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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 8 2714-2721
Copyright © 2000 by The Endocrine Society

Original Studies

Regulation of Insulin-Like Growth Factor-Binding Protein-1 by Nitric Oxide under Hypoxic Conditions1

J. Sugawara, D.-S. Suh, G. H. Faessen, L.-F. Suen, T. Shibata, F. Kaper, A. J. Giaccia and L. C. Giudice

Departments of Gynecology and Obstetrics (J.S., D.-S.S., G.H.F., L.-F.S.) and Radiation Oncology (T.S., F.K., A.J.G.), Stanford University Medical School, Stanford, California 94305-5317

Address all correspondence and requests for reprints to: Linda C. Giudice, M.D., Ph.D., Department of Gynecology and Obstetrics, Stanford University Medical Center, Room HH-333, Stanford, California 94305. E-mail: giudice{at}stanford.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide (NO) is believed to play an important, but as yet undefined, role in regulating hypoxia inducible gene expression. Recently, we have reported evidence suggesting that the human insulin-like growth factor-binding protein-1 (IGFBP-1) gene is directly regulated by hypoxia through the hypoxia-inducible factor-1 pathway. The goal of the current study was to investigate NO regulation of hypoxic induction of IGFBP-1 gene expression using HepG2 cells, a model system of hepatic gene expression. We report that a NO generator, sodium nitroprusside, significantly diminishes hypoxic activation of IGFBP-1 protein and messenger ribonucleic acid expression. Furthermore, these effects are independent of guanylate cyclase/cGMP signaling, as two different inhibitors, LY 83583, a specific inhibitor of guanylate cyclase, and KT 5823, a protein kinase G inhibitor, had no effect on IGFBP-1 induction by hypoxia. Hypoxic induction of a reporter gene containing four tandemly ligated hypoxia response elements was completely blocked by sodium nitroprusside, but not by 8-bromo-cGMP, an analog of cGMP. These results suggest that NO blocks hypoxic induction of IGFBP-1 by a guanylate cyclase/cGMP-independent pathway, possibly at the level of oxygen sensing. The impaired hypoxia regulation of IGFBP-1 by nitric oxide may play a key role in the hyperinduction of IGFBP-1 observed in pathophysiological conditions such as fetal hypoxia and preeclampsia where dysregulation of NO has been observed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN HUMANS, insulin-like growth factor-binding protein-1 (IGFBP-1) is primarily expressed in the adult liver, the liver and kidney of the developing fetus, and the maternal endometrium of pregnancy (the decidua) (reviewed in Ref. 1). It is elevated in the circulation and liver in fetuses with in utero hypoxia and intrauterine growth restriction (IUGR) (2, 3, 4, 5, 6, 7, 8, 9) and is believed to contribute to IUGR by inhibiting IGF-mediated fetal growth. In preeclampsia, a human pregnancy disorder complicated by hypertension, edema, proteinuria, and hypoxia at the maternal:fetal interface (10), levels of IGFBP-1 are elevated in the maternal circulation (11, 12, 13). The source of the observed elevated maternal IGFBP-1 levels has not yet been determined, but may be hepatic in origin or from the decidua (13). In preeclampsia elevated levels of an endogenous inhibitor of nitric oxide (NO) synthase have been postulated to contribute to increased maternal vascular resistance (14). Recently, we demonstrated that hypoxia regulates IGFBP-1 gene induction (15), and work from others has demonstrated multiple regulatory elements in the IGFBP-1 promoter (16) relevant to fetal growth and implantation. These include an insulin response element that decreases IGFBP-1 gene expression and two glucocorticoid response elements, a cAMP response element, and a steroid response element, that increase IGFBP-1 expression. In addition, we identified three consensus sequences for the hypoxia response element (HRE) in intron 1 of the human IGFBP-1 gene and demonstrated that at least one is hypoxia responsive with regard to IGFBP-1 gene expression (15). IGFBP-1 induction by hypoxia is mediated via hypoxia-inducible factor-1 (HIF-1) (15), important in the response of other hypoxia-inducible genes (17).

