Calpain/talin/MLCP axis in pulmonary endothelial barrier regulation

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PROJECT SUMMARY Acute lung injury (ALI) is characterized by lung vascular endothelial cell (EC) barrier compromise resulting in pulmonary edema. While bacterial infections induced by E.coli or Streptococcus pneumoniae (Spn), and their respective bacteria toxin lipopolysaccharides (LPS) and pneumolysin (PLY) are the major causes for ALI, the molecular mechanisms involved in LPS- or PLY-induced ALI are ill-defined. The Scientific Premise comes from our novel preliminary data, which show that both LPS and PLY activate endopeptidase calpain in human lung microvascular ECs (HLMVECs) in ERK-dependent manner and that specific calpain inhibition prevents LPS- and PLY-induced disruption of EC barrier in HLMVECs and LPS-induced pulmonary edema in ALI. Further, we found that ERK-mediated calpain phosphorylation at Ser-50 and calpain activity were much higher in murine lung microvascular ECs isolated from lungs exposed to LPS, indicating that ERK-calpain pathway is highly related to ALI. We show that LPS/PLY induces talin cleavage into head and rod domain and talin phosphorylation in HLMVECs and that overexpression of calpain causes talin cleavage, and Rho-mediated inhibition of myosin light chain phosphatase (MLCP). Talin is activated through either 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 activation of focal adhesions (FAs), leading to RhoA-mediated MLCP inhibition and increased lung EC permeability. We found that MLCP, which opposes EC barrier compromise in ALI, is down- regulated in lungs of ARDS patients, highlighting the importance of MLCP inhibition in human ALI. MLCP binds talin and induced talin dephosphorylation, which may lead to talin degradation followed by FAs disassembly. Interestingly, MLCP dephosphorylates another calpain substrate, ezrin, leading to its deactivation and withdrawal from membrane cytoskeleton. Activation of talin/ezrin axis is involved in FA formation and our data suggest that talin is upstream of ezrin in LPS-induced EC cytoskeletal remodeling. It was shown that ezrin depletion decreases calpain activity suggesting positive feedback effect of MLCP inhibition on LPS/PLY-induced calpain activation and EC barrier compromise via ezrin phosphorylation/membrane attachment. These data led to our novel hypothesis that calpain/MLCP crosstalk coordinates talin activation leading to lung EC barrier disruption in ALI. To test this hypothesis we will employ unique molecular tools such as murine lung-targeting gene delivery in vivo, EC-specific inducible calpain and MLCP KO mice and ERK insensitive knockin mouse (Capn2S50A). We will: (1) evaluate whether ERK-mediated calpain activation is involved in lung microvascular EC barrier disruption and cytoskeletal reorganization in ALI induced by bacterial toxins (LPS, PLY), live bacteria (E.coli, Spn) and in sepsic conditions; (2) determine whether Gram- and Gram+ bacteria, their toxins and sepsis induce talin activation (cleavage/phosphorylation) and FA strengthening leading to lung microvascular EC barrier disruption; (3) examine whether MLCP activity affects calpain activation induced by LPS/E.coli, PLY/Spn and sepsis.