Regulation Of Intestinal Epithelial Permeability By Myosin Phosphatase Target Subunit 1(MYPT1)

Background:The pathogenesis of inflammatory bowel disease(IBD)remains unclear.Previous studies have shown that the occurrence of IBD is closely related to the impairment of intestinal epithelial barrier function.The intestinal mucosal barrier can resist the invasion of foreign harmful substances into the intestinal tract.The barrier function of the intestinal mucosa is mainly regulated by the permeability of the tight junctions between epithelial cells and the integrity of the epithelial cells.Myosin phosphatase(or myosin light chain phosphatase,MLCP)is composed of the catalytic subunit PP1cδand the regulatory subunit MYPT1(myosin phosphatase target subunit 1).MYPT1can target the catalytic subunit PP1cδ to the substrate and enhance the catalytic activity of PP1cδ.MLCP can mediate the dephosphorylation of the myosin light chain(MLC)and can regulate the contraction of the actomysoin ring,which may be associated with the permeability of intestinal epithelial tight junction.However,the regulatory role of MLCP,especially MYPT1,in intestinal epithelial tight junction permeability and intestinal mucosal barrier function remains unclear.Aims:In previous experiments,we conducted experiments with intestinal epithelialspecific Mypt1 knock-down mice(Mypt1+/f;villin-Cre,Myptl haploinsufficiency).We found that the intestinal mucosal recovery of Mypt1+/f;villin-Cre mice were impaired after DSS-induced colitis.We speculated that MYPT1 knock-down may decrease the tightness of intestinal epithelial cell tight junction,thus hinder the repair of the injured intestinal mucosa and aggravate enteritis symptoms in mice.This study further clarifies the molecular mechanism of MYPT1 in the regulation of intestinal epithelial permeability and discusses the relationship between MYPT1 and the occurrence and development of colitis,which provided a theoretical basis for the clinical use of MYPT1 as a target to treat IBD.Methods:1.At the cellular level,Caco-2BBe cells were infected with lentivirus to obtain Caco-2BBe-shMYPT1 cells in which the expression of Mypt1 was knocked down.Using Caco-2BBe-shMYPT1 cells,the influence of Myptl knockdown on the intestinal permeability and tight junction protein expression was investigated by fluorescein isothiocyanate(FITC)-dextran trans-epithelial permeability assay,transepithelial electrical resistance(TER),and western blot.These experiments were carried out to explore the molecular mechanism of MYPT1 in intestinal permeability regulation.2.In animal experiments,Mypt1+/f;villin-Cre mice model were used to detect the gene of inflammatory factors and cell necroptosis proteins,and the pathological section analysis of intestinal tissue were analyzed to explore the effect of Mypt1 on the intestinal tract of mice.In vitro experiments,we use the intestinal tissues to examine the effect of decreased Mypt1 expression on intestinal epithelial permeability and tight junction proteins expression.3.Use Dextran sulfate sodium(DSS)to make a colitis model experiment on Mypt1+/f;villin-Cre mice,record the weight change of the mice every day,and make a weight change chart.In vitro molecular detection,we use the intestinal tissues and intestinal epithelial cells to detect the expression of inflammatory factors,tight junction proteins.Results:1.In Caco-2BBe cells,knock down of Mypt1 reducedTER significantly(cells transfected with shControl,CTR,202.0± 4.9 Ohm*cm2;cells transfected with shMYPT1,152.7 ± 3.2 Ohm*cm2;P<0.001),while the FITC-dextran assay showed that its permeability increased(cells transfected with shControl,CTR,0.007 ± 0.002 mg/mL;cells transfected with shMYPT1,0.020±0.002 mg/mL;P<0.05).2.In vitro experiments,the expression of Mypt1 was decreased in Caco-2BBeshMYPT1 cells(cells transfected with shControl,CTR,1.00± 0.07;cells transfected with shMYPT1,0.19 ± 0.05;P<0.0001,data were normalized to CTR).The expression of Occludin and ZO-1 protein reduced significantly(ZO-1:cells transfected with shControl,CTR,1.00 ± 0.07;cells transfected with shMYPT1,0.54 ± 0.11,Occludin:cells transfected with shControl,CTR,1.00 ± 0.08;cells transfected with shMYPT1,0.78 ± 0.04;P<0.05,data were normalized to CTR).Real-Time qPCR results showed that pore-forming protein Claudin2 was significantly increased(cells transfected with shControl,CTR,1.00 ± 0.04;cells transfected with shMYPT1,1.55± 0.12;P<0.01,data were normalized to CTR).3.In animal experiments,there is no difference between wild type mice and Mypt1+/f;villin-Cre mice on inflammatory factors(eg.TNF-α、IL-2、IL-1β、IL-4、IL-23、IL-5)and cell necroptosis gene(eg.Fadd,Ripk3,Mlkl,and Ripk1).However,the expression of tight junction protein ZO-lin intestinal epithelium decreased(wildtype mice,1.55 ± 0.12;Mypt1+/f;villin-Cre mice,0.42 ± 0.09;P<0.01,data were normalized to wildtype mice),and FITC-dextran expression showed that cell permeability increased in Mypt1+/f;villin-Cre mice(wildtype mice,1.00± 0.05;Mypt1+/f;villin-Cre mice,1.84±0.26;P<0.05,data were normalized to wildtype mice).4.When induced with DSS to make a colitis model,Mypt1+/f;villin-Cre mice were more sensitive to colitis,and intestinal mucosal recovery was impaired,with elevated inflammatory factor expression.The expression of inflammatory factors TNF-α,IL-2,IL-1β increased comparedcompared with wild mice(TNF-α:wildtype mice,1.00 ± 0.33;Mypi1+/f;villin-Cre mice,3.15±0.24;IL-1β:wildtype mice,1.00±0.24;Mypt1+/f;villin-Cre mice,2.95±0.60;IL-2:wildtype mice,1.00±0.25;Mypt1+/f;villinCre mice,3.79±0.81;P<0.05,data were normalized to wildtype mice).5.In cell experiments,western blot showed that MLC protein expression was decreased(cells transfected with shControl,CTR,1.00 ± 0.25;cells transfected with shMYPT1,0.24± 0.05;P<0.01,data were normalized to CTR),and p-MLC/MLC ratio increased(cells transfected with shControl,CTR,the p-MLC/MLC ratio was 1.00 ±0.49;cells transfected with shMYPT1,the p-MLC/MLC ratio was 3.69±1.82;P<0.05,data were normalized to CTR).The Mlc mRNA expression was also decreased in Mypt1+/f;villin-Cre mice(wildtype mice,1.00± 0.22;Mypt1+/f;villin-Cre mice,0.07±0.04,P<0.01,data were normalized to wildtype mice).Conclusions:In this study,cell and animal experiments show that knocking down Myptl can down-regulate the expression of tight junction protein,increase the permeability of intestinal epithelial cells,and thus lead to the damage of the intestinal mucosal barrier.The decrease of MYPT1 expression leads to the increase of MLC phosphorylation level,the contraction of the actomyosin ring,the increase of epithelial tight junction permeability,and the indirect decrease of tight junction protein.Under normal physiological conditions,the decrease of MYPT1 expression does not directly lead to colitis,but is very sensitive to DSS-induced colitis.