過度糖化最終產物在 RAW 264.7 細胞誘導 iNOS 表現中蛋白質激脢
C 所扮演的角色
Roles of Protein Kinase C on BSA-AGEs-Induced Nitric Oxide
Synthase Expression in RAW 264.7 Macrophages
中文摘要 論文名稱：過度糖化最終產物在 RAW 264.7 細胞誘導 iNOS 表現中蛋白質激脢 C 所扮演的角色 研究所名稱: 台北醫學大學生物醫學技術研究所 研究生姓名: 張鑑熹 畢業時間: 八十九學年度第二學期 指導教授: 李宏謨 博士 台北醫學大學生物醫學技術研究所教授
在 RAW 264.7 巨噬細胞中 BSA-AGEs 可誘導 iNOS 表現。本研究探討 BSA-AGEs 誘導 iNOS 表現過程中之訊息傳遞路徑，尤其是 PKC 異構脢在 BSA-AGE 誘導 NO 釋放及 iNOS 表現所扮演的角色。在 RAW 264.7 巨噬細胞中 BSA-AGEs 以具 劑量和時間的依存性的誘導 NO 釋放和 iNOS 表現。tyrosine kinase 抑制劑 genistein 會減弱 BSA-AGE 誘導 RAW 264.7 細胞的 NO 釋放和 iNOS 表現。除此 之外，PI-PLC 抑制劑 U73122、PC-PLC 抑制劑 D609、以及 PKC 抑制劑，包括 staurosporine、Ro 31-8220 和 Go 6976 都會在 RAW 264.7 巨噬細胞中，依不同濃 度而有不同程度抑制 BSA-AGE 誘導 NO 釋放，及抑制 iNOS 表現，但單獨以 BSA 處理 RAW 264.7 細胞，不會引起 iNOS 表現。加入 polymyxin B（LPS 抑制劑） 亦不會減弱 BSA-AGEs 誘導 RAW 264.7 細胞之 iNOS 表現。PI-PLC 抑制劑 U73122 及 tyrosine kinase 抑制劑 genistein 皆會抑制 BSA-AGEs 在 RAW 264.7 細 胞引起 PIP2 的水解反應。以 AGEs 刺激 RAW 264.7 細胞時，細胞內 PKC-a, - b1, -d, 和-h 會從細胞質移到細胞膜。表示在 RAW 264.7 巨噬細胞中，PKC-a, - b1, -d, 和-h 有一種或數種參與 AGEs 誘導 NO 產生和 iNOS 的表現。同時 U73122、 D609、及 genistein 也減弱 PKC-a, -b1, -d, 和-h 的移位表現。
這些數據表示，在 RAW 264.7 巨噬細胞，BSA-AGEs 經過上游 tyrosine kinase， 活化了 PI-PLC 和 PC-PLC，產生 DAG，然後活化 PKC 異構脢，並誘導 iNOS 的 表現和 NO 的釋放。其中一種或多種 PKC 異構脢（-a, -b1, -d, -h ）參與調控 AGEs 誘導 iNOS 表現。
英文摘要
Title of Thesis: Roles of protein kinase C on BSA-AGEs-induced nitric oxide synthase expression in RAW 264.7 macrophages.
Thesis advised by: Horng-Mo Lee Ph.D.
We previously found that Advanced Glycosylation End Products (AGEs) induced nitric oxide synthaseⅡ (iNOS) expression in RAW 264.7 cells. The signaling pathway involved in protein kinase C activation and the roles of PKC isoforms in AGEs-induced NO production in RAW 264.7 cells were investigated. BSA-AGEs caused a dose- and time-dependent increase in NO release and inducible NO synthase (iNOS) expression, but BSA alone did not induce iNOS expression. The polymyxin B （LPS inhibitor）also did not inhibit BSA-AGEs induced iNOS expression. The tyrosine kinase inhibitor genistein attenuated BSA-AGEs-induced NO release and iNOS expression in RAW 264.7 cells. Additionally, the phosphoinositide-specific phospholipase C (PI-PLC) inhibitor, U73122, the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor, D609, and the PKC inhibitors including staurosporine, Ro 31-8220, and Go 6976 all inhibited the BSA-AGEs-induced iNOS expression. BSA-AGEs stimulated PIP2 turnover was attenuated by PI-PLC inhibitor, U73122 and tyrosine kinase inhibitor, genistein. Western blot analysis revealed that incubation of the RAW 264.7 cells with BSA-AGEs, results in PKC-a, -b1, -d, and -h translocation, indicating the possible involvement of one or all of PKC-a, -b1, -d, and -h in AGEs-mediated effects.
These data suggest that BSA-AGEs activate PI-PLC and PC-PLC via an upstream tyrosine kinase to induce PKC activation, then initiate the expression of iNOS and NO release. PKC isoform-a, -b1, -d, and -h were shown to be involved in the regulation of BSA-AGEs-induced iNOS expression.
縮寫詞彙
AGEs, advanced glycosylation end products.
BSA-AGEs, bovine serum albumin derived advanced glycosylation end products NO, nitric oxide.
iNOS, inducible nitric oxide synthase. PLC, phospholipase C.
PI-PLC, phosphoinositide-specific phospholipase C. PC-PLC, phosphatidylcholine-specific phospholipase C. DAG, diacylglycerol.
IP3, Inositol 1,4,5-trisphosphate.
PIP2, phosphatidylinositol 4,5-bisphosphate . PKC, protein kinase C.
