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Originally published In Press as doi:10.1074/jbc.M301373200 on August 25, 2003

J. Biol. Chem., Vol. 278, Issue 46, 45651-45660, November 14, 2003
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Mechanisms of Hypoxic Gene Regulation of Angiogenesis Factor Cyr61 in Melanoma Cells*

Manfred Kunz{ddagger}§, Steffen Moeller¶, Dirk Koczan¶, Peter Lorenz¶, Roland H. Wenger||, Michael O. Glocker¶, Hans-Juergen Thiesen¶, Gerd Gross{ddagger}, and Saleh M. Ibrahim¶

From the {ddagger}Department of Dermatology and Venereology and Institute of Immunology and Proteome Center, University of Rostock, 18055 Rostock and the ||Carl Ludwig Institute of Physiology, University of Leipzig, 04103 Leipzig, Germany

Received for publication, February 7, 2003 , and in revised form, July 28, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hypoxia has a profound influence on progression and metastasis of malignant tumors. In the present report, we used the oligonucleotide microarray technique to identify new hypoxia-inducible genes in malignant melanoma with a special emphasis on angiogenesis factors. A commercially available Affymetrix® gene chip system was used to analyze five melanoma cell lines of different aggressiveness. A total of 160 hypoxia-inducible genes were identified, clustering in four different functional clusters. In search of putative angiogenesis and tumor progression factors within these clusters, Cyr61, a recently discovered angiogenesis factor, was identified. Cyr61 was hypoxia-inducible in low aggressive melanoma cells; however, it showed constitutive high expression in highly aggressive melanoma cells. Further analyses of transcriptional mechanisms underlying Cyr61 gene expression under hypoxia demonstrated that an AP-1 binding motif within the Cyr61 promoter plays a central role in the hypoxic regulation of Cyr61. It could be shown by use of in vitro luciferase assays, electrophoretic mobility shift assays, and immunoprecipitation that hypoxia-inducible factor-1{alpha} interacts with c-Jun/AP-1 and may thereby contribute to Cyr61 transcriptional regulation under hypoxia. Taken together, the presented data show that Cyr61 is a hypoxia-inducible angiogenesis factor in malignant melanoma with tumor stage-dependent expression. This may argue for a hypoxia-induced selection process during tumor progression toward melanoma cells with constitutive high Cyr61 expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is well accepted that local growth and metastasis of a large variety of malignant tumors are dependent on neoangiogenesis (1–3). The process of tumor angiogenesis is largely based on the production and secretion of so-called angiogenesis factors such as vascular endothelial growth factor (VEGF),1 fibroblast growth factor, and interleukin (IL)-8 (3–5). However, the mechanisms of angiogenesis factor production are still poorly understood. Evidence has been provided that a constitutive high expression of angiogenesis factors in isolated tumor cells or cell clones may be the first step in a selection process toward angiogenesis factor-producing tumors (6). This constitutive high expression in tumor cells may derive from the so-called "angiogenic switch," which is supposed to happen very early during tumor development initiating a selection process (for review, see Ref. 2). In accordance with this, it has been shown that a constitutive high expression of IL-8 promotes tumor growth of melanoma cells in vivo (7, 8). However, the molecular mechanisms underlying the induction of the angiogenic switch in tumors have not been defined so far.

More recent investigations have emphasized that the specific conditions of the tumor microenvironment may have a strong impact on the secretion of angiogenesis factors. In particular, tissue hypoxia has been shown to play a key role for the induction of these factors (9–11). Interestingly, even after neovascularization, tumor areas may remain under low oxygen tension, because of inadequate vascularization after neoangiogenesis (12). Thus, hypoxic areas remain a constant feature of malignant tumors and metastases. Up to now a large series of angiogenesis factors have been identified to be inducible by hypoxia. Among these are fibroblast growth factor, VEGF, platelet-derived growth factor, IL-8, and angiogenin (13–19). The majority of these factors had also been shown to be hypoxia-inducible and expressed in malignant melanoma (15, 17, 19, 20).

The recent findings about hypoxia-inducible gene expression have fostered further molecular analyses of gene regulatory mechanisms in tumor cells under hypoxia (21–27). It could be shown that the transcription factors AP-1 and hypoxia-inducible factor (HIF)-1{alpha} are major contributors to gene transcription of hypoxia-inducible genes (21, 24–27). In search for further upstream activators, signal transduction cascades interfering with transcription factors were analyzed in more detail. It could be shown that protein kinase C and the stress signaling pathway of c-Jun N-terminal kinase might play a central role in hypoxic signaling (28–30). However, the mechanisms underlying hypoxic signal transduction are still poorly defined.

In the present report we used the oligonucleotide microarray technique to analyze the gene expression pattern of malignant melanoma cells in search for new hypoxia-inducible genes involved in tumor angiogenesis. It could be shown that Cyr61, a recently discovered angiogenesis factor, is hypoxia-inducible in malignant melanoma cells. Moreover, Cyr61 showed a tumor stage-dependent expression with a constitutive strong expression in highly aggressive melanoma cell lines. The underlying mechanisms of its transcriptional regulation under hypoxia were further studied. We demonstrated that AP-1 and HIF-1{alpha} may both contribute to hypoxia-induced gene expression of Cyr61.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—The following plasmid constructs were used for co-transfection studies in in vitro luciferase assays: pRc/CMV, pRc/CMV-HIF-1{alpha}, and pARNT/CMV4. The pRc/CMV plasmid (empty control vector) was purchased from Invitrogen (Leek, The Netherlands); the pRc/CMV-HIF-1{alpha} vector was generated by cloning of HIF-1{alpha} cDNA into the pRc/CMV vector. The pARNT/CMV4 plasmid (31, 32) carrying the ARNT cDNA was kindly provided by L. Poellinger (Karolinska Institute, Stockholm, Sweden).

Cell Lines and Culture Conditions—The human melanoma cell lines 1F6, 530, Mel57, BLM, and MV3 were kindly provided by G. van Muijen (Institute of Pathology, University of Nijmegen, Nijmegen, The Netherlands). The metastatic potential of these cell lines has been investigated in nude mice. The cell lines 530 and 1F6 represent low aggressive, non-metastatic cell lines; the cell line Mel57 has been characterized as intermediate to highly aggressive; and BLM and MV3 are both highly aggressive, metastatic cell lines (33–35). Melanoma cell lines and Jurkat cells (which were used in control experiments) were maintained in RPMI 1640 medium (Linaris, Bettingen, Darmstadt, Germany), supplemented with 10% fetal calf serum (FCS; Linaris), 2 mM L-glutamine, 100 units/ml penicillin/streptomycin, and 1% nonessential amino acids. Cells were cultured in a humidified incubator (37 °C in 5% CO2, 95% air). For hypoxic treatment cultures were transferred for different time periods to hypoxic culture conditions (1% O2; mentioned as hypoxia) in an hypoxic incubator as described previously (32). Harvesting of cells was also performed under hypoxic conditions to avoid reoxygenation artifacts. Parallel cultures were constantly kept under normal oxygen conditions (mentioned as normoxia). H-Ras-transformed wild type mouse embryonic fibroblasts (MEF-HIF+/+) and HIF-1{alpha}-null mouse embryonic fibroblasts (MEF-HIF-/-) (36) were kept in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 100 units/ml penicillin/streptomycin.

