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Published as doi: 10.1096/fj.06-6822com.
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(The FASEB Journal. 2007;21:950-961.)
© 2007 FASEB

Nitric oxide activation of peroxisome proliferator-activated receptor gamma through a p38 MAPK signaling pathway

Anetta Ptasinska, Shuibang Wang, Jianhua Zhang, Robert A. Wesley and Robert L. Danner1

Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA

1Correspondence: Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA. E-mail: rdanner{at}cc.nih.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Both nitric oxide (NO•) and peroxisome proliferator-activated receptors (PPARs) protect the endothelium and regulate its function. Here, we tested for crosstalk between these signaling pathways. Human umbilical vein and hybrid EA.hy926 endothelial cells were exposed to S-nitrosoglutathione (GSNO) or diethylenetriamine NONOate (DETA NONOate). Electrophoretic mobility shift assays using PPAR-response element (PPRE) probe showed that NO• caused a rapid dose-dependent increase in PPAR{gamma} binding, an effect that was confirmed in vivo by chromatin immunoprecipitation. Conversely, NG-monomethyl-L-arginine, a NOS inhibitor, decreased PPAR{gamma} binding. NO•-mediated PPAR{gamma} binding and NO• induction of cyclooxygenase-2 (COX-2), diacylglycerol (DAG) kinase alpha (DGK{alpha}), and heme oxygenase-1 (HO-1), genes with well-characterized PPRE motifs, were cGMP independent. NO• dose dependently activated p38 MAPK, and p38 MAPK inhibition with SB202190 or knockdown with siRNA was shown to block NO• activation of PPAR{gamma}. Likewise, p38 MAPK and PPAR{gamma} inhibitors or knockdown of either transcript all significantly blocked NO• induction of PPRE-regulated genes. PPAR{gamma} activation by p38 MAPK may contribute to the anti-inflammatory and cytoprotective effects of NO• in the vasculature. This crosstalk mechanism suggests new strategies for preventing and treating vascular dysfunction.—Ptasinska, A., Wang, S., Zhang, J., Wesley, R. A., Danner, R. L. Nitric oxide activation of peroxisome proliferator-activated receptor gamma through a p38 MAPK signaling pathway.


Key Words: endothelial cells • gene expression • inflammation • peroxisome proliferator response element • vascular homeostasis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE INTEGRITY AND FUNCTIONof endothelial cells are fundamental for normal homeostasis of the vessel wall. In health, the endothelium has an antiinflammatory phenotype that maintains the uninterrupted circulation of leukocytes and platelets, whereas endothelial dysfunction and injury have been associated with disparate, pathological conditions such as atherosclerosis, pulmonary hypertension, and sepsis. The devastating impact of these diseases has led to an interest in identifying signaling molecules and pathways that maintain or perturb vascular health (1) .

Under homeostatic conditions, endothelial nitric oxide (NO•) synthase (eNOS; NOS-3) releases NO•, a reactive messenger that regulates smooth muscle tone (2 , 3) , inflammation (4 , 5) , gene expression (6 7 8) , leukocyte adhesion (9) , and platelet aggregation (10) . Somewhat paradoxically, both under and over production of NO• have been associated with endothelial dysfunction (11 , 12) . First described as a vasodilator, NO• relaxes blood vessels by activating soluble guanylate cyclase, thereby increasing intracellular cGMP (13 , 14) . Since this discovery, many other effects of NO• such as superoxide scavenging (15) , adenylate cyclase inhibition (16) , MAPK pathway activation, mRNA stabilization, and transcription factor regulation (7 , 17 , 18) have been ascribed to cGMP-independent mechanisms. However, the relationships among these various signaling mechanisms and the vascular protective functions of NO• are incompletely understood.

Peroxisome proliferator-activated receptors (PPARs) are transducer proteins belonging to the nuclear receptor superfamily. All three major PPAR isoforms, PPAR{alpha}, PPARß/{delta}, and PPAR{gamma}, are expressed in the endothelium. Activation of PPARs involves ligand-specific conformational changes in the receptor that recruit distinct coactivators or corepressors. These protein-protein interactions lead to PPAR heterodimerization with retinoid X receptor (RXR) and subsequent binding to specific peroxisome proliferator response elements (PPREs) within the promoters of PPAR target genes (19 , 20) . Like NO•, PPAR{gamma} and PPAR{alpha} have been closely associated with endothelial health and vascular protection.

PPAR{gamma} is expressed in monocytes, macrophages, smooth muscle cells, and endothelium (21) and plays a central role in regulating the expression of genes related to lipid trafficking, cell proliferation, and inflammatory signaling (22) . In vitro experiments using human endothelial cells indicate that PPAR{gamma} inhibits IFN-{gamma} induced chemokine expression and decreases lymphocyte chemotaxis (23) . Both PPAR{alpha} and PPAR{gamma} activators induced expression of the superoxide scavenger enzyme Cu2+,Zn2+-superoxide dismutase (SOD; 24 , 25 ). PPAR{gamma} also reduces superoxide radical formation by down-regulating the p22 and p47phox components of the NAD(P)H oxidase system (24 , 25) and enhances NO• production by increasing eNOS phosphorylation at serine1177 and its interaction with heat shock protein 90 (26) . Collectively, these effects act to increase NO• bioavailability.

