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 a
Trang 1Differential 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-Agro`3and Antonello Rossi3 1
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
endocan-nabinoid-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
observa-tions to immortalized human lymphoma U937 cells, where
stimulation of FAAH by leptin (up to 300% of the
controls) involves binding to a leptin receptor (Kd¼
2.0 ± 0.1 nM, Bmax¼ 382 ± 5 fmolÆmg 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 endo-cannabinoid system are not modulated by the two hor-mones Interestingly, human neuroblastoma CHP100 cells also have a leptin receptor ( 110 kDa, Kd¼ 2.2 ± 0.2 nM,
Bmax¼ 339 ± 8 fmolÆmg protein)1), a progesterone rec-eptor ( 120 kDa), STAT3 and Ikaros, yet their FAAH is not activated by leptin or progesterone These data, corro-borated by transient expression and electrophoretic mobil-ity-shift assays, demonstrate an unprecedented cell-specific regulation of the FAAH gene, which has important impli-cations for the control of tone and activity of AEA along the neuroimmune axis
Keywords: endocannabinoids; immune system; leptin; neu-rons; progesterone
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–brain-barrier 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 1 (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 expres-sion 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/obmice [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
acetyl-transferase; 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-acylphosphati-dylethanolamine; 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);
phosphatidylcho-line phosphatidohydrolase (EC 3.1.4.4).
(Received 4 June 2004, revised 31 August 2004,
accepted 8 October 2004)
Trang 2Based 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 on demand
synthesis of AEA through N-acyltransferase [20] and
N-acylphosphatidylethanolamine (NAPE)-specific
phos-pholipase 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
unprece-dented evidence that leptin and progesterone up-regulate
FAAH, but not AMT, NAT or PLD, in human immune
cells, while they do not in human neuronal cells
Therefore, the central actions of these two hormones do
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
rele-vance beyond AEA and related endocannabinoids
Materials and methods
Materials
Chemicals were of the purest analytical grade Leptin
(human recombinant), anandamide
(N-arachidonoyletha-nolamine, AEA), progesterone and mifepristone (RU486)
were purchased from Sigma Chemical Co (St Louis, MO,
USA) [3H]AEA (223 CiÆmmol)1), 125I-labeled leptin
(2200 CiÆmmol)1) and [3H]CP55.940
{5-(1,1¢-dimethyhep-tyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl)
cyclohexyl]-phenol; 126 Ci mmol)1} were from NEN Life Science
Products, Inc (Boston, MA, USA) [3
H]N-arachidonoyl-phosphatidylethanolamine (200 CiÆmmol)1) was from ARC
(St Louis, MO, USA), and 1,2-di[1-14
C]palmitoyl-phos-phatidylcholine (111 mCiÆmmol)1) was from Amersham
Pharmacia Biotech (Uppsala, Sweden) Anti-FAAH
poly-clonal Igs were raised in rabbits against the conserved
FAAH sequence VGYYETDNYTMPSPAMR [25]
conju-gated to ovalbumin, and were prepared by Primm S.r.l
(Milan, Italy) Mouse monoclonal antibodies against actin,
STAT3, phospho-STAT3, leptin receptor (LR) and
pro-gesterone receptor (PR), LR and PR blocking peptides, and
rabbit anti-(Ikaros serum) were from Santa Cruz
Biotech-nology (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 lgÆmL)1 of
sLR are enough to neutralize the effects of 10 nM ( 150 ngÆmL)1) leptin, and in this study we used a two-fold excess of each neutralizing agent [9] Goat anti-rabbit 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 UÆmL)1penicillin, 100 lgÆmL)1streptomycin and 10% heat-inactivated fetal bovine serum [18] Human neuroblastoma CHP100 cells (ATCC, Mannassas, VA, USA) were cultured in a 1 : 1 mixture of MEM (Eagle’s minimal essential medium plus Earle’s salts) and Ham’s F-12 media (Flow Laboratories Ltd, Irvine, UK), supple-mented with 15% heat-inactivated fetal bovine serum, sodium bicarbonate (1.2 gÆL)1), 15 mMHepes buffer, 2 mM
L-glutamine and 1% nonessential amino acids [18] Both CHP100 and U937 cells were maintained at 37C 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 37C in humidified 5% CO2atmosphere, at the indicated concentrations for 24 h In the case of progesterone, cells were treated for 1 h in serum-free medium, then heat-inactivated 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
