Anandamide Inhibits Nuclear Factor- B Activation through a Cannabinoid Receptor-Independent Pathway

Anandamide (arachidonoylethanolamine, AEA), an endogenous agonist for both the cannabinoid CB1 receptor and the vanilloid VR1 receptor, elicits neurobehavioral, anti-inflammatory, immunomodulatory, and proapoptotic effects. Because of the central role of nuclear factorB (NFB) in the inflammatory process and the immune response, we postulated that AEA might owe some of its effects to the suppression of NFB. This study shows that AEA inhibits tumor necrosis factor(TNF )-induced NFB activation by direct inhibition of the I B kinase (IKK) and, to a lesser extent, the IKK subunits of B inhibitor (I B) kinase complex, and that IKKs inhibition by AEA correlates with inhibition of I B degradation, NFB binding to DNA, and NFB-dependent transcription in TNF -stimulated cells. AEA also prevents NFB-dependent reporter gene expression induced by mitogen-activated protein kinase kinase kinase and NFB-inducing kinase. The NFB inhibitory activity of AEA was independent of CB1 and CB2 activation in TNF -stimulated 5.1 and A549 cell lines, which do not express vanilloid receptor 1, and was not mediated by hydrolytic products formed through the activity of the enzyme fatty acid amide hydrolase. Chemical modification markedly affected AEA inhibitory activity on NFB, suggesting rather narrow structure-activity relationships and the specific interaction with a molecular target. Substitution of the alkyl moiety with less saturated fatty acids generally reduced or abolished activity. However, replacement of the ethanolamine “head” with a vanillyl group led to potent inhibition of TNF -induced NFB-dependent transcription. These findings provide new mechanistic insights into the antiinflammatory and proapoptotic activities of AEA, and should foster the synthesis of improved analogs amenable to pharmaceutical development as anti-inflammatory agents. Endocannabinoids are a class of lipid mediators found in several tissues and structurally based on a polyunsaturated fatty acid amide or ester motifs (Di Marzo et al., 1999). Anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoylglicerol (2-AG) are the main endocannabinoids described to date. They act as mediators in the brain and in peripheral tissues mainly through the stimulation of brain (CB1) and peripheral (CB2) cannabinoid receptors. Although AEA preferentially binds to CB1, 2-AG is equipotent at both receptor subtypes. AEA is produced by neurons and other cell types from the hydrolysis of the phospholipid precursor N-arachydonoylphosphatidylethanolamide, catalyzed by a Ca -dependent phospholipase D (Di Marzo et al., 1994). Signal termination for AEA includes cellular reuptake by the AEA membrane transporter and hydrolysis by the fatty acid amide hydrolase (FAAH) (Di Marzo et al., 1994, 1999), a process that generates arachidonic acid and ethanolamine. Synaptic release of AEA is tightly regulated by depolarizing stimuli and glutamate receptor stimulation (Di Marzo et al., 1994). The degradative processes are also subject to regulation, and pharmacological inhibition of FAAH and AEA membrane transporter (Di Marzo et al., 1999; Boger et al., 2000) has been pursued as a way to increase the synaptic levels of AEA. AEA can also interact with the vanilloid receptor type 1 (VR1) (Zygmunt et al., 1999; Smart et al., 2000). This ligandgated cation channel is modulated allosterically by capsaicin and its analogs, and is mainly expressed in primary afferent nociceptive neurons (Caterina et al., 1997). Synthetic AEAThis work was supported by MCyT grant BIO 2000-1091-C01 (to E.M.), by European Union grant QLK3-CT-2000-00463 (to E.M. and G.A.), and by INTAS grant 97/1297 (to V.D.M.). R. S. and M.A.C. contributed equally to this study. ABBREVIATIONS: AEA, anandamide; 2-AG, 2-arachidonoylglicerol; CB, cannabinoid