The α and ηη Isoforms of Protein Kinase C StimulateTranscription of Human Involucrin Gene

Hidetoshi Takahashi, Kazuhiro Asano, Akira Manabe, Motoshi Kinouchi, Akemi Ishida-Yamamoto, andHajime IizukaDepartment of Dermatology, Asahikawa Medical College, Asahikawa, Japan

Involucrin is one of the precursor proteins of the cornifiedcell envelope that is formed beneath the cell membraneduring terminal differentiation of keratinocytes. 12-O-tetradecanoylphorbol-13-acetate (TPA), which is a potentprotein kinase C (PKC) activator, induces terminal differ-entiation of keratinocytes. We previously demonstratedthat involucrin promoter activity is stimulated by TPAin cultured fetal rat skin keratinocytes. PKC is a largefamily of proteins and keratinocytes containing five PKCisozymes: α, δδ, ε, ηη, and ζζ. In order to determine therole of the PKC isozyme(s) on involucrin gene expression,we constructed the chloramphenicol acetyl transferase(CAT)-involucrin promoter expression vector by con-necting the 59-upstream region of the human involucringene containing the untranslated first exon to the CATreporter gene. The CAT-involucrin promoter expressionvector was transfected with various PKC isozyme expres-sion vectors into SV40-transformed human keratinocytes(SVHK cells). Transfection of the CAT-involucrin pro-moter expression vector with PKC-α or PKC-ηη expres-sion vectors resulted in a significant increase in theTPA-dependent involucrin promoter activity. The PKCinhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperaz-ine dihydrochloride, inhibited the promoter activitystimulated by TPA. Transfection of PKC-δδ, -ε, and

The cornified cell envelope (CE) is a highly insolublestructure formed beneath the plasma membrane ofkeratinocytes during terminal differentiation (Hohl,1990; Goldsmith, 1993; Eckert et al, 1993). The CEprovides a protective barrier between the environment

and the living layers of the skin, and is believed to play an importantrole in maintaining the structural integrity of the epidermis. Transgluta-minase 1 catalyzes the glutamyl-lysine cross-linking reaction of variousCE precursor proteins that include involucrin (Eckert and Green,1986), loricrin (Hohl et al, 1991), cystatin A (keratolinin) (Takahashiet al, 1997), small proline-rich protein(s) (Kartasova and van de Putte,

Manuscript received February 21, 1997; revised October 15, 1997; acceptedfor publication November 4, 1997.

Reprint requests to: Dr. Hidetoshi Takahashi, Department of Dermatology,Asahikawa Medical College, 3–11 Nishikagura, Asahikawa 078, Japan.

Abbreviations: CE, cornified cell envelope; H-7, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride; INV-CAT, CAT-involucrin promoterexpression vector; SVHK, SV40-transformed human keratinocytes; TRE, TPAresponsive element.

0022-202X/98/$10.50 · Copyright © 1998 by The Society for Investigative Dermatology, Inc.

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-ζζ had no effect on the involucrin-promoter activity.Although the promoter activity was stimulated by trans-fection of PKC-γγ, TPA did not enhance the promoteractivity in the PKC-γγ-transfected SVHK cells. Previouslywe showed three AP-1 binding sites (AP1–1, –2, and –3)on the involucrin promoter region. Both the basal andthe TPA-stimulated involucrin promoter activities weresuppressed by deleting the AP1–1 site (–119 to –113)that is the most proximal to the transcription start site.The deletion of AP1–2 (–297 to –303) or AP1–3(–447 to –453) did not affect the involucrin promoteractivity. Gel retardation analyses disclosed that TPAstimulated the specific DNA binding of the nuclearprotein(s) of control, PKC-α, or PKC-ηη-transfectedSVHK cells, but not of PKC-γγ-transfected cells. Additionof anti-c-Jun and anti-c-Fos antibodies decreased thespecific protein-DNA complex band with a concomitantappearance of supershifted bands. These results indicatethat PKC, specifically PKC-α and PKC-ηη, mediates theTPA-dependent activation of involucrin gene expressionof SVHK cells. PKC-γγ, which is not present in ker-atinocytes, also induces involucrin gene expression in aTPA-independent manner, when introduced into SVHKcells. Key word: cornified cell envelope. J Invest Dermatol110:218–223, 1998