The oxygen sensing and signal processing that activate HIF-1 are poorly understood. Recent evidence suggests that the oxygen sensor is a heme protein (18, 19), and that heme-binding ligands, such as NO and carbon monoxide (CO) suppress hypoxic induction of several genes by inhibiting activation of HIF-1 binding to the HRE (20, 21, 22). Thus, gaseous molecules, such as NO, may mediate physiological adaptive responses by regulating cellular oxygen sensing and downstream, hypoxia-inducible gene expression. This is in contrast to the classical mechanism of action of NO that activates guanyl cyclase, generating cGMP.

Herein, we report that a NO generator, sodium nitroprusside (SNP), markedly diminishes hypoxic activation of IGFBP-1 protein and messenger ribonucleic acid (mRNA) in Hep G2 cells, a model system for studying IGFBP-1 gene regulation. Furthermore, we demonstrate that these effects are independent of a pathway involving guanylate cyclase/cGMP, suggesting that NO inhibition of hypoxic induction of IGFBP-1 gene expression may be at the level of oxygen sensing. These results suggest that a unique regulatory mechanism may exist within the liver for IGFBP-1 gene expression under hypoxic conditions. Whether this has relevance to elevated IGFBP-1 in fetuses developing in pregnancies complicated by uteroplacental insufficiency or in the maternal circulation of preeclamptic pregnancies awaits further investigation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

HepG2 cells, a human hepatocellular carcinoma cell line (23) known to produce IGFBP-1 (24, 25), were purchased from the American Type Culture Collection (ATCC HB8065, Manassas, VA). Cells were grown at 37 C in 95% air-5% CO2 in 175-mm2 cell culture flasks and were fed every 3–4 days with MEM (Life Technologies, Inc., Grand Island, NY) plus 10% FBS and 50 mg/mL gentamicin (Gemini Bio Products, Inc., Calabasas, CA), 0.1 mmol/L nonessential amino acids, and 1 mmol/L sodium pyruvate (Life Technologies, Inc.). Cultures were passaged weekly when confluent at a ratio of 1:4 using trypsin-ethylenediamine tetraacetate (Life Technologies, Inc.).

Materials

SNP and 8-bromo-cGMP (8Br-cGMP) were purchased from Sigma (St. Louis, MO). LY83583 (6-anilino-5,8-quinoline-dione) and KT 5823 were purchased from Calbiochem (La Jolla, CA). Radioactive isotopes were purchased from NEN Life Science Products-DuPont (Boston, MA).

Cell culture and hypoxia treatment

Cells were plated in duplicate in custom-made 60-mm glass plates, with notched sides to allow gas change (26), at 1 x 106 cells and stabilized in serum-free MEM (Life Technologies, Inc.) for 24 h before the following experiments. To examine the effect of SNP and 8Br-cGMP on the expression of IGFBP-1 under normoxic and hypoxic conditions, cells were pretreated with different concentrations of SNP (0.1, 0.5, and 1 mmol/L) and 8Br-cGMP (0.01, 0.1, 0.5, 1.0, and 15 mmol/L) for 1 h. Dishes were placed in specially designed aluminum hypoxia chambers that were prewarmed to 37 C, sealed, and subjected to successive rounds of evacuation followed by flushing with 95% N2/5% CO2 while being slowly agitated as previously described (15). The final oxygen concentration in the medium after one cycle was reduced to 2%, as measured with a Clark-type electrode (Controls Katharobic, Edmonton, Canada). Kinetic studies were conducted in which cells were treated in hypoxia for 2, 6, 12, or 24 h with or without SNP (1 mmol/L). To assess the effect of inhibitors of the nitric oxide/cGMP pathway, LY83583, a specific guanylate cyclase or KT 5823, a specific inhibitor of protein kinase G, was added to Hep G2 cells at final concentrations of 0.01, 0.1, and 1.0 mmol/L. After pretreatment with these inhibitors for 30 min, cells were treated with or without SNP under normoxic or hypoxic conditions for 6 h. At the conclusion of the experiments conditioned media were collected, centrifuged, aliquoted, and stored at -20 C for subsequent analysis. Total cellular RNA was isolated as described below.

