OF THE OVERALL PROGRAM (taken from the application):Physical connections between extracellular matrix (ECM), cytoskeleton, and nuclear matrix may allow osteoblasts to sense mechanical perturbations and translate them into gene expression. Work is proposed to study the pathway in vivo and in vitro in cells at different maturational levels using laminar fluid forces. Project 1 will define the dynamics of osteoblast proliferation after a mechanical stimulus in vivo, and will test the effect of loading frequency on bone cell proliferation. Using two different animal models, they will determine which genes, are influenced indirectly through mechanically-induced prostaglandin and nitric oxide synthesis. The possibility that bone cells accommodate to mechanical loading will be tested using the rat ulnar loading model. Project 2 will determine the interaction of mechanosensitive ion channels (MSCC, VSCC) and intracellular calcium release (iCaR) on fluid shear-induced changes in gene expression in proliferating and differentiated osteoblasts. Regulation of channel function and iCaR by specific integrin binding to different ECM proteins and in response to fluid shear will be measured. Control of channel activation and iCaR by g-proteins in response to fluid shear will be examined using a combination of g-protein activators and inhibitors in conjunction with patch clamp anc Ca2+ imaging techniques. Project 3 will determine the role of actin filament-integrin linkages and integrin-ECM interactions in fluid shear-induced mechanotransduction. It will also determine the role of myosin light chain phosphorylation and the GTP-binding protein rho in fluid shear-induced mechanotransduction. Project 4 will determine the interaction between the poly(dT) sites of the COL1A1 promoter and nuclear matrix architectural transcription factors (NP/NMP4) in mediating the transcriptional response of COL1A1 to fluid shear. It will determine the significance of actin-ECM interactions on NP and NMP4-mediated COL1A1 response to fluid shear. These Projects are supported by three Cores: Administrative and Biostatistics; Cell Biology; Mechanical Loading. Data generated by this Program are intended to define some mechanisms by which fluid forces can regulate gene expression, and will attempt to validate these mechanisms by in vivo studies.
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