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[Cancer Research 65, 4690-4697, June 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

The Proinflammatory Cytokine Interleukin 1ß and Hypoxia Cooperatively Induce the Expression of Adrenomedullin in Ovarian Carcinoma Cells through Hypoxia Inducible Factor 1 Activation

Stilla Frede, Patricia Freitag, Teresa Otto, Christina Heilmaier and Joachim Fandrey

Institut für Physiologie, Universität Duisburg-Essen, Essen, Germany

Requests for reprints: Joachim Fandrey, Institut für Physiologie, Universität Duisburg-Essen, Hufelandstrasse 55, 45147 Essen, Germany. Phone: 49-201-723-4600; Fax: 49-201-723-4648; E-mail: joachim.fandrey{at}uni-essen.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adrenomedullin (ADM) is a potent hypotensive peptide produced by macrophages and endothelial cells during ischemia and sepsis. The molecular mechanisms that control ADM gene expression in tumor cells are still poorly defined. It is known, however, that hypoxia potently increases ADM expression by activation of the transcription factor complex hypoxia inducible factor 1 (HIF-1). Proinflammatory cytokines produced by tumor invading macrophages likewise activate expression of ADM. Herein, we show that apart from hypoxia, the proinflammatory cytokine interleukin 1ß (IL-1ß) induced the expression of ADM mRNA through activation of HIF-1 under normoxic conditions and enhanced the hypoxia-induced expression in the human ovarian carcinoma cell line OVCAR-3. IL-1ß significantly increased accumulation and nuclear translocation of HIF-1{alpha} under normoxic conditions and amplified hypoxic HIF-1 activation. IL-1ß treatment affected neither HIF-1{alpha} mRNA levels nor the hydroxylation status of HIF-1{alpha} and, thus, stability of the protein. Instead cycloheximide effectively prevented the increase in HIF-1{alpha} protein, indicating a stimulatory effect of IL-1ß on HIF-1{alpha} translation. Finally, treatment of HIF-1{alpha} with short interfering RNA revealed a significant role for HIF-1 in the IL-1ß–dependent stimulation of ADM expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Rapid growth of solid tumors often results in the development of a poorly nourished and hypoxic microenvironment (1). Under these conditions, a wide variety of tumor- and host-derived factors are expressed promoting tumor growth, degradation of extracellular matrix, vascularization, angiogenesis, and metastasis (2). However, the molecular mechanism and the relative importance of specific factors and microenvironmental conditions for these processes are not well characterized. Adrenomedullin (ADM), a hypotensive peptide, was originally isolated from pheochromocytoma (3). ADM acts as a modulator of vascular tone, vascular homeostasis, and cell growth (4–6). ADM is expressed in a variety of tissues and cell types under different conditions. Elevated blood ADM concentrations have been described in heart failure and myocardial ischemia (7), sepsis (8), and endotoxic shock (9). A link between hypoxic or ischemic conditions and enhanced ADM expression was suggested by the observation that hypoxia increased the expression of ADM in different tumor cell lines (10). Analysis of the regulatory parts of the ADM (ADM) gene showed at least 20 putative binding sites for the transcription factor complex hypoxia inducible factor 1 (HIF-1). HIF-1 is the key regulator of hypoxia-inducible genes like erythropoietin, vascular endothelial growth factor (VEGF), as well as a growing number of glycolytic and metabolic enzymes (11). The heterodimeric HIF-1 is composed of an oxygen-sensitive HIF-1{alpha} and a constitutive HIF-1ß subunit, which both belong to the family of basic helix-loop-helix and PAS domain proteins (12, 13). HIF-1 activity is primarily regulated by the abundance and activity of the {alpha} subunit. HIF-1{alpha} is degraded under normoxic conditions, which is initiated by hydroxylation of the proline residues 402 and 564 by oxygen-dependent proline hydroxylases (14). Binding of the von Hippel-Lindau tumor supressor gene product (pVHL) to hydroxy-proline HIF-1{alpha} (OH-HIF-1{alpha}) targets the subunit for proteasomal degradation (15). In addition, hydroxylation of the asparagine residue 803 within the COOH-terminal transactivating domain of human HIF-1{alpha} by the O2-sensitive asparagyl-hydroxylase factor inhibiting HIF-1 regulates the interaction of HIF-1{alpha} with transcriptional coactivators like CBP/p300 (16). Under hypoxic conditions, HIF-1{alpha} is stabilized, translocates into the nucleus where it dimerizes with HIF-1ß, and transactivates genes containing hypoxia-response elements within their promoter or enhancer.