In type 2 diabetes mellitus, long-term treatment with thiazolidinediones (TZD), drugs that activate PPAR{gamma} not only lower plasma levels of insulin and glucose but also reduce inflammatory markers of cardiovascular risk while improving vascular function (27) . Conversely, loss of function mutations in the ligand-binding domain of human PPAR{gamma} have been associated with early onset hypertension and accelerate atherosclerosis (28) . Importantly, PPAR{gamma} has been associated with several vascular-protective functions that overlap with those of NO•, such as the repression of endothelin-1 (29) and vascular cell adhesion molecule (30) . Consequently, natural mechanisms of PPAR{gamma} activation in the vasculature and potential interactions between PPAR{gamma} and NO• signaling are of considerable interest.

TZDs such as rosiglitazone and cigitazone are the most potent exogenous PPAR{gamma} ligands yet described (31) , but physiologically relevant PPAR{gamma} ligands have remained elusive. Tissue and plasma levels of known endogenous PPAR{gamma} ligands appear to be lower than those required to activate PPAR{gamma} (31) . These findings suggest that regulatory interactions between PPAR{gamma} and coactivator or corepressor proteins may form the basis for transducing signaling events through this pathway and contribute to ligand and target gene specificity. Consistent with this notion, signal transduction pathways such as the activation of PPAR{gamma} by p38 MAPK during adipogenesis (32) seem likely to be promulgated by effects on cofactors rather than the release of specific ligands. Similarly, NO• activation of p38 MAPK might function as a connecting point between NO• and PPAR{gamma} signaling in endothelium, allowing crosstalk between these pathways.

Here, we investigate interactions between NO• and PPAR signaling in endothelial cells. NO• is shown to specifically activate PPAR{gamma} and thereby induce genes with PPRE promoter motifs. These NO• effects were found to occur through a cGMP-independent mechanism that required p38 MAPK activation. This pathway may account, at least in part, for the shared vascular protective functions of NO• and PPAR{gamma}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Human umbilical vein endothelial cells HUVECs (Clonetics, Cambrex, Walkerville, MD, USA) were cultured in EGM-2 bullet kit medium (Clonetics), containing endothelial cell basal medium-2 supplemented with 2% FBS, human epidermal growth factor, hydrocortisone, human fibroblast growth factor, vascular endothelial growth factor, recombinant insulin-like grown factor 1, and ascorbic acid. HUVECs were used at passages 2 to 4. EA.hy926 cells, an immortalized HUVEC cell line, created from the A549 epithelial cell line and HUVECs were generously contributed by Dr. John B. Graham, University of North Carolina. Cell culture was carried out in Dulbecco’s modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA, USA) with 10% (v/v) FBS, and HAT medium supplement (Invitrogen). All experiments were performed when cells were 80–90% confluent.

Electrophoretic Mobility Shift Assay
HUVECs or EA.hy926 cells were treated for 1 h with one of the following reagents: degraded DETA NONOate (control), fresh diethylenetriamine NONOate (DETA NONOate; Cayman Chemical, Ann Arbor, MI, USA), glutathione (GSH; Sigma St. Louis, MO, USA), or S-nitrosoglutathione (GSNO; Calbiochem, San Diego, CA, USA), as indicated, all at a concentration of 100 µM. To look at the effect of endogenous NO• on PPAR{gamma} binding, HUVECs were pretreated for 1 h with N-monomethyl-L-arginine (L-NMA; 500 µM), a NOS inhibitor, and then were incubated for another 1 h in the presence or absence of 250 µM DETA NONOate. In separate experiments, GSNO and DETA NONOate dose responses (up to 500 µM for each) were tested by EMSA. In other experiments, EA.hy926 were cultured for 1 h with a cell-permeable cGMP analog: 8-Br-cGMP (25 or 100 µM). Nuclear extracts were prepared using CelLytic Nuclear Extraction kit (Sigma). Double-stranded oligonucleotide probes (Santa Cruz Biotechnology, Santa Cruz, CA, USA), containing the PPRE motif (5'-CAAAACTAGGTCAAAGGTCA-3'), were labeled with biotin using Biotin 3'-End DNA Labeling Kit (Pierce, Rockford, IL, USA). PPAR{gamma} binding activity was determined using the light shift chemiluminesencent EMSA kit (Pierce). The nucleoprotein binding reaction was performed by combining 5 µg of nuclear extract and labeled probe for 60 min on ice in the presence or absence of a 200-fold excess of unlabeled specific PPRE probe or unlabeled mutated PPRE probe (5'-CAAAACTAGCACAAAGCACA-3') as indicated. For supershift experiments, extracts were preincubated with 2 µg of polyclonal anti-PPAR{gamma} (Cell Signaling Technology, Danvers, MA, USA) for 30 min before adding labeled probe. Protein DNA mixtures were run on 6% acrylamide gels (Invitrogen), and chemiluminescence was measured using the Image Station 440 charge-coupled device camera system (Eastman Kodak Co., New Haven, CT, USA).

Chromatin Immunoprecipitation Assay
EA.hy926 cells were incubated with DETA NONOate (250 µM) or degraded DETA NONOate (control) for 1 h as indicated. The ChIP assay was performed according to the manufacturer’s instructions (Upstate Cell Signaling Solutions, Charlottesville, VA, USA). The chromatin was sheared by sonication four times for 40 s at one-third of the maximum power with 1 min cooling on ice between each pulse. The same antibody (Ab) used for the EMSA experiments were used for chromatin immunoprecipitation assay (ChIP). Presence of PPAR{gamma} promoter sequence was determined using the following polymerase chain reaction (PCR) promoter-specific primers: COX-2 (-4297 to –3532), 5'-AGTGAGCCTGTGCCTATGAACG- 3' and 5'-AAGGGAAAGAGAGTGTGAGGTGG-3'. The PCR products were analyzed by electrophoresis on 0.8% agarose gels (Invitrogen), stained with ethidium bromide, and quantified with the Image Station 440 charge-coupled device camera system (Eastman Kodak Co.).

