siRNA-mediated knockdown of Fyn inhibits sphingosylphosphorylcholine (SPC)-induced contraction of vascular smooth muscle cells.

Abstract

In contrast to the Ca²⁺-dependent contraction of vascular smooth muscle (VSM) which regulates physiological vascular tone, the Rho-kinase (ROK)-mediated Ca²⁺-sensitization of VSM contraction contributes to abnormal VSM contraction such as vasospasm. We previously found that SPC is an upstream messenger for the ROK-mediated Ca²⁺-sensitization and that inhibitors of Src family tyrosine kinase (Src-TK) blocked the SPC-induced contraction and activation of ROK. In the present study, we attempted to determine the enzyme molecule in a family of Src-TK which contributes to the Ca²⁺-sensitization mediated by a SPC/ROK pathway. In order to accomplish this purpose, we performed the selective knockdown of the target molecule by siRNA, which was transfected into the human coronary artery smooth muscle cells (CASMC) with the transfection efficiency of about 100%. The siRNA-mediated knockdown of Fyn inhibited the contraction of CASMC induced by SPC, without affecting the expression of other proteins such as MAP kinase, whereas non-silencing control siRNA lacked any effect. In contrast, siRNA-mediated knockdown of MAP kinase had no effect on the expression of Fyn and the cell shape of CASMC. These results produce the first direct evidence that Fyn mediates the Ca²⁺-sensitization of VSM contraction induced by a SPC/ROK pathway. In poster presentation, the effects of transfection of Fyn constructs (wild and mutated) on the VSM contraction of CASMC will be also discussed. [Jpn J Physiol 55 Suppl:S70 (2005)]