Title: Progesterone Activates Fatty Acid Amide Hydrolase (FAAH) Promoter in Human T Lymphocytes through the Transcription Factor Ikaros
Abstract: Physiological concentrations of progesterone stimulate the activity of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) in human T lymphocytes, up to a ∼270% over the untreated controls. Stimulation of FAAH occurred through up-regulation of gene expression at transcriptional and translational level and was specific. Indeed, neither the activity of the anandamide-synthesizing N-acyltransferase and phospholipase D, nor the activity of the anandamide transporter, nor the binding to cannabinoid receptors were affected by progesterone under the same experimental conditions. The activation of FAAH by progesterone was paralleled by a decrease (down to 60%) of the cellular levels of anandamide and involved increased nuclear levels of the transcription factor Ikaros. Analysis of the FAAH promoter showed an Ikaros binding site, and mutation of this site prevented FAAH activation by progesterone in transient expression assays. Electrophoretic mobility shift and supershift assays further corroborated the promoter activity data. Furthermore, the effect of progesterone on FAAH promoter was additive to that of physiological amounts of leptin, which binds to a cAMP response element-like site in the promoter region. Taken together, these results suggest that progesterone and leptin, by up-regulating the FAAH promoter at different sites, enhance FAAH expression, thus tuning the immunomodulatory effects of anandamide. These findings might also have critical implications for human fertility. Physiological concentrations of progesterone stimulate the activity of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) in human T lymphocytes, up to a ∼270% over the untreated controls. Stimulation of FAAH occurred through up-regulation of gene expression at transcriptional and translational level and was specific. Indeed, neither the activity of the anandamide-synthesizing N-acyltransferase and phospholipase D, nor the activity of the anandamide transporter, nor the binding to cannabinoid receptors were affected by progesterone under the same experimental conditions. The activation of FAAH by progesterone was paralleled by a decrease (down to 60%) of the cellular levels of anandamide and involved increased nuclear levels of the transcription factor Ikaros. Analysis of the FAAH promoter showed an Ikaros binding site, and mutation of this site prevented FAAH activation by progesterone in transient expression assays. Electrophoretic mobility shift and supershift assays further corroborated the promoter activity data. Furthermore, the effect of progesterone on FAAH promoter was additive to that of physiological amounts of leptin, which binds to a cAMP response element-like site in the promoter region. Taken together, these results suggest that progesterone and leptin, by up-regulating the FAAH promoter at different sites, enhance FAAH expression, thus tuning the immunomodulatory effects of anandamide. These findings might also have critical implications for human fertility. Anandamide (arachidonoylethanolamide, AEA) 1The abbreviations used are: AEA, anandamide (N-arachidonoylethanolamine); 2-AG, 2-arachidonoylglycerol; AMT, AEA membrane transporter; CAT, chloramphenicol acetyltransferase; CBR, cannabinoid receptors; CP55.940, 5-(1,1′-dimethyheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl)cyclohexyl]phenol; CRE, cAMP-response element; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; FAAH, fatty acid amide hydrolase; GAR-AP, goat anti-rabbit antibodies conjugated with alkaline phosphatase; Ik, Ikaros; L, leptin; NAT, N-acyltransferase; P, progesterone; RT, reverse transcriptase; PLD, phospholipase D; NAPE, N-acylphosphatidylethanolamines.1The abbreviations used are: AEA, anandamide (N-arachidonoylethanolamine); 2-AG, 2-arachidonoylglycerol; AMT, AEA membrane