This decrease correlated with an increase in the phosphorylated form of PPAR and an increase in p-ERK1/2 expression in the lung at 18 h after CLP (see Figure 3)
This decrease correlated with an increase in the phosphorylated form of PPAR and an increase in p-ERK1/2 expression in the lung at 18 h after CLP (see Figure 3). In vehicle-treated rats, polymicrobial sepsis resulted in significant lung injury. In the lung and PBMCs, nuclear levels of PPAR were decreased and associated with an increase in phosphorylated PPAR and phosphorylated ERK1/2 levels. Treatment with the MEK1 inhibitor increased the antiinflammatory plasma adipokine adiponectin, restored PPAR expression in PBMCs and lung, and decreased lung injury. The inflammatory effects of sepsis cause changes in PPAR expression and activation, in part, because of phosphorylation of PPAR by ERK1/2. This phosphorylation can be reversed by ERK1/2 inhibition, thereby improving lung injury. INTRODUCTION Peroxisome Dynamin inhibitory peptide proliferatorCactivated receptor (PPAR)- is a ligand-activated transcription factor. Activation of PPAR plays a role in controlling the inflammatory response. Several studies have demonstrated that activation of PPAR by Dynamin inhibitory peptide specific ligands significantly improves survival in clinically relevant models of septic shock (1C3). The beneficial effect of PPAR activation is likely to be secondary to inhibition of the production of several inflammatory mediators, as shown in septic rodents (1C3) and in activated macrophages and monocytes (4). Sepsis and other inflammatory states affect PPAR expression and correlate with the inflammatory response. We have previously demonstrated that PPAR expression is downregulated in the lung and vascular endothelium in rodent models of septic shock and that treatment with PPAR ligands reverses the sepsis-induced reduction (1). In adipose tissue, PPAR expression decreased after mice were challenged with endotoxin, and cytokine-induced suppression of PPAR was Dynamin inhibitory peptide reversed with synthetic agonists (5,6). However, it remains unclear what mechanisms lead to a decrease in PPAR activity in sepsis. Posttranslational modifications are mechanisms that regulate the function of PPAR and may contribute to the downregulation of PPAR in sepsis (7). The activation function (AF)-1 domain of PPAR 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 PPAR2) leads to inhibition of PPAR 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 PPAR by the ubiquitin-proteasome system (10). In cultured adipocytes, using a specific ERK inhibitor reverses the reduction in PPAR (11). Therefore, in this study, we investigated the kinetics of altered PPAR 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 PPAR expression is affected, we investigated the effects of polymicrobial sepsis on the phosphorylation of PPAR by ERK1/2. Furthermore, we investigated whether inhibition of MAPK/ERK kinase (MEK)-1 by PD98059 may restore PPAR expression and afford protective effects in sepsis. MATERIALS AND METHODS The primary antibodies for PPAR and -tubulin were obtained from Thermo Fisher Scientific (Rockford, IL, USA). The primary antibodies for p-PPAR, 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). Rat Model of Cecal Ligation and Puncture The investigation conformed to the published by the Dynamin inhibitory peptide 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 175C250 g, by cecal ligation and puncture (CLP) as previously described (1). Rats were anesthetized Dynamin inhibitory peptide 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 (= 3C6 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 Rabbit Polyclonal to NUCKS1 replace the fluid and blood loss during the operation. Whole blood, plasma and lungs were collected for the biochemical studies described below. Histopathological Analysis Lungs were fixed in 4% paraformaldehyde and.