A. Miyauchi et al., NOVEL IPRIFLAVONE RECEPTORS COUPLED TO CALCIUM INFLUX REGULATE OSTEOCLAST DIFFERENTIATION AND FUNCTION, Endocrinology, 137(8), 1996, pp. 3544-3550
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.