UTP induces osteopontin expression through a coordinate action of NFkappaB, activator protein-1, and upstream stimulatory factor in arterial smooth muscle cells.
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.
Renault
Marie-Ange
MA
INSERM, U441 Athérosclérose, Bordeaux 33600, France.
Jalvy
Sandra
S
Potier
Mylène
M
Belloc
Isabelle
I
Genot
Elisabeth
E
Dekker
Lodewijk V
LV
Desgranges
Claude
C
Gadeau
Alain-Pierre
AP
D000818:Animals; D001158:Arteries / Q000473:pathology*; D001665:Binding Sites; D015153:Blotting, Western; D002465:Cell Movement; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017353:Gene Deletion; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D008156:Luciferases / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008954:Models, Biological; D009130:Muscle, Smooth / Q000166:cytology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019255:NADPH Oxidases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D053495:Osteopontin; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D012795:Sialoglycoproteins / Q000096:biosynthesis* / Q000235:genetics; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015854:Up-Regulation; D051720:Upstream Stimulatory Factors; D014544:Uridine Triphosphate / Q000378:metabolism*
D004268:DNA-Binding Proteins; D016328:NF-kappa B; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C497182:Usf1 protein, rat; D053495:Osteopontin; D004247:DNA; D008156:Luciferases; D019255:NADPH Oxidases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D014544:Uridine Triphosphate