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• W2027185670 abstract "Nerve growth factor induces innervation and epidermal hyperplasia in inflammatory skin diseases like psoriasis. Nerve growth factor production by keratinocytes is increased in the inflammatory lesions. Nerve growth factor induces histamine release from mast cells. We examined the in vitro effects of histamine on nerve growth factor production in human keratinocytes. Histamine enhanced nerve growth factor secretion, mRNA expression, and promoter activity in keratinocytes. Two TPA-response elements on the nerve growth factor promoter were responsible for the activation by histamine. Histamine enhanced transcriptional activity and DNA binding of activator protein 1 at the two TPA-response elements. It shifted the TPA-response-element-binding activator protein 1 composition from c-Jun homodimers to c-Fos/c-Jun heterodimers. Histamine transiently induced c-Fos mRNA expression, which was not detectable in unstimulated keratinocytes, whereas c-Jun mRNA expression was constitutive and was not altered by histamine. Histamine-induced enhancement of nerve growth factor secretion, promoter activity, activator protein 1 transcriptional activity, and c-Fos expression was suppressed by H1 antagonist pyrilamine, protein kinase C inhibitor calphostin C, and PD98059, an inhibitor of mitogen-activated protein kinase kinase 1. Histamine induced the translocation of protein kinase C activity from cytosol to membrane, which was suppressed by phospholipase C inhibitor U73122. It stimulated the phosphorylation of extracellular signal-regulated kinase, which was blocked by pyrilamine, calphostin C, and PD98059. These results suggest that histamine may enhance nerve growth factor production by inducing c-Fos expression in keratinocytes. These effects may be mediated by the H1-receptor-induced signaling cascade of phospholipase C-protein kinase C-mitogen-activated protein kinase kinase 1-extracellular signal-regulated kinase. Nerve growth factor induces innervation and epidermal hyperplasia in inflammatory skin diseases like psoriasis. Nerve growth factor production by keratinocytes is increased in the inflammatory lesions. Nerve growth factor induces histamine release from mast cells. We examined the in vitro effects of histamine on nerve growth factor production in human keratinocytes. Histamine enhanced nerve growth factor secretion, mRNA expression, and promoter activity in keratinocytes. Two TPA-response elements on the nerve growth factor promoter were responsible for the activation by histamine. Histamine enhanced transcriptional activity and DNA binding of activator protein 1 at the two TPA-response elements. It shifted the TPA-response-element-binding activator protein 1 composition from c-Jun homodimers to c-Fos/c-Jun heterodimers. Histamine transiently induced c-Fos mRNA expression, which was not detectable in unstimulated keratinocytes, whereas c-Jun mRNA expression was constitutive and was not altered by histamine. Histamine-induced enhancement of nerve growth factor secretion, promoter activity, activator protein 1 transcriptional activity, and c-Fos expression was suppressed by H1 antagonist pyrilamine, protein kinase C inhibitor calphostin C, and PD98059, an inhibitor of mitogen-activated protein kinase kinase 1. Histamine induced the translocation of protein kinase C activity from cytosol to membrane, which was suppressed by phospholipase C inhibitor U73122. It stimulated the phosphorylation of extracellular signal-regulated kinase, which was blocked by pyrilamine, calphostin C, and PD98059. These results suggest that histamine may enhance nerve growth factor production by inducing c-Fos expression in keratinocytes. These effects may be mediated by the H1-receptor-induced signaling cascade of phospholipase C-protein kinase C-mitogen-activated protein kinase kinase 1-extracellular signal-regulated kinase. 