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• W2146990599 abstract "Several nuclear factors, called coactivators, such as CREB (cAMP response element binding protein)-binding protein (CBP) and p300/CBP associated factor (P/CAF), have intrinsic histone acetyltransferase (HAT) activity. Recent studies have shown that, in addition to histones, transcriptional regulatory molecules are also targets of HATs, and nuclear acetylation is thought to be involved in several biological events. We observed that a high concentration of calcium induced HAT activity in the keratinocyte cell line, HaCaT. The steady-state level of specific acetylated nuclear proteins changed in a dynamic fashion in HaCaT cells induced with 1.2 mmcalcium. One (∼97-kDa acetylated protein designated as ap97) was transiently induced, one (ap78) was induced and then continuously expressed, and one (ap70) disappeared with time. Although the up-regulation of ap70 and ap78 was not influenced by GF109203X, a specific inhibitor of protein kinase C (PKC), the calcium-induced accumulation of ap97 and the induction of P/CAF HAT activity were similarly attenuated by GF109203X. Notably, mutant P/CAF lacking HAT activity repressed the expression of ap97 and involucrin, a keratinocyte differentiation marker. Our results suggest that P/CAF HAT activity and induction of ap97 are involved in calcium-dependent keratinocyte differentiation. Several nuclear factors, called coactivators, such as CREB (cAMP response element binding protein)-binding protein (CBP) and p300/CBP associated factor (P/CAF), have intrinsic histone acetyltransferase (HAT) activity. Recent studies have shown that, in addition to histones, transcriptional regulatory molecules are also targets of HATs, and nuclear acetylation is thought to be involved in several biological events. We observed that a high concentration of calcium induced HAT activity in the keratinocyte cell line, HaCaT. The steady-state level of specific acetylated nuclear proteins changed in a dynamic fashion in HaCaT cells induced with 1.2 mmcalcium. One (∼97-kDa acetylated protein designated as ap97) was transiently induced, one (ap78) was induced and then continuously expressed, and one (ap70) disappeared with time. Although the up-regulation of ap70 and ap78 was not influenced by GF109203X, a specific inhibitor of protein kinase C (PKC), the calcium-induced accumulation of ap97 and the induction of P/CAF HAT activity were similarly attenuated by GF109203X. Notably, mutant P/CAF lacking HAT activity repressed the expression of ap97 and involucrin, a keratinocyte differentiation marker. Our results suggest that P/CAF HAT activity and induction of ap97 are involved in calcium-dependent keratinocyte differentiation. protein kinase C acetylated lysine bovine serum albumin CREB binding protein cAMP response element binding protein 6′-diamidino-2-phenylindole factor acetyltransferase fetal calf serum fluorescein isothiocyanate hemagglutinin histone acetyltransferase Immunoprecipitation nuclear acetylation p300/CBP associated factor promyelocytic leukemia pml gene product tetramethylrhodamine isothiocyanate Tris-HCl buffer saline with 0.02% Tween 20 cytomegalovirus phosphate-buffered saline dithiothreitol phenylmethylsulfonyl fluoride electrophoretic mobility shift assay The role of calcium in the regulation of cell growth and metabolism has been studied primarily in cells of mesenchymal origin, such as mouse 3T3 and human WI-38 fibroblasts (1Boynton A.L. Whitfield J.F. Isaacs R.J. Morton H.J. In Vitro. 1974; 10: 12-17Crossref PubMed Scopus (67) Google Scholar, 2Boynton A.L. Whitfield J.F. Isaacs R.J. Tremblay R. J. Cell. Physiol. 