transporter; CAT, chloramphenicol acetyltransferase; CBR, cannabinoid receptors; CP55.940, 5-(1,1′-dimethyheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl)cyclohexyl]phenol; CRE, cAMP-response element; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; FAAH, fatty acid amide hydrolase; GAR-AP, goat anti-rabbit antibodies conjugated with alkaline phosphatase; Ik, Ikaros; L, leptin; NAT, N-acyltransferase; P, progesterone; RT, reverse transcriptase; PLD, phospholipase D; NAPE, N-acylphosphatidylethanolamines. belongs to a group of endogenous lipids, which include amides, esters, and ethers of long chain polyunsaturated fatty acids, collectively termed "endocannabinoids" (1Mechoulam R. Prostaglandins Leukot. Essent. Fatty Acids. 2002; 66: 93-99Abstract Full Text PDF PubMed Scopus (66) Google Scholar). It binds to cannabinoid receptors (CBR) in the central nervous system and in peripheral cells, thus having many central actions (2Fride E. Prostaglandins Leukot. Essent. Fatty Acids. 2002; 66: 221-233Abstract Full Text PDF PubMed Scopus (156) Google Scholar). Among the peripheral activities of AEA, the regulation of fertility (3Paria B.C. Song H. Wang X. Schmid P.C. Krebsbach R.J. Schmid H.H. Bonner T.I. Zimmer A. Dey S.K. J. Biol. Chem. 2001; 276: 20523-20528Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) and immune function (4Parolaro D. Massi P. Rubino T. Monti E. Prostaglandins Leukot. Essent. Fatty Acids. 2002; 66: 319-332Abstract Full Text PDF PubMed Scopus (116) Google Scholar) has attracted growing interest. These biological actions of AEA are terminated by cellular uptake through an AEA membrane transporter (AMT) (5Fowler C.J. Jacobsson S.O.P. Prostaglandins Leukot. Essent. Fatty Acids. 2002; 66: 193-200Abstract Full Text PDF PubMed Scopus (64) Google Scholar), followed by degradation to ethanolamine and arachidonic acid by the enzyme fatty acid amide hydrolase (FAAH) (6Deutsch D.G. Ueda N. Yamamoto S. Prostaglandins Leukot. Essent. Fatty Acids. 2002; 66: 201-210Abstract Full Text PDF PubMed Scopus (184) Google Scholar). Human lymphocytes have functional CBR, AMT, and FAAH, and the latter enzyme has been shown to play a critical role in regulating human pregnancy (7Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. Lancet. 2000; 355: 1326-1329Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Progesterone (P) is a hormone essential for the maintenance of pregnancy and is also known to modulate immune function (8Correale J. Arias M. Gilmore W. J. Immunol. 1998; 161: 3365-3374PubMed Google Scholar) and to elicit an immunological response critical for normal gestation (9Szekeres-Bartho J. Faust Zs. Varga P. Szereday L. Kelemen K. Am. J. Reprod. Immunol. 1996; 35: 348-351Crossref PubMed Scopus (142) Google Scholar). Indeed, P has been shown to favor the development of human T lymphocytes producing type 2 T-helper (Th2) cytokines (interleukins 4 and 10), which inhibit Th1-type cytokines (interleukin-12 and interferon-γ), thus allowing the survival of fetal allograft and therefore a successful pregnancy (10Piccinni M.P. Giudizi M.G. Biagiotti R. Beloni L. Giannarini L. Sampognaro S. Parronchi P. Manetti R. Annunziato F. Livi C. Romagnani S. Maggi E. J. Immunol. 1995; 155: 128-133PubMed Google Scholar, 11Piccinni M.P. Beloni L. Livi C. Maggi E. Scarselli G. Romagnani S. Nat. Med. 1998; 4: 1020-1024Crossref PubMed Scopus (561) Google Scholar). In this context, the activity of FAAH in maternal peripheral lymphocytes is under control of progesterone, which binds to its intracellular receptor and activates FAAH expression (12Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (137) Google Scholar). As a consequence, the release of cytokines critical for fertility, such as the leukemia inhibitory factor, is favored (12Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (137) Google Scholar). Moreover, lymphocyte FAAH has been shown to control the levels of blood AEA in pregnant women, where low FAAH activity implies high AEA levels, leading to spontaneous abortion (7Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. Lancet. 