Protein Kinase C Regulates Keratinocyte Transglutaminase (TGK) Gene Expression in Cultured Primary Mouse Epidermal Keratinocytes Induced to Terminally Differentiate by Calcium

Protein Kinase C Regulates Keratinocyte Transglutaminase (TGK) Gene Expression in Cultured Primary Mouse Epidermal Keratinocytes Induced to Terminally Differentiate by Calcium Andrzej A. Dfugosz and Stuart H. Yuspa Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Cancer Etiology, National Cancer Institute, Bethesda, Maryland, U.S.A.

During thefinalstage of epidermal differentiation, activation of keratinocyte transglutaminase results in covalent crosslinking of a variety of proteins to form highly protective cornified cell envelopes. We have studied the regulation of keratinocyte transglutaminase (TG^) gene expression in murine epidermal keratinocytes induced to terminally differentiate in vitro by increasing the level of extracellular Ca"*^ or treatment with the protein kinase C (PKC) activator 12O-tetradecanoylphorbol-13-acetate (TPA). Raising extracellular Ca"*"*" induces squamous differentiation of cultured keratinocytes and elicits a concentration-dependent increase in expression of TG^ mRNA; keratinocytes grown for 24 h in 0.12 mM Ca"*"*" medium express ~ 12 times as much TG^ mRNA as basal cells (grown in 0.05 mM Ca"*"*" medium), whereas cultures exposed to 1.4 mM Ca"*"*" express —17 times as much. TPA induces squamous differentiation and TG^ mRNA even in basal keratinocyte cultures grown in 0.05 m-M Ca"*"*" medium, suggesting that expression of this differentiation marker is regulated by the PKC signaling pathway.

T

he barrier function of mammalian skin can be attributed largely to the stratum corneum, a protective layer of terminally differentiated keratinocytes that provides the interface between organism and external environment. The stratum corneum is the most superficial of four cellular compartments in the epidermis, each expressing a unique pattern of keratinocyte differentiation markers. The keratin pairs K5/K14 and Kl/KlO are expressed in basal and spinous cells, respectively; loricrin, filaggrin, and involucrin are expressed in granular cells [1,2]. During the final stages of epidermal differentiation, epidermal transglutaminase generates e-()'-glutamyl) lysine bonds that cross-link substrates such as loricrin and involucrin to form a highly protective cornified cell envelope (reviewed in [3,4]). T h e analysis of keratinocytes isolated from mouse and human epidermis has identified Ca"*"*" as a key regulator of terminal differentiation in vitro [5]. Keratinocytes require medium with a reduced extracellular Ca"*^ concentration (0.05 mM) to maintain a proliferative, basal cell-like phenotype [6]. Raising Ca"*"*" in the medium to Manuscript received September 1, 1993; accepted for publication November 12, 1993. Reprint requests to: Dr. Andrzej A. Dtugosz, LCCTP/NCI/NIH, Building 37/Room 3B25, 9000 Rockville Pike, Bethesda, MD 20892. Abbreviation: TGR, keratinocyte transglutaminase.

0022-202X/94/$07.00

Induction of TG^ mRNA in response to TPA treatment is transient, reaching a peak at 6-8 h and returning to baseline by 24 h. In contrast, elevation of TG^ mRNA levels in response to Ca"^ persists for at least 24 h. The increased abundance of TGR mRNA reflects increased transcription of the TGK gene, based on nuclear run-on analysis or Ca"*"*"- and TPA-treated keratinocytes. Induction of TG^ mRNA by either TPA or Ca"*"*" is blocked in the presence of cycloheximide, suggesting that a PKC-dependent protein factor is required for TGjc gene expression in response to both stimuli. Furthermore, the accumulation of TG^ mRNA in keratinocytes treated with TPA or Ca++ is blocked in cells treated with the PKC inhibitor GF 109203X or bryostatin. These results suggest that the induction of TG^ gene expression by Ca"*^ is dependent on PKC, providing further support for the hypothesis that PKC plays a central role in regulating the late stages of epidermal differentiation. Key words: transcription / phorbol ester / GF 109203X / bryostatin. J Invest Dermatol 102:409-414, 1994

