PPARβ/δ Augments IL-1β-Induced COX-2 Expression and PGE2 Biosynthesis in Human Mesangial Cells via the Activation of SIRT1

Peroxisome proliferator-activated receptor β/δ (PPARβ/δ), a ligand-activated nuclear receptor, regulates lipid and glucose metabolism and inflammation. PPARβ/δ can exert an anti-inflammatory effect by suppressing proinflammatory cytokine production. Cyclooxygenase-2 (COX-2)-triggered inflammation plays a crucial role in the development of many inflammatory diseases, including glomerulonephritis. However, the effect of PPARβ/δ on the expression of COX-2 in the kidney has not been fully elucidated. The present study showed that PPARβ/δ was functionally expressed in human mesangial cells (hMCs), where its expression was increased by interleukin-1β (IL-1β) treatment concomitant with enhanced COX-2 expression and prostaglandin E2 (PGE2) biosynthesis. The treatment of hMCs with GW0742, a selective agonist of PPARβ/δ, or the overexpression of PPARβ/δ via an adenovirus-mediated approach significantly increased COX-2 expression and PGE2 production. PPARβ/δ could further augment the IL-1β-induced COX-2 expression and PGE2 production in hMCs. Moreover, both PPARβ/δ activation and overexpression markedly increased sirtuin 1 (SIRT1) expression. The inhibition or knockdown of SIRT1 significantly attenuated the effects of PPARβ/δ on the IL-1β-induced expression of COX-2 and PGE2 biosynthesis. Taken together, PPARβ/δ could augment the IL-1β-induced COX-2 expression and PGE2 production in hMCs via the SIRT1 pathway. Given the critical role of COX-2 in glomerulonephritis, PPARβ/δ may represent a novel target for the treatment of renal inflammatory diseases.

Glomerulonephritis represents the leading cause of kidney failure among east Asian populations [24,25]. It is generally believed that glomerular mesangial cells (MCs) play an important role in the pathogenesis of glomerulonephritis [26,27]. As specialized, smooth muscle cells, mesangial cells support the capillaries of the renal glomerulus and regulate the glomerular filtration rate. Moreover, MCs have many other functions, including the generation of prostaglandins (PGs), which serve as major mediators of inflammation. Cyclooxygenases (COXs) are key enzymes in the conversion of arachidonic acid to PGs and other eicosanoids. Thus far, two COX subtypes have been identified, i.e., a constitutive one named COX-1 and an inducible one called COX-2. Classically, COX-2 is induced in inflammatory conditions with significant increase in the production of cyclooxygenase-derived prostaglandin E2, a well-known dominant proinflammatory lipid mediator [16,28,29]. In a rat model of mesangioproliferative glomerulonephritis, selective COX-2 inhibition by either rofecoxib or celecoxib led to more profound mesangiolysis and albuminuria [30]. Moreover, several studies have reported that, in cultured MCs of rodents and humans, COX-2 expression and prostaglandin synthesis could be stimulated by IL-1β, a critical proinflammatory cytokine that plays an important role in glomerular inflammation and injury [31][32][33][34].
Both in vitro and in vivo studies have clearly shown that the nuclear receptor PPARβ/δ plays an important role in the progression of inflammatory diseases, and the activation of PPARβ/δ can upregulate the expression of COX-2 in tumor cells [35,36]. To date, the role of PPARβ/δ in glomerular inflammation is not clear. Whether PPARβ/δ is involved in glomerular COX-2 expression and PGE2 production in mesangial cells also remain largely unknown. The current study aims to clarify the potential role and the underlying mechanism of PPARβ/δ in IL-1β-induced COX-2 expression and PGE2 biosynthesis in cultured human mesangial cells.

