Diversity of cytokeratins

.I. J~ol.

Riol.

(I 9X1 ) 153. 933-959

Diversity of Cytokeratins Differentiation Specific Expression of Cytokeratin Polypeptides in Epithelial Celis and Tissues

/)i~vision of Membrane In&it,& German

I)-6900

DitYsion

of Ga&roenterologic

lT~~iwrsity

Ijepartment C7)liwwity

Riologsy

Research Center Federal Republic qf Oermn~nq

DENK,

Ikpartmen,t

Tu’mor

Cancer

Heidelberg, HEIAVT

and Bioche,mistry

Biology

of Cell and

REINHAKD Pathology

of Pathology, of Vienna,

KREPLEK.

A-1090

of Nedici?ze

Viennu,

AND BEATRIX

I’LATZER

of Dermatology.

School

of Innjsbrwk,

Hepatopath~ology

and

School

A lrstria

of Aledicine

A -6020 Innsbruck.

A rcatria

Epithelial cells contain a cytoskeletal system of intermediate-sized (7 to 11 nm) filaments formed by proteins related to epidermal keratins (cytokeratins). Cytoskeletal proteins from different epithelial tissues (e.g. epidermis ant1 basaliomas. cornea, tongue, esophagus, liver. intestine, uterus) of various species (man. cow, rat. mouse) as well as from diverse cultured epithelial cells have been analyzed by one and two-dimensional gel electrophoresis. Major cytokeratin polypeptides are identified by immunological cross-reaction and phosphorylat)ed cytokeratins by ] 32P]phosphate labeling in I&IO. It is shown t,hat different epithelia exhibit different patterns of cytokeratin polypeptides varying in molecular weights (range : 40,000 to 68.000) and electrical charges (isoelectric pH range: 5 to 85). Basic cytokeratins, which usually represent the largest cytokeratins in those cells in which they occur, have been found in all stratified squamous epit,helia examined, and in a murine keratinocyte line (HEL) but not in hepatocytes and intestinal cells, and in most other cell cultures including HeLa cells. Cell type-specificity of cytokeratin patterns is much more pronounced than species diversity. Anatomically related epithelia can express similar patterns of cvtokrratin polypeptides. Carcinomas and cultured epithelial cells oft,en contmue to synthesize cytokeratins characteristic of their tissue of origin but may also produce. in addition or alternatively, other cytokeratins. It is concluded : (1) unlike other t.vpes of intermediate-sized filamenm, cytokeratin filaments are ‘r Author t,o whom all correspondence OO22-2836/81/360933-27

$02.00/O

should be addressed. 9’3‘3 ..* i? 1981 Academic Press Inc. (London)

Ltd

934

WI:. W. FRASKE

ET AI,

highly htlterogcneous in r-omposition and (‘WI c~ontain basic polypeptitirs : (2) structurally indistinguishable filaments of t’he same class, i.e. cytokeratin filaments, are formed, in different epit)helial cells of the same species. bv different proteins of the cytokeratin family ; (3) vertebrate genomes c-ontain relatively large numbers of different cytokeratin genes which are expressed in programs caharacsteristic of specific routes of epithelial differentiation : (4) individual cytokeratins provide tissue- or cell type-sperific markers that are useful in the definition and identification

of the relatedness

or t,he origin

of epithelial

and csarcinoma

c~ells.

1. Introduction Jlorpholo~ic and immunolo# ohsrrvations allow the detinition of a class of cells that occur in various tissues of the vertebrate body and are characterized by the presence of a cytoskeletal complex consisting of differentiated regions of the surface membrane. the desmosomes. and attached intermediate-sized (7 to 11 nm) filaments related to epidermal prekeratin. Desmosomes are junctional complexes different from other kinds of junctions (Farquhar & Palade. 1963: Staehelin, 1971), and the filaments containing proteins immunoloyirally cross-reacting with epidermal prekeratins (cytokeratins) are different in composit’ion from other types of intermediat’e-sized filament’s (Franke et rrl., 1978n,b: Sun Kr Green. 197%; Lazarides. 1980). While cytokerat’in filaments appear to be confined to epithelial cells, cultured ones included (Franke et nl.. 197%r,h.l979a: Schmid et crl., 1979: Sun rt nl., 1979: Green. 1989: Schlegel rf nl., 198&x). it is still unclear whet)her they are present in all “true” epithelia and whether they are involved in epithelial func%ions (for problems of definition of “epithelia” see Hay. 1977). Several authors have ohserved that prekeratin filaments from different layers or regions of epidermis of the same organism cont’ain polypeptides of different sizes (Dale rt rxl.. 1976: Baden B Lee. 1978: Drochmans et nl., 1978: Franke el al., 197%: Skerrow & Hunt,er. 1978: Fuchs H: Green. 1978.1979,1980: Lee et r/Z.. 1979: Steinert et al., 1980). Furt,her differences in the patt’erns of constituent polypeptides have become apparent, from gel electrophoretic and immunological comparisons of epidermal prekeratins wit,h the cytokeratins present in other epithelial cells. cultured keratinocytes included (e.g. see Sun H: (ireen. 1977.19786: Franke et al.. 1978a,c.1979b.c,g,1981a;Fuchs $ Green. 1978: Steinert’ & Yuspa. 1978: Sun et al.. 1979: Milstone, 1981). Therefore, we have compared the composition of cyt,okeratin filaments from diverse kinds of epithelial tissues and carcinomas as well as from several cultured cells of epithelial origin. We report that cytokeratin filaments of different epit,helial cells are formed by different prot,eins of the cytokeratin family. and the patterns of cgt,okeratin polypeptides are characterist’ic of the specific types of epithelial cells.

2. Materials and Methods Albino fractions obtained

strains of mice and rats were used. Intestinal tonofilaments and cytoskeletal from liver were prepared as described (Franke et al.. 1981a,b). Epidermal tissue was from rat lips (cf. Franke et al., 1981b) or from dorsal skin of neonatal rats and mice.

I)IVERSITY

OF CYTOKERATISS

93.5

.1lternatively, epidermal material was used that had been prepared after t.reatment with ac,etica acid (Wint,er rl al., 1980). Bat and mouse corneae were removed from the eyes of freshly decapitated animals and rinsed with PBS (phosphate-buffered saline). Esophageal epithelium was obtained from mid portions of PBS-rinsed esophageal tubes by scraping with a sc~alpel. Epithelial preparat,ions from various regions of uterus and portio vaginalis W~IYJ also obtained by gentle srraping. mice were injected of cytoskeletal proteins For st udirs of phosphorylat,ion intrapcrit,oneally with 5 mC”i [ 3LP]orthophosphate each (50 to loo0 m(‘i/mmol : New England Smlear. Bost,on) and allowed to incorporate for 24 h. Thereafter, the livers were removrtl inid used for isolation of radioactively labeled cyt,oskeletons (Franke rt ~1.. 1981a). Bovine hoof and muzzle epidermis was prepared as described (Drochmans rt al.. l!)‘iH: I”rank(~ ot r/l.. t!)SXrr. 19816). Eyes from calves or covvs vvere rinscbd \\.ith PBS ancl (~rnea~ N’(‘u’ rcsmo\-r*d with a sc*alpel. Esophageal and uterine epithelia were obtained as described for r,ld,~nts.

Human soft epidermal t)issue rich in stratum spinosum was obtained from mamillar iemored during surgery. taking thin slicses parallel to the surfacbe with a cryocut knife. Sormal. i.e. moderately caornified epidermis was obtained in 2 different sets of sampling. Epidermal slices of non-mamillar regions from breast skin obtained during mammary surgerv were prepared using the cryocut knife as a plane. From patients suffering from basal carlI epitheliomas (“basaliomas”) superficially growing nodular basaliomas were excised, in a wedge-shaped fashion, from various regions (back. chest, neck. nose, scalp). For comparison normal epidermal tissue was t,aken from non-malignant skin regions surrounding the lesions. Samples were either extracted directly or were frozen in isopemane cooled in liquid nitrogen until preparation of cyt,oskeletons. Supertirial tissue from t,ongue mucosa was prepared. iising a cryocut knife. from autopsy cases (myocardial infarctions). Esophageal epithelial tissue was prepared from the same corpses, using a procedure similar to that, described for bovine tissue. Uterine tissues were obtained, usually by scraping with a scalpel. from samples prepared for hist,ologiral examinations (within 30 min after excision),during surgery of Iriomyomas. Epithelium of porfio angina/i.s was removed by tangential superficial sections rising a tine scalpel. (c) C’ulturrd

cells

Monolayer cultures of human HeLa cells and rat kangaroo PtK, cells were as describrcl (Franke et al.. t979d). Bovine kidney epithelial cells (MDBK rells: ATCC CCL 22). murine HEL ~rlls. a keratinocyt,e line derived from neonatal mouse skin with cahararteristirs of transformed cell growth (Franke et al., 19796) and rat hepatocytes et al.. (Franke 1!179d.l981c) were also used for comparison. For radioactive labeling of cytoskeletal proteins (~(~11 monolayers were washed twice in methionine-free MEM (minimal essential medium) and labeled for 18 to 14 h in MEM containing 0.2 of the normal methionine concentration and 50 p(*i/ml 1“%lmet~hioninr (spec. radioact. 1000 (‘i/mmol: Xew England Nurlear).

