Differentiation specific functions in cultured and transplanted mouse keratinocytes:

Differentiation

Differentiation (1984) 26: 154169

$3 Springer-Verlag 1984

Differentiation specific functions in cultured and transplanted mouse keratinocytes : Environmental influences on ultrastructure and keratin expression Dirk Breitkreutz *, Axel Bobnert, Erika Herzmann, Paul E. Bowden, Petra Boukamp and Norbert E. Fusenig Division of Differentiation and Carcinogenesis in vitro, Institute of Biochemistry, German Cancer Research Center, Im Neuenheimer Feld 280, D-6900 Heidelberg, Federal Republic of Germany

Abstract. Keratinocytes from neonatal mouse back skin, growing in primary culture (PEC) under conventional conditions (immersed), exerted a reduced programme of differentiation as indicated by cell morphology and organisation, ultrastructure and keratin composition. Four major keratins were found in cytoskeletal extracts (mol.wt. 60K, 59K, 53K, 49K) of primary cultures, together with a minor 51K protein and some residua! actin. This “culture-type” keratin profile remained stable and little variation was observed after repeated treatment with various agents, such as 12-0tetradecanoylphorbol-13-acetate(TPA), retinoic acid (RA) or dimethylsulphoxide (DMSO). The profile was unaltered by long-term growth of primary cultures in low Ca2+ (0.1 mM) medium or on 3T3 feeder-layers. Nevertheless, TPA, RA and low Ca2+ did alter the morphology and filament architecture (as observed by indirect immunofluorescence microscopy with anti-keratin antibodies). Comparison of keratinocytes from primary culture with basal cells isolated from epidermis revealed similarities in electrophoretic profile, with the 60K and 53K keratins being common to both. The other in vivo keratins had a characteristic spatial distribution; 67K and 58K keratins were present in suprabasal cells (spinous and granular), while 64K, 62K, 58.5K and 57.5K keratins were present only in stratum corneum. None of these keratins were found in cultured cells grown under regular conditions. Several morphological features of epidermal differentiation could be restored by the growth of PEC on collagen gels exposed to the atmosphere (“organotypic” culture) without influencing the keratin profile. Almost complete restoration of epidermal function was achieved after transplantation of PEC onto adult syngeneic mice. In this in vivo environment, well-structured epithelia developed which resembled interfollicular epidermis. Restoration of both typical ultrastructure and in vivo type keratin expression occurred within 2 or 3 weeks. Thus, although keratinocytes in primary culture differ considerably from those in vivo, they have not irreversibly lost the capacity for complete differentiation.

*

To whom correspondence should be addressed

Introduction Keratinisation in epidermal cell cultures, when compared to the situation in vivo, has been shown to be incomplete. This is true of gross morphology, ultrastructure [16, 24, 34,44, 52, 62, 681 and the expression of specific differentiation products, for example, the keratins [I, 9, 11, 18, 20, 21, 24, 25, 55, 631. However, there is good evidence to show that cultured keratinocytes have not irreversibly lost their capacity for complete differentiation. Several laboratories have reported that cultured keratinocytes transplanted onto suitable hosts develop stratified epithelia with epidermal-like morphology [3, 33, 70, 71, 731. Keratinising cysts produced in nude mice after injection of cultured cells have indicated that keratin expression can be altered towards that found in vivo [15]. Additionally, it has been recently demonstrated that human keratinocyte cultures depleted of vitamin A will express (although at a low level) keratins characteristic of terminally differentiating cells in vivo [22]. However, the extent to which keratins in cultured keratinocytes differ from those in vivo, at the molecular level, is not well documented. It is established that both in culture and in vivo, keratins are encoded by individual mRNA species [5, 20, 21, 28, 54, 55, 561. However, it has become apparent that the complexity of epidermal keratins in vivo is due to a combination of sequential gene activation and proteolytic modification of existing keratin polypeptides in the maturing keratinocytes [7, 8, 21, 26, 401. This proteolytic modification appears to be absent in culture, as does the expression of the largest keratin (67K). Evidence from antibody studies suggests that two keratins (67K, 58K) are specific for terminally differentiating cells in vivo and are not produced in culture [4, 42, 50, 65, 67, 691. While two of the keratins (60K, 53K) appear common to both culture and in vivo, two others might be culture specific (59K, 49K). Thus, while some keratins are expressed both in vivo and in culture, others appear specific to one or the other [l, 9, 11, 18, 20, 21, 24, 50, 631; (for a review, see [48]). In the present study, we have observed both changes in ultrastructure and keratin expression during the growth

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of mouse keratinocytes under various culture conditions and after transplantation onto subdermal granulation tissue of C,H mice. In such a way, we were able to correlate the influence of environmental factors on both the ultrastructure and the composition of a differentiation-specific product, the keratin family. Preliminary reports of this work have been presented previously [lo, 25,261. Methods

Indirect immunojluorescencemicroscopy

Cells grown on glass cover-slips were fixed €or 5 min with methanol at -2O"C, air dried and rinsed with PBS. The specimens were incubated with guinea-pig antiserum against bovine prekeratin at 37" C for 30 min, rinsed and further incubated with fluorescein-labelled goat anti-guinea pig IgG antiserum [17, 181. The cover-slips were rinsed, mounted with Aquamount (Lerners Lab, New Haven, Conn, USA) and examined with a Zeiss IM35 microscope equipped with epifluorescence optics.

