Possible Role of Transcriptional Coactivator P/CAF and Nuclear Acetylation in Calcium-induced Keratinocyte Differentiation*

Several nuclear factors, called coactivators, such as CREB (cAMP response element binding protein)-bind-ing 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 dy-namic fashion in HaCaT cells induced with 1.2 m M cal- cium. One ( (cid:1) 97-kDa acetylated protein designated as ap97) was transiently induced, one (ap78) was induced and then

In these cells calcium concentrations below 0.5 mM cause G 1 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 (3). 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 (3). 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 (4,5). An increase in extracellular calcium concentration also induces these markers (6,7) along with activation of protein kinase C (PKC) 1 (8).
Several nuclear factors, called coactivators, play crucial roles in the coordination and integration of the transcription of eukaryotic genes (9 -11). 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 (12)(13)(14). The other is their intrinsic enzymatic activity, histone acetyltransferase (HAT), which transfers an acetylbase to the ⑀-portion of lysine residues on histones (15)(16)(17)(18). HAT activities have been implicated in the regulation of transcription (19). 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 (20,21). 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 (22)(23)(24)(25).
The status of histone acetylation is thought to influence cell growth and differentiation (26,27). Numerous studies have shown associations between nuclear acetylation/deacetylation and differentiation/development (28 -32). Calcium plays critical roles in muscle differentiation by regulating repression of the transcription of myocyte enhancer factor 2, which interacts with histone deacetylase (33)(34)(35).
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 ⑀-acetylated lysine (AK) (36). The interactions between PKC, HAT, and NA were also evaluated by in vitro and cell transfection experiments.
Anti-AK Antibody-Anti-AK antibody against the ⑀-portion of lysine residues was developed as reported previously (36). 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 ⑀-portion of AK but not with the ␣-portion of AK or non-AK (36).
Cells and Culture-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 (37). The cells were grown as described previously (37) with some modifications. They were cultured at 37°C in a 5% CO 2 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 (6). The calcium concentration in the medium was adjusted to various levels by adding an appropriate volume of 280 mM calcium chloride.
DNA Constructs and Transient Transfection-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 Tyr 597 /Phe 598 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 (38). For transient transfection, 2 g of DNA was diluted in 100 l of Opti-MEM (Invitrogen). 10 l of LipofectAMINE and 8 l of plus reagent (Invitrogen) were also diluted in 100 l 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.
HAT Assay-Liquid assays for HAT activity were performed as de-scribed by Brownell and Allis (39) with some modification. Nuclear extracts were prepared as described previously (40)  Western Blotting-Western blotting was performed as described previously (12) with some modifications. Briefly, 10 g 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.).
Indirect Immunofluorescence-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 Ϫ20°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 TRITCconjugated 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.
Immunoprecipitation-IP was performed as described previously (12,13). Briefly, ϳ500 g of nuclear extracts was incubated for 1 h with 5 g of anti-CBP or anti-P/CAF antibody in IP buffer (50 mM Tris-HCL (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 5% glycerol). Complexes were bound to 25 l 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.
Electrophoretic Mobility Shift Assays-EMSAs were performed as described previously (41). Briefly, DNA-protein reaction mixtures (20 l) consisted of buffer (5 mM Hepes, pH 7.9, 10% glycerol, 25 mM KCl, 0.05 mM EDTA, 0.125 mM PMSF), 15 g of nuclear extract, 1 g 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 Ϫ80°C. 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.

HAT Activity Induced by Calcium in HaCaT
Induction of NA by Calcium in HaCaT Cells-Because HAT activity is correlated with NA (36), using anti-AK antibody we next examined the time course of NA resulting from high calcium (1.2 mM) treatment. As shown in Fig. 2A, two types of acetylated nuclear proteins (ϳ70 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. 2B, left panel).
Calcium induces epidermal cell differentiation and keratinocyte differentiation markers such as involucrin, filaggrin, loricrin, and transglutaminase (4,5,42). HaCaT cells show normal keratinization and expression of differentiation markers, including involucrin, in high calcium medium (37). 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 (43). We examined the effects of calcium using antibodies against involucrin and AK. As shown in Fig. 2B (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 Inhibition of HAT Activity by PKC Inhibitors-Calcium induces the expression of keratinocyte differentiation marker

