Differential regulation of fatty acid amide hydrolase promoter in
human immune cells and neuronal cells by leptin and progesterone
Mauro Maccarrone
1,2
, Valeria Gasperi
3
, Filomena Fezza
1
, Alessandro Finazzi-Agro
`
3
and Antonello Rossi
3
1
Department of Biomedical Sciences, University of Teramo, Italy;
2
IRCCS C. Mondino, Mondino-Tor Vergata-Santa Lucia
Center for Experimental Neurobiology, Rome, Italy;
3
Department 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 e xpression o f the e ndocan-
nabinoid-degrading enzyme fatty acid amide hydrolase
(FAAH), through S TAT3 (signal transducer and activator
of transcription 3) and its CRE (cAMP response element)-
like t ranscriptional t arget i n t he FAAH p romoter [Maccar-
rone, M., Di Rienzo, M., Finazzi-Agro
`
,A.,&Rossi,A.
(2003) J. Biol. C hem. 278, 13318–13324]. We have also

max
¼ 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 t he control o f tone a nd activity of AEA along the
neuroimmune axis.
Keywords: endocan nabinoids; i mmune system; l eptin; neu-
rons; progesterone.
Leptin is the 16-kDa nonglyco sylated product of the obese
gene, which is secreted by adipose cells, is released into
the circulation, and transported across the blood–brain-
barrier into t he central nervous system, where it regulates
energy homeostasis [1]. Leptin also serves systemic
functions, a part 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) m embers have a
prominent role [7,8]. Recently, we have shown that leptin

dylethanolamine; NAT, N-acyltransferase; PL D, ph ospholipase D;
PR, progesterone receptor; STAT, signal transducer and activator of
transcription.
Enzymes: Fatty acid amide hydrolase (E C 3.5.1.4); phosphatidylcho-
line phosphatidohydrolase (EC 3.1.4.4).
(Received 4 June 2004, revised 31 August 2004,
accepted 8 October 2004 )
Eur. J. Biochem. 271, 4666–4676 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04427.x
Based o n 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 inves tigated whether
leptin and progesterone can modulate the Ôon demandÕ
synthesis of AEA through N-acyltransferase [20] and
N-acylp hosphatidylethanolamine (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, A MT, NAT and P LD 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, w hile they do not in human neuronal cells.
Therefore, the central actions of these two hormones do
not seem to involve the endocannabinoid system but

)1
} were from NEN Life Science
Products, Inc. (Boston, MA, USA). [
3
H]N-arachidonoyl-
phosphatidylethanolamine (200 CiÆmmol
)1
)wasfromARC
(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, a nd were prepared by Primm S.r.l.
(Milan, Italy). M ouse monoclonal antibodies against actin,
STAT3, phospho-STAT3, leptin receptor (LR) and pro-
gesterone re ceptor (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 m anufacturer’s instructions,  1.5 lgÆmL
)1
of
sLR are enough to n eutralize the effects o f 10 n

M
Hepes buffer, 2 m
M
L
-glutamine and 1% nonessential amino acids [18]. Both
CHP100 and U937 cells were maintained at 37 °Cin
humidified 5% CO
2
atmosphere. Incubation of U937 or
CHP100 cells with leptin, alone or in the presence of
different compounds, or with progesterone was performed
at 37 °C in humidified 5% CO
2
atmosphere, 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 w ith v ehicles 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 h ydrolase (EC 3.5.1.4; FAAH) activity
was a ssayed a t pH 9.0 by the reversed-phase HPLC method
described previously [18] using 1 0 l
M
[
3
H]AEA as substrate.
Cell homogenates (20 lg per lane) were prepared as

