- 學位論文
Ras、Raf和ERK路徑在Thrombin誘導肺部巨噬細胞一氧化氮合成?表現的角色探討
The Role of Ras/Raf/ERK Pathway in Thrombin-Mediated Inducible Nitric Oxide Synthase Expression in Alveolar Macrophages
摘要
Thrombin為一個多功能的絲胺酸蛋白?，可從受傷的血管釋放出來，為參與凝血反應重要的凝血因子。此外，thrombin也可以?生多方面的細胞生理反應，其中包括調節發炎反應。根據先前研究指出，在肺部巨噬細胞中，thrombin可誘導一氧化氮合成? (inducible nitric oxide synthease, iNOS) 的表現，然而其中的作用機制仍不清楚。本論文主旨在探討thrombin刺激肺部巨噬細胞產生一氧化氮合成?表現是否透過Ras/Raf/ERK及I?B kinase (IKK)/nuclear factor-?B (NF-?B) 路徑而來。實驗結果證實，thrombin會隨時間及濃度增加而誘導 NR8383肺部巨噬細胞一氧化氮合成?的表現增加。Thrombin誘導一氧化氮合成?的表現會被 Ras抑制劑 (Manumycin A)、 功能性突變之Ras (Ras N17)、 Raf抑制劑 (GW5074)，及MEK抑制劑 (PD98059) 所抑制。Thrombin所誘導 Ras活性的增加會被manumycin A所抑制。Thrombin誘導 Raf-1在 Serine 338位置磷酸化的現象也會被manumycin A及GW5074所抑制，相同地，thrombin所誘導ERK磷酸化也會被manumycin A、GW5074及PD98059所抑制。接著進一步證實，NF-?B 抑制劑 (PDTC)、I?B phosphorylation抑制劑 (Bay117082)及過度表現 I?BM (I?B mutant)皆可抑制thrombin所誘導的一氧化氮合成?的表現。NR8383細胞經由 thrombin刺激會促使IKK?/?磷酸化、I?B?磷酸化、I?B?降解以及?B-luciferase活性增加。Thrombin所誘導 IKK?/?磷酸化及 ?B-luciferase的活化同樣也會被manumycin A、GW5074及 PD98059所抑制。綜合以上的實驗結果，顯示在NR8383肺部巨噬細胞中，thrombin可經由 Ras/Raf/ERK及 IKK?/?/NF-?B之訊息傳遞路徑來媒介誘導性一氧化氮合成?的表現。
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. Previous reports showed that thrombin induced inducible nitric oxide synthase (iNOS) expression in lung macrophages; however, the signal pathway is still unclear. This study investigated the Ras/Raf/ERK and I?B kinase (IKK)/nuclear factor-?B (NF-?B) signaling pathways involved in iNOS expression by thrombin in NR8383 alveolar macrophage. Thrombin caused increase in iNOS expression in a time- and concentration- dependent manner. Thrombin-induced iNOS expression was inhibited by Manumycin A (Ras inhibitor), dominant negative matant of Ras (Ras N17), GW5074 (Raf inhibitor), and PD98059 (MEK inhibitor). The thrombin-induced increase in Ras activity was inhibited by manumycin A. Raf-1 phosphorylation at serine 338 residue by thrombin was inhibited by manumycin A and GW5074. The thrombin-induced ERK phosphorylation was also inhibited by manumycin A, GW5074, and PD98059. Furthermore, pretreatment of PDTC (NF-?B inhibitor) or Bay117082 (I?B phosphorylation inhibitor) or overexpression of I?BM (??I?B mutant)? all inhibited thrombin-induced iNOS expression. Stimulation of NR8383 cells with thrombin induces increase in IKK?/? phosphorylation, I?B phosphorylation, I?B degradation, and ?B-luciferase activity. The thrombin-induced increase in IKK?/? phosphorylation and ?B-luciferase activity was inhibited by manumycin A, GW5074, and PD98059. These results indicated the Ras/Raf/ERK and IKK?/?/NF-?B signaling pathways involved in thrombin-induced iNOS expression in NR8383 cell.