Anandamide Regulates Keratinocyte Differentiation by Inducing DNA Methylation in a CB1 Receptor-dependent Manner
2007; Elsevier BV; Volume: 283; Issue: 10 Linguagem: Inglês
10.1074/jbc.m707964200
ISSN1083-351X
AutoresAndrea Paradisi, Nicoletta Pasquariello, Daniela Barcaroli, Mauro Maccarrone,
Tópico(s)Pancreatic function and diabetes
ResumoAnandamide (arachidonoylethanolamide, AEA) belongs to an important class of endogenous lipids including amides and esters of long chain polyunsaturated fatty acids, collectively termed “endocannabinoids.” Recently we have shown that AEA inhibits differentiation of human keratinocytes, by binding to type-1 cannabinoid receptors (CB1R). To further characterize the molecular mechanisms responsible for this effect, we investigated the expression of epidermal differentiation-related genes after AEA treatment. We observed that keratin 1 and 10, transglutaminase 5 and involucrin are transcriptionally down-regulated by AEA. Most importantly, we found that AEA is able to decrease differentiating gene expression by increasing DNA methylation in human keratinocytes, through a p38, and to a lesser extent p42/44, mitogen-activated protein kinase-dependent pathway triggered by CB1R. An effect of AEA on DNA methylation because of CB1R-mediated increase of methyltransferase activity is described here for the first time, and we believe that the importance of this effect clearly extends beyond the regulation of skin differentiation. In fact, the modulation of DNA methylation by endocannabinoids may affect the expression of a number of genes that regulate many cell functions in response to these substances. Anandamide (arachidonoylethanolamide, AEA) belongs to an important class of endogenous lipids including amides and esters of long chain polyunsaturated fatty acids, collectively termed “endocannabinoids.” Recently we have shown that AEA inhibits differentiation of human keratinocytes, by binding to type-1 cannabinoid receptors (CB1R). To further characterize the molecular mechanisms responsible for this effect, we investigated the expression of epidermal differentiation-related genes after AEA treatment. We observed that keratin 1 and 10, transglutaminase 5 and involucrin are transcriptionally down-regulated by AEA. Most importantly, we found that AEA is able to decrease differentiating gene expression by increasing DNA methylation in human keratinocytes, through a p38, and to a lesser extent p42/44, mitogen-activated protein kinase-dependent pathway triggered by CB1R. An effect of AEA on DNA methylation because of CB1R-mediated increase of methyltransferase activity is described here for the first time, and we believe that the importance of this effect clearly extends beyond the regulation of skin differentiation. In fact, the modulation of DNA methylation by endocannabinoids may affect the expression of a number of genes that regulate many cell functions in response to these substances. Anandamide (arachidonoylethanolamide, AEA) 4The abbreviations used are:AEAarachidonoylethanolamideCB1/2Rtype-1/2 cannabinoid receptorFAAHfatty acid amide hydrolaseNAPEN-acyl-phosphatidylethanolaminesNAPE-PLDNAPE-hydrolyzing phospholipase D2-AG2-arachidonoylglycerolESendocannabinoid systemTPA12-O-tetradecanoylphorbol-13-acetate5AC5-azacytidineNADAN-arachidonoyldopamineK1/10keratin 1/10TGase 5transglutaminase 5MSPmethylation-specific PCRDNMTDNA methyltransferaseEDCepidermal differentiation complexACEAarachidonoyl-2-chloroethylamideMAPKmitogen-activated protein kinaseRTreverse transcription. belongs to an important class of endogenous lipids including amides and esters of long chain polyunsaturated fatty acids, collectively termed “endocannabinoids” (1De Petrocellis L. Cascio M.G. Marzo Di V. Brit. J. Pharmacol. 