ZIPK Regulates Endothelial Cell Permeability By Regulating MLC Phosphorylation

Endothelial cel(EC)contraction increases the intercellular gap and increases the endothelial permeability,but the mechanism of regulation of EC contraction remains unclear.Studies have reported that EC contraction is initiated by the phosphorylated myosin light chain(MLC)catalyzed through myosin light chain kinase(MLCK).However,our previous studies on MLCK knockout mouse showed that MLCK is not required for MLC phosphorylation and EC permeability.Our study investigated the effect of zipper-interacting protein kinase(ZIPK)on the regulat io n of MLC phosphorylation and hence contraction of ECs.Inhibition of ZIPK by pharmacological inhibitor-Staurosporine(STS),si RNA knockdown ZIPK,it was found that a significantly decrease in both MLC mono-and di-phosphorylation and the contractile response to thrombin,showing an important role of ZIPK in EC paracellular permeability.Because of embryonic lethality in endothelium-specific knockout mouse lines(Tie2-cre),in order to study the in vivo function of ZIPK in endothelium,we used tamoxifen-induced systemic Zipk knockout mouse lines(UBC-cre ^{ERT 2};Zipk^fl/fl)to establish The mouse middle cerebral artery occlusion(MCAO)model.The results by the TTC(2,3,5-triphenyltetrazolium chloride)staining showed that the lesion area of the brains of the deletion of ZIPK mouse was significantly smal er than that of control littermates after 24 hours of reperfusion at 1 hour of ischemia.Also,Evans blue staining showd that the damage of the blood-brain barrier in the deletion of ZIPK mouse was significantly smal er.Fourthermore,the results of neurobehavioral studies showed that the deletion of ZIPK had a significant protective effect on ischemia/reperfusio n-induced nerve injury.In summary,the results of this study indicate that ZIPK is a key molecule in the regulation of endothelial cell contraction and MLC phosphorylation.ZIPK may be a prospective therapeutic target for ischemic/reperfusion injury and other human endothelial hypermeability related diseases.