Title: Histamine Enhances the Production of Granulocyte-Macrophage Colony-Stimulating Factor via Protein Kinase Cα and Extracellular Signal-Regulated Kinase in Human Keratinocytes
Abstract: The production of granulocyte-macrophage colony-stimulating factor (GM-CSF) in keratinocytes is related to the chronicity of atopic dermatitis. Mast cell-derived histamine contributes to the cross-talk between mast cells and keratinocytes. We examined the effects of histamine on GM-CSF production in human keratinocytes. Histamine increased GM-CSF secretion, mRNA stability and promoter activity. Activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) elements on the promoter were responsible for the activation by histamine. Histamine enhanced transcriptional activity and DNA binding of AP-1 and NF-κB. Histamine shifted AP-1 composition form c-Jun homodimers to c-Fos/c-Jun heterodimers, and transiently expressed c-Fos protein. Histamine rapidly induced the phosphorylation and degradation of inhibitory κB. Histamine induced membrane translocation of protein kinase Cα. Histamine-induced GM-CSF production was completely abolished by H1 antagonist pyrilamine and conventional protein kinase C inhibitor Gö6976, and partially suppressed by PD98059 which inhibits the activation of extracellular signal-regulated kinase. Gö6976 and PD98059 suppressed histamine-induced c-Fos expression and AP-1 activation. Gö6976 and PD98059 suppressed histamine-induced enhancement of NF-κB transcriptional activity. Histamine-induced phosphorylation and degradation of inhibitory κB was suppressed by Gö6976, but not by PD98059. These results suggest that histamine may enhance GM-CSF production at transcriptional and posttranscriptional levels via H1 receptor, protein kinase Cα and extracellular signal-regulated kinase. The production of granulocyte-macrophage colony-stimulating factor (GM-CSF) in keratinocytes is related to the chronicity of atopic dermatitis. Mast cell-derived histamine contributes to the cross-talk between mast cells and keratinocytes. We examined the effects of histamine on GM-CSF production in human keratinocytes. Histamine increased GM-CSF secretion, mRNA stability and promoter activity. Activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) elements on the promoter were responsible for the activation by histamine. Histamine enhanced transcriptional activity and DNA binding of AP-1 and NF-κB. Histamine shifted AP-1 composition form c-Jun homodimers to c-Fos/c-Jun heterodimers, and transiently expressed c-Fos protein. Histamine rapidly induced the phosphorylation and degradation of inhibitory κB. Histamine induced membrane translocation of protein kinase Cα. Histamine-induced GM-CSF production was completely abolished by H1 antagonist pyrilamine and conventional protein kinase C inhibitor Gö6976, and partially suppressed by PD98059 which inhibits the activation of extracellular signal-regulated kinase. Gö6976 and PD98059 suppressed histamine-induced c-Fos expression and AP-1 activation. Gö6976 and PD98059 suppressed histamine-induced enhancement of NF-κB transcriptional activity. Histamine-induced phosphorylation and degradation of inhibitory κB was suppressed by Gö6976, but not by PD98059. These results suggest that histamine may enhance GM-CSF production at transcriptional and posttranscriptional levels via H1 receptor, protein kinase Cα and extracellular signal-regulated kinase. analysis of variance activator protein-1 AU-rich element electrophoretic mobility shift assay extracellular signal-regulated kinase glyceraldehyde-3-phosphate dehydrogenase granulocyte-macrophage colony-stimulating factor inhibitory κB mitogen-activated protein kinase MAPK kinase nuclear factor of activated T cells nuclear factor-κB protein kinase C phospholipase C Granulocyte macrophage colony-stimulating factor (GM-CSF) plays an important role in the development and perpetuation of atopic dermatitis (Pastore et al., 1997Pastore S. Fanales-Belasio E. Albanesi C. Chinni L.M. Giannetti A. Girolomoni G. Granulocyte macrophage colony-stimulating factor is overproduced by keratinocytes in atopic dermatitis. Implications for sustained dendritic cell activation in the skin.J Clin Invest. 