(d) .-I ,//il/odir.v

For tests of immunological cross-react,ion of polypeptides separat,ed by gel electrophoresis the following guinea pig antibody preparat,ions were used. (1) Antibodies raised against total prrkeratin from desmosome-attached tonotilaments of bovine muzzle (Fig. 1 (a)) which showed a broad range of cross-tea&ion among different epidermal prekerat,in polypeptides (Fig. l(b), slot 1’; caf. Franke rt ol., 198Oa,1981a,b). (2) Antibodies raised against cytokeratin caomponent D of mouse liver cytoskeletons that cross-reacted with, at least some, epidermal prekeratins and with (Aytokeratins present in other epithelial tissues (Fig. l(b), slot I”: Franke et al.. 1981a). (3) Various antibody preparations against protein of indivridual

936

W. W. FRASKE

ET AL.

polypeptide bands of bovine hoof and muzzle prekeratins, some of which showed a broad range of cross-reaction with ot,her prekeratin polypeptides (Freudenstein et al., 1978; Franke et al., 1980a) or reacted only with specific cytokeratin polypeptides (e.g. see Fig. l(b), slot I”‘). For comparison, guinea pig antibodies against murine and human vimentin (Franke ef al.. 1978b,1979d) and galline gizzard desmin (Franke et al., 198Oo) were used. Antibodies were applied either in full sera or as an immunoglobulin G (IgG) fraction purified by chromatography on DEAE-cellulose. In some experiments, antibodies purified by affinity chromatography as described (Franke et al., 198Ou; Osborn et al., 1980) were used with identical results. In immunoreplica and immunoblotting tests none of our antibody preparations against prekeratins and cytokeratins showed cross-reaction with other int,ermediate filament proteins such as desmin and viment,in. (e) Prepamfions of cytoskeletons and cell fractions Desmosome-attached tonofilaments from bovine muzzle were prepared by extraction with high salt buffer and Triton X-100 as described (Franke et al., 1978a,l98lb: cf. Drochmans ef al., 1978). Cytoskeletal fractions were prepared from freshly collected or deep-frozen ( - 70°C) tissue samples. Tissue pieces or minced tissue samples were incubated in buffer A (150 mmNaCl, 5 mM-EDTA, 94 mhr-phenylmethylsulfonyl fluoride, 10 mivr-Tris. HCI, pH 7.4) and homogenized for 1 min using a Polytron homogenizer (Fa. Kinematica, Lucerne. Switzerland) at setting 3. After centrifugation at 35OOg for 5 min t’he pellet obtained was rehomogenized in buffer B (10 mM-Tris. HCl, pH 7.4, 0.6 M-KI, 1.0% (v/v) Triton X-100, EDTA and phenylmethylsulfonyl fluoride as in buffer A) and extracted for 20 min by magnetic stirring in the cold room (0 to 4°C). After pelleting for 5 min at 3500g the material was resuspended and extracted in buffer C (1.5 M-KC] instead of 96 M-KI, other components as for buffer B) for 30 min (same conditions). After centrifugation for 20 min at, 3500 g the pellet obtained was washed twice by resuspension and another centrifugation in 10 mM-Tris. HCI (pH 7.4) containing the same roncent,rations of EDTA and phenylmethylsulfonyl fluoride. Final pellets were either analyzed directly or deep-frozen until use. Alternatively, tissue samples were extracted by a similar procedure using buffers that contained 61 mM-dithiothreitol or 2 miw-2-mercaptoethanol exactly as described for tonofilament fractions from intestinal brush border (Franke et al., 1981b). Cytoskeletons from monolayer cell cult,ures were prepared using a modification of the procedure described (Franke e/ al., 1978a.c.1979j’). Cells rinsed several times with PBS were lysed by brief (3 to 5 min) incubation with 140 mrv-NaCl, 10, Triton X-100, 5 mM-EDTA, 10 mu-Tris. HCl (pH 7.6). Lysis buffer was decanted and cell residues were incubated directly for 30 min in high salt buffer (1.5 M-KCl, 05:/, Triton X-100, other components as in lysis buffer). Residual cell mat,erial was scraped off with the aid of a rubber policeman and was pelleted by centrifugation for 20 min at 3500g. The pellets obtained were washed twice by resuspension in PBS and centrifugation. and the final cytoskeletal pellets were analyzed directly or stored frozen. For control and further purification of filament proteins, in some experiments cytoskeletal material was denatured and dissolved in urea or guanidinium. hydrochloride, cleared by ultracentrifugation. and filamenm were allowed to reform (Tezuka & Freedberg, 1972: Strinert et al.. 1976) by dialysis against buffers as described (Franke et al.. 1978a,1981h; Renner et al., 1981).

(f) Ccl electrophoresis Sodium dodecyl sulfate/polyacrSylamide gel rlectrophoresis was according to Laemmli (1970), using some modifications (cf. Franke et al., 1978a,1981a). For 2-dimensional gel electrophoresis using isoelectric focusing in the first dimension (O’Farrell, 1975; DeRobertis it al., 1977) the material was solubilized by one of the different procedures described (Franke et al., 198lb). Alternatively, non-equilibrium pH gradient electrophoresis according to O’Farrell et al. (1977) was used for separation in t’he first’ dimension. For this, samples were

DIVERSITY

OF CYTOKERATINS

!)37

examined in parallel using both lysis buffers (without and with OYW~ SDst: final concn o25%) recommended by O’Farrell et al. (1977). Reference proteins used in co-electrophoresis are specifically mentioned in Results. Alcohol dehydrogenase and 3-phosphoglyceric phosphokinaae were used as markers for approx. pH 7~0. Pancreatic ribonuclease was assumed to be isoelectric at approx. pH 9.0. Gels containing proteins labeled with [ 35S]methionine or [32P]phosphate were processed for autoradiofluorography. Polypeptide bands were excised from SDS/polyacrylamide gels, and protein was eluted and precipitated as described (Franke et al., 198Oa). Denatured polypeptides thus prepared and washed several times with cold ( -20°C) acetone/water (9 : 1, v/v), followed by a final wash in cold acetone and drying, showed, on isoelectric focusing, unaltered isoelectric pH values. (g) Immunological

identijcafion

of polypeptides

Polypeptides separated by l- or 2-dimensional gel electrophoresis were transferred to nitrocellulose paper sheets by blotting (Townbin et al., 1979), using the following modification. Before blotting gels were soaked in water or in 3 x concentrated electrode buffer (Laemmli, 1970) without SDS for 15 min. Blotting was performed in the same buffer by diffusion for 24 h, and SDS was removed from the nitrocellulose paper sheets by a 1 h incubation in 150 mi+-NaCl, 10 mM-Tris. HCl (pH 7.5), @l% Triton X-100. Blot sheets were then soaked in 1% (w/v) bovine serum albumin in PBS for 12 h at, room temperature, rinsed with 150 mM-Nacl, 10 mM-Tris. HCl (pH 75) and incubated, with gentle shaking, for 1 h at room temperature with the specific solution of guinea pig antibodies (1 to 20 pg IgC/ml) in PBS containing 2% bovine serum albumin. Excess antibodies were washed off with 5 changes of buffer, the last wash buffer containing @l% bovine serum albumin, and the paper sheets were incubated for 2 h at room temperature with 1251-labeled protein A from &zphylococcu,s aureu,s (spec. radioact. 30 mCi/mg) diluted with buffer containing O.lo& bovine serum albumin and 0.1% Triton X-100 to give a total radioactivity of 05 &i/sheet. The paper sheets were washed with buffer containing @5% Triton X-100 at C”C, with 5 changes of buffer, and thoroughly dried between sheets of filter paper at 80°C for 20 min. The blots were exposed to a Kodak X-Omat R film at -70°C. Immunoreplicae of polypeptides separated on polyacrylamide gels using agarose gel overlays were made as described (cf. Franke et al., 1979df). (h) N icroscopy Phase contrast and indirect immunofluorescence light microscopy of frozen tissue sections and cultured cells grown on cover slips, using guinea pig antibodies and fluorescein-coupled rabbit or goat antibodies against guinea pig globulins, were as described (e.g. see Franke et al., 1978a198Ou). For electron microscopy, cystoskeletal pellets were fixed and processed for se&ioning and negative staining as described (Franke et al., 1978c,19816). 3. Results (‘yt,oskeletal preparations obtained after extractions in buffers containing high salt concentrations and Triton X-100 are enriched in intermediate-sized filaments. Electron micrographs showing the purity of such fractions have been presented (Drochmans et al., 1978; Franke et al., 197&,1979c,1981b). With some tissues a considerable source of contamination of preparations of cytoskeletal residues is insoluble extracellular matrix, notably collagen. However, this does not interfere wit,h t,he characterization of prekeratin-like components by gel electrophoresis and t Abbreviation

used : SDS, sodium dodecyl sulfate.