receptor; FAAH, fatty acid amide hydrolase; VR1, vanilloid receptor type 1; NFB, nuclear factorB; I B, B inhibitor; IKK, I B kinase; IKC, I B kinase complex; NIK, NFB inducing kinase; TNF , tumor necrosis factor; MEKK, mitogen-activated protein kinase kinase kinase; COX-2, cyclooxygenase 2; HIV-LTR, HIV long terminal repeat; mAb, monoclonal antibody; ATFMK, arachidonoyl trifluoromethyl ketone; N-AVAM, N-acylvanillamide; DTT, dithiothreitol; NP-40, Nonidet-P40; EMSA, electrophoretic mobility shift assay; RT-PCR, reverse transcriptase-polymerase chain reaction; bp, base pair(s); ERK, extracellular signal-regulated kinase; cyPG, cyclopentenone prostaglandin; SR141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3carboximide hydrochloride. 0026-895X/03/6302-429–438$7.00 MOLECULAR PHARMACOLOGY Vol. 63, No. 2 Copyright © 2003 The American Society for Pharmacology and Experimental Therapeutics 1898/0136101 Mol Pharmacol 63:429–438, 2003 Printed in U.S.A. 429 at A PE T Jornals on Jne 5, 2017 m oharm .aspeurnals.org D ow nladed from capsaicin “hybrids” have been synthesized, and one of them (arvanil) was found to bind to both VR1 and CB1 receptors (Melck et al., 1999a). Over the past few years, there has been a growing awareness that AEA and certain synthetic vanilloids exert CB1and VR1-independent biological activities (Di Marzo et al., 1999, 2000a). Thus, in CB1 / mice, the cannabimimetic effects of AEA are not affected by a selective CB1 receptor antagonist (Adams et al., 1998), whereas in CB1 / mice AEA stimulates guanosine 5 -O-(3-thio)triphosphate binding in brain membranes (Di Marzo et al., 2000b), and arvanil induces inhibition of spasticity and pain via a VR1-independent pathway (Brooks et al., 2002). In addition to the effects on peripheral and central nervous systems, AEA also shows anti-inflammatory, immunomodulatory, and proapoptotic activities (Berdyshev et al., 1997; Maccarrone et al., 2000). Despite the pharmacological relevance of these activities, their mechanistic basis has so far remained elusive. The transcription factor NFB is one of the key regulators of genes involved in the immune/inflammatory response and in survival from apoptosis (Karin and Ben Neriah, 2000). NFB is an inducible transcription factor made up of homoand heterodimers of p50, p65, p52, relB, and c-rel subunits that interact with a family of inhibitory I B proteins, of which I B is the best characterized (Scheme 1). In most cell types, these proteins sequester NFB in the cytoplasm by masking its nuclear localization sequence. Stimulation of cells with a variety of physiological or pathogenic stimuli leads to phosphorylation, ubiquitination, and the subsequent degradation of I B proteins. The degradation of I B results in the translocation of NFB from the cytoplasm to the nucleus. Phosphorylation of I B at serines 32 and 36 is a key step involved in the activation of NFB complexes. This event is mediated by I B kinases (IKKs) (Woronicz et al., 1997), which are formed by a high-molecular-weight complex (IKC) containing at least two kinase subunits (IKK and IKK ) and the associated modulatory protein NEMO/IKK (Scheme 1). The activation of IKK by different stimuli requires distinct signaling proteins, like the mitogen-activated protein kinase kinase kinase family members NIK, MEKK1, MEKK2, and MEKK3, and also TAK1 and AKT/TPKB kinases (Karin and Ben Neriah, 2000; Hagemann and Blank, 2001). The physiological role of these kinases in signaling through the tumor necrosis factor (TNF) receptor type I has not yet been clarified (Baud and Karin, 2001). Recent data suggest that IKK is absolutely required for I B phosphorylation and subsequent degradation in TNF -induced NFB activation, whereas IKK is responsible for the processing of NFB2/p100 in a more specialized pathway (Senftleben and Karin, 2002). NFB is highly activated at