17β-Estradiol Potently Suppresses cAMP-induced Insulin-like Growth Factor-I Gene Activation in Primary Rat Osteoblast Cultures

Abstract

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 estrogendependent regulation of IGF-I 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 1 of the rat IGF-I gene (IGF-I P1), used exclusively in these cells. As reported, 1 M PGE 2 increased IGF-I P1 activity by 5-fold. 17␤-Estradiol alone had no effect, but dose-dependently suppressed the stimulatory effect of PGE 2 by up to 90% (ED 50 ϳ0.1 nM). This occurred within 3 h, persisted for at least 16 h, required ER, and appeared specific, since 17␣-estradiol was 100 -300-fold less effective. By contrast, 17␤-estradiol stimulated estrogen response element (ERE)-dependent reporter expression by up to 10-fold. 17␤-Estradiol also suppressed an IGF-I P1 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 IGF-I P1, suggesting novel downstream interactions in the cAMP pathway that normally enhances IGF-I expression in 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. Co-treatment with both hormones caused a gel shift greatly diminished in intensity, consistent with their combined effects on IGF-I 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.Although postmenopausal osteoporosis is one of the most prevalent age-related skeletal disorders, our understanding of the role of estrogen, which has a critical role in maintaining bone mass, remains incomplete. The principal laboratory animal model of postmenopausal osteoporosis is the ovariectomized (OVX) 1 rat. One consistent observation with this model is an increase in the rate of bone remodeling after OVX-induced estrogen depletion (1-5). Furthermore, consistent with a cause and effect relationship, estrogen administration re-establishes a reduced rate of bone remodeling in the OVX rat.Bone remodeling consists of two opposing events, i.e. bone resorption and formation (6). Skeletal integrity in adults relies on closely coupled remodeling where there is a balance between these catabolic and anabolic processes. Net bone loss in postmenopausal o...

Insulin-like growth factor-1 (IGF1) genotype predicts breast volume after pregnancy and hormonal contraception and is associated with circulating IGF-1 levels: implications for risk of early-onset breast cancer in young women from hereditary breast cancer families

“…The IGF1 polymorphism is located in the promoter region of the IGF1 gene (Rosen et al, 1998). This microsatellite polymorphism is located one kilobase upstream from the IGF1 transcription start site (Rotwein et al, 1986;Weber and May, 1989) and contains specific regulatory elements (McCarthy et al, 1997). In vitro studies of rat osteoblasts have shown a 100 -300-fold difference in their ability to suppress the stimulatory effect of prostaglandin E 2 in response to 17-alpha and 17-beta estradiol when they bind to the oestrogen response elements within the IGF1 promoter region (McCarthy et al, 1997).…”

Runx2 Integrates Estrogen Activity in Osteoblasts

“…To account for competition among plasmids for limiting transcriptional components, control cultures were transfected with a compensating amount of empty vector. Transfection efficiency was assessed in parallel in cells transfected with positive and negative reporter plasmids as described previously (16,23,(35)(36)(37).…”

“…In some samples, nuclear extract was pre-incubated with antiserum for 1 h before adding 32 P-labeled probe. Radioactive complexes were resolved on a 4% nondenaturing polyacrylamide gel and visualized by autoradiography (16,23,37).…”

“…Nuclei and cytoplasm were separated by centrifugation. Nuclei were extracted in hypertonic buffer with glycerol and phosphatase and protease inhibitors, and the nuclear proteins released in this way were separated from insoluble material by centrifugation (16,23,37).…”

“…Estrogen also regulates factors associated with bone formation. For example, estrogen suppresses IGF-I expression in osteoblasts stimulated with hormones that increase cAMP generation and activate transcription factor C/EBP␦ by decreasing C/EBP␦-dependent activation of the IGF-I gene promoter (23). However, estrogen may also enhance bone formation through inhibitory effects on osteoblast apoptosis or stimulatory effects on other osteoblast transcription factors (24 -27).…”