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Peroxisome proliferator–activated receptor (PPAR)-γ is a ligand-activated transcription factor and regulates inflammation. Posttranslational modifications regulate the function of PPARγ, potentially affecting inflammation. PPARγ contains a mitogen-activated protein kinase (MAPK) site, and phosphorylation by extracellular signal-regulated kinase (ERK)-1/2 leads to inhibition of PPARγ. This study investigated the kinetics of PPARγ expression and activation in parenchymal and immune cells in sepsis using the MAPK/ERK kinase (MEK)-1 inhibitor, an upstream kinase of ERK1/2. Adult male Sprague Dawley rats were subjected to polymicrobial sepsis by cecal ligation and puncture. Rats received intraperitoneal injection of vehicle or the MEK1 inhibitor PD98059 (5 mg/kg) 30 min before cecal ligation and puncture. Rats were euthanized at 0, 1, 3, 6 and 18 h after cecal ligation and puncture. Control animals used were animals at time 0 h. Lung, plasma and peripheral blood mononuclear cells (PBMCs) were collected for biochemical assays. In vehicle-treated rats, polymicrobial sepsis resulted in significant lung injury. In the lung and PBMCs, nuclear levels of PPARγ were decreased and associated with an increase in phosphorylated PPARγ and phosphorylated ERK1/2 levels. Treatment with the MEK1 inhibitor increased the antiinflammatory plasma adipokine adiponectin, restored PPARγ expression in PBMCs and lung, and decreased lung injury. The inflammatory effects of sepsis cause changes in PPARγ expression and activation, in part, because of phosphorylation of PPARγ by ERK1/2. This phosphorylation can be reversed by ERK1/2 inhibition, thereby improving lung injury.
Peroxisome proliferator–activated receptor (PPAR)-γ is a ligand-activated transcription factor. Activation of PPARγ plays a role in controlling the inflammatory response. Several studies have demonstrated that activation of PPARγ by specific ligands significantly improves survival in clinically relevant models of septic shock (1–3). The beneficial effect of PPARγ activation is likely to be secondary to inhibition of the production of several inflammatory mediators, as shown in vivo in septic rodents (1–3) and in vitro in activated macrophages and monocytes (4).
Sepsis and other inflammatory states affect PPARγ expression and correlate with the inflammatory response. We have previously demonstrated that PPARγ expression is downregulated in the lung and vascular endothelium in rodent models of septic shock and that treatment with PPARγ ligands reverses the sepsis-induced reduction (1). In adipose tissue, PPARγ expression decreased after mice were challenged in vivo with endotoxin, and cytokine-induced suppression of PPARγ was reversed with synthetic agonists (5,6). However, it remains unclear what mechanisms lead to a decrease in PPARγ activity in sepsis.
Posttranslational modifications are mechanisms that regulate the function of PPARγ and may contribute to the downregulation of PPARγ in sepsis (7). The activation function (AF)-1 domain of PPARγ contains a consensus mitogen-activated protein kinase (MAPK) site, and phosphorylation by extracellular signal-regulated kinase (ERK)-1/2 at serine residue 82 (or 112 for PPARγ2) leads to inhibition of PPARγ transactivation (8,9). This phosphorylated-induced repression is due to conformational changes that can lead to altered affinity for ligands and cofactors (8,9). In addition, phosphorylation promotes degradation of PPARγ by the ubiquitin-proteasome system (10). In cultured adipocytes, using a specific ERK inhibitor reverses the reduction in PPARγ (11).
Therefore, in this study, we investigated the kinetics of altered PPARγ expression and activation in immunologic and parenchymal cells from rats subjected to polymicrobial sepsis. To gain a better understanding of the molecular mechanism by which PPARγ expression is affected, we investigated the effects of polymicrobial sepsis on the phosphorylation of PPARγ by ERK1/2. Furthermore, we investigated whether in vivo inhibition of MAPK/ERK kinase (MEK)-1 by PD98059 may restore PPARγ expression and afford protective effects in sepsis.
