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J. Biol. Chem., Vol. 280, Issue 17, 17286-17293, April 29, 2005

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Activated Protein C Mediates Novel Lung Endothelial Barrier Enhancement

ROLE OF SPHINGOSINE 1-PHOSPHATE RECEPTOR TRANSACTIVATION^*

James H. Finigan, Steven M. Dudek, Patrick A. Singleton, Eddie T. Chiang, Jeffrey R. Jacobson, Sara M. Camp, Shiu Q. Ye, and Joe G. N. Garcia

From the Center for Translational Respiratory Medicine, Division of Pulmonary and Critical Care Medicine, Johns Hopkins University, Baltimore, Maryland 21224

Increased endothelial cell (EC) permeability is central to the pathophysiology of inflammatory syndromes such as sepsis and acute lung injury (ALI). Activated protein C (APC), a serine protease critically involved in the regulation of coagulation and inflammatory processes, improves sepsis survival through an unknown mechanism. We hypothesized a direct effect of APC to both prevent increased EC permeability and to restore vascular integrity after edemagenic agonists. We measured changes in transendothelial electrical resistance (TER) and observed that APC produced concentration-dependent attenuation of TER reductions evoked by thrombin. We next explored known EC barrier-protective signaling pathways and observed dose-dependent APC-mediated increases in cortical myosin light chain (MLC) phosphorylation in concert with cortically distributed actin polymerization, findings highly suggestive of Rac GTPase involvement. We next determined that APC directly increases Rac1 activity, with inhibition of Rac1 activity significantly attenuating APC-mediated barrier protection to thrombin challenge. Finally, as these signaling events were similar to those evoked by the potent EC barrier-enhancing agonist, sphingosine 1-phosphate (S1P), we explored potential cross-talk between endothelial protein C receptor (EPCR) and S1P₁, the receptors for APC and S1P, respectively. EPCR-blocking antibody (RCR-252) significantly attenuated both APC-mediated barrier protection and increased MLC phosphorylation. We next observed rapid, EPCR and PI 3-kinase-dependent, APC-mediated phosphorylation of S1P₁ on threonine residues consistent with S1P₁ receptor activation. Co-immunoprecipitation studies demonstrate an interaction between EPCR and S1P₁ upon APC treatment. Targeted silencing of S1P₁ expression using siRNA significantly reduced APC-mediated barrier protection against thrombin. These data suggest that novel EPCR ligation and S1P₁ transactivation results in EC cytoskeletal rearrangement and barrier protection, components potentially critical to the improved survival of APC-treated patients with severe sepsis.

Received for publication, November 3, 2004 , and in revised form, January 24, 2005.

^* This work was supported by Grant HL 58064 from the NHLBI, National Institutes of Health, the Johns Hopkins Eudowood fellowship, and the Dr. David Marine Endowment. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of Grant F1K-US-V010 from Eli Lilly and Co.

To whom correspondence should be addressed: Dept. of Medicine, University of Chicago Pritzker School of Medicine, 5841 S. Maryland Ave., W604, Chicago, IL 60637. Tel.: 773-702-1051; Fax: 773-702-4427; E-mail: jgarcia{at}medicine.bsd.uchicago.edu.

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