1997; 99: 3009-3017Crossref PubMed Scopus (170) Google Scholar). In the skin lesions with atopic dermatitis, GM-CSF is overproduced by keratinocytes (Pastore et al., 1997Pastore S. Fanales-Belasio E. Albanesi C. Chinni L.M. Giannetti A. Girolomoni G. Granulocyte macrophage colony-stimulating factor is overproduced by keratinocytes in atopic dermatitis. Implications for sustained dendritic cell activation in the skin.J Clin Invest. 1997; 99: 3009-3017Crossref PubMed Scopus (170) Google Scholar). GM-CSF promotes the migration, survival, and antigen-presenting capacity of epidermal Langerhans cells and dermal dendritic cells, and supports the differentiation into dendritic cells from peripheral blood precursors (Pastore et al., 1997Pastore S. Fanales-Belasio E. Albanesi C. Chinni L.M. Giannetti A. Girolomoni G. Granulocyte macrophage colony-stimulating factor is overproduced by keratinocytes in atopic dermatitis. Implications for sustained dendritic cell activation in the skin.J Clin Invest. 1997; 99: 3009-3017Crossref PubMed Scopus (170) Google Scholar). GM-CSF also promotes the survival of eosinophils and monocyte/macrophages, and enhances the proliferation of keratinocytes, contributing the perpetuation of inflammation and epidermal hyperplasia in the chronic lesions with atopic dermatitis (Braunstein et al., 1994Braunstein S. Kaplan G. Gottlieb A.B. GM-CSF activates regenerative epidermal growth and stimulates keratinocyte proliferation in human skin in vivo.J Invest Dermatol. 1994; 103: 601-604Crossref PubMed Scopus (92) Google Scholar;Bratton et al., 1995Bratton D.L. Hamid Q. Boguniewicz M. Doherty D.E. Kailey J.M. Leung D.Y.M. Granulocyte macrophage colony-stimulating factor contributes to enhanced monocyte survival in chronic atopic dermatitis.J Clin Invest. 1995; 95: 211-218Crossref PubMed Google Scholar). In the skin lesions with atopic dermatitis, mast cells are increased in the dermis and may be involved in the development of inflammation (Damsgaard et al., 1997Damsgaard T.E. Olesen A.B. Sorensen F.B. Thestrup-Pedersen K. Schiotz P.O. Mast cells and atopic dermatitis. Stereological quantification of mast cells in atopic dermatitis and normal human skin.Arch Dermatol Res. 1997; 289: 256-260Crossref PubMed Scopus (68) Google Scholar;Ackermann and Harvima, 1998Ackermann L. Harvima I.T. Mast cells of psoriatic and atopic dermatitis skin are positive for TNF-α and their degranulation is associated with expression of ICAM-1 in the epidermis.Arch Dermatol Res. 1998; 290: 353-359Crossref PubMed Scopus (132) Google Scholar). Activated mast cells release histamine, and local histamine concentration is also increased in the lesions with atopic dermatitis (DeMora et al., 1996DeMora F. Garcia G. Puigdemont A. Arboix M. Ferrer L. Skin mast cell releasability in dogs with atopic dermatitis.Inflamm Res. 1996; 45: 424-427Crossref PubMed Scopus (28) Google Scholar). The mast cell-derived histamine contributes to the cross-talk between mast cells and keratinocytes. Histamine acts on keratinocytes and promotes their production of adhesion molecules like intercellular adhesion molecule-1 or of proinflammatory cytokines or chemokines like interleukin (IL)-6, IL-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), or nerve growth factor (Kanda and Watanabe, 2003bKanda N. Watanabe S. Histamine enhances the production of nerve growth factor in human keratinocytes.J Invest Dermatol. 2003; 121: 570-577Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). It is thus plausible that histamine may also induce GM-CSF production in keratinocytes. Previous study reported that histamine in vitro enhanced GM-CSF production by human conjunctival (Weimer et al., 1998Weimer L.K. Gamache D.A. Yanni J.M. Histamine-stimulated cytokine secretion from human conjunctival epithelial cells: Inhibition by the histamine H1 antagonist emedastine.Int Arch Allergy Immunol. 