Objective-We recently reported that the peroxisome proliferator-activated receptor ␥ (PPAR␥) ligands 15-deoxy-⌬ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) and ciglitazone increased cultured endothelial cell nitric oxide (NO) release without increasing the expression of endothelial nitric oxide synthase (eNOS). The current study was designed to characterize further the molecular mechanisms underlying PPAR␥-ligand-stimulated increases in endothelial cell NO production. Methods and Results-Treating human umbilical vein endothelial cells (HUVEC) with PPAR␥ ligands (10 mol/L 15d-PGJ 2 , ciglitazone, or rosiglitazone) for 24 hours increased NOS activity and NO release. In selected studies, HUVEC were treated with PPAR␥ ligands and with the PPAR␥ antagonist GW9662 (2 mol/L), which fully inhibited stimulation of a luciferase reporter gene, or with small interfering RNA to PPAR␥, which reduced HUVEC PPAR␥ expression. Treatment with either small interfering RNA to PPAR␥ or GW9662 inhibited 15d-PGJ 2 -, ciglitazone-, and rosiglitazone-induced increases in endothelial cell NO release. Rosiglitazone and 15d-PGJ 2 , but not ciglitazone, increased heat shock protein 90-eNOS interaction and eNOS ser 1177 phosphorylation. The heat shock protein 90 inhibitor geldanamycin attenuated 15d-PGJ 2 -and rosiglitazone-stimulated NOS activity and NO production. Key Words: peroxisome proliferator-activated receptor ␥ Ⅲ nitric oxide Ⅲ endothelium Ⅲ endothelial nitric oxide synthase Ⅲ thiazolidinedione E ndothelium-derived nitric oxide (NO) is a key molecule in vascular biology that decreases vascular tone, smooth muscle cell proliferation, leukocyte adhesion, and platelet aggregation. [1][2][3][4][5][6] Endothelial dysfunction, characterized by impaired endothelial NO production, participates in the pathogenesis of atherosclerotic disease and is associated with risk factors for vascular disease, including hypercholesterolemia, diabetes mellitus, insulin resistance, and obesity. 7 Our recent studies demonstrate that the peroxisome proliferator-activated receptor ␥ (PPAR␥) ligands 15-deoxy-⌬ 12,14 -PG J 2 (15d-PGJ 2 ) and ciglitazone stimulate NO release from endothelial cells (ECs). 8 Understanding the mechanisms of PPAR␥ ligand-induced stimulation of EC NO release may provide novel insights into the vascular protective effects of PPAR␥ ligands.In ECs, type III endothelial nitric oxide synthase (eNOS) produces NO from the amino acid L-arginine. eNOS is regulated not only at the level of expression, 9 -11 but also post-translationally by mechanisms including interactions of eNOS with other proteins 12-15 and eNOS phosphorylation. 16 -19 For example, specific stimuli including vascular endothelial growth factor (VEGF), histamine, and shear stress have been shown to activate eNOS by promoting the interaction of eNOS with heat shock protein 90 (hsp90), a molecular chaperone protein. 14 Hsp90 has been shown to increase eNOS activity by (1) recruiting Akt, the serine protein kinase B, to phosphorylate eNOS at ser 1177 , 20 (2) facilitating the displa...

The conversion of stromal fibroblasts into contractile myofibroblasts is an essential feature of the wound-healing response that is mediated by transforming growth factor ␤1 (TGF-␤1) and accompanied by transient activation of the vascular smooth muscle ␣-actin (Sm␣A) gene. Multiple positive-regulatory elements were identified as essential mediators of basal Sm␣A enhancer activity in mouse AKR-2B stromal fibroblasts. Three of these elements bind transcriptional activating proteins of known identity in fibroblasts. A fourth site, shown previously to be susceptible to single-strand modifying agents in myofibroblasts, was additionally required for enhancer response to TGF-␤1. However, TGF-␤1 activation was not accompanied by a stoichiometric increase in protein binding to any known positive element in the Sm␣A enhancer. By using oligonucleotide affinity isolation, DNA-binding site competition, gel mobility shift assays, and protein overexpression in SL2 and COS7 cells, we demonstrate that the transcription factors Sp1 and Sp3 can stimulate Sm␣A enhancer activity. One of the sites that bind Sp1/3 corresponds to the region of the Sm␣A enhancer required for TGF-␤1 amplification. Additionally, the TGF-␤1 receptor-regulated Smad proteins, in particular Smad3, are rate-limiting for Sm␣A enhancer activation. Whereas Smad proteins collaborate with Sp1 in activating several stromal cell-associated promoters, they appear to operate independently from the Sp1/3 proteins in activating the Sm␣A enhancer. The identification of Sp and Smad proteins as essential, independent activators of the Sm␣A enhancer provides new insight into the poorly understood process of myofibroblast differentiation.

The mouse vascular smooth muscle alpha-actin (SMA) gene enhancer is activated in fibroblasts by transforming growth factor beta1 (TGFbeta1), a potent mediator of myofibroblast differentiation and wound healing. The SMA enhancer contains tandem sites for the Sp1 transcriptional activator protein and Puralpha and beta repressor proteins. We have examined dynamic interplay between these divergent proteins to identify checkpoints for possible control of myofibroblast differentiation during chronic inflammatory disease. A novel element in the SMA enhancer named SPUR was responsible for both basal and TGFbeta1-dependent transcriptional activation in fibroblasts and capable of binding Sp1 and Pur proteins. A novel Sp1:Pur:SPUR complex was dissociated when SMA enhancer activity was increased by TGFbeta1 or Smad protein overexpression. Physical association of Pur proteins with Smad2/3 was observed as was binding of Smads to an upstream enhancer region that undergoes DNA duplex unwinding in TGFbeta1-activated myofibroblasts. Purbeta repression of the SMA enhancer could not be relieved by TGFbeta1, whereas repression mediated by Puralpha was partially rescued by TGFbeta1 or overexpression of Smad proteins. Interplay between Pur repressor isoforms and Sp1 and Smad coactivators may regulate SMA enhancer output in TGFbeta1-activated myofibroblasts during episodes of wound repair and tissue remodeling.