TransAM PPAR{gamma} ELISA
Nuclear extracts (30 µg of each sample) from EA.hy926 cells (10x106) were prepared as described previously. TransAM PPAR{gamma} ELISA Kits (Active Motif Inc. Carlsbad, CA) were used to examine time and dose responses to NO• exposure. GSNO (100 µM), which releases NO• rapidly, was studied for up to 4 h. DETA NONOate (100 µM), which releases NO• over longer time periods, was studied for up to 24 h. In dose-escalation experiments, 0–500 µM concentrations were tested at 1 h for GSNO and at 1 and 12 h for DETA NONOate. TransAM PPAR{gamma} ELISA detects human PPAR{gamma}1 and PPAR{gamma}2 binding to PPRE consensus sequence and does not cross-react with PPAR{alpha} or PPARß.

Quantitative Real-Time PCR (qRTPCR)
HUVECs or EA.hy926 cells (1x106) were cultured and exposed to escalating concentrations of DETA NONOate (0 to 500 µM) for 12 h. To test the effect of cGMP on the baseline expression and NO•-mediated induction of genes with PPRE promoter motifs, EA.hy926 cells were incubated with 8-Br-cGMP (0 to 100 µM) for 12 h. In other experiments, SB202190 (1 µM; Calbiochem, San Diego, CA, USA), a p38 MAPK inhibitor, or T0070907 (5 µM; Cayman), a specific PPAR{gamma} inhibitor, was added to cell cultures 4 h before NO• donors. Where indicated, siRNA was used to knockdown p38 MAPK (see below). Total RNA was extracted using QIAshredder columns and RNeasy kits and was treated with DNase (all obtained from Qiagen, Valencia, CA, USA). Cyclooxygenase-2 (COX-2), DAG kinase alpha (DGK{alpha}), heme oxygenase-1 (HO-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was measured by qRTPCR (TaqMan) using the ABI Prism 7900 sequence detection system (Applied Biosystem, Foster City, CA, USA) and commercially available probe and primer sets (Applied Biosystem). Reverse transcription was performed with an RT kit (La-Roche, Basel, Switzerland) and TaqMan Universal PCR master mix (Applied Biosystem) according to the manufacturer’s manuals. Relative gene expression was calculated as fold-induction compared with control.

Transfection with Small Interfering RNA
EA.hy926 cells grown in 6-well plates were transfected with 100 nM small interfering RNA (siRNA) duplexes of SMARTpools p38 MAPK or PPAR{gamma} siRNA, or their control scrambled siRNA (Upstate USA, Chicago, IL, USA), using Nucleofector electroporation (Amaxa, Gaithersburg, MD, USA). Knockdown of these genes was confirmed by detecting protein expression using Western blotting as described below. The effect of p38 MAPK knockdown on PPAR{gamma} activation was assessed by EMSA as described above. To further test the mechanism by which NO• regulates COX-2, DGK{alpha} and HO-1, cells were stimulated with DETA NONOate (0–500 µM) for 12 h after p38 MAPK knockdown, followed by the measurement of mRNA expression. In separate experiments, cells were exposed to DETA NONOAte (250 µM) for 24 h after PPAR{gamma} knockdown and the expression of COX-2 and HO-1 protein was examined by Western blotting.

Western blotting
Western blotting and densitometry analysis of blots were performed following a previously described procedure (18) . Rabbit polyclonal anti-p38 or anti-phospho-p38 (Cell Signaling Technology, San Jose, CA, USA), anti-HO-1 (Upstate, Lake Placid, NY, USA), and anti-eNOS (BD Biosciences, San Jose, CA, USA) were used after 1:1000 dilution. Rabbit polyclonal anti-PPAR{gamma} (Upstate) and anti-COX-2 (Cayman Chemical) were employed after 1:500 dilution. Mouse monoclonal anti-{alpha} tubulin (Santa Cruz Biotechnology) was used after 1:5000 dilution.

Statistical analysis
Data are mean ± SE. P values <0.05 are considered significant. NO• donor time-responses were examined using a repeated measures ANOVA, blocked by experiment, followed when appropriate by post hoc Dunnett’s tests for comparing 0 h to subsequent times. NO• dose response on PPAR{gamma} binding was analyzed using blocked (by experiment) ANOVA, followed by the more general Games-Howell post hoc tests for comparing various pairs of doses. Both of these methods address the problem of multiple comparisons. NO• dose effects on p38 MAPK activation and on gene induction were analyzed using the Spearman-Mann nonparametric D-test for trend (a variation on Spearman’s correlation). The impact of cGMP on NO•-induced gene expression was analyzed similarly, except that two-sample t tests were applied, as appropriate. To assess the effect of SB202190 and T0070907, data were analyzed using multiway ANOVA followed by post hoc Dunnett’s test (see Results). To assess the effect of p38 MAPK knockdown, a preliminary ANOVA was run to show that the 2 control treatments (without siRNA and control siRNA) were very similar (P ≥ 0.37 for all); these were then pooled and compared to p38 MAPK knockdown via ANOVA (see Results).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
NO•-donors up-regulate PPAR{gamma} binding
To investigate the relationship between NO• and PPAR signaling, we first evaluated the effects of NO• on PPAR binding by EMSA. NO• increased the PPRE DNA binding activity of nuclear extract from HUVECs and EA.hy926 cells (Fig. 1 A, B, respectively). The sequence specificity of NO•-stimulated PPAR activity was demonstrated by inhibition of binding with an excess of unlabeled PPRE probe, whereas binding was not affected by the same molar excess of oligonucleotide with mutated PPRE motif. The identity of the PPAR-oligonucleotide complex was verified by supershift analysis. PPAR{gamma} Ab shifted the protein-oligonucleotide complex (supershift; Fig. 1A-C ). PPAR{alpha} and ß Ab had no effect on the PPAR-oligonucleotide complex (data not shown). Like exogenous NO• donors, endogenous NO• produced by eNOS in HUVECs also appeared to increase PPAR{gamma} binding. L-NMA, a specific NOS inhibitor, was found to reduce PPAR{gamma} binding while DETA NONOate restored PPAR{gamma} binding above baseline even in the presence of L-NMA (Fig. 1C ). NO•-mediated increases in PPAR{gamma} binding to PPRE-containing promoters were confirmed in vivo by ChIP assays using EA.hy926 cells (Fig. 1D ).