1977; 92: 241-247Crossref PubMed Scopus (98) Google Scholar). In these cells calcium concentrations below 0.5 mm cause G1 arrest and consequently a reduction in cell proliferation. However, epidermal keratinocytes cease to differentiate and proliferate when the calcium concentration is reduced to 0.05–0.1 mm (3Stanley J.R. Yuspa S.H. J. Cell Biol. 1983; 96: 1809-1814Crossref PubMed Scopus (90) Google Scholar). Cultured epidermal monolayer cells in low calcium medium are induced to terminal differentiation by increasing the calcium concentration to 1.2 mm, the usual concentration in the culture medium (3Stanley J.R. Yuspa S.H. J. Cell Biol. 1983; 96: 1809-1814Crossref PubMed Scopus (90) Google Scholar). Human epidermal keratinocytes differentiate from the proliferative basal component to upper cornified cells accompanied by alterations in the expression of epidermal differentiation markers such as involucrin, filaggrin, and loricrin (4Rice R.H. Green H. Cell. 1979; 18: 681-694Abstract Full Text PDF PubMed Scopus (647) Google Scholar,5Fuchs E. J. Cell Biol. 1990; 111: 2807-2814Crossref PubMed Scopus (588) Google Scholar). An increase in extracellular calcium concentration also induces these markers (6Hennings H. Michael D. Cheng C. Steinert P. Holbrook K. Yuspa S.H. Cell. 1980; 19: 245-254Abstract Full Text PDF PubMed Scopus (1507) Google Scholar, 7Yuspa S.H. Kilkenny A.E. Steinert P.M. Roop D.R. J. Cell Biol. 1989; 109: 1207-1217Crossref PubMed Scopus (515) Google Scholar) along with activation of protein kinase C (PKC)1 (8Dlugosz A.A. Yuspa S.H. J. Invest. Dermatol. 1994; 102: 409-414Abstract Full Text PDF PubMed Google Scholar). Several nuclear factors, called coactivators, play crucial roles in the coordination and integration of the transcription of eukaryotic genes (9Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (469) Google Scholar, 10Janknecht R. Hunter T. Nature. 1996; 383: 22-23Crossref PubMed Scopus (348) Google Scholar, 11Montminy M. Nature. 1997; 387: 654-655Crossref PubMed Scopus (3) Google Scholar). The well characterized coactivators, CREB (cAMP response element binding protein)-binding protein (CBP) and p300/CBP associated factor (P/CAF), integrate gene expression with many transcription activators through two distinct mechanisms. One is the association with and recruitment of the transcriptional machinery, which targets promoters (12Nakajima T. Uchida C. Anderson S.F. Lee C.G. Hurwitz J. Parvin J.D. Montminy M. Cell. 1997; 90: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar, 13Nakajima T. Uchida C. Anderson S.F. Parvin J.D. Montminy M. Genes Dev. 1997; 11: 738-747Crossref PubMed Scopus (212) Google Scholar, 14Cho H. Orphanides G. Sun X. Yang X.J. Ogryzko V. Lees E. Nakatani Y. Reinberg D. Mol. Cell. Biol. 1998; 18: 5355-5363Crossref PubMed Scopus (247) Google Scholar). The other is their intrinsic enzymatic activity, histone acetyltransferase (HAT), which transfers an acetyl-base to the ε-portion of lysine residues on histones (15Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2409) Google Scholar, 16Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2419) Google Scholar, 17Mizzen C.A. Allis C.D. Cell. Mol. Life. Sci. 1998; 54: 6-20Crossref PubMed Scopus (191) Google Scholar, 18Struhl K. Genes Dev. 1998; 12: 599-606Crossref PubMed Scopus (1557) Google Scholar). HAT activities have been implicated in the regulation of transcription (19Wolffe A.P. Pruss D. Cell. 1996; 84: 817-819Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Their involvement has been explained by the destabilization of the interaction of hyperacetylated histone with DNA causing modification of chromatin structure, which facilitates the access of transcriptional activators to their cognate DNA-binding sites (20Hebbes T.R. Thorne A.W. Crane-Robinson C. EMBO J. 1988; 7: 1395-1402Crossref PubMed Scopus (715) Google Scholar, 21Lee D.Y. Hayes J.J. Pruss D. Wolffe A.P. Cell. 1993; 72: 73-84Abstract Full Text PDF PubMed Scopus (967) Google Scholar). Recent studies have shown that HATs, acting as factor acetyl transferases (FATs), also acetylate molecules other than histones, such as p53, lymphoid enhancer-binding factor, and transcription factor (for RNA polymerase) II E, which often results in increased transcriptional activity (22Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2189) Google Scholar, 23Imhof A. Yang X.J. Ogryzko V.V. Nakatani Y. Wolffe A.P. Ge H. Curr. Biol. 1997; 7: 689-692Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar, 24Lill N.L. Grossman S.R. Ginsberg D. DeCaprio J. Livingston D.M. Nature. 1997; 387: 823-827Crossref PubMed Scopus (596) Google Scholar, 25Waltzer L. Bienz M. Nature. 