Title: Nitric Oxide Production and Regulation of Endothelial Nitric-oxide Synthase Phosphorylation by Prolonged Treatment with Troglitazone
Abstract: Recently, peroxisome proliferator-activated receptor γ (PPARγ) ligands have been reported to increase endothelial NO, but the signaling mechanisms involved are unknown. Using troglitazone, a PPARγ ligand known as an antidiabetic compound, we investigated the molecular mechanism of its effect on NO production in bovine aortic endothelial cells. Troglitazone increased endothelial NO production in a dose- and time-dependent manner with no alteration in endothelial nitric-oxide synthase (eNOS) expression. The maximal increase (∼3.1-fold) was achieved with 20 μm troglitazone treatment for 12 h, and this increase was accompanied by increases in the expression of vascular endothelial growth factor (VEGF) and its receptor, KDR/Flk-1, and in Akt phosphorylation. Analysis with antibodies specific for each phosphorylated site demonstrated that troglitazone (20 μm treatment for 12 h) significantly increased both the phosphorylation of Ser1179 of eNOS (eNOS-Ser1179) and the dephosphorylation of eNOS-Ser116 but did not alter eNOS-Thr497 phosphorylation. Treatment with anti-VEGF antibody to scavenge the increased VEGF induced by troglitazone partially inhibited troglitazone-stimulated NO production. This was accompanied by the attenuation of troglitazone-stimulated increases in the phosphorylation of Akt and eNOS-Ser1179 with no alteration in eNOS-Ser116 dephosphorylation. We also found that bisphenol A diglycidyl ether, a PPARγ antagonist, partially inhibited troglitazone-stimulated NO production with a concomitant reduction in VEGF-KDR/Flk-1-Akt-mediated eNOS-Ser1179 phosphorylation but with no alteration in eNOS-Ser116 dephosphorylation induced by troglitazone. Taken together, our results demonstrate that prolonged treatment with troglitazone increases endothelial NO production by at least two independent signaling pathways: PPARγ-dependent, VEGF-KDR/Flk-1-Akt-mediated eNOS-Ser1179 phosphorylation and PPARγ-independent, eNOS-Ser116 dephosphorylation. Recently, peroxisome proliferator-activated receptor γ (PPARγ) ligands have been reported to increase endothelial NO, but the signaling mechanisms involved are unknown. Using troglitazone, a PPARγ ligand known as an antidiabetic compound, we investigated the molecular mechanism of its effect on NO production in bovine aortic endothelial cells. Troglitazone increased endothelial NO production in a dose- and time-dependent manner with no alteration in endothelial nitric-oxide synthase (eNOS) expression. The maximal increase (∼3.1-fold) was achieved with 20 μm troglitazone treatment for 12 h, and this increase was accompanied by increases in the expression of vascular endothelial growth factor (VEGF) and its receptor, KDR/Flk-1, and in Akt phosphorylation. Analysis with antibodies specific for each phosphorylated site demonstrated that troglitazone (20 μm treatment for 12 h) significantly increased both the phosphorylation of Ser1179 of eNOS (eNOS-Ser1179) and the dephosphorylation of eNOS-Ser116 but did not alter eNOS-Thr497 phosphorylation. Treatment with anti-VEGF antibody to scavenge the increased VEGF induced by troglitazone partially inhibited troglitazone-stimulated NO production. This was accompanied by the attenuation of troglitazone-stimulated increases in the phosphorylation of Akt and eNOS-Ser1179 with no alteration in eNOS-Ser116 dephosphorylation. We also found that bisphenol A diglycidyl ether, a PPARγ antagonist, partially inhibited troglitazone-stimulated NO production with a concomitant reduction in VEGF-KDR/Flk-1-Akt-mediated eNOS-Ser1179 phosphorylation but with no alteration in eNOS-Ser116 dephosphorylation induced by