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• W2062752021 abstract "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) plus 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 plus 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, which may have important implications in epidermal differentiation and skin development. 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) plus 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 plus 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, which may have important implications in epidermal differentiation and skin development. Endocannabinoids are amides, esters, and ethers of long-chain polyunsaturated fatty acids, found in several human tissues (1Maccarrone M. Finazzi-Agrò A. Vitam. Horm. 2002; 65: 225-255Crossref PubMed Google Scholar). Anandamide (N-arachidonoylethanolamine, AEA) 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, arachidonoyltrifluoromethylketone; CB1/2R, type 1/2 cannabinoid receptors; CP55.940, 5-(1,1′-dimethyheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl)cyclohexyl]phenol; FAAH, fatty acid amide hydrolase; GAM(R)-AP, goat anti-mouse (rabbit) antibodies conjugated to alkaline phosphatase; MAFP, methylarachidonoyl fluorophosphonate; NHEKs, normal human epidermal keratinocytes; PLD, phospholipase D; RT, reverse transcriptase; 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-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide; TGase, transglutaminase; NAPE, N-acylphosphatidylethanolamine; TPA, 12-O-tetradecanoylphorbol 13-acetate; PMSF, phenylmethylsulfonyl fluoride; PKC, protein kinase C; CE, cornified envelope; CAT, chloramphenicol acetyltransferase; VR1, vanilloid receptor 1.1The 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, arachidonoyltrifluoromethylketone; CB1/2R, type 1/2 cannabinoid receptors; CP55.940, 5-(1,1′-dimethyheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl)cyclohexyl]phenol; FAAH, fatty acid amide hydrolase; GAM(R)-AP, goat anti-mouse (rabbit) antibodies conjugated to alkaline phosphatase; MAFP, methylarachidonoyl fluorophosphonate; NHEKs, normal human epidermal keratinocytes; PLD, phospholipase D; RT, reverse transcriptase; 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-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide; TGase, transglutaminase; NAPE, N-acylphosphatidylethanolamine; TPA, 12-O-tetradecanoylphorbol 13-acetate; PMSF, phenylmethylsulfonyl fluoride; PKC, protein kinase C; CE, cornified envelope; CAT, chloramphenicol acetyltransferase; VR1, vanilloid receptor 1. and 2-arachidonoylglycerol (2-AG) are the main endocannabinoids described to date (2Sugiura T. Kobayashi Y. Oka S. Waku K. Prostaglandins Leukot. Essent. Fatty Acids. 2002; 66: 173-192Abstract Full Text PDF PubMed Scopus (295) Google Scholar). They bind to both brain (CB1) and peripheral (CB2) cannabinoid receptors, thus mimicking some of the central and peripheral effects of Δ9-tetrahydrocannabinol, the psychoactive principle of hashish and marijuana (3Mechoulam R. Panikashvili D. Shohami E. Trends Mol. Med. 2002; 8: 58-61Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Recently, AEA has also been shown to activate vanilloid receptors (4De Petrocellis L. Bisogno T. Maccarrone M. Davis J.B. Finazzi-Agrò A. Di Marzo V. J. Biol. Chem. 2001; 276: 12856-12863Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 5Jordt S.E. Julius D. Cell. 2002; 108: 421-430Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar). 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 (6Hillard C.J. Jarrahian A. Chem. Phys. Lipids. 2000; 108: 123-134Crossref PubMed Scopus (170) Google Scholar) and FAAH (7Ueda N. Puffenbarger R.A. Yamamoto S. Deutsch D.G. Chem. Phys. Lipids. 2000; 108: 107-121Crossref PubMed Scopus (175) Google Scholar) have been characterized in several mammalian cells and tissues. Moreover, the main player in AEA synthesis is thought to be a N-acylphosphatidylethanolamines (NAPEs)-hydrolyzing phospholipase D (PLD) (8Hansen H.S. Moesgaard B. Hansen H.H. Petersen G. Chem. Phys. Lipids. 2000; 108: 135-150Crossref PubMed Scopus (205) Google Scholar). In addition, the pathways for the biosynthesis and degradation of 2-AG are different from those of AEA (2Sugiura T. Kobayashi Y. Oka S. Waku K. Prostaglandins Leukot. Essent. Fatty Acids. 