The Role Of Cystic Fibrosis Transmembrane Regulator(CFTR) Chloride Channel In Epithelial Cell Wound Healing

Epithelial wound repair is a continuous,complex and well-defined process that is initiated by proliferation and migration of cells bodering the wound.Physiologicl processe of wound healing involves several physiological mechanisms that precisely regulated by cytokines and other signaling molecules.Rencently,several reports showed that cystic fibrosis transmembrane conductance regulator(CFTR)plays important roles in the process of wound healing,although precise molecular mechanisms remains largely unclear.CFTR is a cAMP-activated chloride channel protein that presents in a wide range of plasma membrane of epithelial cells that involve in fluid secretion and absorption.In the present study,role of CFTR chloride channel in epithelial wound healing was investigated using the Fisher rat thyroid epithelial cells(FRT)expressing wild-type and F508 del mutant CFTR.Because asthma is accompanied by airway epithelial injury and repair,we established an ammonia damage model of airway for further in vivo study of the issue.Role of CFTR in FRT cell proliferation and migration was initially measured using MTT assay and cell wound healing assay,respectively.Cell apoptosis was measured using flow cytometry,Hoechst 33342 staining and western blotting methods.The results showed that:(1)Proliferation rate of FRT cells was significantly affected by types of CFTR expressed in the cells,in which: FRT/wt-CFTR > FRT/F508del-CFTR > FRT/CFTR-null;(2)CFTR specific inhibitor GlyH-101 significantly inhibited the proliferation rate of FRT/wt-CFTR cells,and the effect manifested time-and dose-dependent manners;(3)Hoechst 33342 staining results showed that GlyH-101 caused intracellular DNA fragmantention and apoptotic bodies formation in the FRT/wt-CFTR cells.(4)Western blotting studies indicated that GlyH-101 induced down-regulation of the expression of Bcl-2 protein and upregulation of the expression of Bax,caspase-3,caspase-8 and PARP proteins;(5)PI staining results showed that GlyH-101 induced FRT/wt-CFTR cell cycle arrest at S phase;(6)Western blotting experiments showed that GlyH-101 induced down-regulation expression of cell cycle-related proteins CDK4,CDK6,Cyclin D1 and Cyclin E proteins;(7)GlyH-101 significantly inhibited FRT/wt-CFTR cells migration in time-and dose-dependent manners;(8)Western blotting experiments showed that GlyH-101 down-regulated the intracellular expression of MMP-2 protein;(9)Successfully made ammonia-induced airway epithelial damage model inmice,which provide technique basis for further in vivo study the role of CFTR in wound healing.In conclusion,the present study indicated that CFTR chloride channel play important roles in cell proliferation and migration.CFTR specific inhibitor GlyH-101 inhibited FRT/wt-CFTR cells proliferation through cell cycle arrest and mitochondrial apoptosis induction pathways and inhibited FRT/wt-CFTR cell migration by down-regulation the expression of MMP-2 protein.