1988), elafin (Steinert and Marekov, 1995), and envoplakin (Ruhrberget al, 1996). Recent evidence suggests that involucrin is an earlycomponent of CE and provides a scaffold onto which several otherprecursor proteins are incorporated (Steinert and Marekov, 1995;Ishida-Yamamoto et al, 1997).

Human involucrin gene contains a 59 exon of 43 bp and a 39 exonof 2107 bp that are separated by an intron of 1188 bp (Eckert andGreen, 1986). The structure is characterized by a central segmentcomposed of 39 tandem repeats of 10 amino acids, which is flankedby N- and C-terminal segments. The repeating structure of the centralportion is highly conserved in all higher primates (Eckert and Green,1986), suggesting recent evolutionary origin of involucrin. Involucrinis expressed in the squamous tissue, and is detected in the upperspinous layer and the granular layer of normal human epidermis (Banks-Schlegel and Green, 1981).

Although considerable information is available regarding the functionand the evolution of involucrin protein (Eckert et al, 1993), less isknown about the regulation of the involucrin gene expression. Trans-genic animal studies have shown that 2.5 kb of the 59 upstreamsequence of the involucrin gene is sufficient to confer to differentiation-

VOL. 110, NO. 3 MARCH 1998 REGULATION OF INVOLUCRIN GENE BY PKC 219

dependent and tissue-specific expression of the involucrin (Crishet al, 1993, Carrol et al, 1993). 12-O-tetradecanoylphorbol-13-acetate(TPA), a potent protein kinase C (PKC) activator, induces involucringene expression through a nuclear factor, AP1. The AP1, which is acomplex consisting of jun and fos family proteins, binds to TPA-responsive elements (TRE) and regulates the TPA-inducible genes.The promoter of the human involucrin gene has at least five TRE(Takahashi and Iizuka, 1993; Welter et al, 1995; Lopez-Bayghen et al,1996). These AP1 binding sites are designated as AP1–1 to AP1–5;AP1–1 is the most proximal to the transcription start site and AP1–5is the most distal. We and others have also demonstrated that TEF-1,POU factors, and YY-1 decrease the transcription of the involucrin gene(Takahashi et al, 1995; Welter et al, 1996; Lopez-Bayghen et al, 1996).

PKC is a large family of proteins that consists of at least 11 isozymes(Nishizuka, 1992). PKC-α, -βI, -βII, and -γ are the classical PKC,which depend on calcium and diacylglycerol. PKC-δ, -ε, -η, -θ, and-µ are the novel PKC, which do not require calcium for activation.PKC-ζ and -τ are the atypical PKC, which require neither calciumnor diacylglycerol for the activation. TPA activates classical and novelPKC but not atypical PKC. The keratinocytes contain PKC-α, -δ,-ε, -η, and -ζ (Dlugosz et al, 1992). The specific function of eachPKC isozyme, especially in terms of involucrin gene expression,remains unknown at present.

SV40-transformed human keratinocytes (SVHK cells) are well-established immortalized cell lines sharing features of normal humankeratinocytes (Steinberg and Defendi, 1983; Takahashi et al, 1990).SVHK cells express relatively high amounts of involucrin comparedwith other cell lines, such as A431 and SCC13 (data not shown). Inthis study, we constructed the CAT-involucrin promoter expression(INV-CAT) vector, which was subcloned from the 59-upstream regionof the involucrin gene with the untranslated first exon and wasconnected to the chloramphenicol acetyl transferase (CAT) reportergene. Using the INV-CAT vector, we investigated the effects of PKCisozymes on the involucrin promoter activity of SVHK cells.

