Leptin Activates the Anandamide Hydrolase Promoter in Human T Lymphocytes through STAT3
2003; Elsevier BV; Volume: 278; Issue: 15 Linguagem: Inglês
10.1074/jbc.m211248200
ISSN1083-351X
AutoresMauro Maccarrone, Marianna Di Rienzo, Alessandro Finazzi‐Agrò, Antonello Rossi,
Tópico(s)Regulation of Appetite and Obesity
ResumoPhysiological concentrations of leptin stimulate the activity of the endocannabinoid-degrading enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH) in human T lymphocytes up to ∼300% over the untreated controls. Stimulation of FAAH occurred through up-regulation of gene expression at transcriptional and translational levels and involved binding of leptin to its receptor with an apparent dissociation constant (Kd) of 1.95 ± 0.14 nm and maximum binding (Bmax) of 392 ± 8 fmol·mg protein−1. Leptin binding to the receptor triggered activation of STAT3 but not STAT1 or STAT5 or the mitogen-activated protein kinases p38, p42, and p44. Peripheral lymphocytes of leptin knock-out (ob/ob) mice showed decreased FAAH activity and expression (∼25% of the wild-type littermates), which were reversed to control levels by exogenous leptin. Analysis of the FAAH promoter showed a cAMP-response element-like site, which is a transcriptional target of STAT3. Consistently, mutation of this site prevented FAAH activation by leptin in transient expression assays. Electrophoretic mobility shift and supershift assays further corroborated the promoter activity data. Taken together, these results suggest that leptin, by up-regulating the FAAH promoter through STAT3, enhances FAAH expression, thus tuning the immunomodulatory effects of anandamide. These findings might also have critical implications for human fertility. Physiological concentrations of leptin stimulate the activity of the endocannabinoid-degrading enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH) in human T lymphocytes up to ∼300% over the untreated controls. Stimulation of FAAH occurred through up-regulation of gene expression at transcriptional and translational levels and involved binding of leptin to its receptor with an apparent dissociation constant (Kd) of 1.95 ± 0.14 nm and maximum binding (Bmax) of 392 ± 8 fmol·mg protein−1. Leptin binding to the receptor triggered activation of STAT3 but not STAT1 or STAT5 or the mitogen-activated protein kinases p38, p42, and p44. Peripheral lymphocytes of leptin knock-out (ob/ob) mice showed decreased FAAH activity and expression (∼25% of the wild-type littermates), which were reversed to control levels by exogenous leptin. Analysis of the FAAH promoter showed a cAMP-response element-like site, which is a transcriptional target of STAT3. Consistently, mutation of this site prevented FAAH activation by leptin in transient expression assays. Electrophoretic mobility shift and supershift assays further corroborated the promoter activity data. Taken together, these results suggest that leptin, by up-regulating the FAAH promoter through STAT3, enhances FAAH expression, thus tuning the immunomodulatory effects of anandamide. These findings might also have critical implications for human fertility. leptin leptin receptor anandamide (arachidonoylethanolamide) AEA membrane transporter chloramphenicol acetyltransferase cannabinoid receptors 5-(1,1′-dimethylheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl) cyclohexyl]-phenol cAMP-response element enzyme-linked immunosorbent assay electrophoretic mobility shift assay fatty acid amide hydrolase goat anti-rabbit/mouse antibodies conjugated with alkaline phosphatase insulin-like growth factor I receptor soluble leptin receptor mitogen-activated protein kinase rabbit anti-goat antibodies conjugated with alkaline phosphatase reverse transcriptase signal transducer and activator of transcription Leptin (L)1 is the 16-kDa non-glycosylated product of the obese gene, which is secreted by adipose cells, is released into the circulation, and transported across the blood-brain barrier into the central nervous system where it regulates energy homeostasis (1Ahima R.S. Flier J.S. Annu. Rev. Physiol. 2000; 62: 413-437Crossref PubMed Scopus (1495) Google Scholar). Leptin also serves systemic functions apart from those related to food intake and energy expenditure in mammals, including regulation of fertility (2Cunningham M.J. Clifton D.K. Steiner R.A. Biol. Reprod. 1999; 60: 216-222Crossref PubMed Scopus (441) Google Scholar) and modulation of immune response (3Matarese 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 (178) Google Scholar). These two actions might be interconnected in humans because leptin alters the production from T lymphocytes of T helper 1 and 2 cytokines (4Lord G.M. Matarese G. Howard J.K. Baker R.J. Bloom S.R. Lechler R.I. Nature. 