Cyclooxygenase-2 (COX-2) is an inducible enzyme that drives inflammation and is the therapeutic target for widely used nonsteroidal antiinflammatory drugs (NSAIDs). However, COX-2 is also constitutively expressed, in the absence of overt inflammation, with a specific tissue distribution that includes the kidney, gastrointestinal tract, brain, and thymus. Constitutive COX-2 expression is therapeutically important because NSAIDs cause cardiovascular and renal side effects in otherwise healthy individuals. These side effects are now of major concern globally. However, the pathways driving constitutive COX-2 expression remain poorly understood. Here we show that in the kidney and other sites, constitutive COX-2 expression is a sterile response, independent of commensal microorganisms and not associated with activity of the inflammatory transcription factor NF-κB. Instead, COX-2 expression in the kidney but not other regions colocalized with nuclear factor of activated T cells (NFAT) transcription factor activity and was sensitive to inhibition of calcineurin-dependent NFAT activation. However, calcineurin/NFAT regulation did not contribute to constitutive expression elsewhere or to inflammatory COX-2 induction at any site. These data address the mechanisms driving constitutive COX-2 and suggest that by targeting transcription it may be possible to develop antiinflammatory therapies that spare the constitutive expression necessary for normal homeostatic functions, including those important to the cardiovascular-renal system. cyclooxygenase | nonsteroidal antiinflammatory drugs | prostacyclin | cardiovascular | Vioxx C yclooxygenase (COX) converts arachidonic acid to prostanoids, which include prostaglandins (PGs), prostacyclin, and thromboxane. Prostanoids are important mediators that regulate diverse functions in the cardiovascular, gastrointestinal, urogenital, and nervous systems, as well as playing critical roles in immunity, inflammation and resolution of inflammation. There are two COX

Objective-Hyperglycemia is a recognized risk factor for cardiovascular disease in diabetes. Recently, we reported that high glucose activates the Ca 2ϩ /calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in arteries ex vivo. Here, we sought to determine whether hyperglycemia activates NFAT in vivo and whether this leads to vascular complications. Methods and Results-An intraperitoneal glucose-tolerance test in mice increased NFATc3 nuclear accumulation in vascular smooth muscle. Streptozotocin-induced diabetes resulted in increased NFATc3 transcriptional activity in arteries of NFAT-luciferase transgenic mice. Two NFAT-responsive sequences in the osteopontin (OPN) promoter were identified. This proinflammatory cytokine has been shown to exacerbate atherosclerosis and restenosis. Activation of NFAT resulted in increased OPN mRNA and protein in native arteries. Glucose-induced OPN expression was prevented by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. Key Words: NFAT Ⅲ diabetes Ⅲ hyperglycemia Ⅲ UTP Ⅲ vascular complications Ⅲ inflammation T he matrix cytokine osteopontin (OPN) is emerging as a key regulator of chronic inflammatory diseases, including vascular disease. Plasma OPN levels are associated with the presence and extent of coronary artery disease, 1 restenosis after balloon angioplasty, 2 and human abdominal aortic aneurysm. 3 OPN is highly expressed in human atherosclerotic lesions and is not only a marker of inflammation but also an active player in the progression of atherosclerosis and restenosis. 4 While OPN deficiency has been shown to result in reduced atherosclerotic lesion areas, 5,6 OPN overexpression is associated with enhanced aortic lesion size. 7 Atherosclerotic vascular disease is a major complication in diabetic patients, and the levels of OPN in vivo have been clinically associated with the progression of vascular complications. Plasma levels of OPN significantly correlate to the progression of diabetic nephropathy, 8 and OPN levels in the vitreous are enhanced in patients with diabetic retinopathy. 9 Furthermore, OPN expression is increased in the media of diabetic arteries. 10,11 Thus, mapping the signaling pathway leading to changes in OPN expression may reveal novel pharmacological targets for prevention of vascular disease.In the context of vascular remodeling, soluble factors, cytokines, hormones, and extracellular nucleotides have been shown to induce OPN expression. 12,13 In particular, UTP has been demonstrated to effectively increase OPN protein production by enhanced transcription and stabilization of OPN mRNA. 14,15 We and others have shown that stimulation of G-protein-coupled receptors effectively activates the Ca 2ϩ /calcineurin-dependent transcription factor NFAT in arterial smooth muscle. 16,17 More recently, we have shown that high glucose activates NFAT in intact arteries ex vivo by a mechanism involving the release of extracellular nucleotides (ie, UTP, UDP) acting on P2...