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

Abstract

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 NFκB site located at -1800 and an Ebox located at -1768. Supershift electrophoretic mobility shift assay and chromatin immunoprecipitation assays identified NFκB and USF-1/USF-2 as the DNA binding proteins induced by UTP, respectively, for these two sites. Using dominant negative mutants of IκB kinase and USF transcription factors, we confirmed that NFκB 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 NFκB is up-regulated through PKCδ signals. Finally, our work suggests that the UTP- stimulated OPN expression involves a coordinate regulation of PKCδ-NFκB, ERK1/2-USF, and ERK1/2/ NAD(P)H oxidase AP-1 signaling pathways.