The protective role of MLCP-mediated ERM dephosphorylation in endotoxin-induced lung injury in vitro and in vivo

Abstract

The goal of this study was to investigate the role of MLC phosphatase (MLCP) in a LPS model of acute lung injury (ALI). We demonstrate that ectopic expression of a constitutively-active (C/A) MLCP regulatory subunit (MYPT1) attenuates the ability of LPS to increase endothelial (EC) permeability. Down-regulation of MYPT1 exacerbates LPS-induced expression of ICAM1 suggesting an anti-inflammatory role of MLCP. To determine whether MLCP contributes to LPS-induced ALI in vivo, we utilized a nanoparticle DNA delivery method to specifically target lung EC. Expression of a C/A MYPT1 reduced LPS-induced lung inflammation and vascular permeability. Further, increased expression of the CS1 beta (MLCP catalytic subunit) also reduced LPS-induced lung inflammation, whereas the inactive CS1 beta mutant increased vascular leak. We next examined the role of the cytoskeletal targets of MLCP, the ERM proteins (Ezrin/Radixin/Moesin), in mediating barrier dysfunction. LPS-induced increase in EC permeability was accompanied by PKC-mediated increase in ERM phosphorylation, which was more prominent in CS1 beta-depleted cells. Depletion of Moesin and Ezrin, but not Radixin attenuated LPS-induced increases in permeability. Further, delivery of a Moesin phospho-null mutant into murine lung endothelium attenuated LPS-induced lung inflammation and vascular leak suggesting that MLCP opposes LPS-induced ALI by mediating the dephosphorylation of Moesin and Ezrin.