Preparation of tissue and cells

Back skin epidermis from newborn mice (C,H) was separated after incubation in either 10 mM ethylenediaminotetraacetate (EDTA) at 37" C for 2 h, 10 mM dithioerythritol (DTE)at 22" €or 4 h or 0.5% acetic acid at 4" C for up to 15 h. After treatment with acetic acid or DTE, the epidermis could be further split into a lower living layer and an upper part, which consisted of upper granular cells and the stratum corneum [2]. Pure stratum corneum was isolated from trypsin-stable residues after gentle shaking in phosphate-buffered saline (without Ca2+ , Mg2+) containing 10 mM EDTA (PBS-EDTA) at 37" C. After incubation of skin in 0.2% trypsin at 37" C for 20 min, a split at the basal-suprabasal junction within the epidermis was made, and basal cells were gently scraped from the dermal part [14, 381. Basal cells were further enriched by centrifugation on a discontinuous gradient using Percoll (Pharmacia, Uppsala, Sweden) according to Brysk et al. [13], as modified by M. Gross et al. (in preparation). Keratinocytes for tissue culture were isolated by the trypsinization procedure previously described [23]. Culture conditions

Primary cultures were started by plating approximately 5 x lo5 cells/cm2 in Falcon tissue culture dishes and grown in modified Eagle's MEM containing 17% fetal calf serum, as described previously, except that Ca2+ concentration was 1.3 mM (4X-MEM, see [23]). Usually, cells were plated at 37" C and, after 12-15 h, cultures were incubated at 30" C in gassed incubators. Some cultures were shifted to low Ca2+-medium 18 h after plating, adjusted to 0.09-0.1 mM Ca Z f [32], supplemented with 10% fetal calf serum decalcified on Chelex-100 (Bio-Rad, Richmond, Calif, USA) and incubated at 30" C. Cellular protein was labelled by incubating cultures with 10 pCi/ml 3H-leucine (Amersham, Buckinghamshire, UK) in media containing one-tenth of the usual concentration of cold L-leucine. Cultures treated with 12-O-tetradecanoylphorbol-13acetate (TPA) or retinoic acid received the indicated dose twice a week with every medium change. TPA and retinoic acid were added as the stock solutions dissolved in acetone (final concentration of 0.05% acetone in the medium). "Organotypic" cultures were grown at the air-liquid interphase on type-I collagen (mouse tail tendon) which was gelatinized in the lower part of silicon chambers and used for transplantation as described [24, 271. Transplantation of cultured cells was performed as described by Worst et al. [70, 711 and Fusenig et al. [26]. Either suspensions of cultured cells or intact cultures growing on collagen substratum were transplanted onto an artificially induced subdermal granulation tissue in C,H mice and were protected by a silicon chamber.

Electron microscopy

Specimenswere rinsed in PBS, fixed in 2.5% glutaraldehyde (in 50 mM cacodylate buffer, pH 7.4) for 1-2 h, postfixed in 2% osmium tetroxide (in 25 mM cacodylate buffer) and stained en bloc with 1% uranylacetate for 15-16 h. Dehydrated specimens were embedded in Epon 812, ultrathin sections were stained with 2% uranyl acetate and lead citrate, and examined with a Zeiss EM 109 electron microscope at 50 kV. Protein extraction

( 1 ) Totalproteins. Tissue or cells were thoroughly washed with Dulbecco's PBS (including Ca", Mg2+)at 22" C, followed by PBS containing 3 mM MgCI,, 2 mMethyleneglycol-bis-2-aminoethylether tetracetate (EGTA) and 0.2 mM phenylmethylsulfonylfluoride (PMSF). In some experiments, tissue was pre-extracted with cold acetone. Washed tissue was suspended in electrophoresis sample buffer containing 5% sodium dodecylsulfate (SDS), 5% 8-mercaptoethanol (ME), 8% glycerol, 0.002% bromophenylblue, 2 mM EDTA, 1 mM PMSF, 10 mM Na-phosphate, pH 7.2, heated (5 min at 100" C) and homogenized @ounce type). After agitation for 2-4 h, the extracts were cleared by centrifugation (20,000 g for 30 min at 22" C). In order to separate noncrosslinked and disulphide crosslinked protein, samples were sequentially extracted, first in buffered SDS and then in the previously described buffer including a reducing agent (usually B-mercaptoethanol) . ( 2 ) Cytoskeletal proteins. Cytoskeletons were prepared, as described previously [9, 111, using high salt and low salt buffers and the non-ionic detergent, Triton X-100. Tissue samples were homogenized and sonicated in icecold NTM buffer (150 mMNaCl, 10 mM Tris-HC1, pH 7.4, 3 mM MgCl,, 2 mM EGTA, 1 mM PMSF) containing 1% Triton X-100 (TX), and the crude cytoskeletonswere centrifugated at 5,000 g. The first step was repeated and followed by two extractions with KTM (NTM + 1.5 M KC1) which contained 0.5% TX, one with NTM 0.5% TX and, finally, one with TM (10 mM Tris-HC1, pH 7.4, 3 m M MgCI,, 2 mM EGTA) containing 0.5% TX. Cell cultures were extracted by the same procedure, but directly in the culture dish, leaving cytoskeletons attached. Some cultures were incubated with DNase, RNase (each 50pg/ml in NTM; Sigma, St. Louis, USA, 1400 Kunits units; Serva, Heidelberg, FRG, 100 Kunitz units/mg, respectively) for 5-1 5 min on ice, between the isotonic (NTM) and high salt step (KTM), in order to decrease the viscosity during the later SDS extraction. The cytoskeletons were extracted in electrophoresis sample buffer as previously detailed.