2000; 355: 1326-1329Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 12Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (137) Google Scholar, 13Maccarrone M. Bisogno T. Valensise H. Lazzarin N. Fezza F. Manna C. Di Marzo V. Finazzi-Agrò A. Mol. Hum. Reprod. 2002; 8: 188-195Crossref PubMed Scopus (163) Google Scholar). Taken together, these data have suggested a cross-talk between steroid hormones, cytokines, and the peripheral endocannabinoid system in lymphocytes, which is implicated in regulating immunity and fertility in humans (14Maccarrone M. Falciglia K. Di Rienzo M. Finazzi-Agrò A. Prostaglandins Leukot. Essent. Fatty Acids. 2002; 66: 309-317Abstract Full Text PDF PubMed Scopus (53) Google Scholar). Also leptin (L), the 16-kDa non-glycosylated product of the obese gene, serves systemic functions, which include regulation of fertility (15Cunningham M.J. Clifton D.K. Steiner R.A. Biol. Reprod. 1999; 60: 216-222Crossref PubMed Scopus (433) Google Scholar) and modulation of immune response (16Matarese G. La Cava A. Sanna V. Lord G.M. Lechler R.I. Fontana S. Zappacosta S. Trends Immunol. 2002; 23: 182-187Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Recently, L has been shown to reduce the levels of AEA in the hypotalamus of ob/ob mice, suggesting that this compound partakes of the neural circuitry regulated by L (17Di Marzo V. Goparaju S.K. Wang L. Liu J. Batkai S. Jarai Z. Fezza F. Miura G.I. Palmiter R.D. Sugiura T. Kunos G. Nature. 2001; 410: 822-825Crossref PubMed Scopus (1348) Google Scholar). In addition, L enhances FAAH expression by activating a CRE (cAMP response element)-like site in the promoter region (18Maccarrone M. Di Rienzo M. Finazzi-Agrò A. Rossi A. J. Biol. Chem. 2003; 278: 13318-13324Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Based on this background, we sought to investigate whether P might regulate AEA metabolism in human T lymphocytes. In fact, we show an enhancement of FAAH activity and expression by P, triggered through overexpression of the Ikaros (Ik) transcription factor (19Wargnier A. Lafaurie C. Legros-Maida S. Bourge J.-F. Sigaux F. Sasportes M. Pauli P. J. Biol. Chem. 1998; 273: 35326-35331Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 20Sun L. Heerema N. Crotty L. Wu X. Navara C. Vassilev A. Sensel M. Reaman G.H. Uckun F.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 680-685Crossref PubMed Scopus (168) Google Scholar) and subsequent Ik-dependent up-regulation of promoter activity. We also show that the effect of P is synergistic with that of L. Materials—Chemicals were of the purest analytical grade. AEA, P, mifepristone (RU486), L (human recombinant), and 1,2-dipalmitoyl-N-palmitoylphosphatidylethanolamine were purchased from Sigma. [3H]AEA (223 Ci/mmol) and [3H]CP55.940 (5-(1,1′-dimethyheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl)cyclohexyl]phenol; 126 Ci/mmol) were from PerkinElmer Life Sciences. 1,2-Dioleoyl-3-phosphatidyl[2-14C]ethanolamine (55 mCi/mmol) and 1,2-di[1-14C]palmitoylphosphatidylcholine (111 mCi/mmol) were from Amersham Biosciences (Uppsala, Sweden). Anti-FAAH polyclonal antibodies were elicited in rabbits against the conserved FAAH sequence VGYYETDNYTMPSPAMR (21Giang D.K. Cravatt B.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2238-2242Crossref PubMed Scopus (387) Google Scholar) conjugated to ovalbumin and were prepared by Primm S.r.l. (Milan, Italy). Rabbit anti-Ik antiserum was from Santa Cruz Biotechnology (Santa Cruz, CA), and goat anti-rabbit antibodies conjugated to alkaline phosphatase (GAR-AP) were from Bio-Rad. Isolation and Treatment of T Lymphocytes—Blood samples (20 ml per donor) were drawn from the antecubital vein of healthy donors (age range 28–35 years), who gave informed consent to the study, and were collected into heparinized sterile tubes. Clearance