Mechanism of LPS-induced sFlt-1 Expression in Macrophage

人體內單核球細胞(monocytes)受到外來感染原刺激時會分化成吞噬細胞(macrophages)，同時分泌許多細胞激素(cytokines)以引發適當的免疫反應來清除感染原。對於外來感染原的免疫反應過度時反而會引起敗血病(sepsis)，造成多種器官衰竭和死亡。在美國平均每年約有近百萬人罹患敗血病，每年在敗血疾病的醫藥花費更高達數億美元，所以研發出更有效治療敗血病的方法是具有其急迫性的。先前我們在吞噬細胞的研究結果發現以脂多醣(lipopolysaccharides; LPS)刺激吞噬細胞可以使其分泌出大量的可溶性血管內皮生長因子受器(sFlt-1)；在敗血病動物模式研究結果發現，注射sFlt-1可以降低LPS 所造成老鼠敗血病的死亡率，然而目前關於LPS誘導吞噬細胞分泌sFlt-1的分子機制尚不清楚。本論文研究結果發現廣效性的PKC抑制劑GF109203X可以抑制LPS誘導老鼠吞噬細胞RAW264.7或是人類周邊血液的吞噬細胞分泌sFlt-1，但促進了LPS所誘導VEGF的分泌。此外將細胞以專一性PKCδ抑制劑Rottlerin處理，或在細胞內表現無酵素活性的PKCδ (K376R)突變蛋白也可以得到相同的結果，顯示PKCδ在LPS誘導sFlt-1和VEGF分泌的分子機制中，分別扮演了正面與負面調控的角色。 Statin是 HMG-CoA reductase的抑制劑，臨床上主要用來抑制中間產物mevalonate的生成，進而中斷膽固醇之合成。除了在降低血脂的功能外，目前有許多研究報告發現statin也具有抗發炎的效果；在臨床統計上發現平時有服用statin的人發生敗血病的機率較無服用者較低，可是目前關於statin抗發炎功能的分子作用機制尚未完全了解。在本論文研究結果發現先以低劑量simvastatin (1 μM) 刺激RAW264.7細胞可以提高LPS所誘導sFlt-1的表現，若多加入mevalonate或其下游中間產物geranylgeranyl pyrophosphate (GGPP)則可抑制simvastatin的作用，顯示mevalonate 代謝路徑也參與了LPS誘導sFlt-1表現機制之調控。先前研究發現simvastatin可以活化吞噬細胞內ERK訊息傳遞路徑，在本論文研究中我們發現經過simvastatin的前處理可以延長LPS所誘導ERK1/2磷酸化的時間，如果對吞噬細胞先施予ERK1/2抑制劑U0126前處理，則可以抑制simvastatin對sFlt-1的影響。綜合以上的結果，本論文實驗結果證實了PKCδ訊息傳遞路徑的活化和mevalonate 代謝路徑在LPS誘導吞噬細胞分泌sFlt-1的調控機制中皆扮演了很重要的角色。Upon pathogen infection, the activated monocytes or macrophages secret a variety of cytokines to orchestrate a complicated immune response in the host. However, overwhelming systemic inflammation may result in septic diseases which cause organ dysfunction and eventually lead to death. Nearly one million cases of severe sepsis are diagnosed and cost approximately ten billion US dollars each year in the United States. Development of effective therapies for septic diseases is a high priority task. Our previous studies found that LPS treatment could induce sFlt-1 secretion in macrophage and exogenous sFlt-1 treatment could decrease the mortality in LPS-induced septic mouse model. However, the molecular mechanism of LPS-induced sFlt-1 expression is still not understood. In this thesis, we found that in the murine RAW264.7 cells, as well as in primary human monocytes/macrophages, pretreatment with a general PKC inhibitor GF109203X or with a novel PKCδ inhibitor rottlerin or overexpression of a kinase-inactive form of PKCδ (K376R) eliminated LPS-induced sFlt-1 expression and augmented LPS-induced VEGF expression at both the protein and the transcription levels. These data suggest that PKCδ signaling is involved in LPS-induced sFlt-1expression and serves as a negative mediator in LPS-induced VEGF expression in macrophages.tatin, an inhibitor of HMG-CoA reductase, exert pleiotropic effects independent of cholesterol reduction. It is noted that the mortality and morbidity of sepsis patients treated with statin are reduced. However, the molecular mechanism of anti-inflammatory effect for this drug is not fully understood. In this thesis, we demonstrated that simvastatin enhanced both LPS-induced sFlt-1 mRNA and protein expressions in murine RAW 264.7 macrophages. Co-treatment with mevalonate or its metabolites geranylgeranyl pyrophosphate (GGPP), but not farnesyl pyrophosphate (FPP), blocked the effect of simvastatin. These data suggested that protein geranylgeranylation may play a crucial role in the regulation of sFlt-1 in response to LPS. We also found that simvastatin pretreatment prolonged ERK1/2 phosphorylation in response to LPS stimulation. Furthermore, blockage of ERK signaling by U0126 inhibited sFlt-1 secretion in response co-treatment of LPS and simvastatin. Our results indicate that both PKCδ activation and mevalonate pathway play important roles in LPS-induced sFlt-1 expression in murine RAW 264.7 macrophages.