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Fig. 2a-d. Indirect immunofluorescence microscopy of keratinocyte cultures, using guinea-pig antibodies to bovine prekeratin. a Primary culture growing in regular medium; centrally located “basal” cells contain a fine meshwork of filaments. bKeratinocytes cultured at low Ca’+ concentration; prekeratin-positive filaments of these cuboid cells are mainly oriented around the nucleus. c Keratinocytes growing in media containing 0.33 pM retinoic acid; cellular contacts and filament orientation exhibit irregularities. d Cultures treated with 1 pM TPA for 20 h; cells show irregular contours, reduced cellcell contacts and radially oriented filaments. Bur represents 50 pm, x 360

Polyacrylamide gel electrophoresis Proteins were analysed on SDS slabgels using the Laemmli system [37] with minor modifications [9, 111. Samples were boiled for 1 min and cleared by centrifugation before elec-

trophoresis. Routinely, 10% acrylamide gels were used but, in a few cases, 7.5%-17.5% gradient gels were used for comparison [7], as the mobility of keratins varies considerably on different electrophoretic systems [MI.Gels were stained with 0.05% coomassie blue R250 and scanned with

Fig. 1a-g. Phase-contrast micrographs of mouse keratinocyte cultures. n Primary culture (high density, day 7),showing stratification and proliferation of lower cell layers. b Keratinocyte culture (lower density) after treatment with 0.5% Triton X-100(20 min); filamentous structures are distinct and mitotic figures still preserved (arrow). c Keratinized culture after Triton X-100and high salt (1.5 M KCI) buffer treatment showing superficial horny cells. d Primary cultures in low Ca’+ (0.09 mM); cells form a monolayer, from which some proliferating cells become spherical and detach. e-g Low CaZ+cultures after transfer to regular medium (1.3 mM Ca”). e Early morphological changes 8 h after transfer. 1Cross-striation pattern in differentiating cultures 24 h after transfer, resembling regular cultures 2 or 3 days after plating. g Superficial (parakeratotic) horny cells in culture 41 days after Ca” shift. Most keratinid squames have thickened membranes, small granules and nuclear remnants. Bur represents 50 pm. x 260

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Fig. 3a-d

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a Zeiss KM3 at 575 nm. Gels of radio-labelled proteins were stained and photographed and then processed for fluorography, according to the method Bonner and Laskey [6], and exposed to Kodak X-omat film at - 70” C. Results

morphology (Fig. 1 e, f). Initially, proliferation continued in areas with high cell density, but the majority of cells eventually entered terminal differentiation (Fig. 1g). After longer periods (exceeding 4 weeks) at low Ca2+, cultures contained areas resembling clonal foci where growth persisted, often giving rise to permanent cultures (cf. [26, 361).

Morphology of cultured keratinocytes

Indirect immunofuorescence microscopy

The initial attachment of keratinocytes was optimal at 37” C but, after 12-15 h, the cultures were incubated at 30” C, which improved survival time and differentiation [26]. When growing immersed in medium on plastic substratum, cells formed multiple, superficially keratinizing layers (Fig. 1a, b, c). Treatment with Triton X-100containing buffers revealed a fine filamentous network, particularly in “suprabasal” cells, and polygonal contours of cornified cells (Fig. 1b, c). The cornified squames generally contained remnants of cell nuclei which resembled parakeratotic differentiation in vivo. Under conventional conditions, homeostasis between proliferating and differentiating cell compartments was poorly maintained over an extended period. The life span of keratinocytes in vitro could be prolonged without changing this pattern of differentiation, either by dense central plating in larger dishes (261, by supplementing with 0 5 2 % DMSO, or by growth on a feeder layer of irradiated 3T3 cells [52]. Cells could also be maintained for longer time periods in a “low differentiating state” by treatment with retinoic acid, but no dramatic increase of survival time occurred. Changing calcium levels in the medium influenced proliferation and differentiation. At an intermediate Ca2+-level of about 0.4 mM, cultures could be maintained at an equilibrium between proliferation and terminal differentiation for several months. At low Ca2+-levels (below 0.1 mM), only monolayers formed which showed no tendency to stratify (Fig. I d ; see also [31, 321). Proliferation did not cease at higher cell density, but numerous rounded-up translucent cells of variable size detached from the monolayers and many contained nuclear remnants and granular or filamentous material. Such cells were occasionally observed under regular conditions in primary culture and in derived permanent cell lines. The decrease in Ca2+ did not appear to interfere directly with cellular differentiation per se (as far as cornified envelope formation is concerned), but rather with cell shape, intercellular cohesion and stratification. This phenomenon resembled cornified envelope formation induced by suspending keratinocytes in methocel [29, 531. In addition, low Ca’+conditions produced binucleated cells, probably due to interference with cytokinesis in mitotic cells and enhanced structural chromosomal aberrations [26]. When shifted to higher Ca2+-levels(1.4 mM), most cells remained attached and resumed characteristic

