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The Endocannabinoid System in Human Keratinocytes. Evidence that
Anandamide Inhibits Epidermal Differentiation through CB1 Receptor-
Dependent Inhibition of Protein Kinase C, Activating Protein-1 and
Transglutaminase*
Mauro Maccarrone‡¶, Marianna Di Rienzo§, Natalia Battista§, Valeria Gasperi§, Antonello
Rossi§, and Alessandro Finazzi-Agrò§
From the ‡Department of Biomedical Sciences, University of Teramo, Piazza A. Moro 45,
64100 Teramo, Italy, and the IRCCS C. Mondino, Mondino-Tor Vergata- Santa Lucia
Center for Experimental Neurobiology, Via Ardeatina 306, 00179 Rome, Italy, and the
§Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor
Vergata, Via Montpellier 1, 00133 Rome, Italy
Running title: The endocannabinoid system in human keratinocytes
*This study was partly supported by Ministero dell’Istruzione, dell’Università e della
Ricerca (Cofin 2002) and by Agenzia Spaziale Italiana (contract I/R/098/00), Rome. The
costs of publication of this article were defrayed in part by the payment of page charges. This
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: Department of Biomedical Sciences,
University of Teramo, Piazza A. Moro 45, 64100 Teramo, Italy. Tel.: 39-0861-266875; fax:
39-0861-412583; e-mail: [email protected] .
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Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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SUMMARY
Anandamide (AEA), a prominent member of the endogenous ligands of cannabinoid
receptors (endocannabinoids), is known to affect several functions of brain and peripheral
tissues. A potential role for AEA in skin pathophysiology has been proposed, yet its
molecular basis remains unknown. Here we report unprecedented evidence that spontaneously
immortalized human keratinocytes (HaCaT) and normal human epidermal keratinocytes
(NHEK) have the biochemical machinery to bind and metabolize AEA, i.e. a functional type-
1 cannabinoid receptor (CB1R), a selective AEA membrane transporter (AMT), an AEA-
degrading fatty acid amide hydrolase (FAAH), and an AEA-synthesizing phospholipase D
(PLD). We show that, unlike CB1R and PLD, the activity of AMT and the activity and
expression of FAAH increase while the endogenous levels of AEA decrease in HaCaT and
NHEK cells induced to differentiate in vitro by 12-O-tetradecanoylphorbol 13–acetate (TPA)
+ calcium. We also show that exogenous AEA inhibits the formation of cornified envelopes, a
hallmark of keratinocyte differentiation, in HaCaT and NHEK cells treated with TPA +
calcium, through a CB1R-dependent reduction of transglutaminase and protein kinase C
activity. Moreover, transient expression in HaCaT cells of the chloramphenicol
acetyltransferase reporter gene under control of the loricrin promoter, which contained a
wild-type or mutated activating protein-1 (AP-1) site, showed that AEA inhibited AP-1 in a
CB1R-dependent manner. Taken together, these data demonstrate that human keratinocytes
partake in the peripheral endocannabinoid system, and show a novel signaling mechanism of
CB1 receptors, that may have important implications in epidermal differentiation and skin
development.
Keywords: Cornified envelope; Differentiation; Endocannabinoids; Hydrolysis; Receptor;
Signal Transduction; Skin; Synthesis; Transport.
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INTRODUCTION
Endocannabinoids are amides, esters and ethers of long chain polyunsaturated fatty acids,
found in several human tissues (1). Anandamide (N-arachidonoylethanolamine, AEA)1 and
2-arachidonoylglycerol (2-AG) are the main endocannabinoids described to date (2). They
bind to both brain (CB1) and peripheral (CB2) cannabinoid receptors, thus mimicking some
of the central and peripheral effects of ∆9-tetrahydrocannabinol, the psychoactive principle of
hashish and marijuana (3). Recently, AEA has been also shown to activate vanilloid receptors
(4, 5). The effect of AEA via CB1R and CB2R depends on its extracellular concentration,
which is controlled by i) cellular uptake by a specific AEA membrane transporter (AMT), and
ii) intracellular degradation by the AEA-hydrolyzing enzyme fatty acid amide hydrolase
(FAAH). AMT (6) and FAAH (7) have been characterized in several mammalian cells and
tissues. Moreover, the main player in AEA synthesis is thought to be a N-acyl-
phosphatidylethanolamines (NAPE)-hydrolyzing phospholipase D (PLD) (8). In addition, the
pathways for the biosynthesis and degradation of 2-AG are different from those of AEA (2),
and include a recently identified monoglyceride lipase as the primary mechanism for 2-AG
inactivation (9). Together with AEA and congeners all these proteins form the
“endocannabinoid system”. Endocannabinoids play a number of roles in the central nervous
system (10) and in peripheral tissues (1). In particular, peripheral AEA acts as cardiovascular
and immune systems modulator, shows anti-inflammatory activity and inhibits human cancer
cell proliferation, being more generally involved in the control of cell survival and death (11).
AEA has been also shown to attenuate the pain sensation produced by chemical damage to
cutaneous tissue, by interacting with CB1-like cannabinoid receptors located outside the
central nervous system (12). In this line, growing evidence has been accumulated suggesting a
role for the endocannabinoid system in the peripheral control of pain initiation (13, 14),
leading also to the hypothesis that CBRs possibly present in epidermal cells might act as
cutaneous nociceptors (15). As a matter of fact, mouse epidermal cells contain endogenous
AEA, which mediates cellular response to UVB irradiation (16), and a stable analogue of
AEA has been shown to inhibit the proliferation in vitro of rat epithelial cells, through a
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CB1-dependent mechanism (17). In addition, a recent report has shown that skin tumors of
mice and men express CB1R and CB2R, that healthy human skin expresses CB1R, and that
synthetic agonists of CBRs inhibit tumor growth (18). Moreover, isolated rat paw skin
responds to an in vitro model of neuropathic pain according to a CB1-dependent mechanism
(19). This background prompted us to investigate whether human keratinocytes have the
biochemical machinery to bind and metabolize AEA, and how the endocannabinoid system
might be implicated in the control of epidermal cell growth and differentiation.
