Pulmonary Endothelial Cell Barrier Enhancement by Sphingosine 1-Phosphate: ROLES FOR CORTACTIN AND MYOSIN LIGHT CHAIN KINASE

Steven M Dudek,Jeffrey R Jacobson,Eddie T Chiang,Konstantin G Birukov,Peiyi Wang,Xi Zhan,Joe G.N Garcia

doi:10.1074/jbc.m313969200

Abstract

We recently reported the critical importance of Rac GTPase-dependent cortical actin rearrangement in the augmentation of pulmonary endothelial cell (EC) barrier function by sphingosine 1-phosphate (S1P). We now describe functional roles for the actin-binding proteins cortactin and EC myosin light chain kinase (MLCK) in mediating this response. Antisense down-regulation of cortactin protein expression significantly inhibits S1P-induced barrier enhancement in cultured human pulmonary artery EC as measured by transendothelial electrical resistance (TER). Immunofluorescence studies reveal rapid, Rac-dependent translocation of cortactin to the expanded cortical actin band following S1P challenge, where colocalization with EC MLCK occurs within 5 min. Adenoviral overexpression of a Rac dominant negative mutant attenuates TER elevation by S1P. S1P also induces a rapid increase in cortactin tyrosine phosphorylation (within 30 s) critical to subsequent barrier enhancement, since EC transfected with a tyrosine-deficient mutant cortactin exhibit a blunted TER response. Direct binding of EC MLCK to the cortactin Src homology 3 domain appears essential to S1P barrier regulation, since cortactin blocking peptide inhibits both S1P-induced MLC phosphorylation and peak S1P-induced TER values. These data support novel roles for the cytoskeletal proteins cortactin and EC MLCK in mediating lung vascular barrier augmentation evoked by S1P.

Highlights

We recently reported the critical importance of Rac GTPase-dependent cortical actin rearrangement in the augmentation of pulmonary endothelial cell (EC) barrier function by sphingosine 1-phosphate (S1P)
Cortactin and EC myosin light chain kinase (MLCK) in S1P Barrier Enhancement tion
Barrier-enhancing Stimuli Translocate Cortactin to the EC Periphery—We explored cortactin’s role in EC barrier regulation via immunofluorescent (IF) cellular localization of cortactin in cells exposed to agents known to alter EC permeability

Summary

Introduction

We recently reported the critical importance of Rac GTPase-dependent cortical actin rearrangement in the augmentation of pulmonary endothelial cell (EC) barrier function by sphingosine 1-phosphate (S1P). Direct binding of EC MLCK to the cortactin Src homology 3 domain appears essential to S1P barrier regulation, since cortactin blocking peptide inhibits both S1P-induced MLC phosphorylation and peak S1Pinduced TER values. These data support novel roles for the cytoskeletal proteins cortactin and EC MLCK in mediating lung vascular barrier augmentation evoked by S1P. The functional role of phosphorylated cortactin is unclear, but we recently reported an increased direct association of phosphocortactin with the barrier regulatory enzyme, EC myosin light chain kinase (MLCK), relative to nonphosphorylated cortactin [8].

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Conclusion