Title

凝血酶在關節炎致病機轉中扮演的角色

Translated Titles

The role of thrombin in the pathogenesis of arthritis

Authors

黃俊寅

Key Words

凝血酶風濕性關節炎 ; 退化性關節炎 ; 造骨細胞 ; 軟骨細胞 ; Thrombin ; Rheumatoid Arthritis ; Osteoarthritis ; Osteoblasts ; Chondrocytes ; CCL2 ; MMP-13

PublicationName

中國醫藥大學臨床醫學研究所博士班 學位論文

Volume or Term/Year and Month of Publication

2014年

Academic Degree Category

博士

Advisor

湯智昕

Content Language

英文

Chinese Abstract

第一部分 凝血酶為刺激纖維蛋白沉積,血管生成和促進發炎過程的一個關鍵因子。在這些過程中的異常為類風濕關節炎(RA)的主要特徵。這裡,我們研究了人類成骨細胞內參與凝血酶誘導的CCL2表現及細胞中的訊息傳遞。用定量PCR和ELISA方法來進行凝血酶誘導CCL2的表現評估。對於凝血酶不同的細胞訊息傳遞路徑的作用機制進行了研究,是採用西方點墨法。再利用siRNA基因轉殖來抑制PAR蛋白的表現。以染色體免疫沉澱分析法來研究在細胞內結合c-Jun後啟動CCL2表現。也利用暫時性基因轉殖來檢查AP-1的活性。凝血酶誘導人類成骨細胞和MG-63細胞的CCL2表現。藉由使用藥物抑製劑或活化劑或基因抑制PAR及基因轉殖siRNA顯示,是經由PAR1的受體,而非其他的PAR受體參與凝血酶調節CCL2的表現。凝血酶介導的CCL2表現,會被凝血酶抑製劑(PPACK),PKCδ抑製劑(rottlerin),c-Src抑製劑(PP2),表皮生長因子受體抑製劑(AG1478),MEK抑製劑(PD98059和U0126),或AP-1抑製劑(curcumin and tanshinone IIA)所減低表現。給予凝血酶刺激細胞後,PKCδ,C-Src,EGFR,MEK,ERK激活增加。給予凝血酶處理成骨細胞也增加c-Jun的磷酸化,AP-1的螢光素酶活化,而且c-Jun會結合在CCL2啟動因子的AP-1的元素上。我們的研究結果證明,在人類成骨細胞中凝血酶和PAR1之間的相互作用會增加CCL2表現,是經由PKCδ,c-Src,EGFR,MEK,ERK,c-Jun,和AP-1的細胞訊息轉遞路徑。 第二部分 凝血酶(Thrombin)是纖維蛋白沉積,血管生成和炎症過程中的一種重要介質。在這些過程中的異常是類風濕關節炎和骨關節炎的主要特徵。基質金屬蛋白酶-13 (MMP-13)可能會導致關節炎過程中關節軟骨的破壞。然而,凝血酶導致軟骨細胞生產MMP-13的作用機轉並不清楚。在這項研究中,我們使用定量PCR, western blotting和ELISA來偵測人類軟骨細胞中凝血酶誘導MMP-13的表現,及參與細胞內的信號傳導通路。我們發現,凝血酶刺激培養的人類軟骨細胞,會導致MMP-13分泌的增加。此外,凝血酶誘導MMP-13的生產,在蛋白酶激活受體1和3 (PAR1和PAR3)的siRNA轉染後會降低,但不受PAR4 siRNA 的影響,證明凝血酶誘導的MMP-13表現是受PAR1/PAR3介導的。使用PKCδ,c-Src, EGFR, PI3K,Akt,或AP-1的特異性抑製劑或相對應的siRNA處理後,會減低凝血酶介導的軟骨細胞MMP-13生產表現。此外,凝血酶刺激軟骨細胞,會增加PKCδ,c-Src, EGFR, PI3K,Akt,及AP-1的磷酸化活化表現。更進一步,凝血酶介導的AP-1活化,會受PKCδ,c-Src, EGFR, PI3K,Akt的特異性抑製劑或相對應的siRNA所抑制。我們的結果證明,凝血酶作用是透過PAR1/PAR3受體並激活PKCδ,c-Src, EGFR, PI3K,Akt,和最後的AP-1對MMP-13啟動子,從而促進關節炎中軟骨的破壞。