RNA Extraction and Gene Chip Hybridization—Melanoma cell lines 530, 1F6, Mel57, BLM, and MV3, respectively, were kept under normoxic conditions or exposed to 24 h of hypoxia (1% oxygen). Total RNA was isolated using the total RNeasy kit (Qiagen, Hilden, Germany). RNA concentrations were determined spectrophotometrically at 260 nm. Probes for chip hybridization derived from isolated RNA samples were generated according to the instructions from the supplier (Affymetrix, Santa Clara, CA). The HuGeneFL ArrayTM (Affymetrix) with a capacity of 5600 human full-length characterized genes was used for mRNA expression profiling. Hybridization and washing of gene chips were performed according to the instructions from the supplier and as described earlier by Lockhart and co-workers (37). A laser scanner (Hewlett-Packard Gene ScannerTM) was used for analysis of gene chips, and the expression levels were calculated using a commercially available software provided by Affymetrix (Microarray Suite®).

Biostatistical Analysis of Gene Chip Data—Before cluster analysis genes were pre-selected for those that showed increased expression under hypoxia in at least one out of five cell lines to encompass all hypoxia-inducible genes of our experiments. Pre-selection resulted in a list of 1349 candidate genes. These were subjected to hierarchical clustering using Eisen software as recently described (38). Clustering tools were downloaded from www.rana.lbl.gov/EisenSoftware.htm. Hierarchical clustering was performed using average linkage differences. The "-fold change" of gene expression under hypoxia was used as clustering parameter. By this means four different clusters (cluster I–IV) were generated overall covering 160 genes. Cluster IV was re-clustered to analyze absolute gene expression with the average difference (representing the absolute value of gene expression) as clustering parameter. In the latter analyses, gene expression values were normalized to the mean value of all genes in this experiment.

Generation of a Cyr61 cDNA Probe—A Cyr61 cDNA probe for Northern hybridization was generated by reverse transcriptase-PCR. For this purpose total RNA was extracted from BLM melanoma cells using the total RNeasy kit (Qiagen). One µ g of total RNA was reverse transcribed by superscript reverse transcriptase (Invitrogen, Eggenstein, Germany) using hexamer priming. Primers for PCR amplification were purchased from ARK Scientific Biosystems (Darmstadt, Germany). These had the following recently published sequences (39): 5' primer, 5'-TGTGGAACTGGTATCTCCACACGA-3'; 3' primer, TCTTTTCACTGAATATAAAATTAAAA-3'. PCR amplification generated a cDNA probe of 1039 bp length. PCR conditions: an initial 5-min denaturation step was followed by 10 cycles of 30 s of denaturation at 94 °C, 30 s of annealing at 58 °C, 1 min of primer extension at 72 °C. After that, 20 cycles of 30 s of denaturation at 94 °C, 30 s of annealing at 60 °C, and 1 min of primer extension at 72 °C were carried out. A terminal primer extension step of 10 min at 72 °C was added. The PCR products were separated by agarose gel electrophoresis. The specific fragment of 1039 bp was cut out, purified using the QIAExII gel extraction kit (Qiagen), cloned into the pGEM-T vector (Promega, Heidelberg, Germany), and sequenced using an automated capillary sequencer (PerkinElmer, Weiterstadt, Germany). For Northern hybridization the cDNA probe was cut out from the pGEM-T vector by restriction enzyme digest and radioactively labeled.

RNA Extraction and Northern Blot Analysis—Total cytoplasmic RNA was isolated using the total RNeasy kit (Qiagen). RNA concentration was determined spectrophotometrically at 260 nm. Fifteen micrograms of RNA/sample were denatured in 50% formamide in gel running buffer (0.1 M MOPS, pH 7.0, 40 mM sodium acetate, 5 mM EDTA, pH 8.0) for 15 min at 65 °C, fractionated on a 1% agarose gel in formaldehyde buffer, and subsequently transferred to a nylon membrane (Hybond N+, Amersham Biosciences) in 20x SSC. As a probe for Cyr61 mRNA, a specific 1039 cDNA fragment was used. For Northern hybridization the purified fragment was labeled to high specific activity with [32P]dATP using a random primer labeling system (Roche Molecular Biochemicals, Mannheim, Germany). Membranes were cross-linked by ultraviolet light irradiation and prehybridized in a dextran sulfate buffer (100 g/liter dextran sulfate, 0.6 M NaCl, 0.2 M Na2HPO4, 6 mM EDTA, 1.75% lauroylsarcosinate, 50 µg/ml salmon sperm DNA, pH 6.2) for 1 h at 65 °C. Hybridization was carried out in the same prehybridization solution containing 5 x 106 cpm/ml of labeled probe. After hybridization for 16 h at 65 °C and 6 h at 60 °C, membranes were washed twice with 2x SSPE, 0.1% sodium dodecyl sulfate (SDS) at room temperature; once with 1x SSPE, 0.1% SDS at 60 °C; and once again with 0.2x SSPE, 0.1% SDS. Membranes were then exposed to HyperfilmTM (Amersham Biosciences) with intensifying screens at -80 °C for 3 days. Northern blots for ribosomal L28 RNA were performed, which served as control for equal loading (40).

