內皮細胞中Focal adhesion kinase參與在鞘氨醇1-磷酸鹽引起的 血管新生和發炎反應中的關係

Abstract

Focal adhesion kinase (FAK)是一種重要的訊息分子，可調控integrin以及生長因子受器的下游訊號傳遞，進而影響包括了內皮細胞等多種細胞的貼附、移動、生長和存活。鞘氨醇1-磷酸鹽和內皮細胞膜上的Edg family G-protein-coupled receptors結合在發炎反應時調控細胞的生理現象，例如細胞的生長、移動及血管形成。人類臍靜脈內皮細胞FAK上的Tyr397會受到鞘氨醇1-磷酸鹽刺激而增加，但此刺激會受到PI3K (i.e. LY294002)、PLC (i.e. U73122)和MEK 1 (i.e. PD98059)的抑制劑存在而減少。另外，我們也發現當鞘氨醇1-磷酸鹽在刺激FAK的活性時細胞貼附於基質是一個必要的條件。如果抑制FAK和表現過多Y397F突變之FAK或FRNK時，會減少血管生成並使細胞移動減少及生長速度減緩；相對地，當表現更多正常的FAK時則會促進細胞移動及血管的生成。這些結果顯示FAK參與在鞘氨醇1-磷酸鹽所誘發的血管生成過程中。 除此之外，原本受到鞘氨醇1-磷酸鹽刺激而增加的interleukin-1β (IL-1β)及intercellular adhesion molecule 1 (ICAM-1)會受到FAK抑制劑的影響而不受鞘氨醇1-磷酸鹽的調控，這符合發炎反應是調控血管新生開啟的一種新生前因子。因此，我認為在內皮細胞鞘氨醇1-磷酸鹽會經由調控FAK而活化發炎反應路徑，進而影響血管生成作用。

Focal adhesion kinase (FAK) is a key signal transmitter downstream of integrins and growth factor receptors. It regulates cell adhesion, spreading, migration, proliferation and survival in various cell types including endothelial cells (ECs). Sphingosine 1-Phosphate (S1P) is a bioactive lysophospholipid ligand that binds to the Edg family G-protein-coupled receptors in ECs. It regulates cell physiology during inflammation processes as well as important for promoting cell proliferation, migration and tube formation. In this study, we found that the phospho-Tyr397 of FAK was enhanced by S1P stimulation in human umbilical cord vein endothelial cells (HUVECs) and the induction could be inhibited by the inhibitor of PI3K (i.e. LY294002), PLC (i.e. U73122) or MEK 1 (i.e. PD98059). Furthermore, we also found that all adhesion is required for S1P-induced FAK activation. Accordingly, upon FAK knockdown or overexpression of dominant negative FAK mutants, such as Y397F and FRNK, tube formation, migration and proliferation of HUVECs were reduced in response to S1P stimulation. In contrast, overexpression of wile-type FAK augmented the ability of tube formation and cell migration. These results suggest the involvement of FAK in S1P-induced angiogenesis. In addition, the expression level of interleukin-1β (IL-1β) and intercellular adhesion molecule 1 (ICAM-1) by S1P induction were inhibited in the inactivation of FAK in HUVECs, which is consistent with the fact that inflammatory responses are the pro-angiogenic signals to turn on angiogenesis processes. Hence, here we proposed a new mechanism for FAK in angiogenesis through regulating inflammation pathways in endothelial cells.