LY294002及wortmannnin對於誘發型一氧化氮合成酶蛋白質穩定度及其二聚體形成之效應探討

Differential effects of LY294002 and wortmannin on inducible nitric oxide synthase protein stability and its dimer formation in glomerular mesangial cells

林佳靜(Chia-Ching Lin)

指導教授 ： 林庭慧

中山醫學大學/醫學科技學院/生物醫學科學學系碩士班/碩士(2011年)

https://doi.org/10.6834/CSMU.2011.00058

摘要

一氧化氮(nitric oxide，NO)是一個構造簡單的氣體分子，可經由一氧化氮合成酶(nitric oxide synthase，NOS)催化後產生。當細胞受到細胞激素或是內毒素的刺激時，誘導型一氧化氮合成酶即大量表現且與病理或是發炎反應有關。而許多先前發表的文獻，其研究重點在於調控iNOS基因表現以達降低iNOS蛋白質產生的NO所造成細胞毒性。於本論文中，利用腎絲球細胞株(glomerular mesangial cells，MES-13 cells) 探討phosphatidylinositol 3-kinase (PI3K)抑制劑，對於lipopolysacchride (LPS)/interferon-γ (IFN-γ) 刺激下所產生的iNOS蛋白質之調控機轉。在不影響細胞存活率的情況下，5 μM LY294002可明顯降低NO，iNOS mRNA及其蛋白質，效果較1 μM wortmannin顯著。此外，在訊息傳遞路徑的部分則是發現，LY294002與wortmannin雖然皆是PI3K抑制劑，但是LY294002無法抑制Akt的磷酸化。除了觀察Akt磷酸化之外， STAT-1α的磷酸化則不受到LY294002或是wortmannin的影響；反之，利用Dual luciferase reporter assay則是觀察到NF-κB啟動的Luciferase活性，在LY294002的作用下有30%的下降。經由cycloheximide-blocking experiment，觀察到MES-13細胞處理LPS/IFN-γ/5 μM LY294002，其iNOS蛋白質半衰期改變；反之，細胞處理LPS/IFN-γ/1 μM wortmannin則是不影響iNOS蛋白質半衰期。因此，更進一步利用low temperature SDS-PAGE 證實LY294002導致iNOS蛋白質半衰期改變是因為降低了iNOS蛋白質二聚體的形成，因此造成iNOS蛋白質穩定度改變。雖然LY294002以及wortmannin皆為PI3K抑制劑，但是兩者在MES-13細胞中所導致的效應不同，LY294002可抑制NF-κB的活化及iNOS蛋白質二聚體形成，最終導致iNOS蛋白質加速降解，達到抑制NO的目的。

關鍵字

一氧化氮 ； 誘導型一氧化氮合成酶phosphatidylinositol 3-kinase (PI3K) ； LY294002 ； wortmannin ； 蛋白質穩定度 ； 二聚體/單元體比例 ； 腎絲球細胞

並列摘要

Nitric oxide (NO) is simple molecule that produced by nitric oxide synthase (NOS). When cells were stimulated with cytokines or endotoxin, iNOS was expressed and associated with the pathophysiology of inflammatory events. Numerous studies have focused on the regulation of NO production by iNOS to reduce NO-mediated cytotoxicity. In the present study, we demonstrated the differential effects of LY294002 and wortmannin (phosphatidylinositol 3-kinases (PI3K) inhibitors), on lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-induced NO production in glomerular mesangial cell line, MES-13 cells. At dosages without affecting cell viability of MES-13 cells, 5 μM LY294002 showed more significantly inhibitory effect on LPS/IFN-γ-induced NO production, iNOS protein and gene expressions than 1 μM wortmannin. Akt phosphorylation in MES-13 cells declined upon the addition of wortmannin, but not upon treatment with LY294002. Neither LY294002 nor wortmannin reduced IFN-γ-induced STAT-1α phosphorylation. LY294002 exhibited superior inhibitory effect on NF-κB promoter activity when compared with wortmannin. Moreover, LY294002, but not wortmannin perturbed LPS/IFN-γ-induced iNOS protein stability in MES-13 cells as demonstrated via cycloheximide (CHX)-blocking experiment. LY294002, but not wortmannin decreased iNOS dimer to monomer ratio as demonstrated via low-temperature SDS-PAGE. Although both LY294002 and wortmannin are known as PI3K inhibitors, their differential effects on iNOS expression in MES-13 cells indicated the effects of LY294002 on inhibition of NF-κB activation and accelerating iNOS protein degradation are through mechanism independent of PI3K.

並列關鍵字

nitric oxide (NO) ； inducible nitric oxide synthase (PI3K) ； phosphatidylinositol 3-kinase (PI3K) ； LY294002 ； wortmannin ； protein stability ； dimer/monomer ratio ； MES-13 cells

參考文獻

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