Analysis of the effects of Gla-containing proteins and their receptors on the proliferation of vascular smooth muscle cells.

我们从Hep G2基因文库中克隆的Sky、Ax1/UFO和Mer/Eyk等新型受体酪氨酸激酶，形成了一个基于独特胞外结构域的亚家族。最近，含有γ-羧基谷氨酸(Gla)的蛋白S和Gas6被报道为AX1/Sky受体酪氨酸激酶亚家族的生理配体。在这项研究中，我们试图阐明这种配体-受体系统介导的新的信号通路在血管平滑肌细胞增殖中的生理意义。人蛋白S和大鼠Gas6都不能诱导大鼠动脉血管平滑肌细胞的增殖，但正如Nakano等人所报道的那样，大鼠Gas6增强了凝血酶的有丝分裂活性。S或Gas6蛋白与Ax1/Sky受体亚家族的相互作用具有物种特异性，我们正在分析人蛋白S对牛和人血管平滑肌细胞增殖的影响。通过对融合到免疫球蛋白G1的Fc结构域的受体胞外区组成的人AX1、人Sky和小鼠Mer的可溶性受体的结合能力的分析，发现大鼠Gas6而不是人蛋白S与AX1、Sky和Mer结合。Gas6的C端性激素结合球蛋白样域是Gas6与Ax1结合所必需的，而N端的GLA结构域在相互作用中起着调节作用。我们用炎症细胞因子IL-1β慢性处理冠状动脉外壁，建立了冠状动脉痉挛伴内膜增厚的猪模型。为了了解AX1/Sky受体家族结合蛋白S和Gas6介导的新的信号通路是否与血管痉挛病变的形成有关，我们进一步检测了这些分子在血管平滑肌细胞增殖的病变中的表达。

Novel receptor tyrosine kinases including Sky, which we have cloned from a Hep G2 cDNA library, Axl/Ufo and Mer/Eyk form a subfamily based on a unique extracellular domain. Recently gamma-carboxyglutamic acid (Gla)-containing proteins, protein S and Gas6, have been reported to be physiological ligands for the Axl/Sky subfamily of receptor tyrosine kinases. In this study, we tried to elucidate the physiological importance of the novel signaling pathway mediated by this ligand-receptor system in the proliferation of vascular smooth muscle cells.Both human protein S and rat Gas6 failed to induce proliferation of rat arterial vascular smooth muscle cells, however, rat Gas6 potentiated the mitogenic activity of thrombin as reported by Nakano et al. Species specificity has been reported to exist in the interaction between protein S or Gas6 and Axl/Sky receptor subfamily, we are now analyzing the effect of human protein S on the proliferation of bovine and human vascular smooth muscle cells.Analysis of the binding ability of the soluble receptors of human Axl, human Sky and mouse Mer composed of the extracellular domain of receptors fused to the Fc domain of immunoglobulin G1 showed that rat Gas6, but not human protein S,bound to Axl, Sky and Mer. The C-terminal sex hormone-binding globulin-like domain of Gas6 was indispensable for the binding of Gas6 to Axl and the N-terminal Gla domain had a regulatory role on the interaction.We developed a swine model of coronary spasms with intimal thickening by chronically treating the outside of the coronary arteries with a inflammatory cytokine, IL-1beta. In order to know whether the novel signaling pathway mediated by binding protein S and Gas6 to Axl/Sky receptor family is implicated in formation of the vasospastic lesions, we proceed to determine the expression of these molecules in the lesions with proliferation of vascular smooth muscle cells.