Figure 1
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  		Figure 1. Effect of NO• on PPAR{gamma} binding to PPRE sequence. HUVECs (A) or EA.hy926 (B) cells were stimulated with 100 µM DETA NONOate or GSNO for 1 h, then nuclear extracts were prepared for EMSA using biotin-labeled PPRE probe. Unlabeled PPRE sequence or its mutant were added as specific or non-specific competetors as indicated. C) HUVECs were pretreated 1 h with or without L-NMA (500 µM), a NOS inhibitor, and then incubated another 1 h in the presence or absence of 250 µM DETA NONOate for EMSA (top panel). Expression of eNOS and {alpha}-tubulin were measured by Western blotting (bottom panel). D) ChIP assay to detect the in vivo association of PPAR{gamma} with the PPRE site of the COX-2 promoter. EA.hy926 cells were treated with DETA NONOate (250 µM), or decomposed DETA NONOate (control) as indicated in the top panel. Densitometric measurements from ChIP assay were normalized to input chromatin (bottom panel). NO• significantly increased PPAR{gamma} binding to the PPRE site of the human COX-2 promoter compare to unstimulated cells (P<0.05, one sided). Data are mean ± SE of 3 independent experiments.

Time-course analysis showed that NO•-induced increases in PPAR{gamma} binding were apparent by 10 min, reaching a maximum effect by 1 h (Fig. 2 A, B). GSNO, which produces relatively high levels of NO• for short periods of time, increased PPAR{gamma} binding by >4-fold (Fig. 2A ). DETA NONOate produces lower levels of NO•, but over a longer time frame. Accordingly, DETA NONOate-induced changes in PPAR{gamma} binding were ~2-fold but persisted for up to 8 h (Fig. 2B ). NO•-induced PPAR{gamma} binding was also found to be dose dependent. Observed peak effects on PPAR{gamma} binding varied with donor and incubation time (Fig. 2C-E ). After 1 h incubation, maximum effects on PPAR binding occurred at 100–250 µM for GSNO, a short half-life, fast-releasing NO• donor (Fig. 2C ), and at 50–500 µM for DETA NONOate, a slow-releasing NO• donor (Fig. 2D ). Interestingly GSNO, but not DETA NONOate, decreased PPAR{gamma} binding at high (500 µM) concentrations. After a longer incubation (12 h), the maximum effect of DETA NONOate occurred at ≥500 µM (Fig. 2E ), suggesting that higher doses of this donor produced more prolonged effects on PPAR{gamma} binding. Dose-dependent effects of NO• on PPAR{gamma} binding measured by ELISA (Fig. 2C-E ; top panels) were confirmed by EMSA (Fig. 2C-E ; bottom panels).


Figure 2
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  		Figure 2. Time and dose dependence of NO•-induced effects on PPAR{gamma} binding. A) GSNO time-dependent activation of PPAR{gamma} as determined by TransAM PPAR{gamma} ELISA kit using nuclear extracts of EA.hy926 cells treated for up to 4 h. GSNO (100 µM) time-dependently increased PPAR{gamma} binding (overall ANOVA, P<0.0001); significant effects occurred by 30 min, peaked at 1 h, and were not detectable after 2 h (*P<0.05). Data are mean ± SE of 6 separate experiments. B) DETA NONOate time-dependent activation of PPAR{gamma} as determined by TransAM PPAR{gamma} ELISA kit using nuclear extracts of EA.hy926 cells treated for up to 12 h. DETA NONOate (100 µM) time dependently increased PPAR{gamma} binding (overall ANOVA, P<0.0001); significant effects occurred by 10 min, peaked at 1 h, and were not detectable after 8 h (*P<0.05). Data are mean ± SE of 6 separate experiments. C) GSNO dose-dependent activation of PPAR{gamma} as determined by TransAM PPAR{gamma} ELISA kit (top panel) or EMSA (bottom panel) using nuclear extracts of EA.hy926 cells exposed for 1 h. GSNO dose-dependently increased PPAR{gamma} binding (top panel; overall ANOVA, P<0.001); significant effects occurred at 50 µM and above (*P<0.05). However, the highest dose examined (500 µM) decreased PPAR{gamma} activation relative to effects at 100 and 250 µM (P<0.05). Data are mean ± SE of 3 separate experiments. EMSA results (bottom panel) are similar to those by ELISA. D, E) DETA NONOate dose-dependent activation of PPAR{gamma} as determined by TransAM PPAR{gamma} ELISA kit (top panel) or EMSA (bottom panel) using nuclear extracts of EA.hy926 cells exposed for 1 h or 12 h. DETA NONOate dose dependently increased PPAR{gamma} binding after 1 or 12 h incubation, (top panels of D and E; overall ANOVA, P<0.0001 for both); significant effects occurred at different doses for the 2 incubation times: 50 µM and above after 1 h incubation (D, top panel; *P<0.05), and 250 µM and above after 12 h incubation (E, top panel; *P<0.05). Data are mean ± SE of 6 separate experiments. Both EMSA results (bottom panels of D and E) are similar to those by ELISA.