4-(3-chloroanilino)-6,7-dimethoxyquinazoline activator protein 1 electrophoretic mobility shift assay extracellular signal-regulated kinase guanine nucleotide-binding protein N-[2-((p-bromocinnamyl)aminoethyl)-5-isoquinolinesulfonamide mitogen-activated protein kinase MAPK kinase TPA response element 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione Nerve growth factor (NGF) plays an important role in the development of inflammatory skin diseases like psoriasis, atopic dermatitis, or allergic contact dermatitis (Raychaudhuri et al., 1998Raychaudhuri S.P. Jiang W.-Y. Farber E.M. Psoriatic keratinocytes express high levels of nerve growth factor.Acta Derm Venereol (Stockh). 1998; 78: 84-86Crossref PubMed Scopus (111) Google Scholar;Kinkelin et al., 2000Kinkelin I. Motzig S. Koltzenburg M. Brocker E.-B. Increase in NGF content and nerve fiber sprouting in human allergic contact eczema.Cell Tissue Res. 2000; 302: 31-37Crossref PubMed Scopus (85) Google Scholar;Toyoda et al., 2002Toyoda M. Nakamura M. Makino T. Hino T. Kagoura M. Morohashi M. Nerve growth factor and substance P are useful plasma markers of disease activity in atopic dermatitis.Br J Dermatol. 2002; 147: 71-79Crossref PubMed Scopus (283) Google Scholar). In these skin diseases, NGF production in keratinocytes is enhanced (Raychaudhuri et al., 1998Raychaudhuri S.P. Jiang W.-Y. Farber E.M. Psoriatic keratinocytes express high levels of nerve growth factor.Acta Derm Venereol (Stockh). 1998; 78: 84-86Crossref PubMed Scopus (111) Google Scholar;Kinkelin et al., 2000Kinkelin I. Motzig S. Koltzenburg M. Brocker E.-B. Increase in NGF content and nerve fiber sprouting in human allergic contact eczema.Cell Tissue Res. 2000; 302: 31-37Crossref PubMed Scopus (85) Google Scholar;Toyoda et al., 2002Toyoda M. Nakamura M. Makino T. Hino T. Kagoura M. Morohashi M. Nerve growth factor and substance P are useful plasma markers of disease activity in atopic dermatitis.Br J Dermatol. 2002; 147: 71-79Crossref PubMed Scopus (283) Google Scholar). NGF potentiates the proliferation of neurons, which may result in hyperinnervation and itch sensation in inflammatory skin diseases (Kinkelin et al., 2000Kinkelin I. Motzig S. Koltzenburg M. Brocker E.-B. Increase in NGF content and nerve fiber sprouting in human allergic contact eczema.Cell Tissue Res. 2000; 302: 31-37Crossref PubMed Scopus (85) Google Scholar). NGF also enhances the growth of keratinocytes having the high affinity NGF receptor trkE (Di Marco et al., 1993Di Marco E. Mathor M. Bondanza S. Cutuli N. Marchisio P.C. De Cancedda R. Luca M. Nerve growth factor binds to normal human keratinocytes through high and low affinity receptors and stimulates their growth by a novel autocrine loop.J Biol Chem. 1993; 268: 22838-22846Abstract Full Text PDF PubMed Google Scholar;Pincelli et al., 1994Pincelli C. Sevianani C. Manfredini R. et al.Expression and function of nerve growth factor and nerve growth factor receptor on cultured keratinocytes.J Invest Dermatol. 1994; 103: 13-18Abstract Full Text PDF PubMed Google Scholar), which may lead to epidermal hyperplasia associated with psoriasis or atopic dermatitis (Toyoda et al., 2002Toyoda M. Nakamura M. Makino T. Hino T. Kagoura M. Morohashi M. Nerve growth factor and substance P are useful plasma markers of disease activity in atopic dermatitis.Br J Dermatol. 