Northern blot hybridization

Total RNA was isolated with a modification of the method described by Chomczynski and Sacchi (27), using TRIzol (Life Technologies, Inc.) according to the manufacturer’s instructions. Ten micrograms of total RNA were loaded per lane for 1.2% agarose-formaldehyde gel electrophoresis. Gels were then stained with ethidium bromide to asses loading of total RNA in each lane. RNA was transferred onto nitrocellulose membranes by capillary transfer, and the filters were irradiated in a Stratagene UV cross-linking apparatus (Stratagene, La Jolla, CA). A 938-bp EcoRI fragment of the human IGFBP-1 complementary DNA (cDNA) (28) was labeled with [{alpha}-32P]deoxy-CTP (NEN Life Science Products), using a random priming kit (Pharmacia Biotech, Piscataway, NJ). Membranes were blocked at 42 C in 50% formamide, 5 x SSC (standard saline citrate), 0.3% SDS, 5 x Denhart’s solution, and 100 mg/mL salmon sperm DNA and then probed overnight at 42 C using 1 x 106 cpm/mL of the random primed probe. Filters were washed twice at room temperature in 5 x SSC and 0.5% SDS, twice at 37 C in 1 x SSC and 0.5% SDS, and twice at 65 C in 0.1 x SSC and 1.0% SDS. Films were exposed at -70 C using DuPont enhancing screens. Molecular sizes were confirmed using a RNA marker (Promega Corp., Madison, WI) and ribosomal RNA (18S and 28S) as standards. The hybridization signals on the blots were analyzed quantitatively on a laser scanning densitometer (LKB, Bromma, Sweden). The intensity of the blots was normalized with 18S ribosomal RNA on ethidium bromide staining, and the relative intensity was calculated as a fold of the mean RNA value compared to controls.

IGFBP-1 immunoradiometric assay (IRMA)

IGFBP-1 levels in conditioned medium from duplicate cultures were analyzed by IRMA, according to the manufacturer’s instructions (Diagnostics Systems Laboratories, Inc., Webster, TX). The sensitivity was 0.01 ng/mL, and the intra- and interassay coefficients of variation were 4.7% and 2.3%, respectively. IGFBP-1 levels were normalized to total protein in conditioned medium by the method of Bradford (29).

Western ligand blotting

Western ligand blotting was performed as previously described (30). Seventy-five microliters of conditioned medium were electrophoresed on 12% SDS-PAGE with a 5% stack at 50 V. The size-fractionated proteins were electroblotted onto nitrocellulose at 160 mA for 1 h and 20 min. Blocking was with 1% BSA in saline for 2 h. The filter-immobilized proteins were incubated with 1.5 x 106 cpm/mL [125I]IGF-I and [125I]IGF-II overnight at 4 C, washed, air-dried, and exposed to autoradiography film (NEN Life Science Products) at -80 C. The intensity of the blots was quantitated by laser scanning densitometry, as previously described (15).

Plasmid construction

Complementary oligonucleotides representing the IGFBP-1 HRE (nucleotides 706–756) were synthesized with BamHI-BglII-compatible ends. HRE 4-Luc, a vector consisting of four copies of the HRE of IGFBP-1 gene, was prepared by ligating precut, double stranded oligonucleotide to the BamHI-BglII sites of PGL3 promoter plasmid (Promega Corp.), which contains a heterologous simian virus 40 promoter and luciferase gene. mHRE4-Luc containing four copies of mutated HRE was also constructed. (The structures of the mutant and wild-type constructs are shown in Fig. 3Go with the relevant data.)