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EXPERIMENTAL PROCEDURES
Materials - Chemicals were of the purest analytical grade. Anandamide (AEA), 12-O-
tetradecanoylphorbol 13–acetate (TPA), sodium nitroprusside (SNP), minimum essential
medium (MEM), phenylmethylsulfonyl fluoride (PMSF), N,N’-dimethylcasein and
putrescine were purchased from Sigma Chemical Co. (St. Louis, MO). N-(4-
Hydroxyphenyl)-arachidonoylamide (AM404), arachidonoyl-trifluoromethyl-ketone
(ATFMK) and 2-arachidonoylglycerol (2-AG) were from Research Biochemicals
International (Natick, MA). 3-Morpholinosydnonimine (SIN-1) was from Alexis
Corporation (Läufelfingen, Switzerland). VDM11 was from Tocris-Cookson (Bristol, United
Kingdom), and methyl-arachidonoyl fluorophosphonate (MAFP) was from Cayman
Chemicals (Ann Arbor, MI). Capsazepine and the protein kinase C substrate myelin basic
protein fragment 4-14 (MBP 4-14) were from Calbiochem (La Jolla, CA). Cannabidiol was a
kind gift of Dr. M. van der Stelt (Utrecht University, The Netherlands). N-Piperidino-5-(4-
chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR141716)
and N-[1(S)-endo-1,3,3-trimethyl-bicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-
methylphenyl)-1-(4-methyl-benzyl)pyrazole -3-carboxamide (SR144528) were kind gifts
of Sanofi Recherche (Montpellier, France). [3H]AEA (223 Ci/mmol) and [3H]CP55.940 (5-
(1,1’-dimethyheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl) cyclohexyl]-phenol; 126
Ci/mmol) were from NEN DuPont de Nemours (Köln, Germany). 1,2-Dioleoyl-3-
phosphatidyl[2-14C]ethanolamine (55 mCi/mmol), [3H]putrescine (22 Ci/mmol) and
adenosine 5’-[γ-32P]triphosphate (5000 Ci/mmol) were from Amersham Pharmacia Biotech
(Buckinghamshire, United Kingdom). [3H]Resinferatoxin (48 Ci/mmol) was a kind gift of Dr.
Vincenzo Di Marzo (Consiglio Nazionale delle Ricerche, Pozzuoli, Italy). Anti-FAAH
polyclonal antibodies were elicited in rabbits against the conserved FAAH sequence
VGYYETDNYTMPSPAMR (20) conjugated to ovalbumin, and were prepared by Primm
S.r.l. (Milan, Italy). Rabbit anti-CB1R and anti-CB2R polyclonal antibodies were from
Cayman Chemicals, and mouse anti-actin monoclonal antibodies were from Santa Cruz
Biotechnology (Santa Cruz, CA). Goat anti-rabbit and goat anti-mouse antibodies conjugated
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to alkaline phosphatase (GAR-AP and GAM-AP) were from Bio-Rad (Hercules, CA).
Cell Culture and Treatment – HaCaT cells (21) were kindly provided by Prof. N.E.
Fusenig (German Cancer Research Center, Heidelberg, Germany), and were grown in a 1:1
mixture of MEM and Ham’s F-12 medium (Gibco, Berlin, Germany) supplemented with
10% fetal calf serum and 1% nonessential aminoacids, at 37°C in a 5% CO2 humidified
atmosphere (22). Cryopreserved normal human epidermal keratinocytes (NHEK) from
newborn foreskin were obtained from Clonetics (San Diego, CA) and were grown in dishes
coated with calf skin collagen type III (100 mg per ml) in serum-free keratinocyte growth
medium (Gibco), as reported (23). Third passage NHEK cells were used for each experiment.
Cell differentiation was induced by treating both HaCaT and NHEK cells with TPA (10
ng/ml) + CaCl2 (1.2 mM) for 6 h, 24 h or 5 days (22, 23). AEA and related compounds were
added directly to the culture medium at the same time as TPA + calcium. Controls were
treated with vehicle alone. After each treatment, cell viability was determined by the MTT
test and by Trypan Blue dye exclusion, as reported (24).
Binding to Cannabinoid Receptors - For cannabinoid receptor studies, membrane fractions
were prepared from HaCaT cells (25x106/test) as reported (24), and were used in rapid
filtration assays with the synthetic cannabinoid [3H]CP55.940 (24). Apparent dissociation
constant (Kd) and maximum binding (Bmax) values of [3H]CP55.940 were calculated from
saturation curves through nonlinear regression analysis with the Prism 3 program (GraphPAD
Sofware for Science, San Diego, CA) (24). CBR binding was assayed also in NHEK cells
(25x106/test), using 200 pM [3H]CP55.940. Binding of [3H]AEA to HaCaT cells was
evaluated with the same filtration assays used for [3H]CP55.940 (24), and apparent Kd and
Bmax values were calculated through nonlinear regression analysis of saturation curves. Also
binding of [3H]resinferatoxin to HaCaT cells was evaluated by rapid filtration assays,
performed as described (25). In all experiments, unspecific binding was determined in the
presence of 10 µM “unlabeled” agonist (24, 25). The expression of CB1R and CB2R in
HaCaT cells was assessed by Western blot analysis, performed as detailed below for FAAH,
using anti-CB1 or anti-CB2 polyclonal antibodies (each diluted 1:250), and GAR-AP
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(diluted 1:2000) as second antibody (26). Saturation curves of [3H]CP55.940 binding and
Western blot analysis of CB1R and CB2R were performed under the same experimental
conditions on mouse brain and mouse spleen extracts.