Generation of Cyr61 Promoter Luciferase Constructs—A 883-bp and a 605-bp fragment, respectively, of the recently published promoter region of the Cyr61 gene (Ref. 41; EMBL GenBankTM, accession no. HSA249826) were generated by PCR amplification. The first fragment covers almost the complete Cyr61 promoter region of 935 bp beginning at the transcription start site. Further 5' sequences (beyond position -883) carry no binding motifs for known transcription factors. The second fragment (605 bp) covers the promoter region (also beginning at the transcription start site) ending immediately downstream of an AP-1 binding motif (TGACTCA) at position -624. For fragment generation of the Cyr61 promoter, genomic DNA was isolated from BLM melanoma cells using a commercially available DNA isolation kit (Qiagen). PCR amplification was carried out using the following primers: 883-bp fragment: 5' primer (5'-CGGGTACCTAAAGTGGGAACCTCCA-3') and 3' primer (5'-CCGCTCGAGTCTCGCTCGCGGTCTGCC-3'); 605-bp fragment: 5' primer (5'-CGGGGTACCTCTTCCCCGTTCTACTC-3') and 3' primer (5'-CCGCTCGAGTCTCGCTCGCGGTCTGCC-3'). Both pairs of primers generated a KpnI restriction site at the 5' end and an XhoI restriction site at the 3' end of the amplified fragments. These sites were used for further cloning of the fragments into the pGL3 vector (Promega, Heidelberg, Germany). The pGL3 promoter constructs were sequenced using an automated capillary sequencer (PerkinElmer Life Sciences). The promoter construct carrying the 883-bp fragment was termed cyr-900-luc, and the promoter construct with the 605-bp construct was termed cyr-600-luc.

In Vitro Mutagenesis of Cyr61 Promoter—In vitro mutagenesis was performed to generate mutated cyr-900-luc constructs. A promoter construct with a mutated AP-1 binding motif was termed cyr-900APmut-luc. A promoter construct where all four HRE-like motifs (position -68, -371, -400, -653) mutated was termed cyr-900HIFmut-luc. For mutagenesis of the AP-1 binding motif, TGACTCA, this motif was changed to TGACTAC, which results in an inhibition of transcription factor binding (42). For mutagenesis of the four HRE-like binding motifs, present on the promoter as CACG, this motif was changed to CTTT. A commercially available mutagenesis kit was used (QuikChangeTM site-directed mutagenesis kit, Stratagene, La Jolla, CA), and mutagenesis was performed according to the specifications from the manufacturer. The mutagenesis primers used were as follows.

For AP-1 motif mutagenesis, primers were 5'-GAAGTCCACAAATATTCCTGACTACGAGACACACTCCTC-3' and 5'-GAGGAGTGTGTCTCGTAGTCAGGAATATTTGTGGACTTC-3'. For mutagenesis of HRE-like motifs at position -68, primers were 5'-ACGTCACTGCAACTTTCGGCGCCTCCGC-3' and 5'-GCGGAGGCGCCGAAAGTTGCAGTGACGT-3'; at position -371, 5'-CATCACCACCATCTTTCCCAAAGAACC-3' and 5'-GGTTCTTTGGGGAAAGATGGTGGTGATG-3'; at position -400, 5'-CCCCTCGCCCCTCTTTACCCTCCAACTA-3' and 5'-TAGTTGGAGGGTAAAAGGGGCGAGGGG-3'; and at position -653, 5'-ACTTGTTCCGAACTTTCCTCTTTGAAGT-3' and 5'-ACTTCAAAGAGGAAAGTTCGGAACAAGT-3'.

Successful mutagenesis was confirmed by sequencing using an automated capillary sequencer (PerkinElmer Life Sciences).

In Vitro Luciferase Assays—Cyr61 promoter luciferase constructs were used for in vitro luciferase assays. For this purpose the melanoma cell line 1F6 was transfected with 2 µg of plasmid DNA (of wild type and mutated promoter constructs) using the DMRIE-CTM (1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide) reagent (Invitrogen) according to the specifications from the manufacturer. Briefly, cells were kept under serum-free medium for at least 16 h. 2 µg of plasmid DNA and DMRIE-CTM reagent, respectively, were diluted in 500 µl of OptiMEM (Invitrogen). Both mixtures were put together and incubated at room temperature for 30 min. Culture medium was removed and the lipid-DNA overlaid onto cells and incubated overnight. Subsequently, medium was replaced by RPMI medium (10% FCS) and after additional 24 h changed to low serum conditions (1% FCS) for another 24 h. Subsequently, cells were exposed to hypoxic conditions for indicated time points.

For luciferase assays, total cell extracts were prepared. Briefly, cells were harvested in 100 µl of lysis buffer (50 mM NaMES, pH 7.8, 50 mM Tris-HCl, pH 7.8, 10 mM dithiothreitol, 2% Triton X-100). The crude cell lysates were cleared by centrifugation, 50 µl of cleared cell extracts were added to 50 µl of luciferase assay buffer (125 mM NaMES, pH 7.8, 25 mM magnesium acetate, 2 mg/ml ATP), and activity was measured after injection of 50 µl of 1 mM D-luciferin (AppliChem, Darmstadt, Germany) in a Berthold luminometer (Berthold, Bad Wildbach, Germany). Total protein concentration was measured by the Bradford technique (Bio-Rad, München, Germany). The luciferase activities were normalized on the basis of protein content as well as on {beta}-galactosidase activity of cotransfected Rous sarcoma virus-{beta}-gal vector. The {beta}-galactosidase assay was performed with 20 µl of precleared cell lysate according to a standard protocol, as described earlier (43). For calculation of the -fold induction of luciferase activities, parallel cultures under normoxia and hypoxia were analyzed. Mean ± S.D. of four independent experiments are shown.

To analyze the effect of HIF-1{alpha}/ARNT on c-Jun-dependent transcription, a commercially available trans-reporting system (PathDetect®, Stratagene, Heidelberg, Germany) was used. This system analyses the effects of upstream molecules (e.g. signaling kinases) on the transcriptional activity of c-Jun in an in vitro luciferase assay system. In our experiments, 1F6 melanoma cell were transfected with the fusion transactivator plasmid, pFA2-c-Jun, which contains the activation domain of c-Jun fused with the yeast GAL4 binding domain. The co-transfected pFR-Luc reporter plasmid carries five tandem repeats of a GAL4 binding site linked to the firefly luciferase gene. Co-transfection was performed with a combination of both HIF-1{alpha}/ARNT-carrying plasmids or an appropriate control plasmid, which was used for mock transfection (pFC-dbd). Parallel experiments were performed by co-transfection of mitogen-activated extracellular signal-regulated protein kinase kinase (MEKK), which served as a positive control. Total cell extracts were prepared for luciferase assays as described above.