0.12 mM triggers stepwise changes in gene expression similar to those observed in epidermis; induction of the structural markers Kl and KIO is followed by the appearance of loricrin and filaggrin [7]. Epidermal transglutaminase is also activated in 0.12 mM Ca"*^ medium; however, higher transglutaminase activity is detected when cells are exposed to medium containing 1.4 mM Ca"""*" [8]. Combined with these in vitro findings, the demonstration of a Ca"*^ gradient in mouse and human epidermis, with elevated Ca++ levels detected in differentiating cell layers [9-11], suggests that Ca++ provides a physiologic signal for keratinocyte differentiation. 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inducer of epidermal transglutaminase activity and cornified envelopes both in vitro and in vivo [8,12,13], indicating that f)harmacologic activation of PKC can trigger the terminal stage of keratinocyte differentiation. Furthermore, cultured keratinocytes grown in the presence of elevated extracellular Ca"*"*" exhibit increased levels of^inositol ihosphates [14-16], intracellular Ca"*"*" [17], and diacylglycerol 16,18], suggesting that PKC is also involved in Ca"'~'"-mediated keratinocyte differentiation. Consistent with this hypothesis, Ca'*^-dependent accumulation of loricrin and filaggrin is blocked in cells where PKC has been inactivated, whereas expression of mRNA encoding these markers is enhanced by PKC activators [19]. In this report, we have used pharmacologic agents combined with changes in extracellular Ca'*"^ to determine the involvement of PKC

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Ca^^* (mM) Figure 1. The steady-state level of T G K mRNA is increased during Ca""^mediated keratinocyte differentiation. A) Primary keratinocytes grown as basal cells (in 0,05 mM Ca"*^ medium) were exposed to media with the indicated Ca"*"*" concentration for 24 h to induce terminal differentiation, Poly(A)'^ RNA was analyzed by Northern blotting using T G R and GAPDH cDNA probes, as described in Materials and Methods. T h e T G K transcript is ~3,3 kb in lengtb based on residual ribosomal RNA as markers for 5 kb and 2 kb, NB indicates RNA isolated from newborn mouse epidermis used as a positive control. Similar results were obtained in two additional experiments, B) Quantitation of Ca"'~'"-induced T G K mRNA expression by scanning densitometry, T G K mRNA at each Ca"'^ concentration was normalized to GAPDH and the fold-induction expressed relative to the level in basal cell cultures. Values represent data from three experiments db SEM,

in regulating keratinocyte transglutaminase (TG^) gene expression in vitro. Our findings indicate that activation of PKC is botb necessary and sufficient to induce TG^ gene expression in cultured keratinocytes, strongly supporting tbe concept that late stages of epidermal differentiation are regulated through Ca'^-dependent activation of the PKC pathway, MATERIALS AND METHODS Cell Culture Primary keratinocytes were isolated from skin of newborn BALB/c mice as previously described [6] and cultured iri 60-mm tissue-culture dishes (Costar, Cambridge, MA), Cells were grown in Eagle's minimum essential medium (without Ca"*^ or Mg""^) supplemented with 8% Ca''^-depleted fetal calf serum [6] and 0,25% penicillin-streptomycin solution (GIBCO, Grand Island, NY), The Ca"*^ concentration in the medium was adjusted to specific levels by adding an appropriate volume of 280 mM CaCl2, Cells grown in medium with 0,05 mM Ca"*^ exhihited a basal cell-like phenotype; terminal differentiation was induced by exposure to medium witb higher extracellular Ca"*^ concentrations, as described in the figure legends.