PPARβ/δ Is Functionally Expressed in hMCs
The expression of PPARβ/δ mRNA and protein was evident as assessed by qRT-PCR and Western blot assays ( Figure 1A,B). An immunofluorescence study further demonstrated that PPARβ/δ protein was mainly localized in the nuclei of hMCs, with little expression in the cytoplasm ( Figure 1C). A PPRE-luciferase reporter assay further indicated that the PPARβ/δ-specific agonist GW0742 significantly increased the activity of the PPREdriven luciferase reporter by~five-fold, suggesting that the hMCs had functional PPARβ/δ transcriptional activity ( Figure 1D). Collectively, these results revealed that PPARβ/δ was constitutively expressed and functional in hMCs. The nuclear localization of PPARβ/δ in human mesangial cells as assessed by immunofluorescence. Red color represents PPARβ/δ immunoreactivity. The nuclei were stained with DAPI in blue. IgG was used as a negative control. (D) The effect of the PPARβ/δ agonist GW0742 on PPRE-luciferase activity. The cells were cotransfected with PPRE-luciferase plasmid and β-galactosidase vector for 24 h, followed by treatment with GW0742 (1 μM) for 6 h. The luciferase activity was measured as described in the experimental procedure. ** p < 0.01 compared to DMSO; n = 3. PPARβ: PPARβ/δ.

PPARβ/δ Increases COX-2 Expression and PGE2 Production in hMCs
Previous studies have demonstrated that PPARβ/δ activation upregulates COX-2 expression in tumor cells [37,38]. However, the effect of PPARβ/δ on COX-2 expression in other cell types remains unknown. In the present study, we found that the treatment of hMCs with GW0742, a specific PPARβ/δ agonist, dose-dependently upregulated COX-2 protein expression with little effect on COX-1 expression (Figure 2A). Consistent with the increased COX-2 expression, the PGE2 production was also significantly increased by the GW0742 treatment ( Figure 2B). Meanwhile, GW0742 increased the COX-2 expression and PGE2 production in a time-dependent manner ( Figure 2C,D). To further confirm the effect of PPARβ/δ on COX-2 expression, PPARβ/δ was overexpressed in the hMCs via an adenovirus-based approach. The results showed that PPARβ/δ overexpression also significantly increased the COX-2 protein levels and PGE2 release ( Figure 2E,F). Together, these results strongly indicated that PPARβ/δ can increase the expression of COX-2 and the production of PGE2 in human mesangial cells. The nuclear localization of PPARβ/δ in human mesangial cells as assessed by immunofluorescence. Red color represents PPARβ/δ immunoreactivity. The nuclei were stained with DAPI in blue. IgG was used as a negative control. (D) The effect of the PPARβ/δ agonist GW0742 on PPRE-luciferase activity. The cells were cotransfected with PPRE-luciferase plasmid and β-galactosidase vector for 24 h, followed by treatment with GW0742 (1 µM) for 6 h. The luciferase activity was measured as described in the experimental procedure. ** p < 0.01 compared to DMSO; n = 3. PPARβ: PPARβ/δ.

PPARβ/δ Increases COX-2 Expression and PGE2 Production in hMCs
Previous studies have demonstrated that PPARβ/δ activation upregulates COX-2 expression in tumor cells [37,38]. However, the effect of PPARβ/δ on COX-2 expression in other cell types remains unknown. In the present study, we found that the treatment of hMCs with GW0742, a specific PPARβ/δ agonist, dose-dependently upregulated COX-2 protein expression with little effect on COX-1 expression (Figure 2A). Consistent with the increased COX-2 expression, the PGE2 production was also significantly increased by the GW0742 treatment ( Figure 2B). Meanwhile, GW0742 increased the COX-2 expression and PGE2 production in a time-dependent manner ( Figure 2C,D). To further confirm the effect of PPARβ/δ on COX-2 expression, PPARβ/δ was overexpressed in the hMCs via an adenovirus-based approach. The results showed that PPARβ/δ overexpression also significantly increased the COX-2 protein levels and PGE2 release ( Figure 2E,F). Together, these results strongly indicated that PPARβ/δ can increase the expression of COX-2 and the production of PGE2 in human mesangial cells.

IL-1β Induces PPARβ/δ Expression in hMCs
Given the important role of PPARβ/δ in the regulation of inflammation in many cell types, it is reasonable to hypothesize that PPARβ/δ may also be involved in IL-1β-induced inflammatory process in hMCs. To explore the effect of IL-1β on PPARβ/δ expression, the hMCs were stimulated with various concentrations of IL-1β for 12 h. The results showed that IL-1β significantly increased both the mRNA and protein expressions of PPARβ/δ in a dose-dependent manner ( Figure 3A,B). Unlike PPARβ/δ, the PPARα and PPARγ protein levels remained unaffected by the IL-1β treatment ( Figure 3B). Moreover, IL-1β (5 ng/mL) significantly upregulated the PPARβ/δ expression in a time-dependent manner ( Figure 3C,D). These findings demonstrated that IL-1β can induce PPARβ/δ expression in hMCs. The cells were incubated with IL-1β (5 ng/mL) for the indicated time periods. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control (0 ng/mL or 0 h); n = 4. PPARβ: PPARβ/δ.