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  		Figure 3. Effect of SNP on reporter gene expression. A, Sequence of the sense strands of oligonucleotides used. HRE, The conserved core of the HIF-1 binding site (ACGT) is in bold; MHRE, mutated bases are in small letters and are underlined. B, Schematic representation of the wild-type (HRE4-Luc) and mutant (mHRE4-Luc) constructs. The four copies of oligonucleotides HRE and mHRE, shown in A, were subcloned 5' of the PGL3 promoter vector containing a simian virus 40 promoter. C, Effect of SNP on reporter gene expression. HepG2 cells were transfected with the plasmid HRE4-Luc or mHRE4-Luc and incubated in normoxia or hypoxia in the presence or absence of 1 mmol/L SNP. The resulting relative luciferase activity (the ratio of firefly to control Renilla luciferase activity) was compared to the untreated normoxia control value. Results are the mean ± SEM for at least three independent experiments for each construct.

 
Transient transfection and luciferase assay

HepG2 cells were seeded onto 60-mm plates at a density adjusted to reach 50% confluence before transfection. Cells were cotransfected with Superfect reagent (QIAGEN, Valencia, CA), 5 µg HRE4-Luc, (or mHRE 4-Luc, PGL3 promoter), and 50 ng Renilla luciferase internal control vector (pRL-SV40) according to manufacturer’s instructions. After 18 h of incubation, cells were washed twice with PBS and treated with either SNP or 8Br-cGMP in a serum-free medium for 6 h under normoxic or hypoxic conditions. Luciferase reporter activity was measured with a luminometer and was normalized for Renilla luciferase activity (dual luciferase assay system, Promega Corp.).

Statistical analysis

Results were presented as the mean ± SEM. Differences between groups were examined by one-way ANOVA followed by Scheffe’s F test, and significance was assigned at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of SNP on IGFBP-1 expression under hypoxic conditions

Dose response. SNP, a NO donor, is commonly used to investigate the effects of NO on cellular function. HepG2 cells were treated with or without SNP under hypoxic or normoxic conditions for 6 h, and total RNA was isolated and hybridized to the IGFBP-1 cDNA probe by Northern blotting. A 1.5-kb transcript (Fig. 1Go), identical in size to the IGFBP-1 mRNA previously described (31), was observed. In the absence of SNP, IGFBP-1 mRNA levels were increased an average of 2.8-fold by hypoxic conditions compared to those under normoxic conditions. Hypoxic induction of IGFBP-1 mRNA was markedly suppressed by SNP treatment in a dose-dependent fashion. In contrast, SNP showed little effect on IGFBP-1 mRNA expression in normoxia.



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  		Figure 1.
 
IRMA analysis of conditioned medium from hypoxic cells revealed that IGFBP-1 protein levels increased 3.3-fold (Fig. 1BGo). Under normoxic conditions, IGFBP-1 protein levels were not affected by any concentration of SNP tested. However, under hypoxic conditions, 1 mmol/L SNP inhibited IGFBP-1 levels 73% compared to those in untreated hypoxic controls. IGFBP-1 in conditioned medium was also analyzed by Western ligand blotting (Fig. 1CGo). In normoxia, SNP showed little effect on IGFBP-1 levels, whereas in hypoxic conditions, induction of IGFBP-1 levels was markedly inhibited in a dose-dependent manner, with 1 mmol/L SNP inhibiting levels by 77%. No additional IGFBPs were affected by hypoxia and/or SNP (Fig. 1CGo) under these conditions. These results suggest that NO plays an important role in signaling IGFBP-1 induction under hypoxic conditions.

Time course. To evaluate the kinetics of IGFBP-1 mRNA expression in the presence of SNP under hypoxic conditions, HepG2 cells were preincubated in serum-free medium for 24 h and then cultured in fresh serum-free medium under hypoxic conditions with or without 1 mmol/L SNP for 2, 6, 12, and 24 h. In hypoxic conditions, IGFBP-1 mRNA increased rapidly and reached maximum stimulation at 12 h of incubation (Fig. 2AGo). With the addition of SNP to cells under hypoxic conditions, induction of IGFBP-1 mRNA was decreased throughout the 24 h of culture. IRMA analysis (Fig. 2BGo) of the conditioned medium indicated that SNP completely inhibited hypoxic induction of IGFBP-1 compared to that in untreated hypoxic cells, which exhibited a rapid increase in IGFBP-1 after 6 h and reached maximum stimulation at 12 h.



