Regulation of Endothelial Barrier Integrity via Store-Operated Ca2+ entry

The edema genic factor thrombin increases the vascular permeability via disassembly of endothelial adherens junctions (AJs). VE-PTP stabilizes endothelial AJs through dephosphorylation of VE-cadherin. In the first part of my thesis, I investigated the role of STIM1 activation of store-operated Ca2+ entry (SOCE) in regulating adherens junction assembly. I observed that SOCE activated Pyk2 in human lung microvascular endothelial cells (ECs) and induced tyrosine phosphorylation of VE-PTP at Y1981. VE-PTP phosphorylation promoted Src binding to VE-PTP, Src activation, and subsequent VE-cadherin phosphorylation, and thereby increased the endothelial permeability. The increase in permeability was secondary to disassembly of AJs. In second part of my thesis, I focused on the intrinsic signaling mechanisms that mediate the reassembly of endothelial AJs. I showed that TAK1 activation downstream of SOCE signals the reassembly of endothelial AJs. I observed that STIM1 deficiency in ECs prevented thrombin-induced TAK1 phosphorylation (i.e., activation), indicating that SOCE is essential for thrombin-induced TAK1 activation. TAK1 inhibition, resulted in sustained permeability increase both in vitro and in intact lung microvessels in response to thrombin receptor protease-activated receptor 1 (PAR-1) activation. I also observed that thrombin-induced SOCE was augmented in TAK1 deficient ECs. Since EC-restricted TAK1 knockout mice are embryonically lethal, to study the in vivo role of EC-expressed TAK1, I generated tamoxifen-inducible EC-restricted TAK1 knockout (TAK1iΔEC) mice. Surprisingly, I observed that reduced expression of VE-cadherin and its associated β-catenin in lungs of TAK1iΔEC mice compared with their wild type littermates. In addition, the basal and PAR-1-mediated lung vascular permeability was augmented in TAK1iΔEC mice. Since, Glycogen-Synthase Kinase-3β (GSK-3β) regulates β-catenin expression by phosphorylation, I hypothesized that TAK1 activation promotes inactivation of GSK-3β to stabilize β-catenin. Therefore, I generated inducible EC-restricted GSK-3β knockout (GSK-3β iΔEC) mice and found that augmented β-catenin expression and inhibition of PAR-1-induced lung vascular permeability in GSK-3βiΔEC. These findings support the notion that TAK1 signaling in endothelial cells is required to maintain lung vascular barrier integrity. Thus, targeting the Pyk2 and TAK1 activation pathways may be potentially important strategies to combat vascular leak associated pulmonary edema in sepsis.