Mechanical loading of bone may be transmitted to osteocytes and osteoblasts via shear stresses at cell surfaces generated by the flow of interstitial fluid. The stimulated production of prostaglandins, which mediates some effects of mechanical loading on bone, is dependent on inducible cyclo-oxygenase 2 (COX-2) in bone cells. We examined the fluid shear stress (FSS) induction of COX-2 gene expression in immortalized MC3T3-E1 osteoblastic cells stably transfected with ؊371/؉70 base pairs (bp) of the COX-2 5-flanking DNA (Pluc371) and in primary osteoblasts (POBs) from calvaria of mice transgenic for Pluc371. Cells were plated on collagen-coated glass slides and subjected to steady laminar FSS in a parallel plate flow chamber.

The discovery of a calcium receptor has stimulated interest in the signaling events underlying extracellular calcium ( ] o -promoted chemotaxis of osteoblasts appears to utilize a unique signaling mechanism via a calciumsensing receptor.Multiple cell types sense extracellular calcium (1). Cellular responses to alterations in external calcium are diverse and cell-specific. Elevations in extracellular calcium signal parathyroid cells to reduce parathyroid hormone secretion (2). Renin production by kidney cells is inhibited by high [Ca 2ϩ ] o (3). During bone remodeling, osteoclasts (bone-resorbing cells) and osteoblasts (bone-forming cells) function in local environments characterized by dramatic fluctuations in [Ca 2ϩ ] o concentrations (4). Elevated external calcium in the resorption lucunae acts as a negative feedback on osteoclasts, inhibiting their resorptive capacity (5). In contrast, high [Ca 2ϩ ] o concentrations enhances DNA synthesis and promotes chemotaxis of osteoblasts (6, 7). The signal transduction mechanisms underlying [Ca 2ϩ ] o -stimulated mitogenesis and chemotaxis have yet to be defined; however, [Ca 2ϩ ] o is capable of producing a transient elevation of intracellular calcium in osteoblasts at concentrations that initiate these two physiological processes (8). Since the transient rise in intracellular calcium was not a result of a [Ca 2ϩ ] o influx, the existence of a cation-sensing receptor on the surface of osteoblasts was suggested, similar to that reported earlier in parathyroid cells (9).Recently, calcium receptor cDNA has been cloned and transcripts of this gene were recovered from bovine and human parathyroid, kidney, thyroid and brain cells (10). This 120-kDa polyvalent receptor, BoPCaR1, consists of a seven-transmembrane region and belongs to the G-protein-linked receptor superfamily (10). Recent reports indicate that MC3T3-E1 osteoblast-like cells may have a [Ca 2ϩ ] o -sensing receptor that is homologous, yet different, to the BoPCaR1 (11).The aim of this report is to define the initial post-receptor signal transduction events in [Ca 2ϩ ] o -stimulated chemotaxis of the murine osteoblast clonal cell line MC3T3-E1. Platelet-derived growth factor (PDGF) 1 also promotes chemotaxis in these cells, and we examined the effects of perturbations of various signaling pathways on both PDGF-and [Ca 2ϩ ] o -promoted chemotaxis. In previous studies of other cells, the signaling mechanisms underlying PDGF-induced chemotaxis were examined by mutating specific PDGF receptor tyrosine residues, and it was observed that PDGF-stimulated chemotaxis relies on both phospholipase C and the phosphoinositide 3-kinase (12). Hepatocyte growth factor also activates both PI 3-kinase and PLC, and both signaling events are necessary for hepatocyte growth factor-promoted chemotaxis (13,14). However, not all chemoat-

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