Báo cáo khoa học: Differential regulation of fatty acid amide hydrolase promoter in human immune cells and neuronal cells by leptin and progesterone pdf

Eur J Biochem 271, 4666–4676 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04427.x Differential regulation of fatty acid amide hydrolase promoter in human immune cells and neuronal cells by leptin and progesterone ` Mauro Maccarrone1,2, Valeria Gasperi3, Filomena Fezza1, Alessandro Finazzi-Agro3 and Antonello Rossi3 Department of Biomedical Sciences, University of Teramo, Italy; 2IRCCS C Mondino, Mondino-Tor Vergata-Santa Lucia Center for Experimental Neurobiology, Rome, Italy; 3Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Rome, Italy We have shown recently that in human T lymphocytes, leptin stimulates activity and expression of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH), through STAT3 (signal transducer and activator of transcription 3) and its CRE (cAMP response element)like transcriptional target in the FAAH promoter [Maccar` rone, M., Di Rienzo, M., Finazzi-Agro, A., & Rossi, A (2003) J Biol Chem 278, 13318–13324] We have also shown that progesterone, alone or additively with leptin, up-regulates the FAAH gene in human T-cells, through the Ikaros transcription factor [Maccarrone, M., Bari, M., Di ` Rienzo, M., Finazzi-Agro, A., & Rossi, A (2003) J Biol Chem 278, 32726–32732] Here, we extend these observations to immortalized human lymphoma U937 cells, where stimulation of FAAH by leptin (up to  300% of the controls) involves binding to a leptin receptor (Kd ¼ 2.0 ± 0.1 nM, Bmax ¼ 382 ± fmolỈmg protein)1, appar- ent molecular mass of  110 kDa), and stimulation by progesterone involves an intracellular receptor of  120 kDa Unlike FAAH, the other proteins of the endocannabinoid system are not modulated by the two hormones Interestingly, human neuroblastoma CHP100 cells also have a leptin receptor ( 110 kDa, Kd ¼ 2.2 ± 0.2 nM, Bmax ¼ 339 ± fmolỈmg protein)1), a progesterone receptor ( 120 kDa), STAT3 and Ikaros, yet their FAAH is not activated by leptin or progesterone These data, corroborated by transient expression and electrophoretic mobility-shift assays, demonstrate an unprecedented cell-specific regulation of the FAAH gene, which has important implications for the control of tone and activity of AEA along the neuroimmune axis Leptin is the 16-kDa nonglycosylated product of the obese gene, which is secreted by adipose cells, is released into the circulation, and transported across the blood–brainbarrier into the central nervous system, where it regulates energy homeostasis [1] Leptin also serves systemic functions, apart from those related to food intake and energy expenditure in mammals, including regulation of fertility [2] and modulation of immune response [3] These two actions might be interconnected in humans because leptin alters the production from T lymphocytes of T helper (Th1) and Th2 cytokines [4], which are critical in regulating embryo implantation and materno-fetal exchanges [5,6] Leptin signaling is mediated by the long isoform of the leptin receptor (LR) via different pathways, among which those mediated by the signal transducer and activator of transcription (STAT) members have a prominent role [7,8] Recently, we have shown that leptin reduces the levels of the endocannabinoid anandamide (N-arachidonoylethanolamine, AEA) in human peripheral T lymphocytes by up-regulating the activity and expression of the AEA hydrolase (fatty acid amide hydrolase, FAAH) in these cells [9] The effect of leptin occurred through a STAT3-dependent activation of a CRE (cAMP response element)-like site in the promoter region of FAAH gene [9] Remarkably, leptin has been shown to reduce the levels of AEA also in the hypothalamus of ob/ob mice [10], suggesting that this compound partakes in the neural circuitry regulated by leptin [11,12] We have also shown that the activity and expression of FAAH in human T cells is up-regulated by progesterone, which increases the nuclear levels of the Ikaros transcription factor [13,14], and hence its binding to a specific site in the promoter region of the FAAH gene [15] Progesterone is a critical hormone both for fertility and immune response [5,6] In addition, progesterone is known to regulate a number of activities in the central nervous system [16,17] Correspondence to M Maccarrone, Department of Biomedical Sciences, University of Teramo, Piazza A Moro 45, 64100 Teramo, Italy Fax: +39 0861 412583; Tel.: +39 0861 266875; E-mail: Maccarrone@vet.unite.it Abbreviations: AEA, anandamide (N-arachidonoylethanolamine); AMT, AEA membrane transporter; CAT, chloramphenicol acetyltransferase; CRE, cAMP-response element; FAAH, fatty acid amide hydrolase; GAR/M-AP, goat anti-rabbit/mouse Igs conjugated with alkaline phosphatase; IGF-IR, insulin-like growth factor I receptor; Ik, Ikaros; (s)LR, (soluble) leptin receptor; NAPE, N-acylphosphatidylethanolamine; NAT, N-acyltransferase; PLD, phospholipase D; PR, progesterone receptor; STAT, signal transducer and activator of transcription Enzymes: Fatty acid amide hydrolase (EC 3.5.1.4); phosphatidylcholine phosphatidohydrolase (EC 3.1.4.4) (Received June 2004, revised 31 August 2004, accepted October 2004) Keywords: endocannabinoids; immune system; leptin; neurons; progesterone Ó FEBS 2004 Differential regulation of the human FAAH promoter (Eur J Biochem 271) 