Northern Blot and Nuclear Run-On Analysis Total RNA was isolated from primary keratinocytes by lysis in 4 M guanidine isotbiocyaiiate followed hy ultracentrifugation through a cesium chloride gradient [20], In some experiments, poly(A)"'" RNA was isolated directly from cell lysates as previously described [21], Transcripts were separated by electrophoresis in a 1% agarose/0,66 M formaldehyde gel, transferred to reinforeed nitrocellulose (BA-S NC; Schleicber & Schuell, Keene, NH), an
Figure 2. TPA induces T G R mRNA expression. A) Keratinocyte cultures grown in 0,05 mM Ca"*"*" medium were exposed to TPA at the indicated concentrations for 4 h. Total RNA was isolated and Northern blot analysis performed as described in Materials and Methods. B) Scanning densitometry of Northern hlot in (A), The level of T G K mRNA was normalized to GAPDH and changes in abundance expressed relative to the DMSO-treated control.

vacuum oven [7], Conditions for Northern blotting were previously described [22], except that pre-hybridization and hybridization solutions contained 6 X sodium citrate/sodium chloride buffer (SSC), 5 X Denhardt's solution, 0,5% sodium dodecyl sulfate (SDS), 100 H%/m\ sheared salmonsperm DNA, and 50% formamide [23], T G K rnRNA was identified by hybridization to rat or human T G K C D N A fragments (~2 kb) [24], kindly provided by Dr, Robert Rice (University of California, Davis, CA), The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was a fulllength rat cDNA in pUC18 [25], Probes were labeled with ^^P-CTP by nick-translation. Filters were routinely washed at a maximum stringency of 0,5 X SSC with 0,2% SDS, at 65°C, Transcript levels were quantified using a scanning laser densitometer and ImageQuant software (Molecular Dynamics, Sunnyvale, CA), The abundance of T G K mRNA was normalized to GAPDH transcripts. In vitro transcription reactions were performed as previously described [25] witb modifications [19], Briefly, nuclei were isolated from ~ 1 X 10^ cells and stored at - 7 0 ° C in 200/xl of buffer containing 50 mM tris, pH 8,3, 5 mM MgClj, 0,1 mM ethylenediaminetetraacetic acid (EDTA), and 40% glycerol. Nuclei were added to 200 //I of 2 X reaction buffer that contained 10 mM tris, pH 8,0, 5 mM MgClj, 0,3 M KCl, 200 U/ml RNAsin (Promega, Madison, WI), 500 fiM. ATP, CTP, and GTP, 10 //M UTP, and 200 or 250/iCi ^^P-UTP (800 Ci/mmol, New England Nuclear [NEN], Boston, MA), Nascent transcripts were elongated for 30 min at 30°C, Nuclei were lysed in 4 M guanidine isothiocyanate and total RNA isolated as described above. Ten micrograms of plasmid DNA (rat T G K *nd GAPDH, described above) was immobilized on nitrocellulose filters and bybridization to radiolabeled transcripts performed as previously described [19], "'I-Epidermal Growth Factor (EGF) Binding Assay Binding of'"Ilabeled EGF to primary keratinocytes was determined as previously described [27], witb minor modifications. Following treatment, cells were washed twice with ice-cold binding buffer (Dulbecco's minimum essential medium with 50 mM N,N-bis-[2-hydroxyethyl]-2-aminosulfonic acid [pH 7,4] and 1 mg/ml hovine serum albumin), then incuhated with '^^I-EGF (0,1 /iCi/ml/well; NEN) ± excess unlabeled EGF (1 ^g/tnl, receptor grade; CoUahorative Research, Bedford, MA) in 1 ml of binding buffer for 5 h on a bed of ice. Cultures were washed four times with ice-cold binding buffer and cells harvested in two 500 jA volumes of lysis buffer (0,1 M tris, pH 7,4,0,5% SDS, 1 mM EDTA), Radioactivity was determined hy scintillation counting; non-specific binding was < 200 cpm in all groups, :^

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Reagents TPA was obtained from LC Services (Woburn, MA), bryostatin (bryostatin 1) was a gift from Dr. George Pettit (Arizona State University, Tempe, AZ), and the PKC inhibitor GF 109203X was kindly provided by Dr. Jorge Kirilovsky (Glaxo Pharmaceuticals, Les Ulis, France). TPA and GF 109203X stocks were in dimethylsulfoxide; bryostatin was in ethanol.