NO• effects not mediated by changes in PPAR{gamma} expression or cGMP signaling
To test the possibility that NO• regulates PPAR{gamma} expression, Western blotting was performed in EA.hy926 cells (Fig. 3 A). Neither GSNO (25–500 µM) nor DETA NONOate (25–500 µM) exposure for up to 16 h induced PPAR{gamma} protein expression. Activation of soluble guanylyl cyclase with subsequent signal transduction by the second messenger cGMP is a well-established signaling pathway for NO•. To determine whether cGMP might be involved in NO• activation of PPAR{gamma}, we exposed EA.hy926 cells to the lipophilic cyclic GMP analog 8-Br-cGMP (100 µM) for 1 h. Unlike the significant effect of NO•, high-dose 8-Br-cGMP did not substantially activate PPAR{gamma} binding (Fig. 3B, C ). This result suggested that NO•-mediated increases in PPAR{gamma} binding were independent of cGMP.


Figure 3
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  		Figure 3. Effect of NO• not mediated by changes in PPAR{gamma} expression or by cGMP signaling. A) NO• effect on PPAR{gamma} expression. EA.hy926 cells were incubated with 0–500 µM of GSNO (top panel) or DETA NONOate (bottom panel) for 16 h. Cell lysates were assayed by Western blotting with anti-PPAR{gamma} Ab. NO•-donors did not affect PPAR{gamma} protein expression level. B) Effect of 8-Br-cGMP, a cell permeable analog of cGMP, on PPAR{gamma} activation. EA.hy926 cells were incubated with DETA NONOate (500 µM) or 8-Br-cGMP (25 or 100 µM) for 1 h. Activation of PPAR{gamma} was determined by EMSA. C) Densitometric measurements from EMSA. NO• increased (P<0.01), but high-dose 8-Br-cGMP did not substantially, affect PPAR{gamma} binding (P=NS for both concentration of 8-Br-cGMP). Effects of NO•, in the presence or absence of 8-Br-cGMP, on the expression of PPRE-containing genes: COX-2 (D), DGK{alpha} (E), HO-1 (F). EA.hy926 cells were incubated with DETA NONOate (0–500 µM) and 0 or 100 µM of 8-Br-cGMP for 12 h. DETA NONOate exposure dose-dependently increased the expression of all three PPRE containing genes, COX-2 (P<0.03), DGK{alpha} (P<0.02), and HO-1 (P<0.02), as measured by qRTPCR. NO•-induction of these genes was unaffected by cGMP analog, 8-Br-cGMP (P=NS for all). Data are mean ± SE of 3 separate experiments.

NO•, but not cGMP, induces genes with PPRE promoter motifs
Next, we sought to determine whether NO•-mediated changes in PPAR{gamma} binding altered the expression of genes with PPRE promoter motifs. PPRE-containing genes, regulated by PPAR{gamma}, were identified from the literature and an examination of their promoter sequences. Three were chosen, COX-2, DGK{alpha}, and HO-1, for further analysis. EA.hy926 cells were treated with DETA NONOate for 12 h, and mRNA expression of the selected PPAR{gamma} target genes was measured by qRTPCR. NO• was found to increase the expression of COX-2, DGK{alpha} and HO-1 in a dose-dependent manner (Fig. 3D-F ). In contrast, 8-Br-cGMP, a cell-permeable cGMP analog, had no affect on either baseline expression or NO•-mediated inductions of any of these target genes (Fig. 3D-F ).

NO• activation of p38 MAPK: role in regulating PPAR{gamma} binding
NO• has been shown to activate the p38 MAPK pathway in a variety of cells. To further explore the mechanism by which NO• activates PPAR{gamma} signaling, we tested whether NO• triggers p38 MAPK activation in EA.hy926 cells. Preliminary experiments indicated that exposure to NO• donors for 30 min was sufficient to produce detectable effects on p38 MAPK phosphorylation (data not shown). Here, representative Western blots and densitometry from independent experiments show that both GSNO and DETA NONOate dose-dependently activate p38 MAPK as measured by increased amounts of its phosphorylated form while quantities of total p38 MAPK remain unchanged (Fig. 4 A, B, respectively). NO• activation of p38 MAPK was then assessed as a possible mechanism for changes in PPAR{gamma} binding. As shown in Fig. 5 A, treatment with SB202190, a specific p38 MAPK inhibitor, blocked NO•-dependent activation of PPAR{gamma}. Also in Fig. 5A , NO• is simultaneously shown to activate p38 MAPK, while SB202190 prevents this effect in the same cells. To further evaluate the importance of p38 MAPK in PPAR{gamma} transactivation, p38 MAPK knockdown was performed using siRNA. Knockdown of p38 MAPK was shown to reduce phosphorylated p38 MAPK and to simultaneously suppress PPAR{gamma} transactivation by NO• (Fig. 5B ). These results support the notion that the NO•-induced increase in PPAR{gamma} binding to PPRE consensus sequence is dependent on p38 MAPK activation.