2004; 141: 765-774Crossref PubMed Scopus (416) Google Scholar, 2Bari M. Battista N. Fezza F. Gasperi V. Maccarrone M. Mini Rev. Med. Chem. 2006; 6: 257-268Crossref PubMed Scopus (102) Google Scholar). AEA is released from depolarized neurons, endothelial cells, and macrophages (3Marzo Di V. Trends Pharmacol. Sci. 2006; 27: 134-140Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), and mimics the pharmacological effects of Δ9-tetrahydro-cannabinol, the active principle of hashish and marijuana (4Howlett A.C. Handb. Exp. Pharmacol. 2005; 168: 53-79Crossref PubMed Scopus (27) Google Scholar). Extracellular AEA binds to type-1 and type-2 cannabinoid receptors (CB1R and CB2R) (4Howlett A.C. Handb. Exp. Pharmacol. 2005; 168: 53-79Crossref PubMed Scopus (27) Google Scholar), thus playing many actions in the central nervous system and in the periphery (2Bari M. Battista N. Fezza F. Gasperi V. Maccarrone M. Mini Rev. Med. Chem. 2006; 6: 257-268Crossref PubMed Scopus (102) Google Scholar, 3Marzo Di V. Trends Pharmacol. Sci. 2006; 27: 134-140Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The endogenous concentration of AEA is controlled in vivo through degradation by fatty acid amide hydrolase (FAAH) (5McKinney M.K. Cravatt B.F. Annu. Rev. Biochem. 2005; 74: 411-432Crossref PubMed Scopus (565) Google Scholar), preceded or not by cellular uptake through a putative AEA membrane transporter (6Battista N. Gasperi V. Fezza F. Maccarrone M. Therapy. 2005; 2: 141-150Crossref Google Scholar, 7Glaser S.T. Kaczocha M. Deutsch D.G. Life Sci. 2005; 77: 1584-1604Crossref PubMed Scopus (117) Google Scholar). The main checkpoint in AEA synthesis seems to be the N-acyl-phosphatidylethanolamines (NAPE)-hydrolyzing phospholipase D (NAPE-PLD), which releases on demand AEA from membrane NAPEs (8Okamoto Y. Morishita J. Tsuboi K. Tonai T. Ueda N. J. Biol. Chem. 2004; 279: 5298-5305Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar). However, additional metabolic routes seem to contribute to the synthesis of AEA (9Liu J. Wang L. Harvey-White J. Osei-Hyiaman D. Razdan R. 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Drug Targets CNS Neurol. Disord. 2005; 4: 615-623Crossref PubMed Scopus (74) Google Scholar). Together with AEA, 2-AG and congeners, the proteins that bind and metabolize these substances form the endocannabinoid system (ES) (3Marzo Di V. Trends Pharmacol. Sci. 2006; 27: 134-140Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 14Paradisi A. Oddi S. Maccarrone M. Curr. Drug Targets. 2006; 11: 1539-1552Crossref Scopus (30) Google Scholar). Full and functional ES has been found virtually in all tissues and its relevance within the central nervous system has been clearly demonstrated (15Piomelli D. Nat. Rev. Neurosci. 2003; 4: 873-884Crossref PubMed Scopus (1625) Google Scholar). Peripheral endocannabinoids seem to play a crucial role in modulating the autonomic nervous, reproductive, endocrine, and immune systems (16Marzo Di V. Matias I. Nat. Neurosci. 2005; 8: 585-589Crossref PubMed Scopus (641) Google Scholar, 17Klein T.W. Nat. Rev. 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CRC Press, Boca Raton, FL2006: 451-466Google Scholar). In this context, we have shown that human keratinocytes have a functional ES that enables them to bind and metabolize AEA; moreover, ES was shown to be implicated in the control of epidermal differentiation, through a CB1R-dependent mechanism (24Maccarrone M. Di Rienzo M. Battista N. Gasperi V. Guerrieri P. Rossi A. Finazzi-Agrò A. J. Biol. Chem. 2003; 278: 33896-33903Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The epidermis, which forms the uppermost compartment of the skin, represents a barrier against the environment, provided by terminally differentiating keratinocytes (25Nemes Z. Steinert P.M. Exp. Mol. Med. 1999; 31: 5-19Crossref PubMed Scopus (454) Google Scholar, 26Kalinin A. Marekov