0026-895X/11/7904-786–792$20.00 MOLECULAR PHARMACOLOGY Vol. 79, No. 4 Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics 70201/3676458 Mol Pharmacol 79:786–792, 2011 Printed in U.S.A. Proinflammatory Stimuli Control N-Acylphosphatidylethanolamine- Specific Phospholipase D Expression in Macrophages Chenggang Zhu, Carlos Solorzano, Saurabh Sahar, Natalia Realini, Ernest Fung, Paolo Sassone-Corsi, and Daniele Piomelli Department of Biological Chemistry, University of California, Irvine, California (C.Z., D.P.); Department of Pharmacology, University of California, Irvine, California (C.S., S.S., N.R., E.F., P.S.C., D.P.); and Drug Discovery and Development, Italian Institute of Technology, Genova, Italy (D.P.) Received November 28, 2010; accepted January 12, 2011 ABSTRACT Palmitoylethanolamide (PEA) is an endogenous lipid amide that transcription factor Sp1 is involved in regulating baseline modulates pain and inflammation by engaging peroxisome pro- NAPE-PLD expression but not in the transcriptional suppres- liferator-activated receptor type-␣. Here, we show that the sion induced by LPS. The ability of LPS to down-regulate PEA proinflammatory bacterial endotoxin lipopolysaccharide (LPS) biosynthesis is impaired in peritoneal macrophages from mu- decreases PEA biosynthesis in RAW264.7 macrophages by tant NAPE-PLD-deficient mice, in which PEA is produced suppressing the transcription of N-acylphosphatidylethanol- through a compensatory mechanism distinct from NAPE-PLD. amine-specific phospholipase D (NAPE-PLD), which catalyzes Moreover, NAPE-PLD-deficient mice fail to mount a normal the production of PEA and other lipid amides. Using a luciferase inflammatory reaction in response to carrageenan administra- reporter construct and chromatin immunoprecipitation, we fur- tion in vivo. Our findings suggest that proinflammatory stimuli ther show that LPS treatment reduces acetylation of histone suppress NAPE-PLD expression and PEA biosynthesis in mac- proteins bound to the NAPE-PLD promoter, an effect that is rophages and that this effect might contribute to the inflamma- blocked by the histone deacetylase inhibitor trichostatin A. The tory response. Introduction vating protein-1, and inhibitor of ␬B kinase complex (Glass and Ogawa, 2006). PEA is a naturally occurring lipid amide that activates the Endogenous PEA levels undergo striking changes during nuclear receptor peroxisome proliferator-activated recep- inflammation. Stimulation with proinflammatory agents tor-␣ (PPAR-␣) (LoVerme et al., 2005). This interaction is such as LPS or carrageenan decreases PEA content in vari- probably responsible for the profound anti-inflammatory ef- ous cells and tissues of rodents, including intestine (Capasso fects of PEA (Mazzari et al., 1996; Calignano et al., 1998; et al., 2001), skin (LoVerme et al., 2005), and leukocytes O’Sullivan, 2007), which are abrogated by genetic deletion of PPAR-␣ and closely mimicked by synthetic PPAR-␣ agonists (Endocannabinoid Research Group et al., 2010; Solorzano et (LoVerme et al., 2005, 2006). Although best known for its al., 2009). Adding clinical relevance to these findings, it was roles in the transcriptional regulation of lipid metabolism shown that synovial fluid from patients with rheumatoid (Schoonjans et al., 1996), PPAR-␣ has been also implicated in arthritis and osteoarthritis contains lower amounts of PEA the control of the inflammatory process, which it may influ- compared with control subjects (Richardson et al., 2008). The ence by modulating the activities of nuclear factor-␬B, acti- possibility, suggested by these results, that endogenous PEA might participate in the inflammatory process is supported by experiments showing that pharmacological inhibition of This work was supported by the Sandler Asthma Foundation [Grant 02- PEA hydrolysis prevents the decrease in leukocyte PEA lev- 0075]; and the National Institutes of Health National Institute on Drug Abuse [R01-DA012413]. els induced by inflammatory triggers and concomitantly C.Z. and C.S. contributed equally to this work. blocks inflammation through a PPAR-␣-dependent mecha- Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. nism (Solorzano et al., 2009). A plausible interpretation of doi:10.1124/mol.110.070201. these results is that endogenous PEA acting at PPAR-␣ pro- ABBREVIATIONS: PPAR-␣, peroxisome proliferator-activated receptor-␣; PEA, palmitoylethanolamide; NAPE-PLD, N-acylphosphatidylethano- lamine-specific phospholipase D; LPS, lipopolysaccharide; HDAC, histone deacetylase; NAAA, N-acylethanolamine-hydrolyzing acid amidase; FAAH, fatty-acid amide hydrolase; FBS, fetal bovine serum; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride; MS, mass spectrometry; LC/MS, liquid chromatography/mass spectrometry; ChIP, chromatin immunoprecipitation; bp, base pair; TLR4, Toll-like receptor-4. 