1998; 115: 288-293Crossref PubMed Scopus (59) Google Scholar), nasal (Terada et al., 1999Terada N. Maesako H.K.-I. Hiruma K. Hohki G. Suzuki K. Diesel exhaust particulates upregulate histamine receptor mRNA and increase histamine-induced IL-8 and GM-CSF production in nasal epithelial cells and endothelial cells.Clin Exp Allergy. 1999; 29: 52-59Crossref PubMed Scopus (85) Google Scholar), bronchial (Takizawa et al., 1995Takizawa H. Ohtoshi T. Kikutani T. Histamine activates bronchial epithelial cells to release inflammatory cytokines in vitro.Int Arch Allergy Immunol. 1995; 108: 260-267Crossref PubMed Scopus (49) Google Scholar), or tracheal epithelial cells (Churchill et al., 1992Churchill L. Friedman B. Schleimer R.P. Proud D. Production of granulocyte-macrophage colony-stimulating factor by cultured human tracheal epithelial cells.Immunology. 1992; 75: 189-195PubMed Google Scholar). But it has not been examined if histamine may enhance GM-CSF production by human epidermal keratinocytes. Besides it is unknown which level of GM-CSF production, i.e., translational or transcriptional, histamine may potentiate. In addition, previous studies did not show intracellular signaling events responsible for the GM-CSF induction by histamine. Histamine binds to cell surface receptors coupling to guanine nucleotide-binding 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 (568) 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 cAMP signal; 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: 1411-1419Crossref PubMed Scopus (25) Google Scholar). We have recently found that histamine binds to H1 receptor on human keratinocytes, and induces PLC/PKC/mitogen-activated protein kinase (MAPK) kinase 1 (MEK1)/extracellular signal-regulated kinase (ERK) signaling cascade (Kanda and Watanabe, 2003bKanda N. Watanabe S. Histamine enhances the production of nerve growth factor in human keratinocytes.J Invest Dermatol. 2003; 121: 570-577Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In this study, we examined the effects of histamine on GM-CSF production in cultured human keratinocytes. We found that histamine enhanced GM-CSF production in keratinocytes via H1 receptors at both transcriptional and post-transcriptional levels. We further examined the precise mechanism for these effects focusing on the histamine-induced intracellular signaling pathways. Keratinocytes constitutively secreted a low amount of GM-CSF (mean±SEM 15.5±1.8 pg per mL, n=5), and the secretion was concentration dependently increased by histamine; the stimulatory effect of histamine was manifested at 0.1 μM, and was maximized at 1 μM which increased the secretion 6.1-fold of controls Figure 1. H1 receptor antagonist pyrilamine blocked the stimulatory effect of histamine while H2 antagonist cimetidine or H3 and H4 antagonist thioperamide did not. These results suggest that H1 receptor but not H2, H3, or H4 receptors may mediate histamine-induced enhancement of GM-CSF secretion in keratinocytes. Since 1 μM of histamine was optimal for GM-CSF induction, this concentration will be used in the following experiments. As examined by ELISAs, constitutive histamine secretion by keratinocytes was mean±SEM 9.55±0.91 nM (n=5) which was less than the threshold (100 nM) for GM-CSF induction Figure 1. Pyrilamine, cimetidine, and thioperamide did not reduce the constitutive GM-CSF secretion in the absence of exogenous histamine (data not shown). These results suggest that endogenous histamine secreted by keratinocytes may not contribute to GM-CSF secretion. We next examined if histamine may increase steady-state GM-CSF mRNA level in keratinocytes. At 3 h of incubation, histamine increased GM-CSF mRNA level 6.2-fold of controls, which was blocked by