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  		Figure 2. Kinetics of IGFBP-1 expression in Hep G2 cells treated with SNP under hypoxic conditions. A, Northern blot. HepG2 cells were pretreated with SNP (1 mmol/L) for 1 h, and total RNA was extracted at the indicated times (0, 2, 6, 12, and 24 h) and analyzed by Northern blot hybridization. Relative IGFBP-1 mRNA levels are shown. Densitometric values were normalized to 18S ribosomal RNA values obtained from the ethidium bromide staining. IGFBP-1 mRNA levels were compared to levels at the 0 h point and are expressed as fold induction in the graph. Data are the mean ± SEM from three independent experiments. B, IRMA. IGFBP-1 levels in conditioned medium were quantitated by IRMA (nanograms per mL) and normalized by total protein concentrations (micrograms per mL). Values shown are the mean ± SEM of three different experiments. {circ}, Hypoxia control; {square}, hypoxia and SNP treatment (1 mmol/L).

 
SNP suppresses the hypoxic induction of reporter gene expression

To investigate whether the inhibitory effects of NO occur at the transcriptional level, the effects of SNP on reporter gene expression were investigated. We generated a HRE multimer construct containing four copies of the human IGFBP-1 HRE (HRE4-Luc) or mutated HRE (mHRE4-Luc), inserted 5' of the PGL-3 vector containing a simian virus 40 promoter (Fig. 3Go, A and B). After transfection into HepG2 cells, SNP (1 mmol/L) was added, and the cells were incubated under normoxia or hypoxia for 6 h. In HepG2 cells transfected with HRE4-Luc, hypoxia significantly increased luciferase expression by 2.2-fold (P < 0.05; Fig. 3CGo). SNP markedly suppressed hypoxic induction of the reporter gene expression (Fig. 3CGo). The luciferase activities of mHRE4-Luc and PGL-3 vector were not significantly induced by hypoxia, and SNP displayed little effect on luciferase gene expression (data not shown).

Guanylate cyclase/cGMP pathway and IGFBP-1 expression

Several biological actions of NO are mediated by the activated soluble guanylate cyclase/cGMP pathway, which could be a mechanism involved in the observed suppression of the hypoxic induction of IGFBP-1 by NO. The effect of 8Br-cGMP, an important second messenger of guanylate cyclase, on IGFBP-1 mRNA expression and protein secretion by HepG2 cells was investigated. After 6 h of incubation, cGMP showed no effect on IGFBP-1 mRNA expression (Fig. 4AGo) or protein production (Fig. 4BGo) under hypoxic or normoxic conditions. This was also observed at supraphysiological concentrations (15 mmol/L) of cGMP (data not shown). We also investigated the effects of 8Br-cGMP on reporter gene expression using the constructs described above. After transfection into HepG2 cells, 8Br-cGMP (1 mmol/L) was added, and the cells were incubated under normoxic or hypoxic conditions for 6 h. In HepG2 cells transfected with HRE4-Luc, hypoxia significantly increased luciferase expression, whereas 8Br-cGMP had little effect (Fig. 4CGo).



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  		Figure 4. Effect of cGMP on the hypoxic induction of IGFBP-1. A, Effect of 8Br-cGMP on IGFBP-1 mRNA expression. Cells were treated with 8Br-cGMP (1 mmol/L) for 6 h under normoxia and hypoxia. Total RNA was extracted and analyzed by Northern blot hybridization. The 1.5-kb IGFBP-1 mRNA and ethidium bromide staining of 18S ribosomal RNA are shown. The densitometry results of the 1.5-kb IGFBP-1 mRNA were normalized to the 18S ribosomal RNA values obtained with ethidium bromide staining. IGFBP-1 mRNA levels were compared to the normoxia control and expressed as fold induction in the graph. Data are the mean ± SEM from three independent experiments. B, IRMA analysis. IGFBP-1 levels in conditioned medium were quantitated by IRMA (nanograms per mL) and normalized by total protein concentrations (micrograms per mL). Values compared to the normoxic control are the mean ± SEM of three different experiments. C, Effect of 8Br-cGMP on reporter gene expression. HepG2 cells were transfected with the plasmid HRE4-Luc or mHRE4-Luc and incubated in normoxia or hypoxia in the presence or absence of 1 mmol/L 8Br-cGMP. The resulting relative luciferase activity (the ratio of firefly to control Renilla luciferase activity) was compared to the untreated normoxia control value. Results are the mean ± SEM for at least three independent experiments for each construct.