The primary antibodies for PPARγ and α-tubulin were obtained from Thermo Fisher Scientific (Rockford, IL, USA). The primary antibodies for p-PPARγ, p-ERK1/2 and ERK1/2 and the oligonucleotide for PPARs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).
The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and was reviewed and approved by our Institutional Animal Care and Use Committee. Polymicrobial sepsis was induced in male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA, USA), weighing 175–250 g, by cecal ligation and puncture (CLP) as previously described (1). Rats were anesthetized with thiopentone sodium (70 mg/kg) injected intraperitoneally. After opening the abdomen, the cecum was exteriorized and ligated with a 3.0 silk suture at its base without obstructing the intestinal continuity. The cecum was punctured twice with an 18-gauge needle and returned to the peritoneal cavity. The abdominal incision was closed with 3.0 silk running sutures.
Animals underwent intraperitoneal injection of vehicle (dimethyl sulfoxide [DMSO]) or the MEK1 inhibitor PD98059 (5 mg/kg) 30 min before CLP. Rats were sacrificed at 0, 1, 3, 6 and 18 h after CLP (n = 3–6 for each group). In the control group (CLP 0 h), surgery was performed, but the cecum was neither ligated nor punctured. Saline solution (0.9%, 5 mL) was given subcutaneously to replace the fluid and blood loss during the operation. Whole blood, plasma and lungs were collected for the biochemical studies described below.
Lungs were fixed in 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin and evaluated by three independent observers unaware of the experimental protocol. Specifically, lung injury was analyzed by a semiquantitative score as previously reported (12) based on the following histologic features: (a) alveolar congestion, (b) hemorrhage, (c) infiltration or aggregation of neutrophils in the airspace or vessel wall and (d) thickness of alveolar wall/hyaline membrane formation. Each feature was graded from 0 to 4 (i.e., no injury, minimal, mild, significant or severe). The four variables were summed to represent the lung injury score (total score, 0–16).
Peripheral blood mononuclear cells (PBMCs) were isolated by Percoll density gradient centrifugation (Amersham Biosciences, Piscataway, NJ, USA). Whole blood was layered onto a Percoll gradient, and samples were centrifuged at 1,500g for 30 min at 4°C. After centrifugation, the buffy coat layer was removed and washed in Hanks solution with bovine serum albumin. Samples were centrifuged again, and the pellet was re-suspended in Hanks solution and stored at −80°C.
PBMCs were rinsed and lung tissue was homogenized with a cold buffer containing 0.32 mmol/L sucrose, 10 mmol/L Tris-HCl, 1 mmol/L EGTA, 2 mmol/L EDTA, 0.2 μmol/L NaN3, 50 mmol/L NaF, 20 μmol/L leupeptin, 0.15 μmol/L pepstatin A and 0.2 mmol/L phenylmethylsulfonyl fluoride (PMSF). The cell suspension or tissue homogenate was centrifuged (1,000g at 4°C for 10 min). The supernatant (cytosol + membrane extract) was collected and stored at −80°C. The pellet was solubilized in Triton buffer (1% Triton X-100, 150 mmol/L NaCl, 10 mmol/L Tris-HCl [pH 7.4], 1 mmol/L EGTA, 1 mmol/L EDTA, 0.2 mmol/L sodium orthovanadate, 20 μmol/L leupeptin A and 0.2 mmol/L PMSF). The lysates were centrifuged (15,000g at 4°C for 30 min), and the supernatant (nuclear extract) was collected and stored at −80°C. The amount of protein was quantified by the Bradford assay.
