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 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 approximately 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 (approximately 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.
Sugawara
J
J
Department of Gynecology and Obstetrics, Stanford University Medical School, California 94305-5317, USA.
Tazuke
S I
SI
Suen
L F
LF
Powell
D R
DR
Kaper
F
F
Giaccia
A J
AJ
Giudice
L C
LC
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D000242:Cyclic AMP / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D006207:Half-Life; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D015592:Immunoradiometric Assay; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000096:biosynthesis* / Q000235:genetics; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012334:RNA, Neoplasm / Q000096:biosynthesis / Q000235:genetics; D014407:Tumor Cells, Cultured
D018970:Insulin-Like Growth Factor Binding Protein 1; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP