CYTOSKELETAL FUNCTION IN OSTEOBLAST MECHANOTRANSDUCTION

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The long range goal of this R03 proposal is to define how application of fluid shear forces to osteoblasts promotes gene transcription and new bone formation. Mechanical stimulation of bone induces new bone formation that is preceded by increased expression of genes necessary for deposition of bone matrix proteins. Fluid shear of bone cells, in culture, causes reorganization of the actin cytoskeleton and stimulation of gene expression. We propose that osteoblasts detect and respond to mechanical stimuli in bone using a sensing apparatus involving integrins at the cell surface that link to the internal actin cytoskeleton. This sensing apparatus may translate mechanical stimuli into changes in actin filament organization that increase internal force development which, in turn, signals the nucleus to increase expression of genes necessary for new bone matrix formation. MC 3T3-E1 osteoblasts cells will be used as a model to test the hypothesis that mechanotransduction in bone involves increased intracellular tension that occurs as a result of: 1) reorganizing actin filaments into contractile stress fibers inside bone cells, and 2) anchoring the actin filaments in stress fibers to the inner face of the plasma membrane. Two specific aims are proposed to test this hypothesis. In aim 1 we will determine if disrupting the linkage between actin filaments and integrins inhibits fluid shear- induced signaling. This aim will determine if actin-membrane anchorage is part of the mechanotransduction pathway leading to stress fiber formation, and increased expression of cyclooxygenase-2 (Cox-2) and c- fos. In aim 2 we will examine the mechanisms that regulate stress fiber formation in response to fluid shear. Specifically, we will determine the role of myosin light chain phosphorylation and the GTP-binding protein rho in fluid shear-induced signaling. This aim will test the role of the enzyme myosin light chain kinase, and activation of the GTPase rho, in the formation of stress fibers and expression of Cox-2 and c-fos. These studies are designed to improve our understanding of the mechanisms of mechanotransduction in promoting formation of new bone.