Identification of the cAMP response element that controls transcriptional activation of the insulin-like growth factor-I gene by prostaglandin E2 in osteoblasts.
Insulin-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.
Thomas
M J
MJ
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52246, USA.
Umayahara
Y
Y
Shu
H
H
Centrella
M
M
Rotwein
P
P
McCarthy
T L
TL
D000818:Animals; D001483:Base Sequence; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D003513:Cycloheximide / Q000494:pharmacology; D015342:DNA Probes / Q000378:metabolism; D015232:Dinoprostone / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D007334:Insulin-Like Growth Factor I / Q000235:genetics*; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012045:Regulatory Sequences, Nucleic Acid; D013379:Substrate Specificity; D014158:Transcription, Genetic
D017362:Cyclic AMP Response Element-Binding Protein; D015342:DNA Probes; D012333:RNA, Messenger; D007334:Insulin-Like Growth Factor I; D003513:Cycloheximide; D015232:Dinoprostone