環孢素在牙齦上皮細胞中經由轉化生長因子β及離氨基氧化酶樣蛋白2 誘導上皮-間質轉換

Abstract

實驗目的和背景： 環孢素(Cyclosporin A)是一種常用在器官移植患者上的免疫抑制藥物，大約有七成服用此藥的病人會產生牙齦過度增生(Gingival overgrowth，GO)的副作用，進而影響到說話、吞嚥、咀嚼、口腔衛生、美觀等等，影響生活品質。轉化生長因子-β(Transforming growth factor，TGF-β) 在牙齦腫大的致病機轉中扮演著主要的角色，先前的研究中發現環孢菌素可經由TGF-β傳導路徑誘發上皮-間質轉換(epithelial-mesenchymal transition，EMT)現象，而使患者牙齦增生。其中離氨基氧化酶樣蛋白2 (Lysyl Oxidase-like protein 2, LOXL-2)：除催化膠原蛋白與彈性纖維的交叉共價鍵結(cross-linking)外，在一些研究中指出和EMT現象有關連。本研究期望能更加了解環孢素是否經由LOXL-2誘導人類牙齦上皮細胞產生EMT。 實驗方法： 本研究利用西方墨點法來檢測環孢素-A以及不同抑制劑處理後人類牙齦上皮細胞OECM-1 及 Ca9-22 之LOXL-2的表現以及EMT 標識蛋白Slug及E-cadherin的變化。 實驗結果： 於 OECM-1 及 Ca9-22 細胞加入環孢素素處理後，其 LOXL-2 的表現量隨之增加，OECM-1 細胞在4小時後達到最高值；CA9-22 細胞是在6小時後達到最高值。前處理TGF-β 中和抗體 (20μg/ml)、ALK5 抑制劑SB431542 (20μg/ml)、Smad-3 抑制劑SIS3 (5 μg/ml)，可以抑制OECM-1及CA9-22細胞受環孢素誘導的LOXL-2表現。顯示環孢素是經由TGF-β訊息傳導路徑誘導 OECM-1 及 CA9-22 之LOXL-2 的表現。前處理LOXL-2 的抑制劑 BAPN，可以顯著的抑制環孢素誘導的OECM-1 及 Ca9-22細胞上皮-間質轉換標識蛋白Slug的表現增加及E-cadherin的減少。顯示LOXL-2在Cyclosporin A誘導的EMT扮演重要的角色。 結論： 環孢素經由TGF-β及LOXL-2誘導EMT的產生。

Objectives: Cyclosporin A (CsA) is an immunosuppressive drug commonly used in organ transplant patients. Approximately 70% of the patients show gingival overgrowth (GO). Transforming growth factor (TGF-β) plays a major role in the GO. Previous studies have found that Cyclosporin A can induce epithelial-mesenchymal transition (EMT) via TGF-β signaling pathway, which then plays important roles in the pathogenesis of GO. Lysyl oxidase-like protein 2 (LOXL-2) : catalyzes the cross-linking of collagen and elastin to maintain the rigidity and stability of the extracellular matrix protein. Previous studies have shown LOXL-2 plays important roles in EMT. This study investigated the signaling pathways involved in the CsA-induced EMT. Material and Methods: Western blotting was used to examine the levels of LOXL-2 protein and the EMT marker protein E-cadherin and Slug in human gingival epithelial OECM-1 and Ca9-22 cells after treatment with CsA and various inhibitors. Results: CsA induced LOXL-2 in OECM-1 and Ca9-22 cells in a dose- and time- dependent manner. Pretreatment with TGF-β neutralizing antibody, ALK5 inhibitor SB431542 and Smad-3 inhibitor SIS3 almost completely inhibit cyclosporin A-induced LOXL-2 expression in OECM-1 and CA9-22 cells. These results suggest that CsA-induced LOXL-2 expression in gingival epithelial cells is mediated through TGFβ1 signaling. Pretreatment with LOXL-2 inhibitor BAPN significantly reduced CsA-induced Slug protein levels and reversed CsA-reduced E-cadherin expression in OECM-1 and CA9-22 cells. Conclusion: Cyclosporin A induced EMT through TGFβ signaling and LOXL-2 protein expression.