1998; 395: 521-525Crossref PubMed Scopus (327) Google Scholar). The status of histone acetylation is thought to influence cell growth and differentiation (26Marks P.A. Richon V.M. Rifkind R.A. J. Natl. Cancer. Inst. 2000; 92: 1210-1216Crossref PubMed Scopus (1085) Google Scholar, 27Zhou Q. Melkoumian Z.K. Lucktong A. Moniwa M. Davie J.R. Strobl J.S. J. Biol. Chem. 2000; 275: 35256-35263Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Numerous studies have shown associations between nuclear acetylation/deacetylation and differentiation/development (28Puri P.L. Sartorelli V. Yang X.J. Hamamori Y. Ogryzko V.V. Howard B.H. Kedes L. Wang J.Y. Graessmann A. Nakatani Y. Levrero M. Mol. Cell. 1997; 1: 35-45Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 29Kawasaki H. Eckner R. Yao T.P. Taira K. Chiu R. Livingston D.M. Yokoyama K.K. Nature. 1998; 393: 284-289Crossref PubMed Scopus (302) Google Scholar, 30Shi Y. Mello C. Genes Dev. 1998; 12: 943-955Crossref PubMed Scopus (127) Google Scholar, 31Sartorelli V. Puri P.L. Hamamori Y. Ogryzko V. Chung G. Nakatani Y. Wang J.Y. Kedes L. Mol. Cell. 1999; 4: 725-734Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 32Thomas T. Voss A.K. Chowdhury K. Gruss P. Development. 2000; 127: 2537-2548PubMed Google Scholar). Calcium plays critical roles in muscle differentiation by regulating repression of the transcription of myocyte enhancer factor 2, which interacts with histone deacetylase (33Miska E.A. Karlsson C. Langley E. Nielsen S.J. Pines J. Kouzarides T. EMBO J. 1999; 18: 5099-5107Crossref PubMed Scopus (472) Google Scholar, 34McKinsey T.A. Zhang C.L. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14400-14405Crossref PubMed Scopus (428) Google Scholar, 35Youn H.D. Grozinger C.M. Liu J.O. J. Biol. Chem. 2000; 275: 22563-22567Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). We investigated the association between calcium-induced HAT activation and the differentiation of keratinocytes. Nuclear acetylation (NA) in response to calcium stimulation was examined using a specific antibody against ε-acetylated lysine (AK) (36Kawahara K. Watanabe S. Ohshima T. Soejima Y. Oishi T. Aratani S. Nakata M. Shibata M. Inoue K. Amano T. Fujii R. Yanai K. Hagiwara M. Fukamizu A. Maruyama I. Nakajima T. Biochem. Biophys. Res. Commun. 1999; 266: 417-424Crossref PubMed Scopus (26) Google Scholar). The interactions between PKC, HAT, and NA were also evaluated by in vitro and cell transfection experiments. Dulbecco's modified Eagle's medium containing high glucose, penicillin G, streptomycin, non-essential amino acids, glutamine, and ascorbic acid were purchased from Invitrogen (Grand Island, NY), FCS from HyClone Inc. (Logan, UT), and calcium chloride from Wako pure chemical industries Ltd. (Kobe, Japan). The protein kinase C inhibitors, Go6983 and GF109203X, were purchased fromCalbiochem-Novabiochem Corp. (San Diego, CA); anti-human involucrin monoclonal antibody was from Novocastra Laboratories Ltd. (Newcastle, UK); and anti-human CBP antibody, anti-P/CAF antibody, and anti-promyelocytic leukemia (pml) gene product (PML protein) antibody were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). [14C]Acetyl CoA and [32P]dATP were from Amersham Biosciences, Inc. (Buckinghamshire, UK), and double-stranded oligonucleotide probes containing AP-1 binding element were from Promega (Madison, WI). Anti-AK antibody against the ε-portion of lysine residues was developed as reported previously (36Kawahara K. Watanabe S. Ohshima T. Soejima Y. Oishi T. Aratani S. Nakata M. Shibata M. Inoue K. Amano T. Fujii R. Yanai K. Hagiwara M. Fukamizu A. Maruyama I. Nakajima T. Biochem. Biophys. Res. Commun. 1999; 266: 417-424Crossref PubMed Scopus (26) Google Scholar). Briefly, keyhole limpet hemocyanin-conjugated AK was mixed with Freund's complete adjuvant, and the suspension was injected intradermally into 10 female Japanese white rabbits. An immunogen of the same quality was also administered five times, once every other week. The IgG fraction was collected from serum using a protein A column. This antibody reacts specifically with the ε-portion of AK but not with the α-portion of AK or non-AK (36Kawahara K. Watanabe S. Ohshima T. Soejima Y. Oishi T. Aratani S. Nakata M. Shibata M. Inoue K. Amano T. Fujii R. Yanai K. Hagiwara M. Fukamizu A. Maruyama I. Nakajima T. Biochem. Biophys. Res. Commun. 