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assays (EMSA)—Melanoma cells (1F6 cell line) were exposed to hypoxic conditions for 1, 2, 4, 8, and 12 h, respectively. Parallel cultures were kept under normoxia. Nuclear extracts were prepared as previously described (17). Briefly, cells were washed with ice-cold phosphate-buffered saline and pelleted. Supernatants were removed, and cells were resuspended in 500 µl of buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) and allowed to swell for 10 min. Cells were pulled 10–15 times through a 26-gauge 3/8 needle for cell membrane disruption, and nuclei were pelleted in a microcentrifuge. Nuclei were washed twice in buffer A and resuspended in 50 µl of buffer C (20 mM Hepes, 20% glycerol, 0.4 M NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride) and incubated on ice for 45 min with occasional shaking. After centrifugation, supernatants were harvested, frozen, and stored at -70 °C. The following double-stranded oligonucleotide was used as probe (derived from Cyr61 promoter region including the AP-1 binding sequence (underlined)): 5'-AATATTCCTGACTCAGAGACACA-3' and 5'-TGTGTCTCTGAGTCAGGAATATT-3'. In control experiments a commercially available STAT-1 probe (sc-2573; Santa Cruz Biotechnology, Heidelberg, Germany) with the following sequence was used (STAT-1 binding sequence underlined): 5'-CATGTTATGCATATTCCTGTAAGTG-3' and 5'-CACTTACAGGAATATGCATAACATG-3'. For EMSA, 3 µg of nuclear proteins were incubated at room temperature for 20 min in a 20-µl binding reaction mixture containing 20 mM Hepes, 50 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, 2 µg of poly(dI-dC), and 50,000–100,000 cpm of 32P-end-labeled probe. The protein/DNA complexes were subjected to gel electrophoresis on a 5% (or 4% for supershift analyses) non-denaturing polyacrylamide gel in 0.5% TBE buffer. The polyclonal rabbit antibodies {alpha}-c-Jun (sc-1694 X), {alpha}-c-Fos (sc-7202 X), {alpha}-ATF2 (sc-242 X), {alpha}-JunB (sc-46 X), {alpha}-JunD (sc-74 X), anti-STAT3 (sc-482 X), and mouse monoclonal antibody anti-HIF-1{alpha} for supershift analyses were purchased from Santa Cruz Biotechnology. Gels were dried at 80 °C for 1 h and exposed to HyperfilmTM (Amersham Biosciences) at -70 °C.

Immunoprecipitation and Western Blot Analysis—For immunoprecipitation melanoma cells were lysed using a radioimmune precipitation assay lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholic acid, 0.1% SDS, 1 mM dithiothreitol, 1 mM sodium orthovanadate, supplemented with proteinase inhibitors (Roche Molecular Biochemicals). 500 µg of total cellular protein were mixed with an {alpha}-c-Jun antibody (sc-1694, rabbit polyclonal antibody; Santa Cruz Biotechnologies), or an {alpha}-HIF-1{alpha} antibody (sc-13515, mouse monoclonal antibody, Santa Cruz Biotechnologies), or as negative control an {alpha}-glutathione S-transferase (GST) antibody (sc-459, rabbit polyclonal antibody; Santa Cruz Biotechnologies), and incubated for 2 h at 4 °C with occasional shaking. After that, the mixture of cell lysate and {alpha}-c-Jun, or {alpha}-HIF-1{alpha}, or {alpha}-GST antibody were incubated with 30 µl of Protein A-agarose (for {alpha}-c-Jun and {alpha}-GST) or Protein G-agarose (for {alpha}-HIF-1{alpha}) (Roche Molecular Biochemicals) for 1 h at 4 °C on an automatic rotator. The mixture was then centrifuged for 5 min at 6000 rpm. The pellet was resuspended and washed three times using radioimmune precipitation assay lysis buffer, followed by one step of washing with phosphate-buffered saline. Immunoprecipitated proteins were denatured in electrophoresis sample buffer for 5 min at 95 °C and subjected to SDS-polyacrylamide gel electrophoresis (PAGE). Gels were electroblotted onto nitrocellulose membranes (Highbond ECL®, Amersham Biosciences, Braunschweig, Germany) and subjected to immunodetection. For detection of HIF-1{alpha}, the above mentioned mouse monoclonal antibody was used; for detection of c-Jun, the above mentioned rabbit polyclonal antibody was used. Secondary antibodies were coupled to horseradish peroxidase (Pharmingen, Hamburg, Germany; Promega, Heidelberg, Germany). A standard ECL (enhanced chemiluminescence) reaction (Amersham Biosciences) was performed for signal visualization.

Statistical Analysis—The data of -fold changes of luciferase activity in in vitro luciferase assays are given as mean ± S.D. Student's t test was performed for statistical analysis, and p <= 0.05 was regarded as statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Microarray Analysis of Hypoxia-inducible Genes Identifies Cyr61 as a New Hypoxia-inducible Angiogenesis Factor with Tumor Stage-dependent Expression—The HuFL oligonucleotide microarray carrying 5600 full-length characterized genes was used for expression profiling of hypoxic melanoma cells of different aggressiveness. Genes that showed hypoxia-induced expression in at least one out of five cell lines were included in the further analyses. By means of this pre-selection step, 1349 hypoxia-inducible genes were identified. These where subjected to further cluster analysis as recently described by Eisen and co-workers (38). Hierarchical clustering using the -fold increase of gene expression as clustering parameter generated four different functional clusters. Cluster one contained 33 genes, cluster two contained 11, cluster three 62, and cluster four 54 genes, for an overall total of 160 genes. One cluster (cluster IV) was analyzed in more detail as, in this cluster, genes displayed hypoxia-inducible gene expression in the low aggressive cell lines (530, 1F6; Fig. 1A, two left columns), decreased expression in the cell line of intermediate aggressiveness (Mel57; Fig. 1A, median column), and no or little change in gene expression in the highly aggressive cell lines (MV3, BLM; Fig. 1A, two right columns). This expression pattern was highly suggestive for genes that may have undergone a selection process. These genes and their products might thus be of importance for tumor progression. Within this cluster two new angiogenesis factors for malignant melanoma, Cyr61 and B61 (ephrin-A1) were identified. Re-clustering of cluster IV was performed using the average differences (which represent the absolute expression levels of genes) as clustering parameter (Fig. 1B). These analyses further underlined the outstanding role of Cyr61. Cyr61 was indeed one of a very few genes that showed constitutive high absolute expression in the aggressive cell lines (MV3 and BLM) both under normoxia and hypoxia (Fig. 1B). Absolute values of B61 (ephrin-A1) expression were comparatively low. Interestingly, well known hypoxia-inducible angiogenesis factors such as angiogenin, VEGF, and IL-8 did not cluster in the mentioned clusters (data not shown). However, as expected these angiogenesis factors were found among the total set of 1349 hypoxia-inducible genes. The non-clustering of angiogenin, VEGF, and IL-8 might be explained by the fact that these three angiogenesis factors showed no particular tumor stage-dependent up-regulation under hypoxia. Taken together, the expression pattern of Cyr61 in the presented analyses was suggestive for an important role of this angiogenesis factor in malignant melanoma.