L.N. Steinert P.M. J. Cell Sci. 2001; 114: 3069-3070Crossref PubMed Google Scholar). Epidermal differentiation begins with the migration of keratinocytes from basal layer, composed of proliferating cells, and ends with the formation of the cornified cell envelope, an insoluble protein structure found in differentiated keratinocytes (27Candi E. Schmidt R. Melino G. Nat. Rev. Mol. Cell. Biol. 2005; 6: 328-340Crossref PubMed Scopus (1297) Google Scholar). Cell proliferation and differentiation occur sequentially and are characterized by the expression of specific proteins, such as keratins and transglutaminases (28Fuchs E. Cleveland D.W. Science. 1998; 279: 514-519Crossref PubMed Scopus (836) Google Scholar, 29Lorand L. Graham R.M. Nat. Rev. Mol. Cell. Biol. 2003; 4: 140-156Crossref PubMed Scopus (1223) Google Scholar). Activation of several keratinocyte differentiation genes requires the opening of chromatin structure and demethylation of specific genomic promoter regions. Variation in overall DNA methylation between differentiated and undifferentiated cells has been reported in a number of different models (30Ehrlich M. Gama-Sosa M.A. Huang L.H. Midgett R.H. Kuo K.C. McCune R.A. Gehrke C. Nucleic Acids Res. 1982; 10: 2709-2721Crossref PubMed Scopus (777) Google Scholar, 31Lyon S.B. Buonocore L. Miller M. Mol. Cell. Biol. 1987; 7: 1759-1763Crossref PubMed Google Scholar), and DNA of differentiated keratinocytes has been shown to contain less 5-methylcytosine than DNA of undifferentiated keratinocytes (32Veres D.A. Wilkins L. Coble D.W. Lyon S.B. J. Investig. Dermatol. 1989; 93: 687-690Abstract Full Text PDF PubMed Scopus (11) Google Scholar). Moreover, agents known to inhibit DNA methylation (i.e. 5-azacytidine, 5AC) and histone deacetylation (i.e. sodium butyrate, NaB) are also known to inhibit growth and to promote differentiation of keratinocytes (33Rosl F. Durst M. Hausen Zur H. 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On the other hand, it has been shown that endocannabinoids regulate neuritogenesis, axonal growth, and synaptogenesis in differentiated neurons (36Rueda D. Navarro B. Martinez-Serrano A. Guzman M. Galve-Roperh I. J. Biol. Chem. 2002; 277: 46645-46650Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 37Galve-Roperh I. Aguado T. Rueda D. Velasco G. Guzman M. Curr. Pharm. Des. 2006; 12: 2319-2325Crossref PubMed Scopus (80) Google Scholar), leading to the hypothesis that endocannabinoids are general signaling cues responsible for the regulation of cellular proliferation and differentiation. To evaluate the molecular mechanisms underlying the influence of endocannabinoids, and in particular of AEA, on cell differentiation, we sought to investigate the effects of exogenous AEA on the gene expression pattern of differentiating human keratinocytes. arachidonoylethanolamide type-1/2 cannabinoid receptor fatty acid amide hydrolase N-acyl-phosphatidylethanolamines NAPE-hydrolyzing phospholipase D 2-arachidonoylglycerol endocannabinoid system 12-O-tetradecanoylphorbol-13-acetate 5-azacytidine N-arachidonoyldopamine keratin 1/10 transglutaminase 5 methylation-specific PCR DNA methyltransferase epidermal differentiation complex arachidonoyl-2-chloroethylamide mitogen-activated protein kinase reverse transcription. Materials—Chemicals were of the purest analytical grade. AEA, 12-O-tetradecanoylphorbol-13-acetate (TPA), 5-azacytidine (5AC), and N-arachidonoyldopamine (NADA) were purchased from Sigma. S-Adenosyl-l-[methyl-3H]methionine was from Amersham Biosciences (Buckinghamshire, UK), 2-arachidonoylglycerol (2-AG) was from Research Biochemicals International (Natick, MA). Arachidonoyl-2-chloroethylamide (ACEA) was purchased from Cayman Chemical (Ann Arbor, MI). PD98059 and SB203580 were from Calbiochem (San Diego, CA). N-Piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR141716) was