NOVEL IPRIFLAVONE RECEPTORS COUPLED TO CALCIUM INFLUX REGULATE OSTEOCLAST DIFFERENTIATION AND FUNCTION

Ipriflavone (7-isopropoxyisoflavone) is an effective antiresorptive ag ent used to treat osteoporosis. However, the mechanism of its action o n osteoclasts and their precursor cells is not well understood, To det ermine whether the mechanism involves direct effects on osteoclasts or their precursors, we examined the effects of ipriflavone on cytosolic free calcium ([Ca2+](i)) in osteoclasts and their precursors and meas ured specific binding of H-3-labeled ipriflavone. Highly purified chic ken osteoclast precursors, which spontaneously differentiate into mult inucleated osteoclasts in 3-6 days, were loaded with fura-2, and the s ubcellular [Ca2+](i) distribution was monitored by videoimaging. Iprif lavone induced a rapid increase in [Ca2+](i) followed by a sustained e levation [EC(50) = 5 X 10(-7) M, 263 +/- 74 nM (SE) (n = 8) above basa l levels, by 10(-6) M ipriflavone, sustained phase]. The responses wer e the same in differentiated chicken osteoclasts and isolated rabbit o steoclasts. An influx of extracellular Ca2+ is likely to be responsibl e for the ipriflavone-induced change in [Ca2+](i) because the response was abolished by 0.5 mM LaCl3, or by Ca-free medium containing EGTA. Moreover, high [Ca2+](i) levels were detected adjacent to the cell mem brane after ipriflavone addition. Ipriflavone induced Ca influx mainly through dihydropyridine-insensitive Ca2+ channels, because nicardipin e (10(-7) M) and verapamil(10(-7) M) had no effects on ipriflavone-ind uced [Ca2+](i) responses. [H-3]Ipriflavone binding studies indicated t he presence of specific ipriflavone binding sites (two classes), both in precursor cells [dissociation constant (K-d), 7.60 x 10(-8) M, 2.67 x 10(-6) M] and in mature osteoclasts (K-d, 4.98 x 10(-8) M, 3.70 x 1 0(-6) M). Specific ipriflavone binding was not displaced by various mo dulators of avian osteoclast function, such as estradiol (10(-6) M) or retinoic acid(10(-6) M), indicating that ipriflavone receptors differ from the receptors for these Ca-regulating hormones. The fusion of os teoclast precursor cells was significantly inhibited by ipriflavone, w hich led to dose-dependent inhibition of bone resorption and tartrate- resistant acid phosphatase activity. Novel specific ipriflavone recept ors that are coupled to Ca2+ influx were demonstrated in osteoclasts a nd their precursor cells. These ipriflavone receptors may provide a me chanism to regulate osteoclast differentiation and function.