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  		FIG. 1.
Microarray analysis and gene clustering of hypoxia-inducible genes in malignant melanoma cell lines. The melanoma cell lines 530, 1F6, Mel57, MV3, and BLM (representing different stages of tumor progression) were exposed to 1% hypoxia for 24 h. Control cultures were kept under normoxia. Gene chip hybridization on oligonucleotide microarrays was performed using the HuFL gene chip (Affymetrix®) carrying 5.600 full-length characterized genes. Hierarchical clustering (for details, see "Materials and Methods") was performed after pre-selection of genes for those which showed inducible expression under hypoxia in at least one out of five cell lines. A, cluster analysis of gene cluster IV. This cluster showed the most striking expression pattern in terms of stage-dependent gene expression. The -fold increase of gene expression under hypoxia compared with normoxia was used as clustering parameter. B, re-clustering of cluster IV using the absolute values of gene expression from microarray data as parameter for clustering. In A, red squares indicate the degree of up-regulated gene expression, and green squares indicate the degree of down-regulated gene expression upon hypoxia. Maximum and minimum values of -fold changes are given under the standard color bar. In B, red and green squares indicate the degree of absolute gene expression compared with mean of all genes analyzed in this experiment. Maximum and minimum values are given under the standard color bar. Numbers in A and B above the clusters indicate the melanoma cell lines: 1, 530; 2, 1F6, 3, Mel57; 4, MV3; 5, BLM. N, normoxia; H, hypoxia; acc., GenBankTM accession number.

 
Cyr61 mRNA Is Induced by Hypoxia in a Time-dependent Manner and Shows Tumor Stage-dependent Expression—To further substantiate our microarray analyses, time-course experiments were performed for Cyr61 expression using Northern blot analysis. A Cyr61 cDNA probe was generated by reverse transcriptase-PCR as described in the Materials and methods section. As shown in Fig. 2A, Cyr61 mRNA expression was inducible as early as 12 h after exposure of 1F6 melanoma cells to hypoxia and showed strong up-regulation at 36 h. Northern blots of all five melanoma cell lines before and after exposure to 24 h of hypoxia confirmed that Cyr61 was inducible by hypoxia in the low aggressive cell line 1F6 (Fig. 2B). In contrast, it showed down-regulation in the cell line of intermediate aggressiveness, Mel57. Constitutive high expression, which was not further inducible by hypoxia, was observed in the two highly aggressive variants of melanoma cell lines, BLM and MV3. Together, these data confirm the hypoxia inducibility of Cyr61 gene expression and the differential expression dependent on the tumor stage of the melanoma cell lines.



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  		FIG. 2.
Northern blot analysis of hypoxia-induced Cyr61 expression in melanoma cell lines. Melanoma cell lines 530, 1F6, Mel57, MV3, and BLM were exposed to 1% hypoxia for indicated times (H12, H24, H36). Control cultures were kept under normoxia (N). Total RNA was extracted and used for Northern blot hybridization. A, time course of Cyr61 mRNA expression in 1F6 melanoma cells exposed to indicated times of hypoxia. B, Cyr61 mRNA expression in melanoma cell lines of different aggressiveness under normoxia (N) or 24 h of hypoxia (H). 530 and 1F6 are both low aggressive, non-metastatic variants, Mel57 is a cell line of intermediate aggressiveness, and MV3 and BLM are both highly aggressive, metastatic variants. L28 Northern blots are shown (lower panel in A and B) to confirm equal loading of RNA.

 
AP-1 and HIF-1{alpha} Are Involved in the Transcriptional Regulation of Cyr61 under Hypoxia—To address the question of the molecular mechanisms underlying enhanced Cyr61 gene expression under hypoxia, reporter gene analyses were performed using Cyr61 promoter luciferase constructs. As shown in Fig. 3A, hypoxia significantly induced Cyr61 promoter activity in 1F6 melanoma cells (cyr-900-luc construct). Hypoxic gene induction was completely abolished in experiments using the cyr-900APmut-luc construct where the AP-1 binding motif at position -624 is mutated (see "Materials and Methods"). Essentially the same results were obtained using the truncated promoter construct (cyr-600-luc), carrying promoter sequences starting immediately downstream of the AP-1 binding site reaching to the transcriptional start site. Thus, hypoxia-induced transcription appears to be critically dependent on an intact AP-1 binding motif conferring AP-1 transcription factors a central role in hypoxic gene induction of Cyr61. Mutations in all four HRE-like motifs in the Cyr61 promoter (construct termed cyr-900HIFmut-luc) had no influence on its activity under hypoxia. This construct still displayed strong inducibility under hypoxic conditions (Fig. 3A). Essentially the same results were obtained by use of constructs with single mutations of each of the four HRE-like motifs (data not shown). To rule out that constitutive high expression of Cyr61 mRNA in MV3 and BLM melanoma cells might be the result of mutations in the AP-1 binding motif, this region was sequenced in all cell lines. No mutations could be detected in the AP-1 binding motif in all five melanoma cell lines (data not shown).



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  		FIG. 3.
Firefly luciferase assays of melanoma cells and mouse embryonic fibroblasts exposed to hypoxia using Cyr61 promoter luciferase constructs. A, 1F6 melanoma cells were transfected with 2 µg of Cyr-900-luc, Cyr600-luc, Cyr-900APmut-luc, and Cyr900HIFmut-luc promoter luciferase constructs, respectively. Melanoma cells were exposed to different times of hypoxia as indicated (H8, H16, H24). Control cultures were kept under normoxia (N). -Fold increase of firefly luciferase activity in cell extracts of 1F6 melanoma cells under hypoxia compared with parallel cultures under normoxia is shown. B, 1F6 melanoma cells were transfected with 2 µg of Cyr-900-luc, Cyr600-luc, Cyr-900APmut-luc, and Cyr900HIFmut-luc promoter luciferase constructs, respectively. -Fold increase of firefly luciferase activity in cell extracts after co-transfection with HIF-1{alpha} and ARNT carrying plasmids alone or in combination is shown. Transfection with empty vector (mock transfection) served as control. C, wild type mouse embryonic fibroblasts (MEF-HIF+/+) and HIF-1{alpha} null mouse embryonic fibroblasts (MEF-HIF-/-) were exposed to different times of hypoxia. -Fold increase of firefly luciferase activity in cell extracts of MEF-HIF+/+ and MEF-HIF-/- under hypoxia compared with parallel cultures under normoxia is shown. The luciferase activities in A–C were normalized on the basis of protein content and {beta}-galactosidase activity of co-transfected Rous sarcoma virus-{beta}-gal vector. Mean and standard deviations of four independent experiments are shown. Statistical significances of differences between base-line and induced luciferase activity are indicated by asterisks; p < 0.05.