pyrilamine but not by cimetidine or thioperamide Figure 2. Thus, histamine increased GM-CSF production via H1 receptors at pretranslational level. We then examined if histamine may enhance GM-CSF mRNA stability. Keratinocytes were treated with actinomycin D after incubation with or without histamine for 3 h, and the decay of GM-CSF mRNA was chased Figure 3. The estimated half-life of GM-CSF mRNA in histamine-treated cells was mean±SEM 42.3±2.5 min (n=4), which was significantly higher than that in controls (23.1±2.2 min, p<0.05 by paired t test). The histamine-induced increase of the half-life was blocked by pyrilamine but not by cimetidine or thioperamide. These results indicate that histamine may stabilize GM-CSF mRNA via H1 receptors. We then examined if histamine may enhance GM-CSF transcription in keratinocytes. It is reported that human GM-CSF gene contained cis-elements homologous to NF-κB, AP-1, and nuclear factor of activated T cells (NF-AT) sites Figure 4a, and these may act as enhancer elements for GM-CSF transcription (Tsuboi et al., 1994Tsuboi A. Muramatsu M. Tsutsumi A. Arai K. Arai N. Calcineurin activates transcription from the GM-CSF promoter in synergy with either protein kinase C or NF-κB/AP-1 in T cells.Biochem Biophys Res Commun. 1994; 199: 1064-1072Crossref PubMed Scopus (46) Google Scholar;Jenkins et al., 1995Jenkins F. Cockerill P.N. Bohmann D. Shannon F. Multiple signals are required for function of the human granulocyte-macrophage colony-stimulating factor gene promoter in T cells.J Immunol. 1995; 155: 1240-1251PubMed Google Scholar;Pastore et al., 2000Pastore S. Giustizieri M.L. Mascia F. Giannetti A. Kaushansky K. Girolomoni G. Dysregulated activation of activator protein 1 in keratinocytes of atopic dermatitis patients with enhanced expression of granulocytemacrophage-colony stimulating factor.J Invest Dermatol. 2000; 115: 1134-1143Crossref PubMed Scopus (41) Google Scholar). We transiently transfected GM-CSF promoter (-620/+37 bp) linked to luciferase reporter into human keratinocytes. Histamine treatment increased wild-type GM-CSF promoter activity 3.1-fold of controls, and the effect was blocked by pyrilamine (Figure 4b, first three columns). Thus histamine appeared to enhance GM-CSF transcription in addition to mRNA stabilization. The mutation of AP-1-like element reduced the basal promoter activity and completely abrogated histamine-induced enhancement of the promoter activity (Figure 4b, third three columns). The mutation of NF-κB-like element reduced basal promoter activity and the fold-increase by histamine; however, the stimulatory effect of histamine still remained (Figure 4b, second three columns). The mutation of both NF-κB- and AP-1-like elements completely abrogated the basal and histamine-induced promoter activities (Figure 4b, fourth three columns). On the other hand, the mutation of NF-AT-like element did not affect either basal or histamine-induced promoter activities (Figure 4b, last three columns), indicating that this element may be dispensable for basal and histamine-induced GM-CSF transcription. These results suggest that basal and histamine-induced GM-CSF transcription may be dependent on AP-1- and NF-κB-like elements, and the contribution of the former may be higher. We then analyzed if histamine may enhance transactivation capacities through AP-1- or NF-κB-like elements. Keratinocytes were transiently transfected with luciferase reporter linked to four repeats of NF-κB- or AP-1-like elements in front of TATA box. Histamine potently enhanced transcription through AP-1-like element (4.5-fold of controls), and modestly increased that through NF-κB-like element (2.3-fold) Figure 5. These effects of histamine were counteracted by pyrilamine, indicating the involvement of H1 receptors. We then examined if histamine may enhance DNA binding of transcription factors at AP-1-like site on GM-CSF promoter in parallel with the transactivation capacity. At 