分裂生长因子网络随后诱导一系列细胞内事件，导致SMC的表型改变。在这里，我们研究了一种具有RAC GTP酶激活蛋白活性的新型佛波酯受体Beta2-嵌合体在生长因子刺激的SMC中的作用。用RT-PCR和免疫组织化学方法检测培养的人SMC内源性β2嵌合体的表达。接下来，用重组腺病毒(ADV-CH)在培养的大鼠SMC中过表达HA标记的野生型人β2嵌合体。共聚焦显微镜显示，在PKC抑制剂(GF109203X)存在的情况下，12-O-十四酰佛波醇13-乙酸酯(TPA)诱导外源性β2-嵌合体从细胞质移位到质膜。ADV-CH(10-200MOI)对10%FCS、bFGF(25 ng/ml)和PDGF(10 ng/ml)刺激的SMC增殖有抑制作用，而对对照病毒(ADV-LacZ)则无抑制作用。用纤维连接蛋白包被膜的改良Boyden小室实验表明，ADV-CH(200MOI)可抑制PDGF诱导的SMC迁移约25%。这些数据提示，β2-嵌合体可能调节受体酪氨酸激酶下游的SMC的增殖和迁移。β2-嵌合体系统可能参与人类动脉粥样硬化的形成，是潜在的治疗靶点。

Phenotypic change of vascular smooth muscle cells (SMC) is an important aspect of atherogenesis. Activated cytokinegrowth factor network in the injured vessels subsequently induces a number of intracellular events resulting in the phenotypic modulation of SMC. Here, we investigated the role of beta2-chimaerin, a novel phorbol ester receptor with Rac GTPase-activating protein activity, in growth factor-stimulated SMC. Endogenous expression of beta2-chimaerin was detected in cultured human SMC by RT-PCR and immunohistochemistiy. Next, an overexpression of HA-tagged wild type human beta2-chimaerin was attempted in cultured rat SMC with a recombinant adenovirus (Adv-Ch). Confocal microscopy revealed 12-O-tetradecanoyl phorbol 13-acetate (TPA)-induced translocation of exogenous beta2-chimaerin from cytoplasm to plasma membrane in the presence of the PKC inhibitor (GF109203X). Proliferation of SMC stimulated by 10% FCS, bFGF (25ng/ml), and PDGF (10ng/ml) were inhibited by the infection with Adv-Ch (10-200MOI), but not with control viruses (Adv-LacZ), which was measured by cell counting and BrdU incorporation assays. PDGF-induced SMC migration was inhibited by the infection with Adv-Ch (200MOI) by about 25% in a modified Boyden chamber assay with the fibronectin-coated membrane. These data suggested that beta2-chimaerin might regulate proliferation and migration of SMC at the down-stream of the receptor tyrosine kinases. Beta2-chimaerin system may be involved in human atherogenesis, as is a potential therapeutic target.