From the Department of Biomedical Sciences,
University of Teramo, Piazza A. Moro 45, 64100 Teramo, Italy, and
the ¶ Department of Experimental Medicine and Biochemical
Sciences, University of Rome Tor Vergata, Via Montpellier 1, I-00133 Rome, Italy
Received for publication, November 4, 2002, and in revised form, January 22, 2003
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ABSTRACT |
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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 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 (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 and 2 cytokines (4), which are
critical in regulating embryo implantation and materno-fetal exchanges
(5, 6). 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
(1-4). Leptin signaling is mediated by the leptin receptor (LR), which
exists in at least six different isoforms (1). 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 (7-11). 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 of
ob/ob mice, suggesting that this compound partakes of the
neural circuitry regulated by leptin (12).
AEA belongs to a group of endogenous lipids, which include amides,
esters, and ethers of long chain polyunsaturated fatty acids,
collectively termed "endocannabinoids" (13). It binds to
cannabinoid receptors (CBR) in the central nervous system and in
peripheral immune cells, thus having many central actions (14). Together with the congeners 2-arachidonoylglycerol (12, 15) and
oleoylethanolamide (16), AEA has been implicated in the regulation of
appetite. Among the peripheral activities of AEA, the regulation of
fertility (17) and immune function (18) has attracted growing interest.
These biological actions of AEA are terminated by cellular uptake
through an AEA membrane transporter (AMT) (19), followed by degradation
to ethanolamine and arachidonic acid by the enzyme AEA hydrolase (fatty
acid amide hydrolase, FAAH) (20). Human lymphocytes have functional
CBR, AMT, and FAAH, and the latter enzyme has been shown to play a
critical role in regulating human pregnancy (21). 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 (22). 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
(21-23). 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 (24). 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.
Materials--
Chemicals were of the purest analytical grade.
Leptin (human recombinant for T-cell studies, mouse recombinant for
ob/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 (25) 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).
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 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 (22).
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 (22). 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/ob mice (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 (12). 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.
Anandamide Hydrolase Activity and Expression--
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
(26). Cell homogenates (20 µg/lane) were prepared as described (26)
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
(22). Actin was immunodetected with anti-actin monoclonal antibodies
(1:500), using GAM-AP diluted 1:2000 as second antibody (22).
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
(22). 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
(22). 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 (22). 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 (22). 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 (22).
Analysis of Anandamide Uptake, Cannabinoid Receptor, and Leptin
Receptor--
The uptake of 200 nM [3H]AEA
by intact T lymphocytes (2 × 106/test) through the
AMT was studied as described (26). For CBR studies, membrane fractions
were prepared from T lymphocytes (10 × 106) as
reported (26), were quickly frozen in liquid nitrogen, and stored at
Western Blot Analysis of Protein Phosphorylation--
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 (11). 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 (22). Protein content
was normalized before loading onto the gel, and equal loading of
extracts was verified by Ponceau staining (22).
Construction of Chloramphenicol Acetyltransferase (CAT)
Expression Vectors and Transient Transfection--
Sequence
information for the upstream regulatory region of the
FAAH 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
Nuclear Extracts, EMSA, and Gel Supershift Assays--
Nuclear
extracts were prepared according to Schreiber and co-workers (28) with
the modifications reported by Lee and co-workers (29). EMSA experiments
were performed as described (29), deriving the sequence for the
wild-type cAMP responsive element (CRE)-like site bandshift from
the FAAH promoter region: 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).
Leptin Stimulates FAAH Activity and Expression in Human T
Lymphocytes--
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 proteins. The same
anti-FAAH antibodies were used to further quantify FAAH content by
ELISA, which showed that L 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 showed a single band of the expected molecular size
for FAAH gene, which increased dose dependently in
L-treated cells (Fig. 2B). Densitometric analysis of the
autoradiographic film shown in Fig. 2B (representative of
triplicate experiments) indicated that FAAH mRNA increased to 120, 165, and 200% of the control (100% = 2500 ± 280 units/mm2) in cells treated for 24 h with 1, 5, or 10 nM L, respectively. Under the same experimental conditions,
the expression of the 18 S rRNA gene was unaffected (Fig.
2B). Liquid scintillation counting of RT-PCR products showed
that L increased time dependently FAAH mRNA in human T lymphocytes
in a way parallel to that of enzymic activity and protein content (Fig.