938

W. W. FRANKE

ET AL.

subsequent immunological identification. Cytokeratins can also be further enriched by reconstitution of filaments in vitro (for examples see Tezuka & Freedberg, 1972 : Lee & Baden, 1976; Steinert et al., 1976: Sun & Green, 19786: Franke et al.. 1978a,1981b; Gipson & Anderson, 1980; Milstone, 1981: Renner et al., 1981). In all preparations described in this study results on cytokeratin polypeptides of reconstituted filaments were identical t’o those obtained with original cytoskeletal preparations. A special problem of separation and identification of keratin-like proteins on gel electrophoresis is their limited solubility. even in the presence of high concentrations of urea or SDS. Therefore, we have also employed a preparative way by which prot,eins are first solubilized by boiling in high concentrations of SDS, followed by removal of SDS in acetone (Franke et al., 1981b). This procedure also minimizes proteolytic breakdown by proteases which are known to degrade other intermediate filament proteins such as vimentin (Gard et al., 1979; Franke et al., 1980b).

(a) Epideymal

prekeratim

Prekeratins from bovine hoof and snout epidermis have been st,udied extensively by various authors using one-dimensional gel electrophoresis (e.g. see Skerrow et al.. 1973: Mat,oltsy, 1975; Kt’einert & Idler, 1975: Steinert ef ~1.. 1976,198Ou: Drochmans et al.. 1978; Franke et aE.. 1978a,1981a,b: Lee et aZ.,1979). Prekeratins from both bovine epidermal tissues reveal a number of polypeptides of relahive molecular weights ranging between 48,000 and 68,000. including some that are common and others that are different in the two epidermal tissues (Drochmans et al., 1978: Franke et aE., 1978a,1979e,1981a: Lee et al., 1979: Steinert et al., 198Ou). The typical polypeptide pattern of bovine muzzle prekeratin is shown in Figure l(a). The conclusion that the polypeptides I to VII are prekeratin molecules related to each other is based on similar amino acid composition and some, albeit limited, similarities of proteolytic cleavage patterns (St’einert et rrl., 1980n.b: Milstone B McGuire. 1981 : Schmid et al.. 1982), on their inclusion in lilament,s reconst’ituted irt vitro (Lee & Baden, 1976 ; Steinert et al., 1976 ; Sun & Green, 19786 : Milstone. 1981: Renner et nl., 1981) and on their reaction with antibodies raised against purified individual cytokeratin components from epidermal or non-epidermal tissues (Fig. l(b)). For reasons not’ understood most of our preparations of antibodies to prekeratin polypeptides from various sources showed reaction with polypeptides I to VI but not with components VII and VIII, i.e. the smallest prekeratins of bovine epidermis (e.g. Fig. l(b); cf. Franke et al., 1978a), indicating greater divergence of antigenic determinants between these small prekeratins and the larger ones (for similar observations in human prekeratins see Fuchs & Green, 1978). An even greater complexity of the pattern of prekeratin polypeptides from bovine muzzle epidermis is observed on two-dimensional gel electrophoresis (Fig. 2(a) and (b)). The unfolded prekeratin polypeptides are not only distinguished by their apparent Jfr values but also in their electrical charges (Fig. 2(a) to (f)). Many epidermal prekeratins appear with “satellite spots”, indicative of t’he presence of isoelectric variants. From our findings of phosphorylated variants of

I)IVERSITY

“I *?

939

OF CYTOKERATISS

1

1'

I"

11"

Frc:. 1, SI)S/E”)l~ac,r?lamide gel electrophoresis of prekeratin polypeptides from hovine muzzle. (a) 85”, acrylamide gel. 1(Jo S US : for relative molecular weights of romponents la to VII see Franke e/ ol. (1978n,l981r(). (b) Slot 1, 8% acrylamide, 0.1% SDS; slots 1,-l”‘. reaction. after blotting on nitrocellulose paper, with antibodies raised against a preparation of total prekeratins from desmosomeattached tonofilaments of the same tissue (slot 1’); antibodies against cytokeratin component D from mouse liver (slot 1”) : and antibodies specific to polypeptide VI and VII (slot 1”‘). Slots 1’ to 1”’ present radiofluorographs after reaction with [ “‘1 ]p rotein A from Staphy1ocowu.v nureu.s.

cyt,okeratins of mouse liver and various cultured cells (see below) as well as demonstrations of phosphorylated forms in prekeratins of cultured human keratinorytes (Sun & Green, 19786) and in other intermediate filament proteins sucdh as vimentin and desmin (O’Connor et al., 1979: Cabral & Gottesman. 1979: Lazarides, 1980) we interpret such isoelectric variants as representing various degrees of phosphorylation. the least acidic spot usually being the nonphosphorylated molecule. In general. the larger prekeratins (components I to I\‘) display charges corresponding to isoelectric points in the range from pH 6.5 to approximately 84 (Fig. 2(b)). In contrast to this group of large and basic prekeratins. the smaller bovine prekeratins (VI to VIII) are all very acidic, with apparent isoelectric point values lower than that of co-electrophoresed ST-actin (Fig. 2(a) and (1))). Detailed analysis further shows that all thrre major polypeptides of

IEF rm SDS

940

W. W. FRANKE

ET AL.

NEPHG-

FIG:. 2. Two-dimensional gel electrophoreses (SDS. direction of second dimension). using either isoelectric focusing (IEF; (a), (c) and (e)) or non-equilibrium pH gradient electrophoresis (NEPHG: (b), (d) and (f)) of epidermal prekeratins from bovine muzzle ((a) and (b)), human non-mamillar breast-skin ((c) and (d)) and neonatal mouse back skin ((e) and (f’)). together with co-electrophoresed reference proteins (BSA, bovine serum albumin, M, - 68,000, IEP of major variant, pH 634; A. ti-actin. M, - 42,000, IEP, pH 5.4: AI)H, alcohol dehydrogenase. M, - 43,000, IEP re-determined as pH 7.0). Major cytokeratin components are numbered (for bovine keratins see Fig. l(a)). The insert in (a) presents resolution of bovine prekeratins Via, VIb and VII. Estimated M, values of human prekeratins are: I, 68,000; II, 59,000; III, 58,000; IV, 56,500; V, 56,000; VI, 50,000. The arrowhead in (d) denotes a minor very acidic cytoskeletal component consistently seen in human epidermis and basaliomaa. Arrows for NEPHG point from basic to acidic values (except in Fig. 10).

this group of small and acidic prekeratins differ in their isoelectric point values: component VIb(M, - 54,500) is more acidic (isoelectric point pH 5.25, i.e. below that of co-electrophoresed mammalian vimentin; cf. Franke et al., 1980b) than components Via (M, - 55,500) and VII (M, - 50,000) which is only slightly more acidic than a-a&in.