 
To further investigate the putative role HIF-1{alpha}/ARNT for Cyr61 transcriptional regulation, co-transfection of 1F6 cells with both HIF-1{alpha} and ARNT carrying plasmid constructs was performed. As shown in Fig. 3B, co-transfection of HIF-1{alpha} and ARNT led to a strong induction of the luciferase activity of the cyr-900-luc construct (up to 3.5-fold). Isolated transfection with each of both factors, HIF-1{alpha} or ARNT, induced significant, however, much lower promoter activation (1.5–2-fold). The use of the mutated construct and the truncated version of the Cyr61 promoter, cyr-900APmut-luc and cyr-600-luc, respectively, dramatically reduced promoter activity. However, the truncated promoter construct remained slightly responsive to co-transfected HIF-1{alpha}/ARNT. The promoter construct with the four mutated HRE-like binding motifs, cyr-900HIFmut-luc, was still strongly inducible by co-expression of HIF/ARNT (Fig. 3B).

The influence of HIF-1{alpha} on Cyr61 gene regulation was further studied in H-Ras-transformed wild type mouse embryonic fibroblasts (MEF-HIF+/+) and HIF-1{alpha}-null mouse embryonic fibroblasts (MEF-HIF-/-) (31). Fig. 3C shows that wild type cells responded to experimentally induced hypoxia with an induction of the Cyr61 promoter using the cyr-900-luc construct. In contrast, however, both the cyr-900APmut-luc and cyr-600-luc construct were not inducible by hypoxia. Interestingly, in HIF-1{alpha} null cells (MEF-HIF-/-), the Cyr61 promoter constructs were not hypoxia-inducible. Taken together, these data suggest that AP-1 transcription factor binding is critical for hypoxic gene induction of Cyr61, and both AP-1 and HIF-1{alpha}/ARNT may interact.

EMSA Analysis Shows That c-Jun Is the Major Component of Hypoxia-induced AP-1 Binding—EMSA analyses were performed to further investigate whether AP-1 complexes are induced by hypoxia in 1F6 melanoma cells and to identify transcription factor subunits that might contribute to hypoxia-induced gene expression. The molecular probe used for EMSA analyses was derived from the Cyr61 promoter, harboring the AP-1 binding motif at position -624. As shown in Fig. 4A, under normoxic conditions low constitutive binding was observed in the upper AP-1 protein/DNA complex (complex I), which was strongly inducible after 4, 8, and 12 h, respectively, of hypoxia. The lower complex (complex II) showed no changes under hypoxia. Supershift analyses using a series of antibodies directed against components of the AP-1 complex revealed that the hypoxia-inducible complex contained c-Jun as a major component (Fig. 4B). An {alpha}-STAT3 antibody served as negative control in supershift analyses and did not supershift both complexes (Fig. 4B). The AP-1 complex could also be supershifted using a monoclonal antibody against HIF-1{alpha} (Fig. 4C). A slight, but clearcut band appeared immediately above the super-shifted c-Jun band (Fig. 4C, lanes 2 and 3). A commercially available molecular probe for STAT-1 served as negative control for hypoxic induction of transcription factor binding (Fig. 4D). None of the two STAT-1 protein/DNA complexes (complexes I and II) was inducible by hypoxia. Further EMSA experiments were performed for PMA-stimulated 1F6 melanoma cells and PMA-stimulated Jurkat cells. These experiments were carried out to demonstrate specificity of c-Jun supershifts in hypoxia-stimulated 1F6 melanoma cells. As shown in Fig. 4E, supershift analysis detected c-Jun and JunB as major components of the AP-1 complex in PMA-stimulated 1F6 melanoma cells. In PMA-stimulated Jurkat cells, the shifted complex contained c-Fos and JunB as major components (Fig. 4F). Further EMSA experiments were performed with two probes carrying the core binding motif and flanking sequences of the HRE-like motifs at position -371 and -400 of the Cyr61 promoter, respectively. Neither showed enhanced HIF binding under hypoxia in EMSA (data not shown). However, by use of a commercially available probe carrying the classical HRE binding motif, strong induction of HIF binding was observed (data not shown). Taken together, in melanoma cells hypoxia induces binding of an AP-1 complex to the AP-1 binding motif in the Cyr61 promoter. This hypoxia-inducible complex contains c-Jun and HIF-1{alpha}.



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  		FIG. 4.
EMSA for AP-1 and STAT-1 of nuclear extracts from 1F6 melanoma cells and Jurkat cells under normoxia and hypoxia. A, melanoma cells were exposed to hypoxia for 1, 2, 4, 8, and 12 h (H1, H2, H4, H8, and H12, respectively). Control cultures were kept under normoxia (N). For EMSA of AP-1, the following 32P-labeled double-stranded oligonucleotide derived from the Cyr61 promoter region was used: 5'-AATATTCCTGACTCAGAGACACA-3' (AP-1 binding sequence underlined). Competition experiments were carried out with 10- and 100-fold, respectively, molar excesses of cold probe. B, AP-1 complexes of 1F6 cells after 8 h of hypoxia were analyzed by supershift analysis using {alpha}-c-Jun, {alpha}-c-Fos, {alpha}-ATF2, {alpha}-JunB, {alpha}-JunD, and anti-STAT3 Abs, respectively. C, AP-1 complexes of 1F6 cells after 8 h of hypoxia were analyzed by supershift analysis using {alpha}-c-Jun and anti-HIF-1{alpha} Abs, respectively. D, for EMSA of STAT-1 (control experiments), the 32P-labeled double-stranded oligonucleotide had the sequence 5'-CATGTTATGCATATTCCTGTAAGTG-3' (STAT-1 binding sequence underlined). Competition experiments were carried out with 10- and 100-fold, respectively, molar excesses of cold probe. E, AP-1 complexes of 1F6 melanoma cells induced by stimulation with PMA for 4 h were analyzed by supershift analysis using {alpha}-c-Jun, {alpha}-c-Fos, {alpha}-ATF2, {alpha}-JunB, and {alpha}-JunD Abs, respectively. F, AP-1 complexes of Jurkat cells induced by stimulation with PMA for 4 h were analyzed by supershift analysis using the same Abs as in E. Free probe is indicated. n.s., nonspecific. I and II, complexes I and II.