a kind gift from Sanofi-Aventis (Montpellier, France). Cell Culture and Treatments—HaCaT cells 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. Cell differentiation was induced by treating HaCaT cells with TPA (10 ng/ml) plus CaCl2 (1.2 mm) for 5 days (38Candi 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 (93) Google Scholar). AEA and related compounds were added at the indicated concentrations directly to the serum-free culture medium, at the same time as TPA plus calcium (24Maccarrone M. Di Rienzo M. Battista N. Gasperi V. Guerrieri P. Rossi A. Finazzi-Agrò A. J. Biol. Chem. 2003; 278: 33896-33903Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Culture medium containing freshly prepared AEA and the other reagents was changed daily during the treatment. Culture medium containing vehicles alone was added to controls under the same conditions (24Maccarrone M. Di Rienzo M. Battista N. Gasperi V. Guerrieri P. Rossi A. Finazzi-Agrò A. J. Biol. Chem. 2003; 278: 33896-33903Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). After each treatment, cell viability was determined by Trypan Blue dye exclusion. The treatment of differentiating HaCaT cells with 5AC was performed by seeding 3 × 106 cells in 100-cm2 tissue culture flasks. After 24 h, cells were exposed to 1 μm 5AC for 5 days. Real-time PCR Assay—RNA was extracted using the RNeasy extraction kit (Qiagen, Crawley, UK) from proliferating and differentiating HaCaT cells, following the manufacturer's instructions. RT-PCR reactions were performed using the RT-PCR SuperScript III Platinum Two-Step qRT-PCR Kit (Invitrogen, Carlsbad, CA). 1 μg of total RNA was used to produce cDNA with 10 units/μl SuperScript III reverse transcriptase, in the presence of 2 units/μl RNaseOUT, 1.25 μm oligo(dT)20, 1.25 ng/μl random hexamers, 5 mm MgCl2, 0.5 mm dNTP mix, and DEPC-treated water. The reaction was performed using the following RT-PCR program: 25 °C for 10 min, 42 °C for 50 min, 85 °C for 5 min, then, after addition of 0.1 units/μl of Escherichia coli RNase H, the product was incubated at 37 °C for 20 min. For expression studies, the target transcripts were amplified in ABI PRISM 7700 sequence detector system (Applied Biosystems, Foster City, CA), using the following primers: keratin 10 (K10) F1 (5′-ACGAGGAGGAAATGAAAGAC-3′), K10 R1 (5′-GGACTGTAGTTCTATCTCCAG-3′); keratin 1 (K1) F1 (5′-AGAAAGCAGGATGTCTGG-3′), K1 R1 (5′-AAACAAACTTCACGCTGG-3′); involucrin (INV) F1 (5′-CTCTGCCTCAGCCTTACT-3′), INV R1 (5′-GCTGCTGATCCCTTTGTG-3′); transglutaminase 5 (TG5) F1 (5′-TCAGCACAAAGAGCATCCAG-3′), TG5 R1 (5′-TTCAGGGAGACTTGCACCAC-3′); β-actin F1 (5′-TGACCCAGATCATGTTTGAG-3′) and β-actin R1 (5′-TTAATGTCACGCACGATTTCC-3′). Actin was used as housekeeping gene for quantity normalization. One microliter of the first strand cDNA product was used for amplification in triplicate in a 25-μl reaction solution containing 12.5 μl of Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) and 10 pmol of each primer. The following PCR program was used: 95 °C for 10 min; 40 amplification cycles at 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s. Immunoblotting Analysis—HaCaT cell protein extracts (20 μg per lane) were loaded onto 10% SDS-polyacrylamide gels and blotted onto polyvinylidene difluoride sheets (Amersham Biosciences). Filters were blocked with 10% nonfat dried milk and 5% bovine serum albumin for 2 h, and then were incubated for 2 h with rabbit anti-K10 (diluted 1:1000 in blocking solution; Berkeley Antibody Company, Richmond, CA) and mouse anti-actin (1:1000 in blocking solution; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies. After three washes with phosphate-buffered saline + 0.05% Tween 20, filters were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (1:2000 in blocking solution; Santa