Anandamide hydrolase activity and expression 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, Bucking-hamshire, UK) were phosphorylase b (97.4 kDa) and bovine serum albumin (66.0 kDa) For immunochemical analysis, gels were electroblotted onto 0.45 lm nitrocellu-lose filters (Bio-Rad), and FAAH was visualized with anti-FAAH polyclonal Ig (1 : 200), using GAR-AP diluted
1 : 2000 as second antibody [9] Actin was immunodetected with monoclonal anti-actin Ig (1 : 500), using GAM-AP diluted 1 : 2000 as second antibody [9] The same anti-FAAH Igs were used to further quantify anti-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
1 : 300), as first antibody, and with GAR-AP, diluted
1 : 2000, as second antibody [9] Color development of the alkaline phosphatase reaction was measured at 405 nm, using p-nitrophenyl phosphate as substrate The A405values 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
Trang 30–50 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· 106cells) by means of the
SNAprogesteroneTMTotal 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: 2 min
at 95C, 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
electro-phoresed 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
deter-mination and sequencing, as described previously [18]
Analysis ofN-acyltransferase, phospholipase D,
anandamide transporter and leptin receptor binding
An N-acyltransferase (NAT) assay was performed as
described [20], using 1,2-di[1–14
C]palmitoyl-phosphatidyl-choline (1· 106d.p.m per test) as substrate and measuring
the formation of N-[14
C]palmitoyl-phosphatidylethanol-amines by high performance TLC on silica gel plates
(Sigma Chemical Co.)
1,2-Dipalmitoyl,N-palmitoyl-phos-phatidylethanolamine was used as a standard, and NAT
activity was expressed as pmol
N-palmitoyl-phosphatidyl-ethanolamine formed per min per mg protein [20] The
activity of phospholipase D (phosphatidylcholine
phos-phatidohydrolase, EC 3.1.4.4; PLD) was assayed in cell
homogenates as described [21], by measuring the release of
[3H]AEA from [3
H]N-arachidonoylphosphatidylethanol-amine (100 lM) as substrate [26] PLD activity was
expressed as pmol AEA released per min per mg protein
The uptake of 500 nM[3H]AEA by intact cells (2· 106per
test) through AMT was studied as described [18], and was
expressed as pmol AEA taken up per min per mg protein
The binding of125I-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 nMcold 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 1 : 500) as first antibody, and GAM-AP diluted
1 : 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]
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 (Bio-Rad), as described above for FAAH For immunodetection, the specific first antibody was diluted 1 : 1000, and the appropriate second antibody (GAM-AP or GAR-AP) was diluted 1 : 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), respect-ively, 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 Gen-ome Project) and the proximal promoter region of basepairs from +1 to )107 (+1 being the first nucleotide of the FAAHmRNA) was assembled using synthetic oligonucleo-tides (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 promo-ter 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 manu-facturer’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 2 lg of total DNA and the TransFastTMTransfection Reagent, at a charge ratio of 1 : 1 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 acetyl-transferase (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
Trang 4(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
GCGTGGGATCCCGGCTG-3¢)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
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
FAAHpromoter The complexes were resolved on
non-denaturing 6% polyacrylamide gels in 0.5· TBE buffer
(0.45M Tris/borate, 10 mM EDTA, pH 8.0) for 1 h at
14 VÆcm)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
Statis-tical analysis was performed using the nonparametric
Mann–Whitney U-test, elaborating experimental data by
means of theINSTAT3 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-depend-ent 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 ngÆmL)1) was fully prevented by soluble leptin
receptor (sLR), used at neutralizing concentration of
3 lgÆmL)1 Instead, mock antibodies against IGF-IR were
ineffective at the same concentration (not shown) Also,
progesterone increased the activity of FAAH, in a