A positive reaction with anti-keratin antisera provides a means of distinguishing keratinocytes from other cell types in skin that are often present in primary isolates, e.g. melanocytes, fibroblasts and endothelial cells (all keratin-negative and vimentin-positive). Anti-keratin immunofluorescence showed that, under “normal” culture conditions, a system of intracellular filament bundles connected by intercellular bridges formed a continuous sheet of interwoven fibres (Fig. 2a). The density and number of filaments increased as cells further stratified and keratinised (Fig. 2a, lower left), but once keratinised squames were formed, a more diffuse staining was observed. Reactivity with antisera and structural architecture were largely maintained in substratum-attached cytoskeletons after treatment with buffered Triton X-100 (not shown). Cells grown at reduced Ca2+-levelsrevealed a more diffuse organisation, perinuclear orientation of filaments and a large reduction of intercellular contacts associated with filaments (Fig. 2b). This probably facilitates the continuous detachment of superficial (differentiating)cells from monolayer cultures observed under low CaZ+-conditions. Alterations in the regular arrangement of intracellular filaments were also noted in the presence of retinoic acid (0.33 pM;Fig. 2c) and the phorbolester TPA (1 pA4; Fig. 2d). The filament network was finer, less interdigitations were observed and cell-to-cell contacts were significantly reduced. Non-toxic doses of phorbolester apparently delayed rather than prevented terminal differentiation, whereas retinoic acid mainly promoted the detachment of keratinizing squames from the lower layers of proliferating cells [24, 35,471. Anti-vimentin positive filaments were not detected in the keratinocytes of these primary or early sub-cultures under any of these conditions (see [26]). Ultrastructure of high-density and organotypic cultures

Prolonged survival and improved structural organisation could be obtained by plating keratinocytes centrally at high density [26]. Nevertheless, unlike in vivo, “basal” cells in vitro tend to spread on the substratum and acquire a flattened shape. The most striking characteristic of high-density cultures was the higher incidence of keratohyalin granule formation (Fig. 3a, b), a rare event in epidermal cultures. However, the granules were exclusively of the compact

Fig. 3a-d. Electron micrographs of perpendicular sections through primary keratinocyte cultures. a Centrally plated cultures (day 9); three living cell layers containing tonofilament bundles and keratohyalin (KH)-granules ; superficial dead horny cells have cornifed envelopes. b Transition zone (living to cornified cell) at higher magnification (central urea frumed in a); tonofilament bundles connected to desmosomal plaques (urruwheuds), KH-granule associated with ribosomes, and outermost cell with cornified envelope (arrows).c Keratinocytes grown for 16 days in “organotypic” culture; living cells have perinuclear orientation of filament bundles. Lowermost horny cells are tightly condensed, containing material of intermediate electron density, whereas superficial horny cells appear translucent. d Transition zone at higher magnification; in the lower cells, there are interdigitationsof lateral cell membranes, desmosomes (urrowheadr) and tonofilament bundles (arrows) are seen in cross section. Adjacent horny cells possess cornified envelopes and electron-denseintracellular material of fibrillar nature. Burs represent (a, c) 10 pm; (b, d) 1 pm.(a) x 5,900; (b) x 42,000; (c) x 4,500; (d) x 54,000

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round type. An increasing density of tonofilament bundles was observed in the upper cell layers. The superficial horny cells had cornified envelopes, but were still lacking the typical “keratin-pattern of stratum corneum cells. An inherent morphological and functional feature of epithelia is their polarity which defines a lower, basal and upper, apical side. In the epidermis, functional polarity is maintained throughout the multiple strata, with the outermost cell layer being exposed to the atmosphere. To create a similar micro-environment in culture, techniques have been developed to grow keratinocytes at an air-liquid interphase, providing the nutrients exclusively from the lower side [24, 27, 41, 511. In such an “organotypic” culture, keratinocytes grew on thick collagen gels and formed multiple cell layers. Cross-sections of paraffin-embedded or frozen specimens revealed an improved architecture of the cell layers and superficial areas of densely keratinised squames ~51. At the ultrastructural level, regular stratification was significant,with the living cells containing desmosome-associated tonofilament bundles (Fig. 3c). Interdigitations of lateral cell membranes were common and electron-dense granules were observed in granulosum-like cells. Horny cells possessed cornified envelopes, electron-dense deposits in the intercellular space and intracellular material of fibrillar nature, which resembled the keratin-pattern of the stratum corneum in vivo (Fig. 3d). Despite the detection of basal-lamina components (laminin and type-IV collagen) by immunofluorescence [26], a defined continuous basal lamina and hemidesmosomes were not formed (A. Bohnert et al., in preparation). Thus, although the organotypic culture had many morphological features of epidermis in vivo, the differentiation programme exhibited by these cells was still incomplete. More recently, a significant improvement in the general organisation of strata and the presence of both a dense keratin-pattern and abundant keratohyalin granules was demonstrated by placing freshly separated living dermis below the collagen gel [43]. ”

