G Protein-coupled Receptor 40 (GPR40) and Peroxisome Proliferator-activated Receptor γ (PPARγ)

Background: PPARγ ligands are used to treat type 2 diabetes mellitus, but signaling by these drugs is incompletely understood. Results: Rosiglitazone activation of GPR40 markedly enhanced PPARγ-dependent transcription through downstream effects on p38 MAPK, PGC1α, and EP300. Conclusion: GPR40 and PPARγ can function as an integrated two-receptor signal transduction pathway. Significance: Future drug development should consider the effects of prospective ligands at both receptors. Peroxisome proliferator-activated receptor γ (PPARγ) ligands have been widely used to treat type 2 diabetes mellitus. However, knowledge of PPARγ signaling remains incomplete. In addition to PPARγ, these drugs also activate G protein-coupled receptor 40 (GPR40), a Gαq-coupled free fatty acid receptor linked to MAPK networks and glucose homeostasis. Notably, p38 MAPK activation has been implicated in PPARγ signaling. Here, rosiglitazone (RGZ) activation of GPR40 and p38 MAPK was found to boost PPARγ-induced gene transcription in human endothelium. Inhibition or knockdown of p38 MAPK or expression of a dominant negative (DN) p38 MAPK mutant blunted RGZ-induced PPARγ DNA binding and reporter activity in EA.hy926 human endothelial cells. GPR40 inhibition or knockdown, or expression of a DN-Gαq mutant likewise blocked activation of both p38 MAPK and PPARγ reporters. Importantly, RGZ induction of PPARγ target genes in primary human pulmonary artery endothelial cells (PAECs) was suppressed by knockdown of either p38 MAPK or GPR40. GPR40/PPARγ signal transduction was dependent on p38 MAPK activation and induction of PPARγ co-activator-1 (PGC1α). Silencing of p38 MAPK or GPR40 abolished the ability of RGZ to induce phosphorylation and expression of PGC1α in PAECs. Knockdown of PGC1α, its essential activator SIRT1, or its binding partner/co-activator EP300 inhibited RGZ induction of PPARγ-regulated genes in PAECs. RGZ/GPR40/p38 MAPK signaling also led to EP300 phosphorylation, an event that enhances PPARγ target gene transcription. Thus, GPR40 and PPARγ can function as an integrated two-receptor signal transduction pathway, a finding with implications for rational drug development.

Peroxisome proliferator-activated receptor ␥ (PPAR␥) ligands have been widely used to treat type 2 diabetes mellitus. However, knowledge of PPAR␥ signaling remains incomplete. In addition to PPAR␥, these drugs also activate G protein-coupled receptor 40 (GPR40), a G␣ q -coupled free fatty acid receptor linked to MAPK networks and glucose homeostasis. Notably, p38 MAPK activation has been implicated in PPAR␥ signaling. Here, rosiglitazone (RGZ) activation of GPR40 and p38 MAPK was found to boost PPAR␥-induced gene transcription in human endothelium. Inhibition or knockdown of p38 MAPK or expression of a dominant negative (DN) p38 MAPK mutant blunted RGZ-induced PPAR␥ DNA binding and reporter activity in EA.hy926 human endothelial cells. GPR40 inhibition or knockdown, or expression of a DN-G␣ q mutant likewise blocked activation of both p38 MAPK and PPAR␥ reporters. Importantly, RGZ induction of PPAR␥ target genes in primary human pulmonary artery endothelial cells (PAECs) was suppressed by knockdown of either p38 MAPK or GPR40. GPR40/PPAR␥ signal transduction was dependent on p38 MAPK activation and induction of PPAR␥ co-activator-1 (PGC1␣). Silencing of p38 MAPK or GPR40 abolished the ability of RGZ to induce phosphorylation and expression of PGC1␣ in PAECs. Knockdown of PGC1␣, its essential activator SIRT1, or its binding partner/co-activator EP300 inhibited RGZ induction of PPAR␥-regulated genes in PAECs. RGZ/GPR40/p38 MAPK signaling also led to EP300 phosphorylation, an event that enhances PPAR␥ target gene transcription. Thus, GPR40 and PPAR␥ can function as an integrated two-receptor signal transduction pathway, a finding with implications for rational drug development.
Obesity-associated type 2 diabetes mellitus has reached epidemic proportions in the United States and is a major risk factor for coronary artery disease and stroke (1). Thiazolidinediones (TZDs), 4 synthetic insulin-sensitizing drugs that activate peroxisome proliferator-activated receptor ␥ (PPAR␥), have been widely used to treat this disease (2,3). In addition to lowering glucose levels (3), TZDs also lower blood pressure (4), improve lipid profiles (5), and reduce vascular inflammation (6). These effects in non-adipose tissues have raised the possibility that PPAR␥ ligands may be more broadly useful in vascular disorders, such as atherosclerosis (7), pulmonary arterial hypertension (8), and septic shock (9). However, side effects, including weight gain, fluid retention, congestive heart failure, and bone fractures, have been linked to TZDs (10). These adverse effects underscore our still incomplete understanding of PPAR␥ signaling and the need to develop safer, more effective PPAR␥ ligands (10).
