Serine Phosphorylated Claudin-7 Affects Epithelial Barrier In Human Colonic Cells And Modulated By Transient Receptor Potential Vanilloid 4

Background:UC is characterized by inflammation that comprises the colon epithelial barrier.Tight junction is an essential structure of colonic epithelium and Claudin-7 play a significant role.It has been reported that the abnormal phosphorylation of tight junction proteins can change the way of Claudin connection.In this way,the epithelial cell permeability will change,which is an important factor affecting the epithelial barrier.TRPV4 is one of the transient receptor potential superfamily(transient receptor potential vanilloid,TRPV4),intracellular Ca2+ will increased significantly when TRPV4 was activated.Increased Ca2+ combined with calmodulin kinase and affect the serine phosphorylation of lnfluencing the function of epithlium barrier.Therefore,we investigated the effect of serine phosphorylation of Claudin-7 on the function of colonic epithelial barrier and the regulation of TRPV4 channel on it.Objective:To investigate the effects of serine phosphorylation of Claudin-7 on colonic epithelial barrier function;To charify the regulation of TRPV4 channel on serine phosphorylation of Claudin-7 and affect the colonic epithelial barrier.Method: Western Blot was used to detect the expression of TRPV4 in healthy colonic tissues,UC patients colonic tissues and colonic epithelial cells;Co-immunoprecipitation to examine the interaction between TRPV4 and Caudin-7;NCM460 cells were stable transfected with wild-type Claudin-7 plasmid.Cell groups were named Vector,Cld7-wild,Cld7-m S204,Cld7-m S206 and Cld7-m S207;The location of Claudin-7 on the cells membrane was observed after TRPV4 agonist/inhibitor treatment by confocal microscopy;The levels of FD20 were detected through the monolayer of stable NM460 cells after TRPV4 agonist/inhibitor treatment;Stable NCM460 cells were grown to confluency and investigate the migration of cells by Wound healing assay.Result:(1)The TRPV4 were over expressed in colonic mucosas from IBD patients compared with control colonic mucosas.Cld7-m S204 group decreased the TRPV4 expression as compared to vector group and Cld7-m S204 group and Cld7-m S207 decreased the TRPV4 expression as compared to Cld7-m S206 group;(2)Compared with vector group,Cld7-m S204 group decreased the permeability for in monolayer colonic cells,while Cld7-m S206 and Cld7-m S207 were increased.Vector,Cld7-wild,Cld7-m S206 and Cld7-m S207 group increased the permeability for FD20 when TRPV4 agonist treated,monolayer colonic cells treated with agonist and inhibitor decreased the FD20 permeability in respective groups.In Cld7-m S207 group,no difference of FD20 permeability was found between agonist and inhibitor;(3)Cld7-m S204 and Cld7-m S207 group increased the TER as compared with vector and Cld7-wild.Inhibitor treatment significantly increased the TER in vector cells,cld7-wild cells and cld7-m S206 cells,as compared to agonist treated respective groups.However,in cld7-m S204 group and cld7-m S207 group,no difference of TER was found between GSK treatment and HC treatment;(4)Inhibitor treatment significantly decreased the migration in vector cells,cld7-wild cells and cld7-m S206 cells,as compared to agonist treated respective groups.However,in cld7-m S204 group and cld7-m S207 group,neither agonist nor inhibitor affected the TER in monolayer cells.Conclusion: In human colonic epithelial cells,mutation of Claudin-7 at position 204 increased the permeability of epithelial barrier for FD20;In colonic epithelial cells,TRPV4 regulate the serine phosphorylation of Claudin-7 at position 204 and 207 and change the level the serine phosphorylation of Claudin-7,to effect the permeability of epithelial barrier and migration of cells.