Cell transplants A dramatic improvement in structural organisation was achieved by transplanting isolated keratinocytes on graft beds in syngeneic or allogeneic host mice [25, 70, 711. These cell transplants formed stratified epithelia that strongly resembled interfollicular epidermis in histological section. The similarity was further substantiated by electron microscopy, which revealed multiple strata of well-oriented nucleated and keratinising cells (Fig. 4a). At higher magnification, cells in the transition zone (Fig. 4b) contained numerous keratohyalin granules of the amorphous type often associated with tonofilament bundles. In the horny cells (with

well-developed cornified envelopes), the filaments were densely packed, giving rise to the typical ‘keratin-pattern’ (Fig. 4c). Finally, high magnification of the dermal-epiderma1 junction zone revealed the presence of a continuous, well-outlined basal lamina with hemidesmosomes at the basal-cell plasma membrane (Fig. 4d). These “in vivo” features were expressed by the transplanted keratinocytes, whether they were in direct contact with host mesenchyme or separated from it by a layer of collagen gel. Epidermal keratins

Keratins were extracted from the epidermis of newborn mouse (back skin), immediately after separation by acetic acid from the dermis. This was achieved either by boiling in a buffer containing SDS and mercaptoethanol (total extract) or after removing triton X-100 and low/high-salt soluble proteins (tissue cytoskeleton). Both extracts were characterised by electrophoresis (Fig. 5 ) and contained five major (apparent mol. wt. 67K, 64K, 62K, 58.5K, 57.5K) and three minor keratins (60K, 53K, 49K). These were present in a similar ratio in both types of extract but, although tissue cytoskeleton preparations were enriched for the keratins, some actin (42K) still co-purified. However, extraction of freshly separated epidermis with SDS in the absence of a reducing agent produced a different keratin profile (Fig. 5 , lane 3). This was characterised by a quantitative increase in the 60K, 53K and 49K keratins relative to the 67K and 58K keratins, a significant reduction or absence of the 64K, 62K, 58.5K and 57.5K keratins and the presence of both a 65K protein and actin. After removal of all SDS-soluble protein, the residue was extracted with SDS-mercaptoethanol (Fig. 5, lane 4), which resulted in solubilization of disulphide-linked keratins absent from the SDS extract (64K, 62K, 58.5K, 57.5K). No actin was observed in these extracts. When this serial extraction was performed on tissue cytoskeletons, a variable amount of keratin was soluble in SDS alone (Fig. 5, lane 5 ; cf. lane 3) and extraction of the residue with SDS-mercaptoethanol led to a typical “totalcytoskeleton” profile (cf. Fig. 5, lanes 2, 6). This infers that extensive pre-extraction with triton X-100 and high salt during the cytoskeletal preparation caused artificial disulphide cross-linking of the keratins which are normally kept reduced (as SH) by the conditions within the living cell. The keratin profiles after serial extraction were similar, whether the epidermis was separated by acetic acid (Fig. 5 ) or EDTA (not shown). The spatial location of different keratins within the epidermis was further demonstrated by fractionating into upper and lower layers with acetic acid (Fig. 6a). Keratins in the lower strata (basale, spinosum and lower granulosum) were completely soluble in SDS (Fig. 6a, lanes 1, 2)

Fig. 4a-d. Epidermis-like tissue formed by transplanted keratinocyte cultures after 27 days in the host. a Living cell layers demonstrate the transition from cuboid basal-type cells to differentiated spinous- and granular-type cells. Layers of flattened horny cells contain electron-densematerial. b Higher magnification of transition zone. Stratum granulosum cell with irregularly shaped KH-granule associated with tonofilament bundles and ribosomes ; horny cells show irregularly condensed structures of fibrillar material, disintegrating desmosomes (arrowheadv) and intercellular deposits (arrows). c Enlarged view of horny cells filled with fibrillar material (upper surface on right); electrondense cornified envelopes (arrows) are found on the inner cell membrane, and, at widened intercellularspaces, degenerating desmosomes (arrowheads). d Lower part of basal keratinocyte and underlying mesenchymal fibroblast; basement membrane (lamina lucida and lamina densa) is continuous and keratinocyte plasma membrane is decorated by attachment plaques (arrowheads)of hemidesmosomes with radiating tonofilaments. Below these sites there is some indication of anchoring filaments traversing the basal lamina towards the mesenchyme (arrows). Bars represent (a) 10 pm; (b, d) 1 pm; (c) 0.5 pm. (a) x 4,500; (b) x 30,000;(c) x 97,000;(d) x 30,000

