New Approaches and Concepts in the Study of Differentiation of Oral Epithelia

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DOI: 10.1177/10454411900010030201

1990 1: 167CROBMBeverly A. Dale, Jukka Salonen and Alma H. Jones

New Approaches And Concepts in The Study of Differentiation of Oral Epithelia

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Oral Biology and MedicineNew Approaches And Concepts inThe Study of Differentiation of OralEpitheliaBeverly A. Dale, Jukka Salonen, and Alma H. JonesABSTRACTEpithelial structural proteins, the keratins and keratin-associ-ated proteins, are useful as markers of differentiation becausetheir expression is both region-specific and differentiation-spe-cific. In general, basal cells in all stratified oral epithelia ex-press similar keratins, while the suprabasal cells express aspecific set of markers indicating commitment to a distinctprog ram of differentiation. Critical factors in the regulation ofepithelial protein expression are now under investigation. Thepromoter regions of keratin genes are being characterized todetermine what sequences within the genes are responsible fordifferential expression. One important extracellular factor thatinfluences epithelial protein expression is retinol (vitamin A),which exerts its effects via a group of nu clear receptor proteinsthat may also be expressed in a region-specific manner. Thesemolecular biological approaches enhance our understanding ofthe mechanisms regulating differentiation of oral epithelia andits regional complexity.I. INTRODUCTION

The oral cavity is lined by a mucous membrane that formsthe structural boundary between the body and the externalenvironm ent. The m ucosae are classified into three main types:the masticatory mucosa of the gingiva and hard palate; thelining mu cosa of the cheek and lips, floor of mouth , and ventraltongue; and specialized mucosa of the dorsum of the tongueand vermilion border of the lip. Although each type protectsagainst mechanical damage, the epithelia exhibit considerabledifferences in their histology, thickness, and differentiationsuitable to functional demands of the location, and in theireffectiveness as a barrier to the penetration of noxious or ther-apeutic substances.12 The epithelia are composed of a contin-uously renewing cel l populat ion of kerat inocytes whoseproliferation is confined to the basal cell layer. The suprabasalcells undergo sequential region-specific m orphological and bio-chemical changes. Thus, the maturing cells of masticatory ep-i thel ium undergo the differentiat ion pat tern known askeratinization, whereas that of the lining mucosa d oes not ker-atinize, but has a different pattern of differentiation known asnonk eratinization, and the specialized regions show mixed pat-terns. These regional variations within the oral epithelia havebeen well documented by morphological criteria (see Refer-ences 2 and 3).

The purpose of this review is to present recent data on the

biochemical changes associated with morphological differen-tiation that have resulted from studies using immunologicalmethods and the new tools of molecular biology. These find-ings clarify some concepts of epithelial differentiation. Theemph asis in this review is to explore the region-specific aspectsof epithelial differentiation from a molecular point of view,with emphasis on the expression of structural proteins of theoral epithelium, the keratins and keratin-associated proteins,as markers of differentiation. Expression of these proteins isboth tissue specific and differentiation specific, responds toextracellular influences, and may be dependent on the actionof retinoids. The results from these studies have significantlyimproved our understanding of the relationship between struc-ture and function of the normal oral mucosa. This is essentialto understand changes that occur in disease. Some new mo-lecular biological approaches that will permit future studies onthe molecular mechanism s important for differentiation of oralepithelia and its regional complexity are also reviewed.II . CYTOPLASMIC STRUCTURALPROTEINS ARE USEFUL MARKERS TOSTUDY EPITHELIAL DIFFERENTIATION

Epithelial cells of the oral mucosa, like all epithelial cells,contain keratin intermediate filaments (IF) as a major com-ponent of their cytoskeleton. Because of the size and diversityof the keratin family, these proteins have become useful astools to study differentiation. ' T h e analysis of keratin com-position and other structural proteins within a given epithelialtissue provides reliable information about its program of dif-ferentiation and the extent of differentiation of cells within thet issue." 4 Keratins that are commonly used as markers of var-ious types of epithelial differentiation are summ arized in Table1. Many antibodies to keratins are now available (see Table2) , and keratin cDNAs and gene sequences are rapidly becom-ing available, allowing this multigene family to be used forinvestigation s of tissue-specific and differentiation-specific as-pects of gene expression. Th e proximity of areas with differentfunctional features and patterns of differentiation within theoral cavity suggests exquisite regulation of differentiation. Thus,oral tissues are especially useful for studies of tissue-specificdifferentiation and for studies of the factors that regulate dif-ferentiation.

B . A. Dale (corresponding author), Ph.D., Professor, Departmentsof Oral Biology and Periodontics, Rm. D664 Health Sciences Bldg.SM44, University of Washington, Seattle, Washington 98195. J. Sa-lonen, D. Odont., Institute of Dentistry, Periodontics, University ofTurku, 20520 Turku, Finland. A. H. Jones, M.S., Ph.D., GraduateResearch Assistant, Department of Oral Biology, Rm., B225 HealthSciences Bldg., University of Washington, Seattle, Washington 98195.

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Table 1Keratins as Markers

Type of differentiationStratified epithelia

KeratinizedNonkeratinized4*Hyperproliferative''Cornea

Simple epithelia

of DifferentiationKeratin-

Type II(basic)

51/24638

Type I(acidic)1410/1113161218 , 19

Critical Reviews In

Numbered according to Moll et al., 1982.

A. The Keratin Fam ily of Intermediate Filament Proteins1.Keratin Subfamilies

The keratins are a multigene family of approximately 30pro t e i ns .5 '9 They form the intermediate fi laments of thecytoskeleton of all epithelial cells and their appendages (hairfollicles, sweat glands, salivary glands, etc.). This becameevident approximately 10years agowhen a variety of epitheliawere tested for reaction with polyclonal antibodies to epidermalkerat ins.1011 Positive reactions in all epithelia led to a flurryof activity in thecharacterization andclassification ofkeratins.Biochemical characterization,61 2 cDNA hybridization,13 andthe use of monoclonal antibodies to specific subsets of keratinproteins1416 resulted in their division into two subfamilies ofrelatively acidic (type I) andbasic (type II)keratins andgreatly

Table 2Partial SummaryComponents

Marker ofAll epithelia (multiple

keratins)

Stratified epitheliaGeneral

Basal

SuprabasalCornified

SuprabasalNoncornified

* 'Hyperproliferative' '

CornealHair-type

Simple epithel ium

of Antibodies to Keratins, Associated

Antibody"AE 1 K10, 11, 14, 15,

19 (most type I)A E 3 K 1 , 2, 3,4, 5, 6,

7, 8 (all type II)LP3434BE12Anti-InvolucrinTransglutaminase dLoricrinePKK1PKK2AE 2SC10KL 1LH2, 3RKSE608.60AKH1AE 81C72D 76B10GB10LMM3Anti-psi3AE 5AE13LE41, M20CAM5.2

Antigen"

K5

K 1 9 ( a l s o K 8 ,1 8 )K1 9K l , 10K l5557 kd keratinK10K10K1 0Filaggrin8K13K1 3K1 3K4K4K1 6

K3

K8K8

Cytoskeietai Proteins, and Cornified

Tissue staining patternbPalate

(Basal) suprabasalAll layersAll layersAll layersSpin-gran-corncSpin-gran-cornNTBasal