I

DIVERSITY

OF CYTOKERATISS

01 I

Co-electrophoresis of both hoof and muzzle prekeratins (not shown) has illustrated that in bovine epidermis of these two locations at least ten different prekeratins, not including the specific isoelectric variants. can be distinguished (cf. St,einert et al.. 198Oa). Prekeratins from moderately cornified human skin exhibit polypeptide patterns reminiscent of those of bovine muzzle prekeratins (Fig. 2(c) and (d)). The N, values assigned to the major polypeptides are similar to those reported by Fuchs & Green (1980) for plantar epidermis, with the exception of the presence of only trace amounts of polypeptides of M, 46,000 to 47,000 in the epidermal samples examined by us. ITsing the various prekeratin antibody preparations strong react’ion has been observed with all the large polypeptides ($I* - 58,000 to SS.OOO), demonstrating t)heir relatively close immunologic relatedness (cf. Fuchs & Green, 1978). The small prekeratin component) of -Wr - 50,000 has been identified by its react(ion with some keratin antibodies such as those raised against) bovine prekerat,ins VT and VT1 and those against cyt*okerat(in component I> from murine liver (not shown). We have compared, by two-dimensional gel electrophoresis. prekeratins from a broad range of human skin locations, including scalp skin and plucked hair follicles (not, shown). Besides several identical components, remarkable differences have been noted. -Assuming that more acidic satellite spots represent phosphorylated variants of t,he specific polypeptide (cf. Sun & Green, 19786) we have ident,ified a total of at least 11 different prekeratin polypeptides in human skin (R. Moll el al. 1 human epidermal prekerat’ins can unpublished results). As bovine prekeratins, br c*lassified according to their sizes and charges int,o (1) basic,-to-neutral prekerat,ins represented by the larger components (e.g. I to III in Fig. 2(c) and (d): of M, - 67,000 to 58,000) and (2) acidic (isoelectric pH range: 52 to 55) prekeratins intermediate (e.g. components IV and V) or low (e.g. components VI and “C’” in Figs 2(c).(d) and 9) molecular weight. Direct comparison of bovine and human epidermal prekerat)ins by coclectrophoresis has allowed separation of most prekeratins, but has also shown that at, least, some of the isoelectric variants of human components II and III can comigrate with bovine prekeratin components III and IV, and that bovine prekeratin VII comigrates with human prekeratin VI (not shown) in the system used. A grossly different polypeptide pattern has been observed in basaliomas. in agreement with recent observations of Kubilus et nl. (1980). For example, we havt> invariably found that the largest basic prekeratin of AN’~- 68,000 (component I) is practically absent in basaliomas (e.g. see Fig. 9(b) and (d)). Likewise. basaliomas lacak the largest acidic prekeratins present in normal human skin (IV and \‘). whereas t,he basic components II and III and the acidic component Vl seem to be maintained in the tumors (e.g. see Fig. 9(b) to (d)). On the other hand, the acidic polypeptide of M, - 47,000 (designated “C” in Fig. 9(b) to (d)) found in all basaliomas examined has not been detected in interfollicular epidermis but has been folmd in scalp skin and plucked hair follicles (Et. Moll et ~2.. unpublished results). Epidermal prekeratins from skin tissues of mice and rats have been studied rxtensivrl;. primarily by SDS/polyacrylamide gel electrophoresis (e.g. see Dale et

W.W.FRANKE

942

ET AL.

al., 1976: Lee et al., 1976; Steinert & Yuspa, 1978; Kteinert et al., 1979: Franke et al., 1979h,1981b; Winter et al., 1980: Yen et al., 1980). Such studies have revealed different patSterns of prekeratin polypeptides in different regions of the rodent body but. in general, the aut,hors have emphasized the predominance of only a few prekeratin polypeptides in a specific tissue sample (e.g. see Fig. 6(a): Steinert & Yuspa, 1978; Seinert et al., 1979: Wint*er et al., 1980). However. two-dimensional gel electrophoresis reveals a greater complexity. The different rodent) prekeratin polypeptides can be classified int’o three major categories (Fig. 2(e) and (f) presents an example of mouse skin). (1) Basic prekeratins which include some of relatively high molecular weights (e.g. component’s I and II of 113,- 66,000 and - 62.000 in murine back skin). (2) Neutral t,o moderately acidic (pH 7.0 to 6.3) prekeratins such as murine component III are usua1l.y minor components. whereas (3) the acidic (pH 5.2 to 5.5) prekeratins such as murine component)s V and VI (B, - 58,000 t,o 59,000: Fig. 2(e) and (f)) represent large proportions of the total epidermal keratin. Resides t,he usual appearance spots, probably of sat’ellite polypeptide phosphorylated forms. at, least, seven different epidermal prekeratins can be distinguished in both rodent species. indicating t)hat the c*omplexit’y of prekeratin polypeptides expressed in rodent epidermal cells is comparable to that observed in bovine and human epidermis.

(b) Cytokeratinx

of strati$ed

squamous

epith,elia

other than epidermis

\Ve have compared epidermal prekeratins with the prekeratin-like proteins present in other stratified squamous epithelia such as bongue mucosa, esophagus, patterns of all four t’issuex cornea and portio z~aginalis (Figs 3 to 5). The cytokeratin are different from epidermal prekeratin patt,erns. On t,he other hand, all five strat,itied squamous epithelial tissues csontain at least some basic cytokeratins and these usually represent the relatively largest) keratin-like components present’ in the specific tissue. In agreement with Milstone 62 MGuire (l!CJl). w.e have found on SDS/ polyacrylamide gel electrophoresis that bovine esophageal epithelium contains two major tonofilament polypeptide bands of M, - 59.000 and - 47,000 (Fig. 3(a) to (c)). In addition. two minor polyprptide bands of M, - 53.000 and - 49.000 are also noted. By immunological test on nitrocellulose paper blots only t,he larger major component of M, - 59.000 has shown strong cross-reaction with the basic* bovine epidermal prekeratins (e.g. see Fig. 3(b)), whereas both major polypeptide component,s cross-react, with murine liver cyt,okeratin component D (Fig. 3(c)). of cytoskeletal prot)eins from bovine Two-dimensional gel electrophoresis esophagus (Fig. 4(a) and (b)) shows that the Mr - 59,000 polypeptide band contains at least t’wo polypeptide components: both are slight’ly basic and appear as a series of variants (apparent pH range: 6.6 to 8.0). Two minor romponents (designated 3 and 4) focus at pH values slightly lower than that of r-a&in (Fig. 4(b)), whereas the major component of M, - 47.000 is resolved into t’wo components (designated z and 5: Fig. l(a) and (b)) which are both very acidic. C’oelectrophoresis with epidermal prekeratins from the same species (not shown) has shown that, t,his M, - 47,000 component, is not identical with any of the epidermal

I)I\‘ERSITP

OF

!bu

CYTOKERATISS

1

1'

(4

prekeratins. whereas the major components 1 and 2 (AZ, -59.000) seem to c.omigratr with rpidermal prekeratins III and I\‘. \l’r have also compared t,he esophageal cytokeratins with those present in anothrr anatomically relat,ed epithelium of different location and embtyonic derivation. the mucosa of the anterior part of the tongue which is of ectodermal origin. (‘ytokrratins of bovine tongue mucosa (Fig. 4(c)) show a similar pattern as thsol)hageal cytokeratins. This is directly demonstrable by co-electrophoresis (Fig. -C(d)) which indicates that the minor components designated x and ,v are onl? tlr+ccted in t,ongue whereas component z is only seen in the esopha,gus. Again. more tsstrrlsive differences of cyt,okeratin polypeptide patterns are observed when tc)tlgu(~ ~r~~~c~osa is c~o-t~l~~c~t~~ol’)ioreseti with rpidertnal prrkeratins (Fig. 4(e)). =\s with esophageal rytokcratins comigration with epidermal c*omponent,s has only been observed for the basic components 1 and 2. (‘ortwa is a stratified epithelium which ront,ains a set of characteristic kerat,inlike proteins (Sun & Green. 1977: Sun f+ ml.. 1979: Doran rf 01.. 1980: Gipson CP

W. W. FRANKE

I--

ET AL

I-IEF

NEPHG *-BSA

1n 3-*

C-+

&

u2

r13 i-rz

-

‘i’pJ

(4

I-

NEPHG Xri Y- 1

-IEF #b*w-

1 *-BSA

-9-

IIl-3

;= -**il,.

(d)

56-

7

w

(4

I--

5

(b) I--NEPHG

*

ADH

1\

NEPHG

NEPHG - -BSA

c

--BSA 111

;(llrr,

i

ADH

(9)

A

NEPHG iy+,

--BSA

*

(h) FIG. 4.