FIG. 3. Effect of PKC inhibitors on calcium-induced HAT activation and ap97 expression.
A, specific PKC inhibitors, GF109203X or Go6983, were added 1 h before stimulation with calcium. GF109203X concentrations were 0, 10, 20, and 50 nM and Go6983 concentrations were 0, 5, 10, and 60 nM. HAT activities after 2 h of stimulation with calcium were assessed in nuclear extracts. B, nuclear extracts from calcium-treated (1.2 mM, 2 h) HaCaT cells, which were pretreated with 0, 10, and 20 nM GF109203X were incubated for 1 h with anti-CBP antibody or anti-P/CAF antibody followed by incubation with protein A/G agarose. Bound materials were eluted into HAT assay reaction mixtures. Purification was confirmed by immunoblotting using specific antibodies to CBP or P/CAF (data not shown). Nuclear extracts from cells cultured in 0.05 mM calcium were used as controls (ctrl). C, HaCaT cells were treated with 20 nM GF109203X for 1 h prior to being treated with 1.2 mM calcium for the indicated periods. Nuclear extracts were subjected to 10% SDS-PAGE and analyzed by Western blotting using anti-AK antibody. genes through the PKC signaling pathway (8), 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. 3A). 20 nM GF109203X 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 nM Go6983 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. 3B). 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. 3C. Although the expression patterns of ap70 and ap78 were very similar to those without GF109203X ( Fig. 2A), surprisingly, ap97 was absent throughout the period examined (Fig. 3C). These results indicate that conventional calcium-dependent PKC is involved in calciumdependent HAT activation and that ap97 is the major component in this process.
Inhibition of Expression of Involucrin and ap97 by Mutant P/CAF-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. 4A, upper panel). Western blot analyses of nuclear extracts from transfected cells confirmed that there was sufficient expression of the ectopic proteins (Fig. 4A, 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. 4B, 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.  4C). 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 (44). 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 activ-ity at 2 h after stimulation with 1.2 mM calcium (Fig. 4D). P/CAF is not only required for the increased transcription of the involucrin gene. 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.
Acetylated Nuclear Protein Colocalized with P/CAF and the PML Nuclear Bodies-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 signalactivated cellular responses (45,46). To investigate whether P/CAF could be a component of PML nuclear bodies, we examined the association between NA and PML nuclear bodies. PML protein is found in PML nuclear bodies and is the key protein required for their formation (47). Overexpression of PML protein leads to the accumulation of endogenous CBP in PML nuclear bodies (48). Although PML nuclear bodies are present in the nuclei of most cell lines (49,50), PML protein was not detected in nuclear extracts of HaCaT cells by Western blotting (data not shown). HaCaT cells were transiently transfected with an expression construct coding for PML protein and then stimulated with 1.2 mM calcium. As shown in Fig. 5 (lower panel), PML nuclear bodies were detected as several speckled foci at 2 h. Notably, the foci of PML nuclear bodies clearly overlapped with the NA sites.