Suprabasal

GranularSpotty suprabasal

SuprabasalSuprabasal

Papillae of dorsalIsolated Merkel cells

BuccalBasalAll laversI l l 1UT Vl k?All layersAll layersSuprabasalSuprabasalUpper halfBasal

Negative

Negative or spottySuprabasal

NegativeNT

TongueNegative

Cell Envelope

EpidermisBasalAll 1 a ve r si * l l ld jrW lSAll layersAll layersGranular-cornGranular-cornGranularBasal

Suprabasal

Granular-cornNegative

NegativeNegative

Negative

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Oral Biology and Medicine

Tissue staining pattern1*Marker of

ind some stratified

Antibody"35BH11L E 6 1 , M C 2 8PKK3RCK105LP1KPKK1LP2KAnti-40KKS19.1

Antigen"K8K18K18K7K 7, 8K 8, 18, 19K19K19K19

Palate

Isolated Merkel CellsBasal cells at tips of

rete ridges

Buccal

BasalBasalBasal

Epidermis

NegativeNegativeNegative

Many of these antibodies are commercially available; refer to Linscott's Directory of Immunological and Biological Reagents, 5th ed., 1989.Keratins are identified by catalog number. 6 Other antigens are identified by comm on name s. Data based on References 4 8 , 15, 16, 26, 2 8 4 1 , 44 , 46 464 , 68, 69, 75, 79.Positive in cytoplasm of spinous cells but tending toward cell periphery in granular and cornified layers.Particulate (epidermal-type) form of the enzyme.Component of cornified cell envelope.Tested in rodent soft palate and esophagus, positive in keratohyalin and cornified cell periphery.Antibody reacts wiht profilaggrin and filaggrin of the granular and cornified layers, respectively.

17~20 Specific pairs of keratins are generallyin different types of epithelial tissues with the lowe steight keratins being present in simple and glandular

epithelium, intermediate-sized keratins in stratified epitheliand the largest keratins in cornified (keratinized) epithelia.6Current nomenclature for the keratin family is based on thapparent molecular weight by SDS-polyacrylamide gelectrophoresis or the catalog number according to Moll anco-workers;6 both systems are used in this review.2. Keratin Protein StructureSeveral excellent recent reviews of keratin protein structur

basic Pi acidic19

(50)(50)(48)(46)(45)(40)

FIGURE 1 . Diagram matic representation of two-dimen sional gel electrophoresis of kera-tins. Subfamily A (type II) keratins are relatively acidic, while subfamily B (type I) keratinsare relatively more neutral or basic. Pairs of keratins that are typically expressed in the samecell types are indicated by the symbols; 0, simple epithelia; filled 0, basal cells of stratifiedepithelia; others are expressed in suprabasal cells of tissues showing the patterns of differ-entiation indicated. Keratins are numbered according to the catalog of Moll et al. , 1982;approximate molecular weights in kilodaltons are shown in parentheses. (Adapted from Sun,T. T. , Tseng, S. C. G. , Huang, A. J. W., Cooper, D. , Schermer, A. , Lynch, M. H. , Weiss,R. , and Eichner, R. , Ann. N.Y. Acad. Sci., 455, 307, 1985.)

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Critical Reviews In

7 ' 21 '24 Briefly, all keratins (as well as the proteins that

cal rod portion (70 to 9 5% hom ology), but differ in sequences

-chain unit compo sed of two acidic

7-21-22 Thus, the

ions w ith associated proteins and cellular organelles that

Keratins Are Markers of Differentiation andThe distr ibution of keratins in oral t issues has been

keratins by several gro up s. 2 5 3 4 Such studies35 that cells of the basal layer of all stratified epithelia

in pair k5/14 (50/58 kDa), but upon commitment

ion, it has provided an excellent working model

associated proteins, used for these studies are summarized inTable 2 .1 5 1 6 '3 6 4 1

All stratified epithelia express keratins K5 and K14 and 15in the basal layer, while buccal suprabasal cells start to expressK4/13 (markers for non-keratinization); palate, K6/16 and Kl/10; and epidermis, Kl/10 (markers for keratinization) as theyleave the basal layer. It is interesting to note that the normalexpression of the K 6/16 pair in palate contrasts with epidermisin which the K 6/16 pair is seen only during wound healing42-43or in certain pathologic conditions, most of which are associatedwith thickening or hyperproliferation.4445

The presence of the unique K4/13 keratin pair in buccalepithelium provides biochemical support for the concept thatthe histologic pattern of "n on k eratiniza tion" is a distinct formof differentiation. This is sometimes referred to as "esoph ogeal-type differentiation" because Sun and co-workers first usedesophagus to test antibodies to these keratins. Representativeimmunohistochemical staining reactions are shown in Figure3 for oral-keratinized and nonkeratinized tissues.4. Keratin Expression Reflects the HistologicComplexity of Oral Epithelia

In regions of the oral cavity that exhibit complex patternsof epithelial differentiation, i.e., the keratinized gingiva andadjacent nonkeratinized alveolar mucosa, the palate and adjacentsoft palate, the dorsal tongue, etc., the morphological differencesare reflected in the expression of cytoskeleton components,including keratins and filaggrin. The complexity of keratinexpression in oral epithelia is illustrated here in the gingiva(Figure 4) (summarized from References 26, 28, 30, 32, 46-48) and dorsal tongue (Figure 5) (summarized from References27 and 3 4). The localization of keratins within the gingiva andtongue is based on staining of mouse and human tissues withvarious monoclonal antibodies.a. GINGIVA

The epithelium of attached gingiva has a pattern of keratinexpression similar to palate, while the sulcular epithelium as

involK4.13

FI GU RE 2 . Expr ession of keratins and other structural proteins in buccal and palatal epithelia and epide rmis .



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The junctional epithelium (JE) is a unique tissue and

in basal cells of some stratified tissue s49 K19 is uniformly distributed in the apex of the. Ho wever, within the coronal portion of JE, K1 9 is strongly

ior of the JE. A ntibodies AE1 and PKK 24 8 5 0 T h u s ,

51 areing differentiation. Staining of hum an gingiva with47 and cell surface markers52that cells in the junctional

of JE cells is also seen in a mod el system invitro in which cells attach to a membrane substratum, change

henotype to a more basal appearance, and show a proteinexpression pattern that differs from the suprabasal cells.47b. DORSAL TONGUE

The specialized epithelium of the dorsal tongue is a secondexample of an oral tissue with complex morphology and keratinexpression. The regional variation in differentiation pattern isparticularly dramatic in rodents (Figure 5). 53 The anterior andposterior portions of the filiform papillae and the interpapillaryregion each show unique keratin expression. The posteriorportion of the papillae forms a hard surface and contains uniquekeratins similar to those of hair. The interpapillary region haskeratin expression like that of buccal epithelium. The anteriorpart of the papilla, while histologically most similar to palateand gingiva as evaluated by the expression of keratohyalin andcornification, has keratins similar to buccal epithelium both byimmunohistochemistry and by in situ hybridization, as well assome epidermal-type keratins.2734 The latter, Kl and K10,seem to be expressed separately in different regions of tonguein contrast to their typical pattern of coexpression. K10 mayform complexes with keratins similar to those in hair in theposterior papillae, while Kl may interact with K13 (normallyassociated with nonkeratinized type of differentiation) in theanterior papillae.c. MERKEL CELLS

