title: 17beta-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 E2 (PGE2) 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-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 microM PGE2 increased IGF-I P1 activity by 5-fold. 17beta-Estradiol alone had no effect, but dose-dependently suppressed the stimulatory effect of PGE2 by up to 90% (ED50 approximately 0.1 nM). This occurred within 3 h, persisted for at least 16 h, required ER, and appeared specific, since 17alpha-estradiol was 100-300-fold less effective. By contrast, 17beta-estradiol stimulated estrogen response element (ERE)-dependent reporter expression by up to 10-fold. 17beta-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 PGE2. 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 PGE2 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.
authors.affiliation: Yale University School of Medicine, Section of Plastic Surgery, New Haven, Connecticut 06520-8041, USA. McCarthyTL@maspo3.mas.yale.edu
authors.forename: T L
authors.initials: TL
authors.lastname: McCarthy
authors.forename: C
authors.initials: C
authors.lastname: Ji
authors.forename: H
authors.initials: H
authors.lastname: Shu
authors.forename: S
authors.initials: S
authors.lastname: Casinghino
authors.forename: K
authors.initials: K
authors.lastname: Crothers
authors.forename: P
authors.initials: P
authors.lastname: Rotwein
authors.forename: M
authors.initials: M
authors.lastname: Centrella
mesh_terms: D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D016384:Consensus Sequence; D000242:Cyclic AMP / Q000037:antagonists & inhibitors / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000502:physiology; D015232:Dinoprostone / Q000494:pharmacology*; D004958:Estradiol / Q000494:pharmacology*; D005333:Fetus; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D011960:Receptors, Estrogen / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: D004268:DNA-Binding Proteins; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D004958:Estradiol; D007334:Insulin-Like Growth Factor I; D000242:Cyclic AMP; D008156:Luciferases; D015232:Dinoprostone