Normal bone metabolism requires a balance between bone resorption and bone formation. Cells that resorb bone, osteoclasts, must be tightly coupled to cells that form bone, osteoblasts. Multiple agents have been implicated as coupling agents, primarily growth factors and cytokines. Calcium has recently been shown to stimulate chemotaxis and proliferation in osteoblasts. However, the signal transduction cascade involved in calcium-stimulated chemotaxis in osteoblasts remains unexplored. Using a Boyden chemotaxis chamber, the initial signaling events in calcium-induced chemotaxis were examined based on a model previously established for PDGF-stimulated chemotaxis. According to the PDGF model, both the phospholipase C and the phosphatidylinositol-3-kinase signaling pathways are required for chemotaxis. PDGF (lOng/ml) stimulated chemotaxis of MC3T3-E1 osteoblast-like cells 17.8 times relative to basal. Calcium (SmM) enhanced chemotaxis of the osteoblasts by 7.5 times relative to basal. Wortmannin, an inhibitor of phosphatidylinositol-3-kinase, significantly (p<0.001) reduced PDGF-stimulated chemotaxis of the osteoblasts by 57%. However, wortmannin had no effect on calcium-stimulated chemotaxis. Thus, the signaling mechanism involved in calcium-induced chemotaxis of the MC3T3-E1 cells does not appear to utilize phosphatidylinositol-3-kinase. U-71322, an inhibitor of phospholipase C, significantly (p<0.01) inhibited PDGF-stimulated chemotaxis in the osteoblasts by 70%. Furthermore, U-71322 significantly (p<0.001) reduced calcium-induced chemotaxis by 79%. Therefore, phospholipase C must be involved in calcium-stimulated chemotaxis of MC3T3-E 1 cells. Recently, Brown et al. have cloned a G-linked calcium receptor from kidney and parathyroid cells. It is our hypothesis that the osteoblasts also have a G-linked calcium receptor which is responsible for the initial signaling events found in calcium-stimulated chemotaxis. Future work will focus on cloning the calcium recepto and examining downstream signaling events in calcium-induced chemotaxis.
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