口試委員會審定書………………………………………………..……………….... .i謝…………………………………………………………………….…………..... ii文摘要……………………….…………………………………………………......iiibstract..……………………………………………………………...…………........v錄..............................................................................................................................viibbreviations..............................................................................................................xiiart 1echanism of LPS-induced sFlt-1 Expression in Macrophage...........…………....1iterature Review. Sepsis and Monocyte/Macrophage…….…..…..……………………….........2. VEGF, PlGF, and their Receptors………...........…………………………....4. VEGF expression in Monocyte/ Macrophage…….…………..………….....6. LPS-activated signal transduction pathways……….....…………………....7. Statin in sepsis……………………………………….………………………..9hapter 1ovel PKC signaling is required for LPS-induced soluble Flt-1 expression in macrophages …………………………………………........……………………..11bstract…………………………………………….....………………………. 11ntroduction…………………………………….......………………………….13aterials and Methods…………………..…………………………………....16esults………………………………………………………………………….20PS-induced phosphorylation of multiple PKC isoforms in murine macrophage RAW264.7 cells…………...…………………………………..20ctivation of the PKC signal pathway is required for LPS-induced sFlt-1 secretion in RAW264.7 cells……………………………....………………...20nhibition of the general PKC pathway increases the level of free-form VEGF in culture medium of the LPS-activated RAW264.7 cell……..…..21nhibition of the novel-type but not the conventional or atypical PKC isoforms regulates LPS-induced sFlt-1 and VEGF expressions in RAW264.7 cells…………………………………………......……………….22verexpression of dominant-negative mutant K376R of PKC δ suppresses LPS-induced sFlt-1 secretion in RAW 264.7 cells………....…….………. 23eneral PKC inhibitor GF109203X also regulates LPS-induced sFlt-1 and VEGF expression in human primary macrophages……...…………....….24iscussion…………………………………………………………………...….25hapter 2imvastatin enhances LPS-induced soluble Flt-1 expression in murine macrophages via prolonging ERK activation………........…….........……...…..30bstract…………………………………………………...……………………30ntroduction………………………………………..………………..…………32aterials and Methods…………………………….………………………….35esults……………………………………………….......……………………..39imvastatin augments LPS-induced sFlt-1 expression in RAW 264.7 macrophages…………………………………………..…………………….39evalonate and its metabolite GGPP restore the normal response to LPS in simvastatin-pretreated RAW 264.7 macrophages.…........…….………39imvastatin prolongs LPS-induced ERK phosphorylation in RAW 264.7 macrophages………………………………………………..…………...…..41lockage of ERK signaling inhibits sFlt-1 secretion in response to LPS alone or to co-treatment of LPS and simvastatin in RAW 264.7 macrophages…..….........................................................................................41imvastatin does not affect the LPS-induced VEGF mRNA expressions in RAW264.7 macrophages……………….……………………………….…..42iscussion………………………………………………………………..