Keratinocyte transglutaminase in human skin and oral mucosa:

cytoplasmic localization and uncoupling of differentiation markers

BACH MAI T A \ GEORGE T. GALLAGHER1, RUPA CHAKRAVARTY2 and ROBERT H. RICE2'

'Department of Oral Pathology, Harvard School of Dental Medicine, and the 2Charles A. Dana Laboratory of Toxicology,Harvard School of Public Health, Boston, MA 02115, USA

* Present address for correspondence: Department of Environmental Toxicology, University of California, Davis, CA 95616, USA

Summary

Expression of keratinocyte transglutaminase, aspecific differentiation marker, has been examinedby immunogold-silver cytochemistry in humanepidermis and oral epithelium, and in oral mucosalhyperplasia and neoplasia. Two major findingshave been obtained. First, considerable immuno-reactivity was evident not only at the plasma mem-brane (the site of cross-linked envelope formation)but also in the cytoplasm of spinous cells, sugges-ting a cytoplasmic function for this transglutami-nase. Staining at the cell border was seen princi-pally in the granular layer of orthokeratinizedepithelium (epidermis, hard palate), the outer spi-nous cells of ortho- and parakeratinized epitheliumand in the suprabasal cells showing squamousdifferentiation in benign and malignant neoplasms.By contrast, diffuse cytoplasmic staining was ob-served in the upper spinous layer of the normalepithelium and benign lesions. The cytoplasmicimmunoreactivity, which extended nearly to

the basal layer in hyperkeratosis of the oralmucosa, was evident in two of three verrucouscarcinomas examined. In keeping with their undif-ferentiated character, invasive nests of squamouscell carcinoma and basaloid epithelium in benignand neoplastic lesions were immunonegative fortransglutaminase. The second major finding wasthat lesions of severe oral epithelial dysplasia,immunonegative for transglutaminase, werecapable of expressing involucrin immunoreac-tivity, indicating an uncoupling of keratinocyteprogramming. These results suggest thatimmunogold-silver staining for transglutaminasemay be useful in evaluating the degree of differen-tiation in benign and malignant oral epithelialproliferation.

Key words: keratinocyte, transglutaminase, involucrin.

Introduction

Transglutaminase plays an important role in terminaldifferentiation of the mature keratinocyte. This enzymecatalyzes the formation of e-(y-glutamyl)lysine isopep-tide bonds, and thereby cross-links the soluble proteininvolucrin, certain other proteins and polyamines intoinsoluble cross-linked envelope structures of the cornifiedcells (Rice and Green, 1979; Simon and Green, 1984,1985; Thacherand Rice, 1985; Michel ef al. 1987; Nagaeet al. 1987; Piacentini et al. 1988). Two related forms ofkeratinocyte transglutaminase (particulate and soluble)have been identified in cultured normal human epidermalcells and squamous carcinoma cell lines (Thacher andRice, 1985; Rubin and Rice, 1986). The enzymes chro-matograph identically on DEAE-cellulose, are precipi-tated by three different monoclonal antibodies and dis-play the same apparent molecular weight (92 000) in gelelectrophoresis (Thacher and Rice, 1985). The particu-late form displays post-translational fatty acid acylation,

Journal of Cell Science 95, 631-638 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

accounting for its membrane localization, while thesoluble form lacks this modification (Chakravarty andRice, 1989). Previous work using frozen sections of skinhas shown that the enzyme is expressed maximally in theupper spinous and granular cells of the epidermis, similarto involucrin (Thacher and Rice, 1985; Parenteau et al.1986). However, the existence and localization of thecytosoluble form in epidermis and related epithelia haveremained uncertain, since the monoclonal antibodiesemployed previously are unreactive in formalin-fixedtissues.