PPARβ/δ Activation Augments IL-1β-Induced COX-2 Expression and PGE2 Production in hMCs
To further explore the role of PPARβ/δ in IL-1β-induced COX-2 expression and PGE2 production, human mesangial cells were pretreated with or without GW0742 (1 μM) for 6 h, followed by stimulation with IL-1β (5 ng/mL) for 12 h. The pretreatment of the cells with GW0742 significantly augmented the IL-1β-induced COX-2 mRNA expression compared with GW0742 or IL-1β treatment alone ( Figure 4A). Although GW0742 or IL-1β treatment alone could increase the COX-2 protein levels, the pretreatment with GW0742 further augmented the IL-1β-induced COX-2 protein expression ( Figure 4B,C). Consistently, GW0742 also enhanced the IL-1β-induced PGE2 production in the media of the cultured cells ( Figure 4D). Together, these results indicate that PPARβ/δ activation increased the COX-2 expression and activity both under the basal condition and following the IL-1β treatment.

PPARβ/δ Activation Augments IL-1β-Induced COX-2 Expression and PGE2 Production in hMCs
To further explore the role of PPARβ/δ in IL-1β-induced COX-2 expression and PGE2 production, human mesangial cells were pretreated with or without GW0742 (1 µM) for 6 h, followed by stimulation with IL-1β (5 ng/mL) for 12 h. The pretreatment of the cells with GW0742 significantly augmented the IL-1β-induced COX-2 mRNA expression compared with GW0742 or IL-1β treatment alone ( Figure 4A). Although GW0742 or IL-1β treatment alone could increase the COX-2 protein levels, the pretreatment with GW0742 further augmented the IL-1β-induced COX-2 protein expression ( Figure 4B,C). Consistently, GW0742 also enhanced the IL-1β-induced PGE2 production in the media of the cultured cells ( Figure 4D). Together, these results indicate that PPARβ/δ activation increased the COX-2 expression and activity both under the basal condition and following the IL-1β treatment.

PPARβ/δ Increases the Expression of SIRT1 in hMCs
It was reported that SIRT1 activation increased renal medullary interstitial cell COX-2 expression both in vitro and in vivo, while SIRT1 deficiency-attenuated oxidative stressinduced COX-2 expression in cultured mouse renal medullary interstitial cells [37]. In addition, PPARβ/δ was found to increase SIRT1 gene transcription via a canonical Sp1 binding site [39]. Based on these findings, we hypothesized that a PPARβ/δ-SIRT1-COX-2 axis may exist in human mesangial cells. In fact, we observed a stimulatory effect of PPARβ/δ on SIRT1 expression in hMCs. Following the treatment of hMCs with 1 μM GW0742 for 6 h, the mRNA and protein expressions of SIRT1 were significantly increased ( Figure 5A,B). Similarly, adenovirus-mediated PPARβ/δ overexpression also markedly increased the SIRT1 expression at both the mRNA and protein levels ( Figure 5C,D). These results strongly indicated that PPARβ/δ can elevate SIRT1 mRNA and protein expressions in human mesangial cells.

PPARβ/δ Increases the Expression of SIRT1 in hMCs
It was reported that SIRT1 activation increased renal medullary interstitial cell COX-2 expression both in vitro and in vivo, while SIRT1 deficiency-attenuated oxidative stressinduced COX-2 expression in cultured mouse renal medullary interstitial cells [37]. In addition, PPARβ/δ was found to increase SIRT1 gene transcription via a canonical Sp1 binding site [39]. Based on these findings, we hypothesized that a PPARβ/δ-SIRT1-COX-2 axis may exist in human mesangial cells. In fact, we observed a stimulatory effect of PPARβ/δ on SIRT1 expression in hMCs. Following the treatment of hMCs with 1 µM GW0742 for 6 h, the mRNA and protein expressions of SIRT1 were significantly increased ( Figure 5A,B). Similarly, adenovirus-mediated PPARβ/δ overexpression also markedly increased the SIRT1 expression at both the mRNA and protein levels ( Figure 5C,D). These results strongly indicated that PPARβ/δ can elevate SIRT1 mRNA and protein expressions in human mesangial cells.

