Transcriptional regulation of the human osteopontin promoter: functional analysis and DNA-protein interactions.
Synthesis of cell attachment proteins and cytokines, such as osteopontin (OPN), can promote tumor cell remodeling of the extracellular matrix into an environment that promotes tumor cell attachment and migration. We investigated the transcriptional regulation of OPN in the U-251MG and U-87MG human malignant astrocytoma cell lines. Deletion and mutagenesis analyses of the OPN promoter region identified a proximal promoter element (-24 to -94 relative to the transcription initiation site) that is essential for maintaining high levels of OPN expression in the tumor cells. This element, designated RE-1, consists of two cis-acting elements, RE-1a (-55 to -86) and RE-1b (-22 to -45), which act synergistically to regulate the activity of the OPN promoter. Gel shift assays using nuclear extracts of U-251MG cells demonstrated that RE-1a contains binding sites for transcription factors Sp1, the glucocorticoid receptor, and the E-box-binding factors, whereas RE-1b contains a binding site for the octamer motif-binding protein (OCT-1/OCT-2). Inclusion of antibodies directed toward Myc and OCT-1 in the gel shift assays indicated that Myc and OCT-1 participate in forming DNA-protein complexes on the RE-1a and RE-1b elements, respectively. Our results identify two previously unrecognized elements in the OPN promoter that act synergistically to promote upregulation of OPN synthesis by tumor cells but are regulated by different transcription factors.
Wang
D
D
Department of Pathology, The University of Alabama at Birmingham, 35294, USA.
Yamamoto
S
S
Hijiya
N
N
Benveniste
E N
EN
Gladson
C L
CL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001253:Astrocytes / Q000378:metabolism / Q000502:physiology; D001254:Astrocytoma / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017353:Gene Deletion; D015972:Gene Expression Regulation, Neoplastic; D051863:Host Cell Factor C1; D006801:Humans; D016297:Mutagenesis, Site-Directed; D050811:Octamer Transcription Factor-1; D050813:Octamer Transcription Factor-2; D053495:Osteopontin; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D012795:Sialoglycoproteins / Q000096:biosynthesis / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; D050813:Octamer Transcription Factor-2; C494165:POU2F1 protein, human; C058435:POU2F2 protein, human; C494168:Pou2f1 protein, rat; C489205:Pou2f2 protein, rat; D016271:Proto-Oncogene Proteins c-myc; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D016329:Sp1 Transcription Factor; C506897:Spp1 protein, rat; D014157:Transcription Factors; D053495:Osteopontin