PI3K抑制劑wortmannin增強iNOS表現之分子機制探討

Molecular Mechanisms of PI3K inhibitor wortmannin on the augmentation of iNOS expression in macrophages

羅瑞蓮(Jui-Lien Lo)

指導教授 ： 梁有志

臺北醫學大學/醫學院/醫學檢驗生物技術學研究所/碩士(2007年)

https://doi.org/10.6831/TMU.2007.00118

摘要

構成細胞膜的主要成份是磷脂質，其中磷脂醯肌醇4,5二磷酸（Phosphatidylinositol (4,5)-bisphosphate , PtdIns(4,5)P2）可作為磷脂醯肌醇激酶（phosphatidylinositol 3-kinase,PI3K）和磷脂醯肌醇特異性磷酸脂肪酶（phosphoinositide specific phospholipase C,PI-PLC）的受質，當PtdIns(4,5)P2作為PI3K的受質時，PI3K會將PtdIns(4,5)P2磷酸化生成磷脂醯肌醇3,4,5三磷酸（Phosphatidylinositol (3,4,5)-triphosphate,PtdIns (3,4,5)P3）。當PtdIns(4,5)P2 作為PI-PLC的受質時，PI-PLC會將PtdIns(4,5)P2水解為肌醇-1，4，5-三磷酸（inositol 1,4,5-trisphosphate,Ins(1,4,5)P3）和二醯甘油（diacylglycerol,DG,DAG）。我們的研究主要是探討PI3K抑制劑wortmannin增強誘導型一氧化氮合成酶（induceble nitric oxide synthase, iNOS）表現的分子機制。首先，經由wortmannin處理的巨噬細胞，其Ins(1,4,5)P3產物和iNOS的表現均會增加。而轉殖p85 siRNA（small interfering RNA）的巨噬細胞，因為PI3K的活性消失，也會增強iNOS的表現；相反的，用表現質體ras-p110轉殖的巨噬細胞，因為PI3K的過度活化，使得原本增強的iNOS卻顯著的下降了。此外，在脂質多醣體（lipopolysaccharide, LPS）活化的巨噬細胞裡，過度表現wild-type Akt及dominant-negative Akt，對於增強iNOS的表現並沒有影響。如果使用PI-PLC的抑制劑U73122處理在LPS活化的巨噬細胞，除了iNOS的表現會下降之外，並且也會降低ERK（extracellar signal-regulated kinase,）、JNK（c-Jun N-terminal kinase）、p38 （protein 38）的磷酸化及AP-1（activating protein -1,）的轉錄活性。但是不論「有」和「無」wortmannin處理下，在LPS活化的巨噬細胞，其kappa B 激酶抑制子（inhibitor kappa B, IkappaB）的降解、核因子kappa B（nuclear factor kappa B, NF-kappa B）的結合活性（EMSA）、NF-kappa B的轉錄活性（luciferase reporter assay）均沒有變化；但有wortmannin處理的巨噬細胞其AP-1的結合活性(EMSA)及AP-1的轉錄活性(luciferase reporter assay)均明顯增加。因此我們提出：使用PI3K的抑制劑wortmannin處理的巨噬細胞，其所增強iNOS的表現是透過PI-PLC下游的訊號及轉錄因子AP-1轉錄活性的增強，因而促進iNOS的表現增加。換言之；PI3K的抑制劑wortmannin處理的巨噬細胞，所增強的iNOS表現，並不依賴Akt路徑、及NF-kappa B轉錄因子。

關鍵字

一氧化氮合成酶磷酸脂肪酶二醯甘油 ； 磷脂醯肌醇

並列摘要

Phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2) has been known to serve as substrate for phosphatidylinositol 3-kinase (PI3K) and phosphoinositide-specific phospholipase C (PI-PLC), and that can produce PtdIns(3,4,5)P3 and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) & diacylglycerol (DAG), respectively. To elucidate the role of PI-PLC during activated-macrophages treated with PI3K inhibitor wortmannin, we examined the effects of wortmannin on the enhancement of PI-PLC downstream signals and that leading to increase of inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-activated macrophages. Here, we report that wortmannin inhibited PI3K activity but enhanced PI-PLC downstream signals, and subsequently increased iNOS expression independently of Akt & NF-kB pathways. First, wortmannin treatment enhanced Ins(1,4,5)P3 production and iNOS expression in LPS-activated macrophages. Inhibition of PI3K by p85 siRNA also showed an enhancement of iNOS expression. On the other hand, overexpression of PI3K by ras-p110 expression plasmid significantly decreased iNOS expression in LPS-activated macrophages. In addition, overexpression of wild-type or dominant-negative Akt expression plasmid did not affect the iNOS expression in LPS-activated macrophages. Second, treatment of PI-PLC inhibitor U73122 reversed the enhancement of iNOS expression, the increase of phosphorylation level of ERK, JNK & p38, and the increase of AP-1-dependent gene expression in wortmannin-treated & LPS-activated macrophages. However, I kappa B degradation, NF-kappaB binding activity by EMSA assay, and NF-kappa B-dependent gene expression by luciferase reporter assay did not change during LPS-activated macrophages with or without wortmannin. We propose that the inhibition of PI3K by wortmannin in mouse macrophages enhances the PI-PLC downstream signals, and subsequently increases the LPS induction of iNOS expression independently of Akt pathway, and AP-1 but not NF-kappa B mediates the enhancement of iNOS expression.

並列關鍵字

PI3K ； PtdIns(4,5)P2 ； Ins(1,4,5)P3 ； iNOS ； LPS

參考文獻

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