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: Maccarrone@vet.unite.it.

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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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