SIRT1 Inhibition Blocks the Stimulatory Effect of PPARβ/δ on IL-1β-Induced COX-2 Expression and PGE2 Production in hMCs
To investigate the role of SIRT1 in the effect of PPARβ/δ on the IL-1β-induced upregulation of COX-2, two SIRT1 inhibitors, including NIC (niconinamide) and EX527, were used. The cells were pretreated with GW0742 (1 μM) in the presence or absence of the SIRT1 inhibitor NIC (niconinamide, 5 mM) or EX527 (1 μM) for 6 h, followed by treatment with IL-1β (5 ng/mL) for 12 h. We found that pretreatment with either NIC or EX527 decreased the COX-2 mRNA expression induced by IL-1β and GW0742 ( Figure  6A). Compared with the IL-1β + GW0742 group, the protein abundance of COX-2 was decreased to 43% and 30% in the IL-1β + GW0742 + NIC group and the IL-1β + GW0742+ EX527 group, respectively ( Figure 6B,C). Consistently, PGE2 production in the media of the NIC and EX527 treatment groups was significantly reduced compared with the group

SIRT1 Inhibition Blocks the Stimulatory Effect of PPARβ/δ on IL-1β-Induced COX-2 Expression and PGE2 Production in hMCs
To investigate the role of SIRT1 in the effect of PPARβ/δ on the IL-1β-induced upregulation of COX-2, two SIRT1 inhibitors, including NIC (niconinamide) and EX527, were used. The cells were pretreated with GW0742 (1 µM) in the presence or absence of the SIRT1 inhibitor NIC (niconinamide, 5 mM) or EX527 (1 µM) for 6 h, followed by treatment with IL-1β (5 ng/mL) for 12 h. We found that pretreatment with either NIC or EX527 decreased the COX-2 mRNA expression induced by IL-1β and GW0742 ( Figure 6A). Compared with the IL-1β + GW0742 group, the protein abundance of COX-2 was decreased to 43% and 30% in the IL-1β + GW0742 + NIC group and the IL-1β + GW0742+ EX527 group, respectively ( Figure 6B,C). Consistently, PGE2 production in the media of the NIC and EX527 treatment groups was significantly reduced compared with the group without treatment with the SIRT1 inhibitors ( Figure 6D). Together, these findings demonstrated that the augmentation of PPARβ/δ on IL-1β-induced COX-2 expression and PGE2 production is mediated by SIRT1.
demonstrated that the augmentation of PPARβ/δ on IL-1β-induced COX-2 expression and PGE2 production is mediated by SIRT1.

SIRT1 Knockdown Blocks the Effect of PPARβ/δ on IL-1β-Induced COX2 Expression and PGE2 Production in hMCs
To further support the finding that SIRT1 mediated the stimulatory effect of PPARβ/δ on the IL-1β-induced COX-2 expression, the cells infected with Ad-siSIRT1 (10 MOI) or Ad-siControl (10 MOI) were pretreated with GW0742 (1 μM) for 6 h and then treated with IL-1β (5 ng/mL) for 12 h. As expected, the Ad-siSIRT1 treatment markedly decreased the SIRT1 protein expression. Similar to the findings on SIRT1 inhibition, the knockdown of the SIRT1 expression significantly attenuated the GW0742-and IL-1β-induced COX-2 expression at both the mRNA ( Figure 7A) and protein ( Figure 7B,C) levels. The effects of GW4064 and IL-1β on PGE2 production were also markedly reduced ( Figure 7D). Together, these results further demonstrated that PPARβ/δ enhances the IL-1β-induced COX-2 expression and PGE2 production in hMCs via a SIRT1-dependent pathway.

