Regulation of Insulin-Like Growth Factor-Binding Protein 1 by Hypoxia and 3′,5′-Cyclic Adenosine Monophosphate Is Additive in HepG2 Cells1

Insulin-like growth factor-binding protein 1 (IGFBP-1) is important in regulating minute-to-minute IGF bioavailability in the circulation and is primarily an inhibitor of IGF action systemically and in most cellular systems. Understanding regulation of IGFBP-1 is, thus, important in understanding regulation of IGF actions. The IGFBP-1 promoter contains a cAMP response element, and cAMP stimulates IGFBP-1 gene expression at the transcriptional level. Recently, we have found three consensus sequences for the hypoxia response element in intron 1 of the IGFBP-1 gene. Herein, we have investigated the effects of hypoxia and a cAMP analog, 8-bromoadenosine-3′,5′-cyclic monophosphate (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% pO2) or hypoxia (2% pO2) 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 predominant IGFBP in HepG2-conditioned media, which increased in a dose-dependent manner after incubation with 8-Br-cAMP in normoxia and hypoxia (3-fold and 7-fold at 1 mm, respectively). Under hypoxic, compared with normoxic, conditions, IGFBP-1 protein and messenger RNA (mRNA) levels increased ∼10-fold and 20-fold, respectively. In normoxia, 8-Br-cAMP stimulated IGFBP-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 IGFBP-1 protein and mRNA levels (35-fold and 50-fold at 1 mm) that were time and dose dependent. Primary transcripts of IGFBP-1 mRNA were increased concordantly with IGFBP-1 mRNA. The half-life of the IGFBP-1 mRNA was markedly increased (∼6-fold) by hypoxia, and cAMP minimally enhanced this effect. These results demonstrate that hypoxia and compounds that increase intracellular cAMP additively regulate IGFBP-1 gene expression by transcriptional and posttranscriptional mechanisms. Regulation of IGFBP-1 mRNA and protein by cAMP and hypoxia may be important for understanding the physiologic and pathophysiologic roles of IGFBP-1.