…......44art 2ffects of EBP2 Expression on Cell Growth and Chromosome Stability….........49文摘要..................................................................................................................50bstract………………..………. .……………………………………….……….51iterature Review………………………………………………………………...52. p40/ EBP2/ NoBP………………………………………………………........52. Cyclin E and cancer…..…......….……..........................................................54. Chromosome instability………………………………………………….....56. Role of EBP2 in tumorigenesis…...…………………………………….......57hapter 3ctopic EBP2 expression enhances cyclin E1 expression and induces chromosome instability in HEK293 stable clones……......………………...…..59bstract …………………………………………………...…………….……..59ntroduction………………………………………………......………………..60aterials and Methods………………………………………………………..62esults…………………………………………………………......…………...66eneration of EBP2-EGFP stable clones in HEK293 cells…......………..66ctopic EBP2-EGFP expression increases cyclin E expression…....….…66BP2-EGFP-induced cyclin E expression does not increase Cdk2 activity …………………………………………………………………........67BP2-EGFP induces ATM-p53-p21 pathway……………………..…........68BP2-EGFP expression induces chromosome instability………………...69iscussion……………………………………………………………….……...71able 1: Chromosome number of HEK293 with or without ectopic EBP2-EGFP expression…………………………………...…..........74igures:ig. 1: Phosphorylation of multiple PKC isoforms induced by LPS in murine macrophage RAW264.7 cells………………………...……75ig. 2: GF109203X suppressed sFlt-1 expression in LPS-activated RAW264.7 cells………………………………………………...……76ig. 3: GF109203X increased VEGF expression in LPS-activated RAW264.7 cells……………………………………………………...77 ig. 4: Effects of different PKC isoform inhibitors on LPS-induced sFlt-1 and VEGF expressions in RAW264.7 cells………….......………....78ig. 5: Effects of overexpressing dominant-negative mutant K376R of PKC δ on LPS-induced sFlt-1 and VEGF expressions in RAW 264.7 macrophages…………………………………………..….…..79ig 6. Effects of GF109203X on LPS-induced sFlt-1 and VEGF secretion in primary human macrophages….........……………………….....80ig 7. Simvastatin augmented LPS-induced sFlt-1 secretions in murine RAW264.7 macrophages.………………………..………………..…81ig 8. Mevalonate and geranylgeranylpyrophosphate (GGPP) prevented simvastatin-mediated augmentation of LPS-induced sFlt-1 expression in RAW264.7 cells…………………………………….…82ig 9. Simvastatin prolonged LPS-induced ERK phosphorylation in RAW264.7 cells……..........................……....………………………...83ig 10. ERK inhibitor U0126 inhibited LPS-induced sFlt-1 secretion in RAW264.7 cells………….....………...………………………….......84ig 11. Simvastatin did not affect LPS-induced VEGF mRNA expression in RAW264.7 cells…………...……………………………………....85ig 12: Localization of EBP2-EGFP in HEK293 cells. …………………..86ig 13: Increased cyclin E1 expression in EBP2-EGFP stable clones..….87ig 14: Effects of ectopic EBP2-EGFP expression on cyclins D1, E1, A and B expression using flow cytometry. …………………………….. 88ig 15: Ectopic EBP2-EGFP expression did not alter cell cycle progression........................................................................... …..…..89ig 16. Excess cyclin E1 expression did not influence Cdk2 kinase activity via initiating ATM-p53-p21 system in HEK 293 stable clones. .....90eferences….......…………………………………………………………...... .9