Analysis of Anandamide Uptake - The uptake of [3H]AEA by the AEA membrane
transporter (AMT) of intact HaCaT cells (2x106/test) was performed as described previously
(27). To discriminate non-carrier-mediated from carrier-mediated transport of AEA through
cell membranes, [3H]AEA uptake at 4°C was subtracted from that at 37°C (28). Q10 value of
AMT was calculated as the ratio of AEA uptake at 30°C and 20°C (28). Incubations (15 min)
were also carried out with different concentrations of [3H]AEA, in the range 0-800 nM, in
order to determine apparent Michaelis-Menten constant (Km), maximum velocity (Vmax)
and inhibition constant (Ki) of AMT by nonlinear regression analysis (also in this case, the
uptake at 4°C was subtracted from that at 37°C). The effect of different compounds on the
uptake (15 min) of 200 nM [3H]AEA by AMT was determined by adding each substance
directly to the incubation medium, at the indicated concentrations. AMT activity was assayed
also in NHEK cells (2x106/test), using 400 nM [3H]AEA as substrate. Cell viability after
each treatment was higher than 90% in all cases.
FAAH Activity and Expression - Fatty acid amide hydrolase (E.C. 3.5.1.4; FAAH)
activity, and its apparent Km, Vmax and Ki values were determined in HaCaT cells (20
µg/test) as reported (26). FAAH activity was also assayed in NHEK cells (20 µg/test), using 5
µM [3H]AEA as substrate. HaCaT cell homogenates (20 µg/lane) were prepared as described
(26), and were subjected to SDS-polyacrylamide gel electrophoresis (12%) under reducing
conditions. Rainbow molecular weight markers (Amersham Pharmacia Biotech) 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 were immunoreacted with anti-FAAH
polyclonal (1:200) or anti-actin monoclonal (1:1000) antibodies, using GAR-AP or GAM-
AP (diluted 1:2000) as second antibody, respectively (26). Densitometric analysis of filters
was performed by means of a Floor-S Multi-Imager, equipped with a Quantity One
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software (Bio-Rad). The same anti-FAAH antibodies (diluted 1:300) were used to determine
FAAH protein content also by enzyme-linked immunosorbent assay (ELISA), coating wells
with cell homogenates (20 µg/well) as reported (26). Reverse transcriptase-polymerase chain
reaction (RT-PCR) was performed using total RNA isolated from HaCaT cells (5x106 cells)
by means of the S.N.A.P." Total RNA Isolation Kit (Invitrogen, Carlsbad, CA), as described
(26). The primers were as follows:
(+) 5-TGGAAGTCCTCCAAAAGCCCAG, (-) 5-TGTCCATAGACACAGCCCTTCAG,
for FAAH; (+) 5-AGTTGCTGCAGTTAAAAAGC, (-) 5-CCTCAGTTCCGAAAA
CCAAC, for 18S rRNA.
Five µl of the reaction mixture were electrophoresed on a 6% polyacrylamide gel, which was
then dried and subjected to autoradiography (26). The autoradiographic films were subjected
to densitometric analysis, by means of a Floor-S Multi-Imager equipped with a Quantity
One software (Bio-Rad). Products were validated by size determination and sequencing (26).
Other Biochemical Assays – The endogenous levels of AEA in HaCaT and NHEK cells
(50x106/test) were determined by gas chromatography-electron impact mass spectrometry, as
recently reported (29). The activity of phospholipase D (E.C. 3.1.4.4; PLD) was assayed in
homogenates of HaCaT and NHEK cells (50 µg/test) according to Moesgaard et al. (30),
using 1,2-dioleoyl-3-phosphatidyl-[2-14C]ethanolamine (10 µM) as substrate and
measuring the release of [14C]ethanolamine as described (31). Transglutaminase (E.C.
2.3.2.13; TGase) activity was determined by measuring the incorporation of [3H]putrescine (1
µCi) into N,N’-dimethylcasein, as previously reported (32). HaCaT or NHEK cell extracts
(100 µg in 150 µl per test) were incubated for 20 min at 37°C, then the reaction was stopped
by spotting 100 Μl aliquots onto Whatman 3MM filter paper. Filters were washed to remove
unbound [3H]putrescine, were air-dried and the radioactivity was measured by liquid
scintillation counting in a LKB 1217 Rackbeta spectrometer (Amersham Pharmacia Biotech).
Protein kinase C (E.C. 2.7.1.37; PKC) activity in HaCaT or NHEK cells (10 µg/test) was
determined by measuring the incorporation of [γ-32P]ATP (5 µCi) into the highly selective
substrate MBP 4-14 (25 µM), as reported (22). Reactions were incubated for 10 min at 30°C,
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spotted onto Whatman P81 paper, and washed four times in 75 mM H3PO4. The bound
radioactivity was determined by liquid scintillation counting in a LKB 1217 Rackbeta
spectrometer.
Determination of Cornified Cell Envelopes – Cornified envelopes (CE) were extracted
from HaCaT or NHEK cells (5x106/test) by exhaustive boiling and sonication in 2% sodium
dodecyl sulfate, 20 mM dithiotreitol, 0.1 M Tris-HCl (pH 8.0), and 0.5 mM ethylendiamine
tetraacetate, as previously described (33). CE formation was quantified by spectrophotometry
at 600 nm and was normalized to the protein content (33).
Transient Transfections – Transient transfections were performed in triplicate using
Lipofectin (Gibco), according to the manufacturer’s instructions. HaCaT cells (1x106/test)
were transfected with both wild-type and AP-1 mutated minimal loricrin promoters, placed
upstream of the chloramphenicol acetyltransferase (CAT) reporter gene, as described (34).