MATERIALS AND METHODS

Plasmids constructs The Sca I-digested fragment from the genomic involuc-rin gene was inserted into the Xba I site of promoterless 0-CAT plasmid, whichhad been blunt-ended by Klenow fragment (INV-CAT) (Takahashi and Iizuka,1993). The β-galactosidase expression vector was kindly supplied by Dr. T.Watanabe (Medical Institute of Bioregulation, Kyushu University, Japan). PKCexpression vectors were generous gifts from Dr. S. Ohno (Department ofMolecular Biology, Yokohama City University School of Medicine) (Ohnoet al, 1988). Each deleted fragment was generated by an ‘‘overlap extension’’method (Ho et al, 1989) using polymerase chain reaction (PCR) using oligo-nucleotides (DL–1, 59-CTTATGAGCATGGCATTCCTGAGAA-39; DL–2,59-TTTGAAGATCTTCCCATGAAGGGGT-39; DL–3, 59-TCATGGGAA-GATCTTCA-39; DL–4, 59-TGTGACCAACTTCTGCTCTG-39; DL–5, 59-AGCAGAAGTTGGTCACA-39; DL–6, 59-ACCCCTTCCCCACAGGC-ATC-39; DL–7, 59-TGTCCCTGGACCCTAAAGGGTTTGC; DL–8, 59-AGGAGTCAGGGCACACTTTATATCA). The positions of these sequencesare indicated in Fig 1. In order to construct the AP1–3 deleted vector (T3),we performed two PCR amplifications using DL–1 and DL–2 oligomers orDL–3 and DL–8 oligomers using the INV-CAT vector as the template. Nextwe performed a PCR procedure using DL–1 and DL–8 oligomers using mixedPCR product derived from DL–1 and DL–2 oligomers or DL–3 and DL–8oligomers. The second PCR product, which contained two Pst I sites (Fig 1,double underline), was digested by Pst I restriction enzyme and was subclonedinto the Pst I sites of the deleted INV-CAT vector, which was deleted at theposition of the Pst I sites. Using DL–1 and DL–8 oligomers, we performedsequence analysis and confirmed the correct promoter orientation and thedeleted portion. AP1–2-deleted (T2) and AP1–1-deleted (T1) vectors wereconstructed using DL–1, DL–4, DL–5, and DL–8 oligomers, and DL–1,DL–6, DL–7, and DL–8 oligomers, respectively. Using T1, T2, and T3 vectors,we performed PCR with these oligomers and constructed T12 (AP1–1,–2-deleted), T13 (AP1–1, –3-deleted), T23 (AP1–2, –3-deleted), and T123(AP1–1, –2, –3-deleted) vectors.

Cell culture SV40-transformed human keratinocytes were a generous giftfrom Dr. M.L. Steinberg (Department of Chemistry, City College of the CityUniversity of New York) (Steinberg and Defendi, 1983). Cells were culturedin Dulbecco’s modified Eagle’s medium supplemented with 5% fetal calf serum,100 u penicillin per ml, and 100 µg streptomycin per ml at 37°C in 5% CO2in air.

Figure 1 Four AP1 sites are present on the 59-upstream region of thehuman involucrin gene. DL–1–8 indicate the positions of the primers.Double underlining indicates the positions of the restriction enzyme, Pst I. Thebold letter A is the transcriptional start site. The four shaded boxes indicateputative TRE sites, which are designated as AP1–1 to AP1–4.

Transfection and CAT assay Transfection of plasmid DNA into these cellswas performed by the liposome method using lipofectin (Felgner et al, 1987).Typically, 5 µg of reporter plasmid and 2 µg of β-galactosidase plasmid weretransfected into 1 3 105 SVHK cells. The β-galactosidase plasmid was used asthe internal standard to normalize each transfection efficacy. After 48 h, cellswere collected and CAT assay was performed (Neuman et al, 1987). Theenzyme activity of β-galactosidase in the transfected cell extracts was measuredspectrophotometrically (Maniatis et al, 1989).