troglitazone. Taken together, our results demonstrate that prolonged treatment with troglitazone increases endothelial NO production by at least two independent signaling pathways: PPARγ-dependent, VEGF-KDR/Flk-1-Akt-mediated eNOS-Ser1179 phosphorylation and PPARγ-independent, eNOS-Ser116 dephosphorylation. Troglitazone, the first thiazolinedione (TZD) 1The abbreviations used are: TZD, thiazolidinedione; DM, diabetes mellitus; PPARγ, peroxisome proliferator-activated receptor γ; VSMC, vascular smooth muscle cells; eNOS, endothelial nitric-oxide synthase; iNOS, inducible nitric-oxide synthase; VEGF, vascular endothelial growth factor; BADGE, bisphenol A diglycidyl ether; BAEC, bovine aortic endothelial cells; PKC, protein kinase C; 15d-PGJ2, 15-deoxy-Δ12,14-prostaglandin J2. compound synthesized, was used clinically as an oral antidiabetic drug to improve insulin resistance in patients with type 2 diabetes mellitus (DM) (1.Saltiel A.R. Olefsky J.M. Diabetes. 1996; 45: 1661-1669Crossref PubMed Scopus (0) Google Scholar, 2.Schwartz S. Raskin P. Fonseca V. Graveline J.F. N. Engl. J. Med. 1998; 338: 861-866Crossref PubMed Scopus (334) Google Scholar). However, it was later withdrawn from the market because of fatal hepatic injury (3.Watkins P.B. Whitcomb R.W. N. Engl. J. 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Dilley R.J. Hill M.A. Little P.J. J. Diabetes Compl. 2001; 15: 120-127Crossref PubMed Scopus (39) Google Scholar). Endothelial nitric-oxide synthase (eNOS) is an enzyme essential to the maintenance of cardiovascular integrity by producing NO in vivo, a key molecule with multiple functions, including vasodilation, and many antiatherogenic properties (12.van Haperen R. de Waard M. van Deel E. Mees B. Kutryk M. van Aken T. Hamming J. Grosveld F. Duncker D.J. de Crom R. J. Biol. Chem. 2002; 277: 48803-48807Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Therefore, the dysregulation of eNOS is thought to contribute to the pathogenesis of certain vascular diseases, such as atherosclerosis and hypertension (13.Perrault L.P. Malo O. Bidouard J.P. Villeneuve N. Vilaine J.P. Vanhoutte P.M. J. 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Chem. 2001; 276: 16587-16591Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). Recently, specific sites of phosphorylation have been identified; among these sites, Ser1179 (eNOS-Ser1179; bovine sequence) and eNOS-Thr497 are the most studied. The phosphorylation of eNOS-Ser1179 reduces the Ca2+-calmodulin dependence of the enzyme (20.Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (717) Google Scholar) and increases the rate of electron flux from the reductase domain to the oxygenase domain (21.McCabe T.J. Fulton D. Roman L.J. Sessa W.C. J. Biol. Chem. 2000; 275: 6123-6128Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar), thereby increasing NO production (22.Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3047) Google Scholar, 23.Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2232) Google Scholar, 24.Gallis B. Corthals G.L. Goodlett D.R. Ueba H. Kim F. Presnell S.R. Figeys D. Harrison D.G. Berk B.C. Aebersold R. Corson M.A. J. Biol. Chem. 1999; 274: 30101-30108Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). This phosphorylation is mediated by several specific protein kinases, including protein kinase B (Akt), AMP-activated protein kinase, calmodulin-dependent kinase II, and protein kinase A (20.Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (717) Google Scholar, 25.Boo Y.C. Sorescu G. Boyd N. Shiojima I. Walsh K. Du J. Jo H. J. Biol. Chem. 2002; 277: 3388-3396Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, 26.Bae S.W. Kim H.S. Cha Y.N. Park Y.S. Jo S.A. Jo I. Biochem. Biophys. Res. Commun. 