1 h of incubation, histamine increased the amount of DNA–protein complex with AP-1-containing probe, and the effect was counteracted by pyrilamine (Figure 6a, lanes 2–4). These results suggest that histamine may promote transcription factor binding to AP-1-like site via H1 receptors. In unstimulated keratinocytes, anti-c-Jun did but anti-c-Fos antibody did not supershift the complex (Figure 6a, lanes 5, 6) whereas in histamine-stimulated keratinocytes, both antibodies supershifted the complex (Figure 6a, lanes 7 and 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. We then examined if histamine may increase c-Fos or c-Jun protein levels in keratinocytes. Histamine rapidly and transiently induced c-Fos protein; in unstimulated cells, c-Fos was undetectable, however, after stimulation with histamine, c-Fos was induced at 30 min, then reduced at 1 h, and disappeared at 90 min Figure 6b. c-Jun protein was constitutively detectable and was not increased by histamine. The results indicate that histamine-induced activation of AP-1 may be mainly attributable to the rapid and transient induction of c-Fos. We next examined if histamine may enhance DNA binding of NF-κB as well as AP-1. At 1 h of incubation, histamine increased the amount of DNA–protein complex with NF-κB-containing probe, and the effect was blocked by pyrilamine (Figure 7a, lanes 2–4). Supershift assays showed that the NF-κB complex was composed of p50 and p65 both in unstimulated and histamine-stimulated cells (Figure 7a, lanes 5–8), whereas antibody against c-Rel did not supershift the complex (data not shown). Thus, histamine enhanced DNA binding of NF-κB p50/p65 heterodimers. In resting state, NF-κB p50/p65 dimers are sequestered in cytoplasm and are complexed with inhibitory protein, IκBα. Upon stimulation, IκBα is phosphorylated and released from the complex, and ubiquitinated and degradated by proteasome, which releases NF-κB to undergo nuclear translocation and binding to consensus elements on target genes (Lallena et al., 1999Lallena M.-J. Diaz-Meco M.T. Bren G. Paya C.V. Moscat J. Activation of IκB kinase β by protein kinase C isoforms.Mol Cell Biol. 1999; 19: 2180-2188Crossref PubMed Google Scholar). We thus analyzed if histamine may induce the phosphorylation and degradation of IκBα, necessary for DNA binding of NF-κB. At 10 min of histamine incubation, phosphorylation of IκBα was detected (Figure 7b, upper panel, lane 2), which was also confirmed by slower migrating form of IκBα (Figure 7b, lower panel, lane 2), and at 30 min, the amount of IκBα was reduced (Figure 7b, lower panel, lane 3), indicating histamine-induced degradation of IκBα. At 60 min, IκBα returned to the basal level (Figure 7b, lower panel, lane 4), indicating the re-synthesis of IκBα. These results indicate that histamine may enhance DNA binding of NF-κB p50/p65 heterodimers via phosphorylation and degradation of IκBα. We have recently found that histamine binds to H1 receptor on keratinocytes, and induces membrane translocation of PKC activity dependently on PLC. The histamine-induced activation of PKC also led to the activation of downstream kinases, MEK1 and ERK (Kanda and Watanabe, 2003bKanda N. Watanabe S. Histamine enhances the production of nerve growth factor in human keratinocytes.J Invest Dermatol. 2003; 121: 570-577Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). PKC isoforms are classified into Ca2+ and diacylglycerol-dependent conventional isoforms (PKCα, βI, βII, γ), Ca2+-independent and diacylglycerol-dependent novel isoforms (PKCδ, ε, μ, θ, and η), and Ca2+ and diacylglycerol-independent atypical isoforms (PKCζ, λ/τ) (Reynolds et al., 1994Reynolds N.J. Baldassare J.J. Henderson P.A. Translocation and downregulation of protein kinase C isoenzymes-α and -ε by phorbol ester and bryostatin-1 in human keratinocytes and fibroblasts.J Invest Dermatol. 