of the local Ethics Committee was obtained to sue the human cells. Peripheral lymphocytes were purified by gradient centrifugation, using the density separation medium Lymphoprep (Nycomed Pharma, Oslo, Norway), and then T-cells were isolated from the whole lymphocyte population by means of the Dynal CD2 CELLection kit (Dynal, Olso, Norway), as reported previously (12Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (137) Google Scholar). Purified T lymphocytes were resuspended in RPMI 1640 medium (Invitrogen, Paisley, UK), supplemented with 25 mm Hepes, 2.5 mm sodium pyruvate, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated fetal bovine serum (Invitrogen), at a density of 1.5 × 106 cells/ml in ventilated 25-ml flasks (12Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (137) Google Scholar). Incubation of T lymphocytes with P, alone or in the presence of different compounds, was performed at 37 °C in humidified 5% CO2 atmosphere, at the indicated concentrations and for the indicated periods of time. Cells were treated for 1 h in serum-free medium, and then heat-inactivated fetal bovine serum was added at a final concentration of 10% (12Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (137) Google Scholar). 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. FAAH Activity and Expression—FAAH (arachidonoylethanolamide amidase; EC 3.5.1.4) activity was assayed at pH 9.0 with 10 μm [3H]AEA as substrate, by the reversed phase high performance liquid chromatography method described previously (18Maccarrone M. Di Rienzo M. Finazzi-Agrò A. Rossi A. J. Biol. Chem. 2003; 278: 13318-13324Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). FAAH activity was expressed as pmol of arachidonate released per min per mg of protein. Cell homogenates (20 μg/lane) were prepared as described previously (18Maccarrone M. Di Rienzo M. Finazzi-Agrò A. Rossi A. J. Biol. Chem. 2003; 278: 13318-13324Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) and were subjected to enzyme-linked immunosorbent assay (ELISA), to quantify FAAH protein. Wells were coated with human T-cell homogenates (20 μg/well), which were then reacted with anti-FAAH polyclonal antibodies (diluted 1:300), as first antibody, and with GAR-AP, diluted 1:2000, as second antibody (12Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (137) Google Scholar). In a previous study, the anti-FAAH antibodies have been shown to recognize a single band in human lymphocytes by Western blot analysis (12Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (137) Google Scholar). Color development of the alkaline phosphatase reaction was measured at 405 nm, using p-nitrophenyl phosphate as substrate. The A 405 values could not be converted into FAAH concentrations, because the purified enzyme is not available to make calibration curves. However, the ELISA test was linear in the range 0–50 μg/well of cell homogenate, and its specificity for FAAH was validated by antigen competition experiments (12Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (137) Google Scholar). Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using total RNA isolated from human T lymphocytes (10 × 106 cells) by means of the S.N.A.P.™ total RNA isolation kit (Invitrogen), as described previously (12Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (137) Google Scholar). RT-PCR amplification of FAAH was performed using 100 ng of total RNA, and the EZ rTth RNA PCR kit (PerkinElmer Life Sciences), as reported previously (12Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (137) Google Scholar). The amplification parameters were as follows: 2 min 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 for FAAH were as follows: (+), 5′-TGGAAGTCCTCCAAAAGCCCAG; (–), 5′-TGTCCATAGACACAGCCCTTCAG. Five μl of the reaction mixture were electrophoresed on a 6% polyacrylamide gel, and the RT-PCR products were excised from the gel and counted in a LKB1214 Rackbeta scintillation counter (Amersham Biosciences) (12Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (137) Google Scholar). Products were validated by size determination and sequencing, as described previously (12Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (137) Google Scholar). Analysis of N-Acyltransferase, Phospholipase D, Anandamide Uptake, and Cannabinoid Receptors—N-Acyltransferase (NAT) assay was performed as described previously (22Cadas H. di Tomaso E. Piomelli D. J. Neurosci. 