AhH

d,

DIVERSITY

OF CYTOKERATISS

945

Anderson. 1980: Ramaekers et al., 1980). These are shown after one-dimensional gel rlectrophoresis and immunological examination in Figure 3(a),(b) and (d) and after two-dimensional separation in Figure 4(f). These cells contain two large cytokerat’ins (components 1 and 2: Fig. 4(f)) w h’ICh are basic. show cross-reaction with \rarious cytokeratins, epidermal prekeratin included (Fig. 3(b) and (d)), and migrate to similar positions as epidermal prekeratins I and II (Fig. 4(h)). No suc~h caytokeratins are detected in esophagus and tongue (e.g. see Fig. 4(g)). By cont)rast. cAornea1 cytokeratin 4 (A4, - 60,000) which migrates. on two-dimensional gel electrophoresis. to similar positions as some of the variants of cytokerat’ins 1 and 2 of’ esophagus and tongue and of prekeratins III and 11’ of epidermis (Fig. l(g) ant1 (II)). does not react with the same prekeratin antibodies (Fig. 3(b)): it does react. however. with antibodies to component D from mouse liver (I&g. 3(d)). Finally. the broad cornea1 polypept’ide band of lower size (M, u 56,000. Fig. 3(a) and (d)). is resolved on two-dimensional separations into two polypeptides (designat,ed 5 and (i. Fig. 4(f)). both of which are unusually acidic (isoelectric point about pH 5.0: focusing gel not shown) and seem to be specific for cornea (Fig. 4(g) and (h)). In order to examine possible species differences we have also analyzed c*ytoskeletons from tongue, esophagus and cornea of rodents and human (Fig. 5(a) to (e)). Human and rat esophageal cytoskeletons contain a large cytokeratin band (M, - 59,000) similar to that described for bovine esophagus which. however. on two-dimensional gel electrophoresis displays a greater complexity of individual. slightly basic polypeptides (components 1 t’o 4. in Fig. 5(b) and (d)). (‘hara,cteristically. both rat’ and man also show the presence of a very ac*idic esophageal cytokerat)in corresponding to cytokeratin 5 of bovine esophagus (spot 5 this cyt,okeratin is in Fig. .5(a) t’o (c) and spot 3 in Fig. 5(d)). Interestingly, significantly larger in human (Jfr - 54,000) and rat (~11~- 51,000) than in bovine (,\I, - 49.000) esophagus. Moreover, patterns of cyt,oskeletal polypeptides are also similar in human tongue mucosa and human esophagus (Fig. 5(b) and (c)), and the cytokeratins of rat tongue mucosa resemble t)hose of rat esophagus although the tongue epithelium shows a number of minor components (designated x. y. 1. 3. .i and 6) which have no counterparts in the esophageal cytoskeletons (Fig. 5(d) and (c)). Likewise. comparison of tongue mucosa cytokeratins of the three species (Pigs 4(c) and 5(c) and (e)) shows a similar pattern of major basic and acidic polypeptides although co-electrophoresis (not shown) allows the demonstration ot interspecies differences in the detailed distribution of polypeptide spots (compare also Fig. 4(c) with Fig. 5(c) and (e)).

PI{;. 4. T~l,-dilnensional gel electrophoresis (for design&ions and reference proteins. see Fig. 2) of bovine c,ytoskeletal proteins from esophagus ((a) and (b): insert in (b) shows co-electrophoresed a-a&n. A). tongue ~UCOSB((c): insert shows separation of individual variants of component 5) and cornea (1) as wt~ll as (,~)-electrophoresis of cytoskeletal proteins from esophagus with those from tongue mucow ((1) and from cornea (g). In addition. co-electrophoresis with bovine muzzle prekeratins is shown for cytwkeletons of tongue macosa (e) and cornea (h). Major polypeptides are numbered. letters indicat,e sonw minor apparently tissue-specitic components. Obviously tissue-specific polypeptides identified 1)~ co~elrctrol)h~lresis of cytoskeletal proteins from tongue mucosa with those of esophagus (d) and tnuz& el~idrrmis (c) are designated in (d) and (e). Comigrating cytoskeletal pol,vpeptides present in cornea and wophagus (g) ax well as in cornea and muzzle epidermis (h) are denoted by brackets.

946

W. W. FRAXKE

r

IEF

rfl,

H;A L

f

nl

T”

4

r

ET

3

NEPHG e-BSA

11-1

r

NEPHG HSA-

4-u

(b) 0 -6SA

ln

6-

u7

w-BSA

I--(4

a

AtlH

I

** ‘I 2ADH

(d)

Y-* 9

(4 I-NEPHG

NEPHG

lt

,BSA

64-5

ADH’

/J -7

IEF I t

7’. 213

BSA =

FIG. 5. Two-dimensional ntratified squamous rpithelia

gel elwtrophoretic~ cemparison of c~vtoskeletal prot&ns from various of human and rat t~issuen (separation’in thca 1st dimension was either 113

DIVERSITY

947

OF CYTOKERATISS

Pronounced correspondence of pat,terns of cytoskeletal polypeptides has a,lso been found in cornea from man, cow, rat and mouse, showing the presence of basic. components comparable to polypeptides 1 to 4 of bovine cornea (cf. Fig. 4(f)) as well as c*omponents that are as acidic as components 5 and 6 of bovine cornea (not shown : these cornea-specific components exhibit some small interspec& differences of electrophoretic mobility in the presence of SDS). The coincident occurrence of neut)ral-to-basic cyt,okeratins. predominant’ly thosta of relatively high Mr values. and very acidic ones that are usually of lower molrc+ular weight is also observed in other stratified epithelia. One example showing major cytoskeletal proteins of epithelium of human portio vaginalis is presented in Figure 5(f) and (a).

(c) C’ytokemtins

of hepatocytes

and

intestinal

epithelium

(‘ytoskeletons from liver tissue or freshly dissociated hepatocytes of rodents caontain major polypeptides which differ from the major epidermal prekeratins of rodent, skin (Fig. 6(a)) but, share some common antigenic determinants with epidermal prekeratin as recognized by antibodies to hepatic cytokeratin component D (compare Fig. I(b) and Fig. 6(b): cf. Franke et al.. 1979g,1981a.c). The t,wo prominent cytokeratins designated A and D (Figs 6(a) and (b) and 7(a)) appear as a series of isoelectric variants which, after labeling mice by int,raperitoneal injection of [32P]phosphate, have been identified as one major notIphosphor.vlated form and several more acidic phosphorylated modifications (Fig. 7(a) and (b)). The hepatocyte-typical cytoskeletal polypeptides A, C and I) are continued to be expressed in rat hepatocytes dissociated and kept in short-term monolayer cell culture (Fig. 7(c)) and in cells of a rat hepatoma-derived cell line. MH,(‘, (Franke et al.. 1981d). Cytokeratins similar to components A and D of rodent’ liver but not identical to them (slightly lower M, and isoelectric point values) are also prominent in bovine hepatocytes (not shown) and in hepatocytes and hepatocellular carcinomas of human origin (H. Denk. R. Krepler & W. R’. Franke, unpublished data). Tonofilaments isolated from brush border fractions of absorptive cells of small intest,ines of rat,s, mice and cows (Franke et al.. 1981h) contain cytokeratins of similar sizes and charges as cytokeratins A to D of hepatocytes (Figs 6(c) and (d) and 7(d)) which bind antihodies against certain bovine epidermal prekeratins ( Franke et al., 19X1 b) as well as antibodies to component D from mouse hepatocytes (Fig. 6(r)). Furthermore. when [ 35Slmethionine-labeled cvtoskeletal proteins of rat hcpatocytes kqt in short-t,erm culture (Fig. 7(c)) and unlabeled tonofilament-ricsh

isoelectric focusing, in (a) and (f). or by non-equilibrium pH gradient electrophoresis, in (b) to (e) and (g). (a) and (b) Human esophagus: (c) human tongue; (d) rat esophagus; (e) rat tongue; (f) and (g) human portio vu&n&~. Reference proteins and designations as for Fig. 2. HSA, human serum albumin : /3. residual ,3-actin. Numbers indicate major polypeptides; letters in (c) and (e) denote minor tonguespecific components. Note the occurrence of neutral-to-basic cytokeratin polypeptides in all tissues shown. Note also the presence of some polypeptides of similar electrophoretic properties. especially in esophagus and tongue. (b) to (e).

948

W. W. FRANKE

T-

(4

b)

ET AL

1’

(cl

4)

FIG. 6. Sl)S/polyacrylamide gel electrophoresis and immunoblotting tests of cytoskeletnl proteins from mouse liver ((a) and (b)) and apical tonolilaments from rat small intestine ((c) and (d)) visualized by staining with Coomassie blue ((a); (b) slot 1 ; (c) slots 1 and 2; (d)), or radiofluorography of ‘251-labeled protein A from S. MTCUS ((b) slot 1’: (c) slot 2’). Cytoskeletal preparations from rodent liver contain 2 major polypeptide bands (components A and I), denoted by arrowheads in (a), slot 1) which am different from the major polypeptides of epidermal prekeratin from the same species ((a) slot 2. presents prekeratins from murine back skin: major bands are denoted by horizontal bars). For comparison. reference proteins are shown ((a) slot 3 from top to bottom : p-galactosidase. phosphorylase a. bovine serum albumin. transferrin. glutamate dehydrogenase. act,in). The two major polypeptide components of mouse liver cytoskeletons ((b) slot 1) are identified as cytokeratins by their reaction, after blotting on nitrocellulosr paper, with antibodies to purilied murine cytokeratin 1) ((b) slot 1’) which also react with authentic prekeratins of bovine (cf. Fig. l(b) slot 1”) and human epidermis. Fractions of brush border tonotilaments reveal three major cytoskeletal polypeptides (denoted by arrowheads in (c) slot, 2: (c) slot 1 shows. for comparison. epidermal prekerutins from bovine muzzle). Immunoblotting tests ((c) slot 2’) using antibodies to cytokeratin I) from mouse liver show strong reaction of the 2 larger components and a weak. but signiticant reaction with component M, 10.000. Higher resolution gel electrophoresis of intestinal brush border tonotilament polypeptides. using gels containing l’YO Sl)H. allows the separation of four cytokeratin bands (arrowheads in (d): cf. Franke et trl.. 1981h). A. residual actin.

cytoskeletons from rat intestinal brush border (Fig. 7(d)) are co-electrophoresed (Fig. 7(c) and (e)) components A, C and D of both samples seem to comigrate, indicating that these cytokeratins are similar in hepatocytes and intestinal cells. None of these components, including the M,40,000 polypeptide specific to intestine, comigrates on two-dimensional gel electrophoresis with any of the major epidermal prekeratins of the same or other species (mouse, rat, cow, man; Franke et al., 1981b).

