Identification of novel intracellular signaling molecules and clarification of their signal transduction mechanism which regulate abnormal vascular contraction.

在日本，血管疾病的死亡率与癌症相当。据报道，C^<2+>-依赖性收缩（Ca^<2+>-敏化）在血管疾病中观察到的血管异常收缩中起关键作用。本研究的目的是鉴定新的细胞内信号分子，并阐明其信号转导机制。在第一年，我们发现鞘酰基磷酰胆碱（SPC）是一种诱导血管异常收缩的新介质。我们发现SPC通过依次激活Src家族酪氨酸激酶和rho激酶诱导Ca^<2+>敏化。第二年，为了寻找Src家族酪氨酸激酶的下游介质，我们重点研究了SPC诱导酪氨酸磷酸化的变化，并成功提取了一些作为Src家族酪氨酸激酶下游介质的分子。在第三年，我们获得了以下结果：1。建立了具有收缩能力的血管平滑肌细胞培养体系。sirna介导的Fyn的下调抑制了SPC诱导的培养血管平滑肌的收缩，表明Fyn参与了SPC诱导的信号转导。功能蛋白质组学分析发现p60、p160和p200是Fyn的下游介质。根据这些结果，我们确定Fyn是一种介导异常血管收缩的细胞内信号分子。此外，我们提出Fyn通过酪氨酸磷酸化p60、p160和/或p200诱导血管异常收缩。

Mortality of vascular diseases is comparable to that of cancer in Japan. It has been reported that C^<2+>-dependent contraction (Ca^<2+>-sensitization) plays a pivotal role in abnormal vascular contraction observed in vascular diseases. The purpose of this study was to identify novel intracellular signaling molecules and to clarify their signal transduction mechanisms.In the first year, we identified sphingosylphosphorylcholine (SPC) as a novel mediator to induce abnormal vascular contraction. We found that SPC induces Ca^<2+> psensitization through the activation of Src family tyrosine kinase and Rho-kinase in this orderIn the second year, in order to find the downstream mediators of Src family tyrosine kinase, we focused on the changes in tyrosine phosphorylation induced by SPC and we succeed in extracting some molecules as downstream mediators of Src family tyrosine kinase.In the third year, we obtained the following results :1.We established the cell culture system of vascular smooth muscle which has contractile ability.2.The siRNA-mediated knock down of Fyn inhibited the SPC-induced contraction of the cultured vascular smooth muscles, indicating the involvement of Fyn in the SPC induced signal transduction.3.The functional proteomics analysis identified p60, p160, and p200 as the downstream mediators of Fyn.From these results, we identified Fyn as an intracellular signaling molecule which mediates abnormal vascular contraction. In addition, we propose that Fyn induces abnormal vascular contraction through the tyrosine phosphorylation of p60, p160, and/or p200.