The Role of CFTR on Tendon Differentiation

肌腱是一種力學敏感性組織，其主要的功能是將力從肌肉傳導到骨骼，從而帶動人體的運動。臨床上，常見的肌腱損傷疾病通常是由於肌腱的過度使用而引起的， 比如肌腱炎和肌腱斷裂。然而，由於目前有關肌腱生物學方面的了解十分有限，如何更好地治療肌腱損傷一直是臨床上的難題。CFTR (cystic fibrosis transmembrane conductance regulator) 是一種常見的氯離子通道，發生在編碼該蛋白序列上的基因突變會導致一種名為囊腫型纖維化的疾病 (Cystic Fibrosis)，並好發於具有北歐血統的人群。該基因突變會導致合成的CFTR 蛋白由內質網運輸至細胞膜上的過程受阻，從而在內質網上降解。據報導，患有該疾病的人群中，其骨骼肌系統也呈現出各種症狀，常見的有骨量降低、軟骨畸形、肌肉無力等。最近，這種離子通道被證明同時具有力學敏感特性，說明其在力學傳導中也發揮一定作用。基於CFTR 的力學敏感性和其在骨骼肌系統的重要作用，本課題旨在研究CFTR 在肌腱分化和肌腱損傷治療中的作用。我們發現，在用力學刺激肌腱分化的過程中CFTR 呈明顯上升趨勢；同時肌腱組織在CFTR 突變小鼠 (DF508) 中其細胞排列呈不規則化，肌腱纖維排列紊亂並且直徑大小各異，力學特性也明顯低於野生型小鼠，同時損傷後的肌腱癒合能力也明顯減弱。另外，通過對比從兩種小鼠體內提取的肌腱幹細胞發現，DF508 小鼠內有關肌腱和其相關的細胞外基質的基因表達水平和膠原形成的能力明顯低於野生型小鼠。通過RNA 測序和免疫共沉澱，我們發現位於細胞膜上的CFTR 可以和胞漿內的β-catenin 直接結合，而這種相互結合的關係在DF508 小鼠中被減弱，從而引起細胞內β-catenin 的表達水平升高，並相繼引起下游基因水平的變化。我們隨後證實pERK1/2 的表達水平也在DF508 小鼠體內的mTDSCs 升高。通過使用pERK1/2 的抑製劑，UO126，我們發現其可促進正常肌腱幹細胞的分化和肌腱損傷的修復；而在DF508 小鼠中，UO126可以部分促進肌腱幹細胞的分化和肌腱損傷的修復，這種現像也再次證明CFTR 通過Wnt/β-catenin 和pERK1/2 這條通路來調控肌腱幹細胞的分化，從而引起肌腱和其相關的細胞外基質的基因的異常表達，进而调节損傷癒合。總的來說，本課題發現了一條新的促進肌腱分化的信號通路。即，由CFTR 調控的Wnt/β-catenin 和pERK1/2 信號通路，通過使用pERK1/2 的抑製劑，UO126，可以促進肌腱幹細胞更好地向肌腱分化並同時促進損傷肌腱的癒合。這為今後有關肌腱疾病的臨床治療和基礎研究方面提供了更好的參考和依據。Tendon is a mechanosensitive tissue which enables it to transmitting forces derived from muscle to bone. Overuse of tendon tissues cause injures such as tendinopathy or tendon rupture. However, the repair of tendon injures remains a big challenge due to limited understanding on tendon biology.The cystic fibrosis transmembrane conductance regulator (CFTR) is a well-known chloride ion channel. In clinical, mutation of CFTR gene caused the disease of cystic fibrosis (CF). The musculoskeletal system also showed abnormal in patients with CF, such as low bone mineral density, cartilage abnormality and muscle wasting. Recently, CFTR has been reported being the mechanosensitive gating apart from the ligand-gated ion channel. Due to the mechanosensitive gating properties and essential roles on musculoskeletal system of CFTR, we hypothesize that CFTR may play a significant role in tendon differentiation and tendon repair.In our study, CFTR was found to be up-regulated during tendon differentiation by mechanical stretching in vitro. Tendons from CFTR mutation mice (DF508) exhibited irregular cell arrangement, uneven fibril diameter distribution, weak mechanical properties and delayed tendon injury healing when compared with wild-type (WT). Moreover, both tendon tissues and tendon derived stem cells (TDSCs) isolated from DF508 mice also showed significantly decreased expression of tendon and extracellular matrix (ECM) markers such as Scleraxis, Col1A1, and Decorin. By RNA-sequencing analysis and co-immunoprecipitation assay, we demonstrated that Wnt family member β-catenin directly interacted with CFTR, and mutation of CFTR could stimulate both Wnt/β-catenin and pERK1/2 signaling pathways. Furthermore, tendon differentiation and tendon injury healing could be promoted by inhibition of pERK1/2 signaling pathway both in vivo and in vitro.Taken together, our study demonstrated that dysfunction of CFTR could inhibit tendon differentiation through activation of both Wnt/β-catenin and pERK1/2 signaling pathways. Disruption of the activated Wnt/β-catenin and pERK1/2 signaling may be helpful to develop a therapeutic strategy for tendon injury.Liu, Yang.Thesis Ph.D. Chinese University of Hong Kong 2016.Includes bibliographical references (leaves ).Abstracts also in Chinese.Title from PDF title page (viewed on …).Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only