2002; 66: 173-192Abstract Full Text PDF PubMed Scopus (295) Google Scholar) and include a recently identified monoglyceride lipase as the primary mechanism for 2-AG inactivation (9Dinh T.P. Carpenter D. Leslie F.M. Freund T.F. Katona I. Sensi S.L. Kathuria S. Piomelli D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10819-10824Crossref PubMed Scopus (1128) Google Scholar). Together with AEA and congeners, all these proteins form the “endocannabinoid system.” Endocannabinoids play a number of roles in the central nervous system (10Fride E. Prostaglandins Leukot. Essent. Fatty Acids. 2002; 66: 221-233Abstract Full Text PDF PubMed Scopus (158) Google Scholar) and in peripheral tissues (1Maccarrone M. Finazzi-Agrò A. Vitam. Horm. 2002; 65: 225-255Crossref PubMed Google Scholar). In particular, peripheral AEA acts as a 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 (11Guzman M. Sanchez C. Galve-Roperh I. Pharmacol. Ther. 2002; 95: 175-184Crossref PubMed Scopus (146) Google Scholar). AEA has also been 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 (12Calignano A. La Rana G. Giuffrida A. Piomelli D. Nature. 1998; 394: 277-281Crossref PubMed Scopus (944) Google Scholar). Consistent with this, growing evidence has been accumulated suggesting a role for the endocannabinoid system in the peripheral control of pain initiation (13Calignano A. La Rana G. Loubet-Lescoulie P. Piomelli D. Prog. Brain Res. 2000; 129: 471-482Crossref PubMed Scopus (28) Google Scholar, 14Walker J.M. Huang S.M. Prostaglandins Leukot. Essent. Fatty Acids. 2002; 66: 235-242Abstract Full Text PDF PubMed Scopus (63) Google Scholar), leading also to the hypothesis that CBRs possibly present in epidermal cells might act as cutaneous nociceptors (15Nakamura A. Shiomi H. Jpn. J. Pharmacol. 1999; 79: 427-431Crossref PubMed Scopus (18) Google Scholar). As a matter of fact, mouse epidermal cells contain endogenous AEA, which mediates cellular response to ultraviolet B irradiation (16Berdyshev E.V. Schmid P.C. Dong Z. Schmid H.H. Biochem. J. 2000; 346: 369-374Crossref PubMed Scopus (91) Google Scholar), and a stable analogue of AEA has been shown to inhibit the proliferation in vitro of rat epithelial cells, through a CB1-dependent mechanism (17Bifulco M. Laezza C. Portella G. Vitale M. Orlando P. De Petrocellis L. Di Marzo V. FASEB J. 2001; 15: 2745-2747Crossref PubMed Scopus (122) Google Scholar). 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 (18Casanova M.L. Blazquez C. Martinez-Palacio J. Villanueva C. Fernandez-Acenero M.J. Huffman J.W. Torcano J.L. Guzman M. J. Clin. Invest. 2003; 111: 43-50Crossref PubMed Scopus (350) Google Scholar). Moreover, isolated rat paw skin responds to an in vitro model of neuropathic pain according to a CB1-dependent mechanism (19Ellington H.C. Cotter M.A. Cameron N.E. Ross R.A. Neuropharmacology. 2002; 42: 966-975Crossref PubMed Scopus (63) Google Scholar). 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. Materials—Chemicals were of the purest analytical grade. Anandamide (AEA), 12-O-tetradecanoylphorbol 13-acetate (TPA), sodium nitroprusside (SNP), minimum essential medium, phenylmethylsulfonyl fluoride (PMSF), N,N′-dimethylcasein, and putrescine were purchased from Sigma Chemical Co. (St. Louis, MO). N-(4-Hydroxyphenyl)arachidonoylamide (AM404), arachidonoyltrifluoromethylketone (ATFMK), and 2-arachidonoylglycerol (2-AG) were from Research Biochemicals International (Natick, MA). 3-Morpholinosydnonimine (SIN-1) was from Alexis Corp. (Läufelfingen, Switzerland). VDM11 was from Tocris-Cookson (Bristol, UK), and methylarachidonoyl fluorophosphonate (MAFP) was from Cayman Chemicals (Ann Arbor, MI). Capsazepine and the protein kinase C substrate myelin basic protein fragment 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-pyrazolecarboxamide (SR141716) and N-[1(S)-endo-1,3,3-trimethylbicyclo[2.2.1]-heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)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 PerkinElmer Life Sciences (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 Biosciences (Buckinghamshire, UK). [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 (20Giang D.K. Cravatt B.