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 NFB site located at ؊1800 and an Ebox located at ؊1768. Supershift electrophoretic mobility shift assay and chromatin immunoprecipitation assays identified NFB and USF-1/USF-2 as the DNA binding proteins induced by UTP, respectively, for these two sites. Using dominant negative mutants of IB kinase and USF transcription factors, we confirmed that NFB 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 NFB is up-regulated through PKC␦ signals. Finally, our work suggests that the UTPstimulated OPN expression involves a coordinate regulation of PKC␦-NFB, ERK1/2-USF, and ERK1/2/ NAD(P)H oxidase AP-1 signaling pathways. Osteopontin (OPN)1 is a multifunctional phosphoprotein secreted by many cell types such as osteoclasts, lymphocytes, macrophages, epithelial cells, and smooth muscle cells (SMCs).Its expression is induced during vessel remodeling, as well as wound healing and bone synthesis (1). Overexpression of OPN can be found in pathological settings, such as immunological disorders, neoplastic transformation, metastasis, and formation of urinary stones. Its role in cell migration has been established in vitro in various cell types, including osteoclasts (2), macrophages (3), endothelial cells (4), and SMCs (5, 6). In vivo studies have established that OPN contributes to SMC recruitment into atherosclerotic plaques (7) and neointimal thickening (8), to osteoclast recruitment during bone resorption, (2) and to macrophage recruitment at inflammatory sites (atherosclerotic plaque, granulomas) (3).In the context of vascular remodeling, soluble factors, such as angiotensin II, basic fibroblast growth factor, plateletderived growth factor, interleukin-1 (9), and the nucleotides ATP and UTP (10), all induce expression of OPN. Moreover, nucleotides also modulate contraction, proliferation, and migration of vascular SMCs (11). They act through G proteincoupled P2Y receptors (12) and activate phospholipase C, resulting in elevated levels of intracellular free Ca 2ϩ and activation of PKC. In addition, they induce ROS production through NAD(P)H oxidase (13). Several transcription factors, such as NFAT, AP-1, cAMP-responsive element-binding protein, serum-responsive factor, STAT, and NFB are induced in UTP-stimulated SMCs (14), contro...

Aldosterone-induced osteopontin gene transcription in vascular smooth muscle cells involves glucocorticoid response element

“…[31][32][33][34] Although there are only a few reports on Aldo-induced OPN production, Irita et al 21 reported the involvement of activator protein-1 and nuclear factor-kB in the Aldo stimulation of rat renal fibroblasts. Contrary to these past investigations, our study revealed that the formation of an Aldo-MR complex and its binding to GRE were pivotal for the upregulation of OPN gene transcription.…”

Retinoic Acid Utilizes CREB and USF1 in a Transcriptional Feed-Forward Loop in Order To Stimulate MKP1 Expression in Human Immunodeficiency Virus-Infected Podocytes

“…Furthermore, the temporal regulation of genes by different transcription factors is not well studied. Only one study has reported that UTP stimulates osteopontin expression via coordinated regulation of the NF-B, USF, and AP-1 signaling pathways (31). While NF-B mediates early activation (15 min), USF1/2 and AP-1 mediate late-phase activation (1 h).…”

Osteoprotegerin, but not osteopontin, as a potential predictor of vascular calcification in normotensive subjects

“…21 Moreover, OPN expression is involved in the coordinated regulation of the NFkB, ERK1/2 and AP-1 signaling pathways, which have been found to be involved in various pathologies, including immunological disorders, neoplastic transformation and renal disorders. 22,23 This pleiotropic pattern may explain why OPN is not a good predictor of vascular calcification specifically, despite that its presence was documented in calcified coronary artery plaques. 8,20 OPN is expressed in adipose tissue and in the livers of obese subjects; thus, obesity-induced inflammation may obscure OPN involvement in vascular calcification.…”

“…26 This process is regulated by the same pathways that mediate vascular inflammation and endothelial dysfunction. 5,22 It is possible that the role of fibrinogen in vascular calcification is strictly associated with inflammation.…”