Despite intensive studies, understanding of the nonhypoxic regulation of HIF-1 is still limited. Bacterial lipopolysaccharides as well as proinflammatory cytokines, such as interleukin 1ß (IL-1ß) and tumor necrosis factor {alpha} (TNF{alpha}), are able to increase HIF-1{alpha} protein and enhance the HIF-1 DNA binding (17, 18). For different cell types it was proposed that mitogen-activated protein kinase (MAPK) signaling pathways and activation of nuclear factor-{kappa}B (NF-{kappa}B) are involved in cytokine-induced HIF-1 stabilization (19, 20). Interestingly, proinflammatory cytokines and lipopolysaccharides inhibit the hypoxic induction of the classically HIF-1–regulated gene erythropoietin despite their HIF-activating effects (21). In contrast to erythropoietin, ADM expression is induced both by hypoxia and by lipopolysaccharides but the role of HIF-1 for both stimuli has not been resolved (22).

In human ovarian carcinoma, a significant correlation exists between expression levels of ADM and clinical outcome of the patients (23). Tumors expressing high levels of ADM are associated with a very low survival time. ADM seems to enhance the proliferation of ovarian carcinoma but stimuli that lead to a dramatic increase in ADM expression in certain types of tumors are not well defined. Hypoxic foci within the tumors as well as high levels of inflammatory cytokines secreted by tumor-associated macrophages have been found in different types of tumors (24).

To study the role of hypoxia and the inflammatory cytokine IL-1ß in the induction of the ADM gene, we used the human ovarian carcinoma cell line OVCAR-3. Hypoxia and IL-1ß induced the accumulation of HIF-1{alpha}, and treatment with short interfering RNA (siRNA) against HIF-1{alpha} revealed the importance of this transcription factor for ADM expression in response to both stimuli.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. The human ovarian carcinoma cells OVCAR-3 were grown in RPMI 1640 (BioWhittaker, Cambrex Company, Verviers, Belgium) supplemented with 10% FCS, penicillin (100 units/mL), and streptomycin (100 µg/mL) in a humidified atmosphere of 5% CO2 in air. For the experiments, cells were cultured to 80% confluence in 35 mm Petri dishes containing 1 mL of medium. To achieve hypoxic conditions, culture dishes were placed in a Heraeus incubator (Hanau, Germany) with 3% O2, 5% CO2, and N2 as balance for the indicated time periods. For reoxygenation experiments cells were exposed to hypoxia (3% oxygen) for 4 hours and then transferred to 21% oxygen for different times. At the end of the experiments, cells were lysed and total RNA was extracted for determination of specific mRNAs by reverse transcription-PCR. Additionally, total cell lysates and nuclear extracts were prepared and submitted for Western blot and electrophoretic mobility shift assay (EMSA). To evaluate the effects of inflammatory cytokines, IL-1ß (500 pg/mL), TNF{alpha} (10 ng/mL), and IL-6 (5 pg/mL; all from Calbiochem, Heidelberg, Germany) were added to the ovarian carcinoma cells for 1 to 24 hours under normoxic and hypoxic conditions. The concentrations used in the experiments were not toxic for the cells as judged by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (25). Dimethyloxalylglycine, as a substrate analogue of {alpha}-ketoglutarate and inhibitor of HIF prolyl hydroxylases, was kindly provided by Eric Metzen.

RNA preparation and reverse transcription-PCR. Total RNA was isolated by the acidic guanidinium thiocyanate-phenol-chloroform extraction method (26). One microgram of total RNA was reverse transcribed into cDNA with oligodT15 as primer for reverse transcriptase (avian myeloblastosis virus reverse transcriptase, Promega, Heidelberg, Germany). Qualitative PCRs were carried out for the housekeeping genes ß-actin, HIF-1{alpha}, and ADM to estimate the amount of specific cDNA before real-time PCR. The resulting PCR fragments were visualized on ethidium bromide–stained 1.5% agarose gels. Primers for real-time PCR were designed with the Primer Express software (Applied Biosystems, Weiterstadt, Germany): amplicon sizes of about 100 bp, annealing temperature of 60°C, and CG content of about 60% were preferred. Primer sequences used for qualitative and quantitative PCR are listed in Table 1.


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  		Table 1. Primer sequences used for qualitative and real-time PCR

 
Real-time PCR was done with SYBR green as fluorescent dye on the Gene Amp 5700 Sequence Detection System (Applied Biosystems). The cDNA standards for real-time PCR were prepared from the specific PCR products using a DNA purification kit (Roche, Mannheim, Germany). The amount of standard cDNA was determined photometrically. Standard concentrations ranged from 100 to 0.01 fg/µL. Quantification was done in a two-step real-time PCR with a denaturation step at 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Amounts of specific cDNA were calculated per microgram of total RNA.

