Ipriflavone down-regulates endothelin receptor levels during differentiation of rat calvarial osteoblast-like cells

Ipriflavone (7-isopropoxy-3-phenyl-4H-1-benzopyran-4-one) is a synthetic flavonoid that has been shown to stimulate the activity of osteoblasts, We show here that ipriflavone also promotes the deposition of calcium and the formation of mineralized nodules by newborn rat calvarial osteoblast-like (ROB) cells as well as the activity of alkaline phosphatase. We reported previously that endothelin-1 inhibits the differentiation of ROB cells [Y, Hiruma et al. (1998) J. Cardiovasc. Pharmacol. 31, S521-S523], Therefore, we examined the effects of ipriflavone on the expression of endothelin receptors in ROB cells by polymerase chain reaction-Southern blot analysis and in binding assays with I-125-labeled endothelin-1. Ipriflavone reduced levels of endothelin ETA receptors (to 48% of the control level) in ROB cells around day 7 in our standard cultures, while it had no apparent effect on the expression of the mRNA for the endothelin ETA receptor. By contrast, treatment with 10(-7) M. endothelin-1 on days 6 through 9 alone suppressed mineralization by ROB cells, Ipriflavone also reduced the ability of endothelin-1 to inhibit mineralization by ROB cells. These results suggest that the acceleration of osteoblastic differentiation by ipriflavone might be due, at least in part, to a time-specific down-regulation of endothelin receptors.