1B). Remarkably, treatment of human lymphocytes with 10 nM leptin for 24 h reduced the levels of AEA in these
cells from ~3.0 to ~0.5 pmol/108
cells.2 On the other hand,
the same concentrations of leptin, which enhanced FAAH activity, and
expression were ineffective on CBR binding and AMT activity in T-cells
(Fig. 1C). Furthermore, FAAH activity and protein content in
peripheral lymphocytes from leptin knock-out (ob/ob) mice
were ~25% of the levels in wild-type controls (Table I).
Intravenous injection of a single dose of mouse recombinant leptin (250 µg/ml) into
ob/ob mice was able to increase FAAH activity and content in
peripheral lymphocytes to control levels, whereas it was ineffective on
FAAH of wild-type mice (Table I).
Human T Lymphocytes Have a Functional Leptin Receptor--
Human
T-cells were able to bind 125I-labeled leptin according to
a saturable process (Fig. 3A),
which yielded an apparent dissociation constant (Kd)
of 1.95 ± 0.14 nM and maximum binding
(Bmax) of 392 ± 8 fmol·mg
protein Activation of Downstream Signals by Binding of Leptin to LR in
T-cells--
The binding of leptin to the long form of its receptor is
known to activate different signaling pathways by phosphorylating either STAT 1, 3, or 5 or p42/p44 MAPK (8, 9, 31). Also activation of
p38 MAPK (10) can be part of the signaling cascade triggered by LR.
Therefore, the levels of these proteins were measured in L-treated T
lymphocytes. The non-phosphorylated, inactive forms of each of the
signals studied were present in T-cells, yet only phosphorylated STAT3
increased dose dependently in L-treated T lymphocytes (Fig.
4B). Densitometric analysis of
filters like that shown in Fig. 4 (representative of triplicate
experiments) showed that phosphorylated STAT3 was ~1.5-, ~2.4-, and
~3.5-fold higher than total STAT3 in T lymphocytes treated for
24 h with 1, 5, or 10 nM L, respectively. On the other
hand, none of the other signaling proteins was activated by L under the
same experimental conditions (Fig. 4, A, C-E).
Consistently, the selective inhibitor of p42/p44 MAPK PD98059 (32) or
the selective inhibitor of p38 MAPK SB203580 (33) were ineffective on
the activation of FAAH induced in T-cells by 10 nM L (Fig.
3B) when used at concentrations known to inhibit the target
enzyme. The lack of inhibitors of STAT1, 3, or 5 did not allow to
further extend the pharmacological experiments to these proteins.
Analysis of the FAAH Promoter--
The human FAAH gene
has been located on chromosome 1 (34), which has been completely
sequenced. Therefore, we 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. 5A).
Moreover, there was another SP1 site in the reverse orientation ~100
nucleotides upstream (Fig. 5A), a feature that resembles the
structure of the mouse proximal promoter (35). Inspection of the
promoter sequence did not show any obvious binding site for STAT3;
however, a new mechanism of transcriptional regulation based on STAT3
tethering to a CRE-like site in the LAP/C/EBP In this study we show that leptin stimulates FAAH activity and
expression in human T lymphocytes through a leptin receptor-mediated activation of STAT3 signaling, which leads to up-regulation of a
CRE-like site in FAAH promoter. Moreover, by using leptin-deficient (ob/ob) mice we also show that leptin tonically controls
FAAH activity in vivo, which opens new avenues for the
management of immune and fertility defects under leptin control in humans.
Leptin modulates FAAH activity and expression in T-cells at the same
circulating levels shown to unbalance cytokine production from these
cells (4). These concentrations of L were found to saturate the binding
sites in T lymphocytes (Fig. 3A) with calculated binding
constants (Kd and Bmax)
similar to those of the leptin receptor (7, 30). This observation
together with the ability of cold L to fully displace
125I-labeled L strongly suggest that T-cells have an
authentic LR, thus extending a previous study that demonstrated that
the same cells express the mRNA for the long isoform of LR (4).