2002; 147: 71-79Crossref PubMed Scopus (283) Google Scholar). NGF also promotes the histamine release of mast cells (Bruni et al., 1982Bruni A. Bigon E. Boarato E. Mietto L. Leon A. Toffano G. Interaction between nerve growth factor and lysophosphatidylserine on rat peritoneal mast cells.FEBS Lett. 1982; 138: 190-192Abstract Full Text PDF PubMed Scopus (136) Google Scholar;Pearce and Thompson, 1986Pearce F. Thompson H.L. Some characteristics of histamine secretion from rat peritoneal mast cells stimulated with nerve growth factor.J Physiol. 1986; 372: 379-393Crossref PubMed Scopus (173) Google Scholar;Matsuda et al., 1991Matsuda H. Kannan Y. Ushio H. Kiso Y. Kanemoto T. Suzuki H. Kitamura Y. Nerve growth factor induces development of connective tissue-type mast cells in vitro from murine bone marrow cells.J Exp Med. 1991; 174: 7-14Crossref PubMed Scopus (217) Google Scholar;Horigome et al., 1993Horigome K. Pryor J.C. Bullock E.D. Johnson E.M.J. Mediator release from mast cells by nerve growth factor. Neurotrophin specificity and receptor mediation.J Biol Chem. 1993; 268: 14881-14887Abstract Full Text PDF PubMed Google Scholar;Horigome et al., 1994Horigome K. Bullock E.D. Johnson E.M.J. Effects of nerve growth factor on rat peritoneal mast cells. Survival promotion and immediate-early gene induction.J Biol Chem. 1994; 269: 2695-2702Abstract Full Text PDF PubMed Google Scholar). In the early phases of psoriasis or atopic dermatitis, mast cells are increased in the dermis (Jiang et al., 2001Jiang W.Y. Chattedee A.D. Raychaudhuri S.P. Raychaudhuri S.K. Farber E.M. Mast cell density and IL-8 expression in nonlesional and lesional psoriatic skin.Int J Dermatol. 2001; 40: 699-703Crossref PubMed Scopus (67) Google Scholar;Alenius et al., 2002Alenius H. Laouini D. Woodward A. et al.Mast cells regulate IFN-γ expression in the skin and circulating IgE levels in allergen-induced skin inflammation.J Allergy Clin Immunol. 2002; 0: 106-113Abstract Full Text Full Text PDF Scopus (58) Google Scholar). Mast cell-derived histamine induces keratinocytes to produce various inflammatory cytokines or chemokines such as interleukin-6 or interleukin-8 (Kohda et al., 2002Kohda F. Koga T. Uchi H. Urabe K. Furue M. Histamine-induced IL-6 and IL-8 production are differentially modulated by IFN-γ and IL-4 in human keratinocytes.J Dermatol Sci. 2002; 28: 34-41Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). It is thus plausible that histamine may also induce NGF production in keratinocytes. A previous study reported that histamine in vitro enhanced NGF production by murine transformed keratinocytes PAM212 and 3T3 fibroblasts (Matsuda et al., 1998Matsuda H. Koyama H. Sato H. et al.Role of nerve growth factor in cutaneous wound healing: Accelerating effects in normal and healing-impaired diabetic mice.J Exp Med. 1998; 187: 297-306Crossref PubMed Scopus (195) Google Scholar). It is unknown, however, what level of NGF production, i.e. translational or transcriptional, histamine may potentiate. In addition, previous studies did not show intracellular signaling events responsible for the NGF potentiation by histamine. Histamine binds to cell surface receptors coupling to guanine nucleotide-binding protein (G-protein) and induces various intracellular signaling pathways. Four isotypes of histamine receptors have been identified: H1, H2, H3, and H4 receptors (Oda et al., 2000Oda T. Morikawa N. Saito Y. Masuho Y. Matsumoto S. Molecular cloning and characterization of a novel type of histamine receptor preferentially expressed in leukocytes.J Biol Chem. 2000; 275: 36781-36786Crossref PubMed Scopus (567) Google Scholar). H1 receptor is linked to the activation of phospholipase C (PLC), which generates diacylglycerol activating protein kinase C (PKC) (Megson et al., 2001Megson A.C. Walker E.M. Hill S.J. Role of protein kinase Cα in signaling from the histamine H1 receptor to the nucleus.Mol Pharmacol. 