The functional −443T/C osteopontin promoter polymorphism influences osteopontin gene expression in melanoma cells via binding of c-Myb transcription factor
✍ Scribed by Julia Schultz; Peter Lorenz; Saleh M. Ibrahim; Günther Kundt; Gerd Gross; Manfred Kunz
- Publisher
- John Wiley and Sons
- Year
- 2009
- Tongue
- English
- Weight
- 261 KB
- Volume
- 48
- Category
- Article
- ISSN
- 0899-1987
- DOI
- 10.1002/mc.20452
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✦ Synopsis
Abstract
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.