Whole cell and nuclear extract preparation. For preparation of whole cell extracts, cells were washed with ice-cold PBS, drained, then lysed on the plates with 100 µL extract buffer [300 mmol/L NaCl, 10 mmol/L Tris (pH 7.9), 1 mmol/L EDTA, 0.1% NP40, 1x Protease-Inhibitor-Cocktail; Roche] for 20 minutes on ice. The extract was spun down in a microcentrifuge (5,000 rpm, 4°C, 5 minutes), quantitated using the Bio-Rad Laboratories protein assay reagent, and stored at –80°C.

Nuclear proteins were prepared from 60 mm dishes of subconfluent cells using the method of Schreiber et al. (27). All procedures were carried out at 4°C. After incubation, cells were washed with ice-cold PBS, drained and scraped from the plates with 150 µL buffer A [10 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF), 0.5 mmol/L DTT, 0.4% NP40 Igepal, 1x protease inhibitor cocktail; Roche], and incubated for 20 minutes on ice. Cell lysates were centrifuged at 5,000 rpm at 4°C for 5 minutes and the supernatants were used as cytoplasmic fraction. Pellets were resolved in 80 µL buffer B [20 mmol/L HEPES (pH 7.9), 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L PMSF, 0.5 mmol/L DTT, 1x protease inhibitor cocktail; Roche] and homogenized on a magnetic stirrer for 30 minutes. After centrifugation at 13,000 rpm at 4°C for 15 minutes, supernatants containing the nuclear proteins were transferred to –80°C. Protein concentrations were determined using the Bio-Rad kit.

Western blot analysis. After addition of 1/4 volume of sample buffer [50 mmol/L Tris (pH 6.8), 2% SDS, 5% ß-mercaptoethanol, 0.0125% bromophenol blue, 1% glycine], 50 to 75 µg of total cell lysate or 10 to 20 µg of nuclear extract per lane were subjected to 7.5% SDS-PAGE and transferred onto a nitrocellulose membrane (0.2 µm pore size, Schleicher and Schuell, Dassel, Germany). The efficiency of protein transfer and equal loading was confirmed by staining the nitrocellulose membrane with Ponceau S (Sigma, Steinheim, Germany). Membranes were blocked overnight at 4°C with 5% nonfat dry milk in TBS-T [10 mmol/L Tris (pH 7.5), 100 mmol/L NaCl, 0.05% Tween 20], then washed in TBS-T and incubated with the respective primary antibody at room temperature for 2 hours with gentle shaking. Primary antibodies used were mouse monoclonal antibody raised against human HIF-1{alpha} (1:250, Transduction Laboratories, Heidelberg, Germany) and mouse monoclonal antibody raised against {alpha}-tubulin (1:500, Santa Cruz Biotech, Heidelberg, Germany). The polyclonal rabbit antibody directed against hydroxylated proline 564 in HIF-1{alpha} has been characterized and described earlier (28). After washing with TBS-T, the blot was incubated with horseradish peroxidase–conjugated anti-mouse or anti-rabbit immunoglobulin G (IgG) antibody (Sigma). Immunoreactive proteins were visualized using a chemiluminescence detection system [100 mmol/L Tris-Cl (pH 8.5), 2.65 mmol/L H2O2, 0.45 mmol/L luminol, 0.625 mmol/L p-coumaric acid] followed by exposure to X-ray films (Agfa, Mortsel, Belgium).

Immunofluorescence. OVCAR-3 cells were fixed in ice-cold methanol and acetone (1:1) for 10 minutes at –20°C, blocked with bovine serum albumin, and incubated with a monoclonal anti–HIF-1{alpha} antibody (1:50, Transduction Laboratories) followed by an Alexa-Fluor 488–conjugated goat anti-mouse IgG (1:400, Molecular Probes, Eugene, OR). The immunostained cells were visualized by fluorescence microscopy.

Hypoxia inducible factor 1{alpha} silencing with short interfering RNA. siRNA sequences were selected according to the instructions from Dharmacon (Lafayette, CO) for specific silencing of HIF-1{alpha}. The siRNA target sequence corresponds to nucleotides 641 to 662 of HIF-1{alpha} mRNA (NM_001530). HIF-1{alpha} siRNA with a 3' overhang of dTdT as well as siRNA directed against firefly luciferase (used as a negative control) was obtained from Dharmacon. Cells were transfected with 200 nmol/L siRNA using oligofectamine (Invitrogen, Karlsruhe, Germany) at 40% confluence of the cell monolayer. Experiments were started 24 hours later at 80% confluence.

Electrophoretic mobility shift assay. [{gamma}-32P]ATP was obtained from ICN (Eschwege, Germany). Oligonucleotides containing the sequence of the HIF-1 binding site from the vascular endothelial growth factor promoter (sense 5'-gttggagcccacgtatgcactgtg-3' and antisense: 5'-cacagtgcatacgtgggctccaac-3') as well as a classic NF-{kappa}B binding motif (sense 5'-agttgaggggactttcccaggc-3' and antisense 5'-gcctgggaaagtcccctcaact-3') were synthesized by Invitrogen. After 5'-end labeling of the sense strand not incorporated, ATP was removed with Bio-gel G30 columns (Bio-Rad, Munich, Germany). The annealing reaction was done in the presence of a 2-fold molar excess of unlabeled antisense oligonucleotide. Binding reactions were set up in a volume of 20 µL. Ten micrograms of nuclear extracts were preincubated on ice for 20 minutes in a binding-buffer [12 mmol/L HEPES (pH 7.9), 4 mmol/L Tris-HCl (pH 7.9), 60 mmol/L KCl, 1 mmol/L EDTA, 1 mmol/L DTT] with 100 ng thymus DNA. After the addition of [{gamma}-32P]ATP, the samples were incubated for 30 minutes at room temperature. For supershift assays, the samples were additionally incubated with an anti-HIF–1{alpha} monoclonal antibody (Transduction Laboratories) for 16 hours at 4°C. Samples were subsequently resolved by electrophoresis on native 5% polyacrylamide gels at room temperature. Gels were dried and directly analyzed by autoradiography.

Statistics. Experiments were done in triplicate. Values of mRNA quantification are given as mean ± SE. Dunnett's test and Tukey-Kramer test were used to calculate statistically significant differences of means between treated and control groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time course of adrenomedullin expression and hypoxia inducible factor 1{alpha} accumulation in OVCAR-3 cells. OVCAR-3 cells were incubated from 1 to 24 hours under hypoxic conditions (3% O2) to determine the time course for the induction of ADM gene expression in OVCAR-3 cells. Under normoxic conditions, a constitutive expression of the ADM gene was observed. The addition of IL-1ß (500 pg/mL) under normoxic (21% O2) conditions significantly enhanced ADM gene expression, resulting in a 12-fold increase of ADM mRNA levels after 24 hours of treatment with IL-1ß when compared with untreated controls (Fig. 1A). After 4 hours of hypoxia (3% O2), induction of the ADM gene expression became significant compared with ADM mRNA levels at the onset of hypoxia. IL-1ß did not significantly increase ADM mRNA levels over hypoxia-induced levels (Fig. 1B). It is of note, however, that hypoxic induction was 5-fold more potent than IL-1ß after 24 hours of stimulation. The inflammatory cytokines TNF{alpha} (10 ng/mL) and IL-6 (5 pg/mL) did not influence expression of the ADM gene in OVCAR-3 (data not shown).



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  		Figure 1. Induction of ADM expression in OVCAR-3 cells. Eighty-percent confluent OVCAR-3 cells were incubated for the indicated time periods. Total RNA was isolated, reverse transcribed into cDNA, and subjected to real-time PCR for ADM cDNA quantification. A, OVCAR-3 cells were incubated under normoxic conditions (NOX: 21% O2) with or without IL-1ß (500 pg/mL). B, OVCAR-3 cells were incubated under hypoxic conditions (HOX: 3% O2) with or without IL-1ß (500 pg/mL). Columns, mean ADM cDNA levels normalized to total RNA from four independent experiments; bars, SE. *, P < 0.05; ***, P < 0.001.

 
Because HIF-1 is the key regulator of hypoxia-induced gene expression, we analyzed whether ovarian carcinoma cells were able to accumulate the oxygen-sensitive subunit HIF-1{alpha} during hypoxia. Cells were exposed to hypoxia (3% O2) for 2, 4, and 8 hours. Significant HIF-1{alpha} accumulation was first detected after 2 hours of hypoxic incubation (Fig. 2A). IL-1ß likewise induced HIF-1{alpha} accumulation even under normoxic conditions after 4 and 8 hours of incubation. At both time points, IL-1ß markedly enhanced the hypoxia-induced HIF-1{alpha} accumulation (Fig. 2A). Immunofluorescence staining of HIF-1{alpha} in OVCAR-3 cells showed that HIF-1{alpha} accumulated and was translocated to the nucleus under normoxic conditions after treatment with IL-1ß. Hypoxia-induced nuclear accumulation of HIF-1{alpha} was considerably amplified by IL-1ß (Fig. 2B).



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  		Figure 2. Accumulation of HIF-1{alpha} in OVCAR-3 cells. A, Western blot analysis for HIF-1{alpha}. OVCAR-3 cells were incubated for 2, 4, and 8 hours under normoxic and hypoxic conditions with or without IL-1ß (500 pg/mL). At the end of the experiments, whole cell extracts were prepared. One-hundred micrograms of total cellular protein were subjected to SDS-PAGE, followed by HIF-1{alpha} analysis. Antibodies against {alpha}-tubulin were used to show equal loading. B, representative immunofluorescene analysis of HIF-1{alpha} accumulation and nuclear translocation. OVCAR-3 cells were incubated for 4 hours. Cells were fixed and immunostained with a mouse monoclonal anti–HIF-1{alpha} antibody, detected by an Alexa-Fluor 488 conjugated anti-mouse antibody. Bar, 10 µm.

