Protection against LPS-Induced Pulmonary Edema through the Attenuation of Protein Tyrosine Phosphatase–1B Oxidation

Abstract

One hallmark of acute lung injury is the disruption of the pulmonary endothelial barrier. Such disruption correlates with increased endothelial permeability, partly through the disruption of cell-cell contacts. Protein tyrosine phosphatases (PTPs) are known to affect the stability of both cell-extracellular matrix adhesions and intercellular adherens junctions (AJs). However, evidence for the role of select PTPs in regulating endothelial permeability is limited. Our investigations noted that the inhibition of PTP1B in cultured pulmonary endothelial cells (ECs), as well as in the vasculature of intact murine lungs via the transient overexpression of a catalytically inactive PTP1B, decreased the baseline resistance of cultured EC monolayers and increased the formation of edema in murine lungs, respectively. In addition, we observed that the overexpression of wild-type PTP1B enhanced basal barrier function in vitro. Immunohistochemical analyses of pulmonary ECs and the coimmunoprecipitation of murine lung homogenates demonstrated the association of PTP1B with the AJ proteins b-catenin, p120-catenin, and VE-cadherin both in vitro and ex vivo. Using LPS in a model of sepsis-induced acute lung injury, we showed that reactive oxygen species were generated in response to LPS, which correlated with enhanced PTP1B oxidation, inhibited phosphatase activity, and attenuation of the interactions between PTP1B and b-catenin, as well as enhanced b-catenin tyrosine phosphorylation. Finally, the overexpression of a cytosolic PTP1B fragment, shown to be resistant to nicotinamide adenine dinucleotide phosphate-reduced oxidase-4 (Nox4)-mediated oxidation, significantly attenuated LPS-induced endothelial barrier dysfunction and the formation of lung edema, and preserved the associations of PTP1B with AJ protein components, independent of PTP1B phosphatase activity. We conclude that PTP1B plays an important role in maintaining the pulmonary endothelial barrier, and PTP1B oxidation appears to contribute to sepsis-induced pulmonary vascular dysfunction, possibly through the disruption of AJs.

SH2 Domain-Containing Protein Tyrosine Phosphatase 2 and Focal Adhesion Kinase Protein Interactions Regulate Pulmonary Endothelium Barrier Function

“…Liposomes dissolved in 5% glucose solution were combined with GFP or SHP2 D61A complementary DNA (cDNA; 50 mg) and injected into the retrobulbar sinus of the orbit of anesthetized C57 BL/6 mice. Confirmation of cDNA overexpression within the pulmonary vasculature was assessed by endothelial cell isolation and immunoblot analysis of GFP, SHP2, and VE-cadherin, as previously described (24). Transfection efficiency was assessed by densitometry of SHP2.…”

“…In additional experiments, at 24 hours after intraperitoneal injection of LPS or vehicle into mice, lung permeability was determined by ex vivo perfusion of lungs, as previously described (24,25). Representative plots for lung filtration coefficients experiments are included in the online supplement.…”

“…Changes in endothelial monolayer permeability of transiently transfected LMVECs were assessed using the electrical cell impedance sensor technique (Applied Biophysics, Troy, NY), as previously described (24,25). Transfection efficiency was assessed by densitometry of phosphorylated SHP2 (Y 542 and Y 580 ) and total SHP2 or GFP.…”

Endothelial adherens junctions at a glance

“…Small GTPases, phosphatases and kinases are implicated, at different levels, in the control of junctional organization and vascular permeability (Noren et al, 2001;Lampugnani et al, 2002;Braga and Yap, 2005;Sakurai et al, 2006;Cain et al, 2010;Stockton et al, 2010;Gloerich and Bos, 2011;Pannekoek et al, 2011). Specific kinases and phosphatases might also associate with junctional proteins and modulate their adhesive properties (Weis et al, 2004;Sui et al, 2005;Grinnell et al, 2010;Broermann et al, 2011;Grinnell et al, 2012;Hatanaka et al, 2012).…”

Genetic disruption of protein kinase Cδ reduces endotoxin-induced lung injury

“…Changes in endothelial monolayer permeability were assessed using the electrical cell impedance sensor technique (Applied Biophysics, Troy, NY), as previously described (11,13,15,21). For analysis of monolayer permeability following transfection of LMVEC with cDNA encoding PKC␦ K376R , Polyjet reagent (SignaGen, Gaithersburg, MD) was used to seed cells to confluence onto collagen-coated electric cell-substrate impedancesensing arrays.…”