specific proteinkinasecisozymes mediate the induction...

Vol. 6, 149-157, February 1995 Cell Growth & Differentiation 149

Specific Protein Kinase C Isozymes Mediate theInduction of Keratinocyte DifferentiationMarkers by Calcium

Mitchell F. Denning, Andrzej A. Dlugosz,Erin K. Williams, Zoltan Szallasi, Peter M. Blumberg, andStuart H. Yuspa’

Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of

Cancer Etiology, National Cancer Institute, Bethesda, Maryland 20892

AbstractThe maturation of epidermal keratinocytes is a tightlyregulated, stepwise process which requires proteinkinase C (PKC) activation. We investigated the effect ofelevated extracellular Ca2’�, a potent differentiationsignal which increases cellular sn-i ,2-diacylglycerollevels, on the PKC isozyme profile of cultured murinekeratinocytes. Five PKC isozymes (a, 8, #{128}, �, and ip)

were detected by immunoblotting. During Ca2’�-induceddifferentiation, total cellular PKCa decreased, PKC#{128}andTJ increased 3-5-fold, and the level of other PKCisozymes was relatively unchanged. PKCa underwent aprogressive translocation from the soluble to theparticulate fraction following elevation of extracellularCa2’. The kinetics of PKCa translocation correspondedwith the induction of keratinocyte differentiationmarkers. Both PKCB and #{128}were selectively lost from thesoluble fraction of keratinocytes exposed to elevatedextracellular Ca2�, resulting in an increase in theproportion of these isoforms in the particulate fraction.PKCtp increased in both the soluble and particulatefractions, while PKCC did not change in amount ordistribution during keratinocyte differentiation. Selectivedown-regulation of PKC isoforms by either 12-deoxyphorbol-1 3-phenylacetate or bryostatin 1 inhibitedCa2’�-induced expression of differentiation markers atdoses most specific for the down-regulation of PKCa.Taken together, these observations suggest that theinduction of keratinocyte differentiation by Ca2� resultsin the activation of specific PKC isozymes.

Introduction

The PKC2 family of phospholipid-dependent protein ki-nases consists of at least 1 1 isozymes with distinct enzy-matic properties and tissue expression (1 , 2). PKC isozymescan be classified into three groups based on structural andenzymatic properties. Classical isozymes are stimulated byboth Ca2� and diacylglycerol/phorbol esters (a, I, �lI, andy); novel isozymes lack the C2 domain and do not require

Ca2� for maximal activation (�, #{128},ij, 0, and ii); and the

Received 8/1 8/94; revised 10/26/94; accepted 1 1/14/94.1 To whom requests for reprints should be addressed, at NCI/DCE/LCCTP,Building 37, Room 3B25, 37 Convent DR MSC 4255, Rockville, MD 20892-

4255.2 The abbreviations used are: PKC, protein kinase C; Ca,,, extracellular Ca2�;TPA, 1 2-O-tetradecanoylphorbol-l 3-acetate; DPP, 12-deoxyphorbol-l 3-phenylacetate; DAG, sn-l ,2-diacylglycerol.

atypical isozymes neither bind diacylglycerol/phorbol es-ters nor are stimulated by Ca2� (� and �) (1-4).

PKC has been classically linked to the mitogenic signaltransduction pathways of phospholipase C-coupled growthfactor receptors by virtue of its stimulation by 1 ,2-diacylg-lycerols and other lipid second messengers (5, 6). Recently,involvement of PKC in specialized cellular functions suchas secretion (7-9), adhesion (10), and differentiation (11-13) has also been demonstrated. Data from these special-ized systems has implicated specific functional roles forindividual PKC isozymes. This functional heterogeneity isreflected in the differences in PKC allosteric effectorrequ irements, subcellular distribution, and tissue-specificexpression.

The differentiation of epidermal keratinocytes is a tightlyregulated process in which basal, proliferating cells detachfrom the basement membrane and move sequentially intothe post-mitotic spinous, granular, and cornified layers ofthe epidermis. Strata-specific markers of differentiationhave been identified, including the spinous keratins Kl andK10 and the granular proteins filaggrin, loricrin, and trans-glutaminase (1 4-1 6). While the differentiation marker tran-scripts are confined to individual epidermal layers, the cor-responding proteins persist throughout the more suprabasalcell layers (17, 18).