1999; 266: 417-424Crossref PubMed Scopus (26) Google Scholar). HaCaT cells were provided by Dr. L. Matrisian (Vanderbilt University, Nashville, TN) with the permission of Dr. Nobert E. Fusening (German Cancer Research Center, Heidelberg, Germany). HaCaT cells are a non-tumorigenic immortalized keratinocyte line, which was derived from normal skin, and they have maintained substantial differentiation potential in culture (37Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. J. Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3493) Google Scholar). The cells were grown as described previously (37Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. J. Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3493) Google Scholar) with some modifications. They were cultured at 37 °C in a 5% CO2 humidified atmosphere in Dulbecco's modified Eagle's medium containing high glucose (without calcium and magnesium), 100,000 units/liter penicillin G, 100 mg/liter streptomycin, 0.1 mm non-essential amino acids, 292 mg/liter glutamine, 50 mg/liter ascorbic acid, and 10% calcium-depleted FCS (6Hennings H. Michael D. Cheng C. Steinert P. Holbrook K. Yuspa S.H. Cell. 1980; 19: 245-254Abstract Full Text PDF PubMed Scopus (1507) Google Scholar). The calcium concentration in the medium was adjusted to various levels by adding an appropriate volume of 280 mm calcium chloride. Mutations in P/CAF and CBP were introduced by site-directed mutagenesis using the Quik-Change mutagenesis system (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Double-stranded oligonucleotides were designed such that the wild type sequence corresponding to amino acids Tyr597/Phe598 in P/CAF cDNA were substituted with alanines to generate a mutant of P/CAF lacking HAT activity (pCMV-PCAF HAT-). A similar strategy was used to obtain mutants of CBP. Mutant P/CAF and CBP were expressed in bacteria and tested for HAT activity using histones as substrates (data not shown). Hemagglutinin-tagged (HA) PML protein expression plasmid was made by reverse transcription-PCR-based cloning into pcDNA3-HA (38Miyagishi M. Fujii R. Hatta M. Yoshida E. Araya N. Nagafuchi A. Ishihara S. Nakajima T. Fukamizu A. J. Biol. Chem. 2000; 275: 35170-37175Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). For transient transfection, 2 μg of DNA was diluted in 100 μl of Opti-MEM (Invitrogen). 10 μl of LipofectAMINE and 8 μl of plus reagent (Invitrogen) were also diluted in 100 μl of Opti-MEM. After 5 min at room temperature, the two solutions were mixed and incubated for 20 min to allow complex formation. The mixture was then directly added to 2 ml of antibiotic-free medium, which was on the cells plated on glass coverslips. The medium was changed 5 h after addition of the transfection mixture, and the cells were grown for 24 h in medium containing 0.05 mm calcium after which the medium was replaced with medium containing 1.2 mm calcium. After transfection, the cells were grown for 2 days before analysis of ectopic protein abundance. Liquid assays for HAT activity were performed as described by Brownell and Allis (39Brownell J.E. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6364-6368Crossref PubMed Scopus (240) Google Scholar) with some modification. Nuclear extracts were prepared as described previously (40Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 176419Crossref PubMed Scopus (3918) Google Scholar) with some modifications. Briefly, cell layers (5 × 106) were harvested with a cell scraper, washed with PBS, and incubated for 15 min at 4 °C in buffer A containing 10 mm HEPES (pH 7.8), 10 mm KCl, 2 mm MgCl2, 1 mm DTT, 0.1 mm EDTA, and 0.1 mmPMSF. Then Nonidet P-40 solution (0.6% final concentration) was added, and the cells were mixed vigorously for 15 s and centrifuged for 30 s at 12,000 × g. Pelleted nuclei were suspended in buffer B (50 mm HEPES (pH 7.8), 50 mm KCl, 300 mm NaCl, 0.1 mmEDTA, 1 mm DTT, 0.1 mm PMSF, and 10% glycerol), mixed for 20 min, and then centrifuged for 5 min at 12,000 × g. The nuclear extract reaction mixtures containing 100 mm Tris-HCl, 2 mm DTT, 2 mm PMSF, 0.2 mm EDTA, 0.1 m NaCl, 0.02 mm butyric acid, and 20% glycerol were