 
HIF-1{alpha} Interacts with the AP-1 Transcription Factor via Binding to c-Jun—The presented findings of the in vitro luciferase assays were highly suggestive for a direct interaction of both transcription factors HIF-1{alpha} and c-Jun/AP-1. To further address this question, immunoprecipitation studies were performed using {alpha}-c-Jun as primary antibody to immunoprecipitate HIF-1{alpha} in cellular extracts of hypoxic melanoma cells. For this purpose 1F6 melanoma cells were transfected with a combination of c-Jun, HIF-1{alpha}, and ARNT carrying plasmid constructs. As shown in Fig. 5, under normoxia no HIF-1{alpha} protein could be immunoprecipitated with an {alpha}-c-Jun antibody in 1F6 melanoma cells. However, after 4 h of hypoxia, a strong signal for immunoprecipitated HIF-1{alpha} was detected (Fig. 5). Parallel performed Western blots of whole cell lysates for HIF-1{alpha} showed HIF-1{alpha} expression under normoxia with a slight increase in expression after 4 h of hypoxia. In the reciprocal experiments using an {alpha}-HIF-1{alpha} antibody to immunoprecipitate c-Jun, strongly increased expression of immunoprecipitated c-Jun was observed after 4 h of hypoxia compared with normoxia. The amount of c-Jun protein in whole cell lysates was similar under normoxia and hypoxia. Immunoprecipitation with an antibody against glutathione S-transferase (GST) served as negative control and immunoprecipitated neither HIF-1{alpha} nor c-Jun (data not shown). Taken together, these data suggest that c-Jun and HIF-1{alpha} interact in melanoma cells under hypoxia and that this interaction might contribute to Cyr61 gene regulation.



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  		FIG. 5.
Immunoblots for HIF-1{alpha} and c-Jun protein expression after immunoprecipitation of cell lysates or from whole cell lysates from melanoma cells exposed to hypoxia. 1F6 melanoma cells were exposed to hypoxia for 2 h (H2) and 4 h (H4), respectively. Control cultures were kept under normoxia (N). Cells were lysed, and total protein was extracted. c-Jun was immunoprecipitated using an {alpha}-c-Jun polyclonal antibody and Protein A-agarose. HIF-1{alpha} was immunoprecipitated using an {alpha}-HIF-1{alpha} mouse monoclonal antibody and Protein G-agarose. Immunoprecipitated proteins were subjected to SDS-PAGE and blotted onto nitrocellulose membranes. Immunoblots were carried out using a monoclonal antibody against HIF-1{alpha} (upper panels) or against c-Jun (lower panels). Immunoblots using whole cell lysates were performed in parallel. Signal detection was performed by standard ECL reaction. IP, immunoprecipitation; Ig, rabbit or mouse immunoglobulin.

 
HIF-1{alpha} Directly Interferes with c-Jun-dependent Transcription—To further substantiate our findings that HIF-1{alpha}/ARNT might functionally interact with c-Jun and impact on c-Jun-dependent transcription, a commercially available c-Jun-dependent trans-reporting system was used. In vitro luciferase assays were performed using a luciferase construct where five tandem repeats of yeast GAL4 bindings sites are fused with the luciferase gene. Co-transfection was performed with plasmid vectors carrying a c-Jun/GAL4 fusion protein and different concentrations of HIF-1{alpha}/ARNT. Parallel performed experiments with MEKK or empty vector served as positive or negative controls. As shown in Fig. 6, co-transfection of HIF-1{alpha}/ARNT dramatically increased luciferase activity in a concentration-dependent manner (up to 10-fold). The maximum level of promoter activity reached by HIF/ARNT co-transfection was similar to that of induction by MEKK, which served as a positive control. To rule out nonspecific activation of the promoter construct by HIF-1{alpha}/ARNT transcription factors, assays were performed without the c-Jun/GAL4 fusion protein. Under these conditions, the promoter base-line activity decreased and was not inducible by co-expression of HIF/ARNT (Fig. 6). Taken together, these results strongly argue for an interaction between HIF-1{alpha}/ARNT and c-Jun, which leads to a strong induction of c-Jun-dependent transcription.



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  		FIG. 6.
Firefly luciferase assays of 1F6 melanoma cells using a GAL4-dependent trans-reporting system. 1F6 melanoma cells were transfected with a plasmid vector carrying a c-Jun-GAL4 fusion protein consisting of the activation domain of c-Jun fused with the yeast GAL4 binding domain. The reporter plasmid carried five tandem repeats of a GAL4 binding motif linked to the firefly luciferase gene. Co-transfection was performed with a combination of HIF-1{alpha}/ARNT carrying plasmids or pFC-dbd control plasmid (mock transfection). Whole cell extracts were prepared and luciferase assays were performed as described (see "Materials and Methods"). Mean and standard deviations of four independent experiments are shown. Statistical significances of differences between base-line and induced luciferase activity are indicated by asterisks; p < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Rapidly growing tumors and tumor metastases are exposed to hypoxic or even anoxic conditions in the tissue microenvironment. Hypoxia in turn has a profound influence on tumor pathophysiology, leading to enhanced tumor aggressiveness (Refs. 44–47; for review, see Ref. 11). Although tissue hypoxia is a well documented phenomenon for a wide variety of tumors, our understanding of the hypoxic tumor cell response is far from being complete. More recent interest focused on hypoxia-induced genes that contribute to tumor angiogenesis, a major prerequisite for enhanced tumor growth.

In the present report we used the oligonucleotide microarray technique for mRNA expression profiling of malignant melanoma cell lines to achieve a more complete understanding of the hypoxic tumor cell response of this tumor. Five cell lines of different aggressiveness were analyzed, and a special emphasis was put on the identification of factors involved in tumor angiogenesis. It could be shown by use of hierarchical gene clustering methods (38) that hypoxia-inducible genes clustered in four different gene clusters, overall encompassing 160 different genes. In searching for angiogenesis factors within these clusters, we identified Cyr61 and ephrin-A1, which clustered in cluster IV. The gene expression pattern in this cluster was characterized by gene induction in low aggressive melanoma cells, down-regulation in a melanoma cell line of intermediate aggressiveness, and no or only slight changes of gene expression in highly aggressive melanoma cell lines. Further analysis of absolute gene expression values in this cluster revealed that Cyr61 showed constitutive high expression in highly aggressive cell lines, whereas ephrin-A1 expression values were low and close to background values. Thus, besides its hypoxia inducibility, a clear stage-dependent expression of Cyr61 in melanoma cells could be detected. Moreover, Cyr61 was among the genes with the highest absolute expression in the highly aggressive melanoma cell lines.