4667 Based on this background, we sought to extend our previous observations on the effect of leptin and progesterone on FAAH activity and expression in primary human T lymphocytes to immortalized human lymphoma U937 cells These are a model of peripheral immune cells shown to dispose AEA through an active FAAH and a selective AEA membrane transporter (AMT) [18,19] In addition, we investigated whether leptin and progesterone can modulate the Ôon demandÕ synthesis of AEA through N-acyltransferase [20] and N-acylphosphatidylethanolamine (NAPE)-specific phospholipase D (PLD) [21] In the light of the manifold actions of AEA in the neuroimmune axis [22,23], we sought to investigate the effect of leptin and progesterone also on FAAH, AMT, NAT and PLD of human neuroblastoma CHP100 cells, a model of neuronal cells shown to metabolize AEA [18,19] We report unprecedented evidence that leptin and progesterone up-regulate FAAH, but not AMT, NAT or PLD, in human immune cells, while they not in human neuronal cells Therefore, the central actions of these two hormones not seem to involve the endocannabinoid system but instead may play a role in the effects of leptin and progesterone in the immune system This differential regulation of FAAH suggests that the same factors can lead to a cell-specific control of the tone [24] and the activity of AEA along the neuroimmune axis, with important pathophysiological implications In addition, bearing in mind that FAAH has amidase or esterase activity on many bioactive substrates [22,24], it can be anticipated that the reported findings might have relevance beyond AEA and related endocannabinoids sLR are enough to neutralize the effects of 10 nM ( 150 ngỈmL)1) leptin, and in this study we used a two-fold excess of each neutralizing agent [9] Goat antirabbit and goat anti-mouse Igs conjugated to alkaline phosphatase (GAR-AP and GAM-AP) were from Bio-Rad (Hercules, CA, USA) Cell culture and treatment Human lymphoma U937 cells (ATCC, Mannassas, VA, USA) were cultured in RPMI 1640 medium (Gibco, Paisley, UK), supplemented with 25 mM Hepes, 2.5 mM sodium pyruvate, 100 mL)1 penicillin, 100 lgỈmL)1 streptomycin and 10% heat-inactivated fetal bovine serum [18] Human neuroblastoma CHP100 cells (ATCC, Mannassas, VA, USA) were cultured in a : mixture of MEM (Eagle’s minimal essential medium plus Earle’s salts) and Ham’s F-12 media (Flow Laboratories Ltd, Irvine, UK), supplemented with 15% heat-inactivated fetal bovine serum, sodium bicarbonate (1.2 gỈL)1), 15 mM Hepes buffer, mM L-glutamine and 1% nonessential amino acids [18] Both CHP100 and U937 cells were maintained at 37 °C in humidified 5% CO2 atmosphere Incubation of U937 or CHP100 cells with leptin, alone or in the presence of different compounds, or with progesterone was performed at 37 °C in humidified 5% CO2 atmosphere, at the indicated concentrations for 24 h In the case of progesterone, cells were treated for h in serum-free medium, then heatinactivated fetal bovine serum was added at a final concentration of 10%, as reported [15] Controls were incubated with vehicles alone Cell viability after each treatment was tested by Trypan blue dye exclusion, and was found to be higher than 90% in all cases Materials and methods Anandamide hydrolase activity and expression Materials Chemicals were of the purest analytical grade Leptin (human recombinant), anandamide (N-arachidonoylethanolamine, AEA), progesterone and mifepristone (RU486) were purchased from Sigma Chemical Co (St Louis, MO, USA) [3H]AEA (223 CiỈmmol)1), 125I-labeled leptin (2200 Ciặmmol)1) and [3H]CP55.940 {5-(1,1Â-dimethyheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl) cyclohexyl]phenol; 126 Ci mmol)1} were from NEN Life Science Products, Inc (Boston, MA, USA) [3H]N-arachidonoylphosphatidylethanolamine (200 CiỈmmol)1) was from ARC (St Louis, MO, USA), and 1,2-di[1-14C]palmitoyl-phosphatidylcholine (111 mCiỈmmol)1) was from Amersham Pharmacia Biotech (Uppsala, Sweden) Anti-FAAH polyclonal Igs were raised in rabbits against the conserved FAAH sequence VGYYETDNYTMPSPAMR [25] conjugated to ovalbumin, and were prepared by Primm S.r.l (Milan, Italy) Mouse monoclonal antibodies against actin, STAT3, phospho-STAT3, leptin receptor (LR) and progesterone receptor (PR), LR and PR blocking peptides, and rabbit anti-(Ikaros serum) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) Anti-human insulin-like growth factor I receptor (anti-IGF-IR) and human leptin receptor/Fc chimeras (soluble LR, sLR) were purchased from R & D Systems (Minneapolis, MN, USA) According to the manufacturer’s instructions,  1.5 lgỈmL)1 of Fatty acid amide hydrolase (EC 3.5.1.4; FAAH) activity was assayed at pH 9.0 by the reversed-phase HPLC method described previously [18] using 10 lM [3H]AEA as substrate Cell homogenates (20 lg per lane) were prepared as described previously [18] and were subjected to SDS/PAGE (12%), under reducing conditions Rainbow molecular mass markers (Amersham Pharmacia Biotech, Buckinghamshire, UK) were phosphorylase b (97.4 kDa) and bovine serum albumin (66.0 kDa) For immunochemical analysis, gels were electroblotted onto 0.45 lm nitrocellulose filters (Bio-Rad), and FAAH was visualized with antiFAAH polyclonal Ig (1 : 200), using GAR-AP diluted : 2000 as second antibody [9] Actin was immunodetected with monoclonal anti-actin Ig (1 : 500), using GAM-AP diluted : 2000 as second