UTP induces osteopontin expression through a coordinate action of NFkappaB, activator protein-1, and upstream stimulatory factor in arterial smooth muscle cells

J Biol Chem. 2005 Jan 28;280(4):2708-13. doi: 10.1074/jbc.M411786200. Epub 2004 Nov 22.


Osteopontin (OPN) is an important chemokinetic agent for several cell types. Our earlier studies have shown that its expression is essential for uridine triphosphate (UTP)-mediated migration of vascular smooth muscle cells. We demonstrated previously that the activation of an AP-1 binding site located 76 bp upstream of the transcription start in the rat OPN promoter is involved in the induction of OPN expression. In this work, using a luciferase promoter deletion assay, we identified a new region of the rat OPN promoter (-1837 to -1757) that is responsive to UTP. This region contains an NFkappaB site located at -1800 and an Ebox located at -1768. Supershift electrophoretic mobility shift assay and chromatin immunoprecipitation assays identified NFkappaB and USF-1/USF-2 as the DNA binding proteins induced by UTP, respectively, for these two sites. Using dominant negative mutants of IkappaB kinase and USF transcription factors, we confirmed that NFkappaB and USF-1/USF-2 are involved in the UTP-mediated expression of OPN. Using a pharmacological approach, we demonstrated that USF proteins are regulated by the extracellular signal-regulated kinase (ERK)1/2 pathway, just as the earlier discovered AP-1 complex, whereas NFkappaB is up-regulated through PKCdelta signals. Finally, our work suggests that the UTP-stimulated OPN expression involves a coordinate regulation of PKCdelta-NFkappaB, ERK1/2-USF, and ERK1/2/NAD(P)H oxidase AP-1 signaling pathways.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arteries / pathology*
  • Binding Sites
  • Blotting, Western
  • Cell Movement
  • Cells, Cultured
  • DNA / metabolism
  • DNA-Binding Proteins / metabolism*
  • Gene Deletion
  • Gene Expression Regulation
  • Genes, Dominant
  • Luciferases / metabolism
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Mitogen-Activated Protein Kinase 3 / metabolism
  • Models, Biological
  • Muscle, Smooth / cytology*
  • Mutagenesis, Site-Directed
  • Mutation
  • NADPH Oxidases / metabolism
  • NF-kappa B / metabolism*
  • Osteopontin
  • Plasmids / metabolism
  • Polymerase Chain Reaction
  • Promoter Regions, Genetic
  • Protein Binding
  • Rats
  • Rats, Wistar
  • Sialoglycoproteins / biosynthesis*
  • Sialoglycoproteins / genetics
  • Signal Transduction
  • Transcription Factor AP-1 / metabolism*
  • Transcription Factors / metabolism*
  • Transcription, Genetic
  • Up-Regulation
  • Upstream Stimulatory Factors
  • Uridine Triphosphate / metabolism*


  • DNA-Binding Proteins
  • NF-kappa B
  • Sialoglycoproteins
  • Spp1 protein, rat
  • Transcription Factor AP-1
  • Transcription Factors
  • Upstream Stimulatory Factors
  • Usf1 protein, rat
  • Osteopontin
  • DNA
  • Luciferases
  • NADPH Oxidases
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Uridine Triphosphate