The functional −443T/C osteopontin promoter polymorphism influences osteopontin gene expression in melanoma cells via binding of c-Myb transcription factor

• Published in: Molecular carcinogenesis Vol. 48; no. 1; pp. 14 - 23
• Main Authors: Schultz, Julia, Lorenz, Peter, Ibrahim, Saleh M., Kundt, Günther, Gross, Gerd, Kunz, Manfred
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.01.2009
• Subjects: Blotting, Western; carcinogenesis; Chromatin Immunoprecipitation; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Neoplastic; gene regulation; genetics; Humans; Immunoblotting; Immunoenzyme Techniques; Melanoma - genetics; Melanoma - metabolism; Melanoma - pathology; Osteopontin - genetics; Osteopontin - metabolism; Polymorphism, Genetic - genetics; Promoter Regions, Genetic - genetics; Protein Binding; Proto-Oncogene Proteins c-myb - antagonists & inhibitors; Proto-Oncogene Proteins c-myb - genetics; Proto-Oncogene Proteins c-myb - metabolism; Regulatory Sequences, Nucleic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Small Interfering - pharmacology; Tumor Cells, Cultured; tumor progression
• ISSN: 0899-1987; 1098-2744
• DOI: 10.1002/mc.20452

In the present report, the possible role of a recently described functional polymorphism of the osteopontin (OPN) promoter at position −443 (−443T/C) for OPN expression in melanoma cells was addressed. As shown by real‐time PCR analysis, melanoma metastases that were homozygous for the −443C allele expressed significantly higher levels of OPN mRNA compared with those that were either heterozygous (−443T/C) or homozygous for the −443T allele. In line with this, immunoblotting showed significantly enhanced baseline and bFGF‐induced OPN protein expression in melanoma cell lines which were homozygous for the −443C allele, compared with cell lines with other allelic variants. Similar results were obtained in in vitro luciferase assays. Chromatin immunoprecipitation (ChIP) demonstrated binding of c‐Myb to the −443 OPN promoter region, and binding could significantly be enhanced after bFGF stimulation. Moreover, as shown by electrophoretic mobility shift assays (EMSA), recombinant DNA‐binding domain of c‐Myb bound in a sequence‐specific manner to this region. Finally, the role of c‐Myb for OPN gene regulation via binding to the −443 promoter region could be further substantiated by ectopic overexpression of c‐Myb in melanoma cells, using different reporter gene constructs. Taken together, it is demonstrated that the −443 promoter region exerts influence on OPN gene expression in melanoma cells, and differential binding of c‐Myb transcription factor appears to play a major role in this process. These findings might be a feasible explanation for different OPN expression levels in metastatic tumors and may also have prognostic and therapeutic relevance. © 2008 Wiley‐Liss, Inc.