antibody [9] The same antiFAAH Igs were used to further quantify FAAH protein by enzyme-linked immunosorbent assay (ELISA) Wells were coated with cell homogenates (20 lg per well), which were then reacted with polyclonal anti-FAAH Igs (diluted : 300), as first antibody, and with GAR-AP, diluted : 2000, as second antibody [9] Color development of the alkaline phosphatase reaction was measured at 405 nm, using p-nitrophenyl phosphate as substrate The A405 values could not be converted into FAAH concentrations because the purified enzyme was not available to make calibration curves However, the ELISA test was linear in the range 4668 M Maccarrone et al (Eur J Biochem 271) 0–50 lg per well of cell homogenate and its specificity for FAAH was validated by antigen competition experiments [9] RT-PCR was performed using total RNA isolated from U937 or CHP100 cells (10 · 106 cells) by means of the SNAprogesteroneTM Total RNA Isolation Kit (Invitrogen, Carlsbad, CA, USA), as described [9] RT-PCR reactions were performed using 100 ng of total RNA, for the amplification of FAAH, or 0.4 ng, for 18S rRNA, and the EZ rTth RNA PCR kit (PerkinElmer, Norwalk, CO, USA) The amplification parameters were as follows: at 95 °C, 45 s at 95 °C, 30 s at 55 °C, and 30 s at 60 °C Linear amplification was observed after 20 cycles The primers were the following: (+) 5¢-TGGAAGTCCTCCA AAAGCCCAG (–) 5¢-TGTCCATAGACACAGCCCTT CAG, for FAAH; (+) 5¢-AGTTGCTGCAGTTAAAA AGC (–) 5¢-CCTCAGTTCCGAAAA CCAAC, for 18S rRNA Five microliters of the reaction mixture were electrophoresed on a 6% polyacrylamide gel, which was then dried and subjected to autoradiography [9] In some experiments, the RT-PCR products were excised from the gel and counted in a LKB1214 Rackbeta scintillation counter (Amersham Pharmacia Biotech) Products were validated by size determination and sequencing, as described previously [18] Analysis of N-acyltransferase, phospholipase D, anandamide transporter and leptin receptor binding An N-acyltransferase (NAT) assay was performed as described [20], using 1,2-di[1–14C]palmitoyl-phosphatidylcholine (1 · 106 d.p.m per test) as substrate and measuring the formation of N-[14C]palmitoyl-phosphatidylethanolamines by high performance TLC on silica gel plates (Sigma Chemical Co.) 1,2-Dipalmitoyl,N-palmitoyl-phosphatidylethanolamine was used as a standard, and NAT activity was expressed as pmol N-palmitoyl-phosphatidylethanolamine formed per per mg protein [20] The activity of phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4; PLD) was assayed in cell homogenates as described [21], by measuring the release of [3H]AEA from [3H]N-arachidonoylphosphatidylethanolamine (100 lM) as substrate [26] PLD activity was expressed as pmol AEA released per per mg protein The uptake of 500 nM [3H]AEA by intact cells (2 · 106 per test) through AMT was studied as described [18], and was expressed as pmol AEA taken up per per mg protein The binding of 125I-labeled leptin to U937 or CHP100 cells was analyzed by rapid filtration assays [27] In this case, apparent dissociation constant (Kd) and maximum binding (Bmax) values were calculated from saturation curves in the range 0–12 nM, elaborating the binding data through nonlinear regression analysis with the PRISM3 program (GraphPAD Sofware for Science, San Diego, CA, USA) [9] Unspecific binding was determined in the presence of 100 nM ÔcoldÕ leptin [27] The expression of leptin receptor (LR) and of progesterone receptor (PR) in human cells was assessed by Western blot analysis, performed as detailed above for FAAH, with monoclonal anti-LR or anti-PR Igs (diluted : 500) as first antibody, and GAM-AP diluted : 2000 as second antibody [9] The specificity of anti-LR and anti-PR Igs was ascertained by using the corresponding blocking peptides in competition assays [18] Ó FEBS 2004 Western blot analysis of protein phosphorylation and nuclear levels of Ikaros For the analysis of total STAT3 and of the corresponding phosphorylated (activated) form, whole cell extracts were prepared as reported previously [8] For the determination of Ikaros isoforms, nuclear extracts were prepared from U937 or CHP100 cell suspensions as reported [13] In all cases, whole cell lysates and nuclear extracts (50 lg protein) were loaded onto 10% SDS/polyacrylamide gels, and were then electroblotted onto 0.45-lm nitrocellulose filters (BioRad), as described above for FAAH For immunodetection, the specific first antibody was diluted : 1000, and the appropriate second antibody (GAM-AP or GAR-AP) was diluted : 2000 [9] Protein content was normalized before loading onto the gel [28], and equal loading of extracts was verified by Ponceau staining [13] Cytosolic levels of phospho-STAT3 and nuclear levels of total Ikaros isoforms were quantified by ELISA, performed by coating each well with 25 lg protein/sample, then reacted with monoclonal anti-(phospho-STAT3 Ig) or polyclonal anti-Ikaros Igs (1 : 1000) and GAM-AP or GAR-AP (1 : 2000), respectively, as reported [15] Construction of chloramphenicol acetyltransferase expression vectors and transient transfection Sequence information for the upstream regulatory region of the FAAH gene was downloaded from GenBank (region: gi|11423254 : 644582–754250, International Human Genome Project) and the proximal promoter region of basepairs from +1 to )107 (+1 being the first nucleotide of the FAAH mRNA) was assembled