Chi H, et al

Chi H, et al. LPA significantly improved the mice ATI-2341 survival ATI-2341 to endotoxemia ( 0.05). LPA injection reduced LPS-induced plasma TNF- production (69 6%, 0.05) and myeloperoxidase (MPO) activity in lung (33 9%, 0.05) as compared to vehicle injection. LPS-induced plasma IL-6 was unchanged by LPA. studies with peritoneal macrophages paralleled results from studies. LPA (1 and 10 M) significantly inhibited LPS-induced TNF production (61 9% and 72 9%, respectively, 0.05) but not IL-6. We further shown the anti-inflammatory effect of LPA was reversed by ERK 1/2 and phosphatase inhibitors, suggesting that ERK 1/2 pathway and serine/threonine phosphatases are involved. Inhibition of phosphatidylinositol 3 (PI3) kinase signaling pathways also partially reversed the LPA anti-inflammatory response. However, LPA did not alter NFB and peroxisome proliferator-activated receptor (PPAR) activation. Inhibitors of PPAR did not alter LPA-induced inhibition of LPS signaling. These studies demonstrate that LPA offers significant anti-inflammatory activities ATI-2341 including activation of ERK 1/2, serine/threonine phosphatases, and PI3 kinase signaling pathways. Intro Septic shock is initiated from the systemic inflammatory response syndrome (SIRS), which is the response to the overactivity of the innate immune system. The inflammatory process begins in the nidus of illness where bacteria proliferate and either invade the bloodstream or release numerous bacterial components, such as lipopolysaccharide (LPS), peptidoglycan, and lipoteichoic acid (1,2). The connection of these microbial cellular parts with macrophages, monocytes, or additional sponsor cells induces the release of inflammatory mediators that induce SIRS and the ensuing of septic shock (3). Heterotrimeric guanine nucleotide-binding regulatory (Gi) proteins modulate LPS signaling pathways and downstream pro-inflammatory gene manifestation (4C7). In addition to toll-like receptor (TLR) 4, LPS also binds to a cluster of receptors in lipid rafts, some of which are Gi protein coupled (8). Our earlier studies shown that LPS-induced inflammatory cytokines and chemokines were augmented and in Gi protein-deficient mice compared with WT mice, suggesting an anti-inflammatory part of Gi proteins (5,6). Gi protein-coupled ERK 1/2 signaling pathway mediates LPS-induced signaling (4). Activation of Gi protein with transforming growth element- (TGF-) activates ERK 1/2, which can suppress NFB and p38 signaling and consequently negatively regulate LPS-induced inflammatory reactions (9,10). It also has been reported that ERK 1/2 negatively regulates p38 signaling through upregulation of MAP kinase phosphatase (MKP)-1, a serine/threonine phosphatase (9). MKP-1 decreases pro- and anti-inflammatory cytokines in innate immune reactions and endotoxic shock (11,12). These findings are consistent with our earlier studies and the findings of others that serine/threonine phosphatase regulates LPS-induced signaling pathways (13C15). PI3 kinase signaling pathways have been shown to be controlled, in part, by Gi protein-dependent pathways. PI3 kinase negatively regulates LPS-induced inflammatory response (16,17). These studies suggest that ligands that activate Gi proteins, ERK 1/2, serine/threonine phosphatase, and PI3 kinase are candidates for negative rules of LPS-induced signaling pathways. Lysophosphatidic acid (LPA, 1-acyl-2-lyso-sn-glycero-3-phosphate) is an abundant extracellular metabolite of lyso-phosphatidylcholine generated by a variety of cells that is typically present in micromolar concentrations in biological fluids at sites of swelling (18). It evokes numerous immunodulatory reactions including prevention of apoptosis, chemotaxis, cytokine and chemokine secretion, platelet aggregation, and enhanced wound healing (19C21). LPA binds to at least four specific G-protein-coupled ATI-2341 receptors (GPCRs): LPA1/EDG2, LPA2/EDG4, LPA3/EDG7, and LPA4/GPR23/p2y9 (22). Rabbit polyclonal to ZNF512 Although LPA1 and LPA2 also activate the Gq and G12/13; LPA3 activates only Gi and Gq (22). Agonist coupling with LPA receptors activates signaling pathways such as adenylyl cyclase, phospholipase C, and Ras-MAP kinase (23). A fourth LPA receptor has been recognized, although its biological significance remains to be established (24). In addition to these GPCRs, LPA also activates the cytoplasm/nuclear receptor PPAR (25). Recent studies shown an anti-inflammatory effect of LPA in endotoxemia by reducing organ injury (26). However, the potential anti-inflammatory signaling pathway triggered by LPA remains to be defined clearly. In the present study, we examined the effect of LPA on LPS reactions by assessment of plasma cytokines, and pulmonary swelling by assessment of cells MPO activity and activation of NFB and PPAR. Because macrophages are regarded as important inflammatory cells in endotoxemia, we also examined the anti-inflammatory effect of LPA on LPS-induced TNF and IL-6 production. Additionally, potential anti-inflammatory signaling pathways triggered by LPA were examined with inhibitors of the ERK 1/2, serine/threonine phosphatase, and PI3 kinase signaling pathways. MATERIALS AND METHODS Reagents LPS, LPA (Oleoyl-L–lysophosphatidic acid 18:1), okadaic acid, and calyculin A were purchased from Sigma (St. Louis, ATI-2341 MO, USA). LPA was suspended in PBS with 2% BSA and sonicated for 15 s. Protein free R595 LPS was provided by Dr. Ernst Reitschel (Borstel, Germany). PD98059 and wortmannin were purchased from Calbiochem (San Diego, CA, USA)..