1997; 17: 1226-1242Crossref PubMed Google Scholar), using 1,2-di[1-14C]palmitoylphosphatidylcholine (1 × 106 dpm/test) as substrate and measuring the formation of N-[14C]palmitoylphosphatidylethanolamines by high performance thin layer chromatography on silica gel plates (Sigma). 1,2-Dipalmitoyl-N-palmitoyl-phosphatidylethanolamine was used as standard, and NAT activity was expressed as pmol of N-palmitoylphosphatidylethanolamine formed per min per mg of protein (22Cadas H. di Tomaso E. Piomelli D. J. Neurosci. 1997; 17: 1226-1242Crossref PubMed Google Scholar). The activity of phospholipase D (PLD; EC 3.1.4.4) was assayed in T-cell homogenates by measuring the release of [14C]ethanolamine from 1,2-dioleoyl-3-phosphatidyl-[2-14C]ethanolamine (10 μm), as described previously (23Moesgaard B. Petersen G. Jaroszewski J.W. Hansen H.S. J. Lipid Res. 2000; 41: 985-990Abstract Full Text Full Text PDF PubMed Google Scholar). PLD activity was expressed as pmol of ethanolamine released per min per mg of protein. The uptake of 200 nm [3H]AEA by intact T lymphocytes (2 × 106/test) through AMT was studied as described previously (18Maccarrone M. Di Rienzo M. Finazzi-Agrò A. Rossi A. J. Biol. Chem. 2003; 278: 13318-13324Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) and was expressed as pmol of AEA taken up per min per mg of protein. For CBR studies, membrane fractions prepared from T lymphocytes (10 × 106) as reported previously (18Maccarrone M. Di Rienzo M. Finazzi-Agrò A. Rossi A. J. Biol. Chem. 2003; 278: 13318-13324Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) were quickly frozen in liquid nitrogen and stored at –80 °C for no longer than 1 week. These membrane fractions were used in rapid filtration assays with the synthetic cannabinoid [3H]CP55.940 (used at 400 pm), as reported previously (18Maccarrone M. Di Rienzo M. Finazzi-Agrò A. Rossi A. J. Biol. Chem. 2003; 278: 13318-13324Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Unspecific binding was determined in the presence of 100 nm "cold" agonist (12Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (137) Google Scholar, 18Maccarrone M. Di Rienzo M. Finazzi-Agrò A. Rossi A. J. Biol. Chem. 2003; 278: 13318-13324Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Receptor binding was expressed as fmol of ligand bound per mg of protein. Analysis of the Endogenous Levels of AEA and 2-Arachidonoyglycerol (2-AG)—The endogenous levels of AEA and 2-AG in human T lymphocytes (50 × 106/test) were assayed at NIAAA/National Institutes of Health by liquid chromatography/mass spectrometry, using an Agilent 1100 series LC-MSD equipped with a thermostated autosampler and column compartment, as reported previously (24Wang L. Liu J. Harvey-White J. Zimmer A. Kunos G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1393-1398Crossref PubMed Scopus (288) Google Scholar). The liquid chromatography/mass spectrometry analysis was also corroborated by gas chromatography-electron impact mass spectrometry, performed in Rome as described previously (25Gubellini P. Picconi B. Bari M. Battista N. Calabresi P. Centone D. Bernardi G. Finazzi-Agrò A. Maccarrone M. J. Neurosci. 2002; 22: 6900-6907Crossref PubMed Google Scholar). Western Blot Analysis of Nuclear Levels of Ikaros—For the determination of Ik isoforms in T lymphocytes, nuclear extracts were prepared from T-cell suspensions (19Wargnier A. Lafaurie C. Legros-Maida S. Bourge J.