Programmed cell suicide, or apoptosis, is an important feature of normal cell homeostasis as well as a number of inflammatory processes including lung injury and neoplastic disorders. Endothelial cells are a prominent cell type apoptose in animal models of disease. For example, tumor necrosis factor-alpha (TNF-alpha) is a cytokine shown to induce both pulmonary injury and endothelial cell apoptosis in vivo and in vitro and is increased during lung injury. Information is limited as to the exact sequence and components involved in cellular apoptosis. However, there is increasing appreciation that the micro- filamentous cytoskeleton is intrinsically involved in the apoptosis process and may play a critical role in the "prelethal" phases, either by spatially modifying protein-protein interactions, regulation of intracellular signaling, or by transmitting death messages to downstream effectors. Myosin light chain (MLC) phosphorylation by MLC kinase (MLCK) is a critical regulatory step in actin-myosin interaction and ensuing cytoskeletal changes. Recently, MLCK has been implicated in serum-starvation and TNF-alpha models of apoptosis. Our preliminary data are consistent with the notion that cytoskeletal changes induced by actin-myosin contraction may be important in the progression of endothelial cell apoptosis induced by TNF- alpha and that myosin light chain (MLC) phosphorylation by myosin light chain kinase (MLCK) is one of the events regulating this process. However, the exact mechanisms by which the cytoskeleton is involved in apoptosis is not known. In this proposal, we will examine the hypothesis that the microfilament-based cytoskeleton has an active and critical role in the physiologically and clinically relevant model of TNF-alpha induced endothelial cell apoptosis. The specific aims of this work are: (1) to determine the role of the microfilament cytoskeleton in TNF-alpha-induced endothelial cell apoptosis, (2) to evaluate MCLK modulation of TNF-alpha-induced endothelial cell apoptosis, and (3) to explore the contribution of MAP kinases to cytoskeletal changes in TNF- alpha-induced endothelial cell apoptosis. We believe that elucidation of how the cytoskeleton participates in the cell's vital decisions for survival or cell death may provide clues about the more general role of endothelial cell apoptosis in the pathogenesis of acute lung injury.

K08HL004396

2000-08-01

2005-07-31