DIVERSITY

IEF

IEF

I-+ SOS

949

OF CYTOKERATINS

SDS f\ ,,, r

(‘4

u-, c l’t

-Dm )“w

(d)

b) Nc:. i. Two-dimensional gel electrophoresis (IEF, direction of isoelectric focusing) of cytoskeletal proteins from mouse liver ((a) and (b)). freshly dissociated rat hepatocytes allowed to grow in culture for 30 h of which the last 4 h were in medium containing [35S]methionine ((c) for conditions of labeling see Materials and Methods). and tonofilaments from brush border fractions of rat small intestine (d). (e) The (‘oomassie blue staining of a mixture of a relatively large amount of protein of tonotilaments from rat intestinal brush border (similar to (d)) co-electrophoresed with the [35S]methionine-labeled cytoskeletal proteins from rat hepatocytes (same gel as in (c)). Mouse hepatocyte cytokeratins labeled in tiw with [“PJphosphate are shown in the same gel ((a) and(b)) visualized by Coomassie blue staining (a) and autoradiography (b). The major cytokeratins A and D show isoelectric variants which are ph<,sphorylated ((a) and (b); the specific non-phosphorylated polypeptides are denoted b,v vertical bars and phosphorylatid modifications bv vertical arrows). &migration of cytoskeletal polypeptides A. (’ and I) is demonstrated in [35S]methionine-labeled cvtoskeletons of short-term rat hepatocyte cultures ((c) fluorography) and tonofllaments from rat intestine ((e) Coomassie blue staining). The additional caomponent of M, 40.000 observed in brush border tonofilament fractions is denoted b,v the lower brackets in (d) and (e). Residual actin is designated fi and y.

950

W. W. FRANKE

(d) Cytokeratins

of cultured

65” AL

epithelial

cells

Filament proteins immunologically and biochemically related to epidermal prekeratins are also expressed in diverse cultured epithelial cells, including keratinocytes (Steinert & Yuspa, 1978: Sun & Green, 1977.19786: Franke et al.. 1978b,c,1979b,c,d: Fuchs & Green, 1978: Sun et al.. 1979). Two epithelial cell lines derived from internal organs in which cytokeratin filaments have been studied in particular detail are human HeLa cells and rat kangaroo PtK cells (Osborn et al., 1977,198O: Franke et al., 1978a,b,c,d,1979c,1981c: Sun et al., 1979). Cytoskeletons from both cells show, besides some residual actin and a component of M,57,000 identified as vimentin. t’wo (PtK) or three (HeLa) major polypeptide bands containing cytokerat.in proteins (Figs 8(a) and IO(a)). On two-dimensional gel electrophoresis of HeLa cytoskeletons (Fig. 8(b) and (c)) one can distinguish, besides vimentin and residual actin. four distinct cytokeratin components : component 1 (N, - 54,000 ; isoelectric pH -5.9). component 2 (AZ, - 52,000 ; isoelectric pH -6.0). component 3 (X, - 48,000: pH - 527) and component 4 -5.8). These four component,s seem to caorrespond to 46,000: pH @4 polypeptide spots 31, 36, 44 and 46 of the two-dimensional “map” of HeLa cell proteins of Bravo et nl. (1981). Immunologically, all four components can be identified as cytokeratins but the presence of different antigenic determinants also allows their immunologic distinct’ion (for example see Fig. 8(a) and (c)). All four polypeptides appear with minor, more acidic sat,ellite spots, and incorporation of [ 32P]phosphate has allowed us to identif) t’hese isoelectric variants as phosphorylated forms (Fig. 8(d)). I n order to examine whether the HeLa qtokeratins are related to those present in human epithelial &sues and carcinomas we have compared, by co-electrophoresis. the 13sS]methionine-labeled cyt)oskeletal prot,eins of HeLa cells with unlabeled cyt,oskeletal proteins from various human organs and tumors (not shown). So far we have found only one cytokeratin of a human tissue to comigrate with a HeLa cytokeratin; this is cytokeratin component C from hair follicles and basaliomas which comigrates with HeLa component 3 (Fig. 9). The known broad cross-species reactivity observed with many antibodies against epidermal prekerat’ins and other cytokeratins (Lee et wl.. 1976; Franke ef aZ., 1978a.6.1979d: Freudenstein et al.. 1978; Sun & Green. 1978a: Sun ef al.. 1979; Weber et al., 1980) has also allowed us to ident*ify both major polypeptides of cytokeratin filaments present in PtK cells, i.e. kidney-derived cells from a marsupial, the rat’ kangaroo, as immunologically related to murine cytokeratin D (Fig:. 10(a)). On two-dimensional gel elec+rophoresis (Fig. IO(b) and (c)) the relat’ively large major cytokeratin (AZ, - 52,000) appears to be almost neut,ral whereas the smaller cytokeratin of M, - 45,000 is somewhat more acidic (almost isoelectric with bovine serum albumin). As with HeLa cells several other epithelial cell lines derived from internal organs also seem to lack relatively large and basic cytokeratins (for examples see Franke et al., 1981c,d). It has also been reported that keratinocytes grown in culture show in particular the cessation of considerable changes of expression of prekeratins, synthesis of the relat,ively large prekeratins found in epidermis (Fuchs K: Green.

DIVERSITY

1

2

2'

OF CYTOKERATINS

951

IEF

r

SDS

(4

r

SDS

(4

(b) IEF

n”

(--IEF

k

L (d)

FIG. X. One- and 2.dimensional gel electrophoresis of cytoskeletal proteins from HeLa cells and t.heir it,rtrlrull)l~)Ri~ll crosweac4ion with cytokeratin antibodies. (a) Sl)Sipol~acr?lamide gel electrophoresis IBM’Hc~1.acytoskelrtons (slot, 2. cytokeratin bands are denoted by horizontal bars. vimentin by an ,,pw, I.iwlc and residual actin by .1; slot 1 contains purified vimentin from RVF-SM cells; cf. Franke PI rrl.. 19806, and the corresponding fluorograph shows the reaction of the upper band with antibodies to cytokeratin 1) from mouse liver (slot 2’)). (b) Cytokeratin components identified by 2-dimensional gel ~,l~~c,trolJl”Irrsis (IEF. isoelectric focusing) are denoted by numbers (1 to 4). (c) Fluorograph showinp qwific, wac.tion of‘ wmponent 3 with antibodies against epidermal prekeratins VT and VII (same preparation as in Fig. l(b). slot 1”‘). (d) Autoradiofluorograph of [32Pfphosphate-Iabeled HeLa c~,vtoskc~k~tlms(arro~v\;s on brackets denote phosphorylatrd modificati~ms. t,he other end of the bra&t Iz~(~kiny a!l arrow tlrwcltc~s the position of t,he specitic major non~ph~,r;ph~)r\latetl polvpeptides).

197X,1979.19X0: Steinert 8~ Yuspa, 1978; Sun & Green, 19786: Franke et al., 1979h: Kubilus ef al., 1979: Doran et al., 1980). An instructive example of an altered pattern of cytokeratin expression in a keratinocyte culture is presented by the murine HEL line which also expresses an additional intermediate-sized filament I)rotein. rimentin (Franke et aZ., 1979b). HEL cytoskeletons reveal, on non-

I

SOS

nv +l +?lmBP -2

‘ij3

nv

FIG. 9. Two-dimensional co-electrophoresis of [3SS]methionine-labeled cytoskeletal proteins from HeLa cells ((a) radiofluorograph; for designations see Fig. 8) and unlabeled cytoskeletal proteins from human basalioma ((b) and (d) Coomassie blue staining: VI. epidermal prekeratin component also observed in normal human epidermis; cf. Fig. 2 (c) and (d); a. b and c denote major cytokeratins observed in basaliomas). A photographic superposition of (a) and (b) is shown in (c) and illustrates that component 3 of HeLa cells and component c of basalioma tissue comigrate ((d) presents coelectrophoresis of basalioma cytokeratins with bovine serum albumin. BSA, and a-actin, A).