using synthetic oligonucleotides (Amersham Pharmacia Biotech) The DNA was gel-purified and subcloned into the PstI/XbaI sites of pCAT3-Basic vector (Promega Corporation, Madison, WI, USA) The same strategy was used to introduce mutations in the recombinant plasmids bearing the promoter region The nucleotide sequences of all constructs were verified by dideoxynucleotide chain termination sequencing with a Sequenase kit 2.0 (USB, Cleveland, OH, USA) Human U937 or CHP100 cells (1 · 106 per test) were transfected in triplicate using TransFastTM Transfection Reagent (Promega Corporation), according to the manufacturer’s instructions Typically, cells were washed in phosphate-buffered saline and resuspended in 0.5 mL of serum-free medium, then they were mixed with 0.5 mL of serum-free medium containing lg of total DNA and the TransFastTM Transfection Reagent, at a charge ratio of : with respect to DNA Transfection efficiency was monitored by use of 0.5 lg thymidine kinase b-galactosidase construct (Clontech, Palo Alto, CA, USA) After transfection, the medium was replaced with complete growth medium, and cells were harvested 48 h later For chloramphenicol acetyltransferase (CAT) activity assays, cellular extracts were prepared as described above for FAAH, and different aliquots were used for CAT assays, for b-galactosidase (b-gal) activity determination, a marker of transfection efficiency, and for protein quantitation CAT activity was determined using the Quan-T-CAT assay system (Amersham Life Sciences), whereas the activity of b-gal was assayed using the b-Galactosidase Enzyme System Ó FEBS 2004 Differential regulation of the human FAAH promoter (Eur J Biochem 271) 4669 (Promega Corporation) The values of CAT activity were normalized to b-gal activity and to the protein content and the relative CAT values were the average of at least three independent experiments, each performed in duplicate Nuclear extracts and electrophoretic mobility-shift assays Nuclear extracts were prepared according to Schreiber et al [29] with the modifications reported by Lee et al [30] Electrophoretic mobility-shift assay (EMSA) experiments were performed as described [30], deriving the sequence for the wild-type CRE-like site bandshift from the FAAH promoter region: )61 5¢-CCCGGC TGATCCAGTCCG-3¢ )44 (site in bold typeface) The sequence for the mutated site was the same used for the transfection experiments, i.e )61 5¢-CCCGGCAAATCAAAGTCCG-3¢ )44 (mutated nucleotides are in italic typeface) The Ikaros oligonucleotide was )76 5¢-AGGCGG GCGTGGGATCCCGGCTG3¢ )54 (site in bold), whereas the oligonucleotides used for the cold competitions were 5¢-CTCGCAGCCTGGGAA GATAAGTGG-3¢ (Ikaros site derived from vasoactive intestinal peptide receptor-1 promoter), and )76 5¢-AGG CGG GCGTTTTTTCCCGGCTG-3¢ )54, which is the mutated site used for the transfection experiments (the mutated nucleotides are in italic tyepface) [31] In all oligonucleotides, the numbers refer to positions in the FAAH promoter The complexes were resolved on nondenaturing 6% polyacrylamide gels in 0.5· TBE buffer (0.45 M Tris/borate, 10 mM EDTA, pH 8.0) for h at 14 VỈcm)1, and were autoradiographed overnight Statistical analyses Data reported in this paper are the mean (± SD) of at least three independent determinations, each in duplicate Statistical analysis was performed using the nonparametric Mann–Whitney U-test, elaborating experimental data by means of the INSTAT3 program (GraphPAD Software for Science) Results Leptin and progesterone stimulate FAAH activity and expression in human U937 cells but not in CHP100 cells In previous studies, we have shown that leptin and progesterone up-regulate FAAH activity and expression in human peripheral lymphocytes [9,15] Here, we extend these observations to human U937 cells, where treatment with leptin for 24 h enhanced FAAH activity in a dose-dependent manner (Fig 1A) FAAH activation reached statistical significance (P < 0.05) at 2.5 nM leptin and a  300% maximum at 10 nM Therefore, the last concentration was chosen to further investigate the effect of leptin on FAAH FAAH activation by 10 nM leptin (corresponding to  150 ngỈmL)1) was fully prevented by soluble leptin receptor (sLR), used at neutralizing concentration of lgỈmL)1 Instead, ƠmockÕ antibodies against IGF-IR were ineffective at the same concentration (not shown) Also, progesterone increased the activity of FAAH, in a dosedependent manner (Fig 1B), at the same concentrations used in the previous investigation [15] At a dose of lM, progesterone enhanced FAAH activity and expression to  240% of the untreated control after 24 h of treatment (Fig 1B), and this effect was fully reversed by the synthetic antiprogestinic compound RU486 (Fig 1B), used at the blocking concentration of 10 lM [15] Western blot analysis of U937 cell extracts showed that specific anti-FAAH Igs recognized a single immunoreactive band of the molecular size expected for FAAH, the intensity of which was higher in U937 cells treated with 10 nM leptin or lM progesterone than in controls (Fig 1C) On the other hand, U937 cells treated with 10 nM leptin or lM progesterone expressed the same levels of actin as the controls (Fig 1C), ruling out that the different levels of FAAH in these cells might be due to unequal loading of proteins The same anti-FAAH Igs were used to further quantify FAAH content by ELISA, which