Transfection efficiency was monitored by using a thymidine kinase β-galactosidase (β-gal)
construct (Clontech, Palo Alto, CA). After transfection, cells were left untreated or were
treated for 6 h with various amounts of AEA, alone or in the presence of 1 µM SR141716 or 1
µM SR144528. Cells were then harvested and CAT activity was assayed using the CAT
Enzyme Assay System (Promega, Madison, WI), according to the manufacturers protocol.
CAT activities were normalized to protein content and β-gal activity.
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
nonparametric Mann-Whitney test, elaborating experimental data by means of the InStat 3
program (GraphPAD Software for Science).
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RESULTS
The Endocannabinoid System in HaCaT Cells - The synthetic cannabinoid [3H]CP55.940,
which has high affinity to both CB1 and CB2 receptors (35), was bound dose-dependently to
spontaneously immortalized HaCaT cells (Fig. 1A). These saturation curves were very close
to those obtained with mouse brain membranes (Fig. 1A), a positive control for CB1R (35,
36), and allowed to calculate Kd values of 610 ± 79 and 598 ± 86 pM, and Bmax values of
1378 ± 81 and 1773 ± 115 fmol.mg protein-1, for HaCaT cells and mouse brain respectively.
On the other hand, binding of [3H]CP55.940 to mouse spleen, a positive control for CB2R
(35, 36), showed saturation curves (Fig. 1A) from which a Kd of 245 ± 32 pM and a Bmax of
277 ± 11 fmol.mg protein-1 could be calculated. The Kd and Bmax values found here for
mouse brain and spleen are in agreement with previous reports (reviewed in ref. 35).
Consistently with the binding data, 1 µM anandamide (AEA) and 0.1 µM SR141716, a
selective CB1R antagonist (35), but not 0.1 µM SR144528, a selective CB2R antagonist (35),
displaced [3H]CP55.940, suggesting that only CB1 receptors were expressed on HaCaT cell
surface (Fig. 1B). In order to further confirm the presence of CB1 receptors, Western blot
analysis of HaCaT cell extracts was performed, and compared with mouse brain and spleen
extracts. Western blot analysis showed that specific anti-CB1R, but not anti-CB2R,
antibodies recognized a single immunoreactive band in HaCaT cells (Fig. 1, C-D), further
corroborating that these cells express functional CB1 receptors only. Moreover, HaCaT cells
were able to bind [3H]AEA according to a saturable process (Fig. 2A), showing apparent Kd
of 169 ± 25 nM and Bmax of 943 ± 44 fmol.mg protein-1. The affinity of AEA for CB
receptors of HaCaT cells was close to that reported for rat forebrain CB1R (35), and
consistently binding of 200 nM [3H]AEA to HaCaT cells was displaced by 1 µM SR141716
(down to 15% of the control value), but not by 1 µM SR144528 (85%) nor by both 1 µM
cannabidiol, antagonist of the “endothelial type” cannabinoid receptor (37), and 1 µM
capsazepine, antagonist of vanilloid receptors (4) (~90% in each case). In the same line,
HaCaT cells were unable to bind [3H]resinferatoxin at concentrations up to 500 pM (bound
radioactivity = 243 ± 72 dpm, compared to 200 ± 60 dpm of unspecific binding; p > 0.05),
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further suggesting that HaCaT cells are devoid of vanilloid receptors.
Intact HaCaT cells were able to accumulate [3H]AEA in a temperature- (Q10 = 1.5),
time- (t1/2 = 5 min) and concentration-dependent manner (not shown). [3H]AEA
accumulation occurred according to a saturable process (Fig. 2B) typical of AMT (6, 27, 28),
showing apparent Km and Vmax values of 346 ± 42 nM and 125 ± 6 pmol.min-1.mg
protein-1, respectively. The uptake of 200 nM [3H]AEA was almost completely inhibited by 10 µM
AM404 (down to 20% of the control value) or 10 µM VDM11 (to 15% of the control),
specific inhibitors of AMT (4, 38). Instead this uptake was doubled by the nitric oxide-donor
SNP (5 mM) and by the peroxynitrite-donor SIN-1 (1 mM). Moreover, the uptake was
inhibited by 50 µM PMSF (35% of the control), by 10 µM ATFMK (25%), or by 100 nM
MAFP (20%), selective inhibitors of FAAH activity (4, 7). Even 2-AG dose-dependently
inhibited [3H]AEA uptake by AMT, acting as competitive inhibitor with an apparent Ki of
400 ± 50 nM.
HaCaT cells showed FAAH activity at pH 9.0, with apparent Km of 12 ± 2 µM and Vmax
of 370 ± 27 pmol.min-1.mg protein-1 (Fig. 2C). The hydrolysis of [3H]AEA by HaCaT cells
at pH 5.0 was hardly detectable (Fig. 2C), ruling out the involvement of the amidase
described recently in lysosomes and mitochondria (7). On the other hand, [3H]AEA
hydrolysis at pH 9.0 was fully inhibited by 50 µM PMSF (25% of the control), by 10 µM
ATFMK (15%), or by 100 nM MAFP (15%). Again 2-AG dose-dependently inhibited
[3H]AEA hydrolysis by FAAH, acting as competitive inhibitor with an apparent Ki of 8 ± 1 µM.