Merkel cells are unique cells within the oral mucosa of

gingiva, lip, and palate.54 They are thought to function as touchreceptors or in relation to innervation of the epithelium and donot contribute to the differentiation pattern of the tissue inwhich they reside.5556 Merkel cells can be identified usingmarkers for neuroendocrine cells.57 59 In addition, antibodiesto keratins stain Merkel cells. T hese cells contain keratins K8/18, and K19, typically found in simple epithelium.6063 Theidentification of keratins in Merkel cells supports the idea ofepithelial, rather than neural crest, derivation of these cells (seeabove references). The distribution of Merkel cells along thetips of rete ridges in the masticatory epithelium of maxillarytuberosity is revealed by staining with antibody 35PH11 tokeratin K8 as shown in Figure 6.5. Biochemical Correlations withImmunohistochemistry and Considerations ofEpitope S pecificity

The distribution of keratins in human oral epithelia has beenconfirmed by analysis of epithelial extracts by SDS-polyacrylamide gel electrophoresis and immunoblotting.26-28An example of keratin polypeptides from buccal and gingivalepithelial extracts is shown in Figure 7. Separation of keratinsby two-dimensional gels is also useful for greater resolutionof these proteins.26Antibodies to keratins have restricted epitope specificity.These epitopes may be species specif ic, or sensit ive todenaturation or the effects of fixation or decalcification. Thiscan lead to apparently contradictory findings when tissue stainingand immunoblots are compared. Thus, keratin K19 was notdetected in primate JE,50 while it is clearly present in humanJE tissue.4764 Another example is antibody AE1, which reactswith most acidic keratins when they are denatured and stainedon immunoblots. However, it stains only the basal layer ofepidermis or buccal epithelium in tissue sec tions, reacting withK14, but not with K10 or K13 in the suprabasal cells. A pparentlythe epitope for K10 and K13 is masked in vivo.15 Conversely,some antibodies react with keratins in tissue sections, but notwith the SDS-denatured protein transferred to the nitrocellulosememb rane for immunoreaction; for example, antibody Ks 18.18does not react on immunoblots because its epitope isconformation-specific and involves the K8/18 pair.65 Thus,positive tissue staining can be readily interpreted, but somecaution is required in interpreting negative findings.Monospecific keratin antibodies are now available for somekeratin polypeptides; some of these are listed in Table 2, andmany more will be available within the next few years. Mon-ospecific antibodies have been elicited by using synthetic pep-tides whose sequences were determined by translation of thecDNA sequence for the unique carboxyterminal region (e.g.,to Kl , K14,66 and to K1667) or by chance in the process ofeliciting monoclonal antibodies (e.g., to K368 or to K1334 ).Use of these antibodies minimizes problems of interpretationthat can occur with antibodies that stain several keratins and

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Critical Reviews In

Palate Buccal

H & E as

AE3 (K1-8)

AE1 (K14.16.19)

FIGU RE 3 . Immunohistochemical staining of buccal epithelium and tuberosity epithelium, representing typical regions ofnonkeratinized and keratinized differentiation patterns.

The keratin intermediate filament network of the cytoskeleton important for the function of the intermediate filament networkand for the shape, stability, and motility of epithelial cells.These proteins are known as intermediate filament-associatedproteins (IFAPs).1. Psi 3Psi 3 is an accessory protein of the cytoskeletal system that



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Oral Biology and MedicinePalate Buccal

.

AE2 (K1)

AE8 (K13)

Anti-40k (K19)

Anti-FG

FIGURE 3B.1990 17



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Critical Reviews In

G E

FI GU RE 4 . Keratin and psi 3 expression in the gingiva showing three distinct regions of different pa tternsof expression in the oral, the marginal and sulcu lar, and the junctiona l ep ithelium .

AP

v - - - . , - m \ %

UR E 5 . Keratin expression in the dorsal tongue of rodent (left). Different patterns of expression reflect the comp lexity of morphological differentiati



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FIG UR E 6. Identification of Merkel cells in gingival tuberosity tissue using antibody 35PH1 1 specific for keratin8. (A) Section showing Merkel cells at the tips of rete ridges. (B) Epithelial sheet showing the distribution of thesecells from the underside.

Gingiva Buccal

AE1 AE8AE3SC10AE5 AE1 AE3FIGURE 7. SDS polyacrylamide gel electrophoresis of keratins from oral epithelia stained withcommassie brilliant blue and various antibodies to keratins.

43 -69 It is not known if psi 3 interacts

47

2. FilaggrinFilaggrin is a cationic protein that aggregates with keratifilaments and may aid in their dense packing within the comifielayer (see recent reviews, References 70, 71). It is synthesizein the granular cell layer as a large precursor molecule, storein keratohyalin granules, and converted to filaggrin upotransition of granu lar cells to fully differentiated cornified c ellsExpression of profilaggrin and its conversion to filaggriinvolves the action of numerous enzymes. Profilaggrin iphosphorylated by several protein kinases and then processe

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Critical Reviews Inby a protein phosphatase and specific proteolytic enzymes intwo distinct steps.7273 Filaggrin itself is degraded within thecornified layer after keratin filament aggregation has occurred.74Details of the biochemical events of the filaggrin pathwaywithin oral tissues are not fully understood and may differ fromthose in the epidermis. Filaggrin or its precursor are readilydetected by immunohistochemistry in the granular layer ofkeratinized oral epithelium, although it is masked in the cornifiedlayer. It is only weakly expressed, if at all, in nonkeratinizedlining epithelium.46 Its expression is enhanced in conditions ofhyperkeratinization. 75 Therefore, it is useful as a molecularmarker to distinguish nonkeratinized and keratinized epithelia(Figures 2, 3, and Reference 46) and is a marker for thekeratinized type of epithelial differentiation.C. Involucrin, Transglutaminase, and Cornified CellEnvelopes

Terminal differentiation in epithelia displaying ortho-keratinization as observed in the epidermis and palate involvesthe formation of a thickened structure on the inner aspect ofthe cornified cell plasma membrane. This is commonly knownas the cornified cell envelope (C CE) .76 It is formed from severalsoluble protein precursors, including involucrin, by enzymaticcross-linking via a paniculate transglutaminase;77 reviewed inReference 78 . Both involucrin and transglutaminase are presentin buccal and palatal epithelia.7579 In buccal epithelium, onlythe basal cells are negative, w hile in the palate, basal cells andparabasal cells of the rete ridges are negative by immuno-histochemistry with antibodies to these markers (Figures 2, 3).The expression of both involucrin and transglutaminase increasesin the spinous and granular cell region of hyperkeratoticlesions.75-79Loricrin is another cornified cell compon ent (from the Latin,"to wrap around")- It is a glycine and cystine-rich protein.24Loricrin is expressed in the granular and surface cells of bothkeratinized and nonkeratinized oral epithelia. Antibody to thisprotein stains in a granular distribution in rodent tissues andmay be the sulfur-rich component of keratohyalin, describedas single granules by Jessen and co-workers, 80 and densehomogeneous deposits by Fukuyama and Epstein.8182Involucrin, transglutaminase activity, and observable CCEsare useful as markers of differentiation in cell culture. 7683-84Loricrin should also be useful for this purpo se.24 Because theseproteins are present in both keratinized and nonkeratinizedepithelia, they cannot be used to distinguish the type ofdifferentiation pattern, but can be used as markers of the extentof cell differentiation.