2003; 306: 981-987Crossref PubMed Scopus (89) Google Scholar, 27.Fleming I. Fisslthaler B. Dimmeler S. Kemp B.E. Busse R. Circ. Res. 2001; 88: E68-E75Crossref PubMed Scopus (604) Google Scholar, 28.Mineo C. Yuhanna I.S. Quon M.J. Shaul P.W. J. Biol. Chem. 2003; 278: 9142-9149Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). In contrast, the phosphorylation of eNOS-Thr497 decreases eNOS activity by increasing Ca2+-calmodulin dependence (20.Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (717) Google Scholar, 27.Fleming I. Fisslthaler B. Dimmeler S. Kemp B.E. Busse R. Circ. Res. 2001; 88: E68-E75Crossref PubMed Scopus (604) Google Scholar, 29.Michell B.J. Chen Z. Tiganis T. Stapleton D. Katsis F. Power D.A. Sim A.T. Kemp B.E. J. Biol. Chem. 2001; 276: 17625-17628Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). Phosphatases such as protein phosphatase 1 and protein phosphatase 2B increase the dephosphorylation of eNOS-Thr497, resulting in an increase in NO production (19.Harris M.B. Ju H. Venema V.J. Liang H. Zou R. Michell B.J. Chen Z.P. Kemp B.E. Venema R.C. J. Biol. Chem. 2001; 276: 16587-16591Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 27.Fleming I. Fisslthaler B. Dimmeler S. Kemp B.E. Busse R. Circ. Res. 2001; 88: E68-E75Crossref PubMed Scopus (604) Google Scholar). Another phosphorylation site, eNOS-Ser116, has also been reported, and its dephosphorylation by protein phosphatase 2B increases eNOS activity (30.Kou R. Greif D. Michel T. J. Biol. Chem. 2002; 277: 29669-29673Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Recently, two other sites, eNOS-Ser635 and eNOS-Ser617, were also identified as phosphorylation targets of protein kinase A and Akt, respectively (31.Michell B.J. Harris M.B. Chen Z.P. Ju H. Venema V.J. Blackstone M.A. Huang W. Venema R.C. Kemp B.E. J. Biol. Chem. 2002; 277: 42344-42351Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). However, the roles of these protein kinases and phosphatases as signaling molecules for eNOS phosphorylation and dephosphorylation at several potential sites are dependent on the experimental conditions used, such as the presence of agonists, the endothelial cell type, and treatment time. For example, vascular endothelial growth factor (VEGF) (32.Gelinas D.S. Bernatchez P.N. Rollin S. Bazan N.G. Sirois M.G. Br. J. Pharmacol. 2002; 137: 1021-1030Crossref PubMed Scopus (134) Google Scholar) and fluid shear stress (25.Boo Y.C. Sorescu G. Boyd N. Shiojima I. Walsh K. Du J. Jo H. J. Biol. Chem. 2002; 277: 3388-3396Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar) stimulated Akt-dependent eNOS-Ser1179 phosphorylation at an earlier time, but they also stimulated protein kinase A-dependent eNOS-Ser635 phosphorylation at a later time (33.Boo Y.C. Hwang J. Sykes M. Michell B.J. Kemp B.E. Lum H. Jo H. Am. J. Physiol. 2002; 283: H1819-H1828Crossref PubMed Scopus (209) Google Scholar). In contrast to VEGF and shear stress, which cause no dephosphorylation of eNOS-Thr497, 8-bromo-cAMP rapidly dephosphorylates eNOS-Thr497. All of these findings suggest that the activity of eNOS in cells may be controlled through a coordinated regulation of, and interaction between, several protein kinases and phosphatases. Most studies of troglitazone have focused on the inhibition of cytokine-induced NO production via a decrease in the expression of inducible nitric-oxide synthase (iNOS) in adipocytes (34.Dobashi K. Asayama K. Nakane T. Kodera K. Hayashibe H. Nakazawa S. Life Sci. 2000; 67: 2093-2101Crossref PubMed Scopus (26) Google Scholar) and VSMC (35.Ikeda U. Shimpo M. Murakami Y. Shimada K. Hypertension. 2000; 35: 1232-1236Crossref PubMed Scopus (41) Google Scholar). However, troglitazone is also reported to increase in vivo forearm blood flow (36.Fujishima S. Ohya Y. Nakamura Y. Onaka U. Abe I. Fujishima M. Am. J. Hypertens. 