Changes in the Endocannabinoid System in Differentiating HaCaT Cells - Treatment of
HaCaT cells with TPA + calcium, typical inducers of keratinocyte differentiation (22, 23), led
to a time-dependent increase in the activity of AMT and FAAH, reaching a statistically
significant increase (~160-180% of the control values) after 24 h and a maximum (~210-
280%) after 5 days (Fig. 3A). Western blot analysis of HaCaT cell extracts showed that
specific anti-FAAH antibodies recognized a single immunoreactive band of the molecular
size expected for FAAH, the intensity of which increased time-dependently upon treatment
with TPA + calcium (Fig. 3B). Densitometric analysis of the filter shown in Fig. 3B indicated
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that FAAH protein content in 5-day-treated cells increased to 260% with respect to the
vehicle-treated cells (100% = 10300 ± 1500 units per mm2). On the other hand, 5-day-
treated and control cells expressed the same levels of actin (Fig. 3B), ruling out that the
different levels of FAAH in these cells might be due to different loading of proteins. The
same anti-FAAH antibodies were used to quantify FAAH content by ELISA, demonstrating
that the increase of enzymic activity in HaCaT cells treated with TPA + calcium (Fig. 3A)
was paralleled by increased FAAH expression (not shown). RT-PCR amplification of HaCaT
cell cDNA showed a single band of the expected molecular size for FAAH gene, which was
not affected by TPA + calcium (Fig. 3C). In fact, densitometric analysis of the
autoradiographic film shown in Fig. 3C indicated that in 5-day-treated cells FAAH mRNA
was ~115% of that in untreated cells (100% = 7200 ± 800 units per mm2). Under the same
experimental conditions, also the expression of the 18S rRNA gene was unaffected (Fig. 3C).
Unlike AMT and FAAH, CBR binding was unaffected by treatment of HaCaT cells with
TPA + calcium, and remained the same as in the controls in 5-day-treated cells (Fig. 3A).
Moreover, HaCaT cells showed PLD activity, which was assayed under conditions found to
be optimal for the N-acyl-phosphatidylethanolamines (NAPE)-hydrolyzing PLD (30). A
radiolabeled phosphatidylethanolamine was used instead of radiolabeled NAPEs, which are
not commercially available (31). This is noteworthy, because NAPE-hydrolyzing PLD
activity is considered responsible for AEA synthesis, although the lack of specific inhibitors
of this enzyme makes it difficult to further extend its analysis and to conclusively assess its
contribution to AEA metabolism (8, 30, 31). At any rate, treatment of HaCaT cells with TPA
+ calcium did not affect PLD activity, which remained the same as in the controls in 5-day-
treated cells (Fig. 3A).
Treatment of HaCaT cells with TPA + calcium time-dependently decreased the
endogenous levels of AEA, reaching statistical significance after 24 h and a minimum (25%
of the control value) after 5 days (Table I). Therefore, the levels of AEA inversely correlated
with those of the AEA-degrading agents AMT and FAAH (compare Table I and Fig. 3A).
The Endocannabinoid System in Resting and Differentiating NHEK Cells - To further
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generalize the effects of AEA on human keratinocytes, the analysis of the endocannabinoid
system was extended to normal human epidermal keratinocytes (NHEK). These cells showed
a full endocannabinoid system, i.e. CBR, AMT, FAAH and PLD (Table II). In particular,
binding of 200 pM [3H]CP55.940 was displaced by 0.1 µM SR141716 (to ~15% of the
controls), but not by 0.1 µM SR144528 (~95%), and anti-CB1R, but not anti-CB2R,
antibodies recognized a single immunoreactive band in NHEK cell extracts (not shown).
These data suggest that also NHEK cells express a functional CB1R, as does healthy human
skin (18). NHEK cells were induced to differentiate under the same experimental conditions
as HaCaT cells. Treatment with TPA + calcium reduced AEA content in NHEK cells in a way
quite analogous to that observed in HaCaT cells (Table I). Also the endocannabinoid system
was modulated by TPA + calcium in the same manner as that of HaCaT cells (Table II).
Indeed, the activity of AMT and FAAH increased time-dependently, reaching ~200% and
~250% of the controls in 5-day-treated cells, whereas CBR and PLD were not significantly
affected (Table II).
Effect of Exogenous AEA on Keratinocyte Differentiation In Vitro - Treatment of HaCaT
cells with TPA + calcium led to a ~400% increase in cornified envelope (CE) formation (Fig.
4A), a hallmark of keratinocyte differentiation (22, 32, 33). The increase in CE formation was
paralleled by increased activity of transglutaminase (TGase) (~330% of the controls) and
protein kinase C (~250%), two enzymes strictly related to keratinocyte differentiation (22, 32,
33). Administration of AEA to HaCaT cells dose-dependently reduced CE formation, TGase
activity and PKC activity induced by TPA + calcium, reaching a maximum effect at 1 µM
(Fig. 4A). At this concentration, CE formation, TGase activity and PKC activity were
respectively only ~150%, ~130% and ~120% of the vehicle-treated controls (Fig. 4A). The
effect of 1 µM AEA was reversed by 0.1 µM SR141716, but not by 0.1 µM SR144528 (Fig.
4A), suggesting that it was mediated by CB1 receptors. Treatment with TPA + calcium also
increased CE formation, TGase activity and PKC activity in NHEK cells, where they reached
~500%, ~400% and ~300% of the vehicle-treated controls, respectively (Table III). In these
cells, 1 µM AEA had the same inhibitory effect on CE formation, TGase activity and PKC
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activity, as that observed in differentiating HaCaT cells (compare Table III and Fig. 4A).
Importantly, AEA-induced reduction of CE formation in differentiating HaCaT and NHEK
cells was not associated with a decrease in viability, which remained > 90% of the TPA +
calcium treated cells in both cases.
To further investigate the effect of AEA on the differentiation of human keratinocytes,
transient transfection studies were performed in HaCaT cells with a vector containing the
CAT gene under the control of the loricrin promoter, which contains activating protein–1
(AP-1) responsive sites (22, 39). Incubation with AEA dose-dependently decreased CAT
activity, down to 40% of vehicle-treated controls at 1 µM (Fig. 4B). SR141716, but not
SR144528 (each used at 0.1 µM), fully prevented the effect of 1 µM AEA (Fig. 4B).
