WORTMANNIN INHIBITS SPREADING AND CHEMOTAXIS OF RAT OSTEOCLASTS IN IN-VITRO

Wortmannin (WT) and 17 beta-hydroxywortmannin (HWT), which are inhibit ors of phosphatidylinositol-3(OH)-kinase (PI3K), have been shown previ ously to inhibit bone resorption in vitro and in vivo, possibly by int erfering with formation of th osteoclast ruffled border. Since migrati on of osteoclasts also plays an important role in the process of bone resorption, we investigated the effects of these inhibitors on osteocl ast morphology and motility. Both HWT and WT caused a sustained decrea se in the planar area of osteoclasts in vitro (half maximal effect at 25 and 165 nM, respectively), with the effect of HWT on cell area more readily reversible than WT. These agents also caused accumulation of intracellular vesicles. Time-lapse video microscopy was used to record the migration of osteoclasts in response to macrophage colony-stimula ting factor (M-CSF) or vehicle, flowing passively from a micropipette positioned 200-400 mu m from the cell. M-CSF caused directed migration of osteoclasts, indicating chemotaxis (over 3 h osteoclasts migrated 96 +/- 14 mu m in response to M-CSF vs. 11 +/- 2 mu m in control exper iments). Both WT (100 or 500 nM) and LY294002 (100 mu M), a specific P I3K inhibitor structurally unrelated to WT, significantly inhibited os teoclast chemotaxis in response to M-CSF. Taken together, these effect s of WT, HWT, and LY294002 are consistent with an important role for P I3K in regulating cytoskeletal function in osteoclasts. The inhibitory effects of WT and HWT on bone resorption may be due, in part, to impa irment of osteoclast motility.