The direct binding of TZDs and other ligands to PPAR␥ activates two major but distinct signal transduction pathways. Cisactivation drives transcription through ligand-dependent recruitment of co-activators, such as PPAR␥ co-activator-1␣ (PGC1␣), PPAR␥ dimerization with the retinoid X receptor (11), and the binding of this complex to peroxisome proliferator response elements (PPREs) in the promoter region of target genes. Trans-repression of inflammatory response genes re-quires the covalent modification of PPAR␥ at lysine 395 by the small ubiquitin-like modifier with subsequent tethering of PPAR␥ to nuclear receptor co-repressor and histone deacetylase complexes at NFB and AP-1 sites (12).
Although the anti-diabetic activity of TZDs was first reported in 1982 (26), recognition as ligand/agonists of the orphan nuclear receptor PPAR␥ came more than a decade later (27,28). More recently, TZDs were found to bind to and activate G␣ q protein-coupled receptor 40 (GPR40) (29 -34), a cell membrane receptor associated with free fatty acid-and glucose-induced insulin secretion (35,36), effects that overlap with those of PPAR␥ (37,38). Importantly, GPR40 signaling causes rapid activation of ERK, p38 MAPK, and JNK (31,33). Whereas we previously found that ⅐ NO activation of p38 MAPK initiated PPAR␥ signaling in human endothelial cells (39), others have also associated p38 MAPK with PPAR␥-related effects in adipocytes (20,40). Collectively, these results suggest that TZD signaling through GPR40 with subsequent activation of p38 MAPK might modulate PPAR␥ transcriptional activity in human endothelium.
This investigation sought to determine whether TZD activation of GPR40 and p38 MAPK influences PPAR␥ signaling in human endothelial cells and, if so, to explore the underlying mechanism. A two-receptor paradigm for PPAR␥ signal transduction is proposed with implications for the development of PPAR␥ therapeutics.
Reporter Gene Assay-EA.hy926 cells (2 ϫ 10 5 /2 ml/well) were seeded in 6-well plates 16 h prior to transfection with 100 ng of PPRE reporter (PPRE-CAT or PPRE-LUC), 100 ng of internal control pRL-TK, and 50 ng of PPAR␥ 2 expression plasmid in the presence or absence of additional expression plasmids, including DN-p38 MAPK, DN-G␣ q , GPR40, PGC1␣, and EP300, as indicated. FuGENE 6 transfection reagent was utilized at a ratio of 3 l/g of DNA. Twenty-four hours after transfection, cells were treated for an additional 24 h as indicated in the corresponding figure legends. Chloramphenicol acetyltransferase and luciferase activities were then measured using the CAT ELISA (Roche Diagnostics) and the Dual-Luciferase reporter assay system (Promega), respectively. In reporter experiments with gene knockdown, cells were co-transfected with siRNA, shRNA, or their controls for 48 h, followed by 24-h stimulation. Non-targeting control or p38␣ MAPK siRNA was transfected using Nucleofector kits (Amaxa, Gaithersburg, MD), as described previously (39). GPR40 shRNA pool or its control plasmid was transfected using FuGENE 6.
Detection of PPAR␥ Binding to Specific DNA Sequence-EA.hy926 cells were treated for 1 h with RGZ (10 M) or vehicle control with or without SB (1 M) pretreatment for 40 min, as indicated. Nuclear extracts (3-4 g) were then prepared for TransAM PPAR␥ ELISA (Active Motif, Carlsbad, CA), which detects human PPAR␥ 1/2 binding to PPRE consensus sequence and does not cross-react with PPAR␣ or PPAR␤.
Total RNA was extracted using the RNeasy kit (Qiagen), and cDNA was synthesized with iScript TM cDNA synthesis kits (Bio-Rad). TaqMan PCR was performed with the Applied Biosystems Vii TM 7 instrument.
Immunoprecipitation-PAECs were transfected with various siRNAs, as indicated. Forty-eight hours post-transfection, cells were treated with RGZ (10 M) or vehicle control for 1 and 4 h to examine effects on PGC1␣ phoshorylation and acetylation, respectively, before preparation of nuclear protein and whole cell lysates. Immunoprecipitation of nuclear protein (40 g) was performed with anti-phosphoserine (catalog no. AB1603) and anti-phosphothreonine (catalog no. AB1607) antibodies from Millipore. Immunoprecipitation of whole cell lysates (500 g) used anti-acetyl-lysine antibody (catalog no. 06-933; Millipore). After incubation with rotation at 4°C overnight, Dynabeads protein G (1.5 mg; Invitrogen) was added. Immunoprecipitates were then subjected to Western blotting with anti-PGC1␣ antibody (catalog no. 101707; Cayman Chemical).