DIVERSITY

OF CYTOKERATINS

953

E‘lo. 10. One- and Z-dimensional gel electrophoresis of cytoskeletal proteins from cultured rat kangaroo cells (PtK,) and immunological cross-reaction with cytokeratin antibodies. (a) SDS/polyacrylamide gel electrophoresis of PtK,) cytoskeletal proteins showing vimentin (V). residual actin (A) and 2 major cytoskeietal polypept,ide bands (slot 1). both of which react with antibodies to cytokeratin 1) from rnurinr liver (slot 1’). (b) Two-dimensional gel electrophoresis (IEF. isoelectric focusing in 1st dimension) showing the same components (major PtK, cytokeratins are numbered 1 and 2). (c) Two-dimensional gel electrophoresis of PtKz cytoskeletal proteins using non-equilibrium pH gradient gel electrophorenis (NEPHG. arrow points from acidic to basic values). Reference proteins co-electrophoresed in (c) have apparent IEP values of pH 5-4 (a-actin), pH 634 (BSA, bovine serum albumin) and pH 7.0 (ADH. alcohol dehydrogenese).

equilibrium pH gradient, gel electrophoresis, two basic prekeratin polypeptides (designated 1 and 2 in Fig. 11) similar in charge to the group I to IV prekerat,ins of murine epidermis (cf. Fig. 2(e) and (f)) but of considerably lower M, values than prekeratins I to 111 (-57,500 and -58,500). In addition, these cells contain more acidic prekeratins (designated 3 to 5 in Fig. 11) which have much lower molecular weights than the major acidic prekeratins of murine epidermis (cf. Fig. 2(e) and (f)). By co-electrophoresis (not shown) none of the major prekeratins of HEL keratinocytes has been found to be identical with any of the major prekeratins of murine skin. HEL cells may represent a rare case but they clearly demonstrate that basic cytokeratins can be expressed in permanently proliferating cultured cells.

4. Discussion Intermediate-sized filaments containing proteins immunologically related to the tilamentous (n-fibrous) component of keratin of epidermis and its appendages appear t,o be specific for true epithelial cells (for absence of this type of filaments from other epithelioid cell layers see Franke et al., 1979a,e; Ramaekers et al., 1980). During mammalian embryogenesis such cytokeratin filaments are not found up to the morula stage but they are already prominent structures in the t’rophectoderm of the blastocyst (Brtilet et al., 1980; Jackson et aZ., 1980: Paulin et al., 198Oa). However, formation of filaments of this type is not confined to functional epithelia.

954

W. W’:. PRASKE

ET AI.

IEF

I-IV -3 I I YP

b) I-+ +

NEPHG &SA

1 L-7

-LA* 2

*nV 3,

(4 FIG:. 11, Two-ditnension~rl gel electrophoresis 4’cyt~~skeletal prot,eins of cultured nlurine keratinocxtes (HEI, cells) either unlabeled ((a) and (c) ( oonmssie blur staining) or labeled with [35S]methionine ((b) fluorograph). When isorlevtric focusing (IEF) is used in the 1xt dimension the acidic cytokeratins (designated 3 and 4) as well as vimrntin (V) and residual act.in (designated /3 and y) are clearly identified. whereas some slightly lalprr polypeptidrs (designated 1 and 2) do not enter the focusing gel. The latter. however. are clearly ~solvrd whvrr non-~(t’“ilibriurrr pH gradient gel electrophoresis (NEPHG) is used in the 1st dimension (c), showing that they are mc,re positively charged than c,o-el~c~tropho~esed alcohol dehydrogenast? (Al)H).

They are also expressed in. epithelia-derived tumors (carcinomas) as well as in cultured epithelial and carcinoma cells (Franke et al., 1978a,b,1979c,g; Sun et aZ.. 1979: Bannasch et (11.. 1980: Schlegel et c/l.. 19806). including certain t,eratocarcinoma cell lines (Paulin ef al.. 198Oa,b). Cytokeratin filament’s are indistinguishable, by electron microscopy and X-ray diffraction, from other t,ypes of intermediate-sized filaments (e.g. see Franke et al., 197%; Steinert, 1978; Steinert et al., 1978,198O; Lazarides, 1980; Renner et d.. 1981).

DIVERSITY

OF ("YTOKEKATISS

!j:j,i

Yet thry can be distinguished from other filaments by antibody tecahniques ant1 several other biochemical criteria. (‘ytokerat’in filament,s can caonsist of different sets of polvpeptides differing widely in molecular weight and charge. including e~-rn Irasic polypeptides which have not been found in other types of intermediate tilarnrnts. Moreover. while the other types of intermediate tilaments appear to br t.orrrJ)ositionaIlg constant in the various cell types in which they occur. cytokerat in tilanrt~ntjs. albeit ident’ical in structure. show comJ~ositiona1 differences in diffcrrnt c~J)rthrlial and carcinoma cells. As shown in this study these c*ornpositional tfifferctnces of q-tokeratin filaments are not erratic, but reveal ~11 type-spcifit. J)atterns of polypeptides. M’e have so far not found two epithelial t,issues \j.ith itlrntic4 Jlat’terns of cytoskeletal polypeptides. although strikingly high degrers of uirnilarity ofcytokeratin patterns have been observed with certain epithelia such as tongue mucosa and esophagus. Differences of cytokeratin comlrosition between (hpidernris and ot’her stratified squamous epithelia frorn various regions of the uppc’r’ digestive tract of rabbit have been noted on one-dimensional gel electrophorrsis I)>. I+hs & Green (1980). Similarly. Doran et 01. (1980) hare found different cytokeratins in rabbit skin, esophagus and cornea. in agreement with observations t Irat major c*yt,okrratins of bovine esophagus and cornea are different from thoscaof hoof eJridermis ((iipson & =\nderson. 1980: Milstone & iVJ&uirr. I!Ml). 1n thti trrajoritv , of chasesexamined such differences of cytokeratin tdotnJ)osition bet\\.eerr tliff’rrcbnt rpithelial tissues. or between different layers of epidermis (Haden & Lre. 1078: Fuchs & Green. 1978.1980). or between different t*ultured epithelial culls. stbrm to represent differences of synthesis and are not dur to Jrroteolytit~ tlcgra~clation. This has been shown in analyses of Jrroducts of translation irr r’itrr) lrsirrg messenger RNA from different epiderrnal layers and wII cultirres of rr1a.n (Fuchs R (Green. 1979.1980) and cow (R. Sornmer & \\‘. \\‘. Franke. unpuhlisht~tl data) as well as from rat liver and HeLa cells (our unpublished results). Therefore. w t~ont*ludr that most of t)he different polypeptides Jrresent in q-tokrratin tilaments of different rpit,helial eelIs of the same species are distinct gene lrrodrrcts 01 a large Jrrotjein family. t,he cytokera,tins which exhibit a dualistic csharacter. i.(b. homologies as well as differences. Such tliffrrrnt~cs t)t’c~otnJrositiorr ofa tsytoskeletal strrrcturtl widespread in tLJritht4i;rl ~~41sare Jrerplexing in view of the much more constant t*omJrosit)ion of othtar architectural protein Jrolvrnt3-s, including other types of intermediat’e tilarnents. Htnvrver. tlist,inct wII type-specific differences, albeit much less extensive. ha\-tt also bcJ?n described for ot,her st,ructure-bound, widespread wII Jrroteins such LS ;rt+in (e.g. see \‘andekerckhove & Weber, 1978,1979). Thus, the question why tht, vtartrbratr organisms bother t’o Jrroduce. in different cells. different J)olypeJrtidc~s of’ 1he sanitl protein farnil) in order t,o form an identical structure, i.e. the tonofilarnent. is not Jrrincipally different from the question why similar but genetically different actins are expressed in different cell types and differ even in tlifferrnt tyJ)es of muscle cells. It seems unlikely that such differences of JrrottGrr constituents in the same type of architect,uraJ element are relat,ed to imJrortant frrnt~tional differences of these structures. Rather. the differential exJrressiorr of different Jn-otjeirrs of the same family ma) reflect different Jrrograrns of’ diffrrrrrtiation.

956

W. W. FRANKE

ET AL.