Figure 4
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  		Figure 4. Effect of NO• on p38 MAPK phosphorylation. EA.hy926-cells were treated with different doses (0–100 µM) of GSNO (A) or DETA NONOate (B) for 30 min. Cells were then lysed and blotted for measurement of phosphorylated and total p38 MAPK. Results from replicates at top of each panel were quantified with laser densitometry and expressed as ratios relative to control values. Representative blots for phospho-p38 MAPK and total p38 MAPK are shown. NO•-donors (100 µM) increased phosphorylated p38 MAPK (for GSNO, P=0.0003 and for DETA NONOate, P=0.003). Data are mean ± SE of 4 (GSNO) and 5 (DETA NONOate) separate experiments, respectively.


Figure 5
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  		Figure 5. Inhibition or knockdown of p38 MAPK blocks NO•-induced increases in PPAR{gamma} binding. A) Effect of SB 202190, a p38 MAPK inhibitor, on PPAR{gamma} activation by NO•. EA.hy926 cells were treated with SB 202190 (1 µM) and then incubated with 100 µM DETA NONOate or degraded DETA NONOate (control) for 1 h. Nuclear extracts were subjected to EMSA using biotin-labeled probe containing the PPRE motif (top panel). SB202190 blocked NO•-induced increases in PPAR{gamma} binding. Cells lysates were assayed by Western blotting with anti-phospho-p38 MAPK and anti-p38 MAPK antibodies (bottom two panels, respectively). SB202190 inhibited the phosphorylation of p38 MAPK. B) Effect of p38 MAPK knockdown with siRNA. Twenty-eight hours after transfection, cells were incubated with 100 µM DETA NONOate or degraded DETA NONOate (control) for 1 h. Nuclear extracts were subjected to EMSA using biotin-labeled probe containing the PPRE motif (top panel). Cell lysates were also prepared and assayed by Western blotting with anti-phospho-p38 MAPK and anti-p38 MAPK antibodies (bottom two panels, respectively). Knockdown of p38 MAPK using siRNA decreased intracellular phosphorylated p38 MAPK and substantially blocked PPAR{gamma} activation by NO•. Results represent 3 separate experiments.

Inhibition of p38 MAPK or PPAR{gamma} block NO• induction of genes with PPRE promoter motifs
To further investigate the NO•-p38 MAPK-PPAR{gamma} pathway as a signal transduction mechanism that regulates genes with PPRE promoter motifs, HUVECs or EA.hy926 cells exposed to escalating doses of DETA NONOate were treated with selective inhibitors of either p38 MAPK (SB202190) or PPAR{gamma} (T0070907). COX-2, DGK{alpha}, and HO-1 induction by NO•, as measured by qRTPCR, was blocked substantially by these inhibitors in both HUVECs (Fig. 6 A–C) and in EA.hy926 cells (Fig. 6D, E, F ). These findings again suggest that p38 MAPK and PPAR{gamma} transduce the effects of NO• to genes with PPRE promoter sequences.


Figure 6
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  		Figure 6. Effect of p38 MAPK inhibition or PPAR{gamma} inhibition on NO•-induced expression of genes with PPRE promoter motifs. HUVECs or EA.hy926 cells were pretreated with medium alone, SB202190 (p38 MAPK inhibitor; 1 µM) or T0070907 (PPAR{gamma} inhibitor; 5 µM) for 4 h, and then exposed to DETA NONOate (0–500 µM) for 12 h. The expression of genes was measured by qRTPCR normalized to GAPDH. In HUVECs, COX-2 (A), DGK{alpha} (B), and HO-1 (C) were significantly induced by NO• exposure (overall ANOVA, P<0.01 for all). Likewise, in EA.hy926 cells, NO• induced COX-2 (D); DGK{alpha} (E), and HO-1 (F) was shown to dose-dependent (P<0.002 for dose-dependent effects on all three genes). Importantly, inhibition of p38 MAPK (SB202190) or PPAR{gamma} (T0070907) altered the NO• dose-responses of COX-2, DGK{alpha}, and HO-1, substantially blocking the induction of all three genes (P<0.05 for all relevant comparisons, except P<0.10 for HO-1/SB202190 in HUVECs). Data are mean ± SE of at least 3 separate experiments.

Knockdown of p38 MAPK or PPAR{gamma} block NO•-induction of COX-2, DGK{alpha}, and HO-1
Finally, p38 MAPK and PPAR{gamma} expression was knocked down in EA.hy926 cells using siRNA to further investigate the mechanism by which NO• regulates PPRE containing genes. Knockdown of p38 MAPK substantially abolished dose-dependent induction of COX-2, DGK{alpha}, and HO-1 mRNA by NO• (Fig. 7 A–C), thus confirming the effects seen with SB202190, a p38 MAPK inhibitor. Similarly, PPAR{gamma} knockdown eliminated NO induction of COX-2 and HO-1 protein (Fig. 8 ), results consistent with those seen using T0070907, a PPAR{gamma} inhibitor. Together, these results show that NO• activates PPAR{gamma} through a p38 MAPK-dependent signal transduction pathway and thereby induces the expression of genes with PPRE promoter motifs.