Moreover, the AEA-induced inhibition of CAT activity of the wild-type loricrin promoter
was completely abolished by its counterpart containing an AP-1 mutated site (Fig. 4B). These
data suggest that the effect of AEA on the promoter was CB1-dependent and required an
intact AP-1 element.
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DISCUSSION
In this investigation we report evidence that human keratinocytes have a functional
“endocannabinoid system”, i.e. AEA and the biochemical machinery to bind, synthesize,
transport and hydrolyze it. We also show that differentiating keratinocytes have decreased
levels of endogenous AEA, due to increased degradation of this lipid through AMT and
FAAH. In addition, we demonstrate that exogenous AEA inhibits keratinocyte differentiation
in vitro, through a CB1-dependent mechanism which involves inactivation of protein kinase
C, activating protein-1 and transglutaminase.
Spontaneously immortalized HaCaT cells express functional type-1 cannabinoid receptors
on their surface, as suggested by: i) the Kd and Bmax values calculated from saturation
binding curves (Figs 1A and 2A), ii) the displacement of [3H]CP55.940 (Fig. 1B) and of
[3H]AEA by SR141716, and iii) the cross-reactivity with specific anti-CB1R antibodies (Fig.
1C). The level of CB1R is constant in differentiating keratinocytes, whereas FAAH activity
increases time-dependently (Fig. 3A), due to a higher gene expression at translational level
(Fig. 3B). The uptake of AEA through its specific carrier (AMT) also increases in
differentiating HaCaT cells (Fig. 3A). However, the molecular properties of AMT are not
known and no probes are available to measure its expression (6). AMT activity was fully
prevented by treatment of HaCaT cells with FAAH inhibitors ATMFK and MAFP, in keeping
with a facilitated transport driven by FAAH (40, 41). It is important to recall that the role of
AMT in AEA degradation is still under debate, because FAAH might not need at all a
transporter to get in contact with AEA (42), while AMT might work “in reverse” to export
(rather than import) AEA (43). Recently even the existence of AMT has been questioned,
suggesting that AEA is transported by a FAAH-driven simple diffusion process (44). Yet,
recent investigations showing that FAAH, but not AMT, is activated by progesterone (26),
that AMT, but not FAAH, is activated by nitric oxide and peroxynitrite (27), and that estrogen
activates AMT while inhibiting FAAH (43), seem to favour the existence of an AEA
transporter distinct from the AEA hydrolase. A similar hypothesis is suggested by different
human pathologic conditions, where FAAH, but not AMT, is modulated (reviewed in ref. 1).
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At any rate, the apparent affinity of HaCaT AMT for AEA is very close to that of human
lymphocytes (26) and of human endothelial cells (27), suggesting that the same carrier might
be present on the surface of different peripheral cells. Moreover, AEA uptake by HaCaT
AMT, like that of other human peripheral cells, was significantly increased by the nitric
oxide-donor SNP and even more by the peroxynitrite-donor SIN-1 (24, 27). Unlike AMT
and FAAH, PLD was not affected by treatment of HaCaT cells with TPA + calcium,
suggesting that the decreased AEA content in differentiating keratinocytes was due to a
greater degradation only. A critical role of FAAH in controlling endogenous levels of AEA in
human keratinocytes is in keeping with the hypothesis that FAAH is the key-regulator of
AEA levels in vivo (42), indeed FAAH knockout mice show ~15-fold higher levels of AEA
than wild-type littermates (45) and AEA levels in human blood inversely correlate with
FAAH activity in peripheral lymphocytes (1). Incidentally, the levels of AEA in untreated
human keratinocytes reported in Table I correspond to ~3 pmoles per mg lipid phosphorus, a
value comparable to that reported in mouse epidermal cells (16).
The upregulation of AMT and FAAH upon keratinocyte differentiation, and the subsequent
down-regulation of intracellular AEA, are major findings of this investigation, which were
further generalized to normal human epidermal keratinocytes (Tables I and II). Accordingly,
exogenous AEA was able to inhibit the formation of cornified envelopes induced in vitro by
TPA + calcium (Fig. 4A). Many biochemical markers of differentiation can be identified in
keratinocytes, namely keratins, involucrin, filaggrin and loricrin. All these differentiation
products are required for the assembly of CE, a specialized structure that provides a barrier
for the organism (46, 47). Therefore, CE formation is widely used as a specific hallmark of
keratinocyte differentiation (22, 32, 46, 47). Here we show that AEA inhibited CE formation
in differentiating human keratinocytes (Fig. 4A). Consistently, AEA reduced the activity of
transglutaminase, which increases in differentiating cells (Fig. 4A; see also ref. 48) where it
catalyzes crosslinking reactions required for the orderly assembly of the CE (23). In addition,
in differentiating keratinocytes AEA reduced the activity of PKC, which also has been
demonstrated to be essential for epidermal differentiation (22). The findings on CE formation
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and on the activity of TGase and PKC were extended to normal human epidermal
keratinocytes, suggesting that they may be physiologically relevant also in vivo (Table III). It
seems noteworthy that PKC is an essential upstream regulator of AP-1 (49), a nuclear
transcription factor playing a major role in epidermal differentiation (34, 39). TGase 1, which
is expressed in human keratinocytes (23), is responsive to AP-1 (32). AEA inhibited
transient expression of CAT reporter gene under control of an intact AP-1 (Fig. 4B),
consistently with the inhibition of PKC (Fig. 4A). Taken together, it can be suggested that
AEA, by inactivating AP-1 and its obligatory activator PKC, may reduce TGase expression,
thus inhibiting CE formation. The anti-differentiating effect of AEA did not imply a reduced
viability of differentiating keratinocytes, suggesting that it was not due to induction of cell
death. This seems of interest, also in the light of a report on the ability of AEA to inhibit
differentiation of rat neuronal cells, which appeared during the preparation of this manuscript
(50). Like in human keratinocytes, AEA-induced reduction of neuronal differentiation was
not associated with a decrease in cell viability, and was mediated by CB1 receptors (50).
