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Identification of the cAMP response element that controls transcriptional activation of the insulin-like growth factor-I gene by prostaglandin E2 in osteoblastsInsulin-like growth factor-I (IGF-I), a multifunctional growth factor, plays a key role in skeletal growth and can enhance bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other agents that increase cAMP activated IGF-I gene transcription in primary rat osteoblast cultures through promoter 1 (P1), the major IGF-I promoter, and found that transcriptional induction was mediated by protein kinase A. We now have identified a short segment of P1 that is essential for full hormonal regulation and have characterized inducible DNA-protein interactions involving this site. Transient transfections of IGF-I P1 reporter genes into primary rat osteoblasts showed that the 328-base pair untranslated region of exon 1 was required for a full 5.3-fold response to PGE2; mutation in a previously footprinted site, HS3D (base pairs +193 to +215), reduced induction by 65%. PGE2 stimulated nuclear protein binding to HS3D. Binding, as determined by gel mobility shift assay, was not seen in nuclear extracts from untreated osteoblast cultures, was detected within 2 h of PGE2 treatment, and was maximal by 4 h. This DNA-protein interaction was not observed in cytoplasmic extracts from PGE2-treated cultures, indicating nuclear localization of the protein kinase A-activated factor(s). Activation of this factor was not blocked by cycloheximide (Chx), and Chx did not impair stimulation of IGF-I gene expression by PGE2. In contrast, binding to a consensus cAMP response element (CRE; 5'-TGACGTCA-3') from the rat somatostatin gene was not modulated by PGE2 or Chx. Competition gel mobility shift analysis using mutated DNA probes identified 5'-CGCAATCG-3' as the minimal sequence needed for inducible binding. All modified IGF-I P1 promoterreporter genes with mutations within this CRE sequence also showed a diminished functional response to PGE2. These results identify the CRE within the 5'-untranslated region of IGF-I exon 1 that is required for hormonal activation of IGF-I gene transcription by cAMP in osteoblasts.
Document ID
20040173147
Acquisition Source
Legacy CDMS
Document Type
Reprint (Version printed in journal)
Authors
Thomas, M. J.
(University of Iowa College of Medicine Iowa City, Iowa 52246, United States)
Umayahara, Y.
Shu, H.
Centrella, M.
Rotwein, P.
McCarthy, T. L.
Date Acquired
August 22, 2013
Publication Date
September 6, 1996
Publication Information
Publication: The Journal of biological chemistry
Volume: 271
Issue: 36
ISSN: 0021-9258
Subject Category
Life Sciences (General)
Funding Number(s)
CONTRACT_GRANT: DK37449
CONTRACT_GRANT: HD20805
CONTRACT_GRANT: DK47421
Distribution Limits
Public
Copyright
Other
Keywords
NASA Discipline Musculoskeletal
Non-NASA Center

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