Insulin-Like growth factor I (IGF-I) and cyclic adenosine 3',5'-monophosphate regulate IGF-binding protein-3 gene expression by transcriptional and posttranscriptional mechanisms in mammary epithelial cells.
Insulin-like growth factor I (IGF-I) is a potent mitogen for both normal and transformed mammary epithelial cells (MEC), and IGF-binding protein-3 (IGFBP-3) potentiates IGF-I action in these cells. The synthesis of IGFBP-3 is stimulated by both IGF-I and agents that increase intracellular cAMP (e.g. forskolin) in the bovine MEC line MAC-T. In addition, the combination of IGF-I and cAMP increases IGFBP-3 messenger RNA to a greater extent than does either treatment alone. The molecular mechanisms responsible for this regulation are not known and therefore represent the focus of this study. The half-life of IGFBP-3 messenger RNA in untreated MAC-T cells was determined to be 11 h. Exposure to IGF-I or forskolin increased the half-life to 27 and 101 h, respectively. Nuclear run-on assays indicated that IGFBP-3 transcription rates were increased 3.5 +/- 0.83-fold (n = 4) in cells treated with a combination of IGF-I and forskolin. To further study this regulation, 1.1 kb of the 5'-flanking region of the IGFBP-3 promoter were fused to a promoterless reporter plasmid encoding luciferase. Transient transfection assays indicated that both IGF-I and forskolin alone produced small, but significant, increases in IGFBP-3 promoter activity of 1.57 +/- 0.12 and 1.59 +/- 0.08-fold (P < 0.01), respectively (mean +/- SE; n = 7). However, the combination of IGF-I and forskolin increased IGFBP-3 promoter activity 2.25 +/- 0.14-fold above control values (P < 0.01), suggesting that these factors activate discrete signaling pathways that act in concert to stimulate IGFBP-3 gene transcription. Deletion analysis indicated that promoter fragments containing as little as 267 bp upstream of the TATA box retained responsiveness to IGF-I and forskolin. This region contains a 200-bp sequence that is approximately 80% homologous between the murine and bovine promoters. It contains several conserved AP-2 and Sp1 consensus binding sequences that may be important for the effects of IGF-I and forskolin on IGFBP-3 promoter activity. In summary, these data indicate that IGF-I and cAMP, working through separate signaling pathways, activate both transcriptional and post-transcriptional mechanisms to stimulate IGFBP-3 synthesis in MEC.
Cohick
W S
WS
Department of Animal Sciences, Rutgers, State University of New Jersey, New Brunswick 08901, USA. cohick@aesop.rutgers.edu
Wang
B
B
Verma
P
P
Boisclair
Y R
YR
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000187:drug effects*; D006207:Half-Life; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D008321:Mammary Glands, Animal / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D017385:Sequence Homology; D015398:Signal Transduction; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection
D018972:Insulin-Like Growth Factor Binding Protein 3; D012333:RNA, Messenger; D005576:Colforsin; D007334:Insulin-Like Growth Factor I; D000242:Cyclic AMP