Taken together, these studies suggest that the anti-differentiating effect of AEA may be more
generally involved in neuronal and epidermal development. Finally, the observation that
binding of AEA to CB1 receptors inhibits PKC in differentiating HaCaT and NHEK cells
(Fig. 4A and Table III) uncovers a novel mechanism of CB1R-dependent signal transduction
(51, 52). Keeping in mind that PKC is required for vanilloid receptor 1 (VR1) activation (53),
whereas it causes CB1R down-regulation (54), a dual regulation of PKC by VR1 or CB1R
might have implications in the control of cell survival and death by AEA (11). In fact,
activation of these receptors by AEA leads to opposite effects: anti-apoptotic, for CB1R, or
pro-apoptotic, for VR1 (24, 55). However, further studies are necessary to assess the
contribution of receptor-mediated PKA signaling to the biological actions of AEA.
In conclusion, we report evidence that human keratinocytes have a functional
“endocannabinoid system”, which may sustain the peripheral actions of AEA at the skin level.
Our findings give biochemical ground to the effects of AEA on epidermal cells, especially in
relation to pain sensation (12), response to UV irradiation (16), cell proliferation (17) and
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tumor growth (18). In this context, the finding that human keratinocytes partake in the
peripheral endocannabinoid system, and that AEA can inhibit epidermal differentiation, opens
new perspectives to the understanding of skin development and to the treatment of human
skin diseases where cell hyperproliferation takes place.
Acknowledgments - We wish to thank Drs Margherita Annichiarico-Petruzzelli and
Monica Bari for their expert assistance, and Dr. Eleonora Candi for helpful discussions.
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FOOTNOTES
1The abbreviations used are: AEA, anandamide (N-arachidonoylethanolamine); 2-AG, 2-
arachidonoylglycerol; AM404, N-(4-hydroxyphenyl)-arachidonoylamide; AMT, AEA
membrane transporter; AP-1, activating protein-1; ATFMK, arachidonoyl-trifluoromethyl-
ketone; CB1/2R, type 1/2 cannabinoid receptors; CP55.940, 5-(1,1’-dimethyheptyl)-2-
[1R,5R-hydroxy-2R-(3-hydroxypropyl) cyclohexyl]-phenol; ELISA, enzyme-linked
immunosorbent assay; FAAH, fatty acid amide hydrolase; GAM(R)-AP, goat anti-mouse
(rabbit) antibodies conjugated to alkaline phosphatase; MAFP, methyl-arachidonoyl
fluorophosphonate; MBP 4-14, myelin basic protein fragment 4-14; NHEK, normal human
epidermal keratinocytes; PLD, phospholipase D; RT-PCR, reverse transcriptase-polymerase
chain reaction; SIN-1, 3-morpholinosydnonimine; SNP, sodium nitroprusside; SR141716,
N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-
carboxamide; SR144528, N-[1(S)-endo-1,3,3-trimethyl-bicyclo[2.2.1]heptan-2-yl]-5-(4-
chloro-3-methylphenyl)-1-(4-methyl-benzyl)-pyrazole-3-carboxamide; TGase,
transglutaminase.
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TABLE I
Endogenous levels of anandamide (AEA) in spontaneously transformed (HaCaT) and normal
(NHEK) human keratinocytes, induced to differentiate by TPA + calcium
Treatment AEA content in HaCaT cells
(pmol.mg protein-1)
AEA content in NHEK cells
(pmol.mg protein-1)
Control 480 ± 90
(100%)
375 ± 75
(100%)
TPA + Ca2+ (6 h) 450 ± 90
(94%)
360 ± 70
(96%)
TPA + Ca2+ (24 h) 280 ± 55
(58%)*
200 ± 40
(53%)*
TPA + Ca2+ (5 d) 120 ± 25
(25%)**
95 ± 20
(25%)**
*Denotes p < 0.05, **denotes p < 0.01 vs controls (p > 0.05 in all other cases).
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TABLE II
Binding to type-1 cannabinoid receptors (CB1R) and activity of anandamide membrane
transporter (AMT), fatty acid amide hydrolase (FAAH) and phospholipase D (PLD) in normal
human keratinocytes (NHEK), induced to differentiate by TPA + calcium
Parameter Control TPA + Ca2+ (24 h) TPA + Ca2+ (5 d)
CB1R bindinga 350 ± 35
(100%)
355 ± 35
(101%)
365 ± 35
(104%)
AMT activityb 60 ± 5
(100%)
95 ± 10
(158%)*
125 ± 12
(208%)**
FAAH activityc 95 ± 10
(100%)
165 ± 16
(174%)*
240 ± 24
(253%)**
PLD activityd 70 ± 7
(100%)
72 ± 7
(103%)
75 ± 6
(107%)
aExpressed as fmol.mg protein-1 (ligand: 200 pM [3H]CP55.940).
bExpressed as pmol.min-1.mg protein-1 (substrate: 400 nM [3H]AEA).
cExpressed as pmol.min-1.mg protein-1 (substrate: 5 µM [3H]AEA).
dExpressed as pmol.min-1.mg protein-1 (substrate: 10 µM 1,2-dioleoyl-3-
phosphatidyl-[2-14C]ethanolamine).
*Denotes p < 0.05, **denotes p < 0.01 vs controls (p > 0.05 in all other cases).