PKC enzyme assay PKC enzyme assay was performed using the proteinkinase C enzyme assay kit (Amersham Japan, Tokyo, Japan). Briefly, 1 3 106

transfected SVHK cells were homogenized in the extraction buffer [50 mM Tris-HCl (pH 7.5), 5 mM ethylenediamine tetraacetic acid, 10 mM ethyleneglycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid, 0.3% (wt/vol) β-mercapto-ethanol, 10 mM benzamidine, 50 µg phenylmethylsulfonyl flouride per ml].The homogenates were centrifuged and the supernatants were assayed accordingto the protocol equipped with the assay kit.

Nuclear extraction and gel retardation analyses Nuclear extraction andgel retardation analyses were performed according to the method previouslydescribed (Takahashi et al, 1995). The oligonucleotide probe corresponds to–125 to –104, which includes the AP1–1 site.

Materials Dulbecco’s modified Eagle’s medium was purchased from Gibco(Grand Island, NY). Penicillin and streptomycin were obtained from M.A.Bioproducts (Walkersville, MD). [3H]-Acetyl coenzyme A, [γ-32P]ATP, and[α-32P]deoxycytidine thiotriphosphate were purchased from Amersham (Tokyo,Japan). Lipofectin was obtained from BRL (Bethesda, MD). Anti-c-jun,-junB, -junD, -c-fos, -NFκB, and -fra–1 antibodies were purchased from SantaCruz Biotechnology (Santa Crus, CA). Various isozyme-specific anti-proteinkinase C antibodies were purchased from Beohringer (Penzberg, Germany).

RESULTS

Transfection of PKC isozyme expression vectors increased PKCactivity in SVHK cells We transfected various PKC isozymeexpression vectors into SVHK cells. The increased expression of PKC-α was confirmed by immunoblot analysis and was shown to be plasmiddose-dependent (Fig 2). These effects were detected by the transfectionof other PKC expression vectors (data not shown). The PKC expressionvector-transfected SVHK cells showed increased PKC activity byaround 2-fold compared with the activity of SVHK cells withoutthe transfection (Table I). TPA stimulated the PKC activity of thetransfected SVHK cells by around 6-fold, except for PKC-ζ-transfectedSVHK cells (Table I).

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Figure 2 PKC-α was expressed in SVHK cells in a concentration-dependent manner by the transfection. One 3 105 SVHK cells weretransfected with various amounts (0–3 µg) of PKC-α expression vector.Transfected cells were incubated for 48 h and western blot analysis wasperformed.

Figure 3 Transfection of PKC-α or PKC-ηη expression vectors increasedinvolucrin promoter activity of TPA-treated SVHK cells. Various PKCisozyme expression vectors (5 µg each) were transfected into 1 3 105 SVHKcells with INV-CAT vector (5 µg), and the transfected cells were incubated for24 h. The transfected cells were then cultured in the presence or absence ofTPA (10 ng per ml) for 24 h. The CAT activity of SVHK cells transfected withINV-CAT alone without TPA stimulation was designated as 1.0. C, controlvector; α, PKC-α; β, PKC-β; δ, PKC-δ; ε, PKC-ε; γ, PKC-γ; ζ, PKC-ζ; η,PKC-η.

Table I. Effects of PKC expression vector on PKC enzymeactivity of SVHK cells

Expression vector PKC enzyme activity (pmoles per min per mg)a

Medium TPA

control vector 58.6 6 13.1 150.3 6 14.5PKC-α 115.5 6 22.3 698.8 6 26.7PKC-β 106.5 6 20.3 647.8 6 29.4PKC-γ 125.3 6 19.2 678.3 6 19.6PKC-δ 111.6 6 17.5 688.2 6 26.9PKC-ε 121.1 6 15.2 700.9 6 30.2PKC-ζ 109.8 6 21.1 199.4 6 28.3PKC-η 105.9 6 23.5 630.4 6 32.1

aResults are the means 6 SEM.