D. In situ Hybridization StudiesImmunohistochemical methods reveal the localization of aprotein in cells and tissues; results imply the site of proteinfunctioning. In situ hybridization reveals the localization ofthe mRNA for a protein of interest and, therefore, identifies

the site of active gene transcription. Localization of mRNAfor a particular keratin does not necessarily correspond tolocalization of the protein via immunostaining because someproteins remain in the cell even in the absence of active synthesis,while mRNA is generally short-lived; alternatively, the mRNAcan be stored and not translated. The mRNAs for K5 and K14are detected in the basal cells of epidermis23 '67 '85-86 and oralmucosa,27 but the levels drop in suprabasal cells, even thoughthe proteins can be detected biochemically.24 Thus, as cellsbecome com mitted to their differentiated pheno type, the mRN Asfor "basal" cell keratins are no longer expressed, and theremaining K5 and K14 mRNA is degraded; conversely, themRN As for differentiation-specific keratins are expressed in atissue-specific manner. The mRNAs for Kl and K10 areexpressed in suprabasal cells of epidermis and palate, thosefor K4 and K13 in some basal and all suprabasal cells ofnonkeratinized epithelia. Filaggrin mRNA87-88 is detected inthe granular layer. Loricrin mR NA is also detected in the upperspinous or granular layer.24The use of specific probes allows detection of individualkeratin mRN As, com parable to the use of monospecific keratinantibodies. Expression of mRNAs for keratins in the mousetongue has been studied by Rentrop and co-workers. 27 Theyshowed that mRNA for K14 (52 kDa in mouse) is expressedin basal cells of both dorsal and ventral tongue. A s the expressionof this mRNA is downregulated, an "impressive divergenceand specialization of the keratin expression is observed." 27The mRNA s for K4 (pKt 57-1 probe) and K13 (pKt 47-1 probe)are expressed in suprabasal cells of ventral tongue. In dorsaltongue expression of K4 mRNA is seen in the interpapillaryregion, while K13 mRNA is present both in the interpapillaryregion and the anterior aspect of the papillae. Results suggestthat K13 pairs with K4 in the interpapillary region, but has adifferent partner in the anterior papillae. The mRNAs for K10(pke 59 probe) and K16 (pkSCC 50 probe) occur in discreteregions within this complex papillae structure.

Three main conclusions can be drawn from studies so far;first, b asal cell-specific keratin mRN As are dow nregulated withdifferentiation; second, expression of differentiation-specifickeratins, as well as associated proteins, is regulated mainly atthe level of transcription of their genes; and, third, distinctkeratin mRN As are differentially expressed reflecting regionalcom plexity and subtle aspects of tissue-specific differentiation.E. Applications to O ral PathologyKeratins are important for tum or typing becau se epitheliallyderived tumo rs retain expression of some k eratins of the tissueof origin and may also express additional keratins.89 This areais beyond the scope of the present review, and the reader isreferred to recent reviews.90 Expression of keratins, filaggrin,and involucrin have all been reported to change in various oralpathologic conditions. These changes in structural proteinexpression reflect a change in the program of cel lulardifferentiation.



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Oral Biology and MedicineF. Summary of Expression of Structural Proteins

Studies on the expression of keratins and other structuralproteins have led to a new understanding of normal and abnormalepithelial differentiation.1. Basal cells of all oral stratified epithelia express similar

keratins (K5/14). In some regions basal cells also expressK19.2. Suprabasal cells exhibit a distinct pattern of keratin

expression that can be used as a marker of commitmentto a program of differentiation. Thu s, buccal cells expressK4/13, palatal cells express Kl/10 and K6/16, and cellsof the dorsal tongue show keratin express in discretemorphological regions. The observation that suprabasalcells of buccal epithelium express different keratins thanthose present within basal cells supports the concept thatcells in this tissue are undergoing a distinct type ofdifferentiation. The histological name, nonkeratinized,is somewhat deceptive in this respect. The observationthat suprabasal cells w ithin the junctional epithelium alsoexhibit a different pattern of protein expression than basalcells also suggests that differentiation is occurring in thistissue.

3. Both keratin proteins and filaggrin can be used as markersto distinguish non-, para-, and orthokeratinization patternsof differentiation that occur in the oral mucosa. In contrast,involucrin and transglutaminase, associated with comifiedenvelope formation, are found in all regions of the oralmucosa. These proteins are useful as markers for celldifferentiation within a particular tissue or in cell culture .

4. Changes in the normal expression pattern of keratins,filaggrin, and involucrin signal alterations in the programof differentiation in pathologic tissue.

III. MOLECULAR APPROACHES TOUNDERSTANDING KERATIN GENEEXPRESSIONStudies that begin to explore the expression of the keratingenes in differentiation at the molecular level are addressingthe following questions: 1. Is expression regulated by gene

transcription and/or by posttranscriptional mechanisms such asmRNA stability and protein translation. 2. How do pairs ofkeratins interact and function. 3. What are the common orunique sequences in keratin gene promoters. 4. What are theenhancers of keratin gene expression and how do they work.5. What elements of these regulatory mechanisms are respon-sible for tissue-specific, developmental-specific, and differ-entiation-specific modes of expression. The first of thesequestions is addressed in Section II.D. Most of the remainingquestions can be approached by two main techniques: trans-fection of D NA constructs into cultured cells and the production

of transgenic m ice. A schematic diagram of the keratin proteinsand some features of their genes are shown in Figure 8.A. How Do Keratin Proteins Interact and FormFilaments In Vivo?Studies on keratin protein interactions have been possibleby expressing introduced genes or the cognate mRNA s encodingepidermal cell keratins or mutant keratins in various cell types.Epidermal keratins were expressed in PtK2 kidney simpleepithelial cells and 3T3 fibroblasts, either from microinjectedmRNA91 or by transient transfection of chimeric genes constructsinto the recipient cell.92 The newly expressed epidermal keratinswere incorporated into the preexisting keratin filament networkof the kidney epithelial cells. Thus, the in situ keratin networkcan accomodate keratin polypeptides not normally expressed.In contrast, keratins are not incorporated into the vimentinnetwork of fibroblasts.

Transient transfection has been used to approach severaladditional questions on keratin expression. Although theefficiency of transfection is low (2 to 5%), the method ispowerful for examining functional aspects of keratins, includingkeratin polypeptide stability, coexpression of keratin pairs, andthe role of portions of the protein in filament formation.Expression of the type II (K6b or K5, basic keratins) infibroblasts results in the expression of a type I (K14, acidickeratin) partner, suggesting some type of cross-pair regulatorysignals in keratin expression.92 This regulation appears to actat the posttranscriptional level.93 This observation is consistentwith the appearance of type II keratin expression prior to thetype I pair during normal differentiation.24 Others have shownthat when keratins K8 and K18 are expressed in fibroblasts ortransformed cells, both are stable and form filaments whenthey are expressed together, but neither polypeptide is stableby itself.9495

Keratin assembly into filaments has been studied using keratinK14 cDNAs containing deletions and driven by the SV40promoter. K14 expression from the transfected cDNA wasdetectable in the PtK 2 cells even in deletion mutants becauseall DNA con structs contained a short (15 amino acid) sequencecoding for a portion of a neuropeptide, substance P, to permitstaining with antibody to substance P. Albers and Fuchs 9697showed that the entire carboxyl terminal nonhelical portion ofK14 could be deleted without altering filament assembly, butthat additional deletion of only six amino acids of the helicaldomain disrupted filaments.

