Endothelin-1媒介慢性阻塞性氣喘結締組織生長因子表現的角色探討

Studies on the Role of Endothelin-1 in Mediating Connective Tissue Growth Factor Expression in Chronic Obstructive Asthma

翁志銘(Chin-Ming Weng)

指導教授 ： 陳炳常

共同指導教授 ： 林建煌(Chien-Huang Lin)

臺北醫學大學/醫學院/醫學科學研究所/博士(2013年)

摘要

下上皮纖維化是慢性阻塞性氣喘中氣道重塑的一個重要病理現象。慢性阻塞性氣喘的纖維球擁有較強的能力可以分化帶有α-smooth muscle actin (SMA) 的肌纖維母細胞，直接貢獻在肺部纖維化現象。結締組織生長因子 (connective tissue growth factor, CTGF) 的過度表現會出現在肺部纖維化的進程之中。內皮素-1 (Endothelin-1, ET-1) 是血管收縮以及組織修復的重要媒介物，可貢獻於下上皮纖維化反應。然而對於ET-1誘導CTGF表現以及纖維球分化的分子機制，目前了解仍不完全。在本論文中，我們除了發現在慢性阻塞性氣喘病人的氣道壁中有纖維球的聚集外，也發現了慢性阻塞性氣喘病人纖維球的CTGF呈現過度表現的情形。經過培養後，相較於正常人或是輕度氣喘的病人，慢性阻塞性氣喘病人纖維球的CTGF表現有大幅增加的趨勢。使用ETAR拮抗劑 (BQ123) 可抑制慢性阻塞性氣喘病人纖維球CTGF、α-SMA表現以及纖維球分化現象。我們也發現慢性阻塞性氣喘病人的血清ET-1與纖維球ETAR表現比正常人和輕度氣喘病人高。更進一步地，使用BQ123或是anti-CTGF抗體可以有效抑制ET-1誘導正常人纖維球α-SMA表現。
我們接下來繼續探討ETAR、JNK與轉錄因子AP-1在ET-1刺激人類纖維母細胞 (fibroblasts) CTGF表現的訊息傳遞機制中所扮演的角色。我們發現在人類胚胎肺部纖維母細胞株 (WI-38) 中，ET-1以濃度與時間相關性來刺激CTGF的蛋白表現。而ET-1誘導的CTGF蛋白表現可以被ETAR拮抗劑 (BQ123)，而不是ETBR拮抗劑 (BQ788) 所抑制。此外，ET-1誘導的CTGF蛋白表現亦可以被JNK抑制劑 (SP600125) 與JNK1/2顯性突變質體 (the dominant-negative mutants, DNs) 和AP-1抑制劑 (curcumin) 所抑制。ET-1會依時間相關性誘發JNK和c-Jun產生磷酸化現象。ET-1會刺激AP-1 luciferase活性增加，並具有濃度相關性，而此現象會被SP600125所抑制。我們也發現ET-1誘導的CTGF luciferase activity大部分受到其promoter上AP-1結合區域所調控，主要的調控區域在轉錄起始區上游-747到-408區域。進一步地，ET-1也能增加AP-1特異性DNA-蛋白複合體，並且吸引c-Jun與c-Fos結合至CTGF promoter。此外，我們發現ET-1誘導的α-SMA表現，可以被BQ123、SP600125、curcumin與anti-CTGF抗體所抑制。
綜合以上的結果，我們的研究第一次證實了ETAR依賴性路徑在CTGF表現過程中扮演重要的角色，以及CTGF會媒介纖維球以及纖維母細胞的分化反應，這些反應可能會參與在慢性阻塞性氣喘的下上皮纖維化過程之中。因此，我們研究得出的結果將提供在慢性阻塞性氣喘伴隨下上皮纖維化的治療性藥物開發過程中一個良好的標的。

關鍵字

內皮素-1 ； 內皮素A受體 ； 結締組織生長因子 ； 纖維球 ； 纖維母細胞 ； 慢性阻塞性氣喘

並列摘要

Subepithelial fibrosis is one important pathological phenomenon of airway remodeling in chronic obstructive asthma (COA). Fibrocytes possess increased differentiability into α-smooth muscle actin (SMA)+ myofibroblasts in COA, and contributes to pulmonary fibrosis. Overproduction of connective tissue growth factor (CTGF) underlies the development of lung fibrosis. Endothelin-1 (ET-1) acts as a key mediator of vasoconstriction and tissue repair, and contributes to subepithelial fibrosis. However, the molecular mechanisms of ET-1 in CTGF expression and fibrocyte differentiation are poorly understood. Herein, we show that not only the accmulation of fibrocytes in bronchial walls of COA patients, but also the overexpression of CTGF in fibrocytes from COA patients. After being cultured, CTGF expression was increased in fibrocytes from patients with COA, but not from those of normal subjects or asthma patients without airway obstruction. Treatment with the ETAR antagonist (BQ123), but not ETBR antagonist (BQ788), reduced the expressions of CTGF and α-SMA in fibrocytes and fibrocyte differentiation in patients with COA. Serum levels of ET-1 and the expression of the ETAR in fibrocytes were significantly higher in patients with COA compared to normal subjects and asthma patients without airway obstruction. Furthermore, treatment with BQ123 or an anti-CTGF antibody attenuated α-SMA expression induced by ET-1 in fibrocytes from normal subjects.
We further explore the role of ETAR, JNK, and transcription factor AP-1 in ET-1-induced CTGF expression in WI-38 cells. ET-1 caused concentration- and time-dependently increases in CTGF expression in human embryonic lung fibroblast cell line (WI-38). ET-1-induced CTGF expression was inhibited by BQ123 (ETAR antagonist), but not BQ788 (ETBR antagonist). Moreover, ET-1-induced CTGF expression was significantly reduced by JNK inhibitor (SP600125), the dominant-negative mutants of JNK1/2 (JNK1/2 DN), and AP-1 inhibitor (curcumin). ET-1 induced phosphorylations of JNK and c-Jun in time-dependent manners. AP-1-luciferase activity was concentration- dependently increased by ET-1, and this effect was attenuated by SP600125. We also found that ET-1-induced CTGF expression was most controlled by the AP-1 binding region of CTGF promoter. In particular, it was predominately controlled by the sequence -747 to -408 bp upstream of the transcription start site on the human CTGF promoter. Furthermore, ET-1 caused the formation of AP-1-specific DNA-protein complex and the recruitment of c-Jun to the CTGF promoter. Moreover, we found that ET-1-induced α-SMA expression was inhibited by BQ123, SP600125, curcumin, and anti-CTGF antibody.
Taken together, our study demonstrates for the first time that the ETAR-dependent pathway plays a crucial role in CTGF expression, and CTGF mediates the differentiation of fibrocyte and fibroblast, which may be involved in subepithelial fibrosis of COA. Therefore, the results from our study provide a target for therapeutic intervention in subepithelial fibrosis associated with COA.

並列關鍵字

Endothelin-1 ； ETA receptor ； connective tissue growth factor ； fibrocyte ； fibroblast ； chronic obstructive asthma

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