RESULTS Ca^^ and TPA Induce TGK mRNA in Cultured Keratinocytes TGK gene expression was examined in pprimary mouse epiy g p ddermal keratinocytes growing i as bbasal l cells ll (i(in 005 M C^' 0.05 mM dium) or induced to terminally differentiate in medium containing 0.12 or 1.4 mM Ca"*"*". Although expression of structural differentiation markers is restricted to extracellular Ca"*"*" ranging from 0.1 to 0.3 mM [7], a squamous phenotype is triggered at all Ca'*^ concentrations > 0.1 mM Ca""^ [8]. In primary epidermal keratinocyte cultures, extracellular Ca'*"'" induces TG^ mRNA in a concentration-dependent manner. Based on densitometric scanning of Northern blots, keratinocytes grown for 24 h in 0.12 niM Ca"'"'' medium express ~12 times as much TG^ mRNA as basal cells (grown in 0.05 mM Ca"'"'" medium), whereas cultures grown in 1.4 mM Ca'*"'" express ~ 17 times as much (Fig 1). In individual experiments, TGK mRNA expression in 1.4 mM Ca"'""'" medium was always higher (by 37% ± 13% SEM, n = 3) than in 0.12 mM Ca"'"^ medium. 1.4 mM Ca"'"^ medium was used in subsequent experiments to induce terminal differentiation, unless otherwise indicated. Because PKC activators can substitute for Ca"'""'" to induce epidermal transglutaminase activity [28], we examined the effects of TPA on TGK gene expression in vitro. In this experiment, treatment of basal cell cultures with TPA for 4 h caused a maximal ninefold increase in the level of TG^ mRNA with a 50% effective dose (ED50) of ~ 10 nM (Fig 2). Similar to Ca"'"'" (Fig IB), the level of T G K mRNA induction by TPA was variable between experiments, probably due to different baseline levels of TG^ mRNA in basal cell cultures. In four experiments, induction of TGK rnRNA following exposure to 100 rLM TPA for 4 h varied from ninefold to 78-fold (average = 46-fold ± 14, SEM). Combined, these results indicate that keratinocyte differentiation induced by either Ca"'""'" or TPA is associated with increased expression of TGK mRNA. To further explore the regulation of TGK gene expression, keratinocytes were treated with Ca"'""'" or TPA alone or with both agents combined. Total RNA was isolated at the start of the experiment and after 1, 2, 4, 6, 10, and 24 h of treatment. Based on densitome-