786 Regulation of NAPE-PLD in Macrophages 787 vides a stop signal that hinders the development of inflam- and glyceraldehyde 3-phosphate dehydrogenase, forward, 5Ј-TCACTG- mation. Such a role is consistent with the phenotype of GCATGGCCTTCC-3Ј, reverse, 5Ј-GGCGGCACGTCAGATCC-3Ј, probe, PPAR-␣-deficient mice, which display a heightened sensitiv- 5Ј-TTCCTACCCCCAATGTGTCCGTCG-3Ј. RNA levels were normalized ity to proinflammatory agents (Devchand et al., 1996), and using glyceraldehyde 3-phosphate dehydrogenase as an internal standard. differentiates PEA from lipid mediators that either enhance Protein Analyses. Protein concentrations were measured using the inflammatory process (e.g., prostaglandins) (Flower, the bicinchoninic acid assay (Pierce Chemical, Rockford, IL). Pro- teins were separated by SDS-polyacrylamide gel electrophoresis and 2006) or terminate it by promoting resolution and tissue transferred to polyvinylidene difluoride membranes (GE Healthcare, healing (e.g., lipoxins and resolvins) (Serhan et al., 2008). Chalfont St. Giles, Buckinghamshire, UK). Overnight incubation in In the present study, we investigated the molecular mech- the presence of a previously characterized anti-NAPE-PLD antibody anism through which the proinflammatory bacterial endo- (1:2000) (Fu et al., 2007) at 4°C was followed by incubation with toxin LPS influences endogenous PEA levels in RAW264.7 horseradish peroxidase conjugated anti-rabbit IgG antibody (1:3000; cells and mouse peritoneal macrophages. We found that LPS Sigma-Aldrich, St. Louis, MO) for1hatroom temperature. The suppresses the expression of the PEA-producing enzyme anti-NAPE-PLD antibody was kindly provided by Dr. Ken Mackie (In- NAPE-PLD by influencing the acetylation state of histone diana University, Bloomington, IN) and was raised in rabbits as de- proteins associated with the promoter region of the Nape-pld scribed previously (Fu et al., 2007). Protein bands were visualized using gene. In contrast, LPS does not alter the expression of two the ECL Plus kit (Amersham Biosciences). lipid amidases involved in PEA degradation: N-acylethano- Enzyme Assays. We measured NAPE-PLD activity at 37°C for 30 lamine-hydrolyzing acid amidase (NAAA) (Tsuboi et al., min in Tris-HCl buffer (50 mM), pH 7.4, containing 0.1% Triton X-100, 2007), and fatty-acid amide hydrolase (FAAH) (McKinney 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich), protein (0.1 mg), and 1,2-dipalmitoyl-sn-glycero-3-phospho-ethanolamine-N- and Cravatt, 2005). Our results further suggest that the down- heptadecenoyl (0.1 mM) as substrate, which was prepared as described regulation of NAPE-PLD expression may be functionally impor- previously (Fu et al., 2007). Reactions were stopped by adding chloro- tant because mutant NAPE-PLD-null mice, in which this reg- 2 form/methanol [2:1 (v/v)] containing [ H4]oleoylethanolamide as inter- ulatory process is defective, are unable to mount a normal 2 nal standard. [ H4]Oleoylethanolamide was prepared as described pre- inflammatory reaction in response to carrageenan. viously (Fu et al., 2007). After centrifugation at 1500g at 4°C for 5 min, the organic layers were collected and dried under N2. The residues were suspended in 50 ␮l of chloroform/methanol [1:3 (v/v)] and analyzed by Materials and Methods liquid chromatography/mass spectrometry (LC/MS), monitoring the [MϩNa]ϩ ions of m/z ϭ 334 for N-heptadecenoylethanolamide and Animals and Cells. RAW264.7 cells were purchased from the m/z ϭ 352 for [2H ]oleoylethanolamide. To measure FAAH activity, cell American Type Culture Collection (Manassas, VA), and cultured in 4 homogenates were incubated at 37°C for 30 min in Tris buffer (50 mM), Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) sup- pH 8.0, containing fatty acid-free bovine serum albumin (0.05%), and plemented with fetal bovine serum (FBS, 10%; Invitrogen). [ethanolamine-3H]anandamide (10,000 dpm; specific activity, 20 Ci/ C57BL/6J NAPE-PLD(Ϫ/Ϫ) mice were generated as described pre- mmol). After stopping the reaction with a mixture of chloroform/ viously (Leung et al., 2006) and backcrossed 10 times to C57BL/6J methanol [1:1 (v/v)], we measured radioactivity in the aqueous layers by wild-type mice (The Jackson Laboratory, Bar Harbor, ME). All pro- liquid scintillation counting. To measure NAAA activity, cell homoge- cedures met the National Institutes of Health guidelines for the care nates were incubated at 37°C for 30 min in 0.2 ml of sodium hydrogen and use of laboratory animals and were approved by the University phosphate buffer (50 mM), pH 4.5, containing 0.1% Triton X-100,