Transient transfection allows analysis of a small subset ofcells. In contrast, stable transfectants can be selected 98 andgrown under conditions that permit differentiation. Futureexperiments of this type will allow investigation of questionsthat have not been addressed to date. For instance,nonkeratinized buccal epithelial cells could be stably transducedby Kl and K10, under the direction of an inducible promoter.Would expression of K l and K10 then result in downregulation



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Critical Reviews In

A. Gene Structure-* Upstream regulatory region Transcribed reg ion-

TR E TR E TATAEnhancers Promoter

E.WLM.M.Z.Coding sequences Tand introns AAAA

B. Keratin Gene PromoterCAPK1 ( B ) CCAAT TATA [CAPK14 ( B ) < S> [SP1] TATA

Suprabasal(palate,epidermis)

Basal

CAPK6b ( B ) TATA Suprabasal(palate)CAPK13 TATA Suprabasal(buccal)

C. Keratin Protein Subunit

K1-8(basic)K9-19

Non-Helical

NN

M Helical

=0 #^=1

Non-HelicalC

Cit it

FIGURE 8. Keratin gene organization. (A) General organization of a gene showing the upstream regulatoryregion with TATA box and tissue response elements (TRE) within the promoter, and transcribed regionbeginning at the CAP site (straight arrow). Translation of the mRNA begins at the ATG codon (waveyarrow). Coding sequences (exons), open boxes; introns, hatched boxes; 5' and 3' untranslated portions ofthe mRNA, dotted boxes; polyadenylation site, AAAA. (B) Diagram of the promoter region for keratinsexpressed in tissue- and differentiation-specific mann er. Various consensus sequen ces are indicated: B,Blessing consensus (see text); SP1, protein binding site; S, SV40 viral consensus. The sequence of the K13promoter is not yet known. (C) Keratin protein subunit structure for type I (K9-19) and type II (Kl-8)keratins. Arrows indicate the conserved position of introns within the genes. Underlined arrows indicateintrons that are found only in type I and type II keratins, but not both. (Adapted from Steinert, P. M. andRoop, D. R., Ann. Rev. Biochem., 57, 593, 1988.)



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thenormal buccal cell keratins K4 and 13, a change in tissueor in the expression of other markers ofInother words, does theexpression of a keratin

is differentiation specific cause subsequent changesssociated with that pattern of differentiation, or is it an effect

B. Studies on the Regulation of Keratin GeneExpressionThe underlying basis for thepattern of expression of individual

keratins in a tissue-specific anddifferentiation-specific manneris anexciting newarea of study. Because thekeratin genes aretightly regulated, sequences within the genes must exist thatare critical for regulation of expression. Soluble protein factorsthat interact with genes and regulate gene expression are calledtrans signals (transcription activators or repressors), while thegenetic elements within the upstream regulatory region of thegenes (response elements) are called cis signals.

1. Keratin G enes and Their Chromosom al LocationSequences of many of the keratin genes are now avail-a b l e . 9 3 " 108 All sequences available so far contain TATA box

(binding site of RNA polymerase II109 ) about 30 bp upstreamfrom the ATG translation initiation codon. Inspection of theupstream sequences does not reveal obvious common sequencesfor keratins expressed in basal cells compared with thoseexpressed in suprabasal cells that would be expected to res-pond to differentiation-specific sig nals. How ever, a consensussequence of AA(Pu)CCAAA is found in nearly all keratinssequenced thus far from human, bovine, and mouse genes.10 5This sequence is also present in thehuman involucrin genes,11 0and in some papilloma virus genes,11 1 but not in keratins fromsimple epithelia. This sequence is presumably important fortissue-specific expression by serving as the binding site for aspecific factor present in stratified epithelia (e.g., the CEI andCEII binding proteins11 2). Other sequences of interest that arefound in a limited numb er of keratin genes have been reviewedrecently.23 Adiagram show ing these features isshown inFigure8. Additional sequences that influence transcription aresometimes located within introns, such as that in the gene forprocollagen a 1 (I).11 3 The first intron in keratin K10 has asequence that is conserved between several species and is acandidate for a regulatory function.11 4 Additional regulatorysequences may respond directly or indirectly to retinoids (seeSection IV), calcium concentration, andother factors that jointlymodulate the expression of keratin pairs.

Clusters of type I and II keratins have been mapped tohumanchromosomes 17 (type I, acidic keratin) and 12 (type II, basickeratin), respectively .108115116 This m eans that the joint expres-sion of a pair of keratins is not a consequence of their positionon the chromosome, but may be regulated at the level of tran-scription via common trans-acting factors, such as might bind

to the sequence AA(Pu)CCAAA noted above. Other types ofregulation must also be considered as suggested by the recentresults ofLersch andco-workers 93 whoshowed that K5expres-sion stabilized that of its pair, K14, at a posttranscription step.Detailed sequence analysis of the upstream regulatory re-gions for keratins specifically expressed in the oral cavity areno t yetpublished. However, abovine keratin gene, K6 * (equiv-alent to human K4), which is expressed in tongue mucosa, ispresent in a chromosome cluster with at least one other basickeratin gene.10 5 Future work on K4 andK13 mayreveal controlelements within the upstream sequences that are responsiblefor oral vs. epidermal expression. Work on the K13 promoterregion is in progress.22 7

2. Studies of Regulatory Sequences Within KeratinGenesTransfection studies and work with transgenic mice havebeen used to explore regulatory sequences within the keratin

genes. Both techniques permit studies of portions of theupstreamregulatory region of genes by deletion analysis. They can beextended to applications on tissue-specific aspects of oralepithelia using appropriate probes.a. TRANSFECTION STUDIESTransfection studies rely on the construction of recombinantDNA clones containing the upstream regulatory elements,combined with either the gene of interest or an easily detected"reporter" gene. The chloramphenicol acetyl transferase (CAT)gene is frequently used.11 7 The CATgene is actively expressedonly if transcriptional activators are present in the cell typeused for transfection, and only when their binding sites areincluded in the transfected construct. This is a useful assay todetermine the size of the promoter region and to identifyenhancer sequences.

The promoter isdirectly upstream from thecoding sequencesof a gene and contains the binding sites necessary for trans-cription in a tissue-, development-, and differentiation-specificmanner. Many genes have promoters on the order of 1000 bpin size. Enhancers are short DNA sequences that may liesomewhat distant from thepromoter, or can be located withinthe promoter, within an intron, or even in the 3' flankingsequences (seeFigure 8A ). Enhancer elements confer increasedtranscription in a position-independent manner, i.e., they canbe placed downstream or further upstream or on the noncodingstrand, and still function. Using CAT reporter gene assays,Blessing andco-workers11 8 showed that thebovine keratin geneIV (similar tohuman K6)contains apromoter of approximately600 bp. It contains a region between position -600 and -180relative to the start of mRNA transcription that confersexpression in bovine and mouse epithelial cells, but notfibroblasts. Thus, it activates cell type-specific expression.Hybrid gene constructs containing thebovine la (Kl) andbovineVIb (K10), under thecontrol of this gene IVpromoter/enhancer



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Critical Reviews Inwere stably transfected into simple epithelial cells and wereexpressed at high levels. The recipient simple epithelial cellsdo not normally express these keratins.