2001; 59: 1012-1021Crossref PubMed Scopus (40) Google Scholar). Stimulation of H2 receptor mostly activates adenylate cyclase, which generates a cAMP signal that activates protein kinase A; however, H2 receptor is also linked to PLC in certain cell types (Del Valle and Ganz, 1997Del Valle J. Ganz I. Novel insights into histamine H2 receptor biology.Am J Physiol. 1997; 273: G987-G996PubMed Google Scholar). H3 and H4 receptors are coupled to the inhibition of adenylate cyclase (Coge et al., 2001Coge F. Guenin S.-P. Rique H. Boutin J.A. Galizzi J.-P. Structure and expression of the human histamine H4-receptor gene.Biochem Biophys Res Commun. 2001; 284: 301-309Crossref PubMed Scopus (137) Google Scholar). These histamine-receptor-mediated signals regulate cytokine or chemokine gene expression in target cells (Kanda and Watanabe, 2002Kanda N. Watanabe S. Histamine inhibits the production of interferon-induced protein of 10 kDa in human squamous cell carcinoma and melanoma.J Invest Dermatol. 2002; 119: 1411-1419Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). In this study, we examined the effects of histamine on NGF production in cultured human keratinocytes. We found that histamine potentiates NGF production in keratinocytes via H1 receptors. We further examined the precise mechanism for this effect focusing on the histamine-induced intracellular signaling pathways. Histamine dihydrochloride, cimetidine, pyrilamine, and calphostin C were obtained from Sigma (St Louis, MO). Thioperamide maleate was from Biomol Research Laboratories (Plymouth Meeting, PA). 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2, 5-dione (U73122), N-[[2-(p-bromocinnamylamino)ethyl]-5-isoquinol-inesulfonamide (H-89), and 4-(3-chloroanilino)-6, 7-dimethoxy-quinazoline (AG1478) were obtained from Calbiochem (La Jolla, CA). Recombinant human tumor necrosis factor α was from R&D Systems (Minneapolis, MN). Antibodies used in the electrophoretic mobility shift assay (EMSA) were from Santa Cruz Biotechnology (Santa Cruz, CA). Human neonatal foreskin keratinocytes were cultured in serum-free KGM medium (Clonetics, Walkersville, MD) consisting of basal medium MCDB153 supplemented with 0.5 μg per ml hydrocortisone, 5 ng per ml epidermal growth factor, 5 μg per ml insulin, and 0.5% bovine pituitary extract. The cells in third passage were used. Keratinocytes (2×104 per well) were seeded in triplicate into 24-well plates in 1 ml KGM and adhered overnight; the medium was then changed to basal KBM depleted of hormones, growth factors, and bovine pituitary xtract, and the cells were incubated for 24 h. The medium was removed and the cells were incubated with histamine in KBM for 24 h. The supernatants were assayed for NGF by ELISA (Promega, Madison, WI). Keratinocytes were incubated as above for indicated periods, and cellular mRNA was extracted using an mRNA purification kit (Pharmacia, Uppsala, Sweden) according to the manufacturer's instruction. cDNA was made from mRNA samples as described previously (Tjandrawinata et al., 1997Tjandrawinata R.R. Dahiya R. Hughes-Fulford M. Induction of cyclo-oxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells.Br J Cancer. 1997; 75: 1111-1118Crossref PubMed Scopus (209) Google Scholar). Primers for amplification and the sizes of respective PCR products were as follows: NGF, 5′-CACACTGAG-GTGCATAGCGT-3′ and 5′-TGATGACCGCTTGCTCCTGT-3′ for 352 bp; c-Fos, 5′-GGCTTCAACGCAGACTACGAGG-3′ and 5′-CTCCT-GTCATGGTCTTCACAACG-3′ for 340 bp; c-Jun, 5′-TTCACCTTCTC-TCTAACTGC-3′ and 5′-TCACTCACTGAGCGCTCTTC-3′ for 580 bp; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-GCAGGGGG-GAGCCAAAAGGG-3′ and 5′-TGCCAGCCCCAGCGTCAAAG-3′ for 566 bp (Lane et al., 1998Lane S.J. Adcock I.M. Richards D. Hawrylowicz C. Barnes P.J. Lee T.H. Corticosteroid-resistant bronchial asthma is associated with increased c-fos expression in monocytes and T lymphocytes.J Clin Invest. 