dose-dependent manner (Fig 1B), at the same concentrations
used in the previous investigation [15] At a dose of 1 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 nMleptin or 1 lMprogesterone than in controls (Fig 1C) On the other hand, U937 cells treated with 10 nMleptin or 1 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 1 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 prog-esterone (Fig 1B) increased FAAH mRNA in a dose-dependent 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 1 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 expres-sion were ineffective on the activity of AMT, NAT and PLD in U937 cells, when used either alone or in combina-tion (Table 2) It should be recalled that U937 cells do 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 combina-tion, 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
125I-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 nMand maximum binding (Bmax)
of 382 ± 5 and 339 ± 8 fmolÆmg protein)1, for U937 cells and CHP100 cells, respectively These values are in agree-ment with previous reports on LR of human hepatic cells [32], and on LR stably transfected into different cell types [27,33] Cold leptin fully displaced125I-labeled leptin from its binding site, when used at 100 nM(Fig 2A) Moreover, Western blot analysis showed that specific anti-LR Igs
Trang 5recognized 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
Fig 1 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 n M ( 150 ngÆmL)1) leptin in the presence of 3 lgÆmL)1soluble leptin receptor (sLR) (100% activity in U937 cells is
350 ± 30 pmolÆmin)1Æmg protein)1; protein content, 0.280 ± 0.040 A 405 and mRNA level,
11 000 ± 1300 c.p.m; 100% activity in CHP100 cells, 25 ± 3 pmolÆmin)1Æmg pro-tein)1) (B) Effect of progesterone on the same samples as in (A) Cells were incubated with progesterone alone, or with 1 l M progesterone
in the presence of 10 l M RU486 In both panels: *P < 0.05 vs control, **P < 0.01 vs control and #P < 0.01 vs 10 n M leptin or vs.
1 l M progesterone, respectively In both pan-els, vertical bars represent SD values (C) Up-per panel, Western blot analysis of U937 cells, treated with 10 n M leptin or 1 l M 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 rep-resentative of three independent experiments.
Table 1 Modulation of FAAH expression in human U937 cells by leptin
and progesterone Activity, 100% ¼ 350 ± 30 pmolÆmin)1Æmg
pro-tein)1; Protein, 0.280 ± 0.040 A 405 ; mRNA level, 11 000 ± 1300
c.p.m.
Treatment of
U937 cells
FAAH Expression (%) Activity Protein mRNA Control 100 100 100
Leptin (10 n M )
for 24 h
300 ± 35* 270 ± 30* 310 ± 35*
Progesterone (1 l M )
for 24 h
240 ± 26* 220 ± 25* 240 ± 25*
Leptin (10 n M ) +
progesterone (1 l M )
for 24 h
500 ± 45*# 450 ± 50*# 490 ± 45*#
*P < 0.01 vs control; #P < 0.01 vs 10 n M leptin or vs 1 l M
progesterone.
Table 2 Effect of leptin and progesterone on the activity of AEA membrane transporter (AMT), N-acyltransferase (NAT) and phos-pholipase D (PLD) in human U937 cells.
Treatment of U937 cells AMT NAT PLD Control 100 a 100 b 100 c
Leptin (10 n M ) for 24 h
100 ± 10 90 ± 10 100 ± 10 Progesterone (1 l M )
for 24 h
90 ± 10 90 ± 10 110 ± 10 Leptin (10 n M ) +
progesterone (1 l M ) for 24 h
100 ± 10 100 ± 10 100 ± 10
a
100%, 150 ± 20 pmolÆmin)1Æmg protein)1;b100%, 14 ± 3 pmolÆ min)1Æmg protein)1; c 100%, 90 ± 10 pmolÆmin)1Æmg protein)1.
Trang 6leptin [9] or progesterone [15] occurs through
phosphory-lation 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 1 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
cells, respectively (Fig 3C) Neither phospho-STAT3 nor Ikaros increased in CHP100 cells under the same experi-mental conditions (Fig 3D)
Analysis of theFAAH promoter The human FAAH gene has been located on chromosome 1 [34] and the latter has been completely sequenced There-fore, 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 FAAHpromoter 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 nMleptin or 1 lMprogesterone 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
32P-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-1promoter [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
Fig 2 Characterization of leptin receptor (LR) and progesterone
receptor (PR) (A) Saturation curves of the binding of125I-labeled
leptin to human U937 or CHP100 cells, alone or in the presence of
100 n M cold leptin (data of the cold 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.
