Disturbances in endothelial cell (EC) barrier regulation is a hallmark of lung inflammation, angiogenesis and cancer. Maintaining of EC barrier is under close regulation by competing contractile and tethering forces whose effects are critically dependent upon EC cytoskelatal rearrangement and is under control of intracellular messengers including free Ca2+ and its major intracellular receptor, calmodulin (CaM). Substantial work, including our own, has verified that Ca2+/calmodulin complex triggered permeability induced by edemagenic agents such as the serine protease thrombin. Thrombin receptor proteolysis is sequentially coupled to intracellular Ca2+ increases, activation of Ca2+/CaM-dependen myosin light chain kinase (MLCK), increases in myosin light chain (MLC) phosphorylation, initiation of actomyosin contraction and finally gap formation and barrier dysfunction. However, the role of Ca2+/CaM targets (other than MLCK) in EC barrier regulation is not completely understood. Our preliminary data indicate that thrombin-mediated endothelial cell permeability is dependent (in part) upon activation of the Ca2+/CaM dependent protein kinase II (CaMKII) as specific CaMKII inhibition significantly attenuates thrombin-induced stress fiber formation and permeability. Paradoxically, thrombin also activates the Ca2+/CaM-dependent phosphatase 2B (PPase 2B or calcineurin) which decreases thrombin-induced MLC phosphorylation providing a regulatory mechanism to potentially reverse or limit thrombin-induced cytoskeletal rearrangement and permeability. In addition, thrombin-induced EC barrier dysfunction is correlated with increased phosphorylation of several cytoskeletal proteins including Ca2+/CaM-dependent regulatory protein caldesmon and CaMKII targets, filamin, tau and vimentin. Together, these data clearly convey the complex involvement of Ca2+/CaM-dependent signaling pathways in the regulation of endothelial permeability. In this proposal, we will explore the role of Ca2+/CaM targets (other than MLCK) in thrombin-mediated EC regulation. In SA 1 we will examine the role of CaMKII activity in agonist-induced cytoskeletal protein phosphorylation, cytoskeletal rearrangement and barrier dysfunction. In SA 2 we will examine the role of PPase 2B activity in EC barrier regulation. In SA 3 we will examine the role of caldesmon in agonist-mediated cytoskeletal rearrangement and barrier regulation. These studies will provide an understanding of novel signaling pathways involved in lung EC barrier regulation and promise new directions and targets for treatment of lung disorders.
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