tric scanning of Northern blots in this experiment, TGK mRNA was increased a maximum 179-fold in TPA-treated cultures (at 6 h) and 8.8-fold in Ca"'"'"-treated cultures (at 10 h). In cultures treated with Ca''""'' -I- TPA for 6 h, TGK transcripts were elevated to a maximum level 201 times greater than in basal cell cultures (Fig 3). Although the initial induction of TGK mRNA was detected earlier in response to TPA than Ca"'^ (2 vs 4 h), TGK rnRNA expression was reduced to baseline after 24 h exposure to TPA both in the presence or absence of Ca''"'" (Fig 3). In contrast, induction of TGK rnRNA in response to Ca"'""'" alone remained maximally elevated through 24 h (Fig 3). TGK mRNA Expression is Regulated at the Transcriptional Level Nuclear run-on analysis was performed to determine whether Ca''"'" and TPA affect TGK gene expression at the transcriptional level. For these experiments, cultures were induced to differentiate in mediuni with 0.12 mM Ca''"'" rather than 1.4 mM Ca"'"'" because of difficulties in the isolation of nuclei from keratinocytes grown at high extracellular Ca"'"'" concentrations. The relative transcription rate of the TGK gene is increased during 0.12 mM Ca"'"'"induced keratinocyte differentiation (Fig 4A). In differentiating keratinocytes exposed to TPA for 3 h, TGK transcription is induced to a greater extent (Fig 4B). These findings suggest that accumulation of TGK mRNA in response to Ca"'""'" or TPA is due at least in part to increased transcription of the TGK gene. TGK niRNA Induction is Dependent on Protein Synthesis To further characterize tbe molecular regulation of TGK gene expression, primary keratinocytes were exposed for 6 h to TPA or solvent, ± Ca''"'", in the presence or absence of 20/zg/ml cycloheximide. This concentration of cycloheximide has previously been shown to block protein synthesis by > 95% in cultured mouse epidermal keratinocytes [28]. Accumulation of TGK mRNA is blocked by cycloheximide in cultures treated with TPA, Ca"'""'", or Ca"'""'" -lTPA (Fig 5), suggesting that a protein factor is required for the induction of TGK gene expression by both stimuli. GAPDH mRNA levels do not appear to be influenced by cycloheximide (Fig 5). Induction of TG^ mRNA by Ca++ or TPA Requires PKC Tbe induction of TGK mRNA by TPA even in basal cell keratinocyte cultures suggested that expression of this marker in response to Ca"'"'' occurs through activation of the PKC signaling pathway. To test this hypothesis, TGK mRNA expression was examined in cultures treated with the selective PKC inhibitor GF

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109203X [29], The effect of this compound on inhibition of ' " I EGF binding by TPA [30] was examined as a measure of its ability to block PKC-mediated events in cultured keratinocytes, GF 109203X blocked this response to TPA in a dose-dependent manner, with control levels of'^^I-EGF binding achieved at S: 1 y/M GF 109203X (Fig 6A), suggesting that this agent is an effective PKC inhibitor in cultured keratinocytes, GF 109203X also inhibited TPA- and Ca"^-mediated accumulation of T G R mRNA (Fig 6B, C). Based on results of scanning densitometry, tbe approximate IC50S

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Figure 6. Induction of T G K mRNA is blocked by the PKC inhibitor GF 109203X, A) GF 109203X blocks TPA-mediated inhibition of '^^I-EGF binding. Primary keratinocytes grown in 12-well dishes were treated with tbe indicated concentration of GF 109203X for 45 min, 100 nM TPA or 0,1% DMSO was added and binding of '^M-EGF determined after a 2-h incubation, as described in Materials and Methods. Triplicate cultures were assayed at each dose, witb the SEM indicated by error bars. Similar results were obtained in an additional experiment, B) Basal cell cultures were treated with tbe indicated concentration of GF 109203X for 1 h, followed by exposure to 100 nM TPA for 6 h. Northern blot analysis was performed using total RNA as described in Materials and Methods. C) Basal keratinocyte cultures were exposed to 1,4 mM Ca++ medium + GF 109203X for 8 h. In two otber experiments, GF 109203X blocked accumulation of T G K mRNA in 0,12 mM Ca"*^ medium at a dose of 5 or ' " " '

for blocking T G R mRNA induced by either Ca++ or TPA are witbin the same order of magnitude. As an additional approach to assess PKC's involvement in TG^ gene expression, keratinocytes were pre-treated witb 60 nM hryostatin to functionally inactivate PKC, This agent selectively blocks a variety of PKC-mediated responses in cultured keratinocytes [ 3 1 33], Both TPA- and Ca++-mediated induction of T G K mRNA is blocked in keratinocytes pre-treated with bryostatin (Fig 7), Similar to the results with GF 109203X (Fig 6), tbe approximate IC50S for blocking T G R gene expression in TPA- or Ca++-treated cultures are within the same order of magnitude, based on densitometric analysis. Combined, the results of these experiments suggest that activation of the PKC pathway is required for the induction of T G R gene expression in response to either TPA or Ca++. DISCUSSION

Figure 5. T G K mRNA induction by Ca"*"*" or TPA is blocked in tbe presence of cycloheximide. Fresh medium ± 20 Hg/m\ cycloheximide was added to basal cell cultures. After 30 min, Ca^^, TPA (100 nM), or botb agents were added to control and cycloheximide-treated cultures. Total RNA was isolated after 6 h for Northern blot analysis. Similar results were obtained in an additional experiment.