Chromatin Immunoprecipitation (ChIP) Assay-PAECs were pretreated with SB (10 M), GW1100 (20 M), or vehicle control (CTRL) for 40 min and then incubated with RGZ (10 M) or vehicle CTRL for another 24 h. The ChIP assay was carried out using the EZ ChIP TM kit (Millipore) as instructed by the manufacturer. Briefly, cells were cross-linked with 1% formaldehyde and then fragmented using a Misonix sonicator with microtip probe 4418 at a power setting of 4 and a 30% duty cycle. Sonication was performed three times for 10 s with a 50-s cooling on ice between pulses, shearing chromatin into 200 -1000-bp fragments. Precipitation was carried out overnight at 4°C with anti-PPAR␥ (catalog no. SC-7196; Santa Cruz Biotechnology, Inc., Dallas, TX), anti-EP300 (catalog no. PA1-848; Thermo Scientific), anti-PGC1␣ (catalog no. ab54481; Abcam Inc., Cambridge, MA), or control mouse IgG. Precipitated protein-DNA complexes were eluted, and cross-linkings were reversed for purification of DNA. PCR was performed using the primers 5Ј-TTTACTATTTCCACCAGCGGTCTC-3Ј and 5Ј-TCCCCCACACAGCACATTACTG-3Ј specific for the Ϫ355/ Ϫ136 region of the human CD36 promoter that contains three potential PPREs. The PCR products were analyzed by electrophoresis on 2% agarose gels (E-Gel, Invitrogen), stained with ethidium bromide, and quantified by densitometry.
Statistical Analysis-Data are presented as means Ϯ S.E. For real-time PCR results, geometric means Ϯ S.E. are plotted. All statistical analyses were carried out on log-transformed or ⌬ cycle threshold data (for real-time PCR) using JMP version 11 (SAS Institute Inc., Cary, NC). To analyze continuous dose and time effects, one-way analysis of variance models were used. To test the effects of nominal factors and their interactions, linear mixed models were used, which consider the correlation within each experiment. Non-significant factors were dropped from statistical models to calculate final p values. Post hoc contrasts were tested for our interested comparisons. All p values are two-sided and considered significant if p was Ͻ0.05.
Previously, we found that ⅐ NO activated p38 MAPK in EA.hy926 cells (39). Therefore, we next examined whether RGZ/GPR40/p38 MAPK signaling might be mediated through ⅐ NO as a second messenger. L-NAME, a ⅐ NO synthase inhibitor, had no effect on RGZ-induced p38 MAPK phosphorylation (p ϭ 0.57 for the main effect of L-NAME; Fig. 2E). Conversely, ⅐ NO activation of p38 MAPK, which is probably related to its free radical biology (47), was not dependent on GPR40 in EA.hy926 cells. DTANO, a ⅐ NO donor, time-dependently induced p38 MAPK phosphorylation (p ϭ 0.008; Fig. 2F); this phosphorylation was not altered by either GW1100, a specific GPR40 antagonist (p ϭ 0.55 for DTANO versus GW1100 plus DTANO; Fig. 2G) or GPR40 knockdown (p ϭ 0.61 for DTANO versus GPR40 shRNA plus DTANO; Fig. 2H).
(p values) between RGZ activation of PPAR␥ and siRNA silencing of GPR40 pathway components on the expression of these target genes in PAECs.
Like EA.hy926 cells, PAECs also express GPR40 as measured by Western blotting, and RGZ did not alter this expression (p ϭ 0.30 for the main effect of RGZ; Fig. 4C). GPR40 siRNA significantly reduced GPR40 protein expression (p ϭ 0.002 for the main effect of GPR40 siRNA; Fig. 4C) but had no effect on PPAR␥ expression in PAECs (p ϭ 0.56 for the main effect of GPR40 siRNA; Fig. 4C). Similar to p38␣ MAPK knockdown, GPR40 siRNA significantly blocked the RGZ-induced PPAR␥ target genes CD36, CYP1A1, and FABP4 (p Յ 0.005 for all three genes, RGZ versus RGZ plus GPR40 siRNA; Fig. 4D). Again, RGZ and GPR40 siRNA silencing appeared to interact at endogenous PPAR␥ target genes ( Fig. 4D and Table 1). This was in contrast to the additive effects of RGZ and GPR40, knockdown, DN-G␣ q mutant, or overexpression on a PPAR␥ reporter (Fig. 3). PAEC donor, passage number, and day of experiment varied between Fig. 4, B and D, possibly accounting for the observed variability in gene expression among the siRNA control conditions. These results further support the existence of a functional pathway in endothelial cells that connects GPR40 on the cell surface to nuclear PPAR␥ signaling, two receptors that can be activated by shared ligands.