SIRT1 Knockdown Blocks the Effect of PPARβ/δ on IL-1β-Induced COX2 Expression and PGE2 Production in hMCs
To further support the finding that SIRT1 mediated the stimulatory effect of PPARβ/δ on the IL-1β-induced COX-2 expression, the cells infected with Ad-siSIRT1 (10 MOI) or Ad-siControl (10 MOI) were pretreated with GW0742 (1 µM) for 6 h and then treated with IL-1β (5 ng/mL) for 12 h. As expected, the Ad-siSIRT1 treatment markedly decreased the SIRT1 protein expression. Similar to the findings on SIRT1 inhibition, the knockdown of the SIRT1 expression significantly attenuated the GW0742-and IL-1β-induced COX-2 expression at both the mRNA ( Figure 7A) and protein ( Figure 7B,C) levels. The effects of GW4064 and IL-1β on PGE2 production were also markedly reduced ( Figure 7D). Together, these results further demonstrated that PPARβ/δ enhances the IL-1β-induced COX-2 expression and PGE2 production in hMCs via a SIRT1-dependent pathway.

Discussion
PPARβ/δ, a ligand-activated nuclear receptor, plays a critical role in regulating lipid and glucose metabolism. Growing evidence also has indicated that PPARβ/δ can exert an anti-inflammatory effect by suppressing proinflammatory cytokine production [38,40]. However, whether PPARβ/δ also suppresses the inflammatory process in glomerulonephritis remains unclear. The present study demonstrated that PPARβ/δ was constitutively expressed in human glomerular mesangial cells, where it promoted COX-2 expression and PGE2 production both under the basal condition and after IL-1β treatment. We further revealed that the stimulatory effect of PPARβ/δ on the IL-1βinduced COX-2 expression was mediated by the activation of SIRT1, a highly conserved NAD(+)-dependent deacetylase. Collectively, our findings indicated that PPARβ/δ may enhance the IL-1β-mediated activation of COX-2 to promote PGE2 production in glomerular mesangial cells.

Discussion
PPARβ/δ, a ligand-activated nuclear receptor, plays a critical role in regulating lipid and glucose metabolism. Growing evidence also has indicated that PPARβ/δ can exert an anti-inflammatory effect by suppressing proinflammatory cytokine production [38,40]. However, whether PPARβ/δ also suppresses the inflammatory process in glomerulonephritis remains unclear. The present study demonstrated that PPARβ/δ was constitutively expressed in human glomerular mesangial cells, where it promoted COX-2 expression and PGE2 production both under the basal condition and after IL-1β treatment. We further revealed that the stimulatory effect of PPARβ/δ on the IL-1β-induced COX-2 expression was mediated by the activation of SIRT1, a highly conserved NAD(+)-dependent deacetylase. Collectively, our findings indicated that PPARβ/δ may enhance the IL-1β-mediated activation of COX-2 to promote PGE2 production in glomerular mesangial cells.
As a nuclear receptor transcriptional factor, PPARβ/δ is involved in many physiological and pathological processes [1][2][3]. Increasing evidence has suggested that PPARβ/δ plays an important role in the regulation of inflammatory responses [10][11][12]. PPARβ/δ agonists have been shown to induce the inducible nitric oxide synthase (iNOS) expression in mesangial cells [49] and to upregulate the expression of COX-2 in colon cancer cells [9,50]. Consistent with our finding, Kim et al. previously reported that interleukin 1 (IL-1) increased PPARβ/δ levels in Hep3B human hepatoma cells [51]. However, they found a significant decrease in the expressions of PPARα and PPARγ. Currently, the reason for this discrepancy is not clear. One possible explanation is that the effect of IL-1β on the expression of each PPAR isoform is cell-type-and context-dependent. These observations suggest that PPARβ/δ may also be involved in the regulation of inflammatory processes in mesangial cells. This speculation is supported by our findings that PPARβ/δ was functionally expressed in human glomerular mesangial cells, where its activation and overexpression markedly enhanced basal and IL-1β-induced COX-2 expression and PGE2 production, which may accelerate glomerulonephritis by promoting the biosynthesis of the proinflammatory chemokines and cytokines responsible for the recruitment of leukocytes from circulation to the glomeruli. To date, the role of PPARβ/δ in the inflammatory process remains controversial. Both pro-and anti-inflammatory activities have been reported depending on the cell type and the mechanism of action. Although it is generally believed that PPARβ/δ exerts an anti-inflammatory effect in most extrarenal cell types, based on our findings, it may promote glomerular inflammation by enhancing COX-2-derived PGE2 in human mesangial cells. SIRT1, a NAD(+)-dependent deacetylase, regulates a broad range of pathogeneses of many chronic diseases, including metabolic and renal diseases [9,52]. SIRT1 can suppress the expression of multiple genes involved in the inflammatory process, including NF-κB, interleukin-6, and MCP-1 [53,54]. It has been previously reported that SIRT1 also contributes to the regulation of COX-2 expression and PGE2 production in many types of cells. SIRT1 activation could increase the COX-2 expression of renal medullary interstitial cells both in vitro and in vivo [37]. In addition, PPARβ/δ activation is capable of inducing SIRT1 expression [55,56]. These findings strongly suggest that SIRT1 might mediate the induction of COX-2 expression and PGE2 production by PPARβ/δ. As expected, we found that PPARβ/δ activation increased the SIRT1 expression, and SIRT1 inhibition or knockdown significantly attenuated the stimulatory effect of PPARβ/δ on the IL-1βinduced COX-2 expression and PGE2 production in human glomerular mesangial cells.
In the present study, we provided compelling evidence that the PPARβ/δ-SIRT1-COX2 axis may play an important role in the pathogenesis and progression of glomerulonephritis ( Figure 8). The interplay of PPARβ/δ and SIRT1 drove a vicious cycle in promoting the IL-1β-induced COX2 expression and PGE2 production in glomerular mesangial cells. Firstly, we provided clear evidence that PPARβ/δ promoted SIRT1 expression. Secondly, we were able to show that the stimulatory effect of PPARβ/δ on the IL-1β-elicited COX-2 expression was dependent on SIRT1. Thirdly, we found that IL-1β markedly upregulated PPARβ/δ expression. Finally, as previously reported, COX-2-derived PGE2 could indirectly transactivate PPARβ/δ through the PI3K/Akt signaling pathway [57]. Therefore, breaking the PPARβ/δ-SIRT1-COX-2 axis may represent an attractive option for the treatment of glomerulonephritis.