 
Regulation of hypoxia inducible factor 1{alpha} protein content. To further characterize the mechanisms leading to enhanced HIF-1{alpha} accumulation during IL-1ß treatment, we first analyzed the expression of HIF-1{alpha} mRNA. Quantitation of HIF-1{alpha} mRNA revealed no significant increase by hypoxia or IL-1ß (Fig. 3A) although IL-1ß–treated cells tended to have somewhat higher HIF-1{alpha} mRNA levels. These results raised the question whether increased amounts of HIF-1{alpha} protein were the result of reduced degradation of the protein, due to effects of IL-1ß on proline hydroxylases, or increased translation of the mRNA.



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  		Figure 3. Regulation of HIF-1{alpha} protein content. A, expression of HIF-1{alpha} mRNA in OVCAR-3 cells. Cells were incubated for 4 hours under normoxic and hypoxic conditions in the presence or absence of IL-1ß (500 pg/mL). Total RNA was isolated, cDNA generated, and HIF-1{alpha} cDNA quantitated by real-time PCR. Columns, means from three separate experiments; bars, SE. B, detection of HIF-1{alpha} protein and OH-HIF-1{alpha} after incubation with IL-1ß for 4 hours. The proteasomal inhibitor MG132 and the prolyl hydroxylase inhibitor dimethyloxalylglycine (MMOG) were added 2 or 1 hour before the end of the experiment, respectively. Total cell extracts were prepared, and OH-HIF-1{alpha} and HIF-1{alpha} were analyzed by Western blotting with {alpha}-tubulin as loading control. C, IL-1ß does not cause changes in HIF-1{alpha} protein stability. OVCAR-3 cells were subjected to hypoxia for 4 hours with or without IL-1ß addition and reoxygenated in 21% O2 thereafter. Whole cell extracts were prepared at different time points after the onset of reoxygenation. HIF-1{alpha} protein was detected by Western blot. D, IL-1ß increases the translation of HIF-1{alpha} mRNA. OVCAR-3 cells were incubated with IL-1ß for 8 hours under normoxic conditions. Sixty and fifteen minutes before the end of the incubation, cycloheximide (CHX, 10 µmol/L) was added. HIF-1{alpha} was detected in whole cell extracts with {alpha}-tubulin as loading control.

 
Direct analysis of the hydroxylation state of HIF-1{alpha} (Fig. 3B) was done using an antibody specifically directed against OH-HIF-1{alpha} (28). If IL-1ß delayed HIF-1{alpha} degradation by inhibiting proline hydroxylases, the amount of OH-HIF-1{alpha} should be decreased upon cytokine treatment. To visualize OH-HIF-1{alpha}, proteasomal degradation was prevented by the proteasome inhibitor MG132 (10 µmol/L). As a positive control for HIF-1{alpha} accumulation due to inhibition of proline hydroxylases, the {alpha}-keto-glutarate analogue and proline hydroxylase inhibitor dimethyloxalylglycine (1 mmol/L) was used. IL-1ß and dimethyloxalylglycine increased HIF-1{alpha} to a similar degree but inhibition of the proteasomes allowed the detection of considerable amounts of OH-HIF-1{alpha} in IL-1ß–treated cells only (Fig. 3B). Thus, IL-1ß treatment did not inhibit proline hydroxylase activity like dimethyloxalylglycine. This finding is supported by unchanged HIF-1{alpha} degradation upon reoxygenation in the presence of IL-1ß (Fig. 3C). Cells were exposed to hypoxia (3%O2) for 4 hours followed by reoxygenation in 21% O2. Although IL-1ß treatment increased the absolute levels of HIF-1{alpha} protein under hypoxia, reoxygenation revealed no increased stability of HIF-1{alpha} in IL-1ß–treated cells (Fig. 3C).

To test whether IL-1ß increased the translation of HIF-1{alpha} mRNA, cycloheximide (10 µmol/L), an inhibitor of translation, was added 60 and 15 minutes before the end of 8-hour incubation with IL-1ß under normoxic conditions. HIF-1{alpha} protein accumulation was clearly reduced by cycloheximide, indicating that HIF-1{alpha} protein accumulation by IL-1ß is mainly the result of enhanced translation of HIF-1{alpha} mRNA (Fig. 3D). Collectively, IL-1ß treatment seemed to increase HIF-1{alpha} protein levels by increasing translation rather than by inhibiting proline hydroxylase–dependent degradation.