Trang 7(Table 1), FAAH activity and expression in human
lym-phoma 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 (Kdand Bmax)
similar to those of the leptin receptor [32,33] This
obser-vation, together with the ability of cold leptin to fully
displace125I-labeled leptin, strongly suggests that U937 cells
have an authentic LR Western blot analysis further
corroborates this hypothesis, showing that specific
anti-LR 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
circula-ting levels of 1 lM( 0.3 lgÆmL)1), on FAAH activity in
U937 cells extend our previous observations on T
lympho-cytes [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
pro-gesterone-responsive genes [17,37,38], this finding gives
ground to the hypothesis that the effects of progesterone on
FAAHgene 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 check point for the effect of both leptin and progesterone These observations are in keeping with the hypothesis that FAAH is the key-regulator 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 phos-phorylated) only STAT3 [9], which is preferentially activa-ted 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] Transfec-tion 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) FAAHpromoter also revealed a binding site for Ikaros, a member of the Kruppel family of zinc finger DNA-binding proteins that acts as critical transcriptional regu-lator 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, 2 and 3, the only isoforms which exhibit high DNA binding affinity [14], have been
Fig 3 Activation of downstream signals by leptin (L) or progesterone (P) Human U937 or CHP100 cells were treated for 24 h with 10 n M
leptin (A) or 1 l M 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 iso-forms Ik1, Ik2 and Ik3 (B) The positions of (phospho) proteins are indicated to the right These data are representative of three inde-pendent 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.
Trang 8detected in the nucleus of human peripheral blood
mono-nuclear cells [13,14], where they are essential in controlling
the activation of granzyme B promoter by the
glucocorti-coid, dexamethasone [13] Here, we demonstrated that
circulating levels of progesterone (1 lM, i.e. 0.3 lgÆmL)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, 2 and 3 is known to greatly increase their
affinity for DNA and their consequent ability to activate
transcription [13,14] Transfection experiments using
FAAHpromoter constructs with mutated Ikaros (mutIk)
revealed that this site indeed confers progesterone respon-siveness (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 transcrip-tion 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 Activa-tion of the FAAH promoter by leptin may be additive to that of progesterone at the two different sites (Table 1 and Fig 4)
Fig 4 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.
Trang 9A 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 pmolÆmin)1Æmg 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 endocann-abinoid system
In conclusion, the results reported here represent the first evidence of a differential regulation of FAAH gene expres-sion 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 do not seem to involve modulation of endocannabinoid metabolism
Acknowledgements
We wish to thank Drs Monica Bari and Natalia Battista for their expert assistance in cell isolation and culture This study was partly supported
by Ministero dell’Istruzione, dell’Universita` e della Ricerca (Cofin 2003) and by Istituto Superiore di Sanita` (III AIDS project), Rome.
References
1 Ahima, R.S & Flier, J.S (2000) Leptin Annu Rev Physiol 62, 413–437.
2 Cunningham, M.J., Clifton, D.K & Steiner, R.A (1999) Leptin’s actions on the reproductive axis: perspectives and mechanisms Biol Reprod 60, 216–222.
3 Matarese, G., La Cava, A., Sanna, V., Lord, G.M., Lechler, R.I., Fontana, S., & Zappacosta, S (2002) Balancing susceptibility infection and autoimmunity: a role for leptin? Trends Immunol 23, 182–187.
4 Lord, G.M., Matarese, G., Howard, J.K., Baker, R.J., Bloom, S.R & Lechler, R.I (1998) Leptin modulates the T-cell immune response and reverses starvation-induced immunosuppression Nature 394, 897–901.
5 Piccinni, M.P., Giudizi, M.G., Biagiotti, R., Beloni, L., Gianna-rini, L., Sampognaro, S., Parronchi, P., Manetti, R., Annunziato, F., Livi, C., Romagnani, S & Maggi, E (1995) Progesterone favors the development of human T helper cells producing Th2-type cytokines and promotes both IL-4 production and membrane
Fig 5 Analysis of FAAH promoter by electrophoretic mobility-shift
assay (EMSA) EMSA experiments were performed with 3 lg of U937
or CHP100 nuclear extracts, prepared before (–) and after (+)
sti-mulation with 10 n M leptin (A) or 1 l M progesterone (B,C) (A)
Complex formation (indicated by an arrow) was performed with
oligonucleotides as32P-labeled probes for mutated (mutL) or wild-type
(wt) CRE-like site (B) Complex formation (indicated by an arrow) in
U937 cells, performed with 32 P-labeled oligonucleotide corresponding
to the Ikaros site Lane O represents32P-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.