Cruz Biotechnology) for 1 h. Detection was performed using West Dura Chemiluminescence System (Pierce, Rockford, IL). DNase I Sensitivity Assay—The procedure for the isolation of nuclei was reported previously (39Lee Y.W. Klein C.B. Kargacin B. Salnikow K. Kitahara J. Dowjat K. Zhitkovich A. Christie N.T. Costa M. Mol. Cell. Biol. 1995; 15: 2547-2557Crossref PubMed Scopus (339) Google Scholar). A total of 5 × 105 nuclei in DNase I buffer (10 mm Tris-HCl, 10 mm NaCl, 3 mm MgCl2, 100 mm CaCl2, pH 7.4) were treated with increasing amounts (0, 0.5, 1, 2, and 10 units) of DNase I (Roche Applied Science) in a reaction volume of 200 μl for 30 min at 25 °C. The reactions were terminated by adding an equal volume of stop solution (1% sodium dodecyl sulfate, 0.1 m NaCl, 50 mm Tris-HCl, pH 8.0, and 10 mm EDTA), containing 1 mg of proteinase K per ml, followed by incubation at 55 °C for 2 h. DNA was extracted with phenol-chloroform and was ethanol-precipitated. The K10 gene was amplified by PCR (50 ng/reaction; 30 cycles) with the primers K10P WF and K10P WR, described below for the methylation-specific PCR. The PCR products were separated on 1.6% agarose gel and stained with ethidium bromide. Bisulfite DNA Modification—Genomic DNA was isolated from HaCaT cells using DNeasy kit (Qiagen, Crawley, UK). Sodium bisulfite treatment of DNA was performed using the CpGenome DNA Modification kit (Chemicon International Inc, Temecula, CA). Briefly, DNA (1 μg) was denaturated by adding NaOH (0.2 m) for 10 min at 50 °C. 550 μl of 3 m sodium bisulfite at pH 5.0 was added and mixed, and samples were incubated at 50 °C for 16 h in a water bath. Modified DNA was then bound to a micro-particulate carrier and was desalted by repeated centrifugation and resuspension in 70% ethanol. The conversion to uracil was completed by alkaline desulfonation, and DNA was finally eluted from the carrier by heating in TE buffer for 15 min at 60 °C. DNA preparations were either used immediately or stored at –20 °C. Methylation-specific PCR—PCR analysis was performed as previously described (40Herman J.G. Graff J.R. Myohanen S. Nelkin B.D. Baylin S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9821-9826Crossref PubMed Scopus (5239) Google Scholar). 2 μl of bisulfite-modified DNA was amplified by using PCR master mix (Promega Corp., Madison, WI), containing 25 units/ml of TaqDNA polymerase, 400 μm dNTPs, 1.5 mm MgCl2, and 0.4 μm of each primer. The amplification program was as follow: 95 °C for 5 min; 35 cycles at 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 5 min. The primers used for K10 amplification (M: methylation specific, U: specific for unmethylated sequence, W: unmodified specific) were the following: K10P MF (5′-AGTTTTCGTTTTCGTAGTCGTC-3′), K10P MR (5′-CGAATATAACCTCACCCCG-3′), K10P UF (5′-GGAGTTTTTGTTTTTGTAGTTGTT-3′), K10P UR (5′-AACCAAATATAACCTCACCCCA-3′), K10P WF (5′-AGCTTCCGCCTCCGTAGCCGCC-3′), and K10P WR (5′-CGAATGTGACCTCACCCCG-3′). PCR products were loaded on a 1.8% agarose gel containing ethidium bromide, and were visualized under UV illumination. Genomic Methylation Level—A modification of the methyl-accepting assay (41Broday L. Lee Y.W. Costa M. Mol. Cell. Biol. 1999; 19: 3198-3204Crossref PubMed Scopus (27) Google Scholar) was used to determine the methylation level of DNA isolated from HaCaT cells. DNA (200 ng) was incubated with 4 units of SssI methylases (New England Biolabs, Ipswich, MA) in the presence of 1.5 mm S-adenosyl-l-[methyl-3H]methionine and 1.5 mm nonradioactive S-adenosylmethionine (New England Biolabs). The reaction mixtures (20 μl), in the manufacturer's buffer containing 0.1 μg of RNase A, were incubated at 37 °C for 4 h. The reactions were terminated by adding 300 μl of stop solution (1% sodium dodecyl sulfate, 2 mm EDTA, 5% 2-propyl alcohol, 125 mm NaCl, 1 mg of