Human Mesangial Cell Culture
An established human mesangial cell (hMC) line was used in all the experiments (kindly donated by Dr. Xiongzhong Ruan, UCL Medical School, London, UK). Human MCs were immortalized by transfection with T-SV40 and H-ras oncogenes, retaining many of the morphological and physiological features of normal human MCs. The cells were cultured in RPMI-1640 medium (GIBCO, New York, NY, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO, New York, NY, USA), 2 mmol/L glutamine, 100 unit/mL penicillin, 100 g/mL streptomycin, 5 g/mL insulin, 5 g/mL human transferrin, and 5 ng/mL sodium selenite at 37 °C in the presence of 5% CO2. The cells were split weekly and the media changed twice a week.

Human Mesangial Cell Culture
An established human mesangial cell (hMC) line was used in all the experiments (kindly donated by Dr. Xiongzhong Ruan, UCL Medical School, London, UK). Human MCs were immortalized by transfection with T-SV40 and H-ras oncogenes, retaining many of the morphological and physiological features of normal human MCs. The cells were cultured in RPMI-1640 medium (GIBCO, New York, NY, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO, New York, NY, USA), 2 mmol/L glutamine, 100 unit/mL penicillin, 100 g/mL streptomycin, 5 g/mL insulin, 5 g/mL human transferrin, and 5 ng/mL sodium selenite at 37 • C in the presence of 5% CO 2 . The cells were split weekly and the media changed twice a week.

Western Blot Analysis
Drug-treated or adenovirus-infected hMCs were firstly rinsed twice with ice-cold PBS and then scraped off the six-well plates and lysed in 100 L ice-cold lysis buffer containing 150 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 7.4), 0.1% SDS, 1 mmol/L EDTA, and 1% Triton X-100. The cell lysate was centrifuged at 12,000 rpm for 5 min at 4 • C after sonication; then, the supernatant was collected. The protein content in the supernatant was quantified with a BCA (bicinchoninic acid) Protein Assay Kit (Vigorous Biotechology Beijing Co., Ltd., Beijing, China). The samples were boiled in SDS sample buffer (313 mmol/L Tris-HCl, pH 6.8, 2% SDS, 50% glycerol, 50 mmol/L dithiothreitol (DTT), and 0.05% bromophenol blue) for ten minutes. An amount of 30 µg total protein was separated using 10% SDS-PAGE, and the protein was electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon; Millipore, Bedford, MA, USA). The membranes were incubated at room temperature for 1 h in TBS-T (Tris-buffered saline containing 0.1% Tween 20) containing 5% skimmed milk for blocking nonspecific binding sites. The blocked membranes were incubated with a 1:1000 dilution of anti-β-actin or anti-COX-2 polyclonal antibodies or a 1:500 dilution of anti-COX-1, anti-SIRT1, anti-PPARα, anti-PPARβ/δ, or anti-PPARγ antibodies at 4 • C overnight. The membranes were washed for 5 min with TBS-T buffer 3-5 times and then incubated with 1:3000 horseradish peroxide (HRP)-conjugated-antirabbit or horseradish peroxide (HRP)-conjugated-anti-mouse secondary antibody at room temperature for one hour. The membranes were washed as described above. Finally, the membranes were developed with ECL. The images were analyzed and quantified with Scion Image software (Scion Corporation, Frederick, MD, USA).