The nuclear or cytosol content of PPARγ, phosphorylated PPARγ (p-PPARγ), ERK1/2, phosphorylated ERK1/2 (p-ERK1/2) and α-tubulin in PBMCs and lung was determined by immuno blot analyses. Extracts were boiled in equal volumes of loading buffer (125 mmol/L Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol), and 50 μg protein was loaded per lane on an 8–16% Tris-glycine gradient gel. Proteins were separated electrophoretically and transferred to nitrocellulose membranes. For immunoblotting, membranes were blocked with 5% nonfat dried milk in Tris-buffered saline for 1 h and then incubated with primary antibodies against PPARγ, ERK 1/2, p-ERK 1/2 and p-PPARγ. The membranes were washed in Tris-buffered saline with 0.1% Tween 20 and incubated with secondary peroxidase-conjugated antibody. Membranes were reprobed with primary antibody against α-tubulin to ensure equal loading. Detection was enhanced by chemiluminescence and exposed to photographic film. Densitometric analysis of blots was performed using ImageQuant (Molecular Dynamics, Sunnyvale, CA, USA).
Electrophoretic mobility shift assay (EMSA) was performed as described previously (1). An oligonucleotide probe corresponding to PPARs consensus sequence (5′-GAA AAC TAG GTC AAA GGT CA-3′) was labeled with [γ-32P]ATP using T4 polynucleotide kinase and purified in Bio-Spin chromatography columns (Bio-Rad, Hercules, CA, USA). A total of 10 μg nuclear protein was preincubated with EMSA buffer (12 mmol/L HEPES, pH 7.9, 4 mmol/L Tris-HCl, pH 7.9, 25 mmol/L KCl, 5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 50 ng/mL poly[d(I-C)], 12% glycerol vol/vol and 0.2 mmol/L PMSF) on ice for 10 min before adding radio-labeled oligonucleotide for an additional 10 min. The specificity of the binding reactions was determined by co-incubating duplicate nuclear extract samples with a 10-fold molar excess of unlabeled oligonucleotides (competitor assays), anti-PPARα, anti-PPARβ or anti-PPARγ antibody (supershift assay). Protein–nucleic acid complexes were resolved using a nondenaturing polyacrylamide gel consisting of 5% acrylamide (29:1 ratio of acrylamide:bisacrylamide) and run in 0.5 × Tris borate-EDTA (45 mmol/L Tris-HCl, 45 mmol/L boric acid and 1 mmol/L EDTA) for 1 h at a constant current (30 mA). Gels were transferred to 3M paper (Whatman, Clifton, NJ, USA), dried under a vacuum at 80°C for 1 h and exposed to photographic film at −70°C with an intensifying screen. Densitometric analysis was performed using ImageQuant (Molecular Dynamics).
Plasma levels of adiponectin were measured with a multiplex assay kit (Millipore, Billerica, MA, USA) using the protocol recommended by the manufacturer.
All values in the figures and text are expressed as mean ± SEM of n observations (n = 3–6 animals for each group). Statistical analysis of damage scores and adiponectin levels were performed using the t test. Densitometric statistical analysis of p-ERK to total ERK was compared with the Kruskal-Wallis analysis of variance with the Tukey test for the vehicle group and Holm-Sidak method for the PD98059 group. The t test was used to compare vehicle-treated versus PD98059-treated groups at 18 h after CLP. A value of P = 0.05 was considered significant.
To determine the effect of sepsis on changes in activation of PPARγ in PBMCs, rats were subjected to CLP and euthanized at various time points. Constitutive nuclear expression of PPARγ (1.05 ± 0.08 relative intensity) was observed in PBMCs in control animals (time 0, before CLP), as evaluated by Western blot analysis (Figure 1A). A marked decrease of nuclear PPARγ was observed at 18 h after CLP (0.35 ± 0.13 relative intensity) when compared with control PPARγ content (P < 0.05) (see Figure 1A). A reduction of PPARγ nuclear expression correlated with a significant reduction of PPAR DNA binding, as evaluated by EMSA (Figure 2A). Specific PPARγ DNA binding was confirmed by supershift analysis (Figure 2B). To gain further insights into the molecular mechanisms involved in the decreased nuclear PPARγ content, we evaluated the effect of CLP on p-ERK1/2. In a similar time course analysis, nuclear expression of p-ERK1/2 increased as early as 3 h after CLP and was further enhanced thereafter (Figure 1A, B).
