AP-1 Is Involved in UTP-Induced Osteopontin Expression in Arterial Smooth Muscle Cells

Abstract

Abstract-Osteopontin (OPN), an RGD-containing extracellular matrix protein, is associated with arterial smooth muscle cell (SMC) activation in vitro and in vivo. Many cytokines and growth factors involved in vessel wall remodeling induce OPN overexpression. Moreover, we recently demonstrated that the extracellular nucleotide UTP also induces OPN expression and that OPN is essential for UTP-mediated SMC migration. Thus, we set out to investigate the mechanisms of OPN expression. The aim of this study was to identify transcription factors involved in the regulation of OPN expression in SMCs. First, we explored the contribution of mRNA stabilization and transcription in the increase of UTP-induced OPN mRNA levels. We show that UTP induced OPN mRNA increases via both OPN mRNA stabilization and OPN promoter activation. Then, to identify transcription factors involved in UTP-induced OPN transcription, we located a promoter element activated by UTP within the rat OPN promoter using a gene reporter assay strategy. The Ϫ96 to ϩ1 region mediated UTP-induced OPN overexpression (ϩ276Ϯ60%). Sequence analysis of this region revealed a potential site for AP-1 located at Ϫ76. When this AP-1 site was deleted, UTP-induced activation of the Ϫ96 to ϩ1 region was totally inhibited. Thus, this AP-1 (Ϫ76) site is involved in UTP-induced OPN transcription. A supershift assay revealed that both c-Fos and c-Jun bind to this AP-1 site. Finally, we demonstrate that angiotensin II and platelet-derived growth factor, two main factors involved in vessel wall pathology, also modulated OPN expression via AP-1 activation. S everal studies suggest that migration and proliferation of arterial smooth muscle cells (SMCs) play a prominent role in vascular pathologies such as atherosclerosis, restenosis, and hypertension. 1 SMC migration and proliferation can be induced by many factors, including growth factors and cytokines. [2][3][4] We have previously shown that extracellular nucleotides are also able to induce cell-cycle progression of SMCs 5,6 and their migration. 7 Thus, we were interested in understanding the mechanisms by which UTP induces SMC migration. Nucleotide-induced SMC activation is mediated via G protein-coupled P2Y receptors. Their activation leads to phospholipidase C activation and consequently to [Ca 2ϩ ] i increase and protein kinase C activation. Our previous work demonstrated that P2Y 2 , P2Y 4 , and P2Y 6 receptors are expressed in cultured SMCs 8 and that P2Y 2 is overexpressed in balloon-injured rat carotids. 9 Moreover, we have shown that these receptors are involved in UTP-induced migration. 8 We also demonstrated that UTP induces the expression of the extracellular matrix protein osteopontin (OPN) 6 and that UTP-induced migration is dependent on OPN expression and binding to ␣ v ␤ 3 integrin. 7 OPN is an RGD-containing extracellular matrix (ECM) phosphoprotein involved in cell attachment 10 and migration 11,12 and prevention of apoptosis. 13 OPN expression is induced by many growth factors, hormones, and cytokines invol...

UTP Induces Osteopontin Expression through a Coordinate Action of NFκB, Activator Protein-1, and Upstream Stimulatory Factor in Arterial Smooth Muscle Cells

“…Luciferase and ␤-galactosidase activities were assayed as described previously (15). For each sample, luciferase activity was normalized to ␤-galactosidase activity to compensate for differences in transfection efficiency.…”

“…The UTP-mediated increase of OPN protein expression results from enhanced gene transcription and stabilization of its transcript (15), but the way chemotactic factors regulate OPN expression remains poorly defined. Vitamin D receptor (16), estrogen receptor ␣ (17), Smad (18), Cbfa (19), and Ets-1 (20) have been shown to regulate OPN expression in osteoclasts, whereas metastasis-associated transcription factor (21), Tcf-1, Ets-1, Ets-2, polyomavirus enhancer A-binding protein-3, c-Jun (22) and acute myeloid leukemia-1 (23) are involved in OPN expression in cancer cells.…”

The Bile Acid Sensor Farnesoid X Receptor Is a Modulator of Liver Immunity in a Rodent Model of Acute Hepatitis

“…Previous studies have shown a regulatory function for the AP-1 consensus site located at Ϫ76 bp in the transcriptional regulation of the OPN gene (43)(44)(45). The importance of this AP-1 site for the transcriptional activity of the OPN promoter has been highlighted by a complete loss of basal and inducible OPN promoter activity after site-directed mutagenesis (43)(44)(45).…”

“…The importance of this AP-1 site for the transcriptional activity of the OPN promoter has been highlighted by a complete loss of basal and inducible OPN promoter activity after site-directed mutagenesis (43)(44)(45). We have previously shown that FXR activation induces a SHP-dependent inhibition of JunD and c-Jun binding to the AP-1 binding site (46,47).…”

The dioxin/aryl hydrocarbon receptor mediates downregulation of osteopontin gene expression in a mouse model of gastric tumourigenesis

“…Alternatively, dysregulation of OPN may represent a further downstream event following activation of the dioxin receptor, for example, by receptor-mediated negative regulation of the activity of the c-jun/c-fos complex (AP-1; Gillesby et al, 1997;Suh et al, 2002). AP-1 has previously been shown to activate the OPN promoter (Bidder et al, 2002;Denhardt et al, 2003;Renault et al, 2003). As discussed below, in either scenario it will be important to establish a causal link between dysregulation of OPN expression and stomach tumorigenesis.…”