Keratinocyte differentiation can be induced in vitro by raising extracellular Ca++ from 0.05 to > 0.1 mM, and elevated Ca++ levels have been demonstrated in vivo in differentiating layers of epidermis, suggesting that this ion provides a physiologic signal regulating epidermal differentiation. Based on analysis of T G K gene expression as a marker for keratinocyte differentiation, tbe results of this study suggest that PKC mediates the Ca++ signal for this aspect of kerati-

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Figure 7. Induction of TG^ mRNA is blocked in keratinocytes where PKC has been inactivated using hryostatin. Basal cell cultures were pre-treated with the indicated concentrations of bryostatin for 22 h, followed by exposure to 100 nM TPA for 6 h (A) or 1.4 mM Ca^^ medium for 23 h (B), ± bryostatin. 60 nM hryostatin blocked induction of TGR mRNA by TPA or 0.12 niM Ca"*^ medium in an additional experiment.

nocyte differentiation: 1) botb Ca"*"*" and TPA induce T G K gene expression in cultured keratinocytes; 2) induction of T G R m R N A by either Ca"*"^ or TPA is dependent on a cycloheximidesensitive factor; 3) pharmacologic inhibition of PKC blocks both Ca"'"*'-and TPA-mediated induction of TG^ gene expression. Together with recent findings implicating PKC in Ca"'""'"-mediated expression of the granular cell markers loricrin and filaggrin [19], these results strongly support the concept that PKC plays a fundamental role in regulating the late stages of epidermal differentiation.

T h e involvement of Ca"*""*" in regulating T G K gene expression differs from that for other keratinocyte-specific markers. In cultured mouse epidermal keratinocytes, spinous (Kl, KIO) and granular (loricrin, filaggrin) markers are induced by extracellular Ca"*""*" in a limited concentration range between 0.10 and 0.16 mM [7]. In medium with 1.4 mM Ca"*""*", these structural markers are not induced [7], whereas expression of T G K mRNA is maximally elevated (Fig 1), consistent with the predominantly squamous phenotype observed under these culture conditions. In light of these in vitro observations it is noteworthy that the highest levels of intracellular Ca"*""*" have been localized to cells in the upper granular layer of both mouse and human epidermis [10,11]. Preferential expression of T G K ^t this stage of epidermal differentiation would favor crosslinking of pre-existing structural proteins into cornified cell envelopes, which are largely responsible for the stratum corneum's protective function. A second distinction between the regulation of T G K and other keratinocyte markers is the ability to induce expression of the former in 0.05 mM Ca++ medium by treating with TPA (Fig 2), suggesting that PKC activation is sufficient to induce expression of TGK. In contrast, expression of the granular cell markers loricrin and filaggrin appears to be dependent on a specific intracellular Ca'^'^ level in addition to activation of the PKC pathway [19]. A direct requirement for Ca"*^ in regulating expression of keratinocy te structural markers is supported by the recent demonstration of a Ca"'"'*"-responsive element in the 3' non-coding region of the human keratin 1 gene [34].