To confirm the physiological role of ERK1/2 activation on the regulation of PPARγ, we treated rats subjected to CLP with a selective inhibitor of the upstream MEK1 kinase, PD98059. Treatment with PD98059 decreased nuclear p-ERK1/2 and restored PPARγ expression in PBMCs from rats subjected to CLP (see Figure 1A, B).
We have previously demonstrated that in polymicrobial sepsis, PPARγ expression exhibits maximal degradation in the lung at 18 h after CLP (1). Therefore, we chose to highlight the changes that occur at this critical time point. Similar to results demonstrated in PBMCs, PPARγ was decreased in lung tissue at 18 h after CLP (Figure 3). This decrease correlated with an increase in the phosphorylated form of PPARγ and an increase in p-ERK1/2 expression in the lung at 18 h after CLP (see Figure 3). In animals that received the MEK1 inhibitor PD98059, PPARγ expression was restored and p-PPARγ was decreased in the lung during polymicrobial sepsis (see Figure 3).
In the lungs of vehicle-treated rats, histologic examination revealed extravasation of red cells and accumulation of inflammatory cells into the air spaces. Margination and adhesion of neutrophils were seen in blood vessels at 18 h after CLP (Figure 4). Vehicle-treated rats at 18 h after CLP had a significantly higher lung injury score (9 ± 1.15) when compared with sham animals (5.3 ± 0.8) (P < 0.05) (Figure 5). However, animals that received PD98059 had a significantly lower lung injury score (3.2 ± 0.46; P < 0.05) and amelioration of inflammatory cells when compared with values of vehicle-treated rats.
A serious consequence of sepsis is the occurrence of multiple organ failure, which is preceded by accumulation of neutrophils in major vital organs (13). We next quantified neutrophil infiltration in the lung by measuring the activity of myeloperoxidase, an enzyme specific to granulocyte lysosomes. Myeloperoxidase activity was significantly elevated at 18 h after CLP in the lung from vehicle-treated rats indicating marked neutrophil infiltration (330.2 ± 46.5 U/100 mg tissue). In contrast, animals that received PD98059 had a significant reduction in myeloperoxidase activity (268.1 ± 25.5 U/100 mg tissue) at 18 h after CLP (P < 0.05) (Figure 6).
Adiponectin has a peroxisome proliferator response element in its promoter region (14). Consequently, changes in PPARγ activity may be reflected as changes in adiponectin (15). To determine the downstream effects of PPARγ activation, we measured plasma adiponectin expression as a biomarker for PPARγ activity. In vehicle-treated rats, there was no significant change in plasma adiponectin levels after CLP. However, in PD98059-treated rats, plasma adiponectin levels were significantly higher at 18 h after CLP when compared with vehicle-treated animals (Figure 7).
The present study demonstrates that PPARγ is reduced in immunomodulatory and parenchymal cells during polymicrobial sepsis. Furthermore, the downregulation of PPARγ involves its phosphorylation by ERK1/2, and inhibition of ERK1/2 causes a decrease in phosphorylated PPARγ and restores PPARγ. This restoration of PPARγ correlates with an increase in plasma levels of the antiinflammatory adipokine adiponectin (Figure 8).
In the present study, we observed that PPARγ is altered in PBMCs and lung parenchymal cells during polymicrobial sepsis. Our findings are in agreement with previous studies that demonstrate that the expression, production and activity of PPARγ are affected in other inflammatory conditions. In adipose tissue, PPARγ mRNA and protein expression decreased after mice were challenged in vivo with endotoxin (5). Previous results in our laboratory support the idea that PPARγ is important in controlling inflammation and correlates with clinical outcomes. In the cardiovascular hypodynamic phase of septic shock, PPARγ expression was downregulated in the lung and in thoracic aortas in rats (1). Furthermore, the sepsis-induced reduction in PPARγ expression was reversed by in vivo treatment with PPARγ ligands. In an experimental model of polymicrobial sepsis, Zhou et al. (16) demonstrate that hepatic PPARγ protein and gene expression is downregulated in the late stages of sepsis. Although many studies confirm the effects of sepsis on decreasing PPARγ expression in tissue, contradictory results exist in immune cells. In porcine white blood cells, PPARγ expression increased in the first 6 hours after in vivo lipopolysaccharide (LPS )challenge (17). However, PPARγ expression normalized to control levels by 8 hours post-LPS. Similarly, the expression of PPARγ was increased in peripheral blood mononuclear cells and T-cells from patients with septic shock and sepsis (15,18).
