Critical Role of S1PR1 and Integrin β4 in HGF/c-Met-mediated Increases in Vascular Integrity

Yulia Ephstein,Patrick A Singleton,Weiguo Chen,Lichun Wang,Ravi Salgia,Prasad Kanteti,Steven M Dudek,Joe G.N Garcia,Jeffrey R Jacobson

doi:10.1074/jbc.m112.404780

Abstract

Vascular endothelial cell (EC) barrier integrity is critical to vessel homeostasis whereas barrier dysfunction is a key feature of inflammatory disorders and tumor angiogenesis. We previously reported that hepatocyte growth factor (HGF)-mediated increases in EC barrier integrity are signaled through a dynamic complex present in lipid rafts involving its receptor, c-Met. We extended these observations to confirm that S1PR1 (sphingosine 1-phosphate receptor 1) and integrin β4 (ITGB4) are essential participants in HGF-induced EC barrier enhancement. Immunoprecipitation experiments demonstrated HGF-mediated recruitment of c-Met, ITGB4 and S1PR1 to caveolin-enriched lipid rafts in human lung EC with direct interactions of c-Met with both S1PR1 and ITGB4 accompanied by c-Met-dependent S1PR1 and ITGB4 transactivation. Reduced S1PR1 expression (siRNA) attenuated both ITGB4 and Rac1 activation as well as c-Met/ITGB4 interaction and resulted in decreased transendothelial electrical resistance. Furthermore, reduced ITGB4 expression attenuated HGF-induced c-Met activation, c-Met/S1PR1 interaction, and effected decreases in S1P- and HGF-induced EC barrier enhancement. Finally, the c-Met inhibitor, XL880, suppressed HGF-induced c-Met activation as well as S1PR1 and ITGB4 transactivation. These results support a critical role for S1PR1 and ITGB4 transactivation as rate-limiting events in the transduction of HGF signals via a dynamic c-Met complex resulting in enhanced EC barrier integrity.

Highlights

Hepatocyte growth factor (HGF) is a potent mediator of endothelial barrier enhancement, the mechanisms are poorly understood
To examine the role of sphingosine 1-phosphate receptor 1 (S1PR1) in hepatocyte growth factor (HGF)-induced endothelial cell (EC) barrier enhancement, human pulmonary artery EC (HPAEC) were challenged with HGF (25 ng/ml, 5 min) and whole cell lysates utilized to immunoprecipitate S1PR1 under non-denaturing conditions
Having established that S1PR1 activation is downstream of HGF/ c-Met/phosphatidylinositol-3 kinase (PI3K) signaling, we investigated whether Rac1, the small GTPase activated by S1PR1 ligation, participates in HGF-induced barrier enhancement [1]

Summary

Background

Hepatocyte growth factor (HGF) is a potent mediator of endothelial barrier enhancement, the mechanisms are poorly understood. We previously reported that hepatocyte growth factor (HGF)-mediated increases in EC barrier integrity are signaled through a dynamic complex present in lipid rafts involving its receptor, c-Met [1]. We extended these observations to confirm that S1PR1 (sphingosine 1-phosphate receptor 1) and integrin ␤4 (ITGB4) are essential participants in HGF-induced EC barrier enhancement. The c-Met inhibitor, XL880, suppressed HGF-induced c-Met activation as well as S1PR1 and ITGB4 transactivation These results support a critical role for S1PR1 and ITGB4 transactivation as rate-limiting events in the transduction of HGF signals via a dynamic c-Met complex resulting in enhanced EC barrier integrity. Agents that protect vascular endothelial cell (EC) barrier properties and

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EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION