Transcriptional regulation of the human osteopontin promoter: functional analysis and DNA-protein interactions

Synthesis of cell attachment proteins and cytokines, such as osteopontin (O PN), can promote tumor cell remodeling of the extracellular matrix into an environment that promotes tumor cell attachment and migration, We investiga ted the transcriptional regulation of OPN in the U-251MG and U-87MG human m alignant astrocytoma cell lines. Deletion and mutagenesis analyses of the O PN promoter region identified a proximal promoter clement (-24 to -94 relat ive to the transcription initiation site) that is essential for maintaining high levels of OPN expression in the tumor cells. This clement, designated RE-1, consists of two cis-acting elements, RE-la (-55 to -86) and RE-lb (- 22 to -45), which act synergistically to regulate the activity of the OPN p romoter, Gel shift assays using nuclear extracts of U-251MG cells demonstra ted that RE-la contains binding sites for transcription factors Spl, the gl ucocorticoid receptor, and the E-box-binding factors, whereas RE-lh contain s a binding site for the octamer motif-binding protein (OCT-1/OCT-2), Inclu sion of antibodies directed toward Myc and OCT-1 in the gel shift assays in dicated that Myc and OCT-1 participate in forming DNA-protein complexes on the RE-la and RE-lb elements, respectively. Our results identify two previo usly unrecognized elements in the OPN promoter that act synergistically to promote upregulation of OPN synthesis by tumor cells but are regulated by d ifferent transcription factors.