Previous data indicate that the activation of PKC modu-lates the expression of genes involved in the terminal stagesof epidermal keratinocyte differentiation (1 2, 19, 20). Inculture, primary keratinocytes can be induced to differen-tiate by elevating the Ca0 from 0.05 mt�i to above 0.1 m�i(21), and this signal may be physiologically relevant con-sidering that an increasing gradient of Ca0 exists from thebasal compartment towards the suprabasal differentiatinglayers of the epidermis (22, 23). Elevation of Ca0 increasesphosphatidylinositol turnover and diacylglycerol levels inkeratinocytes (24-27) and induces the phosphorylation ofan identical subset of proteins, as does treatment with thephorbol ester TPA (28). Activation of PKC by phorbol estersor diacylglycerols stimulates cornified envelope formationand causes the transition from the expression of spinous togranular layer differentiation markers. PKC activation,therefore, inhibits the expression of Kl and KlO and in-duces the expression of filaggrin and loricrin (1 2). Takentogether, these data suggest that biochemical and functionalPKC responses are modulated by Ca,, in keratinocytes. 5ev-eral studies have identified changes in PKC activity (29-31),phorbol ester binding (29, 32, 33), and PKC protein levels(29, 34, 35) in keratinocytes during differentiation, but acomprehensive analysis of all PKC isozymes has been lack-ing. PKC� expression is restricted to stratified differentiatingepithelial cells, suggesting that this isoform may play a rolein epithelial differentiation (35, 36). In a reconstituted hu-man epidermis model, PKC�rj mRNA levels were elevatedduring differentiation, while PKC6 and PKCa decreased(30). PKC mRNA levels are relatively unchanged in primarymouse keratinocyte cultures induced to differentiate by

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In this study, the amount and subcellular distribution ofall known normal mouse keratinocyte PKC isozymes arequantitated by immunoblotting in order to identify isozyme-specific responses during epidermal differentiation. Further-more, isozyme-specific inhibitors are used to evaluate therelation of a PKC isozyme to the differentiation response.Our results demonstrate that PKCa, PKC�, and PKC#{128}un-dergo subcellular redistributions, which increase the frac-tion of particulate PKC in differentiating keratinocytes andthat the presence of PKCa correlates best with the inductionof differentiation markers by Ca2�.

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Fig. 1. Kinetics of Ca0-induced differentiation marker expression in kerati-nocytes. Primary mouse keratinocytes were switched from 0.05 msi Ca,, to

either 0.1 2 mM or 1 .4 mM Ca2� for the indicated period of time. Total proteinlysates were analyzed by immunoblotting with antibodies to Kl , Kl 0, Ki 4,filaggrin, and loricrin as described in “Materials and Methods.” The level of

the basal cell keratin Ki 4 does not change during Ca2�-induced keratinocytedifferentiation.

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ResultsInduction of Keratinocyte Differentiation by Elevated Ca0.Primary mouse keratinocytes cultured in media containing0.05 mM Ca2� proliferate and express the basal epidermalkeratins KS and K14 (21). Differentiation is induced byincreasing the Ca0 to 0.12 mtvi, which is optimal for pro-gressive expression of suprabasal epidermal differentiationmarkers over an 1 8-48 h time period or to 1 .4 mt�i Ca2� inwhich the differentiation markers are induced more rapidlybut to a lesser extent (Fig. 1 ). Cells cultured in either 0.1 2 or1 .4 mt�i Ca0 flatten, stratify, and undergo an irreversiblecessation in DNA synthesis, and the onset of morphologicaldifferentiation is more rapid in 1 .4 mt’.i Ca0 cultures (38).Culturing primary keratinocytes in 0.1 2 mivi Ca0 inducedhigh level expression of spinous cell markers K1 and K10and the granular layer markers, filaggrin and loricrin, from36-48 h. Cells grown in 1 .4 m�t Ca0 expressed the supra-basal differentiation markers as early as 1 2 h; however, from36 to 48 h, the amount of these proteins decreased relative

Fig. 2. Total PKC isozyme levels in keratinocytes during Ca2�-induceddifferentiation. Keratinocytes were induced to differentiate with either 0.12mM or 1 .4 mM Ca,, for the indicated times, and PKC proteins in total SDSlysates were analyzed by immunoblotting with specific antisera. PKC levelsfor each isozyme were quantitated from 3-4 similar experiments for statis-

tical data analysis.

to 0.12 mM Ca0 cultures. The decrease in differentiationmarkers from 36 to 48 h may be due to posttranslationalprocessing such as proteolysis or cross-linking and incor-poration into cornified envelopes. Kl4 levels are notchanged during keratinocyte differentiation and are used tonormalize for equal protein loading.