 
We also assessed the effects of LY 83583, a specific inhibitor of guanylate cyclase, and KT 5823, a protein kinase G inhibitor, on IGFBP-1 mRNA expression and protein production. Cells were pretreated with each inhibitor for 30 min and then exposed to normoxic or hypoxic conditions for 6 h with or without the addition of SNP (1 mmol/L). Although IGFBP-1 mRNA expression (Fig. 5AGo) and protein secretion (Fig. 5BGo) under hypoxic conditions were not affected by LY 83583 or KT 5823, SNP markedly suppressed mRNA expression in the presence of these inhibitors. In normoxia, LY 83583 and KT 5823 exhibited little effect on IGFBP-1 mRNA expression, and IGFBP-1 expression was not affected by the addition of SNP (data not shown). Taken together, these results suggest that NO suppression of IGFBP-1 expression under hypoxic conditions is not mediated via the cGMP pathway.



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  		Figure 5. Effects of guanylate cyclase/cGMP inhibitors on the suppressive effect of hypoxic induction of IGFBP-1. A, Effects on IGFBP-1 expression. Cells were preincubated with LY 83583 and KT 5823 for 30 min, and incubated with or without SNP (1 mmol/L) under normoxia or hypoxia for 6 h. Total RNA was extracted and analyzed by Northern blot. IGFBP-1 mRNA and 18S ribosomal RNA are shown. Densitometry was performed for the 1.5-kb IGFBP-1 mRNA transcript and the 18S ribosomal RNA band observed by ethidium bromide staining. IGFBP-1 mRNA levels were compared to the normoxia control value and are expressed as fold induction in the graph. Data are the mean ± SEM from three independent experiments. B, IRMA. IGFBP-1 levels in conditioned medium were quantitated by IRMA (nanograms per mL) and normalized by total protein concentrations (micrograms per mL). Values compared to normoxic control are the mean ± SEM of three different experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We previously reported that hypoxia induces IGFBP-1 gene expression through a HIF-1-mediated pathway in HepG2 cells (15). Recently, several studies have demonstrated that NO modulates hypoxic gene expression in vitro, seemingly through regulation of HIF-1 (32, 33, 34, 35). Herein, we have presented data demonstrating that SNP, a NO donor, inhibits the hypoxic induction of IGFBP-1 mRNA expression and protein production in Hep G2 cells in a dose-dependent manner. Kinetic studies indicate that the addition of SNP markedly and rapidly inhibits the hypoxic induction of IGFBP-1 gene expression and sustains this inhibition for at least 24 h. In contrast, SNP does not show any effect on IGFBP-1 gene expression under normoxic conditions. Two possible mechanisms accounting for these observations include 1) the effect of NO on hypoxic induction of IGFBP-1 gene expression is mediated through the activation of a second messenger of NO, e.g. the guanylate cyclase/cGMP pathway; or 2) NO affects hypoxia sensing and blocks HIF-1 activation through a proximal step. Most of the biological actions of NO have been reported to be mediated through the guanylate cyclase/cGMP pathway (36). In the current study, 8Br-cGMP, a second messenger of NO, even at supraphysiological concentrations did not affect IGFBP-1 mRNA expression and protein secretion in HepG2 cells under normoxic or hypoxic conditions in the presence or absence of NO. Furthermore, the inhibitory effect of NO was not diminished by LY83583, a specific guanylate cyclase inhibitor, or KT 5823, a protein kinase G inhibitor. Taken together, these findings support the conclusion that the effect of NO on the hypoxic induction of IGFBP-1 is independent of the guanylate cyclase/cGMP pathway and that NO inhibits the hypoxic induction of the IGFBP-1 gene at the level of oxygen sensing.