Many questions remain; for instance, in studies in vitro, thebasal cell kera tin, K1 4, is transcribed preferen tially in epithelialcells and also in fibroblasts, albeit at a lower level.11 9 Thesimple epithelial keratin K18 is promiscously transcribed evenwith 2.4 kb of upstream sequences; therefore, this is notsufficient information for tissue-specific expression.98

Extension of these studies will allow further analysis ofdifferentiation-specific seque nces. It is reason able to assumethat several trans-acting factors may be required to specify thecomplex expression patterns of the keratin genes, analogousto those currently being elucidated in other systems as recentlyreviewed.120 These factors may act in either a positive or negativemanner and may confer expression in response to vitamin Aor hormones and growth factors.

b. STUDIES IN TRANSG ENIC M ICEThe production of transgenic mice by microinjection of clonedDNA into fertilized mouse eggs is an alternative approach toidentify the ds-ac ting regulatory sequences required for tissue-specific gene expression (see Reference 121). Studies havebeen initiated on keratin expression in transgenic mice usingthe human keratin K l gene with approximately 2 kb of upstreamregulatory sequences.24 Expression of the human Kl gene wasseen in skin, but not in stomach, liver, or brain. Within theskin, the human Kl mRNA and protein are found mainly inthe suprabasal cell layers. In addition, the human Kl gene isexpressed in the skin at day 15 of developmen t, the same timeas the mouse Kl gene. Thus, this DNA fragment containingthe Kl gene and 2 kb of upstream sequences has some tissue-specific, differentiation-specific and development-specificsignals that respond to transcriptional activators present in mouseepithelia. Similarly, a keratin K1 4 gene (without introns) withapproximately 2.5 kb usptream flankin g sequences w as regulatedin a tissue-specific manner in transgenic mice. This gene isnorm ally dow nregu lated with differentiation. Su prabasal cellsin epidermis showed effective downregulation in the transgenicmice, while those in tongue and esophagus were morevariable.119 Future studies will determine the location and natureof these regulatory signals via deletion analysis and more refinedmethods such as DNA footprinting and other techniques thatprovide information on the exact contact points of transcriptionalfactors with the DNA sequence.IV. MULTIPLE INFLUENCES ON GENEEXPRESSION AND DIFFERENTIATION

Regulation of differentiation in stratified epithelia is influ-enced by many factors, including the adjacent mesenchymaltissue, compo nents of the extracellular matrix, growth factors,and retinoids. Structural proteins are commonly used as tools

in this work. Th is part of the review touches briefly on severalof these aspects, but it concentrates on new concepts in theaction of retinoids and initial studies in the understanding ofkeratin expression. For information on the culture techniquesutilized, including oral organ culture, recombined epithelial/mesenchymal cultures, and oral cell culture techniques, thereader is refered to recent reviews and publications. 122126A. Role of Connective Tissue1. Adult Oral Ep ithelia

The connective tissue retains an influence over the oralepithelial differential pattern even in the adult. This area isreviewed in depth in a future issue of this journal 12 7 and iscovered only very briefly here. T he clearest evidence to supportthe connective tissue influence has been obtained by heterotypictissue recomb ination25128130 and analysis of tissue architecture,keratin protein profiles, and cell surface antigens. Resultssuggest that the connective tissue may provide both perm issiveand instructive signals to the oral epithelium.The influence of connective tissue in vivo was demonstratedin studies in which palatal connective tissue was submergedin the alveolar mucosa. When the grafts were surgically exposedand allowed to heal, reepithelialization from the noncornifiedlining mucosa resulted in cornified masticatory epithelium overthe palatal connective tissue.13 1 The superficial portion of theconnective tissue appears to be responsible for influencing theexpression of keratins typical for this type of differentiation(appearance of K l and 10 and loss of K4 and 13). 33 In addition,subdermal graft ing studies of Mackenzie and Fusenig 13 2demonstrated a lack of influence of deep connective tissue inmaintaining and directing epithelial differentiation. This work,taken together, suggests that some reciprocal interactions ofepithelia and connective tissue are important for determiningthe factors in the connective tissue that subsequently controlthe epithelial differentiation pattern and gene expression. Furtherstudies in this area are needed to understand the molecularbasis of the epithel ial-mesenchymal interact ions. Kerat inproteins and other markers of differentiation have been extremelyimportant tools in this work.2. Developing Palate

Epithelial-mesenchyme interactions control organogenesis ofmany epithelial tissues and epithelial derived structures duringdevelopm ent (see review s, References 133-136). One interestingmodel system for study is that of the organ culture of palatalshelves and their fusion in vitro via programmed cell death ofthe medial edge epithelium. Although cell death seems to bethe converse of epithelial differentiation, keratins are usefulmarkers in these studies because they are poorly expressed inthe medial edge area of shelf fusion, and differentially expressedin regions that become nasal (K8 and K 18) or hard palate (K10)epithelium on opposite aspects of the shelf (see reviews,References 137 and 138). Several messenger signals are required



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sion and can cause precocious cell death;138139 EG F, which140141 extracellular matrix components, growth

ar matrix compo nents. Collagen type IXactors T G Fa and TG Fp , and it is expressed

regulation of epithelial differentiation,

1. Tissue StudiesThe structures of vitamin A (retinol) and related compoundse important regu lators of epithelial differentiation since thelassic work of Fell and Mellanby 14 2 and Hardy.14 3 Excess

vitamin A produces mucous metaplasia with suppression ofkeratinization in the embryonic skin of chicks14 2 and the skinand oral epithelium of rodent embryos.144145 With treatmentin organ culture, hair follicles assume a branched glandularmorphology, epidermis becomes hyperplastic,14 6 and palatalshelves fail to fuse both in vivo14 7 and in vitro.14*14 9 Retinoicacid effects both mesenchymal proliferation of cultured palatalshelves and fusion of the palatal medial edge epithelium.150151

The medial epithelium undergoes conversion to a more nasal(secretory) type instead of the programmed cell death thatnormally accompanies fusion of the palatal shelves. Increasedexpression of EGF -receptors by retinoic acid is one mechanismwhereby retinoic acid may exert a regulatory influence on theepithelium in vitro.152153Deficiencies in vitamin A in vivo result in enhanced expressionof markers of keratinization in target epithelia, including

squamous metaplasia in trachea and keratinization in thecornea.154155 Molecular mechanisms are being pursued in cellculture by various investigators.2. Retinoids Show Differential Effects on Skin andOral Ep ithelia

Excess retinoids have effects on both fetal and adult oralepithelia and skin; however, oral effects are more profound.In skin, topical application results in hair loss, skin fragility,and parakeratosis depending on dose and the specific retinoidemployed (see review, Reference 156). Topical treatment ofmouse tail reduced expression of two keratins that are associatedwith the parakeratotic scale,15 7 but histological effects wereminimal. However, the hamster cheek pouch undergoes histo-logical changes consistent with mucous metaplasia and inhibitionof keratinization in response to elevated levels of vitaminA i58-i6o o m l and skin epithelia from fetal rodents also showdifferential responses to retinoids. Skin maintained in culturefor 6 to 13 d showed thinning of the epidermis and reducednumber of hair follicles, but keratinization occurred at a rateequivalent to that in vivo. In contrast, explants of palate andtongue first form, then shed, a keratinized layer, and sub-sequently secrete mu cous and form goblet cells.14 5 Thus, excess

p-carotene

vitamin Aretinol

CHO

COOHall-trans retinoic acid 13-cisretinoic acid COOH

FIG UR E 9. Structures of (3-carotene, vitamin A, and active meta bolites. Retinol is transported in the serum , retinoicacid is probably the act ive form in most ce l l types and acts as a morphogen in development . 22 6 Retinyl esters are thestorage form of the vitamin, and retinal is essential for visual processes.