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TABLE III
Effect of anandamide (AEA) on the formation of cornified envelopes (CE) and on the
activity of transglutaminase (TGase) and protein kinase C (PKC) in normal human
keratinocytes (NHEK), induced to differentiate by TPA + calcium
(5 days of treatment)
Parameter Control TPA + Ca2+ TPA + Ca2+
+ AEA (1 µM)
CE formationa 0.080 ± 0.007
(100%)
0.405 ± 0.040
(506%)*
0.115 ± 0.015
(144%)**,#
TGase activityb 435 ± 40
(100%)
1745 ± 170
(401%)*
530 ± 50
(122%)#
PKC activityc 2500 ± 240
(100%)
7500 ± 680
(300%)*
2950 ± 280
(118%)#
aExpressed as A600 units.mg protein-1.
bExpressed as pmol.h-1.mg protein-1.
cExpressed as cpm.min-1.mg protein-1.
*Denotes p < 0.01, **denotes p < 0.05 vs controls; #denotes p < 0.01 vs TPA +
calcium (p > 0.05 in all other cases).
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FIGURE LEGENDS
FIG. 1. Cannabinoid receptors in human HaCaT cells. A) Saturation curves of the binding of
[3H]CP55.940 to mouse brain, mouse spleen and HaCaT cells. Vertical bars represent S.D.
values. B) Displacement of 200 pM [3H]CP55.940 by 1 µM AEA and by CB1 and CB2
receptor antagonists SR141716 and SR144528 (100% as in panel A). Vertical bars represent
S.D. values. *Denotes p < 0.01 vs controls (p > 0.05 in all other cases). C-D) Western blot
analysis of mouse brain, mouse spleen and HaCaT cell extracts (20 µg/lane), reacted with
anti-CB1R (C) or anti-CB2R (D) polyclonal antibodies. Molecular mass markers are shown
on the right side.
FIG. 2. Anandamide (AEA) binding and degradation by human HaCaT cells. A) Saturation
curve of the binding of [3H]AEA to HaCaT cells. B) Dependence of the AEA membrane
transporter (AMT) activity in HaCaT cells on AEA concentration, at 37°C or at 4°C. C)
Dependence of the AEA hydrolase (FAAH) activity in HaCaT cells on AEA concentration, at
pH 9.0 or at pH 5.0. In all panels, vertical bars represent S.D. values.
FIG. 3. Changes of the endocannabinoid system in differentiating HaCaT cells. A) Time-
dependence of cannabinoid receptor (CBR) binding, AEA membrane transporter (AMT)
activity, AEA hydrolase (FAAH) activity and AEA-synthesizing phospholipase D (PLD)
activity in HaCaT cells induced to differentiate by TPA + calcium (see Materials and Methods
for details). 100% = 370 ± 36 fmol.mg protein-1 for CBR binding (substrate: 200 pM
[3H]CP55.940), 70 ± 6 pmol.min-1.mg protein-1 for AMT (substrate: 400 nM [3H]AEA), 105 ±
10 pmol. min-1.mg protein-1 for FAAH (substrate: 5 µM [3H]AEA), and 110 ± 10 pmol.
min-1.mg protein-1 for PLD (substrate: 10 µM 1,2-dioleoyl-3-phosphatidyl-[2-
14C]ethanolamine). Vertical bars represent S.D. values. *Denotes p < 0.05, **denotes p < 0.01 vs
controls (p > 0.05 in all other cases). B) Western blot analysis of FAAH expression in
differentiating HaCaT cell extracts, reacted with anti-FAAH polyclonal (upper panel) or
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anti-actin monoclonal (lower panel) antibodies. Molecular mass markers are shown on the
right side. C) RT-PCR analysis of cDNA of the same samples as in panel B. The expected
sizes of the amplicons (199 bp for FAAH and 258 bp for 18S rRNA) are shown on the right
side.
FIG. 4. Effect of exogenous anandamide (AEA) on keratinocyte differentiation. A) Effect of
various amounts of AEA (0.1, 0.5 or 1 µM), alone or in the presence of 0.1 µM SR141716 or
0.1 µM SR144528, on the differentiation of HaCaT cells induced in vitro by TPA + calcium.
100% = 0.050 ± 0.006 A600 units.mg protein-1 (CE), 375 ± 35 pmol.h-1.mg protein-1
(TGase), and 2000 ± 200 cpm.min-1.mg protein-1 (PKC). B) CAT activity of HaCaT cells
transfected with the AP-1-containing promoter. Cells were transiently transfected with CAT
reporter vectors under the control of the wild-type (wt) or AP-1 mutated (mut) loricrin
promoter. Transfected keratinocytes were left untreated or were treated for 6 h with various
amounts of AEA (0.1, 0.5 or 1 µM), alone or in the presence of 0.1 µM SR141716 or 0.1 µM
SR144528. CAT activity was normalized to β-gal activity and protein content (see Materials
and Methods), and was expressed as percentage of untreated controls, set to 100. In both
panels, vertical bars represent S.D. values. In panel A, *denotes p < 0.01, **denotes p < 0.05
vs controls; #denotes p < 0.05, @denotes p < 0.01 vs TPA + Ca2+; §denotes p < 0.01 vs TPA
+ Ca2+ + AEA (1 µM) (p > 0.05 in all other cases). In panel B, *denotes p < 0.01, **denotes
p < 0.05 vs controls; §denotes p < 0.01 vs AEA (1 µM) (p > 0.05 in all other cases).
28
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and Alessandro Finazzi-AgròMauro Maccarrone, Marianna Di Rienzo, Natalia Battista, Valeria Gasperi, Antonello Rossi
protein kinase C, activating protein-1 and transglutaminaseinhibits epidermal differentiation through CB1 receptor-dependent inhibition of The endocannabinoid system in human keratinocytes. Evidence that anandamide
published online June 18, 2003J. Biol. Chem.
10.1074/jbc.M303994200Access the most updated version of this article at doi:
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