17 beta-estradiol potently suppresses cAMP-induced insulin-like growth factor-I gene activation in primary rat osteoblast cultures

Insulin-like growth factor-I (IGF-I) is a key factor in bone remodeling. In osteoblasts, IGF-I synthesis is enhanced by parathyroid hormone and prostaglandin E-2 (PGE(2)) through cAMP-activated protein kinase. In rats, estrogen loss after ovariectomy leads to a rise in serum IGF-I and an increase in bone remodeling, both of which are reversed by estrogen treatment, To examine estrogen-dependent regulation of IGF-P expression at the molecular level, primary fetal rat osteoblasts were co-transfected with the estrogen receptor (hER, to ensure active ER expression), and luciferase reporter plasmids controlled by promoter I of the rat IGF-I gene (HGF-P PP), used exclusively in these cells, Bs reported, 1 mu M PGE(2) increased IGF-I P1 activity by 5-fold. 17 beta-Estradiol alone had no effect, but dose-dependently suppressed the stimulatory effect of PGE(2) by up to 90% (ED50 similar to-0.1 nM). This occurred within 3 In, persisted for at least 16 h, required ER, and appeared specific, since 17 alpha-estradiol was 100-300-fold less effective, By contrast, 17 beta-Estradiol stimulated estrogen response element (ERE)dependent reporter expression by up to 10-fold, 17 beta-Estradiol also suppressed an HGF-I PP. construct retaining only minimal promoter sequence required for cAMP-dependent gene activation, but did not affect the 60-fold increase in cAMP induced by PGE(2). There is no consensus ERE in rat HGF-H P1, suggesting novel downstream interactions in the cAMP pathway that normally enhances IGF-I expression ist skeletal cells, To explore this, nuclear extract from osteoblasts expressing hER were examined by electrophoretic mobility shift assay using the atypical cAMP response element in IGF-I P1. Estrogen alone did not cause DNA-protein binding, while PGE(2) induced a characteristic gel shift complex. eo-treatment with both hormones caused a gel shift greatly diminished in intensity, consistent with their combined effects on IGF-H promoter activity. Nonetheless, hER did not bind IGF-I cAMP response element or any adjacent sequences,These results provide new molecular evidence that estrogen may temper the biological effects of hormones acting through cAMP to regulate skeletal IGF-I expression and activity,.