Figure 7
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  		Figure 7. Effect of p38 MAPK knockdown on NO•-induced COX-2, DGK{alpha}, and HO-1 mRNA expression. EA.hy926 cells were transfected with p38 MAPK siRNA or control siRNA. Other cells were not transfected as an additional control. After 28 h of recovery or just further incubation, cells were exposed to DETA NONOate (0–500 µM) for 12 h. (A) COX-2, (B) DGK{alpha}, and (C) HO-1 expression were measured by qRTPCR normalized to GAPDH. Again, NO• dose-dependently induced COX-2 (P≤0.0006), DGK{alpha} (P=0.009), and HO-1 (P<0.02) expression in both untransfected and control siRNA transfected cells. Knockdown of p38 MAPK using siRNA altered NO• dose responses compared to the controls for COX-2 (P<0.0001), DGK{alpha} (P<0.03), and HO-1 (P<0.0001), substantially blocking their induction. Data are mean ± SE of 3 separate experiments.


Figure 8
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  		Figure 8. Effect of PPAR{gamma} knockdown on NO•-induced COX-2 and HO-1 protein expression. EA.hy926 cells (1x106) were transfected with PPAR{gamma} siRNA or control siRNA. Other cells were not transfected as an additional control. After 48 h of recovery or just further incubation, cells were incubated with or without DETA NONOate (250 µM) for 24 h, and COX-2 and HO-1 protein expression was measured by Western blotting. DETA NONOate increased both COX-2 and HO-1 protein expression when the cells were either unaltered (without siRNA transfection) or transfected with scrambled control siRNA (control siRNA). However, transfection of specific PPAR{gamma} siRNA, knocked down PPAR{gamma} protein expression, and also abolished the up-regulation of COX-2 and HO-1 by NO•. The protein expression of {alpha}-tubulin, a control housekeeping gene, was not affected by either NO• or siRNA transfection.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Both PPAR{gamma} and NO• regulate endothelial function and protect the vasculature from injury, but mechanisms that interconnect the functionality of these regulatory molecules remain obscure. Here, a novel mechanism is described by which NO• may exert some of its antiinflammatory and cytoprotective effects in the vasculature through PPAR{gamma}. NO• was found to activate PPAR{gamma} and increase its binding to a consensus PPRE sequence in a dose- and time-dependent manner. Low doses of NO• enhanced, whereas high dose (above 250 µM of GSNO) reduced the DNA binding of PPAR{gamma}. These changes in PPAR{gamma} binding occurred as early as 10 min after exposure to NO• donors, with a maximal effect at 1 h. Previous reports (25) as well as our studies demonstrate that NO• did not change PPAR{gamma} expression. The rapid induction of PPAR{gamma} DNA binding activity without a change in its protein expression suggested that NO• was acting through a very fast post-translational mechanism. Further experiments identified p38 MAPK rather than cGMP as the intermediary signal transduction step leading to PPAR{gamma} activation. NO• donors were found to increase p38 MAPK phosphorylation, an effect previously described in human monocytoblastoid cells (18 , 33) . SB202190, a specific p38 MAPK inhibitor, or knockdown of p38 MAPK with siRNA, were both shown to block NO• activation of PPAR{gamma}. Similarly, bone morphogenetic protein 2 has been reported to activate PPAR{gamma} transcriptional activity in undifferentiated mesenchymal cells through a signaling pathway that was partially dependent on p38 MAPK (32) .

The mechanism by which NO•-p38 MAPK signaling activates PPAR{gamma} has not been determined. Direct activation of PPAR{gamma} by p38 MAPK-mediated phosphorylation is unlikely because PPAR{gamma} possesses only one consensus MAPK phosphorylation site at serine 112. Phosphorylation of this site by extracellular signal-regulated kinases (Erks) has been shown to inhibit rather than enhance PPAR{gamma} transcriptional activity (34) . Therefore, it is more likely that p38 MAPK activates PPAR{gamma} by phosphorylating an as yet unidentified target. PPAR{gamma} can interact with a number of other proteins such as PGC-1 (PPAR coactivator-1) and ATF-2 to regulate gene promoter activity, so NO•-p38 MAPK signaling might indirectly activate PPAR{gamma} by modulating one or more of its transcriptional partners. Cytokines have been shown in cultured muscle cells to activate PGC-1 through p38 MAPK-mediated phosphorylation (35) . This led to the increased expression of genes linked to mitochondrial uncoupling. In addition, ATF-2 is a well-known p38 MAPK substrate that crosstalks with other transcriptional factors or coactivators to control gene promoter activity (36) . Clearly, the identification and characterization of coactivator(s) that are the putative target of NO•-p38 MAPK-PPAR{gamma} signaling will be essential for future studies.

In addition to the likely role of protein targets as intermediatory steps in PPAR{gamma} activation by NO• and p38 MAPK, interactions between NO•- and PPAR{gamma}-specific ligands may further modulate crosstalk between these signal transduction pathways. Recently, nitrolinoleic acid and nitrated oleic acid have been shown to be potent endogenous PPAR{gamma} ligand agonists (37) . Their production is significantly increased by stress or inflammation, which themselves are associated with increased NO• production and p38 MAPK activation. Therefore, NO• might activate PPAR{gamma} by both forming molecular adjuncts that are potent ligand agonists and by activating essential cofactors through p38 MAPK.