1998; 11: 1134-1137Crossref PubMed Scopus (71) Google Scholar). Furthermore, the PPARγ ligands 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and ciglitazone were recently shown to increase endothelial NO production (37.Calnek D.S. Mazzella L. Roser S. Roman J. Hart C.M. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 52-57Crossref PubMed Scopus (283) Google Scholar). However, no detailed mechanism underlying this increase has yet been reported. Together with the previous observation that TZDs in vivo increased the expression of VEGF, a well known agonist for endothelial NO production (38.Baba T. Shimada K. Neugebauer S. Yamada D. Hashimoto S. Watanabe T. Diabetes Care. 2001; 24: 953-954Crossref PubMed Scopus (47) Google Scholar), these results prompted us to characterize the molecular mechanism underlying the troglitazone-stimulated increase in endothelial NO production. Our current data demonstrate, for the first time, that troglitazone increases NO production by at least two independent signaling pathways: PPARγ-dependent, VEGF-KDR/Flk-1-Akt-mediated eNOS-Ser1179 phosphorylation and PPARγ-independent, eNOS-Ser116 dephosphorylation. Materials—Troglitazone was obtained as a gift from Sankyo Co. (Tokyo, Japan), and bisphenol A diglycidyl ether (BADGE) was obtained from Sigma. Antibodies against eNOS and Akt were purchased from Transduction Laboratories (Lexington, KY) and New England Biolabs (Beverly, MA), respectively. Antibodies against VEGF and KDR/Flk-1 were purchased from Sigma and Santa Cruz Biotechnology (La Jolla, CA), respectively. Antibodies against Akt phosphorylated at Ser473 (p-Akt-Ser473) and p-eNOS-Ser1179 were obtained from Cell Signaling Technology (Beverly, MA), and those against p-eNOS-Thr497 and p-eNOS-Ser116 were from Upstate Biotechnology, Inc. (Lake Placid, NY). Trizol reagent for RNA extraction and SuperScript™ II RNase H– reverse transcriptase were obtained from Invitrogen. Recombinant Taq (rTaq) DNA polymerase was purchased from TaKaRa Biomedicals (Shiga, Japan), and collagenase (type 2) was purchased from Worthington Biochemical Corporation (Freehold, NJ). Minimal essential medium, Dulbecco's phosphate-buffered saline, newborn calf serum, penicillin and streptomycin antibiotics, l-glutamine, trypsin-EDTA solution, and plasticware for cell culture were purchased from Invitrogen. All other chemicals were of the purest analytical grade. Cell Culture and Drug Treatments—Bovine aortic endothelial cells (BAEC) were isolated exactly as described previously (39.Kim H.P. Lee J.Y. Jeong J.K. Bae S.W. Lee H.K. Jo I. Biochem. Biophys. Res. Commun. 1999; 263: 257-262Crossref PubMed Scopus (266) Google Scholar) and maintained in minimal essential medium supplemented with 5% newborn calf serum at 37 °C under 5% CO2 in air. The endothelial cells were confirmed by their typical cobblestone configuration when viewed by light microscopy and by a positive indirect immunofluorescence test for von Willebrand factor VIII. The cells between passages 5 and 9 were used for all experiments. When BAEC were grown to confluence, the cells were further maintained for the indicated times in minimal essential medium supplemented with 0.5% newborn calf serum containing various concentrations of troglitazone. In some experiments, the cells were co-treated with either anti-VEGF antibody (200 μg/ml) or BADGE (5 μm). RNA Extraction and Semi-quantitative Reverse Transcription-PCR— After treatment with troglitazone for the indicated times, the culture medium was removed, and total cellular RNA was isolated with Trizol reagent (Invitrogen) according to the manufacturer's instructions. A reverse transcription reaction was performed with 2 μg of total RNA in a final volume of 40 μl using 20 pmol