Immunocytochemical investigations have shown in-volucrin to be a useful marker in characterizing thedifferentiation of keratinocyte-containing neoplasms(Warhol et al. 1982; Said et al. 1983; Kaplan et al. 1984;Murphy et al. 1984; Walts et al. 1985). The present studyexplores the usefulness of transglutaminase for thispurpose. Transglutaminase and involucrin have nearlyidentical patterns of expression in normal epidermis(Thacher and Rice, 1985). However, they have now been

631

shown clearly in cultured squamous carcinoma cells todiffer temporally in their induction and to be modulateddifferently by physiological and toxic agents (Rice et al.19886; Rubin et al. 1989). These observations suggestthat their patterns of expression might not be identical inneoplastic lesions, and that an uncoupling of keratinocyteprogramming might be observable in vivo as well as inculture. The present investigation of normal and neoplas-tic epithelia with polyclonal antisera thus permits localiz-ing the transglutaminase in fixed sections and examiningits expression relative to involucrin.

Materials and methods

Biopsy tissueSpecimens were obtained from the pathology files of theHarvard School of Dental Medicine and Tufts School of DentalMedicine. The tissues had been fixed in 10% neutral bufferedformalin for 2-8 h, routinely dehydrated and paraffin embed-ded. The diagnosis was confirmed in each case usinghematoxylin-eosin-stained sections.

AntiseraThe rabbit anti-involucrin antiserum was that prepared andused previously (Rice and Green, 1979; Warhol et al. 1982).Rabbit anti-transglutaminase antiserum was raised against thepaniculate enzyme. To this end, the paniculate fraction fromcultured human epidermal cells was extracted with Emulgennonionic detergent and the extract was clarified by high-speedcentrifugation. Transglutaminase (estimated 40 /ig) was immu-noprecipitated with B.C1 monoclonal antibody (Thacher andRice, 1985) and the washed immune complexes were injectedwith Freund's complete adjuvant. All the experiments wereperformed using a single bleed from one rabbit after four boostsof antigen. Prior to use on the tissue sections illustrated, theantiserum was absorbed with the keratin fraction from culturedepidermal cells and with the Emulgen-soluble paniculate ex-tract adsorbed to nitrocelluose after removal of the transgluta-minase by B.C1 immunoprecipitation. The antiserum reactedstrongly and specifically with keratinocyte transglutaminase inimmunoblots (Towbin et al. 1979) of cultured human epider-mal cell extracts (Fig. 1). For confirmation of the tissue-staining patterns in some instances, specific antibodies wereaffinity purified; the antiserum was absorbed with transglutami-nase blotted onto nitrocellulose (after immunoprecipitation andSDS-polyacrylamide gel electrophoresis) with subsequent elu-tion at pH2.5 and rapid neutralization. The affinity-purifiedantibodies gave the same staining pattern as the absorbedantiserum (above) in immunoblotting and in tissue sections ofnormal skin and normal and hyperkeratotic oral mucosa.

• i- Fig. 1. Immunoblotting of cultured^ * ~"~ human epidermal cell extracts. Samples

(approximately 0.1 mg of protein) ofeither a nonionic detergent extract of theparticulate fraction (A,B) (Chakravartyand Rice, 1989) or a homogenized culture(C) were electrophoresed on 10 %polyacrylamide gels, transferred tonitrocellulose and reacted with a sampleof the absorbed anti-transglutaminaseantiserum at dilutions of 1: 100 (A) or1:1000 (B,C). Preimmune serum showed

f\ D C» no bands.