cells) by means of the
SNAprogesterone
TM
Total RNA Isolation Kit (Invitrogen,
Carlsbad, CA, USA), as described [9]. RT-PCR reactions
were performed using 100 ng of total RNA, for the
amplification of FAAH, or 0.4 ng, for 18S rRNA, and
the EZ rTth RNA PCR k it (PerkinElmer, Norwalk, CO,
USA). 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 the following: (+) 5¢-TGGAAGTCCTCCA
AAAGCCCAG (–) 5¢-TGTCCATAGACACAGCCCTT
CAG, for FAAH; (+) 5¢-AGTTGCTGCAGTTAAAA
AGC (–) 5¢-CCTCAGTTCCGAAAA CCAAC, for 18S
rRNA.
Five microliters o f the reaction mixture were electro-
phoresed on a 6% polyacrylamide gel, w hich was then dried
and subjected to autoradiograp hy [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 of
N
-acyltransferase, phospholipase D,
anandamide transporter and leptin receptor binding
An N-acyltransferase (NAT) assay was performed as
described [20], u sing 1,2-di[1–
14

per
test) through AMT was studied as described [18], and was
expressed as p mol AEA taken up per min p er mg protein.
The binding of
125
I-labeled leptin to U937 or CHP100 cells
was analyzed by rap id filtration assays [27]. In this case,
apparent dissociation constant (K
d
) and maximum binding
(B
max
) values were calculated f rom saturation curves in the
range 0–12 n
M
, elaborating the binding data through
nonlinear regression analysis with the
PRISM
3program
(GraphPAD Sofware for Science, San Diego, CA, USA) [9].
Unspecific binding was determined in the presence of
100 n
M
Ôcold Õ leptin [27]. The expression of leptin receptor
(LR) and of p rogesterone 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 : 5 00) as first antibody, a nd GAM-AP diluted
1 : 2000 as second antibody [9]. The specificity of anti-LR
and anti-PR Igs was ascertained by using t he corresponding

FAAH mRNA) was assembled using synthetic oligonucleo-
tides (Amersham Pharmacia Biotech). The DNA was
gel-purified and subcloned into the PstI/XbaIsitesof
pCAT3-Basic vector ( Promega Corporation, Madis on,
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 S equenase kit 2.0 (USB, Cleveland, OH, USA).
Human U937 or C HP100 cells (1 · 10
6
per test) were
transfected in triplicate using TransFast
TM
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
TransFast
TM
Transfection Reagent, at a charge ratio of 1 : 1
with respect to DNA. Transfection efficiency was monitored
by use o f 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

mutated nucleotides are in italic tyepface) [31]. In all
oligonucleotides, the numbers refer to positions in the
FAAH promoter. The complexes were resolved o n non-
denaturing 6% polyacrylamide gels in 0.5· TBE buffer
(0.45
M
Tris/borate, 10 m
M
EDTA, pH 8.0) for 1 h at
14 VÆcm
)1
, and were autoradiographed overnight.
Statistical analyses
Data reported in t his 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 b y
means of the
INSTAT
3 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, w e 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

of U937 cell extracts s howed 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 w ith 10 n
M
leptin or 1 l
M
progesterone
than in controls (Fig. 1C). On the other hand, U937 cells
treatedwith10n
M
leptin or 1 l
M
progesterone expressed
the same levels of actin as the controls (Fig. 1C), ruling out
that the d ifferent 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 f or FAAH gene, which
increased in cells treated with 10 n
M
leptin or 1 l
M
progesterone (Fig. 1C). Under the same experimental
conditions, the expression of the 18S rRNA gene was
unaffected (Fig. 1C). Liquid scintillation counting of

I-labeled leptin according to a saturable process (Fig. 2A)
that yielded apparent dissociation constants (K
d
)of
2.0 ± 0.1 and 2.2 ± 0.2 n
M
and maximum binding (B
max
)
of 382 ± 5 and 339 ± 8 fmolÆmg protein
)1
, f or 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]. ÔColdÕ leptin fully displaced
125
I-labeled leptin from
its binding site, when used at 100 n
M
(Fig. 2 A). Moreover,
Western blot analysis showed that specific anti-LR Igs
Ó FEBS 2004 Differential regulation of the human FAAH promoter (Eur. J. Biochem. 271) 4669
recognized a s ingle immunoreactive b and 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 a nalysis also showed that specific anti-PR Igs
recognized a single immunoreactive band with the same
intensity i n U937 and CHP100 cells, with an apparent