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Although the role of Ca"*""" in triggering keratinocyte-specific gene expression is well establisbed, tbe biochemical pathways regulating this complex process have not been fully elucidated. Induction of epidermal transglutaminase activity and cornified envelope formation by TPA, both in vitro and in vivo [8], suggested that the terminal stage of keratinocyte differentiation is regulated by PKC. The observation that increased extracellular Ca"*""*" is associated with elevated cellular diacylglycerol levels [16,18] provided a biochemical link coupling the Ca"""*" signal for keratinocyte differentiation to the PKC signaling pathway. The ability to block Ca"'~'"-mediated T G K gene expression by inactivating PKC in mouse (Figs 6,7) as well as human [35] keratinocytes strongly suggests that Ca"*"*" induces this differentiation marker through the PKC signaling pathway. Along with the upregulation of T G K gene expression by PKC, the additional influence oT other factors is likely to be important in determining the final level of expression. For example, whereas dexamethasone also induces T G K mRNA in cultured keratinocytes, retinoic acid blocks accumulation of T G K mRNA in response to TPA, Ca++, or dexamethasone [36]. There are several distinctions between TPA- and Ca"'""'"-induced T G K gene expression (Fig 3) that may be related to the way these two stimuli influence PKC: 1) TPA induces T G K mRNA more rapidly than Ca"*""*". The slower induction of T G K mRNA in Ca"*"*"treated cells may reflect slow accumulation of endogenous diacylglycerol with a corresponding delay in PKC activation [16]. In contrast, TPA directly activates PKC resulting in expression of certain genes {e.g.,fos andftm) within minutes, others (e.g., transin and collagenase) within hours [37]. 2) T G K rnRNA is induced to a higher level by TPA than Ca'*'+. Consistent with this result, phorbol esters are far more effective activators of PKC than physiologic agents (hormones, growth factors, etc.) that generate endogenous diacylglycerols via hydrolysis of membrane phospholipids. 3) Despite the more rapid appearance and greater induction of T G K mRNA in response to T"PA, by 24 h transcript levels return to baseline, whereas in Ca"'""'"-treated keratinocytes T G K mRNA remains maximally elevated. Transient induction of T G K mRNA by TPA may be due to PKC downregulation [38] resulting in a limited activation phase in TPA-treated cultures and brief induction of T G K mRNA. Unlike TPA, Ca++ does not downregulate PKC in cultured keratinocytes* [39], permitting prolonged activation of the PKC pathway in response to this signal. Relative to Ca++-treated cultures, the low level of T G K mRNA in cultures exposed for 24 h to Ca++ + TPA supports the notion that PKC is required for prolonged expression of T G K mRNA in response to Ca"*""*". Inhibition of Ca+"^- and TPA-mediated T G K mRNA induction by cycloheximide (Fig 5) suggests that T G K gene expression is dependent on a protein factor(s) that is regulated by PKC. This result may explain findings reported in an earlier study in which TPAmediated transglutaminase activity was blocked by cycloheximide [28]. Recent documentation of an AP-1 site upstream of the human T G K initiation codon [40] suggests that PKC may regulate transcription of the T G K gene via Fos and Jun family members. Supporting this notion, TPA increases luciferase activity in cells transfected with a rabbit T G K promoter reporter construct, and this increase is blocked by bryostatin [41], Additional studies are needed to determine whether PKC-mediated changes in transcript stability also contribute to the increased levels of T G K mRNA seen in TPAand Ca'*"'''-treated cultures. Ten PKC isoforms have been described [42]; five of these, PKC a, S, e, f, and t], are expressed in cultured mouse epidermal keratinocytes* [43]. Studies in several cell types indicate that different PKC isozymes have distinct functions [44,45], The restricted expression of PKC t] in granular cells suggests that this isoform is involved in regulating late stages of epidermal differentiation [46]. Experiments are currently underway to directly assess the role of individual PKC isozymes in regulating keratinocyte differentiation in vitro. * Denning MF, Dtugosz AA, Yuspa SH: Redistribution of specific protein kinase C isozymes accompany Ca^''"-induced keratinocyte differentiation {zhstr).J Invest Dermatol 100:495, 1993.

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The results of this study may have implications for the treatment of a variety of skin disorders. For example, marked thickening of the stratum corneum is frequently a prominent feature in psoriasis. This change may be secondary to the increased T G K mRNA levels [47] and transglutaminase activity [48] that have been reported in lesional skin. Identification of PKC as a key regulator of T G K gene expression suggests that selective inhibitors of this signaling pathway may be useful in treating dermatoses characterized by hyperkeratosis.

21.

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