of oligo(dT)15 in the presence of 200 units of SuperScript™ II RNase H– reverse transcriptase (Invitrogen). Subsequent PCR amplification of cDNA encoding VEGF was carried out in a total volume of 20 μl containing 0.5 units of rTaqDNA polymerase and 10 pmol of each primer. Primer pairs for PCR were as follows (40.Kang M.A. Kim K.Y. Seol J.Y. Kim K.C. Nam M.J. J. Gene Med. 2000; 2: 289-296Crossref PubMed Google Scholar): for VEGF (475 bp), forward 5′-ACGACAGAAGGGGAGCAGAAAG-3′, reverse 5′-GGAACGTTGCGCTCAGACACA-3′. Amplification of cDNA encoding glyceraldehyde-3-phosphate dehydrogenase (494 bp) was performed for semi-quantitative normalization using the following primers: forward, 5′-ACCACAGTCCATGCCATCAC-3′, and reverse, 5′-TCCACCACCCTGTTGCTGTA-3′. The amplified fragments were separated on a 2% agarose gel containing ethidium bromide and visualized with an image analyzing device (Vilber Lourmat, France) under UV illumination. The bands on the images were quantitated with the image analyzing software, ImageJ (National Institutes of Health, Bethesda, Washington, D. C.). Western Blot Analysis—For Western blot analysis, the cells treated with troglitazone in the absence or presence of various chemicals were washed with ice-cold Dulbecco's phosphate-buffered saline and lysed in lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100, 1 mm EDTA, 1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 10 mm β-glycerophosphate, 1 mm NaF, 1 mm Na3VO4) containing 1× Protease Inhibitor Mixture™ (Roche Applied Science). The protein concentrations were determined with the BCA protein assay kit (Sigma). Equal quantities of protein (30 μg) were separated on sodium dodecyl sulfate-polyacrylamide gel under reducing conditions and then electrophoretically transferred onto nitrocellulose membranes. The blots were then probed with the appropriate antibody directed against VEGF (1:500), KDR/Flk-1 (1:1000), Akt (1:4000), eNOS (1:4000), p-Akt-Ser473 (1:4000), p-eNOS-Ser1179 (1:1000), p-eNOS-Thr497 (1:2000), or p-eNOS-Ser116 (1: 4000), followed by the corresponding secondary antibody and finally developed using ECL reagents (Amersham Biosciences). Measurement of NO Release—NO production by BAEC was measured as nitrite (a stable metabolite of NO) concentration in cell culture supernatants, as described in many previous studies (41.Moon J. Yoon S. Kim E. Shin C. Jo S.A. Jo I. Thromb. Res. 2002; 107: 129-134Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), with minor modifications. Briefly, after cells were treated with troglitazone for the indicated times in the absence or presence of various chemicals, the culture medium was changed to Kreb's solution (pH 7.4; 1.5 ml/60-mm dish), which contained 118 mm NaCl, 4.6 mm KCl, 27.2 mm NaHCO3, 1.2 mm MgSO4, 2.5 mm CaCl2, 1.2 mm KH2PO4, and 11.1 mm glucose, and was equilibrated for 1 h at 37 °C. At the end of the incubation, 200 μl of each supernatant (in Kreb's solution) was carefully transferred into a 96-well plate, with the subsequent addition of 100 μl of Griess reagent (50 μl of 1% sulfanilamide containing 5% phosphoric acid and 50 μl of 0.1% N-(1-naphthyl)ethylenediamine). After color development at room temperature for 10 min, the absorbance was measured on a microplate reader at a wavelength of 520 nm. Each sample was assayed in triplicate wells. A calibration curve was plotted using known amounts of sodium nitrate solution. With this protocol, the measured values represent the amounts of NO produced by the cells during the 1-h incubation in Kreb's solution, following troglitazone treatment of a specified duration in the absence or presence of various chemicals. Therefore, subsequent NO production was solely dependent on eNOS activity at the end of these treatments. Statistical Analysis—All