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Fig. 4a-d

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Fig. 5. SDS-polyacrylamidegel electrophoresis of epidermal keratins from newborn mouse (back skin), SDS-mercaptoethanol(ME) extraction of total epidermis ( T ; lane 1 ) and epidermal cytoskeletal preparation (keratin, K ; lane 2); SDS-extract (without ME) of total epidermis (T-SH; lane 3) and epidermal cytoskeleton ( K - S H ; lane 5), and SDS-ME extraction of residual material (-S-S-) from total epidermis (lane 4) and epidermal cytoskeleton (lane 6). Molecular weights are given on the right ( x

and the profile resembled that obtained from whole epidermis with SDS alone (cf. Fig. 5, lane 3). The total cytoskelePal extract of upper strata contained typical stratum corneum keratins (64K, 62K, 58.5K, 57.5K) and small amounts of the 67K keratin (found in living epidermis), presumably from the upper granular cells present in this fraction (Fig. 6a, lane 3). This keratin band was eliminated by pre-extraction with SDS and only the four “stratumcorneum” keratins remained (Fig. 6a, lane 4). The profile was identical to both that obtained by sequential extraction of whole epidermis (Fig. 5 ) and a total extract of trypsinisolated stratum corneum (not shown). Similar results were obtained with total and cytoskeletal extracts after dithiothreitol separation of the epidermis (Fig. 6b): five major keratins were located in the lower epidermal cells (Fig. 6b, lane 1 ; 67K, 60K, 58K, 53K, 49K) and four in the stratum corneum (Fig. 6b, lane 2; 64K, 62K, 58.5K, 57.5K) which, in this case, was apparently not contaminated with living cells (no 67K band). Trypsin was used to produce a basal-spinous layer split (see Methods) and the basal-cell fraction obtained was purified by percoll gradient centrifugation [13] (M. Gross et al., in preparation). Cytoskeletal preparations of such cell fractions (Fig. 6c) predominantly contained the 60K, 53K and 49K keratins (lane l), while spinous-granular cell fractions mainly contained 67K and 58K keratins (lane 2). The remaining stratum corneum with a few granular cells attached (lane 3) showed a typical disulphide crosslinked keratin profile, together with traces of the 67K keratin. Keratins from cultured keratinocytes

Freshly isolated epidermal cell suspensions were extracted and found to have a keratin profile analogous to that of the living epidermal cells in situ (Fig. 7a, lane 1). Cells which did not attach in culture contained only traces of SDS-extractable keratin (Fig. 7a, lane 2), but further extraction of the residue in the presence of SDS-mercaptoeth-

Fig. 6a-c. Distribution of keratin polypeptides in the different compartments of epidermis. a Extracts of lower ( L ; lanes 1 , 2) and upper epidermal layers (V;Iunes 3, 4 ) after intraepidermal split with acetic acid. SDS-soluble total protein of lower layers (lane 1 ) with no residual crosslinked keratin (x; lane 2); SDS-ME soluble (total) keratin from cytoskeleton (lane 3) and from SDS-insoluble residue of upper layers ( x ; lane 4). Dashes indicate mol. wt. 67K, 60K,58K,53K,49K and 42K.b Cytoskeletal fractions after DTE separation of epidermis and subsequent split into lower ( L ; lane I ) and upper layers (U;lane 2). c Cytoskeletal proteins of cell fractions enriched in basal ( B ; lane I ) and in suprabasal cells (spinous, lower granular cells, S ; lane2) and remaining upper epidermal layers (mainly stratum corneum, C; lane3). Note that the 60K, 53K and 49K polypeptides are major components of basal cells, whereas 67K and 58K increase significantly in the suprabasal cells

anol (Fig. 7a, lane 3) revealed the presence of two major keratins (67K, 58K). They were analogous in size to SDSsoluble keratins found in higher epidermal strata in vivo (see Figs. 5,6), but were disulphide crosslinked, presumably due to the oxidising conditions in these leaky, dying cells. Extraction of attached cells revealed a different profile of SDS-extractable keratins, both in total extracts (Fig. 7a, lane 4) and tissue cytoskeletons (Fig. 7a, lane 6), compared to those in vivo (Fig. 7a, lane 1). The reduction in 67K and 58K keratins observed even after 24 h was paralleled by an increase in “culture-type” keratins (60K, 59K, 53K, 49K) and, by 48 h, the 67K and 58K keratins had virtually disappeared from SDS-soluble material (Fig. 7a, lanes 8, 10). This culture-type keratin profile (49K-60K) then remained stable in primary culture (Fig. 7a, lanes 12, 14). Extraction of SDS-insoluble material with SDS-mercaptoethanol revealed the presence of residual crosslinked keratins (67K, 58K) in primary cultures 24 h after plating (Fig. 7a, lanes 5 , 7). These were probably derived from differentiated epidermal cells still adhering to the monolayer, as after elimination of these cells by vigorous washing, the 67K and 58K keratins disappeared (Fig. 7a, lanes 9, 11). However, at higher plating density, adhering residual cells