In contrast to the activation of PGC1␣ by p38 MAPK-mediated phosphorylation, acetylation inhibits PGC1␣ activity and therefore also plays a key regulatory role in the co-transcriptional activity of PGC1␣ (24,25). SIRT1 binds to and deacetylates PGC1␣ both in vivo and in vitro and appears to be required for PGC1␣ activation (24,25). Therefore, we evaluated the role of SIRT1 in RGZ/GPR40/p38 MAPK/PGC1␣ PPAR␥ signal transduction. RGZ decreased PGC1␣ acetylation in PAECs (p Ͻ 0.001 for RGZ versus control within the control siRNA condition; Fig. 5C), whereas SIRT1-specific siRNA increased PGC1␣ acetylation (p Ͻ 0.001 for SIRT1 siRNA versus control siRNA in the absence and presence of RGZ; Fig. 5C) and abolished the decrease in PGC1␣ acetylation seen with RGZ (p ϭ 0.37 for RGZ versus control within the SIRT1 siRNA condition; Fig. 5C).
As expected and consistent with the importance of PGC1␣ as a PPAR␥ transcriptional co-activator, siRNA silencing of either PGC1␣ or SIRT1 significantly inhibited RGZ-induced PPAR␥ target genes (Fig. 5D, left and right panels, respectively) including CD36, CYP1A1, and FABP4 (p Ͻ 0.001 for all three genes, RGZ versus RGZ plus PGC1␣ or SIRT1 siRNA; Fig. 5D). As seen for p38 MAPK and GPR40 knockdown, the silencing of either PGC1␣ or SIRT1 profoundly reduced the ability of RGZ to induce PPAR␥ target genes ( Fig. 5D and Table 1). Specific siRNA for PGC1␣ and SIRT1 effectively reduced PGC1␣ (p ϭ 0.001 for the main effect of PGC1␣ siRNA; Fig. 5E) and SIRT1 (p Ͻ 0.001 for the main effect of SIRT1 siRNA; Fig. 5F) protein expression in PAECs, respectively. RGZ did not alter SIRT1 protein expression (p ϭ 0.83 for the main effect of RGZ; Fig. 5F) or phosphorylation state (data not shown).
EP300 Contributes to the RGZ/GPR40/p38 MAPK/PGC1␣/ PPAR␥ Signaling Pathway-EP300, a general transcriptional co-activator with intrinsic histone acetyltransferase and chromatin remodeling activity, has been shown to interact with PGC1␣ and PPAR␥, thereby enhancing PPAR␥ transcriptional activity (50,51). Importantly, p38 MAPK has been demonstrated to directly phosphorylate EP300, thereby potentiating its acetyltransferase activity (22,52). Therefore, the role of EP300 in the pathway described here was investigated in human endothelial cells. Consistent with the role of PGC1␣ in PPAR␥ signaling, PGC1␣ overexpression in EA.hy926 cells increased PPRE reporter activity (p Ͻ 0.001 for a plasmid dose effect in the absence of RGZ; Fig. 6A). A similar dose response was seen in the presence of RGZ, but the RGZ effect became smaller with increasing amounts of PGC1␣ plasmid (p Ͻ 0.001 for a negative interaction between RGZ and PGC1␣; Fig. 6A), possibly Knockdown of p38␣ MAPK blocked RGZ-induced p38 MAPK phosphorylation (p ϭ 0.43 for RGZ versus CTRL within the p38␣ siRNA condition; p ϭ 0.009 for an interaction between p38 MAPK siRNA and RGZ) and decreased total p38 MAPK protein (p Ͻ 0.001 for p38␣ siRNA main effect) but did not alter PPAR␥ protein expression (p ϭ 0.10 for p38␣ siRNA main effect). B, p38␣ MAPK siRNA silencing significantly inhibited RGZ-induced expression of PPAR␥ target genes (p Ͻ 0.001 for RGZ versus RGZ plus p38␣ siRNA for all three genes); C, GPR40 siRNA knockdown reduced GPR40 protein expression (p ϭ 0.002 for GPR40 siRNA main effect) without affecting PPAR␥ expression (p ϭ 0.56 for GPR40 siRNA main effect). D, GPR40 siRNA knockdown suppressed RGZ-induced PPAR␥ target genes (p Յ 0.005, RGZ versus RGZ plus GPR40 siRNA for all three genes). PAECs were transfected with specific siRNA or a scrambled CTRL siRNA. At 48 h after transfection, cells were treated with RGZ (10 M) or vehicle CTRL for 30 min prior to the measurement of phosphorylated p38 MAPK (pp38), total p38 MAPK (p38), and PPAR␥ in A and for 24 h prior to the measurement of GPR40 and PPAR␥ in C by Western blotting. In B and D, 48 h after siRNA transfection, cells were treated with RGZ (10 M) or vehicle CTRL for 24 h followed by measurement of PPAR␥ target genes by real-time PCR. Densitometry results of three independent experiments using different PAEC donors and a representative blot are shown in A and C. In B (n ϭ 5) and D (n ϭ 4), results are presented as geometric means Ϯ S.E. (error bars) of independent experiments using different PAEC donors. because RGZ/p38 MAPK-dependent induction and phosphorylation