Transfection of PKC-α or PKC-ηη expression vectors increasedinvolucrin promoter activity in TPA-treated SVHK cells Todetermine the role of PKC isozymes on involucrin gene expression,we performed co-transfection of the INV-CAT vector with variousPKC isozyme expression vectors (Fig 3). Consistent with TPA-inducedactivation of endogenous PKC, involucrin promoter activity wasincreased by TPA in SVHK cells transfected with control vector (Fig 3,lane C). Transfection of PKC-β, -δ, -ε, -ζ had no effect on theinvolucrin promoter activity compared with the transfection of thecontrol vector. Although the transfection of these vectors also increasedthe promoter activity in the presence of TPA, the augmentation wasnot significantly different from that of the control vector-transfectedcells (Fig 3, lanes β, δ, ε, ζ). Involucrin promoter activity, however,

Figure 4 Increased promoter activity by the transfection of PKCexpression vector was suppressed by PKC inhibitor H-7. Following co-transfection of INV-CAT vector and PKC isozyme (α, γ, η) expression vectors,SVHK cells were incubated with TPA (10 ng per ml) or H-7 (100 µM), orboth, for 24 h. The CAT activity of cells transfected with INV-CAT alonewithout TPA stimulation was designated as 1.0.

was significantly stimulated by the co-transfection of PKC-α or PKC-η in a TPA-dependent manner (Fig 3, lanes α, η). The promoteractivity was stimulated by the transfection of PKC-γ in the absence ofTPA (Fig 3, lane γ). TPA did not augment the promoter activity ofPKC-γ-transfected SVHK cells. The PKC inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride (H-7), significantlyinhibited the promoter activity stimulated by TPA in control, PKC-α, and -η expression vector-transfected SVHK cells (Fig 4). Theaddition of H-7 singly to the incubation medium significantly inhibitedthe promoter activity of PKC-γ-transfected SVHK cells, but had noeffect on other PKC vector-transfected SVHK cells (Fig 4). Theseresults indicate that the activation of PKC-α and -η results in stimulationof involucrin promoter activity and that PKC-γ introduced into SVHKcells is endogenously activated in the absence of TPA.

AP1–1 site (–119 to –113) was critical for involucrin promoteractivity The 59-upstream promoter region of the INV-CAT vectorcontains three AP-1 binding sequences (AP1–1, –2, and –3) (Fig 1).In order to determine the critical TRE region(s) in the INV-CATvector, we constructed various TRE-deleted vectors. The transfectionof AP1–2- or AP1–3-deleted vectors had no effect on the involucrinpromoter activity that was stimulated by TPA. The transfection ofAP1–1 deleted vectors (T1, T12, T13, T123), however, showed asignificant decrease in the basal involucrin promoter activity and TPA-dependent stimulation was abolished (Fig 5). In addition, involucrinexpression was not stimulated by TPA in PKC-α- or PKC-η-transfectedSVHK cells, when AP1–1 was deleted from the INV-CAT vector(Fig 6); the deletion of AP1–2 or –3 again had no effect. The putativeAP1–4 (–512 to –505), which is also present in our INV-CAT vector(Welter et al, 1995), was apparently not functional, because our deletionanalysis showed negligible effect on the promoter activity (Figs 5, 6).This is consistent with the results of Welter et al (1995), indicating thatwithin the promoter region up until –811 bp, the AP1–1 region iscritical for the regulation of the basal and PKC-stimulated involucrinpromoter activity in SVHK cells.

