The functional -443T/C osteopontin promoter polymorphism influences osteopontin gene expression in melanoma cells via binding of c-Myb transcription factor.
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.
Schultz
Julia
J
Department of Dermatology and Venereology, University of Rostock, Rostock, Germany.
Lorenz
Peter
P
Ibrahim
Saleh M
SM
Kundt
Günther
G
Gross
Gerd
G
Kunz
Manfred
M
D015153:Blotting, Western; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D053495:Osteopontin / Q000235:genetics* / Q000378:metabolism; D011110:Polymorphism, Genetic / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D020598:Proto-Oncogene Proteins c-myb / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D012045:Regulatory Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D020598:Proto-Oncogene Proteins c-myb; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D053495:Osteopontin