Immunogold-silver staining

Serial 4/im tissue sections were mounted on glass slides(previously coated with 0.5 % chrome alum-gelatin) andstained with hematoxylin-eosin or immunogold-silver (Horis-berger, 1979; Holgateeta/. 1983; Hacker et al. 1985; Parenteauet al. 1986). To this end, the sections were deparaffinized inxylene, rehydrated in a graded ethanol series and distilledwater, and held in 0 . 1 5 M NaCl, 0 .02M Na2HPO4 (pH7.4)(PBS) for 30min. For immunostaining, the sections wereincubated for 30min in 5 % nonimmune goat serum-0.1%bovine serum albumin (BSA)-PBS and then for 2h in anti-transglutaminase diluted 1: 800 or 1 h in anti-involucrin diluted1:500 in PBS-0.1% BSA. After treatment with the primaryantibody, the sections were rinsed three times (these andsubsequent rinses were for 3 min) in PBS-0.1% BSA andincubated for 1 h with Auroprobe goat anti-rabbit IgG coupledto colloidal gold (LM GAR, Janssen Life Sciences, Piscataway,NJ) diluted 1:20 in PBS-0.1% BSA or with protein A-goldprepared as described (Horisberger et al. 1979) diluted 1:80with PBS-0.1% BSA. Sections were given three rinses inPBS-0.1 % BSA and then three rinses in distilled water. Forsilver enhancement of bound colloidal gold, the slides weretreated with enhancing solution (IntenSE II kit, Janssen) andexamined microscopically. At the appropriate times, develop-ment was stopped by immersing the slides in distilled water.Some sections were lightly counterstained with Mayer's hema-lum. The sections were then dehydrated in a graded ethanolseries and mounted in mounting medium. All incubations werecarried out in a moist chamber at room temperature. Negativecontrols performed routinely with each experiment consisted ofsections incubated either with preimmune rabbit serum orwithout primary antibody. Routine positive controls weresections of human foreskin treated in parallel.

Results

Normal epithelium

The transglutaminase immunoreactivity of normal epi-dermis and epithelium of the oral cavity was examined toprovide a basis for comparison with hyperplastic, dys-plastic and neoplastic tissue samples. In the skin, asshown in Fig. 2A, positive staining for transglutaminasewas found principally in the granular layer and the outerspinous layer. At high magnification (not shown), stain-ing in the outer spinous cells appeared almost entirelycytoplasmic, while in the granular layer the cell borderswere heavily stained, usually considerably more heavilythan the cytoplasm. By contrast, as shown previously inother studies using formaldehyde-fixed tissues (e.g. seeMurphy et al. 1984), involucrin staining was evident inthe upper spinous and granular layers but was detectedonly in the cytoplasm (Fig. 2C). Neither antigen wasobserved in the basal layer or the basal half of the spinouslayer, and the cornified layer was virtually unreactive aswell.

The transglutaminase immunoreactivity observed inoral epithelium was similar to that in epidermis. Inregions of orthokeratinization, such as the hard palate(Fig. 3A), the most intense staining was evident at theborders of granular and upper spinous cells and little inthe stratum corneum. Substantial cytoplasmic stainingwas visible in these cells and even in mid-spinous cellslacking peripheral immunoreactivity. Nonkeratinized

632 B. M. Ta et al.

B 3A CT

I

V,

ct

j.,v^:

Fig. 2. Distribution of transglutaminase and involucrinimmunoreactivities in human skin. Sections were stained withanti-transglutaminase (A) or anti-involucrin (C) or thecorresponding preimmune sera (B,D) (X150). In these andsubsequent panels, broken lines indicate the position of thebasement membrane; the noticeable nuclear staining in theepithelia and connective tissue (ct) arises from lighthematoxylin counterstaining.

buccal mucosa stained most intensely at the borders ofthe well-differentiated cells in the middle of the epi-thelium and not in the basal layer. Staining of the mostsuperficial cells was less positive, as illustrated (Fig. 3B).

Lesions of oral mucosaTransglutaminase immunoreactivity was examined inlesions of lichen planus and hyperkeratosis (Fig. 4). Bothconditions showed heavy staining accentuated at the cellperiphery in the granular layer, which appeared thickerthan in the normal epithelium. In addition, the cytoplas-mic staining extended through the spinous layer nearly tothe basal layer (Fig. 4C). One difference noted betweenthe two conditions was that the parakeratotic stratumcorneum of hyperkeratotic lesions (Fig. 4B), althoughless immunoreactive than the underlying granular layer,still stained much more strongly than the virtuallyunreactive mature corneocytes of orthokeratotic lichenplanus (Fig. 4A).

Like the above benign lesions, verrucous carcinomaand keratoacanthoma of the lip were strongly immuno-

B

Fig. 3. Distribution of transglutaminase immunoreactivity inoral epithelia. A. Hard palate (XSOO); B, buccal mucosa(X200); C, preimmune serum, section adjacent to B (X200).

reactive for transglutaminase in broad regions that ap-peared cytologically well differentiated. As illustrated inFig. 5, the staining in verrucous carcinomas was concen-trated at the cell borders. Two of the three samplesexamined (Fig. 5C) also exhibited considerable cytoplas-mic staining in these regions and in adjacent cells withcytoplasmic but not peripheral staining. The othersample (Fig. 5D) exhibited cells in which the stainingwas almost entirely peripheral. The single lesion ofkeratoacanthoma examined (Fig. 5A) showed stainingprimarily at the cell periphery, while more diffusecytoplasmic staining was evident in cells separating thedifferentiated from the more basal-like undifferentiatedcells. The highly differentiated cells in keratin plugs wereonly weakly immunoreactive in both types of lesions. Incontrast to these patterns, lesions of reactive hyperplasia,associated with considerable inflammatory infiltrate,were immunonegative (Fig. 5B). This phenomenon may

Keratinocyte transglutaminase expression 633

4A

r "'ct

Fig. 4. Distribution of transglutaminase immunoreactivity inbenign oral disorders. Representative results are presented forlichen planus of buccal mucosa (A, X150) and gingivalhyperkeratosis (B, X150). C (X300) is a higher magnificationof B.

be analogous to the suppression by inflammation ofinvolucrin expression in immature squamous metaplasiaof the uterine cervix (Warhol et al. 1982).

Comparison of transglutaminase and involucnnThe relative expression and localization of transglutami-nase and involucrin were examined in oral dysplasias andsquamous cell carcinomas. It was clear that undifferen-tiated squamous cell carcinomas of the gingiva expressedlittle if any transglutaminase or involucrin immunoreac-tivity (Table 1). By contrast, well-differentiatedsquamous cell carcinomas were strongly positive for eachantigen. In such samples, as shown in Fig. 6A, transglu-taminase was almost entirely peripheral in cells where itwas expressed. These cells were in regions judged to bedifferentiated by histological criteria (hematoxylin-eosinstaining). The involucrin that was also strongly expressedin the same regions was entirely cytoplasmic (Fig. 6B).Consistent with these observations, moderately differen-tiated squamous cell carcinomas exhibited focal areas ofsquamoid differentiation that were immunoreactive,

yielding patchy involucrin staining essentially as ob-served previously for such lesions in the epidermis(Murphy et al. 1984).

Transglutaminase immunoreactivity and its coordi-nation with that of involucrin were examined in dyspla-sias of the oral mucosa. As shown in Fig. 7A, the degreeof transglutaminase staining was inversely related to thedegree of dysplasia. Thus, in mild dysplasia immuno-reactivity was evident in the upper spinous layer withintensity similar to that observed in keratinized normaloral epithelium and epidermis. In moderate and severedysplasia, the staining was restricted to the outermost celllayers or was virtually absent, respectively. In contrast,involucrin staining was evident through most of theepithelium, even in severe dysplasia, but was increasinglypatchy, forming irregular foci of immunoreactivity as thedegree of dysplasia increased. Illustrating this uncoup-ling of expression, Fig. 7B,C shows a lesion of severedysplasia in which immunoreactivity of involucrin butnot transglutaminase was readily detectable.