CHP100 cells, 25 ± 3 pmolÆmin
)1
Æmg pro-
tein
)1
). (B) Effect of progesterone o n the s ame
samples a s in (A). Cells w ere incubated with
progesterone alone, or with 1 l
M
progesterone
inthepresenceof10l
M
RU486. In both
panels: * P < 0.05 vs. control, **P <0.01vs.
control and #P <0.01vs.10n
M
leptin or vs.
1 l
M
progesterone, r espectively. In both pan-
els, vertical bars represent SD values. (C) Up-
per p anel, 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.

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

Æmg protein
)1
.
4670 M. Maccarrone et al. (Eur. J. Biochem. 271) Ó FEBS 2004
leptin [9] or p rogesterone [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 c ells treated with 1 0 n
M
lepti n
(Fig. 3A). In addition, nu clear l evels o f I karos isoforms Ik1,
Ik2 and Ik3 were increased by treatment of U937 cells, but
not of CHP100 cells, for 24 h with 1 l
M
progesterone
(Fig. 3B). Quantitative a nalysis 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. 3 D).
Analysis of the
FAAH
promoter

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 w ith 10 n
M
leptin or 1 l
M
progesterone. As shown
in Fig. 5A, complex formation was only found with
oligonucleotides containing the CRE-like site of the FAAH
promoter. C omplex formation was not seen when the
mutant oligonucleotide (bearing the same mutation used
for the transient transfection experiment) was used as a
32
P-labeled probe, neither wa sitobservedinCHP100cells
under the same experimental conditions (Fig. 5A). On the
other hand, specificity o f the binding to the Ikaros s ite was
confirmed b y u sing a cold c ompetitor, which corresponds to
an established functional Ik site derived from the vasoactive
intestinal peptide receptor-1 promoter [31], and the mutated
site used for transient transfection experiments. Cold
competitor and m utated site, r espectively, 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. 5 C).
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 prog esterone

)
similar to those of the leptin receptor [32,33]. This obser-
vation, together with the ability of ÔcoldÕ leptin to fully
displace
125
I-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 L R [ 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 a t circula-
ting levels of 1 l
M
( 0.3 lgÆmL
)1
), on FAAH activity in
U937 cells extend our previous observations on T l ympho-
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
FAAH gene expression were mediated through its bin ding
to PR [15]. On the other hand, leptin and progesterone were
ineffective on AEA transport through AMT, and on AEA
synthesis through NAT and PLD in U937 cells (Table 2),
suggesting that FAAH was the only Ôcheck pointÕ for the

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 o f 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 lgofprotein)
shown in (A,B) is reported in (C,D), where
*P < 0.01 vs. control (CTR), and vertical
bars represent SD values.
4672 M. Maccarrone et al. (Eur. J. Biochem. 271) Ó FEBS 2004
detected in the nucleus of human peripheral blood mono-
nuclear cells [13,14], where they are essential in contro lling
the activation of granzyme B promoter by the glucocorti-
coid, dexamethasone [13]. Here, we demonstrated that
circulating levels of progesterone (1 l
M

Ó FEBS 2004 Differential regulation of the human FAAH promoter (Eur. J. Biochem. 271) 4673
A major finding of this investigation is that FAAH
activity and expression in human neuroblastoma CHP100
cells was not up-regulated by leptin and/or progesterone,
although these cells use LR, PR, S TAT3 and Ikaros. Th is
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 l ack of responsiveness of neuronal c ells
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
)1
Æmg protein
)1
with 10 l
M
[
3
H]AEA as
substrate; Fig. 1) and expression (Fig. 1C,D) in CHP100
cells is significantly lower than that in U937 cells seems to

links between networks controlled by leptin o r 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. T his study was partly s upported
by Ministero dell’Istruzione, dell’Universita
`
edellaRicerca(Cofin
2003) and by Istituto Superiore di Sanita
`
(III AIDS project), Rome.
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