Trang 10CD30 expression in established Th1 cell clones J Immunol 155,
128–133.
6 Piccinni, M.P., Beloni, L., Livi, C., Maggi, E., Scarselli, G &
Romagnani, S (1998) Defective production of both leukemia
inhibitory factor and type 2 T-helper cytokines by decidual T cells
in unexplained recurrent abortions Nature Med 4, 1020–1024.
7 Bjorbaek, C., Buchholz, R.M., Davis, S.M., Bates, S.H., Pierroz,
D.D., Gu, H., Neel, B.G., Myers, M.G Jr & Flier, J.S (2001)
Divergent roles of SHP-2 in ERK activation by leptin receptors.
J Biol Chem 276, 4747–4755.
8 Schrager, J.A., Der Minassian, V & Marsh, J.W (2002) HIV Nef
increases T cell ERK MAP kinase activity J Biol Chem 277,
6137–6142.
9 Maccarrone, M., Di Rienzo, M., Finazzi-Agro`, A & Rossi, A.
(2003) Leptin activates the Anandamide Hydrolase promoter in
human T lymphocytes through STAT3 J Biol Chem 278,
13318–13324.
10 Di 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 (2001) Leptin-regulated endocannabinoids are involved in
maintaining food intake Nature 410, 822–825.
11 Harvey, J & Ashford, M.L (2003) Leptin in the CNS: much more
than a satiety signal Neuropharmacology 44, 845–854.
12 Veniant, M.M & LeBel, C.P (2003) Leptin: from animals to
humans Curr Pharm Des 9, 811–818.
13 Wargnier, A., Lafaurie, C., Legros-Maida, S., Bourge, J.-F.,
Sigaux, F., Sasportes, M & Pauli, P (1998) Down-regulation of
human granzyme B expression by glucocorticoids
Dex-amethasone inhibits binding to the Ikaros and AP-1 regulatory
elements of the granzyme B promoter J Biol Chem 273, 35326–
35331.
14 Sun, L., Heerema, N., Crotty, L., Wu, X., Navara, C., Vassilev,
A., Sensel, M., Reaman, G.H & Uckun, F.M (1999) Expression
of dominant-negative and mutant isoforms of the antileukemic
transcription factor Ikaros in infant acute lymphoblastic leukemia.
Proc Natl Acad Sci USA 96, 680–685.
15 Maccarrone, M., Bari, M., Di Rienzo, M., Finazzi-Agro`, A &
Rossi, A (2003) Progesterone activates fatty Acid Amide
Hydrolase (FAAH) promoter in human T lymphocytes
through the transcription factor Ikaros J Biol Chem 278, 32726–
32732.
16 Belelli, D & Herd, M.B (2003) The contraceptive agent Provera
enhances GABA (A) receptor-mediated inhibitory
neuro-transmission in the rat hippocampus: evidence for endogenous
neurosteroids? J Neurosci 23, 10013–10020.
17 Sakamoto, H., Shikimi, H., Ukena, K & Tsutsui, K (2003)
Neonatal expression of progesterone receptor isoforms in the
cerebellar Purkinje cell in rats Neurosci Lett 343, 163–166.
18 Maccarrone, M., van der Stelt, M., Rossi, A., Veldink, G.A.,
Vliegenthart, J.F.G & Finazzi-Agro`, A (1998) Anandamide
hydrolysis by human cells in culture and brain J Biol Chem 273,
32332–32339.
19 Maccarrone, M & Finazzi Agro`, A (2003) The endocannabinoid
system, anandamide and the regulation of mammalian cell
apoptosis Cell Death Differ 10, 946–955.
20 Cadas, H., di Tomaso, E & Piomelli, D (1997) Occurrence and
biosynthesis of endogenous cannabinoid precursor,
N-arachido-noyl phosphatidylethanolamine, in rat brain J Neurosci 17,
1226–1242.