1990 18 1by guest on January 16, 2012 For personal use only. No other uses without permission.cro.sagepub.comDownloaded from


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Critical Reviews Inretinoids seem to have m ore potent effects on the differentiationof oral epithelia than on epidermis. One mechanism that maybe responsible for differential tissue responses is discussed inSection IV.4.3. Retinoids Alter the Expression of Differentiation-Specific Epithelial Products In Vitroa. KERATINSMany markers of differentiation in cultured keratinocytesshow a response to retinoids consistent with a regulatory rolefor vitamin A in terminal differentiation (see reviews, References23 and 161). Early studies of neonatal keratinocytes culturedwith 3T3 feeder cells showed that both stratification andexpression of Kl and K10/11 16 2 are enhanced by removingvitamin A by delipidization of the serum used in cell culture.Similar results were obtained with keratinocytes from epi-dermis, conjunctiva, and vaginal epithelia.16 2 These effectswere specifically reversed by the addition of retinyl acetate.In the presence of excess retinoids, the expression of K13 andK19 increased.162164

Other studies confirmed these observations and extended theconcept of vitamin A regulation of keratin expression to thelevel of transcription of the gene to mRNA. The mRNA forKl and K10 are not produced in keratinocytes cultured in thepresence of retinoids.162163 In contrast, K1 3, 15, and 19 wereshown to be positively regulated by retinoic acid. 163166 K 6,16 , and 17 were slightly downregulated, while K4, 5, and 14were not affected.166 These effects are summarized in Table 3and show that individual keratin proteins have different responsesto retinoids. It is important to note that retinoid effects onkeratin expression are slow,1 6 2 1 6 7 1 6 9 suggesting that retinoidregulation of keratin expression is indirect.

Retinoid effects may operate in association w ith other factorsthat regulate differentiation of cultured cells. For instance , incultures of oral epithelial cells, enhancem ent of K1 3 expressionby retinoids is observed,17 0 but seems to be dependent onthe presence of physiologic concentrations of calcium.17 1Furthermore, retinoid effects on K13 expression are tissuespecific. In squamous cell carcinoma lines, K13 is expressed,apparently due to the increased sensitivity of these cells toret inoids.165166 In tracheal epithelial cells, K13 is a marker ofsquamous differentiation, rather than the normal pseudostratifiedpattern; in this tissue, addition of retinoids downregu lates K 13 ,apparently functioning to enhance the normal pattern ofdifferentiation.17 2

Culture on collagen rafts that can be raised to the air-liquidinterface results in improved stratification and differentiationin vitro with epithelial histology more similar to that invlvo 166,173-176 j n tfc s S y S t e m ? optimal retinoic acid concentrationfor epidermal cell differentiation is 10 ~~9 M. At less than10" 9 M retinoic acid, the histology is suggestive of hyper-keratinization, while at higher retinoid concentrations expressionof both keratins 1 and 10 and filaggrin (see below) is reducedand the cultures become hyperplastic.17 7 Detailed studies of

Table 3Effect of Retinoids on Protein Expression in HumanEpithelial CellsMarker for tissue Effect of added

Protein type retinoid' Ref.b

K lK10FilaggrinK13K13

K1 9

K5K1 4K 6K1 6K1 7InvolucrinK7K8K1 8K1 9

Keratinization

Nonkeratinized oralStratified squamous

tracheal epithelialBasal cells in some

stratified, nonkera-tinized oral

Stratified epithelial

' 'Hyperprolifera-t ive"

Stratified epithelialSimple, mesothelial

i

I*

== or si. i

=t

162178175, 166, 177162, 163, 165,

172

162, 165, 163,164, 166

166, 177

166, 177, 169

177168

a Symbols indicate decrease ( I ) , increase ( f ), or no change (= ) in proteinexpression with added retinoid. Concentration and retinoid used vary. Seereferences for details.

b Results reported to date are for epidermal cells or squamous cell carcinom alines, except for K13 in tracheal cells172 and simple epithelial studies.168

c Uniform suprabasal distribution.166d Squamous cell carcinoma lines are more sensitive to retinoids than normal

epithelia.165169e Species is rabbit. See text for further information.f Expressed in isolated stratifying cells. 166

retinoid effects in well-differentiated raft cultures have not beenreported using oral epithelial cells.b. FILAGGRIN

Retinoids also regulate filaggrin production in kera tinocytes,both at the level of biosynthesis of profilaggrin and post-translational processing . The effect is consistent with the "a nti-keratinizing" role of retinoids. Epidermal keratinocytes exposedto retinoids in culture showed reduced profilaggrin expressionin parallel with the decrease in keratin K l and K10 exp ression,while culture in delipidized serum results in increased pro-filaggrin in both human 161177178 and rodent179180 cultured cells.c. CORNIFIED CELL ENVELOPE

Retinoids suppress formation of the cornified cell envelope,another marker of keratinization,181182 perhaps via effects on182 Volume 1, Issue 3



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Themechanism ofof CCE formation is not clear, but may be related

183184

OTHER EFFECTS IN CULTURED CELLSRetinoids reduce the number of desmosomes, 18 5 enhance186187 alter lipid synthesis,18 8 alter posttrans-

of proteins,18 9 and enhance phosphory-of amembrane associated protein.190 Some of these effects

ay be related to the lipid properties of the retinoids and then-in addition to presumed effects

. How Do Retinoids Function in Regulation ofMost studies of retinoid action in cell culture rely on direct

of the retinoid. However, epithelial cells and fibroblastsnot come into contact with retinoids in the free form in vivoas a complex with serum retinol

in the serum with transthyretintheretinoid interacts with a cell in a target

it enters and is then complexed with another bindingin the cytosol. Retinoic acid enters the nucleus and

via the newly discoveredof these stages is important for

Theproposed mechanism of action of retinoidsin Figure 10.

a. SYSTEMIC CIRCULATIONDietary (3-carotene is taken up by intestinal mucosa. It ismetabolized to retinol, complexed with a cellular retinol bindingprotein (CRBP type II), esterif ied, and transported viachylomicrons to parenchymal cells in the liver. These cellssynthesize sRBP that is secreted with bound retinol (reviewedin References 191 and 192). sRBP is a 21-kDa protein thathas been isolated from several species 193194 and characterizedby X-ray crystallography.19 5 The human sRBP gene has beencloned.19 6 Once in the bloodstream the retinol-sRBP bindsstrongly to transthyretin, a thyroxine binding protein secretedindependently from the liver. When retinol is delivered to thetarget cell surface, transthyretin and sRBP dissociate and arefiltered through the kidneys or are recycled.19 1

b. CELLULAR BINDING PROTEINSTwo intracellular binding proteins for retinoids, cellularretinol-binding protein (CRBP) andcellular retinoic acid-bindingprotein (CRABP), occur in a wide variety of cell types. Bothare proteins of about 15.5 kDa and may facilitate intracellularretinol trafficking.19 7 Similarity in the structure of several ofthe retinoid binding proteins suggests they are members of afamily of related retinoid and fatty acid binding proteins (Table4 ) 198-203 ce llu lar RB Ps have been recently review ed.20 4CRBP is present in low amounts in both epidermis anddermis andoral mucosa.20 5 CRABP is most abundant in tissuesthat are sensitive to the action of retinoic acid. It is found inhigh concentrations in skin, adrenal glands, testis, and

Proposed Mechanism of Action of RetinoidsTTR-SRBP

FIGURE 10. Proposed mechanism of action of retinoids in epithelial cells. Retinoids areassociated with binding proteins. Retinol enters the cell and is either stored as an ester, orconverted to retinoic acid. Retinoic acid binds to nuclear retinoic acid receptors that alter theexpression of unknown target genes.