showed that leptin (Fig 1A) and progesterone (Fig 1B) increased FAAH protein in human U937 cells in parallel to the increase of enzymic activity RT-PCR amplification of cDNA of U937 cells showed a single band of the expected molecular size for FAAH gene, which increased in cells treated with 10 nM leptin or lM progesterone (Fig 1C) Under the same experimental conditions, the expression of the 18S rRNA gene was unaffected (Fig 1C) Liquid scintillation counting of RT-PCR products showed that leptin (Fig 1A) and progesterone (Fig 1B) increased FAAH mRNA in a dosedependent manner in human U937 cells, in a way parallel to that of enzymic activity and protein content The effect of leptin and progesterone on FAAH activity and expression was additive, in fact treatment of U937 cells for 24 h with 10 nM leptin and lM progesterone enhanced FAAH activity, protein level and mRNA content up to  500%, 450% and 490% of the controls, respectively (Table 1) On the other hand, the same concentrations of leptin and progesterone which enhanced FAAH activity and expression were ineffective on the activity of AMT, NAT and PLD in U937 cells, when used either alone or in combination (Table 2) It should be recalled that U937 cells not express functional cannabinoid receptors [18] Unlike U937 cells, FAAH activity and expression in human neuroblastoma CHP100 cells was not affected by leptin and progesterone, used either alone or in combination, neither was the activity of AMT, NAT and PLD in the same cells (Figs 1A,B,D and data not shown) It should be recalled that also CHP100 cells are devoid of cannabinoid receptors [18] Both U937 and CHP100 cells have leptin receptor and progesterone receptor Human U937 and CHP100 cells were able to bind 125 I-labeled leptin according to a saturable process (Fig 2A) that yielded apparent dissociation constants (Kd) of 2.0 ± 0.1 and 2.2 ± 0.2 nM and maximum binding (Bmax) of 382 ± and 339 ± fmolỈmg protein)1, for U937 cells and CHP100 cells, respectively These values are in agreement with previous reports on LR of human hepatic cells [32], and on LR stably transfected into different cell types [27,33] ÔColdÕ leptin fully displaced 125I-labeled leptin from its binding site, when used at 100 nM (Fig 2A) Moreover, Western blot analysis showed that specific anti-LR Igs Ó FEBS 2004 4670 M Maccarrone et al (Eur J Biochem 271) Fig Effect of leptin and progesterone on FAAH activity and expression (A) Effect of leptin on the activity of FAAH in human U937 and CHP100 cells and on the protein content and the mRNA of FAAH in U937 cells These cells were incubated for 24 h with leptin alone, or with 10 nM ( 150 ngỈmL)1) leptin in the presence of lgỈmL)1 soluble leptin receptor (sLR) (100% activity in U937 cells is 350 ± 30 pmolỈmin)1Ỉmg protein)1; protein content, 0.280 ± 0.040 A405 and mRNA level, 11 000 ± 1300 c.p.m; 100% activity in CHP100 cells, 25 ± pmolỈmin)1Ỉmg protein)1) (B) Effect of progesterone on the same samples as in (A) Cells were incubated with progesterone alone, or with lM progesterone in the presence of 10 lM RU486 In both panels: *P < 0.05 vs control, **P < 0.01 vs control and #P < 0.01 vs 10 nM leptin or vs lM progesterone, respectively In both panels, vertical bars represent SD values (C) Upper panel, Western blot analysis of U937 cells, treated with 10 nM leptin or lM progesterone and reacted with specific anti-FAAH (top) or anti-actin (bottom) Igs The positions of FAAH and actin are indicated to the right Lower panel, RT-PCR analysis of cDNA of the same samples as in (C, upper panel) The expected sizes of the amplicons (199 base pairs for FAAH and 258 base pairs for 18S rRNA) are indicated to the right (D) Same as in (C) on CHP100 cells Data shown in (C,D) are representative of three independent experiments Table Modulation of FAAH expression in human U937 cells by leptin and progesterone Activity, 100% ¼ 350 ± 30 pmolỈmin)1Ỉmg protein)1; Protein, 0.280 ± 0.040 A405; mRNA level, 11 000 ± 1300 c.p.m Treatment of U937 cells FAAH Expression (%) Treatment of U937 cells Control Leptin (10 nM ) for 24 h Progesterone (1 lM ) for 24 h Leptin (10 nM ) + progesterone (1 lM ) for 24 h Table Effect of leptin and progesterone on the activity of AEA membrane transporter (AMT), N-acyltransferase (NAT) and phospholipase D (PLD) in human U937 cells Activity Protein mRNA 100 300 ± 35* 100 270 ± 30* 100 310 ± 35* 240 ± 26* 220 ± 25* 240 ± 25* 500 ± 45*# 450 ± 50*# 490 ± 45*# Control Leptin (10 nM ) for 24 h Progesterone (1 lM ) for 24 h Leptin (10 nM ) + progesterone (1 lM ) for 24 h AMT a NAT b PLD 100 100 ± 10 100 90 ± 10 100c 100 ± 10 90 ± 10 90 ± 10 110 ± 10 100 ± 10 100 ± 10 100 ± 10 100%, 150 ± 20 pmolỈmin)1Ỉmg protein)1; b 100%, 14 ± pmolỈ min)1Ỉmg protein)1; c 100%, 90 ± 10 pmolỈmin)1Ỉmg protein)1 a *P < 0.01 vs control; #P < 0.01 vs 10 nM leptin or vs lM progesterone recognized a single immunoreactive band in U937 and CHP100 cell extracts, with an apparent molecular mass of  110 kDa (Fig 2B) This value is consistent with the expected molecular mass of the long form of LR [1,33] Western blot analysis also showed that specific anti-PR Igs recognized a single immunoreactive band with the same intensity in U937 and CHP100 cells, with an apparent molecular mass of  120 kDa (Fig 2C) This value is consistent with the expected molecular mass of the full length form of PR [17] Activation of downstream signals