Expression of PKC Isoforms in Differentiating Keratino-cytes. The total levels of individual PKC isozymes in dif-ferentiati ng kerati nocytes were assessed by i mmu noblotti ngSDS lysates from cells cultured in either 0.1 2 mr’it or 1 .4 mviCa0 for 12-48 h. Fig. 2 shows that the amounts of PKC� and

Cell Growth & Differentiation 151

PKCC in total cell lysates did not change during Ca2�-induced differentiation. Statistical analysis of densitometricdata from three to four immunoblot experiments such asthat in Fig. 2 indicates there was a decrease in PKCa toapproximately 50% of control after 48 h in 0.1 2 mt�’t Ca0,although this decrease was not quite statistically significant(P = 0.055). In contrast, both PKC#{128}and PKC’q increasednoticeably during differentiation, and their rates of increaseover the 48-h time course were significant (P< 0.05) at both0.1 2 and 1 .4 mM Ca0. The rate ofchange in PKC#{128}and PKC�was greater in 1 .4 mM Ca0 than in 0.1 2 mt’�i Ca0. Thestrongest trend observed was the increase in PKCip, whichrose at a rate of 1 34% in 0.1 2 mt�i Ca0 and 164% in 1 .4 mt�iCa0 over a 24-h period. The changes in total PKCa andPKCi’� levels in differentiating keratinocytes are in agree-ment with previous data from immunohistochemical stain-ing of the epidermis (34, 35).

Ca0-stimulated Keratinocyte Differentiation Induces Re-distribution of Specific PKC Isozymes. Because changes inthe total amounts of PKC isozymes do not indicate the stateof activation of PKC during Ca2�-induced differentiation,we analyzed the change in distribution of PKC isozymes insoluble and particulate fractions following exposure tohigher Ca2� media to determine if isozymes were translo-cated. Translocation of PKC to the particulate fraction isconsidered to be an indicator of cellular PKC activation (1).In 0.1 2 mM Ca0, the predominately soluble (95%) PKCaunderwent a progressive redistribution to the particulatefraction, with the largest translocation occurring between24-48 h (Fig. 3). PKC� and PKC#{128}were both distributedapproximately equally between soluble and particulatefractions in basal cells, and 0.12 mtvt Ca2�-induced differ-

entiation reproducibly caused translocation of PKC#{128}at 1 hand a selective decrease in the soluble amounts of theseisoforms at later time points. In 0.1 2 mt�i Ca0, PKCi� in-creased in both the soluble and particulate fractionsthroughout the differentiation time course. PKCC was pre-dominately in the soluble fraction, and no redistribution orchange in the amount of this isoform was detected duringkeratinocyte differentiation (data not shown).

Ca0 (1 .4 mM) caused more rapid and extensive changes inPKC isozyme subcellular distribution than 0.1 2 mt�i Ca0, butthe pattern was the same (Fig. 3). The rate of PKCa trans-location was greatest within the first 6 h after switch to 1 .4mM Ca0. The loss of soluble PKC� and PKC#{128}was alsoaccelerated in 1 .4 mt�.i Ca0, and there were no appreciablechanges in particulate levels of these isoforms. The transientrise in particulate PKC#{128}was also seen in 1 .4 mt�i Ca0. Ca,,(1 .4 mM) caused increases in both soluble and particulatePKC’q, and PKC� did not change in response to elevated Ca0(data not shown).

The data in Fig.3 were recalculated to show the relativedistribution of PKC isoforms between soluble and particu-late fractions at each time point as a percentage of the totalat that time (Fig. 4). The redistribution of PKCa from solubleto particulate during Ca2tinduced differentiation was ev-ident in this analysis. This isoform shows the greatestchange during the induction of differentiation markers, bothfor 0.1 2 and 1 .4 mM Ca2�, but the time course of changewas more rapid in 1 .4 mt�’i Ca2�. The percentages of PKC6and #{128}in the particulate fraction increased 1 h followingelevation of Ca0 to 0.1 2 or 1 .4 m�t Ca2� and increasedfurther between 24-48 h. As in the prior analysis, the extentof this redistribution to the particulate fraction for PKC6 wasmore rapid and of greater magnitude in 1 .4 mt’�i Ca2� than

for 0.1 2 mM Ca2�. This was less evident for PKC#{128},althoughthe trend was there. The relative distribution of PKC’rj be-tween the soluble and particulate fractions was variableamong experiments, and this analysis does not yield a clearconclusion. The analysis in Fig. 4 indicates that the relativeproportions of particulate PKCa, �, and #{128}increase in kera-tinocytes during Ca2�-induced differentiation.

