Thrombin 刺激肺部上皮細胞誘導介白素-8 表現經由 Gβγ2 依賴Rac 及PI3K/Akt 的活化

Thrombin 為一個多功能的絲胺酸蛋白?，可從受傷的血管釋放來，為參與凝血反應重要的凝血因子。此外，它也可?生多方面的胞生理反應，其中包括調節發炎反應。根據先前研究指出，在呼吸道上皮細胞中，thrombin 可誘導前發炎物質IL-8 的產生，然而其中作用機制仍不清楚。本篇論文主要探討主題在thrombin 刺激肺部上皮細胞?生IL-8 表現是否經由Gβγ2、Rac 及PI3K/Akt 之訊息傳導而來。在A549 肺部上皮細胞中，thrombin 誘導IL-8 的釋放呈現濃度相關反應曲線增加，並可利用轉染及報告基因的方法證實thrombin 及受體N 端合成胜?SFLLRN-NH2(PAR1 作用劑)和GYPGQV-NH2PAR4 作用劑)也可誘導IL-8-luciferase 的活性，但TFRGAP-NH2 PAR3 作用劑)卻無法增加IL-8-luciferase 的活性。接著進一步證實，thrombin 誘導IL-8 釋放及IL-8-luciferase 的活性，可被轉染dominant　negative mutant of Rac (Rac N17)和PI3K 抑制劑(LY 294002)及dominant negative mutant of Akt (Akt DN)所抑制。Thrombin 具時間相性誘導Rac 及Akt 的活性，同時thrombin SFLLRN-NH2 及GYPGQV-NH2 也可誘導Akt Ser473 的磷酸化。更進一步證實，thrombin 誘導Akt Ser473 的磷酸化，可被Rac N17 及LY 294002 所抑制；同時thrombin 所誘導Akt 的激?活性，也可被Rac N17、LY 294002及Akt DN 所抑制。此外，thrombin 所誘導的IKKα/β的磷酸化可被Rac N17、LY 294002 及Akt 抑制劑所抑制；同時Rac N17、LY 294002及Akt DN 可抑制thrombin 誘導的IKKα/β的激?活性。再者RacN17、Akt DN 及LY 294002 皆可以抑制thrombin 誘導κB-luciferase的活性。最後更進一步證實thrombin 可以依時間相關性的誘導Gβγ2及Rac 與p85α的結合。綜合以上的實驗結果，可以推測出在A549肺部上皮細胞中，thrombin 可經由Gβγ2/Rac/PI3K/Akt 的路徑活化IKKα/β進一步再活化NF-κB 來調控IL-8 的表現及釋放。

關鍵字

凝血? ；介白素-8 ；發炎及應

並列摘要

Thrombin, a multifunctional serine protease generated at sites of vascular injury, and known for its pivotal role in the coagulation cascade, contributes to tissue repair, but also promotes a wide range of cellular responses including modulation of the inflammatory responses. A previous report showed that thrombin can induce IL-8 release in human epithelial cells, however, the signal transduction is still unclear. This investigated the signaling pathway involved in Gβγ2, Rac and PI3K/Akt in IL-8 expression caused by thrombin in A549 lung epithelial cell.Thrombin caused a concentration-dependent increase in IL-8 release, which was attenuated by cell transfection with dominant negative mutant of Rac (Rac N17), LY 294002 (a PI3K inhibitor), and dominant negative mutant of Akt (Akt DN). Thrombin, SFLLRN-NH2 (a PAR1 agonist peptide), and GYPGQV-NH2 (a PAR4 agonist peptide), all induced an increase in IL-8-luciferase activity, while TFRGAP-NH2 (a PAR3 agonist peptide) had no effect. Treatment of A549 cells with thrombin caused increase in Rac and Akt activities. Similarly, SFLLRN-NH2, and GYPGQV-NH2 but not TFRGAP-NH2 inducedphosphorylation of Akt at Ser473. Pretreatment of A549 cells with LY294002 or transient transfection with Rac N17 inhibitedthrombin-induced Akt activity. In addition, Rac N17 and Akt DN inhibited thrombin-induced IKKα/β kinase activity. Moreover, Rac N17,LY 294002, and Akt DN all inhibited thrombin-induced increase in κB-luciferase activity. Further studies reveals that thrombin induced therecruitment of p85α and Rac to Gβγ2 in time-dependent manner. These results indicate that thrombin activates the Gβγ2/Rac/PI3K/Akt signaling pathway to activate IKKα/β, which in turn initiates NF-κB activation,and ultimately induces IL-8 expression and release in A549 cells.

並列關鍵字

thrombin ； Interleukin-8 ； inflammation

參考文獻

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Thrombin 刺激肺部上皮細胞誘導介白素-8 表現經由 Gβγ2 依賴Rac 及PI3K/Akt 的活化

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