of PGC1␣ was rendered less important by direct PGC1␣ overexpression. EP300 overexpression also increased both basal and RGZinduced PPRE reporter activity (p ϭ 0.003 for EP300 main effect; p ϭ 0.68 for an interaction between EP300 and RGZ; Fig.  6B). Although PGC1␣ overexpression had a stronger overall effect on PPRE reporter activity (p Ͻ 0.02 for PGC1␣ versus EP300 in both the absence and presence of RGZ; Fig. 6C), EP300 overexpression better preserved RGZ effect size and increased PPRE reporter activity additively with PGC1␣ in the presence of RGZ (p Ͻ 0.001 for PGC1␣ and EP300 main effects; p ϭ 0.77 for an interaction between PGC1␣ and EP300; Fig. 6C). Notably, RGZ treatment of EA.hy926 cells increased EP300 FIGURE 5. PGC1␣ transduces RGZ/GPR40/p38 MAPK signaling to PPAR␥ in PAECs. A, RGZ induced PGC1␣ phosphorylation, an effect blocked by siRNA knockdown of p38␣ MAPK or GPR40 (p ϭ 0.001 for RGZ versus CTRL within CTRL siRNA; p Ն 0.45, RGZ versus CTRL within p38␣ or GPR40 siRNA conditions). PAECs were transfected with specific siRNA or scrambled CTRL siRNA, as indicated. At 48 h after transfection, cells were treated with RGZ (10 M) or vehicle CTRL for 1 h prior to the extraction of nuclear protein for immunoprecipitation with phosphothreonine and phosphoserine antibodies. Phospho-PGC1␣ (pPGC1␣) in immunoprecipitates was detected by Western blotting with anti-PGC1␣. B, RGZ induced PGC1␣ protein expression in PAECs, an effect blocked by siRNA knockdown of p38␣ MAPK or GPR40 (p ϭ 0.01 for RGZ versus CTRL within CTRL siRNA; p Ն 0.62, RGZ versus CTRL within p38␣ or GPR40 siRNA). Cells were transfected as in A. At 48 h after transfection, cells were treated with RGZ (10 M) or vehicle CTRL for 4 h prior to the extraction of nuclear protein for measurement of PGC1␣ by Western blotting. C, RGZ slightly decreased (p Ͻ 0.001 for RGZ versus CTRL within CTRL siRNA), but SIRT1 siRNA increased PGC1␣ acetylation in PAECs (p Ͻ 0.001 for SIRT1 siRNA versus CTRL siRNA in the absence and presence of RGZ). At 48 h after transfection, PAECs were treated with RGZ (10 M) or vehicle CTRL for 4 h prior to the extraction of whole cell lysates for immunoprecipitation with anti-acetyl-lysine antibodies. Acetyl-PGC1␣ (Ac-PGC1␣) in immunoprecipitates was detected by Western blotting with anti-PGC1␣. D, siRNA knockdown of PGC1␣ or SIRT1 inhibited RGZ-induced PPAR␥ target genes in PAECs (p Ͻ 0.001 for RGZ versus RGZ plus PGC1␣ or SIRT1 siRNA for CD36, CYP1A1, and FABP4). Results are presented as geometric means Ϯ S.E. (error bars) of five independent experiments using different PAEC donors. E and F, specific siRNA for PGC1␣ and SIRT1 reduced their protein expression in PAECs, respectively (p Յ 0.001, main effects of PGC1␣ and SIRT1 siRNA). Cells were transfected with siRNA for PGC1␣ or SIRT1 or scrambled CTRL siRNA. At 48 h after transfection, PAECs were treated with RGZ (10 M) or vehicle CTRL for 24 h prior to isolation of total RNA for measurement of PPAR␥ target genes by real-time PCR or for 4 h prior to the preparation of nuclear protein or whole cell lysates for measurement of PGC1␣ and SIRT, respectively, by Western blotting. Densitometry results of independent experiments using different PAEC donors (top panels) and a representative blot (bottom panels) are shown in A and B (n ϭ 4), C (n ϭ 5), E (n ϭ 3), and F (n ϭ 4). phosphorylation in EA.hy926 cells (p ϭ 0.01 for RGZ versus control within the control condition; Fig. 6D), an activating event blocked by either p38 MAPK (SB) or GPR40 (GW1100) inhibition (p Ն 0.19 for RGZ versus control in the presence of SB or GW1100; Fig. 6D). Specific siRNA silencing of EP300 inhibited both basal and RGZ-induced PPRE reporter activity in EA.hy926 cells (p Ͻ 0.001 for EP300 siRNA main effect; p ϭ 0.53 for an interaction between EP300 siRNA and RGZ; Fig. 6E). Likewise, siRNA knockdown of EP300 in PAECs significantly inhibited RGZ-induced PPAR␥ target genes, CD36, CYP1A1, and FABP4 (p Ͻ 0.001 for all three, RGZ versus RGZ plus EP300 siRNA; Fig. 6F). Similar to the knockdown of other GPR40 pathway components, EP300 silencing markedly interfered with the ability of RGZ/PPAR␥ signaling to induce CD36, CYP1A1, and FABP4 ( Fig. 6F and Table  1). Efficient knockdown of EP300 mRNA expression was achieved in both EA.hy926 cells and PAECs (p Ͻ 0.001 for both; Fig. 6G).