incubated for 30 min at 30 °C with 125 nCi of [14C]acetyl CoA and 10 mg/ml histones (Sigma-Aldrich, Milwaukee, WI) and then subjected to 12% SDS-PAGE. The dried gel was autoradiographed at −80 °C for 4–7 days using Kodak X-Omat AR films (Eastman Kodak, Rochester, NY). Western blotting was performed as described previously (12Nakajima T. Uchida C. Anderson S.F. Lee C.G. Hurwitz J. Parvin J.D. Montminy M. Cell. 1997; 90: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar) with some modifications. Briefly, 10 μg of whole cell or nuclear extracts from HaCaT cells were subjected to 8 or 10% SDS-PAGE and then transferred to a nitrocellulose membrane. The membrane was incubated for 1 h with blocking solution consisting of 1% BSA and 5% low fat milk in 25 mm Tris-HCl buffer saline containing 0.02% Tween 20 (T-TBS) and then was reacted with the indicated antibodies for 1 h at 25 °C. After washing three times with T-TBS for 10 min each at 25 °C, the membrane was incubated for 30 min at 25 °C with peroxidase-conjugated goat anti-rabbit, anti-mouse, or anti-rat IgG (1:3000 dilution). Proteins were visualized using an enhanced chemiluminescent detection system (Amersham Biosciences, Inc.). Immunostaining with the indicated antibodies was performed with HaCaT cells cultured in four wells of a Lab-Tech chamber slide (Nalge Nunc International, Cambridge, MA). Cells were fixed at −20 °C for 30 min with 100% methanol. The slides were treated for 1 h with a blocking buffer consisting of 1% BSA in PBS, incubated with the indicated antibodies for 1 h, and then washed three times with PBS. The primary antibodies were detected by treating for 30 min with FITC-conjugated goat anti-rabbit IgG for AK, with TRITC-conjugated goat anti-mouse IgG for involucrin and P/CAF and with TRITC-conjugated goat anti-rat IgG for HA (1:200 dilutions). Nuclei were stained with DAPI. IP was performed as described previously (12Nakajima T. Uchida C. Anderson S.F. Lee C.G. Hurwitz J. Parvin J.D. Montminy M. Cell. 1997; 90: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar, 13Nakajima T. Uchida C. Anderson S.F. Parvin J.D. Montminy M. Genes Dev. 1997; 11: 738-747Crossref PubMed Scopus (212) Google Scholar). Briefly, ∼500 μg of nuclear extracts was incubated for 1 h with 5 μg of anti-CBP or anti-P/CAF antibody in IP buffer (50 mm Tris-HCL (pH 7.6), 150 mmNaCl, 1% Nonidet P-40, 1 mm EDTA, 5% glycerol). Complexes were bound to 25 μl of protein A/G-agarose beads (Santa Cruz Biotechnology). The agarose beads were washed three times with IP buffer and were eluted with reaction mixture for HAT assays or boiled in loading buffer for Western blotting. EMSAs were performed as described previously (41Nakajima T. Kitajima I. Shin H. Takasaki I. Shigeta K. Abeyama K. Yamashita Y. Tokioka T. Soejima Y. Maruyama I. Biochem. Biophys. Res. Commun. 1994; 204: 950-958Crossref PubMed Scopus (65) Google Scholar). Briefly, DNA-protein reaction mixtures (20 μl) consisted of buffer (5 mm Hepes, pH 7.9, 10% glycerol, 25 mm KCl, 0.05 mm EDTA, 0.125 mm PMSF), 15 μg of nuclear extract, 1 μg of poly(dI-dC), 0.28 ng of radiolabeled probe of consensus sequence for AP-1 binding element in the presence or absence of a molar excess of cold probe. Mixtures were incubated at 22 °C for 20 min. Samples were loaded onto non-denaturing high ionic strength polyacrylamide gels. After running at 150 V for 1 h, the gels were dried and exposed to X-Omat films (Kodak, Rochester, NY) at −80 °C. To investigate whether HAT enzymes in HaCaT cells are activated by calcium stimulation, cells were cultured in 0.05 mm calcium and the calcium concentration was increased to 1.2 mm. The time course of HAT activity in nuclear extracts after increasing calcium is shown in Fig. 1. Although a trace of HAT activity was detected under low calcium (0.05 mm) conditions, it was induced significantly by 1.2 mm calcium with a maximum activity at 2 h. Although HAT activity gradually decreased after 2 h, it was still detected weakly at 16 h. This result suggests that HAT activation could be involved in the calcium-induced signaling pathway in HaCaT cells. Because HAT activity is correlated with NA (36Kawahara K. Watanabe S. Ohshima T. Soejima Y. Oishi T. Aratani S. Nakata M. Shibata M. Inoue K. Amano T. Fujii R. Yanai K. Hagiwara M. Fukamizu A. Maruyama I. Nakajima T. Biochem. Biophys. Res. Commun. 