Down-Regulation of PKC Isozymes by DPP or Bryostatin1 Inhibits Ca0-induced Keratinocyte Differentiation. Toaddress the issue of PKC isozyme specificity in keratinocytedifferentiation, we used DPP, a potent inhibitor of mouseskin tumor promotion (39), and bryostatin 1 , a macrocycliclactone shown to block several TPA and Ca2�-inducedkeratinocyte responses (1 2, 40). DPP and bryostatin 1 bindand activate partially purified or recombinant PKC in vitrobut paradoxically block certain TPA-induced responses inculture (1 2, 40) or in vivo (39, 41). The antagonistic effectsof these drugs are believed to result from their ability topotently down-regulate PKC in an isozyme-specific manner(42-44). As shown in basal keratinocytes (0.05 m�i Ca2�)previously3 and for Ca2�-induced keratinocytes in Fig. 5,the down-regulation of individual PKC isozymes by DPP inkeratinocytes was also isozyme selective. PKCa was down-regulated at a dose (ED50, 34 nM) 20-fold higher than otherisozymes, except PKCC, which was not down-regulated byany concentration of DPP tested. PKC�, PKC#{128},and PKC’rpwere all down-regulated with an ED50 between 0.1-1 .5 nMDPP. Since the expression of granular cell differentiationmarkers loricrin and filaggrin are closely dependent on PKCactivation (1 2), we used the expression of these proteins asan assay to determine which isoforms are specifically in-volved in the differentiation response. Fig. S shows that,relative to 0.05 mM Ca2�, complete inhibition of 0.1 2 m�iCa2�-induced loricrin and filaggrin expression occurs at1 00-1 000 nM DPP but not 1 0 nM DPP. The sharp inhibitionof marker expression between 10 and 100 nM DPP corre-sponds with the abrupt down-regulation of PKCa between1 0 and 1 00 nM DPP, whereas PKC6 and PKC#{128}are substan-tiallydecreased at 10 nt�i DPP.

Bryostatin 1 had a different selectivity for PKC isozymedown-regulation than DPP in Ca2�-induced keratinocytes(compare Figs. 5 and 6). PKCa was down-regulated with anED50 of 0.2 nM, while the level of PKC6 was biphasic inresponse to bryostatin 1 , being down-regulated completelyat 1 nM but returning to approximately 40% of control at100 nM. PKC#{128}levels were decreased to about 35% by 0.1nM bryostatin 1 , and higher concentrations did not decreasePKC#{128}further. Bryostatin 1 caused a slight decrease in PKC�at 0.1 nM to approximately 60% of control but increasedPKC’q 2-3-fold at 1-1 00 nivi. The level of PKCC was notaffected by any dose of bryostatin 1 . These dose-responsecurves to bryostatin 1 in the keratinocytes are very similar tothose reported for fibroblasts (43) and keratinocytes in 0.05mM Ca2� (44). Analysis of granular cell differentiationmarker expression in Fig. 6 demonstrates inhibition of 0.12mM Ca0-induced loricrin and filaggrin at 0.1 nM bryostatin1 and almost complete inhibition (to the 0.05 mt�’i Ca2�level) at 1 nM and higher. The dose-response for inhibition

3 z. Szallasi, K. Kosa, C. B. Smith, A. A. Dlugosz, E. K. Williams, S. H. Yuspa,and P. M. Blumberg. Differential regulation by the anti-promoting 12-de-oxyphorbol 13-phenylacetate reveals distinct roles of the classical and novelprotein kinase C isozymes in biological responses of primary mouse kerati-nocytes, Mol. Pharmacol., in press.

