The work detailed in this proposal is aimed at further elucidating the mechanisms behind ALI and ARDS, which affect over 200,000 Americans each year and kill over 75,000 annually. Disruption of cell-cell and cell-extracellular matrix (ECM) contacts is a major hallmark of these diseases, leading to increased endothelial permeability and lung edema. Our lab has previously demonstrated a role for protein kinase C8 (PKC8) in enhancing microvascular endothelial basal barrier function through increased focal adhesion formation and modulation of RhoA activity, possibly through a p190RhoGAP (p190)-dependent pathway. Protein tyrosine phosphatases (PTPs) have been shown to affect integrin-mediated cell-cell and cell-ECM adhesion. The SH2 domain-containing PTP, SHP2, has been specifically implicated in regulating endothelial barrier function through inhibition of c-Src and its known effector, p190. The following specific aims will investigate our overall hypothesis that SHP2, activated by PKC8, regulates pulmonary endothelial basal barrier function in vitro and in vivo through inhibition ofc-Src, resulting in maintenance of active RhoA-GTP, and stabilization of adherens and tight junctions.
Aim I will examine if SHP2 regulates endothelial barrier function in vitro through inhibition of c-Src, maintenance of RhoA-GTP, and stabilization of intercellular adherens and tight junctions. Immunoprecipitation (IP) assays in conjunction with immunoblotting (IB), in vitro kinase and phosphatase assays, and RhoA and p190 activity assays, will be employed to assess the interaction of SHP2with c-Src and the effects of this interaction on their respective activities. The electrical cell impendence sensor (ECIS) technique will be used to assess overall monolayer permeability, and immunofluorescent staining will be used to assess junctional integrity.
Aim II will determine if PKC8 indirectly regulates endothelial barrier function through modulation of SHP2. Following manipulation of PKC8, IP and IB will be used to examine activation of SHP2 and c-Src. In vitro activity assays for SHP2, p190, and RhoA will also be performed, as in Aim I. Finally, Aim III will examine SHP2 regulation of lung barrier function in vivo. Mouse lungs will be treated with vehicle or SHP2 inhibitor. Subsequent measurement of in vivo albumin extravasation and wet-to-dry lung weights, along with analysis of capillary filtration in isolated perfused lungs, will be used to assess barrier function and edema formation. This project will help gain insight into the pathways that regulate the barrier function of the lung microvascular endothelium. Through our examination of SHPa, c-Src, p190, and PKC8, we hope to identify targets for potential therapies aimed at treating ALI and ARDS.
| Grinnell, K L; Casserly, B; Harrington, E O (2010) Role of protein tyrosine phosphatase SHP2 in barrier function of pulmonary endothelium. Am J Physiol Lung Cell Mol Physiol 298:L361-70 |
