| 摘要: | 約有10 %的氣喘患者對類固醇治療無反應，並伴隨嚴重的呼吸道重塑及呼吸道阻塞，被定義為嚴重氣喘(severe asthma, SA)。先前有研究指出Interleukin-6 (IL-6)參與在發炎反應及肺部纖維化。Casein kinase 2 (CK2)透過調控β-catenin signaling參與在bleomycin (BLM)誘導的小鼠肺纖維化中。Ying Yang 1 (YY1)在特發性肺纖維化(idiopathic pulmonary fibrosis, IPF)患者中過度表現。但在IL-6誘導CTGF表現的途徑中，CK2及YY1的分子機制仍不清楚。因此我們將探討在人類肺纖維母細胞中，CK2及YY1調控IL-6誘導CTGF表現之角色。在本研究中，我們發現在人類肺纖維母細胞中，CK2抑制劑apigenin抑制IL-6誘導的CTGF表現有濃度關係，且轉染CK2α、CK2α’以及CK2β的siRNA都會抑制IL-6誘導的CTGF表現。進一步研究發現IL-6誘導CK2β以及CK2α/α’磷酸化，且轉染CK2β siRNA可以抑制CK2α及CK2α’磷酸化。利用免疫螢光染色法發現，在IL-6刺激後CK2α會由細胞質進入細胞核中，而CK2α’以及CK2β則不會。此外，轉染YY1 siRNA後能夠抑制IL-6誘導的CTGF表現，進一步發現IL-6可誘導YY1磷酸化。接續利用共同免疫沉澱法發現，在IL-6刺激後CK2α會與YY1結合形成complex，而CK2α’與CK2β則不會與YY1結合。我們進一步利用染色質免疫沉澱法發現以IL-6刺激後CK2α及YY1會結合在CTGF promoter上的YY1 binding site，CK2α’與CK2β則不會。綜上所述，在人類肺纖維母細胞中，IL-6透過活化CK2α及YY1，並使CK2α及YY1形成complex，結合至CTGF promoter上，進而促進CTGF表現。說明CK2α及YY1可能是未來發展肺纖維化治療的標的。
About 10 % of asthma patients have steroids-insensitive along with airway remodeling and airway obstruction, which is defined as severe asthma. Previous studies suggested that interleukin-6 (IL-6) was involved in inflammation and pulmonary fibrosis. Casein kinase 2 (CK2) was involved in bleomycin (BLM)-induced pulmonary fibrosis in mice by regulation of β-catenin signaling. Ying Yang 1 (YY1) was overexpressed in patients with idiopathic pulmonary fibrosis (IPF). However, the molecular mechanism of CK2 and YY1 in IL-6-induced CTGF expression remain unclear. Therefore, we will explore the role of CK2 and YY1 involved in IL-6-induced CTGF expression in human lung fibroblasts. In this study, we found that CK2 inhibitor, apigenin, inhibited IL-6-induced CTGF expression in human lung fibroblasts in a dose-dependent manner. Transfection with CK2α, CK2α’ and CK2β siRNA attenuated IL-6-induced CTGF expression. Furthermore, IL-6 increased CK2β and CK2α/α’ phosphorylation. Transfection of CK2β siRNA inhibited the phosphorylation of CK2α and CK2α’. Using immunofluorescence, we found that IL-6 induced the translocation of CK2α from cytosol to nucleus, but not CK2α’ and CK2β. Moreover, IL-6-induced CTGF expression was decreased by the transfection of YY1 siRNA. IL-6 increased YY1 phosphorylation in time-dependent manner. Subsequently, using the co-immunoprecipitation, we found that IL-6 stimulated CK2α and YY1 formed a transcriptional complex, while CK2α’ and CK2β would not bind to YY1. Moreover, using ChIP assay, we found that IL-6 stimulated CK2α and YY1 binding to YY1 binding site at CTGF promoter, but not CK2α’ and CK2β. In conclusion, IL-6 activates CK2α and YY1, then stimulates formation of CK2α and YY1 complex and recruitment to the CTGF promoter region, and eventually promotes the production of CTGF in human lung fibroblasts. CK2α and YY1 might be a therapeutic target of pulmonary fibrosis. |