EMSAs were done with nuclear extracts from OVCAR-3 cells to evaluate the DNA binding capacity of the IL-1ß–induced HIF-1{alpha} to a typical hypoxia response element. No HIF-1 DNA binding was observed under normoxic control conditions. After treatment with IL-1ß under normoxic conditions, the specific inducible HIF-1 complex was bound to DNA. Hypoxia (3 hours, 3% O2 and 1% O2) also stimulated binding of the HIF-1 complex, which was further enhanced by IL-1ß. The identity of HIF-1{alpha} as part of the inducible complex was confirmed by supershifting the complex with an anti–HIF-1{alpha} antibody (Fig. 4).



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  		Figure 4. Hypoxia- and IL-1ß–induced HIF-1 DNA binding. IL-1ß–treated OVCAR-3 cells were incubated under different degrees of hypoxia. EMSA of nuclear extracts was done with 32P-labeled HIF-1 consensus oligonucleotide. The inducible HIF-1 complex, the constitutive and the unspecific bands are indicated with arrows. Supershift was induced by adding an anti–HIF-1{alpha} antibody.

 
Role of hypoxia inducible factor 1{alpha} in ADM gene expression. To clarify the role of HIF-1{alpha} in the IL-1ß–induced ADM expression under normoxic conditions, HIF-1{alpha} mRNA was reduced by transfecting HIF-1{alpha} siRNA into OVCAR-3 cells (Fig. 5A). After siRNA treatment, hypoxic incubation failed to induce the accumulation of HIF-1{alpha} protein in OVCAR-3 cells (Fig. 5B). Silencing of HIF-1{alpha} had no effect on the constitutive ADM expression but significantly reduced the IL-1ß–induced expression of ADM under normoxia (Fig. 5C). In contrast, HIF-1{alpha} siRNA completely abolished the hypoxic induction of ADM (Fig. 5D). Furthermore, the IL-1ß–dependent increase of ADM expression under hypoxia was significantly reduced by HIF-1{alpha} siRNA treatment. Transfection of OVCAR-3 with siRNA directed against firefly luciferase had no effect on ADM expression under any incubation conditions (data not shown). Thus, HIF-1{alpha} seems to be critically involved in the IL-1ß–induced ADM expression. Interestingly, however, although hypoxic and HIF-1–dependent expression was almost completely abolished by siRNA treatment, IL-1ß still induced ADM expression, indicating that other transcription factors could also be involved in this effect.



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  		Figure 5. Role of HIF-1{alpha} in IL-1ß- and hypoxia-induced ADM expression. A, HIF-1{alpha} mRNA was knocked down by incubating OVCAR-3 cells with 200 nmol/L HIF-1{alpha} siRNA for 24 hours. At the end of the experiments, total RNA was prepared, reverse transcribed, and HIF-1{alpha} cDNA was quantitated by real-time PCR. Columns, means of HIF-1{alpha} cDNA normalized to total RNA from three independent experiments; bars, SE. Statistical significance was calculated against controls not treated with siRNA. B, HIF-1{alpha} siRNA treatment reduced the amount of hypoxia-inducible HIF-1{alpha} protein. HIF-1{alpha} was detected by Western blotting in whole cell extracts after 4 hours of hypoxic incubation. C, induction of ADM expression under normoxic conditions after treatment with HIF-1{alpha} siRNA for 24 hours, followed by an incubation with or without IL-1ß. Columns, means of the ADM cDNA quantifications each in triplicate of four separate experiments; bars, SE. D, inhibition of hypoxia-induced ADM expression by HIF-1{alpha} siRNA. Cells were incubated with 200 nmol/L HIF-1{alpha} siRNA for 24 hours, followed by a 4-hour hypoxic incubation with or without IL-1ß. Columns, means of four independent ADM cDNA quantifications each in triplicate; bars, SE. **, P < 0.01; ***, P < 0.001. Statistical significance was calculated against cells not treated with siRNA.

 
Role of nuclear factor {kappa}B in interleukin 1ß–induced hypoxia inducible factor 1 activation and adrenomedullin expression. Recently, the involvement of NF-{kappa}B in cytokine-induced HIF-1 activation was proposed (20). Therefore, we treated OVCAR-3 cells with the NF-{kappa}B inhibitor pyrrolidinium dithiocarbamate before incubation with IL-1ß. By EMSA we found NF-{kappa}B activation upon stimulation with IL-1ß, which was reduced with pyrrolidinium dithiocarbamate treatment (Fig. 6A). In contrast, however, HIF-1{alpha} protein accumulation as well as HIF-1 DNA binding was not lowered by pyrrolidinium dithiocarbamate (Fig. 6B). To analyze the effect of NF-{kappa}B on IL-1ß–induced ADM expression, OVCAR-3 cells were preincubated with pyrrolidinium dithiocarbamate before treatment with IL-1ß. Quantitation of ADM cDNA showed that pyrrolidinium dithiocarbamate treatment resulted in a reduction of IL-1ß–induced ADM mRNA levels (Fig. 6C). A complete inhibition of IL-1ß–induced ADM expression was achieved by the combined treatment with HIF-1{alpha} siRNA and pyrrolidinium dithiocarbamate (Fig. 6D).