1998; 394: 897-901Crossref PubMed Scopus (1854) Google Scholar), which are critical in regulating embryo implantation and materno-fetal exchanges (5Piccinni 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: 128-133Crossref PubMed Google Scholar, 6Piccinni M.P. Beloni L. Livi C. Maggi E. Scarselli G. Romagnani S. Nature Med. 1998; 4: 1020-1024Crossref PubMed Scopus (579) Google Scholar). In this line, mice genetically defective in leptin (ob/ob knock-out) are obese, infertile, and immunodeficient, and administration of exogenous leptin can reverse these defects (1Ahima R.S. Flier J.S. Annu. Rev. Physiol. 2000; 62: 413-437Crossref PubMed Scopus (1495) Google Scholar, 2Cunningham M.J. Clifton D.K. Steiner R.A. Biol. Reprod. 1999; 60: 216-222Crossref PubMed Scopus (441) Google Scholar, 3Matarese 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 (178) Google Scholar, 4Lord G.M. Matarese G. Howard J.K. Baker R.J. Bloom S.R. Lechler R.I. Nature. 1998; 394: 897-901Crossref PubMed Scopus (1854) Google Scholar). Leptin signaling is mediated by the leptin receptor (LR), which exists in at least six different isoforms (1Ahima R.S. Flier J.S. Annu. Rev. Physiol. 2000; 62: 413-437Crossref PubMed Scopus (1495) Google Scholar). Yet, only the long LR isoform has all intracellular motifs necessary for signaling via the signal transducer, activator of transcription (STAT), and/or the mitogen-activated protein kinase (MAPK) pathways (7Wang Y. Kuropatwinski K.K. White D.W. Hawley T.S. Hawley R.G. Tartaglia L.A. Baumann H. J. Biol. Chem. 1997; 272: 16216-16223Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 8Banks A.S. Davis S.M. Bates S.H. Myers Jr., M.G. J. Biol. Chem. 2000; 275: 14563-14572Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 9Bjorbaek C. Buchholz R.M. Davis S.M. Bates S.H. Pierroz D.D. Gu H. Neel B.G. Myers Jr., M.G. Flier J.S. J. Biol. Chem. 2001; 276: 4747-4755Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 10Wong M. Uddin S. Majchrzak B. Huynh T. Proudfoot A.E. Platanias L.C. Fish E.N. J. Biol. Chem. 2001; 276: 11427-11431Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 11Schrager J.A. Der Minassian V. Marsh J.W. J. Biol. Chem. 2002; 277: 6137-6142Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The relative importance of these divergent signaling events in leptin action is still unknown. Recently, leptin has been shown to reduce the levels of anandamide (arachidonoylethanolamide, AEA) in the hypothalamus ofob/ob mice, suggesting that this compound partakes of the neural circuitry regulated by leptin (12Di 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 (1374) Google Scholar). AEA belongs to a group of endogenous lipids, which include amides, esters, and ethers of long chain polyunsaturated fatty acids, collectively termed “endocannabinoids” (13Mechoulam R. Prostaglandins Leukotrienes Essent. Fatty Acids. 2002; 66: 93-99Abstract Full Text PDF PubMed Scopus (68) Google Scholar). It binds to cannabinoid receptors (CBR) in the central nervous system and in peripheral immune cells, thus having many central actions (14Fride E. Prostaglandins Leukotrienes Essent. Fatty Acids. 2002; 66: 221-233Abstract Full Text PDF PubMed Scopus (159) Google Scholar). Together with the congeners 2-arachidonoylglycerol (12Di 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 (1374) Google Scholar, 15Kirkham T.C. Williams C.M. Fezza F. Di Marzo V. Br. J. Pharmacol. 2002; 136: 550-557Crossref PubMed Scopus (668) Google Scholar) and oleoylethanolamide (16Rodriguez de Fonseca F. Navarro M. Gomez R. Escuredo L. Nava F. Fu J. Murillo-Rodriguez E. Giuffrida A. LoVerme J. Gaetani S. Kathuria S. Gall C. Piomelli D. Nature. 2001; 414: 209-212Crossref PubMed Scopus (597) Google Scholar), AEA has been implicated in the regulation of appetite. Among the peripheral activities of AEA, the regulation of fertility (17Paria 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 (173) Google Scholar) and immune function (18Parolaro D. Massi P. Rubino T. Monti E. Prostaglandins Leukotrienes 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) (19Fowler C.J. Jacobsson S.O.P. Prostaglandins Leukotrienes 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 AEA hydrolase (fatty acid amide hydrolase, FAAH) (20Deutsch D.G. Ueda N. Yamamoto S. Prostaglandins Leukotrienes Essent. Fatty Acids. 2002; 66: 201-210Abstract Full Text PDF PubMed Scopus (188) 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 (21Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. Lancet. 2000; 355: 1326-1329Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Indeed, the expression of lymphocyte FAAH is under control of progesterone and contributes to the release of cytokines critical for fertility, such as the leukemia inhibitory factor (22Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (141) 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 (21Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. Lancet. 