The specificity of the expression of cytokeratin polypeptides in relation to differentiation is demonstrated by our finding that cytokeratin patterns are characteristic of specific epithelia or groups of developmentally related epithelial tissues. Relatively simple cytokeratin patterns characterized by low numbers of medium-sized (M,.!iO,OOOto 56,000), moderately negative (pH 5.8 to 6.6) and small (M,40,000 to 49,000), acidic (pH 5.2 to 5.7) polypeptides occur in embryonal trophectoderm (Jackson et al., 1980), certain teratocarcinoma cells (Paulin et al., 198ob), in hepatocytes and hepatoma cells (Franke et aE., 1981a,c), in mucosa cells of the intestine (Franke et al., 1981b) and in urothelium (Schmid et al., 1982). By contrast, representatives of the subgroup of relatively large and basic cytokeratins have typically been found in stratified squamous epithelia of both ectodermal or entodermal origin (this study) and in mammary gland epithelia (Schmid et aZ., 1982). This also shows that no direct germ layer relationship of cytokeratin expression exists but that histological relationships are important. Interestingly, many of these epithelia expressing relatively large and neutral-to-basic cytokeratins are known to be able to “keratinize”, in a mode resembling epidermal keratinization, in special regions or under certain, sometimes pathological conditions (e.g. see Hackemanrl et aZ., 1968; Zerban & Franke, 1977). Cytokeratins seem to have been considerably conserved during evolution as indicated by their immunological cross-reactivity (Lee et al., 1976; Franke et al., 1978a,b ; Freudenstein et al.. 1978; Sun & Green, 1978a). Our study has also shown that tissue diversity and specificity of t,he cytokeratin pattern is much more pronounced than species differences. For example. similar patterns of cytoskeletal polypeptides are found in corresponding tissues of man, cow and rodents, although minor species-specific differences of molecular sizes or charges in the same tissue can also be noted. It may well be that the diverse cytokeratins observed in different tissues and species contain one or several homologous “core” regions t’hat are especially stable during evolution, and that such conserved regions correspond to the n-helical cores described in epidermal prekeratins as particularly resistant to treatment with trypsin (Skerrow et al., 1973: Matoltsy, 1975; Steinert et aZ., 1980b). The presence of such common R-helical cytokeratin core regions directing the assembly of the diverse cytokeratins into coiled coils of a-helices and into tonofilaments would explain why so differently sized and charged molecules can form the same filament’ structure. Although our study indicates that the number of cytokeratin genes expressed in vertebrates is relatively large (cf. Fuchs & Green, 1978,198O; Steinert et al., 198Oa) it is certainly too premature to estimate the total number of genes of this family. Studies using complementary DNAs to cytokeratin mRKAs as probes are currently being performed in order to estimate the total number of cytokeratin-like sequences in genomes of vertebrates and to examine possible rearrangements of such sequences during epithelial differentiations. ITsing such methods Kemp (1975) has estimated that the chicken genome contains as much as 100 to 240 different genes coding for “feather keratins” which, however, are differently structured, small (M, 10,000 to 12,000) polypeptides apparently unrelated to the cr-filamentous cyk’okeratins.

1)IT’ERGITY

OF CYTOKERATINS

957

Our finding that the patterns of cytokeratin polypeptides are characteristic of specaific tissues or cell types, or of histologically related groups of epithelia, makes them cytoskeletal protein markers that, should allow to distinguish and identify different. epithelia by biochemical criteria. We suggest that patterns of cytokeratins are also of value in characterizing cultured epit’helial cells. including established cell lines. especially in comparison with the putative tissue of origin. Determinations of cytokeratin polypeptide patterns should also be helpful in the characterization of a number of pathological formations, including diseases known to be associated with alterat.ions of the cytokeratin system (e.g. see Denk et nl., 1979: Franke et nl.. 19Xlu). Moreorer, since carcinoma cells maintain the expression of cytoskeletal filaments of the cytokeratin type, both in the tissue and in culture (Franke et al.. I!f7Xh.1!~70h.c.d.l9Xlc: Sun et al., 1979; Bannasch et al., 1980; Kubilus et al., 1980: S(.hlegcl rt ~1.. 19XO~.b: M’inter et ~2.. 1980; Gabbiani et rtl.. 1981). it will be important to clarify to what ext,ent cell t,ype-specific cytokeratins are synthesized in transformed cells and what abnormalities of cytokerat’in expression occur in cwrcGoma cells. As we have shown for human basaliomas (this study) and results) at least some hr~pat~ocellular carcinomas (H. Denk et al., unpublished cytokeratins characteristic of the normal tissue are continued to be expressed in these Ilrimary tumors. We propose that determinations of the cytokeratin pat,terns using antibodies specific for individual of primary tumors or metastases, cyt,okeratins and t,wo-dimensional gel electrophoresis. can be helpful in the charac+erization of neoplastic lesions and the origin of metast,ases. This work has been supported in part by the Deutsche Forschungsgemeinschaft (grant to 1V.U..F.) and the Fonds zur Fijrderung der Wissenschaftlichen Forschung (grant to H.D.). \Vv thank Drs E.-D. .Jarasch, J. Kartenbeck and .J. Schweizer (Heidelberg) for experimental help and valuable discussions.

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ET

AL.

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I)IVISKSITU

OF (‘YTOKERATISS

!I;,!)

t )‘Fartdl. 1’. H. (1!)75). ,/. Mol. (‘herrc. 250. 100-10~1. ()‘Farrc4l. I’. Z.. (Goodman, H. AI. & O’Farrell. 1’. H. (1977). (‘r/l. 12. 1IX-111Z. Oshorn, M.. Franke. W’. W. & Weber. K. (1977). Proc. Xxt. .drctd. Sci.. I’.S..-J. 74,2490-%49-C. ()shorn. II.. Franke. \\.. I\‘. Cy-\\‘eber. K. (1980). E.rp. (‘r/l KPS. 125. 3i 46. l’attlitr. I).. Jsabinrt. C’.. \\‘rber. K. I!? Osborn. nl. (198(h). E.r[). (‘r/l Rrs. 130. “Hi- SW. I’aulitr. I).. Vowst. S. & I’c~rrrau. .J. (l98Oh). ./. Alk)/. Hiol. 144. !&lOl l:;tm;~t~kt~rs. F. (‘. S.. Oslwrn. 11.. Svhnrid. E.. \Z’oher. K.. liltwtnr~ntlal. H. & Frattkts. \\‘. \\’ (I!)XO). F.t,p. (‘o/I /f/w. 127. 309-317. I:cwtlt-r. \\‘.. Ftwtkv. \2’. \V.. Sc.hmid, E.. Geislvr. S.. \VvIw. Ii. & Mattdvlkow. 1’. (I981 ). .I. .I/ol. Niol. 149. r’X.>~mi. Schlty?t~l. It., Banks-Schlegc~l. S. & Pinkus. G. S. (1980~~)./A. /~/WY/. 42. !)lP!Ni. S(.hIv~~l. I<.. Banks-Sc~hltyyl. S.. M~~Leod. .I. A. & I’inkus. (:. S. (l!CWh). .iwrr. ./. /‘u/ho/. 101. 41 4s. Schmid. I*:., Tapscott, S., Bennett. G. S.. Croop. ,J.. Fellini. S. A.. Holtzer. H. di Frankr. I\‘. \V. (I!lT!)). /)i~~~r~,ctic~/io~/. 15. Zi-JO. S~~lttnitl. Id:.. Sc~hillvr. I). L.. Mall. R.. Stadltar. .I. R: Frank?. \V. \\‘. (1982). /)i~~~~r(,~//if//if/l/.itt tlw ,,wss. Sktwo\\.. I). c! Httnttlr. I. (197X). IZiochirrc. Hiophys. A&I. 537. l73-1HJ. Sktvcn\.. I).. Natoltsv. A. (:. & Matoltsv. M. N. (1973). ./. Bid. Phrnt. 248, 18”0- 18%. ~tar+ic~litr. .A (I 97-l). ‘I,,/. h’rc.. (‘ytol. 39.. 19JP18-1-. S:tvinc,rt. I’. 11. (I9iX). ./. Mol. H’iol. 123. -t9-70. Stc~itwrt. I’. & Idlt~r. \\‘. N’. (197.5). Hiocher//. J. 151, 603-Bl1. ,S;tc*itrt~t~t. I’. & Yuspa. S. H. (1977). ~Scir~w. 266, 1491P1193. Stritwt. I’. AI.. Idler. \V. \V. I% Zimmrrman. S. H. (1976). J. X0/. l3id. 108. 547. Mii. SttGnrrt. I’. M.. Zimmwman. S. B.. Starger. .J. hl. & Goldman, R. D. (1978). Pro?. Sat. =Irnd. X.i.. I’.S.A. 75. 6098~6101. StchitrFrt. I’.. Itllc~r. \\‘. I\‘.. Poirier. RI. C‘.. Katoh. Y.. Stoner. (:. I). & \‘uspa. S. H. (I%‘!)). liioch i/H. /~iO/,//~/s. .-Irto. 577. 1 I -“I Strittc~tf. I’. .\I.. Jtllt~r. \V. \V. & \\‘antz. 11. L. (1980~). Hioch~rr/. ./. 187. 91% SIB. Stc~itwrt. I’. 11.. ltllvr. \\.. \\‘. B (:oldman. It. 1). (I!MV)). f’roc. .\irt. .-lctrt/. SC;.. f’.dy..-l. 77. -IT,34 4538. Sutl. ‘I’. ‘I’. A (:twn. H. (1977). S~ltrrr (Lonrlor,). 269, 489~#):1. Suit. ‘I‘. ‘I’.
Oy h’, AYitttotr.u