RGZ-induced Binding of PPAR␥, PGC1␣, and EP300 to the Proximal CD36 Promoter; Dependence on p38 MAPK and GPR40 -PGC1␣ and EP300 remodel and open chromatin for active transcription. Therefore, RGZ/GPR40/p38 MAPK signaling may be critical for certain genes and in some cell types for PPAR␥ transcriptional activation. ChIP assays in human PAECs demonstrated that RGZ increased the binding of endogenous PPAR␥ (p Ͻ 0.001), PGC1␣ (p ϭ 0.004), and EP300 (p ϭ 0.04) to native PPREs in the proximal CD36 promoter (comparing RGZ with control in the absence of SB or GW1100; Fig. 6H). These effects on PPAR␥, PGC1␣, and EP300 binding were all abolished by either p38 MAPK (SB) or GPR40 (GW1100) inhibition (p Ն 0.18 for RGZ versus control in the presence of SB or GW1100; p Յ 0.03 for interactions between RGZ and SB or GW1100 on the binding of PPAR␥, PGC1␣, and EP300; Fig.  6H).

Discussion
Our results demonstrate that GPR40 and PPAR␥ can function together as an integrated two-receptor signal transduction pathway. Besides the direct activation of its canonical receptor PPAR␥, RGZ also required GPR40 to optimally propagate a PPAR␥ nuclear signal in human endothelium (Fig. 7). GPR40 and PPAR␥ appeared to function at least additively and sometimes synergistically to initiate PPAR␥ genomic responses, depending on the transcriptional context. This conclusion is based on the following: 1) RGZ activated p38 MAPK; 2) PPAR␥ DNA binding and reporter activity was at least partially p38 MAPK-dependent; 3) GPR40 inhibition or knockdown blocked RGZ activation of p38 MAPK; 4) inhibition of GPR40 signaling, including use of an antagonist, GPR40 silencing, or expression of a DN-G␣ q mutant, suppressed, whereas GPR40 overexpression further increased, RGZ-induced PPRE reporter activity; 5) RGZ activation of p38 MAPK and the PPRE reporter was independent of ⅐ NO synthase, and ⅐ NO activation of p38 MAPK was likewise GPR40-independent; and importantly, 6) in human primary PAECs, knockdown of p38 MAPK or GPR40 substantially reduced the ability of RGZ to induce PPAR␥ target genes.
In addition to these findings, RGZ treatment of PAECs increased the phosphorylation and expression of PGC1␣, a key co-activator of PPAR␥ (50). Knockdown of either p38␣ MAPK or GPR40 abolished these effects of RGZ on PGC1␣. RGZ also modestly decreased PGC1␣ acetylation, an activating event; knockdown of SIRT1, a deacetylase, eliminated this effect and increased PGC1␣ acetylation. As expected, given its essential role in the formation of a PPAR␥ transcriptional activation complex (50), knockdown of PGC1␣ or its essential activator SIRT1 inhibited RGZ-induced PPAR␥ target gene expression in PAECs. Finally, EP300, an acetylase that docks with PGC1␣ and remodels chromatin to optimize the transcription of PPAR␥ target genes (50,53), was also phosphorylated and activated by p38 MAPK. Like PGC1␣, EP300 appeared to play an important downstream role in RGZ/GPR40/p38 MAPK modulation of PPAR␥ signaling. Collectively, these experiments demonstrate that p38 MAPK, PGC1␣, and EP300 link GPR40 to downstream PPAR␥ genomic signaling. Binding to and activating both GPR40 and PPAR␥ appears to be a common feature of several PPAR␥ agonists (29 -34). This direct connection between GPR40 signaling and PPAR␥ transcriptional activation argues that the effects of these ligands on human endothelium might be best understood as a cognate two-receptor system, integrated by p38 MAPK, PGC1␣, and EP300.
