Khan Rafiqul Islam, Yazawa Takashi, Anisuzzaman Abu Syed Md, Semba Shingo, Ma Yanju, Uwada Junsuke, Hayashi Hisayoshi, Suzuki Yuichi, Ikeuchi Hiroki, Uchino Motoi, Maemoto Atsuo, Muramatsu Ikunobu, Taniguchi Takanobu
Division of Cellular Signal Transduction, Department of Biochemistry, Asahikawa Medical University, Asahikawa, Japan; Department of Pharmacy, University of Rajshahi, Rajshahi, Bangladesh.
Division of Cellular Signal Transduction, Department of Biochemistry, Asahikawa Medical University, Asahikawa, Japan.
Biochim Biophys Acta. 2014 Apr;1842(4):635-45. doi: 10.1016/j.bbadis.2013.12.007. Epub 2013 Dec 21.
Impairment of epithelial barrier is observed in various intestinal disorders including inflammatory bowel diseases (IBD). Numerous factors may cause temporary damage of the intestinal epithelium. A complex network of highly divergent factors regulates healing of the epithelium to prevent inflammatory response. However, the exact repair mechanisms involved in maintaining homeostatic intestinal barrier integrity remain to be clarified. In this study, we demonstrate that activation of M1 muscarinic acetylcholine receptor (mAChR) augments the restitution of epithelial barrier function in T84 cell monolayers after ethanol-induced epithelial injury, via ERK-dependent phosphorylation of focal adhesion kinase (FAK). We have shown that ethanol injury decreased the transepithelial electrical resistance (TER) along with the reduction of ERK and FAK phosphorylation. Carbachol (CCh) increased ERK and FAK phosphorylation with enhanced TER recovery, which was completely blocked by either MT-7 (M1 antagonist) or atropine. The CCh-induced enhancement of TER recovery was also blocked by either U0126 (ERK pathway inhibitor) or PF-228 (FAK inhibitor). Treatment of T84 cell monolayers with interferon-γ (IFN-γ) impaired the barrier function with the reduction of FAK phosphorylation. The CCh-induced ERK and FAK phosphorylation were also attenuated by the IFN-γ treatment. Immunological and binding experiments exhibited a significant reduction of M1 mAChR after IFN-γ treatment. The reduction of M1 mAChR in inflammatory area was also observed in surgical specimens from IBD patients, using immunohistochemical analysis. These findings provide important clues regarding mechanisms by which M1 mAChR participates in the maintenance of intestinal barrier function under not only physiological but also pathological conditions.
在包括炎症性肠病（IBD）在内的各种肠道疾病中，均可观察到上皮屏障受损。众多因素可能导致肠上皮的暂时性损伤。一个由高度不同的因素组成的复杂网络调节上皮的愈合以防止炎症反应。然而，维持肠道屏障稳态完整性所涉及的确切修复机制仍有待阐明。在本研究中，我们证明，M1毒蕈碱型乙酰胆碱受体（mAChR）的激活通过粘着斑激酶（FAK）的ERK依赖性磷酸化，增强了乙醇诱导的上皮损伤后T84细胞单层中上皮屏障功能的恢复。我们已经表明，乙醇损伤降低了跨上皮电阻（TER），同时ERK和FAK磷酸化也减少。卡巴胆碱（CCh）增加了ERK和FAK磷酸化，并增强了TER的恢复，这被MT - 7（M1拮抗剂）或阿托品完全阻断。CCh诱导的TER恢复增强也被U0126（ERK通路抑制剂）或PF - 228（FAK抑制剂）阻断。用干扰素 - γ（IFN - γ）处理T84细胞单层会损害屏障功能，并降低FAK磷酸化。IFN - γ处理也减弱了CCh诱导的ERK和FAK磷酸化。免疫和结合实验显示，IFN - γ处理后M1 mAChR显著减少。使用免疫组织化学分析，在IBD患者的手术标本中也观察到炎症区域M1 mAChR的减少。这些发现为M1 mAChR不仅在生理条件下而且在病理条件下参与维持肠道屏障功能的机制提供了重要线索。