Nuclear factor(s) from PKC-α and -ηη-transfected SVHK cellsspecifically bound to the AP1–1 site that is stimulated byTPA The interaction between the AP1–1 sequence and the AP-1nuclear protein(s) was investigated by gel shift analyses (Fig 7A).Control-vector-transfected SVHK cells showed a specific band indicat-ing the interaction of nuclear protein with AP1–1 consensus sequence,which was stimulated by the addition of TPA. The nuclear factor(s)from PKC-α- and -η-transfected SVHK cells showed a significantlyincreased binding to the probe, which was efficiently competed by theexcess of unlabeled oligomers containing the AP1–1 binding site. Asexpected, PKC-γ-transfected SVHK cells did not show TPA-dependentstimulation of the specific DNA-protein binding. Anti-c-jun and anti-

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Figure 5 AP1–1 site (–119 to –113) was critical for involucrin promoteractivity. Each TRE deleted INV-CAT vector was transfected into SVHK cells,and the cells were treated with TPA (10 ng per ml) for 24 h. The activity ofcells transfected with INV-CAT alone without TPA stimulation was designatedas 1.0. C, INV-CAT vector; T3, AP1–3-deleted INV-CAT vector; T2, AP1–2-deleted INV-CAT vector; T1, AP1–1-deleted INV-CAT vector; T23, AP1–2 and –3-deleted INV-CAT vector; T13, AP1–1- and –3-deleted INV-CATvector; T12, AP1–1- and –2-deleted INV-CAT vector; T123, AP1–1-, –2-,and –3-deleted INV-CAT vector.

Figure 6 The transfection of PKC-α or PKC-ηη expression vectorsstimulated AP1–1 containing INV-CAT promoter activity. Followingco-transfection of TRE-deleted INV-CAT vectors with PKC-α (A) or PKC-η (B) expression vectors, SVHK cells were incubated with or without TPA(10 ng per ml) for 24 h. The activity of cells transfected with INV-CAT alonewithout TPA stimulation was designated as 1.0. C, INV-CAT vector; T3, AP1–3-deleted INV-CAT vector; T2, AP1–2-deleted INV-CAT vector; T1, AP1–1-deleted INV-CAT vector; T23, AP1–2- and –3-deleted INV-CAT vector;T13, AP1–1- and –3-deleted INV-CAT vector; T12, AP1–1- and –2-deletedINV-CAT vector; T123, AP1–1-, –2-, and –3-deleted INV-CAT vector.

c-fos antibody decreased the specific bands, and a supershifted bandappeared near the top of the lane (Fig 7B, lanes 4 and 6). Nosupershifted band was detected by the addition of anti-junB, -junD,or -fra-1 antibodies (Fig 7B, lanes 5, 7, and 8). These results indicatethat TPA stimulates the binding of nuclear factor(s) from PKC-α- and-η-transfected SVHK cells to the AP1–1 site, and the nuclear factor ismost likely composed of c-jun and c-fos.

Figure 7 Nuclear factor(s), which are composed of c-jun and c-fosprotein, from PKC-α and -ηη-transfected SVHK cells specifically boundto AP1–1 site in the presence of TPA. (A) The nuclear extracts (4 µl) fromPKC-transfected SVHK cells were reacted with the synthesized oligomer, whichcontained AP1–1 region. The DNA-protein complex was separated on 4%polyacrylamide gel. C, nuclear extract from control vector transfected SVHKcells; α, nuclear extract from PKC-α expression vector transfected-SVHK cells;η, nuclear extract from PKC-η expression vector transfected-SVHK cells; γ,nuclear extract from PKC-γ expression vector transfected-SVHK cells. Theconcentration of TPA was 10 ng per ml. Competitor indicate 50 3 unlabeledprobe. (B) Lane 1, nuclear extract from TPA-treated SVHK cells; lane 2, nuclearextract from TPA-treated SVHK cells plus 50 3 unlabeled probe; lane 3, nuclearextract from TPA-treated SVHK cells plus the oligomer containing AP-2consensus sequence; lane 4, nuclear extract from TPA-treated SVHK cells plusanti-c-jun antibody; lane 5, nuclear extract from TPA-treated SVHK cells plusanti-junB antibody; lane 6, nuclear extract from TPA-treated SVHK cells plusanti-c-fos antibody; lane 7, nuclear extract from TPA-treated SVHK cells plusanti-junD antibody; lane 8, nuclear extract from TPA-treated SVHK cells plusanti-fra-1 antibody; lane 9, nuclear extract from TPA-treated SVHK cells plusanti-NFκB antibody. The → indicates the supershift band.