1999; 266: 417-424Crossref PubMed Scopus (26) Google Scholar), using anti-AK antibody we next examined the time course of NA resulting from high calcium (1.2 mm) treatment. As shown in Fig.2 A, two types of acetylated nuclear proteins (∼70 and 97 kDa designated ap70 and ap97) were detected by Western blotting of the nuclear extracts of cells grown in 0.05 mm calcium. Subsequently, ap70 gradually decreased. In low calcium ap97 was detected but to a lesser extent than ap70. It was transiently induced by high calcium with a maximum at 2 h after stimulation and then it decreased. The time course of ap97 was fairly consistent with that of HAT activity shown in Fig. 1. When cells were treated with 1.2 mm calcium, acetylation of a 78-kDa protein, designated ap78, was induced. In contrast to ap70 and ap97, ap78 was not detected in low calcium, but was expressed for 16 h in high calcium. Thus, calcium clearly changed the status of NA. Next we examined the morphological changes resulting from calcium stimulation. Phase-contrast microscopy showed that cells growing as monolayers in low calcium (0.05 mm) attached to each other tightly after 16 h of culture with high calcium (1.2 mm), indicating cornified stratification (Fig.2 B, left panel). Calcium induces epidermal cell differentiation and keratinocyte differentiation markers such as involucrin, filaggrin, loricrin, and transglutaminase (4Rice R.H. Green H. Cell. 1979; 18: 681-694Abstract Full Text PDF PubMed Scopus (647) Google Scholar, 5Fuchs E. J. Cell Biol. 1990; 111: 2807-2814Crossref PubMed Scopus (588) Google Scholar, 42Dlugosz A.A. Yuspa S.H. J. Cell Biol. 1993; 120: 217-225Crossref PubMed Scopus (220) Google Scholar). HaCaT cells show normal keratinization and expression of differentiation markers, including involucrin, in high calcium medium (37Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. J. Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3493) Google Scholar). Involucrin, a structural component of mature squamous epithelium, is incorporated into the marginal band as part of the formation of the protein envelope that characterizes squamous cells immediately prior to terminal differentiation (43Murphy G.F. Flynn T.C. Rice R.H. Pinkus G.S. J. Invest. Dermatol. 1984; 82: 453-457Abstract Full Text PDF PubMed Scopus (186) Google Scholar). We examined the effects of calcium using antibodies against involucrin and AK. As shown in Fig. 2 B(right panel), although involucrin was not detected in HaCaT cells after 2 h in both low and high calcium, it was induced after 16 h of culture in high calcium, indicating the differentiation of HaCaT cells. Stronger NA was detected in cells cultured for 2 and 16 h in high calcium compared with those cultured in low calcium. The acetylation status in the nucleus was correlated with calcium-induced involucrin expression. These results again suggest that HAT activation and the consequent NA are associated with calcium-dependent keratinocyte differentiation Calcium induces the expression of keratinocyte differentiation marker genes through the PKC signaling pathway (8Dlugosz A.A. Yuspa S.H. J. Invest. Dermatol. 1994; 102: 409-414Abstract Full Text PDF PubMed Google Scholar), and its concentration affects HAT activity and the NA pattern. These facts prompted us to examine the effects of Go6983 and GF109203X, specific PKC inhibitors, on HAT activity and the NA profile. Cells were incubated with or without Go6983 or GF109203X for 1 h prior to calcium treatment. Nuclear extracts were prepared at the indicated times after treatment with 1.2 mm calcium. HAT activity at 2 h and the time course of NA were examined. HAT activity was inhibited by both Go6983 and GF109203X (Fig.3 A). 