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2238-2242Crossref PubMed Scopus (390) Google Scholar) 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 Biotechnologies (Santa Cruz, CA). Goat anti-rabbit and goat anti-mouse antibodies conjugated to alkaline phosphatase (GAR-AP and GAM-AP) were from Bio-Rad (Hercules, CA). Cell Culture and Treatment—HaCaT cells (21Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. J. Cell Biol. 1998; 106: 761-771Crossref Scopus (3464) Google Scholar) were kindly provided by Prof. N. E. Fusenig (German Cancer Research Center, Heidelberg, Germany) and were grown in a 1:1 mixture of minimum essential medium and Ham's F-12 medium (Invitrogen, Berlin, Germany) supplemented with 10% fetal calf serum and 1% nonessential amino acids, at 37 °C in a 5% CO2 humidified atmosphere (22Savini I. Catani M.V. Rossi A. Duranti G. Melino G. Avigliano L. J. Invest. Dermatol. 2002; 118: 372-379Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). 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/ml) in serum-free keratinocyte growth medium (Invitrogen), as reported (23Candi E. Oddi S. Terrinoni A. Paradisi A. Ranalli M. Finazzi-Agrò A. Melino G. J. Biol. Chem. 2001; 276: 35014-35023Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). 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) plus CaCl2 (1.2 mm) for 6 h, 24 h, or 5 days (22Savini I. Catani M.V. Rossi A. Duranti G. Melino G. Avigliano L. J. Invest. Dermatol. 2002; 118: 372-379Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 23Candi E. Oddi S. Terrinoni A. Paradisi A. Ranalli M. Finazzi-Agrò A. Melino G. J. Biol. Chem. 2001; 276: 35014-35023Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). AEA and related compounds were added directly to the culture medium at the same time as TPA plus calcium. Controls were treated with vehicle alone. After each treatment, cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and by Trypan Blue dye exclusion, as reported (24Maccarrone M. Lorenzon T. Bari M. Melino G. Finazzi-Agrò A. J. Biol. Chem. 2000; 275: 31938-31945Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Binding to Cannabinoid Receptors—For cannabinoid receptor studies, membrane fractions were prepared from HaCaT cells (25 × 106/test) as reported (24Maccarrone M. Lorenzon T. Bari M. Melino G. Finazzi-Agrò A. J. Biol. Chem. 2000; 275: 31938-31945Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar) and were used in rapid filtration assays with the synthetic cannabinoid [3H]CP55.940 (24Maccarrone M. Lorenzon T. Bari M. Melino G. Finazzi-Agrò A. J. Biol. Chem. 2000; 275: 31938-31945Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Apparent dissociation constant (Kd) and maximum binding (B max) values of [3H]CP55.940 were calculated from saturation curves through nonlinear regression analysis with the Prism 3 program (GraphPad, San Diego, CA) (24Maccarrone M. Lorenzon T. Bari M. Melino G. Finazzi-Agrò A. J. Biol. Chem. 2000; 275: 31938-31945Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). CBR binding was assayed also in NHEK cells (25 × 106/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 (24Maccarrone M. Lorenzon T. Bari M. Melino G. Finazzi-Agrò A. J. Biol. Chem. 2000; 275: 31938-31945Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar), and apparent Kd and B max 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 previously (25Ross R.A. Gibson T.M. Brockie H.C. Lesile M. Pashmi G. Craib S.J. Di Marzo V. Pertwee R.G. Br. J. Pharmacol. 2001; 132: 631-640Crossref PubMed Scopus (209) Google Scholar). In all experiments, unspecific binding was determined in the presence of 10 μm “unlabeled” agonist (24Maccarrone M. Lorenzon T. Bari M. Melino G. Finazzi-Agrò A. J. Biol. Chem. 2000; 275: 31938-31945Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 25Ross R.A. Gibson T.M. Brockie H.C. Lesile M. Pashmi G. Craib S.J. Di Marzo V. Pertwee R.G. Br. J. Pharmacol. 2001; 132: 631-640Crossref PubMed Scopus (209) Google Scholar). 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 (diluted 1:2000) as second antibody (26Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanici C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (140) Google Scholar). 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 (2 × 106/test) was performed as described previously (27Maccarrone M. Bari M. Lorenzon T. Bisogno T. Di Marzo V. Finazzi-Agrò A. J. Biol. Chem. 2000; 275: 13484-13492Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). To discriminate noncarrier-mediated from carrier-mediated transport of AEA through cell membranes, [3H]AEA uptake at 4 °C was subtracted from that at 37 °C (28Hillard C.J. Edgemond W.S. Jarrahian A. Campbell W.B. J. Neurochem. 