PEAK1 Deficiency Exacerbated Dss-induced Acute Colitis Through Src/FAK/Paxillin/Cortactin Complex Regulated Tight Junctions In Mice

PEAK1 is a non-receptor tyrosine protein kinase localized at actin cytoskeleton and other parts,but it is still unclear how PEAK1 affects actinanchored tight junctions and tight junctions-regulated ulcerative colitis.Spontaneous colitis phenotype was not observed in PEAK1 deficiency mice.However,loss of PEAK1 exacerbated DSS-induced colitis in mice,which was confirmed by body weight,survival rate,histopathology and inflammatory factors.Immunoblotting,immunofluorescence,immunoprecipitation and other methods were used to explore the underlying molecular mechanism in primary intestinal epithelial cells and intestinal epithelial cell lines according to the analysis of intestinal transcriptome sequencing.Our results showed that the proinflammatory factor IL-6 was increased significantly in PEAK1 deficiency mice with colitis;REG3? was declined in PEAK1-knock out intestinal epithelial cells;the phosphorylation of STAT3 Y705 was also elevated in PEAK1 deficiency intestinal epithelial cells.Hence,we propose the first hypothesis to explain the colitis: loss of PEAK1 decreases REG3? which is an inhibitor of JAK/STAT3 signaling pathway,and activated STAT3 signaling releases a large number of proinflammatory factors such as IL-6 to aggravated colitis.In the emphasized tight junctions-related experiments,knock out of PEAK1 significantly reduced the expression of tight junctions-related protein ZO-1 and occludin with little effect on the adhesion junctions-related protein Ecadherin;knockdown of PEAK1 significantly degraded ZO-1 by BAPTA in NCM460 cells;tyrosine phosphorylation of Src Y416,FAK Y397,Paxillin Y118 and Cortactin Y421 were distinctly declined in PEAK1 deficiency intestinal epithelial cells;PEAK1 combined ZO-1 and Src;overexpression of Src rescued ZO-1,occludin and the phosphorylation of and FAK Y397 and Paxillin Y118 in PEAK1 knock-down NCM460 cells.Therefore,we propose the second hypothesis: scaffold protein PEAK1 deficiency releases Src kinase which recruits its substrates FAK,Paxillin and Cortactin to maintain the tight junctions,and feeble tight junctions-related colitis was exacerbated in PEAK1 deficiency mice.