The adaptive response of bone to mechanical loading is a fundamental tenet of bone biology. The importance of mechanical loading for the homeostasis of bone turnover and the maintenance of skeletal integrity are emphasized in osteoporosis prevention programs. Mechanical stimuli are essential to therapies in orthodontics and orthopedics. Nevertheless, very little is known about the pathways leading from mechanical stimuli to altered gene transcription. A popular hypothesis is that mechanical strains generate interstitial fluid flow within the mineralized bone matrix, which subsequently exerts shear stress on cellular membranes. Previous studies suggest that prostaglandins (PGs) can mediate some of the anabolic effects of mechanical loading on bone, and fluid shear stress (FSS) can stimulate production of PGs via induction of cyclooxygenase-2 (COX-2) expression. The goal of this application is to elucidate pathways by which FSS induces COX-2 in osteoblastic cells and to examine the role of COX-2 in mediating effects of FSS. This research application takes advantage of an unusual large-scale flow chamber and mice transgenic for the COX-2 promoter-fused to a luciferase reporter (Pluc). We will characterize the induction of COX-2 by FSS in osteoblastic MC3T3-E1 cells and in primary osteoblasts. Transcriptional regulation will be studied in MC3T3-E1 cells stably transfected with Pluc constructs and in primary osteoblasts from Pluc transgenic mice. We will test the hypothesis that the FSS induction of COX-2 transcription in osteoblasts occurs via protein kinase C (PKC)-mediated activation of the extracellular regulated kinase (ERK) signaling pathway. Using site-directed mutagenesis and 5'-sequential deletion analysis, we will examine the roles of putative cis-acting sites, including a """"""""shear stress response element"""""""" and an AP-1 binding site, in mediating the FSS induction of COX-2 promoter activity. We will look for effects of FSS on primary osteoblast proliferation and differentiation and expression of factors supporting osteoclastogenesis. To assess the role of COX-2 in these effects, we will use primary osteoblasts from mice with the COX-2 gene disrupted. Finally, we will develop mice transgenic for the COX-2 promoter fused to a green fluorescent protein (GFP). These mice will be subjected to brief periods of loading following hind limb unloading (tail suspension) and histology for GFP fluorescence done to identify bone cells expressing COX-2 in response to loading.
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