Cyr61 was initially described as a growth factor-inducible gene in mouse fibroblasts (48). It belongs to the so-called CCN family of immediate early genes. The abbreviation CCN stands for the names of the best known family members: CCN1 (CYR61), CCN2 (CTGF, connective tissue growth factor), and CCN3 (NOV, or nephroblastoma-overexpressed) (for review, see Ref. 49). Additional members, CCN4, CCN5, and CCN6, have been described more recently (49). A characteristic structural feature of all these proteins is highly conserved cysteine-rich domains. CCN family members exert a wide variety of different functions, mainly related to cellular proliferation and cell adhesion. One of the best characterized factors is in fact Cyr61. Cyr61 is a secreted protein involved in cellular proliferation, differentiation, DNA synthesis, cell adhesion to extracellular matrix, and angiogenesis (49–53). The receptors mediating the diverse functions of Cyr61 have been investigated in more detail, and it could be shown that Cyr61 binds to members of the family of integrins, e.g. {alpha}v{beta}3, {alpha}v{beta}5, {alpha}2b{beta}3, and {alpha}6{beta}1 (54–57). Integrin receptor binding is of importance for endothelial cell activation and proliferation, but may also account for the direct effects of Cyr61 on tumor cells and for its binding to extracellular matrix.

Interestingly, Cyr61 is able to promote tumor formation in experimental mouse models (41). Furthermore, in accordance with our findings, it could be shown in more recent experiments that high Cyr61 expression was associated with an aggressive phenotype of breast carcinoma cells lines and may play a role during tissue invasion of breast carcinoma cells (39, 58). From a functional point of view, these findings may argue for a selection process in vivo, which finally leads to aggressive phenotypes with high Cyr61 expression. It is well accepted that hypoxia may select for aggressive tumor cell phenotypes (Ref. 45; for review, see Ref. 11). Because Cyr61 is strongly hypoxia-inducible, hypoxia might be a central mechanism by which selection toward high Cyr61 expression occurs. Interestingly, another member of the CCN gene family, CTGF, has also been shown to be hypoxia-inducible (59).

The second angiogenesis factor identified in our microarray analyses was ephrin-A1 (B61 gene). It is well established that ephrins are major contributors to embryonic vasculogenesis (60). Their putative role in tumor angiogenesis has been emphasized recently (61). The absolute expression levels in melanoma cells in our investigations were comparatively low. However, ephrin-A1 might play a role in malignant melanoma as its expression had been shown to be associated with melanoma progression (62).

Because Cyr61 might be of importance for melanoma progression, the transcriptional mechanisms of its regulation under hypoxia were further investigated. By means of reporter gene analysis and EMSA, we were able to show that both transcription factors, AP-1 and HIF-1{alpha}, may contribute to the hypoxic induction of Cyr61 in melanoma cells. Interestingly, the core binding motif for HIF-1{alpha}, CGTG, is not present in the published Cyr61 promoter sequence (41). However, the complementary sequence of the HIF-1{alpha} core binding motif, CACG, is present four times in the Cyr61 promoter, up- and downstream of the described AP-1 binding motif at position -624. In our experiments, mutations in the AP-1 binding motif of the Cyr61 promoter construct totally abolished hypoxia inducibility of the reporter gene constructs. In contrast, mutation of all for HRE-like motifs had no influence and transcriptional activity under hypoxia in melanoma cells. However, the presence of HIF-1{alpha} might be of critical importance for Cyr61 transcription, because the Cyr61 promoter was not responsive to hypoxia in mouse embryonic fibroblasts lacking the HIF-1{alpha} gene. Similar results have been recently published by others (63). It had been shown that c-Jun/AP-1-dependent transcription may critically dependent on the presence of HIF-1{alpha} (63). In immunoprecipitation and EMSA studies, we could further show that under hypoxia HIF-1{alpha} directly binds to c-Jun/AP-1 and may thereby interfere with Cyr61 gene activation. These findings are in accordance with very recent data presented by Alfranca and co-workers (64), who showed that HIF-1{alpha} and AP-1 may directly interact. The current knowledge about interaction partners (in particular transcription factors and transcriptional co-activators) of HIF-1{alpha} has recently been summarized (27). We could further show by GAL4-dependent reporter gene assays that co-expression of HIF/ARNT dramatically induced c-Jun-dependent transcription, similar to that observed in positive control experiments with MEKK. This further argues for an interaction between c-Jun/AP-1 and HIF/ARNT in hypoxic gene regulation.

Taken together, in the present report, we show that Cyr61 is hypoxia-inducible and shows tumor stage-dependent expression in malignant melanoma cells. Based on our findings of the microarray analyses, Cyr61 may have an outstanding role in malignant melanoma. The described mechanisms of transcriptional regulation point to new targets for interference with tumor progression. In particular, HIF-1{alpha} may be an interesting target for future melanoma therapy. Indeed, it had been shown that inhibition of HIF-1{alpha} activity by antisense technique was able reduce aggressive tumor growth in vivo (65).


   FOOTNOTES
 
* This work was supported by German Bundesministerium für Bildung und Forschung Grant O31U214A (to M. K. and M. O. G.). 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. Back

§ To whom correspondence should be addressed: Dept. of Dermatology and Venereology, University of Rostock, Augusten Str. 80, 18055 Rostock, Germany. Tel.: 49-381-4949708; Fax: 49-381-4949702; E-mail: manfred.kunz{at}med.uni-rostock.de.

1 The abbreviations used are: VEGF, vascular endothelial growth factor; ECL, enhanced chemiluminescence; DMRIE-CTM, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide; EMSA, electrophoretic mobility shift assay; HIF-1{alpha}, hypoxia-inducible factor-1{alpha}; AP-1, activator protein-1; ARNT, aryl hydrocarbon receptor nuclear translocator; CTGF, connective tissue growth factor; STAT-1, signal transducer and activator of transcription-1; Ab, antibody; IL, interleukin; PMA, phorbol 12-myristate 13-acetate; MEKK, mitogen-activated extracellular signal-regulated protein kinase kinase; GST, glutathione S-transferase; CMV, cytomegalovirus; MOPS, 4-morpholinepropane-sulfonic acid; MES, 4-morpholineethanesulfonic acid; FCS, fetal calf serum. Back


   ACKNOWLEDGMENTS
 
We thank Dr. G. N. P. van Muijen and Prof. Dr. D. J. Ruiter (Institute of Pathology, University of Nijmegen, Nijmegen, The Netherlands) for providing cell lines 530, 1F6, Mel57, MV3, and BLM. We thank R. Waterstradt for excellent technical assistance.



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