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of differentiation markers by bryostatin 1 correlates bestwith the complete down-regulation of PKCa since markerexpression remains suppressed at 1 0-1 00 flM bryostatin 1when keratinocytes have detectable levels of PKC8, PKCe,PKCq, and PKCC. The PKC6 protected from down-regula-tion by high doses of bryostatin 1 is catalytically active (44).PKC6 and PKC� levels are increased by 1 0-1 00 n�i bryo-statin 1, while filaggrin and loricrin levels remain low,suggesting that PKC6 and PKCr� are not major regulators ofthese markers directly. The reduction in expression of dif-ferentiation markers by 0.1 nt�i bryostatin 1 suggests thatPKCB, PKCs, or PKCi� could influence this pathway to someextent, although together with the DPP results, these phar-

macological studies indicate a more important contributionby PKCa.

Discussion

The existence of multiple PKC isoforms having distinct co-factor requirements and tissue distributions suggests spe-cific roles for individual PKC isozymes in cellular physiol-ogy. Epidermal keratinocytes express at least five PKCisoforms, including isozymes which are Ca2�-dependent(PKCa), Ca2�-independent (PKC8, E, and �), and diacylg-lycerol/phorbol ester-independent (PKC�). PKCI3 has beendetected in the epidermis due to its high expression in

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ing Ca2�.induced keratinocytedifferentiation. Primary keratino.cytes were cultured for the mdi.cated time in 0.1 2 or 1 .4 msi Ca0and fractionated into soluble andTriton X-100 solubilized (particu-

late) fractions. PKC isoforms werequantitated by densitometry of rn.munoblots, and the data are cx-pressed as the percentage soluble(0) or particulate (#{149})relative tothe total level as quantitated fromthe sum of soluble plus particulatefractions for each time point.Points, means; bars, SEs fromthree to four separate experi-ments.

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Langerhans cells but is not expressed in keratinocytes (34,45). Immunological detection of PKC’y in epidermal lysateshas also been reported, but this isoform has not been lo.calized to keratinocytes (46, 47). The immunoblot analysisof total extracts from differentiating keratinocytes (Fig. 2)supports data from immunohistochemistry showing in-creases in PKC� (35) and decreases in PKCa (34) duringdifferentiation. The decrease in PKCa may be due to partialdown-regulation since this isoform undergoes a progressivetranslocation, indicative of activation, which is often fol-lowed by accelerated degradation of the PKC enzyme (1).Immunohistochemical localization of PKC� to the upper-most region of the granular layer of the epidermis (35)suggests its involvement in the posttranslational modifica-

tion of differentiation-specific enzymes or structural pro-

teins rather than in the changes in gene expression occur-ring at the junction between spinous and granular layers. Inaddition, the level of PKC,j and the expression of granularlayer differentiation markers did not correlate closely inDPP or bryostatin 1-treated cultures (Figs. 5 and 6).

Ca2� induces rapid turnover of inositol phospholipids,changes in lipid metabolism, and accumulation of choles-terol sulfate in keratinocytes, all of which are capable ofactivating PKC (1 , 5, 6, 48). Additionally, there is a progres-sive increase in the endogenous PKC agonist DAG in cul-tured keratinocytes following elevation of Ca0. This in-crease in DAG is graded to the Ca,, and remains elevated for48 h, suggesting that a sustained activation of PKC may

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Fig. 5. Inhibition of Ca2tinduced differentiation markers and down-regu-

lation of PKC isoforms by DPP. Primary keratinocytes were pretreated for 16h with the indicated DPP concentration in 0.05 msi Ca2’-containing me-dium before the addition of Ca2� to 0.1 2 mst. The cells then were culturedfor 48 h, and total SDS lysates were analyzed for differentiation markers andPKC isozymes as described in Figs. 1 and 2, respectively. Similar results wereobtained in three experiments.