It has been hypothesized that when heme ligands (e.g. NO or CO) bind to the oxygen sensor with higher affinities than O2 (37, 38), the sensor protein locks into a relaxed oxy form configuration, whereas without ligands the sensor keeps the deoxy form, and the hypoxic signal transduction pathway is activated. Recent evidence suggests that NO and other gaseous molecules, e.g. CO, which are heme ligands, suppress hypoxia induction of gene expression by blocking HIF-1 binding to the HRE (20, 21, 22). In the current study, experiments with the HRE4-Luc reporter/promoter construct demonstrate that SNP significantly suppresses hypoxic induction of reporter gene expression. These data suggest that NO blocks HIF-1 activity and diminishes hypoxic activation of the IGFBP-1 gene. Furthermore, 8Br-cGMP does not affect hypoxic induction of IGFBP-1 gene expression at the transcriptional level, suggesting that the blocking effect of SNP on HIF-1 activity is probably mediated through an alternative pathway. In other cell models, e.g. Hep3B, HeLa, and Neuro 2A cells, hypoxia-inducible gene expression of vascular endothelial growth factor and erythropoietin is suppressed by NO or CO at the level of oxygen sensing, independent of the guanylate cyclase/cGMP pathway (20, 22, 33), consistent with results reported herein. However, in contrast, Liu et al. (21), using bovine pulmonary artery endothelial cells and rat aortic smooth muscle cells, reported that the inhibitory effect of NO on VEGF induction is mediated through the guanylate cyclase/cGMP pathway. Thus, regulation of hypoxia-inducible genes by NO is cell- or tissue-specific, and the molecular mechanisms responsible for this dichotomous response remain to be determined.

NO has diverse physiological roles, including neurotransmission and maintenance of vascular tone. The finding that NO is a heme ligand and effectively inhibits hypoxia induction of a variety of genes, including IGFBP-1, suggests that NO may have additional physiological roles, e.g. providing a counterregulatory mechanism for hypoxia-induced gene regulation. These additional roles are likely to be cell- and tissue-specific. There are several pathophysiological states in which circulating IGFBP-1 levels are markedly elevated in the setting of tissue or systemic hypoxia. These include IUGR due to uteroplacental insufficiency and in utero hypoxia, and also preeclampsia, a hypertensive disorder of pregnancy in which there is hypoxia in the feto-placental unit. In preeclampsia, decreased levels of NO are believed to contribute to the elevated systolic blood pressure observed (39), and impairment of NO regulation has been suggested (40, 41). It is tempting to speculate that hypoxia contributes to the observed elevation of IGFBP-1 in these disorders and that dysregulation of NO may permit elevated IGFBP-1 expression. The inhibition by SNP, a NO donor, of hypoxic induction of IGFBP-1 in Hep G2 cells, described herein, provides insight into mechanisms underlying IGFBP-1 gene regulation. The data described suggest that NO regulates IGFBP-1 at the level of gene transcription, that these effects are independent of the guanylate cyclase/cGMP pathway, and that the regulation probably occurs at the level of oxygen sensing. However, whether these mechanisms are operational in pregnancy disorders such as preeclampsia or IUGR due to uteroplacental insufficiency and in utero hypoxia awaits further extensive investigation in vitro with relevant tissues and in vivo using animal models.


   Footnotes
 
1 This work was supported in part by NIH Grant HD-31398 (to L.C.G.), the March of Dimes Birth Defects Foundation (to L.C.G. and A.J.G.), CA-73832 (to A.J.G.), and the Naomi Vanden Horn Cancer Research Fund (to A.J.G.). Back

Received December 9, 1999.

Revised January 31, 2000.

Accepted April 21, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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