Activation of p38 MAPK increases transcription of PPAR␥regulated genes in endothelial cells (39) and adipocytes (20,21,54). TZDs (20,(55)(56)(57) have been long known to activate p38 MAPK independent of PPAR␥ in various cell types, including adipocytes, astrocytes, and epithelial cells. However, the potential role of GPR40 was not appreciated at the time of these early studies, and the underlying mechanisms seemed to be cell typedependent (20,(55)(56)(57). Reactive oxygen species were implicated in astrocytes (55) and adipocytes (20), whereas endoplasmic reticulum stress was implicated in liver epithelial cells (57). Here, RGZ activation of p38 MAPK was directly tied to GPR40 in human endothelium. Both the GPR40 antagonist GW1100 and GPR40 gene silencing significantly blocked RGZ-induced p38 MAPK phosphorylation. This finding is consistent with recent reports that TZDs bind to and activate GPR40 in bron- FIGURE 6. CBP/EP300 participation in RGZ/GPR40/p38 MAPK/PGC1␣/PPAR␥ signaling. A-C, overexpression of PGC1␣, CBP/EP300, or both enhanced PPAR␥ reporter gene activity. In A, PGC1␣ overexpression increased PPRE reporter activity in the absence of RGZ (p Ͻ 0.001 for a plasmid dose effect); the PGC1␣ effect became smaller in the presence of RGZ (p Ͻ 0.001 for a negative interaction between RGZ and PGC1␣). In B, EP300 overexpression also increased both basal and RGZ-induced PPRE reporter activity (p ϭ 0.003 for the main effect; p ϭ 0.68 for an interaction between RGZ and EP300 plasmid). In C, PGC1␣ and EP300 additively enhanced PPRE reporter activity in the absence and presence of RGZ (p Ͻ 0.001 for PGC1␣ and EP300 main effects; p ϭ 0.77 for an interaction between PGC1␣ and EP300). EA.hy926 cells were co-transfected with PPRE-LUC reporter; a PPAR␥ 2 expression plasmid; and either a PGC1␣ expression plasmid, an EP300 expression plasmid, or both, as indicated. DNA amounts were balanced with the empty vector pcDNA3.1 plasmid. At 24 h after transfection, cells were treated with RGZ (10 M) or vehicle CTRL for 24 h prior to the measurement of luciferase activity. D, RGZ induced EP300 phosphorylation in EA.hy926 cells (p ϭ 0.01 for RGZ versus CTRL within CTRL), an effect blocked by SB, a specific p38 MAPK inhibitor, or GW1100, a specific GPR40 antagonist (p Ն 0.19 for RGZ versus CTRL with SB or GW1100). Cells were treated with SB (10 M), GW1100 (20 M), or vehicle CTRL for 40 min and then incubated with RGZ (10 M) or vehicle CTRL for another 15 min prior to the extraction of whole cell lysates for measurement of phosphorylated and total EP300 by Western blotting. Densitometry results of three independent experiments and a representative blot are shown. E, siRNA knockdown of EP300 inhibited PPAR␥ reporter gene activity (p Ͻ 0.001 for EP300 siRNA main effect; p ϭ 0.53 for an interaction between EP300 siRNA and RGZ). EA.hy926 cells were first transfected with EP300 siRNA or scrambled CTRL siRNA for 48 h and then co-transfected with PPRE-LUC reporter and a PPAR␥ 2 expression plasmid for 24 h, followed by an additional 24-h treatment of RGZ (10 M) or vehicle CTRL prior to the measurement of luciferase activity. F, EP300 siRNA inhibited RGZ-induced PPAR␥ target genes (p Ͻ 0.001 for RGZ versus RGZ plus EP300 siRNA for all three genes) in PAECs. G, EP300 siRNA reduced EP300 mRNA in EA.hy926 cells and PAECs (p Ͻ 0.001 for the main effect of EP300 siRNA in both cell types). Cells were transfected with EP300 siRNA or scrambled CTRL siRNA for 48 h and then treated with RGZ (10 M) or vehicle CTRL for 24 h prior to isolation of total RNA for measurement of PPAR␥ target genes and EP300 mRNA by real-time PCR. H, RGZ increased the binding of PPAR␥ (p Ͻ 0.001), PGC1␣ (p ϭ 0.004), and EP300 (p ϭ 0.04) to PPRE in the CD36 promoter (RGZ versus CTRL within the CTRL condition), an effect blocked by SB or GW1100 (p Ն 0. chial epithelial cells (32), osteocytes (33), and GPR40-transfected HEK293 cells (31,58), causing the rapid phosphorylation of p38 MAPK (31,33,58).
Like phosphorylation, reversible acetylation is another key modulator of PGC1␣ (24,25). So far, only two proteins have been unequivocally shown to regulate the reversible acetylation of PGC1␣, the acetyltransferase GCN5 (23) and the NAD ϩ -dependent deacetylase SIRT1 (24,25). GCN5 acetylates and inhibits PGC1␣ activity (23), whereas SIRT1 deacetylates and enhances PGC1␣ activity and in turn induces transcription of its target genes (24,25). Overexpression of Sirt1 in the liver of mice induces gluconeogenic genes under the control of PGC1␣, whereas Sirt1 knockdown attenuates this effect (60). Also, in skeletal muscle, Sirt1 is required for PGC1␣-mediated induction of the fatty acid oxidation pathway (61). In the present study, RGZ was seen to modestly decrease PGC1␣ acetylation in endothelial cells; SIRT1 knockdown eliminated this effect, increasing PGC1␣ acetylation. These results suggest that PGC1␣ phosphorylation might facilitate its deacetylation by SIRT1. It was previously reported that JNK1 phosphorylates SIRT1 and promotes its enzymatic activity in HEK293T cells (62); p38 MAPK has been reported to increase SIRT1 expression in neurons (63) and decrease it in chondrocytes (64). In our study, RGZ did not affect either SIRT1 phosphorylation or expression in endothelial cells. However, consistent with its essential role in activating PGC1␣ via deacetylation, SIRT1 knockdown significantly inhibited RGZ-induced PPAR␥ target gene expression in endothelial cells.
