Novel MLCK-MIF Interaction in Endothelium

内皮细胞构成血管系统和许多组织之间的界面，是通透性、炎症、新生血管和凝血的主要参与者。我们的实验室已经明确了内皮细胞骨架在血管病理生物学中的关键作用。然而，尽管它的重要性得到公认，但在这些过程中细胞骨架调节的确切机制尚不完全清楚。我们的工作证明了钙离子依赖的内皮细胞肌球蛋白轻链激酶(EC MLCK)催化的肌球蛋白轻链(MLC)磷酸化在血管通透性、中性粒细胞跨内皮细胞迁移、细胞运动和细胞凋亡中起着关键作用。我们克隆了这一酶，发现EC MLCK to含有一个在平滑肌MLCK中没有的独特的氨基末端序列。利用酵母双杂交系统，EC MLCK的N端部分被发现与促炎细胞因子-巨噬细胞移动抑制因子(MIF)相互作用，MIF是天然和获得性免疫的调节因子，在ARDS、脓毒症和关节炎中具有病理作用。事实上，抗MIF抗体在海洋内毒素血症模型中是有保护作用的。通过GST-MLCK下拉和免疫沉淀实验，我们证实了EC MLCK和MIF之间这种高度新颖的相互作用的特异性。然而，这种相互作用的生物学意义尚不清楚。在特定的目标#1中，MLCK和MIF将在空间上共存于人的肺动脉内皮细胞内。具体目标#2将利用体外结合分析来表征EC MLCK和MIF的新N末端中相互作用所必需的结构位点。最后，在特定目标#3中，将评估MLCK-MIF相互作用在通透性、中性粒细胞跨内皮细胞迁移、内皮细胞趋化和细胞凋亡方面的生物学意义。总之，这些研究将确定多功能MLCK和高度相关的细胞因子MIF之间的新相互作用在关键的内皮病理过程中的作用。

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.