Calpain-mediated lung endothelial barrier modulation in acute lung injury

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Summary Acute lung injury (ALI) is characterized by lung vascular endothelial (EC) barrier compromise resulting in increased EC permeability and pulmonary edema. The infections of Gram negative bacteria compose the major cause for ALI. Little has been known about how Gram negative endotoxins (i.e. lipopolysaccharides, LPS) induce EC barrier disruption. We found that LPS activates endopeptidase, calpain, in human lung microvascular ECs (HLMVECs) and the specific calpain inhibition prevents LPS-induced disruption of EC barrier function of HLMVECs and LPS-induced pulmonary edema in ALI. Calpain is a family of calcium-dependent non-lysosomal neutral cysteine endopeptidases that act via limited proteolysis of substrate proteins in mammalian cells, including HLMVECs. Our preliminary data show that LPS induces talin cleavage into head and rod domain and talin phosphorylation in HLMVECs and that overexpression of calpain causes talin cleavage and RhoA activation and myosin light chain phosphatase (MLCP) phosphorylation resulting in MLCP inhibition and myosin light chain (MLC) phosphorylation. Talin is activated through talin cleavage or phosphorylation. Talin cleavage separates head from rod domain thus removing auto-inhibition and stimulating talin head binding to integrin and thus induces FA activation, leading to RhoA activation and MLCP inhibition and MLC phosphorylation and increased lung EC permeability. Talin activation through phosphorylation at Ser-425 can be through cyclin-dependent kinase 5 (CDK5). Our data show that calpain inhibition attenuates LPS-induced increase in CDK5 activity and that MLCP is involved in talin dephosphorylation. This proposal is to study a novel hypothesis that calpain/MLCP coordination regulates talin activation (cleavage/phosphorylation) leading to endothelial barrier disruption in ALI. We will determine whether Gram negative endotoxin LPS, E. coli and P. aeruginosa induce calpain activation and calpain leads to lung microvascular endothelial barrier disruption and Rho-mediated MLCP phosphorylation/inhibition and MLC phosphorylation in ALI. We will define whether LPS, E. coli and P. aeruginosa induce talin activation (cleavage/phosphorylation) and FA strengthening, leading to lung microvascular endothelial barrier disruption in vitro and in vivo. We will investigate whether calpain regulates talin activation (cleavage/phosphorylation) in lung microvascular endothelial barrier disruption in ALI induced by LPS, E. coli and P. aeruginosa. We will assess whether plasma from ALI patients with Gram negative sepsis induces HLMVEC barrier compromise via calpain-talin-FA-MLCP pathway. This proposal is novel because it will identify calpain as mediators in lung EC barrier compromise and calpain serves this role by regulating novel talin cleavage/phosphorylation, RhoA activation and MLCP activity in ALI. A better understanding of the mechanistic insight will provide a framework from which novel calpain inhibition and MLCP activation strategies can be developed for intervention and treatment of ALI.