20 nmGF109203X affected calcium-induced HAT activity more than 10 nm GF109203X, but no more significant effect on HAT activity was detected by 50 nm GF109203X than by 20 nm GF109203X. 10 nm Go6983 inhibited HAT activity more than 5 nm Go6983, and 60 nmGo6983 did not inhibit it completely. We performed IP assays to see if CBP and P/CAF are involved in calcium-induced HAT activation, and, if so, whether the HAT activities of CBP and P/CAF are suppressed by inhibition of PKC. CBP and P/CAF were purified from nuclear extracts using monoclonal anti-CBP and anti-P/CAF antibodies. The qualities of the purified proteins were confirmed by Western blotting (data not shown), and they were assayed for HAT activity as described under “Experimental Procedures.” Calcium induced the HAT activities of both CBP and P/CAF at 2 h, and these inductions were inhibited by GF109203X (Fig. 3 B). The inhibition of P/CAF HAT activity by 20 nm GF109203X was much greater than that of CBP. The effects of 20 nm GF109203X on NA stimulated by 1.2 mm calcium are shown in Fig. 3 C. Although the expression patterns of ap70 and ap78 were very similar to those without GF109203X (Fig. 2 A), surprisingly, ap97 was absent throughout the period examined (Fig. 3 C). These results indicate that conventional calcium-dependent PKC is involved in calcium-dependent HAT activation and that ap97 is the major component in this process. To determine more definitively whether P/CAF is important in calcium-induced HAT activation and keratinocyte differentiation, we introduced an active mouse HA-CBP with HAT activity or an inactive HA-CBP mutant and an active mouse FLAG-tagged (FLAG)-P/CAF or an inactive FLAG- P/CAF mutant into HaCaT cells by transient transfections. The transfection efficiency was 85% as indicated by the expression of a linked green fluorescence protein (Fig. 4 A, upper panel). Western blot analyses of nuclear extracts from transfected cells confirmed that there was sufficient expression of the ectopic proteins (Fig. 4 A, lower panel). We examined involucrin expression induced by calcium in the transfected cells after treating them with 1.2 mm calcium for 24 h as described under “Experimental Procedures.” As shown in Fig.4 B, transfection with wild type CBP had little effect on involucrin expression and mutant CBP suppressed involucrin slightly. In contrast, transfection with P/CAF resulted in significant alterations in involucrin expression. Wild type P/CAF increased involucrin expression moderately and when the cells were transfected with mutant P/CAF, involucrin expression was repressed significantly. Furthermore, mutant P/CAF inhibited the expression of ap97 induced by treatment with 1.2 mm calcium for 2 h (Fig. 4 C). The expressions of ap70 and ap78 after stimulation with calcium were not affected by any of the transfections. Calcium-regulated involucrin gene expression is mediated in part by interaction of AP-1 transcription factors with an AP-1 site in the calcium response region of the involucrin promoter (44Ng D.C. Shafaee S. Lee D. Bikle D.D. J. Biol. Chem. 2000; 275: 24080-24088Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). EMSA using double-stranded oligonucleotide probes containing the consensus sequence of the AP-1 site, with nuclear extracts from HAT-active or -dead CBP and P/CAF transfectants, revealed that both mutants reduced AP-1 binding activity at 2 h after stimulation with 1.2 mm calcium (Fig. 4 D). P/CAF is not only required for the increased transcription of the involucringene. However, it is also possible that transcriptional factors other than AP-1 are required for optimal expression of the differentiation markers. These results provide evidence for the involvement of P/CAF in calcium-dependent keratinocyte differentiation via the PKC signaling pathway through acetylation of a 97-kDa nuclear protein. To determine whether P/CAF interacts with NA, we investigated the localization of NA and P/CAF in the nucleoli of HaCaT cells stimulated with 1.2 mm calcium. After 2 h of stimulation, cells were fixed and examined by indirect immunofluorescence staining. P/CAF was detected in HaCaT cells after calcium stimulation and was localized with NA (Fig.5, upper panel). The detection of NA after 2 h of stimulation with 1.2 mm calcium implies the existence of ap97. PML nuclear bodies are thought to be cellular regulatory domains where proteins such as CBP and CBP-interacting molecules may be activated or inactivated to coordinate signal-activated cellular responses (45Hodges M. Tissot C. H" @default.
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