163 Fig. 7a, b and b’. Changes in keratin polypeptides during early stages in culture. a The pattern of a total cell suspension immediately before plating (lane I) is shown in comparison to that of cells not attached (fanes2, 3) and cells attached after 1 day (lanes 4-7), 2 days (fanes8-1 l), 7 days (lanes 12, 13), and 14 days (fanes14, 15) in culture. Total cell extract in lane I; sequential SDSextracts (without ME; where marked x, with ME) of total cell proteins in lanes 2, 4, 8 and 3, 5, 9 respectively, and of cytoskeletons in lanes 6, 10, 12, 14 and 7, 11, 13, 15 respectively. b, b’ (3H)-leucine labelling of proteins in keratinocyte cultures. Upper panel (b), coomassie-blue stained gel and lower panel (b’), corresponding fluorograph. Cytoskeletal extracts of cultures 1 day (lane l), 2 days (lane 2), 3 days (lane 3) and 4 days (lane 4) after plating. Cells were labelled, either immediately after plating for 25 h (lane 1) and for 48 h (lane 2), or for 48 h after the first day (lane 3) and after the second day (fane4). Virtually no label is present in the 67K component (arrowhead). Dashes indicate mol. wt. 67K, 60K, 58K,53K, 49K and 42K

persisted for some time and the 67K and 58K keratins were still found in these primary cultures after 1 week or more. In cultures from which residual epidermal cells had been washed, little disulphide crosslinked material was found during the first few days. However, in older, stratified cultures (1-2 weeks after plating) which contained many “horny-cells”, small amounts of crosslinked keratins were observed (Fig. 7a, lanes 13, 15). In general, these were of the same molecular weight as the culture-type SDS-soluble keratins, but an extra protein was present (58.5K). This was probably generated by intracellular proteolysis of the 60K or 59K keratins in a manner related to that found in vivo (81. Experiments with 3H-leucine incorporation into keratins further substantiated that the alteration of keratin expression associated with keratinocytes becoming established in primary culture was rapid (Fig. 7b). While total cytoskeletal extracts of cells 24 h (Fig. 7b, lane 1) and 48 h (Fig. 7b, lane 2) after plating contained the 67K keratin, this was not labelled in the corresponding fluorograph. After 3 or 4 days (Fig. 7b, lanes 3, 4), all of the major culture-type keratins were labelled and the fluorograph closely resembled the stained gel. Thus, the persistence of the 67K keratin, and probably the 58K keratin also, appeared to be due to a “carry-over” from the isolate and these keratins were not synthesized in primary culture under the conditions used. Also, in the presence of 0.33-3.3 p M retinoic acid or 0.1-1 p M TPA, no major changes in keratin synthe-

sis were observed under the same labelling conditions (electrophoretic profiles not shown here), despite the altered keratin-filament organisation (immunofluorescence). Long-term primary culture Keratinocytes were maintained in primary culture for longer periods (up to 3 months) either in low Caz+-medium (0.09 mM C a Z f )or on 3T3 feeder layers (Fig. 8). The keratin profile remained unaltered whether cells were grown in regular medium (Fig. 8a, lane 1) or low Ca2+-medium (Fig. 8a, lane 3), even though the cells differed considerably in morphology. However, sequential extraction of the attached cells showed that cells grown at low Caz+-levels (Fig. 8 a, 10 days) differed from age-matched cultures in regular medium in that they contained no disulphide crosslinked keratins (cf. Fig. 8a, lanes 2, 4). Under low CaZ+growth conditions, keratinocytes only formed monolayers, but considerable numbers of detached cells were found floating in the medium. These floating cells resembled attached cells in their keratin profile (Fig. 8a, cf. lanes 3, 5), but contained considerable amounts of crosslinked material (Fig. 8a, lane 6, cf. lane 4). Interestingly, the 67K and 58K “in vivo-type” keratins were present in both total and cytoskeletal extracts of these floating cells (Fig. 8a, lane 6). The culture-type keratin profile was maintained in low CaZ+for up to 66 days and was unchanged at 2 days (not

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Fig. 8a-c.Protein profiles of cells grown under conditions for longterm culture. a Sequential SDS-extracts (X with mercaptoethanol) of total protein from 10-day-old cultures grown at regular Ca2+ (lunes I , 2) and at low C a Z + concentration: attached cell layer (1; lanes 3. 4) and detached round cells ( T ; lanes 5, 6). Note the absence of SDS-insoluble cross-linked keratin in the low Ca2+ monolayer (X;lane 4)and the presence of 67K polypeptide apparent in detached cells (lanes5, 6). Dashes indicate mol. wt. 67K, 60K, 58K, 53K, 49K and 42K. b Cytoskeletal preparations from cultures grown for 2 months at low Ca2 and, after shift to regular media, for an additional 4 days (lane I) and 18 days (lane 2). c Cells grown on 3T3 feeder layers for 2 months; cytoskeletons sequentially extracted with SDS (lane I ) and SDS-ME (lane 2) +

shown), 4 days and 18 days after a shift to normal C a Z f levels (Fig. 8 b). In the presence of 3T3 feeder cells, multilayered keratinised cell sheets developed, but the same stable culture-type keratin profile was observed over a period of @days (Fig. 8c). The profile of SDS-soluble keratins was similar in the lower cell layers and cornified sheets (peeled off from above) and, in both fractions, a similar amount of crosslinked material of the same electrophoretic profile was found. Keratinocyte transplants

The only persistent change towards an in vivo keratin profile occurred in keratinocytes transplanted on syngeneic animals. In comparison, no alterations of the culture-type keratins were observed in ‘organotypic’ cultures grown on collagen, with or without 3T3 feeder cells (Fig. 9a). About