Activation of PPAR{gamma} by low-dose NO• is in contrast to the inhibition that we observed at high doses of GSNO but not DETA NONOate. GSNO and DETA NONOate differ substantially in their kinetics of NO• delivery, which appear to be reflected in our findings. Each molecule of DETA NONOate slowly releases two molecules of NO• and the parent compound has a half-life of ≥20 h in cell culture medium (38 , 39) . Concentrations of DETA-NONOate in the micromolar range generate steady-state concentrations of NO• in cell culture medium in the nanomolar range, which is roughly equivalent to NO• production by eNOS under normal physiological conditions (38 , 39) . In contrast, mM concentrations of DETA NONOate release µM amounts of NO•, which is similar to NO• generation by activated macrophages expressing inducible NOS (iNOS). In the present study, micromolar amounts of DETA NONOate were shown to activate PPAR{gamma} through a p38 MAPK-dependent mechanism, suggesting that this signal transduction pathway may be relevant to regulatory events in the vasculature under physiological conditions. Consistent with this possibility, eNOS inhibition in HUVECs was shown to reduce PPAR{gamma} binding as measured by EMSA. Different from DETA NONOate, GSNO releases relatively high levels of NO• over short periods of time and can activate a number of MAPK pathways including Erk1/2 (40) . As already discussed, Erk1/2 phosphorylation of PPAR{gamma} attenuates PPAR{gamma} binding to PPRE promoter motifs and thereby might account for the bimodal effect of NO• on PPAR{gamma} signaling. Similar dichotomous responses of PPAR{gamma} to GSNO have been previously reported in THP-1 and U937 cells (25) . These disparate effects of low and high NO• on PPAR{gamma} might (Fig. 9 ) also somewhat explain the paradoxical roles of eNOS (a low output NO• producer) and iNOS (a high output NO• producer) in conditions such as atherosclerosis (11 , 12) .


Figure 9
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  		Figure 9. Hypothetical model: NO• regulation of PPAR{gamma}-mediated gene transcription through MAPK pathways.

To address the question of whether NO•-p38 MAPK-PPAR{gamma} signaling transcriptionally regulates genes containing PPRE promoter motifs, COX-2, DGK{alpha}, and HO-1 were chosen for further study. Notably, NO• significantly increased the mRNA levels of these genes in both HUVECs and EA.hy926 cells. Pretreatment with SB202190 or T0070907, specific p38 MAPK and PPAR{gamma} inhibitors, respectively, substantially blocked NO• induction of COX-2, DGK{alpha}, and HO-1. The specificity of NO•-p38 MAPK-PPAR{gamma} signaling for these genes was further confirmed by knockdown experiments in which siRNA for p38 MAPK or PPAR{gamma} was shown to attenuate their induction by NO•. Consistent with our results, several other studies have demonstrated that PPAR{gamma} agonists regulate COX-2, DGK{alpha} and HO-1 through PPRE promoter binding sites. It has been reported that PPAR{gamma} activation induces COX-2 through its PPRE motif (41 42 43) . Also, PPAR{gamma} agonists (TZD and PGJ2) increase the expression of DGK{alpha} through a PPRE-dependent mechanism in HUVECs (44) . Finally, the HO-1 promoter contains at least one PPRE motif, located at –623 bp relative to the transcription start site, which is transcriptionally activated by PPAR{gamma} agonists (45) .

Crosstalk between PPAR{gamma} pathways and NO• signaling seems likely to be operative under physiological conditions, and our data show that NO• can activate a number of PPAR{gamma} target genes. COX-2 and eNOS are recognized as key markers of endothelial integrity and health. Their respective products, PGI2 and NO•, share antiadhesive, antithrombotic, and antiproliferative properties and play a major role in the maintenance of vascular homeostasis (46) . DGK{alpha} is an evolutionarily conserved lipid kinase that phosphorylates DAG to yield phosphatidic acid, thereby terminating DAG-PKC signal transduction. PKC has been connected to the pathogenesis of cardiovascular disease, especially in regards to diabetic vasculopathy (44) . Therefore, DGK{alpha} is considered to have an important antiinflammatory role in the maintenance of vascular homeostasis. By up-regulating DGK{alpha}, PPAR{gamma} agonists block DAG-PKC signal transduction (44) . This suggests NO•-p38 MAPK-PPAR{gamma} signaling may have similar protective effects on vascular endothelium that might be additive to other PPAR{gamma} activation strategies.

Our data show that NO• activates PPAR{gamma} through a p38 MAPK signaling pathway, thereby transcriptionally regulating genes with promoters that contain PPRE motifs. Whether NO• -p38 MAPK-PPAR{gamma} signaling has regulatory effects independent of PPRE promoter sites has not been determined. Recent studies have suggested that PPAR{gamma} can regulate other transcription factors such as NF-{kappa}B and CCAAT/enhancer-binding protein delta and thereby affect non-PPRE promoter sites. This capability may account for the broad anti-inflammatory effects of PPAR{gamma} agonists that appear to extend beyond PPRE-containing target genes (47) . Nonetheless, NO• activation of PPAR{gamma} signaling may be responsible for some vascular-protective properties of NO• and explain their overlapping functionality. Further, the interesting observation that low amounts of NO• activates while higher concentrations block PPAR{gamma} binding provides a plausible mechanism that might partially explain the dual nature of NO• in cardiovascular disease. The crosstalk between NO• and PPAR{gamma} described here can be used to identify additional PPAR{gamma} targets genes and may lead to new strategies to promote vascular health.


   ACKNOWLEDGMENTS
 
The authors thank James Shelhamer for critical review of this manuscript and Jennifer Candotti for editorial assistance. This study was supported by intramural National Institutes of Health funds from Critical Care Medicine Department.

Received for publication July 3, 2006. Accepted for publication October 11, 2006.


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 DISCUSSION
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