proteinase K per ml, 0.25 mg of carrier DNA per ml) for 1 h at 37 °C. DNA was extracted with phenol-chloroform and was ethanol-precipitated. The recovered DNA was resuspended in 30 μl of 0.3 m NaOH and incubated for 30 min at 37 °C. DNA was spotted on Whatman GF/C filter discs, dried, and then washed five times with 5% (w/v) trichloroacetic acid followed by 70% (v/v) ethanol. Filters were placed in scintillation vials and incubated for 1 h at 60 °C with 500 μl of 0.5 m perchloric acid. Then, 5 ml of scintillation mixture was added, and the 3H incorporation was determined by a Beckman liquid scintillation counter. Higher levels of [3H]methyl group incorporated into DNA were indicative of lower levels of genomic DNA methylation (41Broday L. Lee Y.W. Costa M. Mol. Cell. Biol. 1999; 19: 3198-3204Crossref PubMed Scopus (27) Google Scholar). Assay of DNA Methyltransferase—Cell extracts were prepared in ice-cold lysis buffer containing 50 mm Tris-HCl, pH 7.8, 1 mm EDTA, 10% glycerol, 0.01% sodium azide, 10% Tween-80, 100 μg/ml RNase A, and 0.5 mm phenylmethylsulfonyl fluoride. De novo methyltransferase activity was measured as previously described (42Xiong Y. Dowdy S.C. Podratz K.C. Jin F. Attewell J.R. Eberhardt N.L. Jiang S.W. Cancer Res. 2005; 65: 2684-2689Crossref PubMed Scopus (136) Google Scholar, 43Adams R.L. Rinaldi A. Seivwright C. J. Biochem. Biophys. Methods. 1991; 22: 19-22Crossref PubMed Scopus (55) Google Scholar). Cellular protein extracts (30 μg) were incubated in the presence of 3 μg of double-stranded oligonucleotides and 2.4 μCi of S-adenosyl-l-[methyl-3H]methionine (Amersham Biosciences), at 37 °C for 1 h. The reaction was terminated by adding 90 μl of stop solution (1% sodium dodecyl sulfate, 2 mm EDTA, 3% (w/v) 4-amino salicylate, 5% butyl alcohol, 0.25 mg/ml calf thymus DNA, and 1 mg/ml proteinase K), and incubating at 37 °C for 45 min. The reaction mixture was then spotted on Whatman GF/C filter paper discs (Fisher Scientific, East Brunswick, NJ), and filters were washed twice with 5% trichloroacetic acid, rinsed in 70% ethanol, and dried at 56 °C for 20 min. Finally, filters were submerged in UltimaGold scintillation mixture (Packard, Meriden, CT) and radioactivity was measured in a Beckman liquid scintillation counter (LS 5000TD). A blank control reaction was done simultaneously using cell extracts that were heated to 80 °C for 15 min to inactivate the methyltransferase activity. The results, expressed as counts per min (cpm), were corrected by subtracting the background level. Statistical Analysis—The data reported in this article are the mean ± S.D. of at least three independent determinations, each performed in duplicate. Statistical analysis was performed by the nonparametric Mann-Whitney U test, elaborating experimental data by means of the InStat 3 program (GraphPad Software for Science, San Diego, CA). AEA Inhibits Keratinocyte Differentiation by Regulating Gene Expression—Spontaneously immortalized keratinocytes (HaCaT cells) can be induced to differentiate by treatment with TPA plus calcium (38Candi 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 (93) Google Scholar, 44Savini I. Catani M.V. Rossi A. Duranti G. Melino G. Avigliano L. J. Investig. Dermatol. 2002; 118: 372-379Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Fig. 1 shows that, as expected, induction of differentiation of HaCaT cells for 5 days determines a significant increase in expression of genes known to be up-regulated during differentiation, as measured by quantitative RT-PCR. Notably, the increase in keratin 1 (K1), keratin 10 (K10), and transglutaminase 5 (TGase 5) (Fig. 1, A, B, D), which are all induced later during epidermal differentiation (38Candi 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 (93) Google Scholar, 45Ming M.E. Daryanani H.A. Roberts L.P. Baden H.P. Kvedar J.C. J. Investig. Dermatol. 