Discussion

Previous studies of transglutaminase localization infrozen sections of rat and human tissues have shown thatthe enzyme, consistent with its known membrane anchor-age by esterified fatty acids in cultured keratinocytes(Chakravarty and Rice, 1989), is localized at the cellperiphery in stratified squamous epithelia (Thacher andRice, 1985; Thacher, 1989). Although immunocyto-chemistry of certain rat tissues (Parenteau et al. 1986),and human middle ear cholesteatoma and meatal epider-mis (Broekaert et al. 1988) in frozen section, has sugges-ted some cytoplasmic localization of the enzyme, thepresent study employing fixed tissue reveals the consider-able extent to which this occurs. A precursor—productrelationship has not yet been established between thekeratinocyte transglutaminase and the cytosoluble 'epi-dermal' transglutaminase identified in skin (and known tobe subject to reduction in size by proteolysis; Negi et al.1985), but such a relationship would be compatible withthe present findings. Presumably, the cytoplasmic ac-tivity is responsible for isopeptide bonding detected inthe keratin fraction of extracted callus (Abernathy et al.1977) and adds rigidity to the mature cells.

According to studies primarily of enveloped animalviruses, the transferrin receptor and the la-associatedinvariant chain, fatty acid acylation of membrane pro-teins occurs in the rough endoplasmic reticulum or Golgiapparatus with subsequent sorting to the proper finaldestination (reviewed by Towler and Gordon, 1988). Inthe normal epithelium examined, translocation to theplasma membrane appears to occur by the time the cellsreach the granular layer, thereby positioning the enzymefor the formation of cross-linked envelope structures.However, the substantial cytoplasmic immunoreactivityin maturing spinous cells suggests that this process is notimmediate and may itself be a function of cell maturation.Alternately, the cytoplasmic form could reflect a type ofrelease from the membrane. For example, the transgluta-

634 B. M. Ta et al.

V-

\ \I \

/ ' ct \Fig. 5. Distribution of transglutaminase in verrucous carcinoma, keratoacanthoma and reactive hyperplasia.A. Keratoacanthoma of the lip (X1S0); B, gingival reactive hyperplasia (X200); representative patterns obtained from verrucouscarcinoma (X500) of the floor of the mouth (C) and tongue (D).

Keratinocyte transglutaminase expression 635

,6A

Fig. 6. Comparison of transglutaminase and involucrin immunoreactivities in well-differentiated gingival squamous cellcarcinoma. A. Peripheral staining of transglutaminase (X150); B, cytoplasmic staining of involucrin (X150).

I

I /

ct

CtB

ct

7A

Fig. 7. Comparison of transglutaminase and involucrin immunoreactivities in dysplasia of palatal mucosa. A. Transglutaminasestaining in moderately (1), severely (2) and mildly dysplastic (3) regions of a single lesion (XlSO); transglutaminase (B) andinvolucrin (C) staining of serial sections of a severe dysplasia (X150).

636 B. M. Ta et al.

Table 1. Transglutaminase immunoreactivity in skin and oral cavity

Diagnosis Number and site Pattern and intensity*

Normal

Lichen planus

Hyperkeratosis

Basal cell hyperplasia

Verrucous carcinoma cells

Keratoacanthoma

Squamous cell carcinomaWell-differentiated

Moderate differentiation

Undifferentiated

DysplasiaMild

Moderate

Severe

3 Foreskin1 Palate1 Buccal

1 Palate2 Gingiva2 Buccal

1 Palate1 Retromolar pad3 Gingiva

2 Gingiva

1 Tongue1 Floor of mouth1 Retromolar pad

1 Lip

1 Gingiva1 Floor of mouth1 Tongue1 Floor of mouth2 Palate1 Tongue

2 Palate2 Buccal1 Palate1 Retromolar pad2 Palate1 Retromolar pad

+ In spinous (cytoplasmic) to + + + in granular/superficial cells (cytoplasnuc andperipheral)

+ + In spinous (accentuated cytoplasmic) to + + + + in superficial cells (cytoplasmicand peripheral)

+ + In spinous (mostly cytoplasmic) to + + + + in superficial cells (cytoplasmic andperipheral)