sites of inflammation in diverse diseases (Tak and Firestein, 2001), where it regulates the transcription of proinflammatory cytokines, chemokines, cytokine receptors, adhesion molecules, and key enzymes in the inflammatory process, such as cycloxygenase-2 (COX-2) and inducible nitric-oxide synthases (Ghosh et al., 1998). Endocannabinoids are also rapidly generated in response to proinflammatory stimulation of immune cells, and they might operate a negative feedback control over the proinflammatory response, possibly by negatively regulating the activation of transcription factors involved in the inflammatory response (Berdyshev et al., 2001b). Furthermore, NFB inhibition results in cell apoptosis in some cell systems (Beg et al., 1995) and might be one of the mechanisms underlying AEA apoptotic effects on both immune and nervous cells (Guzman et al., 2001). Previous studies have shown that, in murine macrophages and splenocytes, cannabinoids and the endocannabinoid 2-AG may either activate or inhibit NFB activity via cannabinoid receptorand protein kinase A-dependent mechanisms (Kaminski, 1996; Daaka et al., 1997). Surprisingly, AEA has not yet been investigated as a potential NFB modulator. We report here that AEA inhibits the TNF -induced signals leading to IKK activation, I B degradation, and NFB activation and that this activity is essentially CB1and VR1-independent. The NFB inhibitory activity of AEA was retained in certain vanillamides, such as the capsaicin-AEA hybrid arvanil and the fatty acid-based vanilloid olvanil, but not in closely related analogs modified on the acyl chain. Materials and Methods Cell Lines and Reagents. The 5.1 clone (obtained from Dr. N. Isräel, Institut Pasteur, Paris, France) line is a Jurkat-derived clone stably transfected with a plasmid containing the luciferase gene Scheme 1. Schematic representation of TNF signaling pathway from tumor necrosis factor receptor type 1 (TNFR1) to transcription factor NFB. TNF binding to TNFR1 results in the recruitment of TNFR1associated proteins, which activate the IKC either directly or by means of the activation of a putative IKK kinase(s) (MEKK-1?). Active IKC phosphorylates I B , thereby leading to subsequent I B ubiquitination and degradation by the proteasome, with subsequent release of NFB that can enter the nucleus and start gene transcription. AEA blockade of NFB activity could be mediated by inhibition of a cytokine-induced second messenger responsible for activation of IKK in IKC. 430 Sancho et al. at A PE T Jornals on Jne 5, 2017 m oharm .aspeurnals.org D ow nladed from driven by the HIV-LTR promoter and was maintained in exponential growth in RPMI 1640 medium (BioWhittaker, VerViers, Belgium) supplemented with 10% heat inactivated fetal calf serum, 2 mM L-glutamine, 1 mM HEPES and antibiotics (Invitrogen, Paisley, Scotland), and 200 g/ml G418. The A549 lung adenocarcinome cell line was obtained from Glaxo SmithKline (London, UK) and was maintained in complete Dulbecco’s modified Eagle’s media. The antiI B mAb 10B was a gift from R. T. Hay (University of St. Andrews, Fife, Scotland), the mAb anti-tubulin was purchased from SigmaAldrich (St. Louis, MO), and the rabbit polyclonal anti-IKK(FL419) was from Santa Cruz Biotechnology, Inc. (San Diego, CA). The CB1 antagonist SR141716A was purchased from Tocris Cookson (Bristol, UK), and the FAAH inhibitor arachidonoyl trifluoromethyl ketone (ATFMK), anandamide (2-arachidonoyl ethanolamide), and arachidonic acid were from Sigma-Aldrich. Arvanil was purchased from Cayman Chemicals (Ann Arbor, MI). The synthesis of the N-AVAMs olvanil, retvanil, retvanil-Ac, farvanil, and ervanil will be published elsewhere (Appendino et al., 2002). N-Palmitoyl-, Nlinolenoyl, and N-docosahexaenoyl-ethanolamines were synthesized from the condensation of the corresponding fatty acid chlorides with ethanolamine. [ -P]ATP (3000 Ci/mmol) was purchased from ICN Pharmaceuticals (Costa Mesa, CA). The KBF-Luc plasmid, which contains three copies of NFB binding site (from major histocompatibility complex promoter), fused to a minimal simian virus 40 promoter driving luciferase. The GST-I B (1–54) plasmid, and the expression vectors for IKK , IKK , MEKK1, and NIK have been described elsewhere (Hehner et al., 1999). Transient Transfections and Luciferase Assays. A549 cells (10/ml) were transiently transfected with the KBF-Luc reporter. The transfections were performed using LipofectAMINE PLUS reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations for 24 h. After incubation with AEA for 30 min, transfected cells were stimulated for 6 h with 2 ng/ml TNF . To determine NFB-dependent transcription of the HIV-LTR-Luc 5.1 cells were preincubated for 30 min with AEA and analogs as indicated, followed by stimulation with 2 ng/ml TNF for 6 h. Then the cells were lysed in 25 mM Tris-phosphate, pH 7.8, 8 mM MgCl2, 1 mM DTT, 1% Triton X-100, and 7% glycerol. Luciferase activity was measured using an Autolumat LB 953 (PerkinElmer Life Science, Boston, MA) following the instructions of the luciferase assay kit (Promega, Madison, WI), and protein concentration was measured by the Bradford method. The background obtained with the lysis buffer was subtracted from each experimental value, the relative luciferase units per microgram of protein were calculated and the specific transactivation expressed as fold induction over untreated cells. All the experiments were repeated at least six times. Statistical analysis was performed using analysis of variance followed by the Student-Newman-Keuls method with values of p 0.05 considered to be significant. Western Blots. 5.1 cells (1 10 cells/ml) were stimulated with TNF in the presence or absence of AEA for the indicated period of time. Cells were then washed with phosphate-buffered saline and proteins were extracted from cells in 50 l of lysis buffer (20 mM HEPES, pH 8.0, 10 mM KCl, 0.15 mM EGTA, 0.15 mM EDTA, 0.5 mM Na3VO4, 5 mM NaFl, 1 mM DTT, 1 g/ml leupeptin, 0.5 g/ml pepstatin, 0.5 g/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) containing 0.5% NP-40. Protein concentration was determined by a Bradford assay (Bio-Rad, Hercules, CA), and 30 g of proteins was boiled in Laemmli buffer and electrophoresed in 10% SDS/ polyacrylamide gels. Separated proteins were transferred to nitrocellulose membranes (0.5 A at 100 V; 4°C) for 1 h. The blots were blocked in Tris-buffered saline solution containing 0.1% Tween 20 and 5% nonfat dry milk overnight at 4°C, and immunodetection of specific proteins was carried out with primary antibodies (anti-I B , anti-IKK , and anti-tubulin) using an ECL system (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK). Densitometry analyses were carried out for the I B and -tubulin blots, and the optical density ratio I B / -tubulin was calculated and assigned the value 1 to untreated cells. IKK Kinase Assay. Cells were lysed in NP-40 lysis buffer [20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, 0.5 mM sodium vanadate, 10 g/ml leupeptine, 10 g/ml aprotinin, 1% (v/v) NP-40, and 10% (v/v) glycerol] for 15 min at 4°C, and after centrifugation for 10 min at 13,000 rpm, the supernatant was incubated with 25 l of protein A/G-Sepharose and incubated for 2 h on a spinning wheel. After centrifugation, the supernatants were incubated with 2 g of anti-IKKantibody and 25 l of protein A/G-Sepharose and incubated for 2 to 4 h on a spinning wheel at 4°C. The precipitate was washed three times in cold lysis buffer and three times in cold kinase buffer (20 mM HEPES/KOH, pH 7.4, 25 mM -glycerophosphate, 2 mM DTT, and 20 mM MgCl2). The kinase assay was performed in a final volume of 20 l of kinase buffer containing 40 M ATP and 5 Ci of -32P-ATP and 2 g of the purified substrate protein GST-I B (1–54). After incubation for 20 min at 30°C, the reaction was stopped by the addition of 5 SDS loading buffer. After separation by SDS-polyacrylamide gel electrophoresis the gel was fixed, dried, and exposed to X-ray film. Isolation of Nuclear Extracts and Mobility Shift Assays. 