1998; 102: 2156-2164Crossref PubMed Scopus (118) Google Scholar;Risse-Hackl et al., 1998Risse-Hackl G. Adamkiewicz J. Wimmel A. Schuermann M. Transition from SCLC to NSCLC phenotype is accompanied by an increased TRE-binding activity and recruitment of specific AP-1 proteins.Oncogene. 1998; 16: 3057-3068Crossref PubMed Scopus (51) Google Scholar;Iannone et al., 2002Iannone F. de Bari C. Dell'Accio F. Covelli M. Patella V. Lo Bianco G. Lapadula G. Increased expression of nerve growth factor (NGF) and high affinity NGF receptor (p140 TrkA) in human osteoarthritic chondrocytes.Rheumatology. 2002; 41: 1413-1418Crossref PubMed Scopus (109) Google Scholar;Kanda and Watanabe, 2002Kanda N. Watanabe S. Histamine inhibits the production of interferon-induced protein of 10 kDa in human squamous cell carcinoma and melanoma.J Invest Dermatol. 2002; 119: 1411-1419Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). PCR involved one denaturing cycle of 95°C for 3 min, 30 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s, and a final extension at 72°C for 3 min. The PCR products were analyzed by electrophoresis on 2.5% agarose gels and stained with ethidium bromide, viewed by ultraviolet light. Densitometric analysis was performed by scanning the bands into Photoshop and performing densitometry with NIH Image Software. The firefly luciferase reporter plasmids driven by human NGF promoter (–600/+250 bp relative to the transcriptional start site) were constructed by PCR and insertion into pGL3 basic vector (Promega) as described previously (Cartwright et al., 1992Cartwright M. Martin S. D'Mello S. Heinrich G. The human nerve growth factor gene: Structure of the promoter region and expression in L929 fibroblasts.Mol Brain Res. 1992; 15: 67-75Crossref PubMed Scopus (35) Google Scholar), and are denoted pNGF luc. Site-specific mutation of the promoter was created by multiple rounds of PCR using primers with altered bases as described previously (Li and Kolattukudy, 1994Li Y.-S. Kolattukudy P.E. Functional role of the cis-acting elements in human monocyte chemotactic protein-1 gene in the regulation of its expression by phorbol ester in human glioblastoma cells.Mol Cell Biochem. 1994; 141: 121-128Crossref PubMed Scopus (20) Google Scholar). p4×TPA response element 1 (TRE1)-TATA-luc and p4×TRE2-TATA-luc were constructed by inserting four copies of TRE1 (5′-GCGTGTGACTCAGGAGT-3′, consensus sequence underlined) or TRE2 (5′-AGCGGTGAGTCA-GGCTG-3′) from human NGF promoter in front of the TATA box upstream of firefly luciferase reporter as described previously (Kanda and Watanabe, in press). Transient transfections were performed with Effectene (Qiagen, Tokyo, Japan) as described previously (Zellmer et al., 2001Zellmer S. Gaunitz F. Salvetter J. Surovoy A. Ressig D. Gebhardt R. Long-term expression of foreign genes in normal human epidermal keratinocytes after transfection with lipid/DNA complexes.Histochem Cell Biol. 2001; 115: 41-47Crossref PubMed Scopus (25) Google Scholar). The efficiency of transfection into keratinocytes by this method was 28.5%±3.1% (mean±SEM, n=9) as determined by flow cytometry using β-galactosidase vector. Effectene produced a higher transfection efficiency than Lipofectin (8.1%±1.2%), Lipofectamine (9.5%±1.1%), or polybrene-dimethylsulfoxide (8.5%±0.9%), and thus was adopted in this study. Keratinocytes were plated in 10 cm dishes and grown to about 60% confluence. Twenty-four hours before the transfection, the medium was changed to KBM. Keratinocytes were incubated for 6 h with 5 μg of pNGF luc or p4×TRE1-TATA-luc or p4×TRE2-TATA-luc and 1 μg of Rous sarcoma