1997; 69: 631-638Crossref PubMed Scopus (315) Google Scholar). The Q10 value of AMT was calculated as the ratio of AEA uptake at 30 °C and 20 °C (28Hillard C.J. Edgemond W.S. Jarrahian A. Campbell W.B. J. Neurochem. 1997; 69: 631-638Crossref PubMed Scopus (315) Google Scholar). Incubations (15 min) were also carried out with different concentrations of [3H]AEA, in the range 0–800 nm, to determine the apparent Michaelis-Menten constant (Km), maximum velocity (V max), 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 (2 × 106/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 (EC 3.5.1.4; FAAH) activity and its apparent Km, V max, and Ki values were determined in HaCaT cells (20 μg/test) as reported (26Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanici C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (140) Google Scholar). 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 (26Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanici C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (140) Google Scholar) and were subjected to SDS-PAGE (12%) under reducing conditions. Rainbow molecular mass markers (Amersham Biosciences) were phosphorylase b (97.4 kDa), bovine serum albumin (66.0 kDa), ovalbumin (46.0 kDa), and soybean trypsin inhibitor (27.0 kDa). For immunochemical analysis, gels were electroblotted onto 0.45-μm nitrocellulose filters (Bio-Rad) and 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 (26Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanici C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (140) Google Scholar). Densitometric analysis of filters was performed by means of a Floor-S™ Multi-Imager, equipped with Quantity One software (BioRad). The same anti-FAAH antibodies (diluted 1:300) were used to determine FAAH protein content also by enzyme-linked immunosorbent assay, coating wells with cell homogenates (20 μg/well) as reported (26Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanici C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (140) Google Scholar). RT-PCR was performed using total RNA isolated from HaCaT cells (5 × 106 cells) by means of the SNAP™ total RNA isolation kit (Invitrogen, Carlsbad, CA), as described (26Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanici C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (140) Google Scholar). The primers were as follows: (+) 5′-TGGAAGTCCTCCAAAAGCCCAG, (–) 5′-TGTCCATAGACACAGCCCTTCAG, for FAAH; (+)5′-AGTTGCTGCAGTTAAAAAGC, (–) 5′-CCTCAGTTCCGAAAACCAAC, for 18 S rRNA. Five microliters of the reaction mixture was electrophoresed on a 6% polyacrylamide gel, which was then dried and subjected to autoradiography (26Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanici C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (140) Google Scholar). The autoradiographic films were subjected to densitometric analysis, by means of a Floor-S™ Multi-Imager equipped with Quantity One software (Bio-Rad). Products were validated by size determination and sequencing (26Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanici C. Finazzi-Agrò A. J. Immunol. 2001; 166: 7183-7189Crossref PubMed Scopus (140) Google Scholar). Other Biochemical Assays—The endogenous levels of AEA in HaCaT and NHEK cells (50 × 106/test) were determined by gas chromatography-electron impact mass spectrometry, as recently reported (29Maccarrone M. Attinà M. Cartoni A. Bari M. Finazzi-Agrò A. J. Neurochem. 2001; 76: 594-601Crossref PubMed Scopus (87) Google Scholar). The activity of phospholipase D (EC 3.1.4.4; PLD) was assayed in homogenates of HaCaT and NHEK cells (50 μg/test) according to Moesgaard et al. (30Moesgaard B. Petersen G. Jaroszewski J.W. Hansen H.S. J. Lipid Res. 2000; 41: 985-990Abstract Full Text Full Text PDF PubMed Google Scholar), using 1,2-dioleoyl-3-phosphatidyl-[2-14C]ethanolamine (10 μm) as substrate and measuring the release of [14C]ethanolamine as described (31Gubellini P. Picconi B. Bari M. Battista N. Calabresi P. Centone D. Bernardi G. Finazzi-Agrò A. Maccarrone M. J. Neurosci. 2002; 22: 6900-6907Crossref PubMed Google Scholar). Transglutaminase (EC 2.3.2.13; TGase) activity was determined by measuring the incorporation of [3H]putrescine (1 μCi) into N,N′-dimethylcasein, as previously reported (32Rossi A. Catani M.V. Candi E. Bernassola F. Puddu P. Melino G. J. Invest. Dermatol. 