- Except for foci of mature keratinocytes

+ In suprabasal to + + + in superficial cells; strong peripheral staining; considerable

cytoplasmic staining except in lesion from tongue

+ + In suprabasal (cytoplasmic) to + + + in superficial cells (cytoplasmic andperipheral)

+ + In suprabasal to + + + + in superficial cells; almost entirely peripheral

- Except for occasional differentiated foci

+ + To + + + in superficial cells (cytoplasmic and peripheral); focal stainingsuprabasally

Focal staining (cytoplasmic and peripheral) in suprabasal and superficialcells

•The degree of reactivity was judged to range from very strong ( + + + + ) to negative (—).

minase can be readily solubilized from the particulatefraction by exogenous or endogenous proteolytic activity(Thacher and Rice, 1985; Rice et al. 1990). In asubstantial fraction of verrucous carcinomas and in mostwell-differentiated squamous cell carcinomas of oralmucosa, present results indicate that the enzyme becomespositioned on the membrane almost immediately follow-ing synthesis, since little of the cytoplasmic form isdetected. This phenomenon could reflect altered timingof the protein sorting activity or lack of endogenousrelease during maturation of cells in such lesions. How-ever, a small proportion (typically 5 %) of the enzymeextracted from cultured epidermal cells is found in thesoluble fraction (Thacher and Rice, 1985) without thefatty acid anchor (Chakravarty and Rice, 1989). Theformal possibility that the enzyme is synthesized in asoluble cytoplasmic form and becomes anchored by theaction of transacylases found in the plasma membraneseems less likely but has not been ruled out.

The present survey of normal and pathological tissuesamples (summarized in Table 1) suggests several diag-nostic applications to oral epithelium. For example, whileone verrucous carcinoma sample resembled well-differ-entiated carcinomas in the lack of obvious cytoplasmicstaining, the other two resembled normal keratinizingepithelium in this respect. In the latter instance, the cellsexhibiting cytoplasmic staining were contiguous withcytologically more mature cells displaying not only per-

ipheral transglutaminase staining but also granular in-clusions. This dichotomy raises the possibility of dis-tinguishing among these lesions for prognostic purposes.Other possible applications include lesions of dysplasiaand reactive hyperplasia. Thus, keratinocyte transgluta-minase may supplement involucrin and keratin K19(Lindberg and Rheinwald, 1989) in diagnostic oral path-ology.

Study of malignant lingual and epidermal keratinocytelines has shown that expression of transglutaminase andinvolucrin can be uncoupled by a variety of physiologicaland toxic agents (Rice et al. 19886; Rubin et al. 1989).Under certain conditions, the cells can express substan-tial amounts of one marker in the virtual absence of theother. It is evident that premalignant keratinocytes canalso be subject to uncoupling in culture as well (Rice etal. 1988a). The present observations with premalignantoral lesions provide strong evidence for this phenomenonin vivo. Thus, in severe dysplasias the absence oftransglutaminase expression provides a clear contrastwith the readily detectable, albeit patchy, staining seenwith involucrin through much of the epithelium. Withreference to responses of neoplastic keratinocytes inculture (Rubin and Rice, 1986; Rubin et al. 1989), whereretinoic acid suppresses transglutaminase much morepowerfully than involucrin, the behavior of dysplasticgingival epithelial cells is analogous to increased sensi-tivity to circulating retinoids. As in the larynx (Kaplan et

Keratinocyte traiisglutaminase expression 637

al. 1984), perturbation of the differentiation disorganizesbut does not fully suppress involucrin expression.

This work was supported in part by PHS grants no. AR27130 from the National Institute of Arthritis, Musculoskeletaland Skin Diseases and CA 46928 from the National CancerInstitute. We thank Ms Qin Qin for assistance in immunoblot-ting.

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(Received 16 October 1989 -Accepted, in revised form, 16 January1990)

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