5.1 or A549 cells (10/ml) were stimulated with the agonists in complete medium as indicated. Cells were then washed twice with cold phosphate-buffered saline and proteins from nuclear extracts isolated as described previously (Sancho et al., 2002). Protein concentration was determined by the Bradford method (Bio-Rad). For the electrophoretic mobility shift assay (EMSA), the consensus oligonucleotide probes NFB, 5 -AGTTGAGGGGACTTTCCCAGG-3 , and the commercial SP1 site (Promega) were end-labeled with [ -P]ATP. The binding reaction mixture contained 3 g of nuclear extract, 0.5 g of poly(dI-dC) (Amersham Biosciences Inc., Piscataway, NJ), 20 mM HEPES, pH 7, 70 mM NaCl, 2 mM DTT, 0.01% NP-40, 100 g/ml bovine serum albumin, 4% Ficoll, and 100,000 cpm of end-labeled DNA fragments in a total volume of 20 l. When indicated, 0.5 l of rabbit anti-p50, anti-p65, or preimmune serum was added to the standard reaction before the addition of the radiolabeled probe. For cold competition, a 100-fold excess of the double-stranded oligonucleotide competitor was added to the binding reaction. After 30-min incubation at 4°C, the mixture was electrophoresed through a native 6% polyacrylamide gel containing 89 mM Tris-borate, 89 mM boric acid, and 1 mM EDTA. Gels were pre-electrophoresed for 30 min at 225 V and then for 2 h after loading the samples. These gels were dried and exposed to X-ray film at 80°C. Reverse Transcriptase-PCR (RT-PCR) Amplification of CB1/ CB2 and FAAH mRNA. Total RNA was prepared from 5.1 and A549 cells by the lithium chloride/urea method and digested with DNase. Retrotranscription of mRNA into cDNA was performed in a 20l reaction mixture according to the SuperScript II RNase H Reverse Transcriptase (Invitrogen) protocol, using 0.5 g of Oligo(dT)12–18 Primer (Invitrogen) for 5 g of mRNA. The reaction mixture was incubated for 50 min at 42°C, and stopped by heating at 70°C for 15 min, cooled in ice, and stored at 20°C. RT-PCR amplification was performed in a 50l PCR reaction mixture containing 0.5 to 2 l of the retro-transcription mixture, 1 PCR buffer, 1.5 mM MgCl2, 200 M dNTPs, 10 M each of 5 and 3 primers, and 2.5 units of recombinant TaqDNA polymerase (Invitrogen). The mixtures were amplified in a MultiGene cycler IR system (Labnet, Woodbridge, NJ). The primers used were as follows: CB1 sense primer, 5 -CGCAAAGATAGCCGCAACGTGT-3 ; CB1 antisense primer, 5 -CAGATTGCAGTTTCTCGCAGTT-3 ; CB2 sense primer, 5 -TTTCCCACTGATCCCCAATG-3 ; CB2 antisense primer, 5 AGTTGATGAGGCACAGCATG-3 ; FAAH sense primer, 5 -GCCTGGGAAGTGAACAAAGGGACC-3 ; FAAH antisense primer, 5 -CCACTACGCTGTCGCACTCCGCCG-3 ; -actin antisense primer, 5 GCAACTAAGTCATAGTCCGC-3 ; and -actin sense primer, 5 CTGTCTGGCGGCACCACCAT-3 . The primers chosen for RT-PCR of FAAH and -actin mRNA amplification included different exons. Therefore, by using these primers any possible DNA contamination Anandamide Inhibits NFB 431 at A PE T Jornals on Jne 5, 2017 m oharm .aspeurnals.org D ow nladed from would be detected by the amplification of a higher size band corresponding to an amplicon containing an intron. The amplification profile consisted of an initial denaturation of 2 min at 95°C and then 20 to 35 cycles of 30 s at 95°C, annealing for 30 s at 55°C (CB1 and -actin) or at 60°C (CB2 and FAAH) and elongation for 1 min at 72°C. A final extension of 10 min was carried out at 72°C. The expected sizes of the amplicons were 244 bp for CB1, 337 bp for CB2, 202 bp for FAAH, and 232 for -actin. PCR products were electrophoresed on a 1% (w/v) agarose gel and detected by UV visualization.