Transient Transfection and Luciferase Assay
The PPARβ/δ transcriptional activity was determined with a luciferase reporter experiment. The cells were transiently cotransfected with PPRE-luciferase plasmid reporter (PPRE-luc) and β-galactosidase vector using lipofectamine-plus reagent (Gibco-BRL, Rockville, MD, USA) for 24 h, followed by different stimulations as indicated. The stimulated cells were washed twice with ice-cold phosphate-buffered saline (PBS) and then harvested in 100 µL reporter lysis buffer (Promega, Madison, WI, USA). The luciferase activities were measured using a luminometer (Auto LUMIcounter Nu1422ES; Nition, Tokyo, Japan).

Adenovirus Infection of hMCS
Subconfluent hMCs were infected with Ad-GFP, Ad-SIRT1, Ad-siControl, or Ad-siSIRT1, separately. The same doses of Ad-GFP and Ad-siControl were used as negative controls in our studies. Particularly, Ad-PPARβ/δ and Ad-TTA without 0.1 g/mL tetracycline were needed to overexpress PPARβ/δ, whereas Ad-PPARβ/δ and Ad-TTA with 0.1 g/mL tetracycline were used as controls. The infected hMCs were cultured for 24 h before the experimental assays. The hMCs were also infected with Ad-siSIRT1 for 24 h following PPARβ/δ activation.

Measurement of Prostaglandin E2 Levels
The medium of the cultured human mesangial cells in a six-well plate was collected, centrifuged, and stored at −80 • C until PGE2 measurement. The PGE2 content in the medium was measured with a PGE2 High-Sensitivity Immunoassay kit (Cayman Chemical, Ann Arbor, MI, USA). The protocol for measuring the PGE2 concentration was described in the manufacturer's directions provided with the kit. The concentration of PGE2 in each cultured well was normalized to the protein content of the cells in the corresponding well.

Immunofluorescence Staining
The hMCs were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min, followed by three washes with PBS. The fixed cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature and washed three times with PBS. The cells were then blocked with 1% bovine serum albumin (BSA) in PBS at room temperature for 10 min. To detect the PPARβ/δ protein expression, a rabbit anti-PPARβ/δ polyclonal antibody (ab8937, 1:100 dilution in 0.5% BSA) was incubated with the cells overnight at 4 • C. After washing three times with PBS, the cells were incubated with appropriate Dylight 594 (1:100) (red)-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature, followed by three washes with PBS. The nuclei were stained with DAPI (1:3000) for 5 min at room temperature. The stained cells were investigated using an Olympus BX-60 fluorescence microscope (Olympus, Tokyo, Japan).

Statistical Analysis
The data were given as means ± SD. The significance of difference was determined with an unpaired, two-tailed Student's t-test or ANOVA. p < 0.05 was considered to be statistically significant.

Conclusions
Our study demonstrated that PPARβ/δ is functionally expressed in human glomerular mesangial cells. IL-1β could induce PPARβ/δ expression in dose-and time-dependent manners accompanied by increased COX-2 expression and PGE2 production. The activation of PPARβ/δ exacerbated the IL-1β-induced COX-2 expression and PGE2 production, essentially, through inducing SIRT1 expression and activity. Our findings suggested a vicious cycle between PPARβ/δ and COX-2 in promoting proinflammatory PGE2 production, which may serve as a target for the treatment of glomerulonephritis.