results are expressed as the means ± standard deviation (S.D.), with n indicating the number of experiments. Statistical significance was determined by Student's t test for two points. All differences were considered significant at a p value of < 0.05. Troglitazone Increases NO Production in BAEC with No Alteration in eNOS Expression—Troglitazone increased NO production by BAEC in a dose- and time-dependent manner, as shown in Fig. 1. The maximal increase in NO levels (3.1 ± 0.42-fold of the control) was observed with 20 μm troglitazone treatment for 12 h. Longer incubation (24 h) of cells with 20 μm troglitazone caused no further increase. Therefore, all of the subsequent experiments were performed using these conditions. Western blot analysis revealed that the troglitazone-stimulated increase in NO production did not result from an increase in eNOS protein expression (Fig. 1B), suggesting that classical intracellular genomic activity is not responsible for the observed effect. Troglitazone Increases NO Production by Up-regulating the Expression of VEGF and Its Receptor, KDR/Flk-1—Because troglitazone has been previously reported to increase the in vivo and in vitro expression of VEGF (42.Emoto M. Anno T. Sato Y. Tanabe K. Okuya S. Tanizawa Y. Matsutani A. Oka Y. Diabetes. 2001; 50: 1166-1170Crossref PubMed Scopus (85) Google Scholar), a well known agonist of NO production in endothelial cells, we next tested whether this troglitazone-stimulated NO increase is mediated by the up-regulation of VEGF. Troglitazone increased the expression of VEGF mRNA in a dose- and time-dependent manner, as shown in Fig. 2 (A and B). The maximal increase (6.23 ± 1.33-fold of the control) was observed with 20 μm troglitazone treatment for 24 h, although a significant increase (4.12 ± 0.53-fold of the control) was also found with the 12-h treatment. In concert with the increase in VEGF mRNA levels, troglitazone also increased VEGF protein expression in a dose-dependent manner (Fig. 2C). The maximal increase (3.73 ± 1.09-fold of the control) was observed with 20 μm troglitazone treatment for 12 h. A similar dose response was also apparent in the troglitazone-stimulated increase in the expression of the VEGF receptor, KDR/Flk-1 (Fig. 2C), peaking at 20 μm. To determine whether these increases in VEGF and KDR/Flk-1 expression mediate troglitazone-stimulated NO production in BAEC, we used anti-VEGF antibody to scavenge the increased VEGF induced by troglitazone. Anti-VEGF antibody partially (∼40%), but significantly, reduced troglitazone-stimulated NO production, as shown in Fig. 2D, suggesting that troglitazone-stimulated NO production in BAEC may be mediated, at least in part, by the VEGF–KDR/Flk-1 signaling pathway. Troglitazone Increases NO Production in BAEC by Increasing Either eNOS-Ser1179 Phosphorylation or eNOS-Ser116 Dephosphorylation—Because phosphorylation of eNOS-Ser1179 is a major mechanism for VEGF-mediated increase in NO production (43.He H. Venema V.J. Gu X. Venema R.C. Marrero M.B. Caldwell R.B. J. Biol. Chem. 1999; 274: 25130-25135Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 44.Hood J.D. Meininger C.J. Ziche M. Granger H.J. Am. J. Physiol. 1998; 274: H1054-H1058PubMed Google Scholar), we examined whether troglitazone increases NO production by the stimulation of the phosphorylation of this residue. Troglitazone (20 μm) increased eNOS-Ser1179 phosphorylation in a time-dependent manner, as shown in Fig. 3, suggesting a potential role for troglitazone in eNOS-Ser1179 phosphorylation. The maximal increase (2.4 ± 0.53-fold of the control) was observed with the 12-h treatment. With longer incubation (24 h) of the cells with troglitazone, however, a slight but significant attenuation was observed (1.8 ± 0.17-fold of the