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  		Figure 6. Role of NF-{kappa}B in IL-1ß- and hypoxia-induced ADM expression. A, EMSA demonstrating the activation of NF-{kappa}B by IL-1ß. Pyrrolidinium dithiocarbamate (50 µmol/L) reduced the specific DNA-binding complex. B, DNA binding (top) and accumulation (bottom) of HIF-1{alpha} by IL-1ß are not influenced by inhibition of NF-{kappa}B. C, pyrrolidinium dithiocarbamate (PDTC) partly inhibits the IL-1ß–induced, but not the hypoxia-induced, ADM expression. OVCAR-3 cells were pretreated with pyrrolidinium dithiocarbamate followed by incubation with IL-1ß for 4 hours under normoxic and hypoxic conditions. D, the combined treatment of cells with HIF-1{alpha} siRNA and pyrrolidinium dithiocarbamate abolished the IL-1ß–induced ADM expression. Columns, means of four independent ADM cDNA quantifications each in triplicate; bars, SE. *, significant difference versus controls; {circ}, statistically significant differences versus IL-1ß–treated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we investigated the regulation of ADM expression in the human ovarian carcinoma cell line OVCAR-3. ADM was initially isolated from pheochromocytoma (3) but its expression was later shown in a variety of other tumors like neuroblastoma, ganglioneuroblastoma, and adrenocortical carcinomas (29). ADM modulates the vascular tone and induces hypotension through activation of its corresponding receptor (ADM-R). In addition, ADM and ADM-Rs were shown to be ubiquitously expressed during embryogenesis. Especially maternal decidual cells and embryonic cytotrophoblast cells show high levels of ADM and ADM-R expression and it has been reasoned that ADM might facilitate the invasion of cells (30–32). With respect to carcinogenesis, ADM may be involved in a similar process during tumor cell invasion (33). Furthermore, in a variety of tumor cells, ADM acts as an autocrine proliferation factor which could be inhibited by neutralizing antibodies (34).

In ovarian carcinoma, high ADM expression has been recognized as a negative prognostic factor for the patients' outcome although the factors and exact conditions involved in the regulation of ADM expression in these tumors are not well characterized (23). The role of HIF-1{alpha} in ADM expression was outlined by Garayoa et al. (10). They showed at least 20 putative HIF-1 binding sites spread over the promoter and enhancer DNA elements. Hypoxia and hypoxia mimetics like desferrioxamine or CoCl2 induced ADM expression in human breast cancer cells. High expression of HIF-1{alpha} and of the hypoxia inducible, HIF-1–dependent VEGF was found by immunohistochemistry in epithelial ovarian carcinoma tissues (35). In that study, a significant correlation between HIF-1{alpha}, VEGF expression, and degree of malignancy of the tumors was reported. Similar observations were published by Hata et al. (23) with respect to ADM expression in ovarian carcinoma. Immunohistochemical staining revealed that the tumor cells themselves were the main source of ADM production.

We and others have previously reported that inflammatory cytokines induce HIF-1{alpha} accumulation and HIF-1 DNA binding in different cell types (17, 36–38). Herein we report that IL-1ß induced the HIF-1–dependent ADM gene in OVCAR-3 cells under normoxic conditions, comparable to its effect in astrocytes and arterial smooth muscle cells (39, 40). In addition, we found no significant amplification with the hypoxic induction of ADM mRNA.

Signaling of IL-1ß most likely occurs via membrane-bound IL-1 receptors leading to the activation of different MAPKs and the transcription factor NF-{kappa}B (41). In contrast, O2-dependent ADM expression is regulated by oxygen-dependent abundance of the {alpha} subunit of the HIF-1 complex. Under normoxic conditions, HIF-1{alpha} is hydroxylated at proline residues 402 and 564 by proline hydroxylases, recognized by pVHL, polyubiquitinated, and degraded by the proteasomes (14). Thus, we initially focused on the question whether IL-1ß treatment enables HIF-1{alpha} to escape from proteasomal degradation. Our data in Fig. 3 clearly show that proline hydroxylase activity was not affected by IL-1ß because the cytokine had no effect on the hydroxylation status of HIF-1{alpha} (Fig. 3B). Consequently, HIF-1{alpha} protein stability was not affected. Moreover, IL-1ß did not significantly induce HIF-1{alpha} mRNA but normoxic accumulation of HIF-1{alpha} protein by IL-1ß treatment was blocked by cycloheximide. Therefore, translation of HIF-1{alpha} mRNA seems to be significantly up-regulated by IL-1ß and overcomes the rate of normoxic degradation. This interpretation is supported by the fact that IL-1ß did not inhibit proteasomal degradation per se because the typical appearance of polyubiquitinated HIF-1{alpha} was lacking, which is observed on treatment with MG132 (compare lanes 2 and 4, Fig. 3B, middle). Moreover, in contrast to dimethyloxalylglycine, which inhibits proline hydroxylation by the proline hydroxylases, OH-HIF-1{alpha} was not reduced when proteasomal degradation was inhibited by MG132 (Fig. 3B, top, lane 6 versus lane 5). Collectively, IL-1ß seems to increase HIF-1{alpha} accumulation via enhanced translation of HIF-1{alpha} mRNA.