virus β-galactosidase vector (pRSV-β-gal), premixed with enhancer, transfection buffer, and Effectene. The transfected cells were washed and incubated in KBM for 18 h and then treated with histamine. After 6 h, cell extracts were prepared and luciferase activities were quantified using the luciferase assay system (Promega). The same cell extracts were assayed for β-galactosidase activity using the chemiluminescent Galacto-Light kit (Tropix, Bedford, MA). All readings were taken using a Lumat 9501 luminometer (Berthold, Wildbach, Germany). The results obtained in each transfection were normalized for β-galactosidase activity and expressed as relative luciferase activity. EMSA was performed as described previously (Cartwright et al., 1992Cartwright M. Martin S. D'Mello S. Heinrich G. The human nerve growth factor gene: Structure of the promoter region and expression in L929 fibroblasts.Mol Brain Res. 1992; 15: 67-75Crossref PubMed Scopus (35) Google Scholar). The probes used were 32P-labeled annealed double-stranded DNA containing TRE1 or TRE2 from human NGF promoter. The sequence of the TRE1 probe is 5′-ATTTGGAGCGTGTGACTCAGGAGTACGGGAG-3′ (consensus sequence underlined) and that of the TRE2 probe is 5′-GGAGCGCAGCGGTGAGTCAGGCTGCCCCGAG-3′. For gel shift assays, 2–5 μg of nuclear protein extracts were incubated at room temperature for 5 min with a mixture containing 6 mM HEPES (pH 7.9), 0.4 mM ethylenediamine tetraacetic acid (EDTA), 125 mM KCl, 10% glycerol, 0.05 μg per μL poly dI-dC, 1 mM dithiothreitol, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 10 mM NaF, 50 μg per ml aprotinin, 50 μg per ml leupeptin. One nanogram of labeled probe was added and the reactions were incubated for another 20 min. In antibody supershift experiments, the nuclear extracts were preincubated with various antibodies for 30 min before the addition of probe. Reactions were then fractionated on a nondenaturing 5% polyacrylamide gel, and visualized with phosphorimager (Molecular Dynamics, Sunnyvale, CA). The dual threonine/tyrosine phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) was assessed by western blot as described previously (Gee et al., 2002Gee K. Lim W. Ma W. Nandan D. Diaz-Mitoma F. Kozlowski M. Kumar A. Differential regulation of CD44 expression by lipopolysaccharide (LPS) and TNF-α in human monocytic cells: Distinct involvement of c-jun N-terminal kinase in LPS-induced CD44 expression.J Immunol. 2002; 169: 5660-5672Crossref PubMed Scopus (55) Google Scholar). Keratinocytes were incubated with 10–6 M histamine or 10 ng per mL tumor necrosis factor α for 10 min. The cells were lyzed with lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 100 mM NaF, 100 mM sodium orthovanadate, and 1 mM ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA, pH 7.7)) and centrifuged for 20 min at 14,000×g at 4°C. The supernatant proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membrane was blocked and incubated with antiphospho-ERK1/2 or antiphospho-JNK antibodies (Santa Cruz Biotechnology) or antiphospho-p38 antibody (New England Biolabs, Mississauga, Ontario, Canada), followed by peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA). The blots were developed with an enhanced chemiluminescence kit (Amersham, Arlington Heights, IL). The blots were stripped and reprobed with rabbit polyclonal antibodies specific for each of unphosphorylated ERK1/2, JNK, and p38 MAPK (Santa Cruz Biotechnology), as described above. PKC activity was analyzed as described previously (Migliaccio et al., 1998Migliaccio S. Washburn T.F. Fillo S. Rivera H. Teti A. Korach K.S. Wetsel W.C. Modulation of estrogen receptor levels in mouse uterus by protein kinase C isoenzymes.Endocrinology. 