Although the effects of RGZ/PPAR␥ and RGZ/GPR40 were mostly additive in PPRE reporter assays, the dual activation of both receptors appeared interdependent and synergistic at PPAR␥-regulated target genes in the chromatin microenvironment (see Table 1 for a summary of interactions between RGZ and GPR40 pathway siRNA silencing). Across all three PPAR␥regulated genes combined, interaction testing between RGZ and each siRNA target indicated that RGZ activation of GPR40 and PPAR␥ function synergistically (Table 1). Likewise, across all siRNA silencing combined, each PPAR␥-regulated gene, CD36 (p Ͻ 0.001), CYP1A1 (p ϭ 0.001) and FABP4 (p Ͻ 0.001), demonstrated significant interactions between RGZ-induced PPAR␥ responses and signaling through the GPR40 pathway (Table 1). Largely additive effects on a PPRE reporter plasmid and evidence for GPR40 and PPAR␥ interdependence at endogenous genes might be explained by the ability of the proposed signaling cascade to actively remodel chromatin. Consistent with this concept, p38 MAPK or GPR40 inhibition both markedly blocked the RGZ-induced binding of PPAR␥, PGC1␣, and EP300 to a PPRE-rich site in the proximal promoter of CD36, a prototypic PPAR␥ target gene.
TZDs, synthetic ligands of PPAR␥, including RGZ, ciglitazone, troglitazone, and pioglitazone, have all been shown to activate GPR40 with subsequent signal transduction through stress kinases (29 -34). RGZ compared with pioglitazone (two FIGURE 7. Proposed GPR40 and PPAR␥ integrated signal transduction pathway in human endothelial cells. In the classical PPAR␥ signaling pathway, RGZ binds directly to PPAR␥, inducing a conformational change that results in its dissociation from co-repressors (not depicted), such as nuclear co-repressor and histone deacetylases, and the recruitment of co-activators, including PGC1␣ and EP300. However, as shown here, RGZ and other PPAR␥ ligands also bind to and activate GPR40 on the cell surface, resulting in p38 MAPK phosphorylation, which in turn phosphorylates and thereby activates both PGC1␣ and EP300. As noted, PGC1␣ deacetylation by SIRT1 is also essential for its activation. Phosphorylation releases PGC1␣ from its repressor p160 MBP and leads to a conformational change, permitting PGC1␣ to dock with PPAR␥ and recruit newly activated EP300. EP300 is a histone acetyltransferase that remodels local chromatin and enhances gene transcription. The activated PPAR␥ complex thus heterodimerizes with retinoid X receptor, which binds to peroxisome proliferator response elements in accessible promoters, inducing the transcription of target genes.
TZDs used to treat type 2 diabetes mellitus) produces a more potent and prolonged activation of ERK1/2 (31), a stress kinase pathway associated with vascular inflammation (65). These differences in the potency and sustainability of ERK1/2 activation could potentially explain some of the efficacy and safety differences among existing synthetic PPAR␥ ligands. Different from TZDs, 15-deoxy-⌬12,14-prostaglandin J2, a natural PPAR␥ ligand (27), did not appear to activate GPR40 in bronchial epithelial cells (32). Conversely, agonists selective for GPR40 have been described with little or no effect on PPAR␥ activity (29,66). Therefore, it may be possible to design drugs that activate PPAR␥ independently of GPR40 or selectively activate GPR40/ p38 MAPK while circumventing GPR40/ERK activation. Such selective agents or biased ligands (67,68) might arguably be less inflammatory and thus have better risk/benefit profiles in patients with vascular disease. In addition, unexplored effects through other unidentified cognate GPR and nuclear receptor pairs, as exemplified by GPR40/PPAR␥, could explain important safety and efficacy differences among nuclear receptordirected drugs.
Author Contributions-S. W. and R. L. D. conceived the study, designed experiments, analyzed and interpreted data, and wrote the paper. S. W. performed transfections, Western blots, PPAR␥ target gene real-time PCR experiments, and the chromatin immunoprecipitation assays. K. S. A. identified the role played by EP300, helped design key experiments related to acetylation, maintained the pulmonary artery endothelial cell cultures, and revised drafts of the manuscript. J. M. E., E. J. D., and G. A. F. contributed to the scientific concept, provided technical advice, raised critical questions, and revised drafts of the manuscript. E. J. D. also lent important expertise in nuclear receptor signaling. J. Y. W. performed the experiments shown in Fig. 2, B-D. A. P. started the study and performed the experiments shown in Fig. 1. R. C. and J. S. performed the statistical analyses. All authors read, edited, and approved the final version of the manuscript.