2000; 355: 1326-1329Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 22Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (141) Google Scholar, 23Maccarrone 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 (169) 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 (24Maccarrone M. Falciglia K. Di Rienzo M. Finazzi-Agrò A. Prostaglandins Leukotrienes Essent. Fatty Acids. 2002; 66: 309-317Abstract Full Text PDF PubMed Scopus (53) Google Scholar). Therefore, we sought to investigate whether leptin might regulate AEA metabolism in human T-cells, thus assuming that the actions of this endocannabinoid on fertility and immunity could be part of the molecular events responsible for the effects of leptin. In fact, we show an enhancement of FAAH activity and expression by leptin, triggered through binding to LR and subsequent STAT3-dependent up-regulation of promoter activity. Chemicals were of the purest analytical grade. Leptin (human recombinant for T-cell studies, mouse recombinant forob/ob mice injections) and anandamide (AEA) were purchased from Sigma. PD98059 and SB203580 were from Calbiochem (La Jolla, CA). [3H]AEA (223 Ci/mmol), 125I-labeled leptin (2200 Ci/mmol) and [3H]CP55.940 (5-(1,1′-dimethylheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl) cyclohexyl]-phenol; 126 Ci/mmol) were from PerkinElmer Life Sciences. Anti-FAAH polyclonal antibodies were elicited in rabbits against the conserved FAAH sequence VGYYETDNYTMPSPAMR (25Giang D.K. Cravatt B.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2238-2242Crossref PubMed Scopus (395) Google Scholar) conjugated to ovalbumin and were prepared by Primm S.r.l. (Milan, Italy). Mouse monoclonal antibodies against actin, STAT1, STAT3, p38 MAPK, and their phosphorylated (activated) forms, monoclonal antibodies against phosphorylated p42 and p44 MAPK, rabbit polyclonal antibodies against p42 and p44 MAPK or against STAT5, goat polyclonal antibodies against phospho-STAT5, and rabbit anti-goat antibodies conjugated to alkaline phosphatase (RAG-AP) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-human leptin and anti-human insulin-like growth factor I receptor (anti-IGF-IR) monoclonal antibodies, and human leptin receptor/Fc chimeras (soluble leptin receptor, sLR) were purchased from R&D Systems (Minneapolis, MN). According to the manufacturer's instructions, ∼100 ng/ml of anti-leptin antibodies or of sLR are enough to neutralize the effects of 1 nm (∼15 ng/ml) leptin, and in this study we used a 2-fold excess of each neutralizing agent. Goat anti-rabbit and goat anti-mouse antibodies conjugated to alkaline phosphatase (GAR-AP and GAM-AP) were from Bio-Rad (Hercules, CA). 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 use 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, Oslo, Norway) as reported (22Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (141) Google Scholar). Purified T lymphocytes were resuspended in RPMI 1640 medium (Invitrogen), 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 (22Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (141) Google Scholar). Incubation of T lymphocytes with leptin 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. 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. Peripheral lymphocytes were isolated by means of Lymphoprep (Nycomed Pharma) also from leptin knock-out (ob/ob) mice and their wild-type littermates, purchased from Jackson Laboratories (Bar Harbor, ME). Wild-type and ob/obmice (6 per group) received a single intravenous injection of 250 μg of mouse recombinant leptin, or vehicle in the controls, and were sacrificed 24 h later by decapitation (12Di 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 (1374) Google Scholar). Blood was immediately collected and peripheral lymphocytes were isolated. All animal experimental protocols were approved by the local Committee on Animal Care and Use and met the guidelines of the National Institutes of Health, detailed in the Guide for the Care and Use of Laboratory Animals, and of the European Community directives regulating animal research. Fatty acid amide hydrolase (EC 3.5.1.4; FAAH) activity was assayed at pH 9.0 with 10 μm [3H]AEA as substrate by the reversed phase high performance liquid chromatography method already described (26Maccarrone M. Lorenzon T. Bari M. Melino G. Finazzi-Agrò A. J. Biol. Chem. 2000; 275: 31938-31945Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Cell homogenates (20 μg/lane) were prepared as described (26Maccarrone M. Lorenzon T. Bari M. Melino G. Finazzi-Agrò A. J. Biol. Chem. 2000; 275: 31938-31945Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar) and