Regulation of insulin-like growth factor-binding protein 1 by hypoxia and 3 ',5 '-cyclic adenosine monophosphate is additive in HepG2 cells

Insulin-like growth factor-binding protein 1 (IGFBP-1) is important in regu lating minute-to-minute IGF bioavailability in the circulation and is prima rily an inhibitor of IGF action systemically and in most cellular systems. Understanding regulation of IGFBP-1 is, thus, important in understanding re gulation of IGF actions. The IGFBP-1 promoter contains a cAMP response elem ent, and cAMP stimulates IGFBP-1 gene expression at the transcriptional lev el. Recently, we have found three consensus sequences for the hypoxia respo nse element in intron 1 of the IGFBP-1 gene. Herein, we have investigated t he effects of hypoxia and a cAMP analog, 8-bromoadenosine-3',5'-cyclic mono phosphate (8-Br-cAMP), on IGFBP-1 expression in HepG2 cells, a model system for IGFBP-1 gene regulation. HepG2 cells were exposed to normoxia (20% pO( 2)) or hypoxia (2% pO(2)) for 24 h in the absence or presence of 8-Br-cAMP (0.1, 0.5, and 1 mM). Western ligand blotting revealed IGFBP-1 as the predo minant IGFBP in HepG2-conditioned media, which increased in a dose-dependen t manner after incubation with 8-Br-cAMP in normoxia and hypoxia (3-fold an d 7-fold at 1 mM, respectively): Under hypoxic, compared with normoxic, con ditions, IGFBP-1 protein and messenger RNA (mRNA) levels increased similar to 10-fold and 20-fold, respectively. In normoxia, 8-Br-cAMP stimulated IGF BP-1 protein and mRNA levels in a dose-dependent manner (7-fold and 10-fold at 1 mM). Hypoxia and 8-Br-cAMP showed additive stimulatory effects on IGF BP-1 protein and mRNA levels (35-fold and 50-fold at 1 mM) that were time a nd dose dependent. Primary transcripts of IGFBP-1 mRNA were increased conco rdantly with IGFBP-1 mRNA. The half-life of the IGFBP-1 mRNA was markedly i ncreased (similar to 6-fold) by hypoxia, and cAMP minimally enhanced this e ffect. These results demonstrate that hypoxia and compounds that increase i ntracellular cAMP additively regulate IGFBP-1 gene expression by transcript ional and posttranscriptional mechanisms. Regulation of IGFBP-1 mRNA and pr otein by cAMP and hypoxia may be important for understanding the physiologi c and pathophysiologic roles of IGFBP-1.