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

Thomas L. McCarthy, Changhua Ji, Hong Shu, Sandra Casinghino, Kristina Crothers, Peter Rotwein, Michael Centrella

    Research output: Contribution to journalArticlepeer-review

    68 Scopus citations

    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 μM PGE2 increased IGF-IP1 activity by 5-fold. 17β-Estradiol alone had no effect, but dose-dependently suppressed the stimulatory effect of PGE2 by up to 90% (ED50 ~0.1 nM). This occurred within 3 h, persisted for at least 16 h, required ER, and appeared specific, since 17α-estradiol was 100300-fold less effective. By contrast, 17β-estradiol stimulated estrogen response element (ERE)- dependent reporter expression by up to 10-fold. 17β-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.

    Original languageEnglish (US)
    Pages (from-to)18132-18139
    Number of pages8
    JournalJournal of Biological Chemistry
    Volume272
    Issue number29
    DOIs
    StatePublished - Jul 18 1997

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

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