No 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...

No abstract

In vertebrates, the actin-binding proteins tropomyosins are encoded by four distinct genes that are expressed in a complex pattern during development and muscle differentiation. In this study, we have characterized the transcriptional machinery of the ␣-tropomyosin (␣-Tm) gene in muscle cells. Promoter analysis revealed that a 284-bp proximal promoter region of the Xenopus laevis ␣-Tm gene is sufficient for maximal activity in the three muscle cell types. The transcriptional activity of this promoter in the three muscle cell types depends on both distinct and common cis-regulatory sequences. We have identified a 30-bp conserved sequence unique to all vertebrate ␣-Tm genes that contains an MCAT site that is critical for expression of the gene in all muscle cell types. This site can bind transcription enhancer factor-1 (TEF-1) present in muscle cells both in vitro and in vivo. In serumdeprived differentiated smooth muscle cells, TEF-1 was redistributed to the nucleus, and this correlated with increased activity of the ␣-Tm promoter. Overexpression of TEF-1 mRNA in Xenopus embryonic cells led to activation of both the endogenous ␣-Tm gene and the exogenous 284-bp promoter. Finally, we show that, in transgenic embryos and juveniles, an intact MCAT sequence is required for correct temporal and spatial expression of the 284-bp gene promoter. This study represents the first analysis of the transcriptional regulation of the ␣-Tm gene in vivo and highlights a common TEF-1-dependent regulatory mechanism necessary for expression of the gene in the three muscle lineages.