by binding to LR or PR In previous studies, we have shown that up-regulation of FAAH activity and expression in human T lymphocytes by Ó FEBS 2004 Differential regulation of the human FAAH promoter (Eur J Biochem 271) 4671 cells, respectively (Fig 3C) Neither phospho-STAT3 nor Ikaros increased in CHP100 cells under the same experimental conditions (Fig 3D) Analysis of the FAAH promoter Fig Characterization of leptin receptor (LR) and progesterone receptor (PR) (A) Saturation curves of the binding of 125I-labeled leptin to human U937 or CHP100 cells, alone or in the presence of 100 nM ÔcoldÕ leptin (data of the ÔcoldÕ leptin experiment with CHP100 cells were superimposable to those of U937 cells, and were omitted for the sake of clarity) Western blot analysis of cell extracts (20 lg per lane), reacted with anti-LR (B) or anti-PR (C) Igs Molecular mass markers and the position of LR and PR are indicated to the right Data shown in (B,C) are representative of three independent experiments In (A), vertical bars represent SD values leptin [9] or progesterone [15] occurs through phosphorylation of STAT3, or increase of the nuclear content of the transcription factor Ikaros, respectively Therefore, the levels of phospho-STAT3 and Ikaros were determined in leptin-treated and progesterone-treated U937 and CHP100 cells The nonphosphorylated, inactive form of STAT3 was present in both cell-types, yet the active phospho-STAT3 increased only in U937 cells treated with 10 nM leptin (Fig 3A) In addition, nuclear levels of Ikaros isoforms Ik1, Ik2 and Ik3 were increased by treatment of U937 cells, but not of CHP100 cells, for 24 h with lM progesterone (Fig 3B) Quantitative analysis of phospho-STAT3 and of total Ikaros isoforms by ELISA corroborated the Western blot data, showing that phospho-STAT3 and Ikaros increased up to  300 and 250% of the untreated controls in leptin-treated or progesterone-treated U937 The human FAAH gene has been located on chromosome [34] and the latter has been completely sequenced Therefore, we have inspected this chromosome to gain insight on the FAAH promoter features Human FAAH promoter was found to lack TATA boxes and, like many genes bearing this feature, it had a proximally positioned SP1 site (Fig 4A) Moreover, there was another SP1 site in the reverse orientation  100 nucleotides upstream (Fig 4A), a feature which resembles the structure of the mouse proximal promoter [35] Inspection of the promoter sequence did not show any obvious binding site for STAT3, however, it had a CRE-like site (Fig 4A), which is a target for transcriptional regulation based on STAT3 tethering [36] In addition, the FAAH promoter had an Ikaros consensus site located at position )66, upstream the CRE-like element (Fig 4A) Transient transfections using constructs containing both the SP1 and the CRE-like ()107 to +1) sites driving the CAT reporter gene in U937, but not in CHP100, cells showed that the )107 construct was up-regulated by leptin (Fig 4B, wt and wt + L) Disruption of this site by mutation abolished the leptin up-regulation (Fig 4B, mutL and mutL + L) Also progesterone up-regulated the reporter CAT gene in U937, but not in CHP100, cells if under control of the region between the two SP1 sites of the FAAH upstream region (Fig 4B, wt and wt + P) Moreover, mutation of the Ikaros consensus site abolished FAAH up-regulation by progesterone (Fig 4B, mutIk and mutIk + P) The effect of leptin and progesterone on FAAH promoter was additive (Fig 4B, wt + L + P) To confirm that leptin and progesterone act through a CRE-like element and an Ikaros consensus site, respectively, EMSA experiments were performed using nuclear extracts prepared from U937 or CHP100 cells, untreated or treated for 24 h with 10 nM leptin or lM progesterone As shown in Fig 5A, complex formation was only found with oligonucleotides containing the CRE-like site of the FAAH promoter Complex formation was not seen when the mutant oligonucleotide (bearing the same mutation used for the transient transfection experiment) was used as a 32 P-labeled probe, neither was it observed in CHP100 cells under the same experimental conditions (Fig 5A) On the other hand, specificity of the binding to the Ikaros site was confirmed by using a cold competitor, which corresponds to an established functional Ik site derived from the vasoactive intestinal peptide receptor-1 promoter [31], and the mutated site used for transient transfection experiments Cold competitor and mutated site, respectively, abolished or had no effect on the retarded complex in U937 cells (Fig 5B) Conversely, no retarded complex was seen in CHP100 cells under the same experimental conditions (Fig 5C) Discussion In this study we show that leptin and progesterone stimulate, independently (Fig 1A,D) or additively 4672 M Maccarrone et al (Eur J Biochem 271) Ó FEBS 2004 Fig Activation of downstream signals by leptin (L) or progesterone (P) Human U937 or CHP100 cells were treated for 24 h with 10 nM leptin (A) or lM progesterone (B), then lysates (50 lg of protein) were immunoblotted with the specific antibody against the inactive (total) or active (phosphorylated, phospho) form of STAT3 (A), or against Ikaros isoforms Ik1, Ik2 and Ik3 (B) The positions of (phospho) proteins are indicated to the right These data are representative of three independent experiments Quantitative analysis by ELISA of the