Mechanisms of hypoxic gene regulation of angiogenesis factor Cyr61 in melanoma cells.
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-1alpha 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.
Kunz
Manfred
M
Department of Dermatology and Venereology and Institute of Immunology and Proteome Center, University of Rostock, 18055 Rostock, Germany. manfred.kunz@med.uni-rostock.de
Moeller
Steffen
S
Koczan
Dirk
D
Lorenz
Peter
P
Wenger
Roland H
RH
Glocker
Michael O
MO
Thiesen
Hans-Juergen
HJ
Gross
Gerd
G
Ibrahim
Saleh M
SM
D020816:Amino Acid Motifs; D015152:Blotting, Northern; D045744:Cell Line, Tumor; D055515:Cysteine-Rich Protein 61; D018076:DNA, Complementary / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D000860:Hypoxia; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017874:Immediate-Early Proteins / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D019169:Jurkat Cells; D008156:Luciferases / Q000378:metabolism; D008545:Melanoma / Q000378:metabolism; D016296:Mutagenesis; D020411:Oligonucleotide Array Sequence Analysis; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism
C501848:CCN1 protein, human; D055515:Cysteine-Rich Protein 61; D018076:DNA, Complementary; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D014157:Transcription Factors; D012313:RNA; D008156:Luciferases