occur in response to the DAG generated (26). The data inFigs. 3 and 4 demonstrate progressive and graded redistri-bution of PKCa, PKC6, and PKC#{128}following the elevation ofCa0, although only PKCa underwent a classical transloca-tion, where a largely soluble form became predominantlyparticulate. Increasing the Ca,, also increases intracellularCa2� (Ca,) levels in keratinocytes, and the high Ca may actsynergistically with DAG to activate the only Ca2�-depen-dent PKC isoform in keratinocytes (PKCa). It is unclear whyPKC� and PKC#{128}do not accumulate in the particulate frac-tion of differentiating keratinocytes, but it may be due todown-regulation of the translocated PKC. Although there isno difference in the in vitro binding of the synthetic DAGanalogue 1 -oleoyl-2-acetylglycerol to various PKC isoforms(49), isozyme-specific down-regulation of PKC� and PKC#{128}has been observed with this DAG analogue (50). The totalamounts of PKC� and PKC#{128}remain constant and increaserespectively during Ca2�-induced differentiation (Fig. 2);however, fractionation of the cells into soluble and partic-ulate fractions revealed an overall decrease in PKC� andPKC#{128}levels relative to 0.05 m�i Ca2� (Fig. 3). This discrep-ancy between the total amounts of PKC� and PKC#{128}detectedin Figs. 2 and 3 may be due to different methods of sample

Fig. 6. Inhibition of Ca2�-induced differentiation markers and down-regu-lation of PKC isoforms by bryostatin. Primary keratinocytes were pretreatedfor 16 h with the indicated bryostatin concentration in 0.05 mxi Ca2�-containing medium before the addition of Ca2� to 0.1 2 mta. The cells then

were cultured for 48 h, and total protein lysates were analyzed for differen-tiation markers and PKC isozymes as described in Figs. 1 and 2, respectively.Similar results were obtained in three experiments.

preparation that could affect the extent of extraction of PKC

or the stability of PKC during sample preparation. A PKC#{128}-related kinase has been detected associated with cytokera-tins (51). If PKC were present in this compartment, it wouldbe detected by SDS extraction but not in soluble or Tritonx-100 solubilized material.

The DPP and bryostatin 1 dose-response studies in con-

cert with the translocation data suggest the involvementPKCa in the Ca2�-induced expression of epidermal granu-lar differentiation markers (Figs. 3-6). PKCa translocationcorrelates with the time course of marker expression, and itsdown-regulation by DPP and 10-100 nt’�i bryostatin 1 isassociated with the inhibition of marker expression. Addi-tionally, the ED50 for inhibition of TPA-induced keratino-cyte transglutaminase mRNA by bryostatin 1 also corre-sponds to the down-regulation of PKCa (20). Since theregulation of keratinocyte differentiation markers by PKCoccurs at the level of mRNA stability and gene transcription(12, 20), our data suggest a specialized role for PKCa inregulating keratinocyte gene expression. PKCa has alsobeen found to mediate the induction of gene expression and


differentiation in mammary (1 3) and myeloid (1 1) cells.Although the translocation and pharmacological data mdi-cate an important role for PKCa in gene expressionchanges, the contribution of additional PKC isoforms(PKC6, PKC#{128},and PKCi�) to other differentiation-relatedchanges cannot be excluded. For example, TGF�32 is ex-pressed in suprabasal, differentiating keratinocytes, and theinduction of TGFj32 has been linked to PKC#{128}overexpres-sion in fibroblasts (52).

The effects of 1 .4 mM Ca0 on PKC levels and redistribu-tion were more rapid and pronounced than the effects of0.1 2 mivi Ca,, (Figs. 2-4), yet differentiation marker expres-sion was optimal in 0.1 2 mr�i Ca,, (Fig. 1). Keratinocytescultured in 1 .4 mM Ca,, undergo more rapid morphologicalchanges, produce more cornified envelopes, and expressmarkers earlier than cells cultured in 0.1 2 mtvt Ca,,. Addi-tionally, rapid activation of PKC by the potent ligand TPAinhibits the expression ofthe spinous keratins Ki and K1 0 inkeratinocytes induced to differentiate by 0.1 2 mt�i Ca0 (12)and stimulates cornified envelope formation. These resultssuggest that a gradual increase in Ca0 and activation of PKCis important for the complete expression of the keratinocytedifferentiation program, and rapid activation of PKC in 1.4mM Ca,, drives keratinocytes directly to a terminal stage ofdifferentiation.

An increased understanding of the biochemical regula-tion of epidermal differentiation can aid in the treatment ofskin disorders, particularly those where aberrant differenti-ation is a major component such as in psoriasis, certainichthyoses, and the keratinaceous plugs of acne. Further-more, aberrant regulation of PKC isoforms has been linkedto skin carcinogenesis (53, 54). The current studies suggestthat PKC isozyme-selective ligands or inhibitors couldprove useful in treating epidermal diseases with a minimumof side effects or toxicity.