1998; 139: 4598-4606Crossref PubMed Scopus (16) Google Scholar). Keratinocytes were incubated with 10–6 M histamine for 10 min. The cells were harvested and homogenized in a buffer containing 20 mM Tris-HCl (pH 7.4), 2 mM EDTA, 10 mM EGTA, 250 mM sucrose, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 0.24 mM leupeptin. Nuclei and debris were removed from the samples by centrifugation (500×g, 5 min) and this post nuclear fraction was then centrifuged (105,000×g, 90 min). The supernatant was saved as the cytosolic fraction. The pellet was homogenized in the same buffer, except for containing 0.1% Triton X-100. The samples were mixed continuously for 1 h at 4°C and then centrifuged as described above. This supernatant was saved as the membrane fraction. The cytosolic or membrane fractions were loaded onto columns packed with diethylaminoethyl-sephacel resin and were washed extensively with a solution of 20 mM Tris-HCl (pH 7.4), 20 mM NaCl, 0.5 mM EDTA, 0.5 mM EGTA, and 10 mM 2-mercaptoethanol. PKC was eluted with the same buffer except that the NaCl concentration was increased to 100 mM. Aliquots of purified PKC from cytosolic or membrane fractions were assayed for protein contents using the Bio-Rad protein determination assay. PKC activity was assessed using a reaction mixture containing 20 mM Tris-HCl (pH 7.4), 8 mM MgCl2, 16 μM ATP, [γ-32P]ATP, and 200 μg per ml histone III-S at 30°C for 3 min. Activity was determined in the presence or absence of 100 μM CaCl2, 8 μg per ml phosphatidyl serine, and 2 μg per ml diolein. Samples were filtered with Whatman GF/C filters, and 32P incorporation into lysine-rich histone was quantified by liquid scintillation. Keratinocytes (2×104 per well) were seeded into 24-well plates in KGM, adhered overnight, and incubated with KBM for 24 h. The medium was replaced with fresh KBM, and the cells were incubated for 24 h. The supernatant histamine amount was measured by ELISA (Cosmo Bio, Tokyo, Japan). Keratinocytes constitutively secreted a low amount of NGF (21.5±2.8 pg per ml, mean±SEM, n=5), and the secretion was concentration-dependently increased by histamine; the stimulatory effect of histamine was manifested at 10–7 M and was maximized at 10–6 M, which increased the secretion 4.5-fold of controls (Figure 1). The stimulatory effect of histamine was not suppressed by pertussis toxin, indicating that the effect may be mediated via pertussis-toxin-resistant G-proteins. H1 receptor antagonist pyrilamine blocked the stimulatory effect of histamine (Figure 1) whereas H2 antagonist cimetidine (Figure 1) or H3 and H4 antagonist thioperamide (data not shown) did not. These results suggest that H1 receptor but not H2, H3, or H4 receptors may mediate histamine-induced enhancement of NGF secretion in keratinocytes. As 10–6 M of histamine was optimal for NGF induction, this concentration was used in the following experiments. As examined by ELISA, constitutive histamine secretion by keratinocytes was 9.75±0.83 nM (mean±SEM, n=5), which was less than the threshold (100 nM) for NGF induction (Figure 1). Pyrilamine, cimetidine, and thioperamide did not reduce the constitutive NGF secretion in the absence of exogenous histamine (data not shown). This suggests that endogenous histamine secreted by keratinocytes may not contribute to NGF secretion. We next examined if histamine may enhance NGF mRNA expression in keratinocytes. At 3 h of incubation, histamine increased the NGF mRNA level 6.1-fold of controls, which was reversed by pyrilamine but not by cimetidine or pertussis toxin (Figure 2). Thus histamine increased NGF production via H1 receptors at pretranslational level. We then examined if histamine may enhance NGF promoter activity in keratinocytes. It is reported that human NGF gene contains