Endothelial cells form the interface between the vasculature and many tissues and are major participants in permeability, inflammation, neovascularization, and coagulation. Our laboratory has defined a critical role for the endothelial cytoskeleton in vascular pathobiology. However, despite its recognized importance, the exact mechanisms operative is cytoskeletal regulation in these processes are incompletely understood. Our work has demonstrated a critical role of phosphorylation of myosin light chains (MLC) catalyzed by the Ca+2 - CaM dependent endothelial cell myosin light chain kinase (EC MLCK) in vascular permeability, neutrophil transendothelial migration, cell motility, and apoptosis. We have cloned this enzyme and found that EC MLCK to contains an unique amino terminal sequence not present in smooth muscle MLCK. Using the yeast two-hybrid system, the N-terminal portion of EC MLCK was found to interact with a proinflammatory cytokine, macrophage migration inhibitory factor (MIF), a regulator of innate and acquired immunity with a pathologic role in ARDS, sepsis, and arthritis. Indeed, anti-MIF antibodies are protective in a marine model of endotoxemia. Using GST-MLCK pull-down and immunoprecipitation assays, we have confirmed the specificity of this highly novel interaction between EC MLCK and MIF. However, the biological significance of this interaction is not known.
In Specific Aim #1, MLCK and MIF will be spatially colocalized within human pulmonary artery endothelial cells.
Specific Aim #2 will characterize the structural sites within both the novel N- terminus of EC MLCK and MIF necessary for interaction using in vitro binding assays. Finally, in Specific Aim #3 the biologic significance of MLCK-MIF interaction in permeability, neutrophil transendothelial migration, endothelial cell chemotaxis, and apoptosis will be evaluated. Together these studies will define the contribution of the novel interaction between the multifunctional MLCK and a highly relevant cytokine MIF in key endothelial pathologic processes.