These findings taken together with the observation that the effects of
L on FAAH activity and expression were fully neutralized by anti-L
antibodies and by soluble LR (Fig. 1A) strongly suggest that
FAAH activation by L occurred through activation of LR. On the other
hand, L was ineffective on CBR binding and AMT activity in T
lymphocytes (Fig. 1C), suggesting that FAAH was the only "check point" for the effect of L. These observations extend
previous studies on the role of FAAH, but not CB receptors or AEA
transporter, in modulating immunoendocrine interactions in early
pregnancy in humans (21, 22). They are also in keeping with the
hypothesis that FAAH is the key regulator of AEA levels in
vivo; indeed FAAH knock-out mice show ~15-fold higher levels of
AEA than wild-type littermates (36), and AEA levels in human blood
inversely correlate with FAAH activity in peripheral lymphocytes (23).
In the same line, we found that 10 nM L, which increased
FAAH in T lymphocytes up to ~300% over controls (Fig.
1A), reduced AEA levels in these cells down to
~15%.2 Interestingly, FAAH has been shown to be
critically linked to drug/alcohol abuse and dependence in humans (37),
again suggesting that this enzyme is pivotal for controlling the
biological activity of AEA and potentially that of other
FAAH-hydrolyzable congeners like 2-arachidonoylglycerol and
oleoylethanolamide (12, 16). In addition, the reduction of FAAH
activity and content in leptin-deficient (ob/ob) compared
with wild-type mice and the ability of exogenous leptin to reverse this
reduction (Table I) speak in favor of a tonic control of FAAH by L
in vivo. This hypothesis is strengthened by the lack of
effect of exogenous L in wild-type mice (Table I), which already have
enough circulating L to saturate LR binding sites (1, 2). In this
context, it seems noteworthy that AEA did not affect L binding to LR
(Fig. 3A), suggesting that AEA could not directly contribute
to the L-mediated regulation of its degradation by FAAH. At any rate,
the up-regulation of FAAH expression by L is a major finding of this
investigation and is associated with higher FAAH activity in
T-cells.
The mechanism of FAAH activation by L was further investigated. Binding
of L to the long isoform of LR is known to trigger different signaling
pathways, which include STAT-dependent and/or MAPK-dependent signal transduction (7-11). In human T
lymphocytes, physiological doses of L activated only STAT3 (Fig. 4,
A-E), which is preferentially activated also in other cell
types (7, 8, 31) and tissues (Ref. 1 and references therein). To
clarify how STAT3 could up-regulate FAAH activity and expression, we
analyzed the FAAH promoter. Like many promoters lacking a TATA box, it had a proximally positioned SP1 site, which was preceded both in the
human and mouse promoters by another SP1 site in reverse orientation
(Fig 5A). The FAAH promoter did not contain STAT3 DNA
binding motifs yet it did contain a CRE-like element between the two
SP1 sites (Fig. 5A). Such CRE-like elements have been recently shown to be tethered by STAT3, thus leading to a novel type of
up-regulation of gene transcription (31). Transfection experiments
using FAAH promoter constructs with mutated CRE-like elements (mutL)
revealed that indeed these sites confer STAT3 responsiveness (Fig. 6).
EMSA analysis and gel supershift assays further corroborated this
conclusion (Fig. 5B). Therefore, it can be concluded that L,
by binding to LR in human T lymphocytes, activates STAT3, which in turn
up-regulates FAAH gene transcription by tethering to
a CRE-like site in the FAAH promoter. The overall regulation of FAAH
promoter by L in human T-cells is depicted in Scheme
I.
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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
(26). The same filtration assays were used to analyze the binding of
125I-labeled leptin to T-cells (9, 27). 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) (26). Unspecific
binding was determined in the presence of 100 nM "cold"
leptin (9, 27).
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
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 TransFastTM
Transfection 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
-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
-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
-galactosidase was assayed
using the
-Galactosidase Enzyme System (Promega). The values of CAT
activity were normalized to
-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.
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 of
32P-labeled oligonucleotide (31). Dye was omitted from the
loading buffer, and the gel was run at 4 °C in 0.2× TBE buffer at 5 V/cm.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of leptin on FAAH activity, CBR
binding, and AMT activity. A, effect of leptin on the
activity (white bars) and the protein content (hatched
bars) of FAAH. Human T lymphocytes were incubated for 24 h
with leptin alone or with 10 nM (~150 ng/ml) leptin in
the presence of 3 µg/ml anti-leptin antibodies, sLR, or anti-IGF-IR
mock antibodies (100% = 140 ± 15 pmol·min 1·mg
protein
1 for the activity or 0.220 ± 0.025 absorbance units at 405 nm for the protein content). B,
time-course of the effect of 10 nM leptin on FAAH activity
(white bars), protein content (hatched bars), and
mRNA level (black bars) in human T-cells (100% as in
A for the activity and the protein content; 100% = 15000 ± 1500 cpm for the mRNA level).