DISCUSSION

Involucrin with an extended rod-shaped structure (Yaffe et al, 1992) isassumed to be initially incorporated into CE, forming a scaffold ontowhich other CE precursors, such as elafin, SPRR, and loricrin, aredeposited (Steinert, 1995; Steinert and Marekov, 1995). The involucringene expression is tissue-specific and differentiation-dependent andnormally detected in the suprabasal epidermal layers (Eckert, 1989).Although SVHK cells make up a transformed cell line, they are muchmore easily manipulated in transfection experiments of involucrin geneexpression. Notably SVHK cells contain similar PKC isozymes (α, δ,ε, η, ζ) as normal human keratinocytes and show a significant involucrinexpression, which is marginally detected in A431 and SCC13 cells(data not shown).

Involucrin gene is TPA-inducible and contains several AP-1 bindingsites on the 59-upstream of the promoter region (Takahashi and Iizuka,1993; Welter et al, 1995; Lopez-Bayghen et al, 1996). Our resultsindicate that PKC-α and PKC-η, when introduced into SVHKcells, mediate the TPA-dependent activation of the involucrin gene

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transcription. The effect was inhibited by the specific PKC inhibitor,H-7. Transfection of PKC-γ also stimulated involucrin gene expressionin the absence of TPA. Previous analyses indicate that keratinocytescontain five PKC isozymes, PKC-α, -δ, -ε, -η, and -ζ (Dlugosz et al,1992). SVHK cells do not contain PKC-γ (data not shown). So farPKC-γ is solely expressed in the nervous system, and we suppose thatPKC-α and -η are responsible for the regulation of the involucringene expression of keratinocytes.

There are several reports concerning the localization of PKCisozymes in the epidermis (Leibersperger et al, 1991; Wever et al, 1992;Koizumi et al, 1993). In normal skin, PKC-η is expressed in theuppermost granular layer (Koizumi et al, 1993), whereas PKC-αmRNA is expressed from basal to spinous layers (Wever et al, 1992).Because involucrin is expressed in the upper spinous layer and thegranular layer, the involucrin gene might be activated initially by PKC-α in the spinous layer and then by PKC-η in the granular layer. Thenuclear factors, such as TEF-1, POU, and YY-1, might suppress theinvolucrin expression in the basal cell layer (Takahashi et al, 1995;Welter et al, 1996; Lopez-Bayghen et al, 1996).

Involucrin is known to be cross-linked by transglutaminase 1. Uedaet al reported that transglutaminase 1 is induced by PKC-η in fetal ratskin keratinocytes (Ueda et al, 1996). They also showed that thetranscription of transglutaminase 1 was increased by the transfection ofPKC-η expression vector even in the absence of TPA stimulation.Although we also detected an increase in PKC activity of around 2-fold in the absence of TPA by the transfection of each PKC expressionvector, the involucrin transcription level did not change in SVHKcells. Ueda et al used luciferase expression vectors for the reportergene, which is known to be more sensitive than the CAT reportergene. In addition, the transfection efficacy of the INV-CAT vectorinto SVHK cells is much less than that into fetal rat skin keratinocytes(Takahashi and Iizuka, 1993). These factors might explain the discrep-ancy of the results of gene expression between the two cell lines interms of TPA-dependency.