-F. Sigaux F. Sasportes M. Pauli P. J. Biol. Chem. 1998; 273: 35326-35331Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), and aliquots (50 μg protein) were loaded onto 10% SDS-polyacrylamide gels and were electroblotted onto 0.45-μm nitrocellulose filters (Bio-Rad), as described previously (19Wargnier A. Lafaurie C. Legros-Maida S. Bourge J.-F. Sigaux F. Sasportes M. Pauli P. J. Biol. Chem. 1998; 273: 35326-35331Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). For immunodetection, anti-Ik antiserum was diluted 1:1000, and the second antibody (GAR-AP) was diluted 1:2000 (12Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (137) Google Scholar). Protein content was normalized before loading onto the gel (18Maccarrone M. Di Rienzo M. Finazzi-Agrò A. Rossi A. J. Biol. Chem. 2003; 278: 13318-13324Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), and equal loading of extracts was verified by Ponceau staining (19Wargnier A. Lafaurie C. Legros-Maida S. Bourge J.-F. Sigaux F. Sasportes M. Pauli P. J. Biol. Chem. 1998; 273: 35326-35331Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Rainbow molecular mass markers (Amersham Biosciences) were bovine serum albumin (66.0 kDa) and ovalbumin (46.0 kDa). Nuclear levels of total Ik isoforms were further quantified by ELISA, performed by coating each well with 25 μg protein/sample, as described above for FAAH, and then reacted with anti-Ik antiserum (1:1000) and GAR-AP (1:2000). Construction of Chloramphenicol Acetyltransferase Expression Vectors and Transient Transfection—Sequence information for the upstream regulatory region of FAAH gene was downloaded from GeneBank™ (region: gi/11423254:644582-754250, International Human Genome Project), and the proximal promoter region of base pairs from +1 to –107 (+1 being the first nucleotide of the FAAH mRNA) was assembled using synthetic oligonucleotides (Amersham Biosciences). The DNA was gel-purified and subcloned into the PstI/XbaI sites of pCAT3-Basic vector (Promega Corp., Madison, WI). 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 (United States Biochemical, Cleveland, OH). Human T-cells (1 × 106 per test) were transfected in triplicate using TransFast™ transfection reagent (Promega Corp.), according to the manufacturer's instructions. Typically, cells were washed in phosphate-buffered saline and resuspended in 0.5 ml of serum-free medium, and then they were mixed with 0.5 ml of serum-free medium containing 2 μg of total DNA and the TransFast™ transfection reagent, at a charge ratio of 1:1 with respect to DNA. Transfection efficiency was monitored by use of 0.5 μg of thymidine kinase β-galactosidase construct (Clontech, Palo Alto, CA). 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 β-galactosidase activity determination, a marker of transfection efficiency, and for protein quantitation. CAT activity was determined using the Quan-T-CAT assay system (Amersham Biosciences), whereas the activity of β-galactosidase was assayed using the β-galactosidase enzyme system (Promega Corp.). The values of CAT activity were normalized to β-galactosidase activity and to the protein content (18Maccarrone M. Di Rienzo M. Finazzi-Agrò A. Rossi A. J. Biol. Chem. 2003; 278: 13318-13324Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Nuclear Extracts, Electrophoretic Mobility Shift, and Supershift Assays—Nuclear extracts were prepared according to Schreiber et al. (26Schreiber E. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3903) Google Scholar) with the modifications reported by Lee et al. (27Lee J.-H. Jang S.-I. Markova N.G. Steinert P.M. J. Biol. Chem. 1996; 271: 4561-4568Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Electrophoretic mobility shift assay (EMSA) experiments were performed as described previously (27Lee J.-H. Jang S.