Phorbol Myristate Acetate Downregulates the Binding Sites for Colony-Stimulating Factor-1 on Osteoclasts Isolated from Rats

Abstract

Colony-stimulating factor-1 (CSF-1) is the growth factor for the cells of the mononuclear phagocytic system and for osteoclasts. We tested whether phorbol myristate acetate (PMA), a phorbol ester activating protein kinase C, modulates the number of binding sites for CSF-1 on isolated rat osteoclasts. PMA decreased binding of CSF-1 to osteoclasts within 60 minutes. The effect of PMA was dose dependent at concentrations between 10(-9) M and 10(-6) M. The inactive phorbol ester, 4alpha-phorbol 12,13-didecanoate, had only a slight effect. Since the osteoclast preparation was contaminated with other cells, the action of PMA on the osteoclasts might have been either direct or indirect. In pure osteoclasts harvested by a micropipette, the downregulation of CSF-1 binding by PMA reached only about three-quarters of that in nonpurified preparations. Addition of osteoblastic osteosarcoma UMR106 cells increased the effect of PMA. Antiserum against CSF-1, which was added to osteoclasts contaminated with other cells, mainly osteoblasts, partially inhibited the effect of PMA, but the antiserum had no effect in pure osteoclasts. These data suggest that the effect mediated by osteoblasts or other contaminating cells is due to the release of CSF-1, which is known to downregulate its binding sites on osteoclasts. The direct action of PMA on osteoclasts decreased the binding only to about 40%, in contrast to CSF-1 which completely downregulated the binding. The data also differ from the published results about macrophages. In these cells, PMA downregulates the binding of CSF-1 completely. The CSF-1 binding sites on osteoclasts recovered within 4 hours after removal of PMA, and cycloheximide, an inhibitor of protein synthesis, inhibited the recovery.