PPARgamma ligand inhibits osteopontin gene expression through interference with binding of nuclear factors to A/T-rich sequence in THP-1 cells

• Published in: Circulation research 2002-02, Vol.90 (3), p.348
• Main Authors: Oyama, Yuko, Akuzawa, Nobuhiro, Nagai, Ryozo, Kurabayashi, Masahiko
• Format: Article
• Language: English
• Subjects: Animals; AT Rich Sequence - physiology; Cell Line; Chromans - pharmacology; COS Cells; Gene Expression - drug effects; Gene Expression - physiology; Gene Expression Regulation - drug effects; Genes, Reporter; Humans; Hypoglycemic Agents - pharmacology; Ligands; Monocytes - cytology; Monocytes - drug effects; Monocytes - metabolism; Mutagenesis, Site-Directed; Nuclear Proteins - metabolism; Osteopontin; Promoter Regions, Genetic; Protein Binding - drug effects; Rabbits; Receptors, Cytoplasmic and Nuclear - metabolism; RNA, Messenger - metabolism; Sialoglycoproteins - antagonists & inhibitors; Sialoglycoproteins - genetics; Sialoglycoproteins - metabolism; Tetradecanoylphorbol Acetate - pharmacology; Thiazoles - pharmacology; Thiazolidinediones; Transcription Factors - metabolism; Transcription Factors - pharmacology; Transfection; Troglitazone
• ISSN: 1524-4571

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily that acts as a key player in adipocyte differentiation, glucose metabolism, and macrophage differentiation. Osteopontin (OPN), a component of extracellular matrix, is elevated during neointimal formation in the vessel wall and is synthesized by macrophages in atherosclerotic plaques. In the present study, we investigated the molecular mechanisms regulating OPN gene expression by PPARgamma in THP-1 cells, a cell line derived from human monocytic leukemia cells. Northern and Western blot analyses showed that exposure of THP-1 cells to PMA (phorbol 12-myristate 13-acetate) increases OPN mRNA and protein levels in a time-dependent manner. PMA-induced OPN expression was significantly decreased by troglitazone (Tro) and other PPARgamma ligands. Transient transfection assays of the human OPN promoter/luciferase construct showed that PPARgamma represses OPN promoter activity, and the PPARgamma-responsive region within the OPN promoter lies between -1000 and -970 relative to the transcription start site. Site-specific mutation analysis and electrophoretic mobility shift assays indicated that a homeobox-like A/T-rich sequence between -990 and -981, which functions as a binding site for PMA-induced nuclear factors other than PPARgamma, mediates the repression of OPN expression by Tro. Furthermore, concatenated A/T-rich sequences conferred the PPARgamma responsiveness on the heterologous promoter. Taken together, these data suggest that PPARgamma ligand inhibits OPN gene expression through the interference with the binding of nuclear factors to A/T-rich sequence in THP-1 cells.