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 posttranscriptional mechanisms to stimulate IGFBP-3 synthesis in MEC.