same samples (25 lg of protein) shown in (A,B) is reported in (C,D), where *P < 0.01 vs control (CTR), and vertical bars represent SD values (Table 1), FAAH activity and expression in human lymphoma U937 cells, but not in human neuroblastoma CHP100 cells FAAH activation occurs via a leptin receptor-mediated activation of STAT3 signaling and/or via a progesterone receptor-mediated increase in nuclear levels of Ikaros, which in turn leads to up-regulation of a CRE-like site or of an Ikaros binding site, respectively, in the FAAH promoter Leptin modulates FAAH activity and expression in U937 cells at the same circulating levels shown to modulate immune cell response [4] These concentrations of leptin were found to saturate the binding sites in U937 cells (Fig 2A), with calculated binding constants (Kd and Bmax) similar to those of the leptin receptor [32,33] This observation, together with the ability of ÔcoldÕ leptin to fully displace 125I-labeled leptin, strongly suggests that U937 cells have an authentic LR Western blot analysis further corroborates this hypothesis, showing that specific antiLR Igs recognized a single band (Fig 2B) of the expected molecular mass of the long isoform of LR [1,27] These data extend our recent observations on primary human T-cells [9] and suggest that regulation by leptin, being conserved in immortalized cells, has a critical role for the immune function Also the effects of progesterone, used at circulating levels of lM ( 0.3 lgỈmL)1), on FAAH activity in U937 cells extend our previous observations on T lymphocytes [15], and here we show for the first time that these cells have the full length form of PR (Fig 2C) As this form is known to function as a transcriptional activator of progesterone-responsive genes [17,37,38], this finding gives ground to the hypothesis that the effects of progesterone on FAAH gene expression were mediated through its binding to PR [15] On the other hand, leptin and progesterone were ineffective on AEA transport through AMT, and on AEA synthesis through NAT and PLD in U937 cells (Table 2), suggesting that FAAH was the only Ôcheck pointÕ for the effect of both leptin and progesterone These observations are in keeping with the hypothesis that FAAH is the keyregulator of AEA levels in vivo, indeed FAAH knockout mice show  15-fold higher levels of AEA than wild-type littermates [39], and AEA levels in human blood inversely correlate with FAAH activity in peripheral lymphocytes [40] The mechanism of FAAH activation by leptin and progesterone was further investigated Binding of leptin to the long isoform of LR is known to trigger different signaling pathways, among which STAT-dependent signal transduction is prominent [7,8] In human T lymphocytes, we have shown recently that leptin activated (i.e phosphorylated) only STAT3 [9], which is preferentially activated also in other cell types [33,36] and tissues ([1] and references therein) Phospho-STAT3 was up-regulated by leptin also in U937 cells (Fig 3A,C), where the FAAH promoter did not contain STAT3 DNA binding motifs, yet it did contain a CRE-like element between the two SP1 sites (Fig 4A) Such CRE-like elements have been shown recently to be tethered by STAT3, thus, leading to a novel type of up-regulation of gene transcription [36] Transfection experiments using FAAH promoter constructs with mutated CRE-like elements (mutL) revealed that indeed these sites confer STAT3 responsiveness (Fig 4B) EMSA analysis further corroborated this conclusion (Fig 5A) FAAH promoter also revealed a binding site for Ikaros, a member of the Kruppel family of Ôzinc fingerÕ DNAbinding proteins that acts as critical transcriptional regulator of lymphocyte ontogeny and differentiation [14] Ikaros comprises eight different isoforms, Ik1 through Ik3 localizing to the nucleus and Ik4 through Ik8 localizing to the cytosol [14] Recently Ik1, and 3, the only isoforms which exhibit high DNA binding affinity [14], have been Ó FEBS 2004 Differential regulation of the human FAAH promoter (Eur J Biochem 271) 4673 Fig Analysis of FAAH promoter by transient expression (A) Proximal upstream region of human FAAH gene Left and right handed arrows denote SP1 sites in the – and + strands, respectively Pentagonal box: Ikaros (Ik) site; oval box: CRE-like site (B) 5¢ Flanking regions of the human FAAH gene were cloned in the PstI/XbaI sites of pCAT3-basic vector, wt: )107 to +1 upstream region containing the two SP1 sites flanking the Ikaros site and the CRE-like site; mutL: same as wt but with the mutated CRE-like sequence; mutIk: same as wt but with mutated Ikaros site; mutated sites are in white Human U937 cells and CHP100 cells were transfected with the aforementioned constructs and left untreated or treated with leptin or progesterone Transfection efficiency was monitored by the use of thymidine kinase b-galactosidase construct The values of CAT activity were normalized to b-gal activity and to the protein content and are expressed as percentage with respect to the activity of the empty vector, pCAT3 basic, which was set to 100% *P < 0.01 vs control, #P < 0.01 vs leptin or progesterone alone, and horizontal bars represent SD values detected in the nucleus of human peripheral blood mononuclear cells [13,14], where they are essential in controlling the activation of granzyme B promoter by the glucocorticoid, dexamethasone [13] Here, we demonstrated