2000; 115: 731-739Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). 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 and air-dried, and the radioactivity was measured by liquid scintillation counting in an LKB 1217 Rackbeta spectrometer (Amersham Biosciences). Protein kinase C (EC 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 myelin basic protein fragment 4-14 (25 μm), as reported (22Savini I. Catani M.V. Rossi A. Duranti G. Melino G. Avigliano L. J. Invest. Dermatol. 2002; 118: 372-379Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Reactions were incubated for 10 min at 30 °C, spotted onto Whatman P81 paper, and washed four times in 75 mm H3PO4. The bound radioactivity was determined by liquid scintillation counting in an LKB 1217 Rackbeta spectrometer. Determination of Cornified Cell Envelopes—Cornified envelopes (CE) were extracted from HaCaT or NHEK cells (5 × 106/test) by exhaustive boiling and sonication in 2% SDS, 20 mm dithiothreitol, 0.1 m Tris-HCl (pH 8.0), and 0.5 mm EDTA, as previously described (33Steven A.C. Steinert P.M. J. Cell Sci. 1994; 107: 693-700Crossref PubMed Google Scholar). CE formation was quantified by spectrophotometry at 600 nm and was normalized to the protein content (33Steven A.C. Steinert P.M. J. Cell Sci. 1994; 107: 693-700Crossref PubMed Google Scholar). Transient Transfections—Transient transfections were performed in triplicate using Lipofectin (Invitrogen), according to the manufacturer's instructions. HaCaT cells (1 × 106/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 (34Rossi A. Jang S.I. Ceci R. Steinert P.M. Markova N.G. J. Invest. Dermatol. 1998; 110: 34-40Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Transfection efficiency was monitored by using a thymidine kinase β-galactosidase 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 manufacturer's protocol. CAT activities were normalized to protein content and β-galactosidase activity. Statistical Analysis—Data reported herein are the mean (±S.D.) of at least three independent determinations, each in duplicate. Statistical analysis was performed by using the nonparametric Mann-Whitney test, elaborating experimental data by means of the InStat 3 program (GraphPad). The Endocannabinoid System in HaCaT Cells—The synthetic cannabinoid [3H]CP55.940, which has high affinity to both CB1 and CB2 receptors (35Pertwee R.G. Pharmacol. Ther. 1997; 74: 129-180Crossref PubMed Scopus (1297) Google Scholar), 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 (35Pertwee R.G. Pharmacol. Ther. 1997; 74: 129-180Crossref PubMed Scopus (1297) Google Scholar, 36Lin S. Khanolkar A.D. Fan P. Goutopoulos A. Qin C. Papahadjis D. Makriyannis A. J. Med. Chem. 1998; 41: 5353-5361Crossref PubMed Scopus (122) Google Scholar), and allowed to calculate Kd values of 610 ± 79 and 598 ± 86 pm, and B max 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 (35Pertwee R.G. Pharmacol. Ther. 1997; 74: 129-180Crossref PubMed Scopus (1297) Google Scholar, 36Lin S. Khanolkar A.D. Fan P. Goutopoulos A. Qin C. Papahadjis D. Makriyannis A. J. Med. Chem. 1998; 41: 5353-5361Crossref PubMed Scopus (122) Google Scholar), showed saturation curves (Fig. 1A) from which a Kd of 245 ± 32 pm and a B max of 277 ± 11 fmol·mg protein–1 could be calculated. The Kd and B max values found here for mouse brain and spleen are in agreement with previous reports (reviewed in Ref. 35Pertwee R.G. Pharmacol. Ther. 1997; 74: 129-180Crossref PubMed Scopus (1297) Google Scholar). Consistently with the binding data, 1 μm anandamide (AEA) and 0.1 μm SR141716, a selective CB1R antagonist (35Pertwee R.G. Pharmacol. Ther. 1997; 74: 129-180Crossref PubMed Scopus (1297) Google Scholar), but not 0.1 μm SR144528, a selective CB2R antagonist (35Pertwee R.G. Pharmacol. Ther. 1997; 74: 129-180Crossref PubMed Scopus (1297) Google Scholar), displaced [3H]CP55.940, suggesting that only CB1 receptors were expressed on HaCaT cell surface (Fig. 1B). 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 ban" @default.
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• W2062752021 title "The Endocannabinoid System in Human Keratinocytes" @default.
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