DISCUSSION
Several previous studies demonstrated a link between HAT activation and differentiation. Various histone deacetylase inhibitors were shown to induce differentiation in a variety of cancer cells (26), and the quinidine-induced differentiation of breast cancer cells coincided with histone H4 hyperacetylation (27). These observations favor our hypothesis that HAT activity plays a pivotal role in keratinocyte differentiation induced by calcium.
In the present study, we demonstrated that calcium induced HAT activation and the presence of three types of acetylated nuclear proteins ap70, ap78, and ap97 in HaCaT cells treated with a high concentration of calcium. Acetylated proteins changed, not in abundance, but in the extent of their acetylation, because Western blotting by anti-AK antibody was done with 10 g of each nuclear extract and cycloheximide treatment had no significant effect on nuclear acetylation (data not shown). ap97 was transiently hyperacetylated in a time-dependent manner with a maximum at 2 h of stimulation with high calcium. No appreciable increase in acetylation of ap97 was evident following treatment with an increased concentration of calcium for 24 and 48 h (data not shown). These results imply that acetylation of ap97 is an indicator of calcium-induced cell differentiation. However, the direct association of acetylation of ap97 with P/CAF HAT activity is not clear, but the connection between P/CAF and NA is evident from the result shown in Fig. 5.
PKC is a large family of protein kinases classified into three subfamilies; conventional PKCs, which are calcium-responsive and include ␣, ␤I, ␤II, and ␥; novel PKCs, which do not respond to calcium and include ␦, ⑀, and ; and atypical PKCs, which are not affected by calcium, phorbol esters, or diacylglycerol and include and (51,52). The PKC isotypes play distinct roles in the regulation of growth and differentiation, depending on which cell types are being examined (53). Of the PKC polypeptides, human skin expresses ␣, ␤I, ␤II, ␦, ⑀, , and , and ␤ and are the major polypeptides in keratinocyte (54). We demonstrated that the PKC inhibitors, Go6983 and GF109203X, repressed HAT activity and that GF109203X blocked ap97 expression. Calcium-dependent conventional PKC ␣ and ␤ are inhibited by 20 nM GF109203X or 10 nM Go6983, and 60 nM Go6983 also inhibits the novel PKC isoforms ␦, ⑀, and (55,56). As shown in Fig. 3A, no significant differences were noted in the inhibition of HAT activity by 10 nM and 60 nM Go6983. This suggests that conventional PKC is involved in calcium-dependent HAT activation. However, it is probable that a signaling pathway other than PKC is involved in calcium-induced HAT activation because of the incomplete inhibition of HAT activity by 60 nM Go6983.
Several recent reports suggested that CBP may be phosphorylated by various kinases that are important in either cell cycle regulation or different signal transduction pathways, and these phosphorylation events have been speculated to affect various CBP activities (57)(58)(59)(60)(61). Yuan and Gambee (62) identified a major p300 phosphorylation site and determined that PKC was responsible for its phosphorylation. It is still uncertain from our study whether PKC directly phosphorylates P/CAF. 20 nM GF109203X almost completely reduced the HAT activity of P/CAF, suggesting that conventional PKC may phoswas analyzed by Western blotting with anti-involucrin antibody (INV.). pCMV was used as controls (ctrl in B and C). C, nuclear extracts from HaCaT cells expressing each ectopic protein were separated by 10% SDS-PAGE, and the status of NA after 2 h of stimulation with calcium was analyzed by Western blotting with anti-AK antibody. D, the binding of nuclear proteins to the consensus sequence of the AP-1 site was evaluated by electrophoretic mobility shift assays of nuclear extracts. phorylate P/CAF as well as CBP. The importance of P/CAF in calcium-dependent keratinocyte differentiation was confirmed by the introduction of mutant CBP and P/CAF into HaCaT cells. Expression of involucrin was enhanced by enforced expression of wild type P/CAF (but not CBP) and was inhibited by an acetylation-defective mutant. Accumulation of ap97 was similarly inhibited by the HAT-dead P/CAF mutant. Although it is conceivable that P/CAF regulates involucrin gene expression independent of its involvement in the differentiation program, mutant-CBP and -P/CAF both reduced calcium-increased AP-1 binding activity, indicating that P/CAF HAT activity maintained calcium-induced keratinocyte differentiation at more than just the level of transcription. In addition to involucrin, inactive P/CAF had little ability to induce K10, another keratinocyte differentiation marker (data not shown).
CBP and P/CAF are immunoprecipitated from complexes containing additional proteins with HAT and transcription stimulation (63)(64)(65). P/CAF can be found in complexes with histone-like TATA-binding protein associated factors but devoid of p300/CBP (66). This indicates that CBP and P/CAF, and possibly other HATs, exert their functions either concomitantly or separately depending on the targets. Indeed, regulated transcription relies on the differential use of the acetyltransferase activity of either p300/CBP or P/CAF, as shown in the present study. Acetyltransferase activity of P/CAF but not of CBP is required by MyoD (28,31) and the retinoic acid receptor (67), which is a nuclear receptor deeply associated with keratinocyte differentiation (68,69). These facts strongly suggest that ap97 functions as a nuclear receptor, which would be acetylated by P/CAF and activate the downstream pathway of signal transduction for keratinocyte differentiation.
The function of PML nuclear bodies is not fully understood; however, several recent studies provide some clues. In the present study, acetylated nuclear protein aggregated with both P/CAF and PML nuclear bodies after 2 h of calcium stimulation. It is probable that, in response to calcium stimulation, activated P/CAF is recruited to PML nuclear bodies and plays significant roles in the regulation of transactivation, including differentiation.
In contrast to ap97, ap70 and ap78 were not affected by PKC inhibitors and mutant CBP or P/CAF. FATs other than CBP and P/CAF could control the expression of ap70 and ap78, and their acetylated form might be involved in cell functions other than differentiation. Because ap70 disappeared by 2 h after stimulation with high calcium, its acetylated form is probably associated with continuous cell proliferation. The time-dependent overexpression of ap78 suggests an association with the cell cycle signaling pathway, because almost all cells were arrested in G 1 after 16 h of stimulation with calcium (data not shown). The cell cycle signaling pathway has been correlated with HAT activity (60).
In conclusion, calcium-induced HAT activity via the PKC signaling pathway and P/CAF, rather than CBP, play critical roles in keratinocyte differentiation, probably in the differentiation program rather than at the level of transcription. Nuclear acetylated proteins, especially ap97, would be pivotal in this signaling pathway. Although the functions of ap70 and ap78 are not evident, some signaling pathway must be employed in their induction.