Role of protein tyrosine phosphatase SHP2 in barrier function of pulmonary endothelium

Abstract

Pulmonary edema is mediated in part by disruption of interendothelial cell contacts. Protein tyrosine phosphatases (PTP) have been shown to affect both cell-extracellular matrix and cell-cell junctions. The SH2 domain-containing nonreceptor PTP, SHP2, is involved in intercellular signaling through direct interaction with adherens junction proteins. In this study, we examined the role of SHP2 in pulmonary endothelial barrier function. Inhibition of SHP2 promoted edema formation in rat lungs and increased monolayer permeability in cultured lung endothelial cells. In addition, pulmonary endothelial cells demonstrated a decreased level of p190RhoGAP activity following inhibition of SHP2, events that were accompanied by a concomitant increase in RhoA activity. Furthermore, immunofluorescence microscopy confirmed enhanced actin stress fiber formation and diminished interendothelial staining of adherens junction complex-associated proteins upon SHP2 inhibition. Finally, immunoprecipitation and immunoblot analyses demonstrated increased tyrosine phosphorylation of VE-cadherin, beta-catenin, and p190RhoGAP proteins, as well as decreased association between p120-catenin and VE-cadherin proteins. Our findings suggest that SHP2 supports basal pulmonary endothelial barrier function by coordinating the tyrosine phosphorylation profile of VE-cadherin, beta-catenin, and p190RhoGAP and the activity of RhoA, signaling molecules important in adherens junction complex integrity.

Fig. 1.

SHP2 inhibition causes endothelial barrier dysfunction. A: changes in resistance across monolayers were measured after transient transfection of equivalent numbers of pulmonary artery endothelial cells (PAEC) with GFP (dashed line) or SHP2^C459S (solid line) for 48 h. Data are presented as means ± SE of the resistance across the SHP2^C459S monolayers normalized to the resistance across the GFP-transfected monolayers. Means ± SE; n = 3; *P < 0.0001 vs. GFP. A representative immunoblot probing for SHP2 is shown. B: changes in resistance across PAEC monolayers were measured following exposure to vehicle or NSC-87877. Arrow indicates addition of treatment. Means ± SE; n = 4; *P < 0.05 vs. vehicle for all doses.

Fig. 2.

Overexpression of catalytically inactive SHP2 promotes stress fiber formation and adherens junction disassembly. Equivalent numbers of PAEC were transfected with GFP or SHP2^C459S for 48 h. Endothelial cells were fixed and immunofluorescently stained for filamentous actin with phalloidin and for β-catenin or VE-cadherin with protein-directed antibodies. Insets are the same endothelial cells viewed for overexpression of GFP (left) or overexpression of SHP2^C459S by costaining with anti-HA antibody (right). Arrows indicate intercellular gapping; arrowheads denote increased stress fiber formation. Scale bars, 20 μM; n = 3.

Fig. 3.

Inhibition of SHP2 leads to increased tyrosine phosphorylation of adherens junction proteins. Equivalent numbers of PAEC were transfected with GFP or SHP2^C459S for 48 h (A and B) or confluent monolayers of lung microvascular endothelial cells (LMVEC) were incubated with 100 μM NSC-87877 for 3 h (C and D). Equal amounts of endothelial cell lysate were immunoprecipitated with antibodies directed against VE-cadherin (A and C) or β-catenin (B and D), and precipitates were immunoblotted for phosphorylated tyrosine. The immunoblots were stripped and reprobed for the immunoprecipitated protein. The data are presented as means ± SE of densitometric values of tyrosine phosphorylated adherens junction protein relative to total adherens junction protein, normalized to GFP (A and B) or vehicle (C and D). N = 3; *P < 0.05 vs. GFP or vehicle, respectively.

Fig. 4.

Overexpression of catalytically inactive SHP2 leads to reduced association of p120-catenin and VE-cadherin. Equivalent numbers of PAEC were transfected with GFP or SHP2^C459S for 48 h. Equivalent amounts of endothelial cell lysate were immunoprecipitated with an antibody directed against p120-catenin, and precipitates were immunoblotted for VE-cadherin. The immunoblots were then stripped and reprobed for p120-catenin. N = 3; *P < 0.05 vs. GFP.

Fig. 5.

Pulmonary edema formation results on SHP2 inhibition. A: filtration coefficients (k_f) were determined in isolated, perfused rat lungs at baseline (open bars) and following a 30-min exposure to vehicle (0 μM) or indicated concentration of NSC-87877 (solid bars). Values are means ± SE; n = 3–6; *P < 0.05 vs. vehicle. B: anesthetized rats were given a bolus of vehicle (PBS; open bar) or 100 μM NSC-87877 (solid bar). The rats were then given 4% Evans blue dye (EBD)-conjugated albumin after 5 min and were euthanized after an additional 45 min. The lungs were harvested, and the amount of extravasated albumin in the lungs was determined spectrophotometrically. Values are means ± SE; n = 9–10; *P < 0.05 vs. vehicle.

Fig. 6.

Inhibition of SHP2 correlates with increased RhoA activity. Lysates from PAEC transfected with SHP2^C459S or GFP (A and B) or from LMVEC monolayers treated with 100 μM NSC-87877 for 3 h (C and D) were incubated with GST-RBD (A and C) or GST-RhoGDI-1α (B and D) conjugated to agarose beads, and precipitates, in parallel with total lysates, were immunoblotted for RhoA. Data are presented as means ± SE; n = 3. *P < 0.05 vs. GFP or vehicle, respectively.

Fig. 7.

Inhibition of SHP2 correlates with decreased p190RhoGAP activity and increased tyrosine phosphorylation of p190RhoGAP. PAEC were transfected with SHP2^C459S or GFP (A and C) or confluent LMVEC monolayers were incubated with 100 μM NSC-87877 for 3 h (B). A and B: equivalent amounts of lysate were incubated with GST-RhoA(Q63L) conjugated to agarose beads, and precipitates, in parallel with total lysates, were immunoblotted for p190RhoGAP. Equivalent amounts of lysates were immunoprecipitated with anti-p190RhoGAP antibody, and the precipitates were immunoblotted for phosphorylated tyrosine (C). Membranes were then stripped and reprobed for total p190RhoGAP. Data are presented as means ± SE; n = 3. *P < 0.05 vs. GFP or vehicle, respectively; §P < 0.08 vs. GFP.