C, CBR binding and AMT activity in T-cells treated for
24 h with 10 nM leptin (100% = 20000 ± 2000 cpm·mg protein
1 for CBR binding or 50 ± 5 pmol·min
1·mg protein
1 for AMT
activity). In A-B, * denotes p < 0.05 versus control and ** denotes p < 0.01 versus control. In all panels, vertical bars
represent S.D. values.
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Fig. 2.
Effect of leptin on FAAH expression.
A, Western blot analysis of human lymphocytes, treated with
different amounts of leptin and reacted with specific anti-FAAH
(upper panel) or anti-actin (lower panel)
antibodies. Molecular mass markers and the positions of FAAH and actin
are indicated to the right. B, RT-PCR analysis of
cDNA of the same samples as in A. The expected sizes of
the amplicons (199 bp for FAAH and 258 bp for 18 S rRNA) are indicated
to the right. These data are representative of three
independent experiments.
FAAH activity and expression in peripheral lymphocytes isolated from
wild-type and leptin knock-out (ob/ob) mice
1·mg
protein
1 for the activity and 0.220 ± 0.025 A405 units for the protein
content).
1. These values are in agreement with previous
reports on LR of human hepatic cells (7, 30) and on LR stably
transfected into different cell types (7, 27). Cold leptin fully
displaced 125I-labeled leptin from its binding site when
used at 100 nM, whereas AEA was ineffective at
concentrations up to 10 µM (Fig. 3A).
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Fig. 3.
Characterization of LR. A,
saturation curves of the binding of 125I-labeled leptin to
human T lymphocytes alone (circles) or in the presence of
100 nM cold leptin (triangles) or of 10 µM anandamide (squares). B, effect
of p42/44 MAPK inhibitor PD98059 and of p38 MAPK inhibitor SB203580
(each used at 10 µM) on the activation of FAAH induced by
a 24-h treatment of T-cells with 10 nM leptin. In
B, ** denotes p < 0.01 versus
control. In both panels, vertical bars represent S.D.
values.
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[in a new window]
Fig. 4.
Activation of downstream signals by
leptin. Human T lymphocytes were treated for 24 h with
different concentrations of leptin, and then lysates (50 µg of
proteins) were immunoblotted with the specific antibody against the
inactive (total) or active (phosphorylated,
phospho) forms of STAT1 (A), STAT3
(B), STAT5 (C), p42/p44 MAPK (D), or
p38 MAPK (E). The positions of phosphoproteins are indicated
to the right. These data are representative of three
independent experiments.
promoter has been
recently reported (31). Such a CRE-like site was indeed present in the
FAAH promoter (Fig. 5A), and transient transfections using
constructs containing only the proximal SP1 site (
33 to +1) or both
the SP1 and the CRE-like (
107 to +1) sites driving the CAT reporter
gene in T lymphocytes showed that the
33 construct worked as an
unregulated promoter (Fig. 6,
min), while the
107 construct was up-regulated by leptin
(Fig. 6, wt). The two SP1 sites in the human FAAH promoter
flank a sequence that resembles a CRE-like site similar to that found
in the LAP/C/EBP
promoter (31). Disruption of this site by mutation
abolished the leptin up-regulation (Fig. 6, mutL and
mutL + leptin). To confirm that leptin acts through this
sequence, EMSA experiments were performed using nuclear extracts
prepared from T lymphocytes untreated or treated for 24 h with 10 nM leptin. As shown in Fig. 5B, complex
formation was found only 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. Gel supershift assays showed the
presence of the STAT3 protein in the shifted complex (Fig.
5B).
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[in a new window]
Fig. 5.
Analysis of FAAH promoter by EMSA.
A, paired proximal upstream regions of human (H)
and mouse (M) FAAH genes. Identical nucleotides
are marked with an asterisk. Left- and
right-handed arrows denote SP1 sites in the and + strands respectively. Oval box, CRE-like site;
rectangular box, estrogen-responsive element
(ERE) site. B, gel shift and supershift (wt + Ab) experiments were performed with 3 µg of T lymphocytes
nuclear extracts prepared before (
) and after (+) stimulation with 10 nM leptin. Shifted and supershifted complexes are indicated
with a big and a small arrow respectively.