1994; 103: 780-784Abstract Full Text PDF PubMed Scopus (49) Google Scholar, 46Candi E. Oddi S. Paradisi A. Terrinoni A. Ranalli M. Teofoli P. Citro G. Scarpato S. Puddu P. Melino G. J. Investig. Dermatol. 2002; 119: 670-677Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), was much higher than that of an early differentiation marker like involucrin (47Eckert R.L. Yaffe M.B. Crish J.F. Murthy S. Rorke E.A. Welter J.F. J. Investig. Dermatol. 1993; 100: 613-617Abstract Full Text PDF PubMed Scopus (154) Google Scholar) (Fig. 1C). Interestingly AEA treatment significantly reduced activation of differentiating genes. In addition, we observed that the mRNA level reduction (∼50%) after AEA treatment was paralleled by a decreased protein level, at least in the case of K10 (Fig. 1E). These findings are well in line with our previous observation that AEA inhibits cornified envelope formation (24Maccarrone M. Di Rienzo M. Battista N. Gasperi V. Guerrieri P. Rossi A. Finazzi-Agrò A. J. Biol. Chem. 2003; 278: 33896-33903Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), and suggest that AEA is able to inhibit keratinocyte differentiation by modifying the gene expression profile of these cells. Inhibition of DNA Methylation Prevents the Effects of AEA on Gene Expression—Because it has been shown that DNA methylation levels change during keratinocyte differentiation (32Veres D.A. Wilkins L. Coble D.W. Lyon S.B. J. Investig. Dermatol. 1989; 93: 687-690Abstract Full Text PDF PubMed Scopus (11) Google Scholar) and that inhibitors of methylation promote this phenomenon (33Rosl F. Durst M. Hausen Zur H. EMBO J. 1988; 7: 1321-1328Crossref PubMed Scopus (57) Google Scholar), we investigated the possibility that AEA was affecting gene expression levels through alteration of DNA methylation. Treatment of HaCaT cells with TPA plus calcium in the presence of 1 μm 5AC, an inhibitor of DNA methylation (48Juttermann R. Li E. Jaenisch R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11797-11801Crossref PubMed Scopus (583) Google Scholar, 49Santi D.V. Norment A. Garrett C.E. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6993-6997Crossref PubMed Scopus (427) Google Scholar), resulted in a ∼2-fold increase in K10 expression, as compared with cells treated only with TPA plus calcium, suggesting that inhibition of DNA methylation allows increased transcription of this gene (Fig. 2). Most importantly, treatment with 1 μm 5AC abolished the effect of AEA on K10 expression levels, which were comparable to those of cells differentiated without AEA (Fig. 2). These data strongly suggest that inhibition of differentiation by AEA occurs through changes in chromatin methylation patterns, because inhibition of DNA methylation is sufficient to prevent AEA effects on keratinocyte differentiation. AEA Decreases Gene Transcription by Inducing DNA Methylation—To validate the hypothesis that AEA could change DNA methylation levels in the K10 locus, we used a DNase I sensitivity assay, by which we tested nuclease accessibility in nuclei isolated from HaCaT cells. Proliferating cells exhibited marked resistance to increasing concentrations of DNase I compared with differentiating cells (Fig. 3A), where K10 gene was completely digested with one enzyme unit. As expected, treatment with 1 μm 5AC enhanced the sensitivity to DNase I treatment by reducing the methylation levels. Consistent with its possible role in regulating methylation levels, treatment of differentiating cells with AEA induced a strong resistance to DNase I digestion. Once again, 5AC was able to revert this effect, confirming a role for methylation in the activity of AEA on keratinocyte differentiation. To further confirm that the observed changes i
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