Prostaglandin E2 rapidly stimulates insulin-like growth factor-I gene expression in primary rat osteoblast cultures: evidence for transcriptional control.
Osteoblast-enriched (Ob) cultures isolated from fetal rat bone synthesize insulin-like growth factor-I (IGF-I), which functions as a locally acting growth and differentiation factor in the skeleton. Consistent with prior studies demonstrating that IGF-I production is enhanced in bone by agents that induce cAMP, prostaglandin E2 (PGE2) stimulates both cAMP synthesis and IGF-I mRNA in Ob cells. However, little is known about how cAMP regulates IGF-I expression in this or any other cell system. In rat tissues, multiple mechanisms influence levels of IGF-I mRNA, including transcription from two promoters, differential RNA splicing and stability, and alternative RNA polyadenylation. To determine how cAMP influences IGF-I gene expression in Ob cultures, we examined the responses of these cells to treatment with PGE2. PGE2 rapidly enhanced the accumulation of both large and small IGF-I transcripts, with increases in IGF-I mRNA detected within 2 h of treatment and persisting for 24 h. Analysis of precursor RNA by a highly specific and sensitive ribonuclease protection assay demonstrated a rise in nascent IGF-I mRNA within 30 min of exposure to PGE2, with a peak stimulation of 4-fold above control levels seen by 2 h and levels remaining elevated for up to 24 h. IGF-I transcripts in Ob cells were directed only by promoter 1, the more 5' of the two rat IGF-I gene promoters. As additionally assessed using the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole, PGE2 treatment had little effect on IGF-I mRNA stability. In aggregate, these studies show that in fetal rat Ob cultures, PGE2 enhances IGF-I gene expression primarily through transcriptional mechanisms that are limited to a single IGF-I gene promoter. Ob cells, therefore, may be an excellent model for determining how cAMP regulates IGF-I gene transcription.
Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110.
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Bichell
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Rotwein
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McCarthy
D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology; D015232:Dinoprostone / Q000494:pharmacology*; D005091:Exons; D005260:Female; D015870:Gene Expression / Q000187:drug effects*; D013006:Growth Hormone / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000235:genetics*; D007438:Introns; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects*
D012333:RNA, Messenger; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D013006:Growth Hormone; D000242:Cyclic AMP; D015232:Dinoprostone