Oligonucleotides as 32P-labeled probes contained mutated
(mutL) or wild-type (wt) CRE-like site.
View larger version (18K):
[in a new window]
Fig. 6.
Analysis of FAAH promoter by transient
expression. The 5' flanking regions of the human FAAH
gene were cloned in the PstI/XbaI sites of
pCAT3-basic vector. Min (black bars), 33 to +1
upstream region containing only the proximal SP1 site; wt
(dark gray bars),
107 to +1 upstream region containing the
two SP1 sites flanking a putative CRE-like site (gray);
mutL (light gray bars), same as wt but
with the mutated CRE-like sequence; mutated sites and nucleotides are
in white and underlined respectively. T
lymphocytes were transfected with the aforementioned constructs and
left untreated or treated with leptin. Transfection efficiency was
monitored by the use of thymidine kinase
-galactosidase construct.
The values of CAT activity were normalized to
-galactosidase
activity and to the protein content and are expressed as % with
respect to the activity of the empty vector, pCAT3-basic, which was set
to 100%. * denotes p < 0.01 versus
control, and vertical bars represent S.D. values.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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[in a new window]
Scheme 1.
Model of the activation of FAAH
promoter by leptin in human T-cells. L binds to its receptor LR in
human T lymphocytes leading to the activation of STAT3 but not of other
typical LR-dependent downstream signals like STAT1, STAT5,
or MAPK, p38, p42, or p44. Phosphorylation of STAT3 results in the
activation of a CRE-like region in the FAAH promoter, thus
up-regulating gene expression.
It seems noteworthy that this is the first characterization of the promoter and the first description of the transcriptional regulation of human FAAH. As yet, two interesting reports have characterized the promoter (38) and the transcriptional regulation (35) of mouse FAAH in neuronal cell lines. In particular, they have shown either putative (38) or imperfect (34) estrogen response elements in the FAAH promoter region, giving some ground to our previous report that estrogen down-regulates FAAH activity in mouse (39). Remarkably, here we show that the human FAAH promoter does not contain an estrogen response element at the same position (Fig. 5A). In this context, we have recently shown that estrogen down-regulates FAAH in human cells according to a non-genomic mechanism (40). These observations suggest a relevant species specificity of FAAH regulation, though the human and mouse FAAH (localized on chromosome 1 and 4, respectively) share 84% sequence identity (25) and have a conserved genomic structure (34). In addition, also a tissue specificity of FAAH promoter activity has been observed (38), which might further contribute to divergent regulation in different species or in different tissues of the same species. The interaction between different transcription factors, some of which have been identified here for the first time, on FAAH regulation awaits for further clarification.
In conclusion, the results reported here represent the first evidence
of a link between the hormone-cytokine networks controlled in T
lymphocytes by leptin and the peripheral endocannabinoid system and
suggest that FAAH, but not anandamide transporter or CB receptors,
might be the target for new therapies of human defects in immunity and fertility.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Judith Harvey-White and Prof. George Kunos (National Institute on Alcohol Abuse and Alcoholism) for the assays of anandamide levels, Dr. Massimo Federici for helpful discussions, and Drs. Monica Bari and Natalia Battista for their expert assistance in cell isolation and culture.
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FOOTNOTES |
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* This study was partly supported by Istituto Superiore di Sanità (IV AIDS project) and by Ministero dell'Istruzione, dell'Università e della Ricerca (COFIN 2002), Rome, Italy.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 39-0861-266875; Fax: 39-0861-412583; E-mail: Maccarrone@vet.unite.it.
Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M211248200
2 M. Maccarrone, J. Harvey-White, G. Kunos, and A. Finazzi-Agrò, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: L, leptin; LR, leptin receptor; AEA, anandamide (arachidonoylethanolamide); AMT, AEA membrane transporter; CAT, chloramphenicol acetyltransferase; CBR, cannabinoid receptors; CP55.940, 5-(1,1'-dimethylheptyl)-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/M-AP, goat anti-rabbit/mouse antibodies conjugated with alkaline phosphatase; IGF-IR, insulin-like growth factor I receptor; sLR, soluble leptin receptor; MAPK, mitogen-activated protein kinase; RAG-AP, rabbit anti-goat antibodies conjugated with alkaline phosphatase; RT, reverse transcriptase; STAT, signal transducer and activator of transcription.
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