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的死亡率相当。据报道，C^<2+>依赖性收缩（Ca^<2+>-敏化）在血管疾病中观察到的异常血管收缩中起关键作用。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，我们鉴定了鞘氨醇磷酸胆碱（SPC）作为一种新的介导剂，可诱导异常血管收缩。我们发现SPC通过依次激活Src家族酪氨酸激酶和Rho-激酶来诱导Ca^<2+> p增敏。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诱导的酪氨酸磷酸化的变化，并成功地提取了一些Src家族酪氨酸激酶的下游介质分子。结果表明：1.建立了具有收缩能力的血管平滑肌细胞培养体系; 2. siRNA介导的Fyn敲低抑制SPC诱导的血管平滑肌收缩，表明Fyn参与SPC诱导的信号转导; 3.功能蛋白质组学分析证实了Fyn的下游调节因子p60、p160和p200，从而证实Fyn是一种介导血管异常收缩的细胞内信号分子。此外，我们建议，Fyn通过p60，p160和/或p200的酪氨酸磷酸化诱导异常血管收缩。

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.