English Abstract

Part I Thrombin is a key factor in the stimulation of fibrin deposition, angiogenesis, and proinflammatory processes. Abnormalities in these processes are primary features of rheumatoid arthritis (RA). Here, we investigated the intracellular signaling pathways involved in thrombin-induced CCL2 expression in human osteoblasts. Thrombin-mediated CCL2 expression was assessed with qPCR and ELISA. The mechanisms of action of thrombin in different signaling pathways were studied using Western blotting. Knockdown of PARs protein was achieved by transfecting of siRNA. Chromatin immunoprecipitation assays were used to study in vivo binding of c-jun to the CCL2 promoter. Transient transfection was used to examine AP-1 activity. Stimulation of human primary osteoblasts and MG-63 cells with thrombin induced CCL2 expression. By using pharmacological inhibitors or activators or genetic inhibition by the PAR, siRNA revealed that the PAR1 receptor but not other PAR receptors is involved in thrombin-mediated up-regulation of CCL2. Thrombin-mediated CCL2 production was attenuated by thrombin inhibitor (PPACK), PKCδ inhibitor (rottlerin), c-Src inhibitor (PP2), EGFR inhibitor (AG1478), MEK inhibitors (PD98059 and U0126), or AP-1 inhibitors (curcumin and tanshinone IIA). Stimulation of cells with thrombin increased PKCδ, c-Src, EGFR, MEK, and ERK activation. Treatment of osteoblasts with thrombin also increased the c-Jun phosphorylation, AP-1-luciferase activity, and c-Jun binding to the AP-1 element on the CCL2 promoter. Our results suggest that the interaction between thrombin and PAR1 increases CCL2 expression in human osteoblasts via the PKCδ, c-Src, EGFR transactivation, MEK, ERK, c-Jun, and AP-1 signaling pathway. Part II Thrombin is a key mediator of fibrin deposition, angiogenesis, and proinflammatory processes. Abnormalities in these processes are primary features of rheumatoid arthritis and osteoarthritis. Matrix metalloproteinase-13 (MMP-13) may contribute to the breakdown of articular cartilage during arthritis. However, the role of thrombin in MMP-13 production in chondrocytes is unknown. In this study, we investigated the intracellular signaling pathways involved in thrombin-induced MMP-13 expression in human chondrocytes by using qPCR, western blotting, and ELISA. We found that stimulation with thrombin led to increased secretion of MMP-13 in cultured human chondrocytes. Further, this thrombin-induced MMP-13 production was reduced after transfection with siRNAs against protease activated receptors 1 and 3 (PAR1 and PAR3), but not with PAR4 siRNA, demonstrating that PAR1/PAR3 mediated the thrombin-induced increase in MMP-13 expression. Treatment with specific inhibitors for PKC???z?nc-Src, EGFR, PI3K, Akt, or AP-1 or with the corresponding siRNAs against these signaling proteins also abolished the thrombin-mediated increase in MMP-13 production in chondrocytes. In addition, stimulation of cells with thrombin increased PKC???z c-Src, EGFR, PI3K, Akt, and AP-1 activation. Furthermore, thrombin-mediated AP-1 activation was inhibited by the specific inhibitors or the specific siRNAs for PKC???z c-Src, EGFR, PI3K, and Akt cascades. Our results provide evidence that thrombin acts through the PAR1/PAR3 receptors and activates PKC?? and c-Src, resulting in EGFR transactivation, activation of PI3K, Akt, and finally AP-1 on the MMP-13 promoter, thereby contributing to cartilage destruction during arthritis.

Topic Category 醫藥衛生 > 社會醫學
醫學院 > 臨床醫學研究所博士班
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