were subjected to SDS-PAGE (12%) under reducing conditions. Rainbow molecular mass markers (Amersham Biosciences) were phosphorylase b (97.4 kDa), bovine serum albumin (66.0 kDa), ovalbumin (46.0 kDa) and soybean trypsin inhibitor (27.0 kDa). For immunochemical analysis, gels were electroblotted onto 0.45 μm nitrocellulose filters (Bio-Rad), and FAAH was visualized with anti-FAAH polyclonal antibodies (1:200), using GAR-AP diluted 1:2000 as second antibody (22Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (141) Google Scholar). Actin was immunodetected with anti-actin monoclonal antibodies (1:500), using GAM-AP diluted 1:2000 as second antibody (22Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (141) Google Scholar). Densitometric analysis of filters was performed by means of a Floor-S MultiImager equipped with a Quantity One software (Bio-Rad). The same anti-FAAH antibodies were used to further quantify FAAH protein by enzyme-linked immunosorbent assay (ELISA). Wells were coated with human T-cell or mouse peripheral lymphocyte 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 (22Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (141) Google Scholar). 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 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 (22Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (141) Google Scholar). Reverse transcriptase (RT)-PCR was performed using total RNA isolated from human T lymphocytes (10 × 106 cells) by means of the SNAPTM Total RNA Isolation Kit (Invitrogen, Carlsbad, CA) as described (22Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (141) Google Scholar). RT-PCR reactions were performed using 100 ng of total RNA for the amplification of FAAH or 0.4 ng for 18 S rRNA and the EZ rTth RNA PCR kit (PerkinElmer Life Sciences). 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 were as follows: (+) 5′-TGGAAGTCCTCCAAAAGCCCAG, (-) 5′-TGTCCATAGACACAGCCC-TTCAG, for FAAH; (+) 5′-AGTTGCTGCAGTTAAAAAGC, (-) 5′-CCTCAGTTCCGAAAACCAAC for 18 S rRNA. Five μl of the reaction mixture were electrophoresed on a 6% polyacrylamide gel, which was then dried and subjected to autoradiography (22Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (141) Google Scholar). The autoradiographic films were subjected to densitometric analysis by means of a Floor-S MultiImager equipped with a Quantity One software (Bio-Rad). In some experiments the RT-PCR products were excised from the gel and counted in a LKB1214 Rackbeta scintillation counter (Amersham Biosciences). Products were validated by size determination and sequencing as described (22Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (141) Google Scholar). The uptake of 200 nm [3H]AEA by intact T lymphocytes (2 × 106/test) through the AMT was studied as described (26Maccarrone M. Lorenzon T. Bari M. Melino G. Finazzi-Agrò A. J. Biol. Chem. 2000; 275: 31938-31945Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). For CBR studies, membrane fractions were prepared from T lymphocytes (10 × 106) as reported (26Maccarrone M. Lorenzon T. Bari M. Melino G. Finazzi-Agrò A. J. Biol. Chem. 2000; 275: 31938-31945Abstract Full Text Full Text PDF PubMed Scopus (321) 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 (400 pm) as described previously (26Maccarrone M. Lorenzon T. Bari M. Melino G. Finazzi-Agrò A. J. Biol. Chem. 2000; 275: 31938-31945Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). The same filtration assays were used to analyze the binding of125I-labeled leptin to T-cells (9Bjorbaek C. Buchholz R.M. Davis S.M. Bates S.H. Pierroz D.D. Gu H. Neel B.G. Myers Jr., M.G. Flier J.S. J. Biol. Chem. 2001; 276: 4747-4755Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 27Tartaglia L.A. Dembski M. Weng X. Deng N. Culpepper J. Devos R. Richards G.J. Campfield L.A. Clark F.T. Deeds J. Muir C. Sanker S. Moriarty A. Moore K.J. Smutko J.S. Mays G.G. Wool E.A. Monroe C.A. Topper R.I. Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3238) Google Scholar). In this case, apparent dissociation constant (Kd) and maximum binding (Bmax) values were calculated from saturation curves in the range 0–15 nm, elaborating the binding data through nonlinear regression analysis with the Prism 3 program (GraphPAD Software for Science, San Diego, CA) (26Maccarrone M. Lorenzon T. Bari M. Melino G. Finazzi-Agrò A. J. Biol. Chem. 2000; 275: 31938-31945Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Unspecific binding was determined in the presence of 100 nm “cold” leptin (9Bjorbaek C. Buchholz R.M. Davis S.M. Bates S.H. Pierroz D.D. Gu H. Neel B.G. Myers Jr., M.G. Flier J.S. J. Biol. Chem. 2001; 276: 4747-4755Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 27Tartaglia L.A. Dembski M. Weng X. Deng N. Culpepper J. Devos R. Richards G.J. Campfield L.A. Clark F.T. Deeds J. Muir C. Sanker S. Moriarty A. Moore K.J. Smutko J.S. Mays G.G. Wool E.A. Monroe C.A. Topper R.I. Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3238) Google Scholar). For the analysis of total STAT1, STAT3, and STAT5 of p38, p42, and p44 MAPK and of the corresponding phosphorylated (activated) forms, whole cell extracts were prepared as reported (11Schrager J.A. Der Minassian V. Marsh J.W. J. Biol. Chem. 2002; 277: 6137-6142Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Cell lysates (50 μg of protein) were loaded onto 10% SDS-polyacrylamide gels and were then electroblotted onto 0.45-μm nitrocellulose filters (Bio-Rad) as described above for FAAH. For immunodetection, the specific first antibody was diluted 1:1000, and the appropriate second antibody (GAM-AP, GAR-AP, or RAG-AP) was diluted 1:2000 (22Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (141) Google Scholar). Protein content was normalized before loading onto the gel, and equal loading of extracts was verified by Ponceau staining (22Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (141) Google Scholar). Sequence information for the upstream regulatory region of theFAAH gene was downloaded from GenBankTM(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 thePstI/XbaI sites of pCAT3-Basic vector (Promega Corporation, 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 (USB, Cleveland, OH). Human T-cells (1 × 106 per test) were transfected in triplicate using TransFastTMTransfection Reagent (Promega) 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 TransFastTM 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 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). The values of CAT activity were normalized to β-galactosidase 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 were prepared according to Schreiber and co-workers (28Schreiber E. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3918) Google Scholar) with the modifications reported by Lee and co-workers (29Lee 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). EMSA experiments were performed as described (29Lee 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), deriving the sequence for the wild-type cAMP responsive element (CRE)-like site bandshift from the FAAH promoter region: −61 5′-CCCGGCTGATCCAGTCCG-3′ −44 (site in bold). The sequence for the mutated site was the same used for the transfection experiments, i.e. −61 5′-CCCGGCAAATCAAAGTCCG-3′ −44 (mutated nucleotides are underlined). The numbers in the oligonucleotides refer to positions in the FAAH promoter. The complexes were resolved on non-denaturing 6% polyacrylamide gels in 0.5 x TBE buffer 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 a mixture of anti-STAT3 polyclonal antibodies, consisting of equimolar amounts of sc-482X, sc-483X, sc-7993X, and sc-8001X (Santa Cruz Biotechnology) before addition of32P-labeled oligonucleotide (31Niehof M. Streetz K. Rakemann T. Bischoff S.C. Manns M.P. Horn F. Trautwein C. J. Biol. Chem. 2001; 276: 9016-9027Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Dye was omitted from the loading buffer, and the gel was run at 4 °C in 0.2× TBE buffer at 5 V/cm. 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). In vitro treatment of human T-cells with L for 24 h enhanced FAAH activity in a dose-dependent manner (Fig. 1A). FAAH activation reached statistical significance (p < 0.05) at 1 nm L and a maximum at 10 nm. Therefore, the last concentration was chosen to further investigate the effect of L on FAAH. FAAH activation by 10 nm L (corresponding to ∼150 ng/ml) was fully prevented by anti-leptin antibodies or by sLR, both used at neutralizing concentrations of 3 μg/ml. Instead, “mock” antibodies against IGF-IR were ineffective at the same concentration (Fig. 1A). Time-course experiments showed that L-induced activation of FAAH was significant (p < 0.05) 12 h after T lymphocytes treatment and reached a maximum at 24 h (Fig. 1B). Western blot analysis of T lymphocyte extracts showed that specific anti-FAAH antibodies recognized a single immunoreactive band of the molecular size expected for FAAH, the intensity of which was dose dependently higher in L-treated than in control cells (Fig. 2A). Densitometric analysis of the filter shown in Fig. 2A(representative of triplicate experiments) indicated that FAAH protein increased to 135, 175, and 235% of the control (100% = 11500 ± 1200 units/mm2) in cells treated for 24 h with 1, 5, or 10 nm L, respectively. On the other hand, T-cells expressed the same levels of actin at all concentrations of leptin (Fig. 2A), ruling out that the different levels of FAAH in these cells might be due to unequal loading of pro
Referência(s)