High efficiency of HIF-1{alpha} mRNA translation is ensured under hypoxic conditions by a formerly characterized internal ribosomal entry site in the 5' untranslated region of HIF-1{alpha} (42). Several growth factors have been found to increase translation of HIF-1{alpha} (43, 44) and we have reported that thyroid hormones stimulate HIF-1{alpha} protein synthesis in the human hepatoma cell line HepG2 (45). Recently, TNF{alpha} was found to increase HIF-1{alpha} by enhanced translation through the internal ribosomal entry site (46). Studies to uncover the signaling pathways involved in nonhypoxic HIF-1{alpha} activation revealed the involvement not only of MAPKs and PI3/Akt kinase in HIF-1{alpha} accumulation and transactivation (19, 47, 48) but also of NF-{kappa}B (49). We confirmed the activation of NF-{kappa}B in OVCAR-3 cells on stimulation with IL-1ß, but inhibition of NF-{kappa}B failed to reduce IL-1ß–induced HIF-1{alpha} accumulation and DNA binding. Therefore, a critical involvement of NF-{kappa}B as an upstream component of the IL-1ß signaling cascade leading to HIF-1{alpha} activation may be excluded in our cells. Functional integrity of NF-{kappa}B signaling was required for the increased translation of HIF-1{alpha} mRNA by TNF{alpha} (46) but, interestingly, herein OVCAR-3 cells neither responded to TNF{alpha} with increased HIF-1{alpha} accumulation nor enhanced expression of the HIF-1 target gene ADM.

Inhibition of NF-{kappa}B significantly reduced IL-1ß–induced ADM expression, which shows an important role for NF-{kappa}B in cytokine-induced ADM expression. In contrast, hypoxic induction was not affected by pyrrolidinium dithiocarbamate. We thus conclude that HIF-1 and NF-{kappa}B independently contribute to the expression of ADM. Neither inhibition of HIF-1 by siRNA nor of NF-{kappa}B by pyrrolidinium dithiocarbamate alone completely inhibited the IL-1ß–induced expression of the ADM gene. To abolish the IL-1ß effect, the combined reduction of HIF-1 and NF-{kappa}B activity was required.

Taking into consideration that the ADM gene and the surrounding regulating DNA segments contain many hypoxia response elements, our siRNA approach seemed to be very effective. We were able to significantly reduce the cooperative activation of ADM by hypoxia and IL-1ß, which is the result of an IL-1ß–dependent increase in HIF-1{alpha} synthesis through enhanced translation and hypoxic stabilization. This results in an almost 350-fold stimulation of ADM expression when comparing normoxic untreated versus hypoxic IL-1ß–treated cells. Overexpression of HIF-1 was shown to induce a variety of factors that facilitate invasion of carcinoma cells, particularly of colon cancers (50). A similar situation may be found in ovarian tumors and contribute to spreading of the disease. HIF-1–dependent expression of ADM in early phases of tumor development could be due to a combination of reduced degradation of its {alpha} subunit in focal areas of moderate hypoxia and increased HIF-1{alpha} translation by inflammatory cytokines, such as IL-1ß, contributed by tumor-associated macrophages. No areas of severe hypoxia due to a fast growing and insufficiently perfused tumor mass are required to induce HIF-1 and HIF-1–dependent genes if IL-1ß from macrophages is present. Increased ADM expression as a result of high HIF-1 levels could contribute to proliferation of tumor, invasion of tumor cells, and metastasis particularly in the early phase of tumor development.


   Acknowledgments
 
Grant support: Deutsche Forschungsgemeinschaft (Fa 225/20-1).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Peter Ratcliffe (Oxford, United Kingdom) for generously providing the OH-HIF-1{alpha} anti-serum, and Eric Metzen (Lübeck, Germany) for kindly providing dimethyloxalylglycine.

Received 10/29/04. Revised 2/17/05. Accepted 3/27/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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