Materials and MethodsCell Culture. Primary keratinocytes were isolated fromnewborn BALB/c mice. Keratinocytes were cultured in Ea-gle’s minimal essential medium containing 8% Chelex (Bio-Rad)-treated fetal bovine serum with the final Ca2� con-centration adjusted to 0.05 mr’�i as described (38). For theelevation of Ca,,, an aliquot of 0.28 M CaCl2 was addeddirectly to the medium to avoid PKC translocation causedby media change. Bryostatin 1 and DPP were obtained fromthe Pharmaceutical Resources Branch of the National Can-cer Institute and LC Services, respectively. Cultures werepretreated with either bryostatin 1 or DPP for 16 h beforethe addition of 0.1 2 mt�i Ca2t

Cell Fractionation. At the indicated times, cells werewashed twice with ice-cold PBS and scraped into PKC lysisbuffer [20 mM Tris-HCI (pH 7.5), S mt�i EDTA, 1 mt�i PMSF,40 �ig/ml Ieupeptin, 0.16 pg/mI Calpain Inhibitor I, and 0.4pg/mI Calpain Inhibitor II]. After brief sonication, the lysatewas centrifuged at 100,000 X gfor 1 h, and the supernatantwas taken as the soluble fraction. The pellet was resus-pended in PKC lysis buffer containing 1 % Triton X-1 00,centrifuged as before, and the supernatant was removed forthe particulate fraction. Cell fractionation was performed inthe presence of the divalent cation chelator EDTA to avoidthe effect of elevated Ca2� on membrane partitioning ofPKC during the fractionation procedure (1, 55). Proteinconcentration was determined by the Bio-Rad proteinassay.

Immunoblotting. For total SDS lysates, the cells werewashed twice with ice-cold PBS and scraped into SDSsample buffer (56), boiled, and run immediately on 8.5%polyacrylamide gels. For fractionated samples, 20 pg solu-ble and 10 pg particulate protein were loaded. Proteinswere transferred to n itrocellulose electrophoretically, andthe membranes were blocked in 5% milk. For detection ofPKC isozymes, the membranes were incubated with anti-PKC antibodies specific for the catalytic subunit of PKCa ata dilution of 1 :500 (Upstate Biotechnology, Inc.), PKC6 at a1 :1 0,000 dilution (Calbiochem), PKC#{128}at a dilution of either1 :1 00 (Santa Cruz Biotechnology, Inc.) or 1 :250 (Transduc-tion Laboratories), PKCi’p at a 1 :1 00 dilution (Santa CruzBiotechnology), and PKCC at a dilution of 1 :5000 (Researchand Diagnostic Antibodies). The keratinocyte differentiationmarkers K1 , K1 0, K1 4, filaggrin, and loricrin were detectedwith antibodies described previously (21 , 57). An additionalband is often detected with our K10 antibody and maycorrespond to Ku (21). Proteins were detected using theECL system (Amersham) with horseradish peroxidase con-jugated secondary antibody (Bio-Rad) at a 1 :5000 dilution.All PKC antibodies were isozyme specific as determined bythe lack of cross-reactivity to purified recombinant PKCisozymes isolated from a baculovirus expression system.Recombinant PKC isozymes were kindly provided by Dr.M. G. Kazanietz of the Molecular Mechanism of TumorPromotion Section of the Laboratory of Cellular Carcino-genesis and Tumor Promotion at the National CancerInstitute.

Data Analysis and Statistics. The immunoblots werequantitated using a Molecular Dynamics personal densi-tometer. For the evaluation of total PKC levels during dif-ferentiation, the rate of change for each isoform after 0.1 2 or1 .4 mt�t Ca0 exposure was evaluated from 3-4 replicateexperiments using multiple linear regression analysis. A rateof 100% indicates a 2-fold increase. For each isoform andeach Ca0 concentration, the logarithm of the isozyme levelwas regressed against time, fitting a different intercept foreach experiment to account for the interexperiment varia-tion in levels at time zero but assuming a constant slopeacross all experiments. The slope and the SE of each slopewere estimated using standard least squares methods.

AcknowledgmentsWe thank Dr. Marcelo G. Kazanietz for providing recombinant PKC isozymecontrols and critical reading of this manuscript and Dr. Robert E. Tarone forstatistical analysis of the data. We also thank Drs. Tamar Tennenbaum,Luowei Li, and Ulrike Lichti for helpful discussions.

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