Grabski Allison D, Shimizu Takuya, Deou Jessie, Mahoney William M, Reidy Michael A, Daum Guenter
University of Washington, Department of Surgery, 815 Mercer Street, Seattle, WA 98109, USA.
Arterioscler Thromb Vasc Biol. 2009 Oct;29(10):1644-50. doi: 10.1161/ATVBAHA.109.191965. Epub 2009 Jul 16.
This study tests the hypothesis that S1P2R regulates expression of SMC differentiation genes after arterial injury.
Carotid ligation injury was performed in wild-type and S1P2R-null mice. At various time points after injury, expression of multiple SMC differentiation genes, myocardin, and S1P receptors (S1P1R, S1P2R, and S1P3R) was measured by quantitative PCR. These experiments demonstrate that at day 7 after injury, S1P2R specifically regulates expression of smooth muscle alpha-actin (SMA) and that this is not mediated by changes in expression of myocardin or any of the S1PRs. In vitro studies using carotid SMCs prepared from wild-type and S1P2R-null mice show that S1P stimulates expression of all SMC-differentiation genes tested, but S1P2R significantly regulates expression of SMA and SM22 alpha only. Chromatin immunoprecipitation assays suggest that S1P-induced recruitment of serum response factor to the SMA promoter and enhancer largely depends on S1P2R. S1P-stimulated SMA expression requires S1P2R-dependent activation of RhoA and mobilization of calcium from intracellular stores. Chelation of calcium does not affect the activation of RhoA by S1P, whereas blockade of Rho by C3 exotoxin partially inhibits the mobilization of calcium by S1P.
The results of this study support the hypothesis that S1P2R regulates expression of SMA after injury. We further conclude that transcriptional regulation of SMA by S1P in vitro requires S1P2R-dependent activation of RhoA and mobilization of calcium from intracellular calcium stores.
本研究旨在验证1-磷酸鞘氨醇2型受体（S1P2R）在动脉损伤后调节平滑肌细胞（SMC）分化基因表达的假说。
对野生型和S1P2R基因敲除小鼠进行颈动脉结扎损伤。在损伤后的不同时间点，通过定量聚合酶链反应（PCR）检测多个SMC分化基因、心肌素以及S1P受体（S1P1R、S1P2R和S1P3R）的表达。这些实验表明，在损伤后第7天，S1P2R特异性调节平滑肌α-肌动蛋白（SMA）的表达，且这一调节并非由心肌素或任何S1P受体的表达变化介导。使用从野生型和S1P2R基因敲除小鼠制备的颈动脉SMC进行体外研究表明，S1P刺激所有检测的SMC分化基因的表达，但S1P2R仅显著调节SMA和SM22α的表达。染色质免疫沉淀分析表明，S1P诱导血清反应因子募集到SMA启动子和增强子主要依赖于S1P2R。S1P刺激的SMA表达需要S1P2R依赖的RhoA激活以及从细胞内储存库动员钙。钙螯合不影响S1P对RhoA的激活，而C3外毒素对Rho的阻断部分抑制了S1P对钙的动员。
本研究结果支持S1P2R在损伤后调节SMA表达的假说。我们进一步得出结论，体外S1P对SMA的转录调节需要S1P2R依赖的RhoA激活以及从细胞内钙储存库动员钙。