that circulating levels of progesterone (1 lM, i.e  0.3 lgỈmL)1) increased the nuclear levels of these Ikaros isoforms in U937 cells  2.5-fold over the controls (Fig 3B,C) This is noteworthy, because homo- and heterodimer formation between Ik1, and is known to greatly increase their affinity for DNA and their consequent ability to activate transcription [13,14] Transfection experiments using FAAH promoter constructs with mutated Ikaros (mutIk) revealed that this site indeed confers progesterone responsiveness (Fig 4B), and EMSA analysis corroborated this conclusion (Fig 5B) Taken together, it can be concluded that in human U937 cells leptin by binding to LR activates STAT3, which in turn up-regulates FAAH gene transcription by tethering to a CRE-like site in the FAAH promoter In addition, progesterone activates the FAAH gene by stimulating the binding of Ikaros to DNA, thus, enhancing its transcriptional activity on the FAAH promoter Activation of the FAAH promoter by leptin may be additive to that of progesterone at the two different sites (Table and Fig 4) Ó FEBS 2004 4674 M Maccarrone et al (Eur J Biochem 271) Fig Analysis of FAAH promoter by electrophoretic mobility-shift assay (EMSA) EMSA experiments were performed with lg of U937 or CHP100 nuclear extracts, prepared before (–) and after (+) stimulation with 10 nM leptin (A) or lM progesterone (B,C) (A) Complex formation (indicated by an arrow) was performed with oligonucleotides as 32P-labeled probes for mutated (mutL) or wild-type (wt) CRE-like site (B) Complex formation (indicated by an arrow) in U937 cells, performed with 32P-labeled oligonucleotide corresponding to the Ikaros site Lane O represents 32P-labeled oligonucleotide alone, which contained the Ikaros site; competitor wild type (wt) was a cold oligonucleotide containing the Ikaros site derived from vasoactive intestinal peptide receptor-1 promoter; competitor mutant (mut) was a cold oligonucleotide containing the mutated Ikaros site used in transfection experiments (C) Same as in (B) with CHP100 cells A major finding of this investigation is that FAAH activity and expression in human neuroblastoma CHP100 cells was not up-regulated by leptin and/or progesterone, although these cells use LR, PR, STAT3 and Ikaros This unprecedented observation gives support to previous reports showing that in mouse hypothalamus FAAH activity is not regulated by leptin [10], whereas it is in mouse peripheral lymphocytes [9] However, the molecular reasons for the lack of responsiveness of neuronal cells remain unclear and different hypotheses can be put forward On one hand, it can be suggested that coactivators triggered by leptin or progesterone in immune cells are missing in neuronal cells On the other hand, it is also possible that silencers of FAAH gene expression are present in neuronal cells but not in immune cells While both hypotheses are in keeping with the tissue-specificity of FAAH promoter regulation observed recently in vitro in mouse cells [41], the observation that background FAAH activity ( 25 vs  350 pmolỈmin)1Ỉmg protein)1 with 10 lM [3H]AEA as substrate; Fig 1) and expression (Fig 1C,D) in CHP100 cells is significantly lower than that in U937 cells seems to speak in favour of a constitutive repression of FAAH gene in the former cells To the best of our knowledge, this is the first demonstration that FAAH is differentially regulated in neuronal and immune cells by the same factors; a finding which may have important implications for the regulation of the tone and activity of AEA in the neuroimmune axis In particular, it seems noteworthy that leptin [42,43] and progesterone [44,45] play roles in survival, death and proliferation of immune and neuronal cells; processes in which AEA and the endocannabinoid system are also involved [19] In particular, leptin and progesterone, by stimulating AEA degradation through FAAH, might prevent apoptosis induced by AEA in immune cells [19], whereas they should be unable to protect by the same mechanism neuronal cells [19] Supporting this is the observation that progesterone enhances AEA hydrolase in immune cells by increasing nuclear levels of Ikaros – this factor is crucial for the in vivo enhancer function of TrkA, the nerve growth factor receptor [46] Trk receptors are suppressed by AEA in breast and prostate cancer cells [47], suggesting a possible functional link between Ikaros, AEA tone and Trk receptors in peripheral cells Finally, it should be pointed out that genes in addition to FAAH may be differentially regulated by leptin and/or progesterone in U937 cells and CHP100 cells, potentially extending the implications of the reported findings beyond the endocannabinoid system In conclusion, the results reported here represent the first evidence of a differential regulation of FAAH gene expression in immune and neuronal cells, suggesting cell-specific links between networks controlled by leptin or progesterone and the peripheral endocannabinoid system Conversely, the central actions of the two hormones not seem to involve modulation of endocannabinoid metabolism 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are present in neuronal cells but not in. .. and proliferation of immune and neuronal cells; processes in which AEA and the endocannabinoid system are also involved [19] In particular, leptin and progesterone, by stimulating AEA degradation