-I. Markova N.G. Steinert P.M. J. Biol. Chem. 1996; 271: 4561-4568Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), using the Ikaros oligonucleotide –76 5′-AGGCGGGCGTGGGATCCCGGCTG-3′–54 (site in bold), whereas the oligonucleotides used for the cold competitions were 5′-CTCGCAGCCTGGGAAGATAAGTGG-3′ (Ikaros site derived from vasoactive intestinal peptide receptor-1 promoter), and –76 5′-AGGCGGGCGTTTTTTCCCGGCTG-3′–54, which is the mutated site used for the transfection experiments (the mutated nucleotides are underlined) (28Dorsam G. Goetzl E.J. J. Biol. Chem. 2002; 277: 13488-13493Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). In all oligonucleotides, the numbers refer to positions in the FAAH promoter. The complexes were resolved on non-denaturing 6% polyacrylamide gels in 0.5 × TBE buffer (0.45 m Tris borate, 10 mm EDTA, pH 8.0) for 1 h at 14 V/cm and were autoradiographed overnight. For gel supershift analysis, nuclear extracts were preincubated overnight at 4 °C with 3 μg of rabbit anti-Ik antiserum, before addition of 32P-labeled oligoucleotide (26Schreiber E. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3903) Google Scholar). Dye was omitted from the loading buffer, and the gel was run at 4 °C in 0.2 x TBE buffer at 5 V/cm. The autoradiographic films were subjected to densitometric analysis by means of a Floor-S MultiImager equipped with Quantity One software (Bio-Rad), as reported previously (18Maccarrone M. Di Rienzo M. Finazzi-Agrò A. Rossi A. J. Biol. Chem. 2003; 278: 13318-13324Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Statistical Analysis—Data reported in this paper are the mean (±S.D.) of at least three independent determinations, each in duplicate. Statistical analysis was performed by the non-parametric Mann-Whitney test, elaborating experimental data by means of the InStat 3 program (GraphPAD Software for Science, San Diego, CA). Progesterone Stimulates FAAH Activity and Expression in Human T Lymphocytes—In vitro treatment of human T-cells with P for 24 h enhanced FAAH activity in a dose-dependent manner (Fig. 1A). FAAH activation reached statistical significance (p < 0.05) at 250 nm P and a maximum at 1 μm. Therefore, the last concentration was chosen to further investigate the effect of P on FAAH. Activation of FAAH by 1 μm P was fully reverted by the synthetic antiprogestinic compound RU486 (29Lebeau M.C. Baulieu E.E. Human Reprod. 1994; 9: S11Crossref Scopus (12) Google Scholar), used at 10 μm (Fig. 1A). Time course experiments showed that P-induced activation of FAAH was significant (p < 0.05) 12 h after T lymphocytes treatment and reached a maximum at 24 h (Fig. 1B). Anti-FAAH antibodies were used to quantify FAAH content in T-cells by ELISA and showed that P increased FAAH protein in human T lymphocytes in parallel to the increase of enzymic activity (Fig. 1, A and B). RT-PCR amplification of cDNA of human T lymphocytes, followed by liquid scintillation counting of RT-PCR products, showed that P increased dose- and time-dependently also FAAH mRNA in human T lymphocytes, in a way parallel to that of enzymic activity and protein content (Fig. 1, A and B). Remarkably, treatment of T-cells with 10 μm RU486 fully prevented the increase in FAAH protein and mRNA levels induced by 1 μm P (Fig. 1, A and B). These data closely resemble our previous report on the effect of P on the whole population of peripheral lymphocytes and are in keeping with the observation that FAAH activity in T-cells accounts for 85% of the activity measured in the whole cell population (12Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (137) Google Scholar). In addition, treatment of T lymphocytes with 1 μm P for 24 h significantly (p < 0.05) reduced the endogenous levels of AEA in these cells to 60% of the controls (Table I). It also reduced to a similar extent (67%) the cellular level of 2-AG, an endocannabinoid that is also cleaved by FAAH (17Di Marzo V. Goparaju S.K. Wang L. Liu J. Batkai S. Jarai Z. Fezza F. Miura G.I. Palmiter R.D. Sugiura T. Kunos G. Nature. 2001; 410: 822-825Crossref PubMed Scopus (1348) Google Scholar), and 10 μm RU486 fully reverted these effects of P (Ta