1994; 103: 364-369Crossref PubMed Scopus (45) Google Scholar). To date, PKCα, δ, ε, η, and ζ have been detected in keratinocytes (Reynolds et al., 1994Reynolds N.J. Baldassare J.J. Henderson P.A. Translocation and downregulation of protein kinase C isoenzymes-α and -ε by phorbol ester and bryostatin-1 in human keratinocytes and fibroblasts.J Invest Dermatol. 1994; 103: 364-369Crossref PubMed Scopus (45) Google Scholar). We thus analyzed which PKC isoforms may be translocated from cytosol to membrane by histamine. In resting keratinocytes, conventional PKCα and novel PKCδ are expressed abundantly in cytosol and to lesser amounts in membrane Figure 8. Histamine induced membrane translocation of PKCα, but not that of PKCδ. The histamine-induced membrane translocation of PKCα was blocked by pyrilamine. Much smaller amounts of novel PKCε, η, and atypical PKCζ were detected only in cytosolic fraction of keratinocytes, and histamine did not induce the membrane translocation of these isoforms. Thus, histamine appeared to selectively activate conventional PKCα via H1 receptors in keratinocytes. We then analyzed if PKCα and downstream kinase ERK may be responsible for histamine-induced GM-CSF expression using specific kinase inhibitors. The concentration of each inhibitor was determined from reported IC50 values (Li et al., 2002Li J. Hellmich M.R. Greeley Jr., G.H. Townsend Jr., C.M. Evers B.M. Phorbol ester-mediated neurotensin secretion is dependent on the PKC-α and -δ isoforms.Am J Physiol Gastrointest Liver Physiol. 2002; 283: G1197-G1206Crossref PubMed Scopus (28) Google Scholar). Conventional and novel PKC inhibitor GF109203X and conventional PKC inhibitor Gö6976 completely blocked histamine-induced enhancement of GM-CSF secretion Figure 9a, promoter activity Figure 9b, and mRNA stability (Figure 9c, data not shown for GF109203X). These were partially suppressed by MEK1 inhibitor PD98059, which inhibits the activation of ERK. On the other hand, novel PKC inhibitor rottlerin, p38 MAPK inhibitor SB203580,or c-Jun N-terminal kinase inhibitor SP600125 did not affect histamine-induced GM-CSF secretion, promoter activation, or mRNA stabilization. These results suggest the requirement of PKCα and ERK for histamine-induced enhancement of GM-CSF production both at transcriptional and post-transcriptional levels. In parallel with GM-CSF transcription, histamine-induced increase of AP-1 transcriptional activity was blocked by Gö6976 Figure 10a, GF109203X (data not shown), and PD98059 Figure 10a although these were not altered by rottlerin, SB203580, or SP600125 (data not shown). Gö6976 and PD98059 also suppressed the enhancement of DNA binding of AP-1 Figure 10b and c-Fos induction by histamine Figure 10c. These results suggest that PKCα and ERK may be required for histamine-induced c-Fos expression and resultant activation of AP-1. As with AP-1, histamine-induced increase of NF-κB transcriptional activity was suppressed completely by Gö6976 Figure 11a and GF109203X (data not shown) and partially by PD98059 Figure 11a, but not by rottlerin, SB203580, or SP600125 (data not shown). These results suggest that histamine-stimulated PKCα and ERK may enhance transcriptional activities of AP-1 and NF-κB, and thus promote GM-CSF transcription dependent on AP-1 and NF-κB. Gö6976 suppressed histamine-induced enhancement of DNA binding of NF-κB Figure 11b, and histamine-induced IκBα phosphorylation and degradation Figure 11c while these were not suppressed by PD98059. These results indicate that histamine-activated PKCα may promote IκBα phosphorylation and degradation and resultantly enhance DNA binding of NF-κB and ERK may enhance transcriptional activity of NF-κB by IκBα-independent mechanism. Histamine enhanced GM-CSF production in human neonatal foreskin keratinocytes at transcriptional and post-transcriptional levels via H1 receptors. We also preliminarily found that histamine similarly increased GM-CSF secretion and mRNA level in keratinocytes from lesional skin of atopic dermatitis (data not shown). Histamine enhanced GM-CSF transcription via the activation of AP-1 and NF-κB and also stabilized GM-CSF mRNA. PKCα and downstream kinase ERK are responsible for both transcriptional and post-transcriptional effects. It is known that PKCα activates c-Raf1, which phosphorylates and activates MEK1, upstream kinase of ERK (Schönwasser et al., 1998Schönwasser D.C. Marais R.M. Marshall C.J. Parker P.J. Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical kinase C isotypes.Mol Cell Biol. 1998; 18: 790-798Crossref PubMed Scopus (664) Google Scholar). In keratinocytes, histamine may thus bind to H1 receptor and possibly trigger PLC/PKCα/c-Raf1/MEK1/ERK signaling cascade. PKCα inhibitor Gö6976 completely blocked histamine-induced GM-CSF production while the inhibitory effect of MEK1 inhibitor PD98059 was partial, indicating that PKCα may promote GM-CSF production by ERK-dependent and -independent mechanisms. Among five PKC isoforms α, δ, ε, η, and ζ, PKCα, δ, ε, and η can be activated by diacylglycerol generated by PLC linked to H1 receptor; however, only PKCα appeared to be activated by histamine. The results are consistent with those in H1 receptor-transfected Chinese hamster ovary cells (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). Possibly, PKCδ, ε, and η may not respond to histamine-induced diacylglycerol due to their lower levels of expression or tight compartmentalization in cytoplasm, or both. It is also reported that individual PKC isoforms can be activated dependently on stimuli added or cell types (Reynolds et al., 1994Reynolds N.J. Baldassare J.J. Henderson P.A. Translocation and downregulation of protein kinase C isoenzymes-α and -ε by phorbol ester and bryostatin-1 in human keratinocytes and fibroblasts.J Invest Dermatol. 1994; 103: 364-369Crossref PubMed Scopus (45) Google Scholar;Li et al., 2002Li J. Hellmich M.R. Greeley Jr., G.H. Townsend Jr., C.M. Evers B.M. Phorbol ester-mediated neurotensin secretion is dependent on the PKC-α and -δ isoforms.Am J Physiol Gastrointest Liver Physiol. 2002; 283: G1197-G1206Crossref PubMed Scopus (28) Google Scholar). GM-CSF transcription in keratinocytes was more highly dependent on AP-1 than on NF-κB; however, the cooperation between AP-1 and NF-κB was required for sufficient transcription Figure 4b. Possibly AP-1 and NF-κB bound to cis-elements on GM-CSF promoter may interact directly or through common co-activators like CBP. Such physical interaction may stabilize the DNA binding of each factor or enhance the interaction of each factor with general transcriptional apparatus like TATA-binding protein (Thomas et al., 1997Thomas R. Tymms M.J. McKinlay L.H. Shannon M.F. Seth A. Kola I. ETS1, NFκB and AP1 synergistically transactivate the human GM-CSF promoter.Oncogene. 1997; 14: 2845-2855Crossref PubMed Scopus (134) Google Scholar). Histamine transiently induced c-Fos protein. The induced c-Fos may heterodimerize with c-Jun and the heterodimers may bind to AP-1-like site on GM-CSF promoter and drive transcription. On the other hand, c-Jun homodimers appeared to bind to AP-1-like site in unstimulated keratinocytes. It is reported that c-Fos/c-Jun heterodimers much more avidly bind to DNA and have much higher transcriptional activity than c-Jun homodimers (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 (34) Google Scholar;Suzuki et al., 1991Suzuki T. Okuno H. Yoshida T. Endo T. Nishina H. Iba H. Difference in transcriptional regulatory function between c-Fos and Fra-2.Nucleic Acids Res. 1991; 19: 5537-5542Crossref PubMed Scopus (192) Google Scholar). Thus, histamine may enhance DNA binding and transcriptional activity of AP-1 by inducing c-Fos and thus shifting AP-1 composition from c-Jun homodimers to c-Fos/c-Jun heterodimers. This effect of histamine also occurred in nerve growth factor gene transcription dependent on AP-1 (Kanda and Watanabe, 2003bKanda N. Watanabe S. Histamine enhances the production of nerve growth f