control). Furthermore, using antibodies specific to p-eNOS-Thr497 and p-eNOS-Ser116, we also observed that troglitazone significantly stimulated the dephosphorylation of eNOS-Ser116 in a time-dependent manner (Fig. 3), whereas the phosphorylation levels of eNOS-Thr497 were not altered by troglitazone treatment for up to 12 h. It should be noted that eNOS-Ser116 dephosphorylation by troglitazone occurred at an earlier time point (6 h) than eNOS-Ser1179 phosphorylation and that a slight but significant increase in the phosphorylation of eNOS-Thr497 was also observed with longer exposure (24 h). Our current results, together with previous findings of a positive role for both eNOS-Ser1179 phosphorylation and eNOS-Ser116 dephosphorylation in increasing NO production (30.Kou R. Greif D. Michel T. J. Biol. Chem. 2002; 277: 29669-29673Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 32.Gelinas D.S. Bernatchez P.N. Rollin S. Bazan N.G. Sirois M.G. Br. J. Pharmacol. 2002; 137: 1021-1030Crossref PubMed Scopus (134) Google Scholar), indicate that troglitazone-dependent coordinated changes in the phosphorylation levels of eNOS at these two different residues are primarily involved in the troglitazone-stimulated NO increase in BAEC. Troglitazone-induced Increases in eNOS-Ser1179 Phosphorylation and eNOS-Ser116 Dephosphorylation Are Mediated by at Least Two Separate Signaling Pathways—We examined whether either the phosphorylation or dephosphorylation of eNOS at the Ser1179 or Ser116 residues, respectively, is the consequence of a troglitazone-stimulated VEGF–KDR/Flk-1-mediated signaling pathway. Co-treatment with anti-VEGF antibody and troglitazone significantly reversed the eNOS-Ser1179 phosphorylation induced by troglitazone, whereas no alteration in the phosphorylation levels of either eNOS-Thr497 or eNOS-Ser116 was observed, as shown in Fig. 4 (A–C). Furthermore, anti-VEGF antibody also completely reversed the troglitazone-stimulated increase in the phosphorylation of Akt, an intermediate signaling molecule that mediates VEGF-eNOS-Ser1179 phosphorylation (Fig. 4D). Troglitazone also stimulated Akt phosphorylation in a time-dependent manner, showing a very similar pattern to the phosphorylation of eNOS-Ser1179 induced by troglitazone (data not shown). These results, together with the finding that anti-VEGF antibody partially attenuates troglitazone-stimulated NO increase (Fig. 2D), suggest that troglitazone increased NO production in BAEC in part by increasing VEGF-KDR/Flk-1-Akt-mediated eNOS-Ser1179 phosphorylation. In contrast, because anti-VEGF antibody did not alter the level of phosphorylation of eNOS-Ser116 (Fig. 4C) and eNOS-Ser116 dephosphorylation has a potential role in the troglitazone-stimulated increase in NO production, we hypothesize that the VEGF-KDR/Flk-1-Akt-mediated pathway is not an upstream signaling pathway for eNOS-Ser116 dephosphorylation. This hypothesis was further examined using BADGE, a PPARγ antagonist (45.Wright H.M. Clish C.B. Mikami T. Hauser S. Yanagi K. Hiramatsu R. Serhan C.N. Spiegelman B.M. J. Biol. Chem. 2000; 275: 1873-1877Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 46.Zander T. Kraus J.A. Grommes C. Schlegel U. Feinstein D. Klockgether T. Landreth G. Koenigsknecht J. Heneka M.T. J. Neurochem. 2002; 81: 1052-1060Crossref PubMed Scopus (126) Google Scholar). BADGE (5 μm) partially (∼40%) attenuated the troglitazone-stimulated increase in NO production, as shown in Fig. 5A. Furthermore, BADGE completely blocked KDR/Flk-1 expression stimulated by troglitazone (Fig. 5B). Like anti-VEGF antibody, BADGE significantly attenuated the phosphorylation of Akt and eNOS-Ser1179 induced by troglitazone but not the phosphorylation of eNOS-Thr497 and eNOS-Ser116 (Fig. 5, C–F). These data further support t