PKC-γ stimulated the transcription of the involucrin gene in a TPA-independent manner. It is known that PKC-γ is less sensitive todiacylglycerol but is markedly stimulated by low concentrations of freearachidonic acid (Sekiguchi et al, 1988); PKC-α and -β isozymesrequire higher concentrations of arachidonic acid for full activation.The transcriptional activation of INV-CAT by PKC-γ in the absenceof TPA might indicate the increased arachidonic acid level in SVHKcells. Consistent with the idea that the introduced PKC-γ is endogen-ously activated in SVHK cells, the effect of PKC-γ was also inhibitedby the protein kinase C inhibitor, H-7.

Co-transfection of various AP-1-deleted expression vectors showedthat AP1–1, which is located on –119 to –113, the most proximal tothe transcription start site, was critical for the TPA-dependent regulationof involucrin gene transcription. These results are consistent withprevious reports showing that AP1–1 is one of the most activeregulatory domains of the involucrin gene (Takahashi and Iizuka, 1993;Welter et al, 1995; Lopez-Bayghen et al, 1996). Welter et al showedthat fra-1, junB, and junD are the binding factors for the AP-1 sitesin the involucrin promoter (Welter et al, 1995). In our analysis,combined c-fos and c-jun overexpression in keratinocytes activatedthe involucrin promoter (Takahashi and Iizuka, 1993) and gel shiftanalysis indicated that the c-fos/c-jun complex specifically bound tothe AP1–1 sites (Fig 7B). It must be mentioned, however, that AP1–1 deletion from the INV-CAT vector did not completely suppress thetranscriptional activity, suggesting the presence of other regulatoryfactor(s) for the involucrin gene transcription.

TPA-treated epidermis shares features of psoriatic hyperproliferativeepidermis. These include increased involucrin expression (Takahashiet al, 1996), increased phospholipase A2 (Bartel et al, 1987), phospho-lipase C (Fisher et al, 1990), and transglutaminase activities (Esmannet al, 1989). Interestingly, diacylglycerol level in the psoriatic epidermisis increased compared with the uninvolved or normal epidermis (Fisheret al, 1990), suggesting that PKC is activated in the psoriatic epidermis.An apparent decrease in PKC activity (Horn et al, 1987) as well as inJun/Fos expression (Basset-Seguin et al, 1991) might reflect downregul-ation of PKC following its activation; PKC-α is sensitive to downregul-

ation and in situ hybridization analysis indicated decreased expressionof PKC-α in psoriasis (Wever et al, 1992). On the other hand,the expression of PKC-η, which is insensitive to downregulation(Murakami et al, 1996), is distributed in the uppermost granular layerin normal epidermis and suprabasal layers in psoriatic epidermis(Koizumi et al, 1993). It is interesting to note that the localization ofPKC-η mostly coincides with that of involucrin in the normal andpsoriatic epidermis, suggesting that PKC-η might be responsible forthe involucrin expression.

Besides involucrin, proteins involved in CE formation includeloricrin, cystatin A, SPRR, and transglutaminase 1 (see Introduction),the expression of which are all stimulated by TPA (Yamanishi et al,1992; DiSepio et al, 1995; Owens et al, 1996).

This suggests a common controlling mechanism for the expressionof CE precursor proteins as well as cross-linking enzyme(s). Thecharacterization of the AP1-dependent signaling pathway via specificPKC isozymes will clarify the nature of the regulation, which will leadus to the understanding of the molecular mechanisms of keratinocytedifferentiation.

This study was supported in part by grants 08457233 (HI) and 08770618 (HT)from the Ministry of Education, Science, Sports and Culture of Japan and from theMinistry of Health and Welfare, Japan. The technical assistance of Mrs. Y Kotani andMrs. K Takahashi and the secretarial assistance of Miss Y Maekawa are greatly appreciated.

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