21 Moesgaard, B., Petersen, G., Jaroszewski, J.W & Hansen, H.S.
(2000) Age dependent accumulation of N-acyl-ethanolamine
phospholipids in ischemic rat brain A (31) P NMR and enzyme
activity study J Lipid Res 41, 985–990.
22 Bisogno, T., De Petrocellis, L & Di Marzo, V (2002) Fatty acid
amide hydrolase, an enzyme with many bioactive substrates.
Possible therapeutic implications Curr Pharm Des 8, 533–547.
23 Parolaro, D., Massi, P., Rubino, T & Monti, E (2002) Endocannabinoids in the immune system and cancer Pros-taglandins Leukot Essent Fatty Acids 66, 319–332.
24 Cravatt, B.F & Lichtman, A.H (2003) Fatty acid amide hydro-lase: an emerging therapeutic target in the endocannabinoid sys-tem Curr Opin Chem Biol 7, 469–475.
25 Giang, D.K & Cravatt, B.F (1997) Molecular characterization of human and mouse fatty acid amide hydrolases Proc Natl Acad Sci USA 94, 2238–2242.
26 Okamoto, Y., Morishita, J., Tsuboi, K., Tonai, T & Ueda, N (2004) Molecular characterization of a phospholipase D generating anandamide and its congeners J Biol Chem 279, 5298–5305.
27 Tartaglia, 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 (1995) Identification and expression cloning of a leptin receptor, OB-R Cell 83, 1263–1271.
28 Bradford, M.M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72, 248–254.
29 Schreiber, E., Muller, M.M & Schaffner, W (1989) Rapid detection of octamer binding proteins with mini-extracts, prepared from a small number of cells Nucleic Acids Res 17, 6419.
30 Lee, J.-H., Jang, S.-I., Markova, N.G & Steinert, P.M (1996) The proximal promoter of the human transglutaminase 3 gene Stratified squamous epithelial-specific expression in cultured cells is mediated by binding of Sp1 and ets transcription factors
to a proximal promoter element J Biol Chem 271, 4561– 4568.
31 Dorsam, G & Goetzl, E.J (2002) Vasoactive intestinal peptide receptor-1 (VPAC-1) is a novel gene target of the hemolympho-poietic transcription factor Ikaros J Biol Chem 277, 13488– 13493.
32 Cohen, B., Novick, D & Rubinstein, M (1996) Modulation of insulin activities by leptin Science 274, 1185–1188.
33 Wang, Y., Kuropatwinski, K.K., White, D.W., Hawley, T.S., Hawley, R.G., Tartaglia, L.A & Baumann, H (1997) Leptin receptor action in hepatic cells J Biol Chem 272, 16216–16223.
34 Wan, M., Cravatt, B.F., Ring, H.Z., Zhang, X & Francke, U (1998) Conserved chromosomal location and genomic structure of human and mouse fatty-acid amide hydrolase genes and evalua-tion of clasper as a candidate neurological mutaevalua-tion Genomics 54, 408–414.
35 Waleh, N.S., Cravatt, B.F., Apte-Deshpande, A., Terao, A & Kilduff, T.S (2002) Transcriptional regulation of the mouse fatty acid amide hydrolase gene Gene 291, 203–210.
36 Niehof, M., Streetz, K., Rakemann, T., Bischoff, S.C., Manns, M.P., Horn, F & Trautwein, C (2001) Interleukin-6-induced tethering of STAT3 to the LAP/C/EBPbeta promoter suggests a new mechanism of transcriptional regulation by STAT3 J Biol Chem 276, 9016–9027.
37 Tora, L., Gronemeyer, H., Turcotte, B., Gaub, M.P & Chambon,
P (1998) The N-terminal region of the chicken progesterone receptor specifies target gene activation Nature 333, 185–188.
38 Wen, D.X., Xu, Y.F., Mais, D.E., Goldman, M.E & McDonnell, D.P (1994) The A and B isoforms of the human progesterone receptor operate through distinct signaling pathways within target cells Mol Cell Biol 14, 8356–8364.
39 Cravatt, B.F., Demarest, K., Patricelli, M.P., Bracey, M.H., Giang, D.K., Martin, B.R & Lichtman, A.H (2001) Super-sensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase Proc Natl Acad Sci USA 98, 9371–9376.