two TREs, TRE1 (–62/–56 bp) and TRE2 (+35/+41 bp), and these may act as enhancer elements for NGF transcription (Cartwright et al., 1992Cartwright M. Martin S. D'Mello S. Heinrich G. The human nerve growth factor gene: Structure of the promoter region and expression in L929 fibroblasts.Mol Brain Res. 1992; 15: 67-75Crossref PubMed Scopus (35) Google Scholar) (Figure 3a). We transiently transfected NGF promoter (–600/+250 bp) linked to luciferase reporter into human keratinocytes. Histamine treatment increased NGF promoter activity 5.1-fold of controls, and the effect was blocked by pyrilamine (Figure 3b). The mutation of TRE1 or TRE2 each reduced the basal promoter activity and histamine-induced enhancement of the activity. The mutation of both TRE completely abrogated the basal and histamine-induced promoter activities. These results suggest that both TRE1 and TRE2 may be required for basal and histamine-induced NGF promoter activities. We then analyzed if histamine may enhance transcriptional activities dependent on TRE1 or TRE2. Keratinocytes were transiently transfected with luciferase reporter linked to four repeats of TRE1 or TRE2 in front of the TATA box. Histamine increased the TRE1- or TRE2-dependent transcriptional activities, and the effects were reversed by pyrilamine (Figure 4). It is known that TRE is mostly bound by transcription factor activator protein 1 (AP-1) (Allegretto et al., 1990Allegretto E.A. Smeal T. Angel P. Spiegelman B.M. Karin M. DNA-binding activity of Jun is increased through its interaction with Fos.J Cell Biochem. 1990; 42: 193-206Crossref PubMed Scopus (33) Google Scholar). We then examined if histamine may enhance DNA binding of AP-1 at TRE on the NGF promoter. At 1 h of incubation, histamine increased the amount of DNA-protein complex with TRE1 probe, and the effect was reversed by pyrilamine (Figure 5, lane 4). This suggests that histamine may increase transcription factor binding to TRE in keratinocytes via H1 receptors. In unstimulated keratinocytes, anti-c-Jun did but anti-c-Fos antibody did not supershift the complex (Figure 5, lanes 5, 6), whereas in histamine-stimulated keratinocytes both antibodies supershifted the complex (Figure 5, lanes 7, 8). Antibodies against the other Fos family (FosB, Fra-1, Fra-2) or Jun family (JunB, JunD) proteins did not supershift the complexes by nuclear extracts from unstimulated or histamine-stimulated keratinocytes (data not shown). These results suggest that histamine may shift the AP-1 composition from c-Jun homodimers to c-Fos/c-Jun heterodimers at TRE1. Similar results were obtained by using the TRE2 probe (data not shown). We then examined if histamine may enhance c-Fos or c-Jun expression in keratinocytes. Histamine rapidly and transiently induced c-Fos mRNA. In unstimulated cells, c-Fos mRNA was undetectable; however, in histamine-stimulated cells, the expression was induced at 15 min, maximized at 30 min, reduced at 1 h, and disappeared at 3 h (Figure 6). c-Jun mRNA expression was constitutively detectable and was not enhanced by histamine. The results indicate that histamine-induced NGF production may be mainly attributable to the induction of c-Fos expression. Histamine is known to induce various intracellular signaling pathways: the PLC-PKC pathway leading to the activation of ERK (Megson et al., 2001Megson A.C" @default.
• W2027185670 created "2016-06-24" @default.
• W2027185670 creator A5015522451 @default.
• W2027185670 creator A5072424866 @default.
• W2027185670 date "2003-09-01" @default.
• W2027185670 modified "2023-10-18" @default.
• W2027185670 title "Histamine Enhances the Production of Nerve Growth Factor in Human Keratinocytes" @default.
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