Our results provide a mechanism through which a decrease in PPARγ in sepsis may be partially explained and are consistent with previous results demonstrated in endotoxic shock (19). We hypothesized that posttranslational modifications, including phosphorylation of PPARγ by ERK1/2, may alter PPARγ in a model of polymicrobial sepsis. Our current data demonstrate that nuclear content of p-ERK1/2 increases in PBMCs after CLP and correlates with a decrease in PPARγ. In the lung, an increase of p-ERK1/2 also correlated with phosphorylation of PPARγ. Posttranslational modifications are mechanisms that regulate the function of PPARγ (7). The AF-1 domain of PPARγ contains a consensus MAPK site, and phosphorylation at serine residue 82 (or 112 for PPARγ2) leads to inhibition of PPARγ transactivation (8,9,20). This phosphorylated- induced repression is due to conformational changes that can lead to altered affinity for ligands and cofactors (8,9). Additionally, phosphorylation of PPARγ promotes its degradation through the ubiquitin-proteasome system (10). In MCF-7 breast cancer cells, inhibition of p-ERK1/2 with α-eleostearic acid correlated with decreased PPARγ in a time-dependent manner (21). An alternative mechanism resulting in decreased nuclear PPARγ expression and activity could occur through direct interaction of nuclear PPARγ with MEKs, resulting in the nuclear export of PPARγ, thereby preventing its nuclear activation (22). Thus, it is possible that during the inflammatory process, alteration of protein conformation by posttranslational mechanisms may affect the expression of the receptor (19).
Adiponectin is an adipocyte-derived protein that is secreted into plasma. Adiponectin has beneficial effects including an antiatherosclerotic action, it improves insulin sensitivity and it activates glucose uptake in skeletal muscle cells (23). Additionally, adiponectin is an antiinflammatory cytokine that inhibits nuclear factor (NF)-κB activation in endothelial cells and macrophages (24,25). Furthermore, adiponectin is induced by PPARγ agonists via direct binding to the peroxisome proliferator response element in the adiponectin promoter (14,23). Tsuchihashi et al. (26) previously demonstrated that plasma adiponectin levels were decreased in rats subjected to polymicrobial sepsis. We demonstrate that adiponectin levels were significantly increased in animals who received the ERK1/2 inhibitor PD98059 during polymicrobial sepsis. This increase in adiponectin reflects the changes in the kinetics of PPARγ after CLP. This finding is in agreement with previous studies that demonstrate that alteration of PPARγ through treatment with PPARγ ligands alters adiponectin expression (27–29). Combs et al. (28) demonstrate that adiponectin levels were significantly increased in healthy male subjects treated with the PPARγ ligand rosiglitazone. Moreover, in patients with the dominant-negative PPARγ mutation, adiponectin levels were lower compared with patients with severe insulin resistance with no mutation.
The inflammatory effects of polymicrobial sepsis cause changes in PPARγ expression and activation in PBMCs and lung tissue in rats. These changes in PPARγ are, in part, due to the phosphorylation of PPARγ by ERK1/2 and can be reversed by ERK1/2 inhibition. Furthermore, adipokines are altered during polymicrobial sepsis and adiponectin plasma levels correlate with PPARγ expression and can be augmented with ERK1/2 inhibition. More studies are necessary to investigate the molecular link between adipokines and the inflammatory response in sepsis.
This work was supported in part by National Institutes of Health Grants R01 GM-067202 (to B Zingarelli), T32 ES-10957 (to JM Kaplan), K12 HD-028827 (to JM Kaplan), and K08GM093135 (to JM Kaplan).
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
Online address: http://www.molmed.org