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Critical Reviews InTable 4Retinoid Binding Proteins

SizeName Abbreviation Ligand (kDa) Ref.Serum retinol binding sRBP Retinol 20.5 193, 194, 196

proteinCellular retinol binding CRBP Retinol 15.7 19 8, 19 9,2 00protein

Cellular retinol binding CRBP type II Retinol 15.6 198, 20 1, 202protein (intestinal)

Cellular retinoid acid CRAB P Retinoic acid 15.5 199binding protein

uterus.204206 In skin, it is localized in epiderm is.205207 It is alsopresent in cultured keratinocytes20 8 and increases dramaticallyin differentiating keratinocytes.20 9 CRABP probably has animportant function in epithelial differentiation,210 possibly tomodulate transfer of retinoic acid to the nucleus. 21 1 However,the presence of large amounts of the binding protein in theepidermis, a tissue that responds to excess retinoids by inhibitionof terminal differentiation, is still a paradox. Perhaps C RAB Psequesters retinoic acid, preventing it from entering the nucleus.Alternatively, the ratio of CRA BP with and w ithout associatedretinoic acid may be critical in the function of the retinoid orits transfer to the nuclear receptor protein (see below).

There is some evidence of differences in the metabolism orresponse of oral mucosa to retinoids. Buccal mucosa containsfour times the level of CRABP of normal adult epidermis, butsimilar low levels of CRBP. Furthermore, the fate of sRBPdiffers in oral tissues. In buccal epithelium active sRBP isretained in the tissue, while in the epidermis the retinoid bindingcapacity is lost.212 -213 Thus, sRBP could be important inmediating the effects of retinoids in tissues. Two studies supportthis idea: treatment with retinoid complexed to sRBP in organculture showed minimal effects in muco us metaplasia assays,21 4and epidermal cells took up 15- to 20-fold less retinol whenpresented with the complex than the free retinoid.21 5 Thus,some impo rtant questions regarding retinoid delivery and tissueutilization are unresolved.c. NUCLEAR RECEPTORS

Retinoic acid moves to the nucleus21 1 where an interactionwith chromatin or nuclear protein has been demo nstrated.21 6'21 7Once delivered to the nucleus, arrays of tissue-dependentdifferentiation-specific genes are either up- or dow nregulated.This process is now thought to involve the newly discoverednuclear retinoic acid receptor proteins.218223 These receptorswere initially cloned from X g tl l cDNA libraries with probesfor the DNA binding region of steroid hormone receptors.Investigators reasoned that retinoid receptors might have DNAbinding domains similar to other nuclear receptors proteins,specifically those of the steroid and thyroid hormone bindingreceptors (reviewed in Reference 224). These receptors are

now known to belong to a superfamily of DNA binding proteins;they are responsive to retinoic acid and called the retinoic acidreceptors (RARs).

Three distinct RARs h ave been cloned from rodent and hum anlibraries (see Reference 222) and additional related receptorsmay exist. Work is actively underway to demonstrate tissuedistribution of the RARa, RARp, and RAR7. RARa isubiquitously expressed; RAR|S is most strongly expressed inkidney, spinal cord, cerebral cortex, prostate, and in theembryo.22 5 RAR7 is the predominant receptor in skin.222 -223RA Rp , but not RA Ra , expression is induced by retinoic acidin hepatoma cells22 5 and in F9 embryocarcinoma cells uponretinoic acid-induced differentiation.22 3 This observation mayhave significance in the function of retinoic acid as a morphogenin development.22 6 The receptors have different affinities forretinoic acid, and the sequence of each of the three receptorsis conserved between species, suggesting that each has a distinctfunctional role. It is extremely likely that tissue-specific anddifferentiation-specific retinoid effects are med iated via thisfamily of nuclear receptors.V. SUMMARY

Differentiation within the oral epithelia shows region-spe-cific patterns of expression of the keratin proteins as well asassociated pro teins. In g eneral, the b asal cell layer in all regionsshows similar keratin expression, while the suprabasal celllayers express a specific set of markers, indicating co mm itmentto a pattern of differentiation. Man y extracellular factors in-fluence expression of the genes for these proteins. One im-portant factor is retinol (vitamin A), which is now known toexert its effect on gene expression by a group of nuclear re-ceptor proteins similar to the steroid hormone and thyroid hor-mone receptors. Some region-specific gene expression may bemediated through the differential expression of these receptorsin the different regions of the oral cavity and skin. More in-formation should be available in the next few years to test thishypothesis. Expression of the keratin genes is currently beinginvestigated by characterization of their regulatory regions inorder to determine what sequ ences within the genes themselvesare responsible for differentiation-, developm ent-, and region-specific expression.

REFERENCES

1. Squier, C. A., The permeability of keratinized and nonkeratinized oralepithelium to horseradish peroxidase, J. Ultrastmct. Res., 43, 160,1973.2. Schroeder, H. E., Differentiation of Human Oral Stratified Epithelia,S. Karger, Basel, 1981.



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Oral Biology and Medicine3. Squier, C. A. and Meyer, J . , Eds . , Current Concepts of theHistology

of Oral Mucosa, Charles C Thomas, Springfield, 1971.4. O'Guin, W. M., Galvin, S., Schermer, A., and Sun, T. T., Patterns

of keratin expression define distinct pathways of epithelial developm entand differentiation, in Current Topics in Developmental Biology, TheMolecular and D evelopmental Biology of Keratins, Vol. 22, Sawyer,R. H., Ed. Academic Press, Orlando, FL, 1986.

5. Franke, W. W., Schiller, D., Moll, R., Winter, S., Schmid, E.,Engelbrecht, I., Denk, H., Krepler, R., and Platzer, B., Diversityof cytokeratins: differentiation specific expression of cytokeratin poly-peptides in epithelial cells and tissues, J. Mol. BioL, 153, 933, 1981.

6. Moll, R., Franke, W. W., Schiller, D. L., Geiger, B., and Krepler,R., The catalog of human cytok eratins: patterns of expression in normalepithelia, tumors, and cultured cells, Cell, 31, 11, 1982.

7 Quinlan, R. R., Schiller, D. L., Hatzfeld, M., Achtstatter, T.,Moll, R., Jorcano, J. L., Magin, T. M., and Franke, W. W.,Patterns of expression and organization of cytokeratin intermediatefilaments, Ann. N.Y. Acad. Sci., 455, 282, 1985.

8 Sun, T. T., Tseng, S. C. G., H uang, A. J. W., Cooper, D., Schermer,A., Lynch, M. H., Weiss, R., and Eichner, R., Monoclonal antibodystudies of mammalian epithelial keratins: a review, Ann. N.Y. Acad.Sci., 455, 307, 1985.

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New Approaches and Concepts in the Study of Differentiation of Oral Epithelia

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