Fig. 9a-c. Re-expression of the “in vivo” type pattern in isolated keratinocytes upon transplantation in adult mice. a Comparison of cytoskeletal preparations from culture and transplants at different stages; “organotypic” culture on collagen gel at day 17 with 3T3 feeder cells growing on the culture dish underneath the collagen gel (oC; lane I), and transplants at day 13 (Tf;lane 2), day 23 (T2; lane ‘3)and day 27 (T3;lane 4). All bands present in epidermis of newborn mouse were also expressed in well-developed transplants. Dashes indicate mol. wt. 67K, 60K, 58K, 53K, 49K and 42K. b Comparison of cytoskeletons from newborn mouse epidermis (nE; lane I) transplant at day 27 (lower loading than a, T3; lane 2), and whole epidermis of adult mouse (back skin, aE; lane 3). c Long-term experiment showing a transplant at day 70 dissected into graft bed (lane f), lower (lane 2) and upper part ( I n 3), which could be pealed off easily from the lower layers. Whereas the larger bands (62K,64K, 67K) were progressively degraded in the cornified layers with the age, these bands reappeared in the layers below with the regeneration of new epidermal-like tissue. The material scraped from the remaining graft beds mainly consisted of collagen bands, occasionally with traces of 57K protein, presumably vimentin (lane 1)

2 weeks after transplantation, the growing transplants became visible to the naked eye and keratins of 67K, 64K, 62K and 58K appeared (Fig. 9a, lane 2). These components clearly increased, together with other polypeptides in the range of 44K to 60K,with the growth of the transplants (Fig. 9a, lanes 3, 4). The majority of bands corresponded to those of lower and upper epidermal layers of the newborn mouse, with some similarity to adult epidermis (Fig. 9b). The keratin profiles truly reflected the morphological state of transplants. In long-term experiments (day 70), the uppermost layers which detached easily during preparation showed progressive degradation and loss of larger polypeptides (Fig. 9c, lane 3), while the epithelia regenerating from below again exhibited the complete keratin profile (Fig. 9c, lane2). No connection was observed to the surrounding

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epidermis of the host and material from the graft beds contained collagen and, occasionally, some vimentin (57K) which is indicative of mesenchymal cells (Fig. 9 c, lane 1). Discussion

’

Although vertebrate skin has been successfully employed to demonstrate the effect of both cell and tissue interactions on phenotypic expression and differentiation (for further references see [12, 43]), a more defined in vitro system is required to study interactions at the molecular level. In the present investigation, we have grown keratinocytes in primary culture under varying environmental conditions and assessed the capability of extrinsic factors to modulate epidermal differentiation. This was judged by correlating alterations in morphology, ultrastructure and keratin composition. In this way, we have demonstrated that although neonatal mouse skin keratinocytes elicit a reduced programme of differentiation in primary culture, they have not lost the capability to produce an organised tissue with the morphological and functional properties of interfollicular epidermis. However, this only occurred when cells were exposed to a “ natural” environment, such as transplantation onto granulation tissue of adult syngeneic mice [25, 26, 70, 711. This complete epidermal differentiation could not be demonstrated under any of the culture systems employed and, therefore, we must conclude that various factors present in, or passing through, living dermis or mesenchymal tissue are required. Keratinocytes grown under conventional conditions do possess certain characteristics of in vivo epidermal differentiation [16,24, 34, 44,52, 62, 681. Primary cultures demonstrate stratification and corneocyte formation, and these keratinocytes contain desmosomes, membrane-coating granules and abundant tonofilaments and, also, develop cornified envelopes. However, certain features are absent or rare in regular culture, such as the formation of hemidesmosomes, a continuous basal lamina, keratohyalin granules and a dense “keratin-matrix” typical of dead horny cells. The nucleus is often retained and corneocytes resemble those found in parakeratotic tissue. Considering these morphological distinctions, it was interesting to examine keratin composition. Keratinocytes grown under conventional culture conditions generally express keratins of smaller size [1, 9, 11, 18, 20, 21, 24, 25, 54, 631, but careful comparison with epidermal extracts showed that more subtle differences, other than the lack of 62-67K keratins, existed. This has also been clearly shown by recent studies on keratin mRNAs using specific cDNA probes [54]. In accordance with previous work [9, 56, 571, newborn mouse epidermis contained eight major keratins (67K, 64K, 62K, 60K, 58.5K, 58K, 57.5K, 53K) and small amounts of smaller proteins (46K-51 K). The major keratins could further be divided into two separate groups: keratins of the living epidermal cells (67K, 60K, 58K, 53K) and disulphide crosslinked keratins of the dead horny cells (64K, 62K, 58.5K, 57.5K). A similar situation has previously been described for human and rabbit epidermis [l, 7, 8, 211. However, we have now achieved a subdivision of the living epidermis into cell-fractions of basal-type, spinous-type and granular-type, by trypsinisation [14, 381 and percoll gradient centrifugation [13]; (M. Gross et al., in preparation). This has demonstrated that a change in keratin expression

occurs as cells pass through the living cell layers, with basalcells being characterised by the presence of two major keratins (60K, 53K) and a variable amount of a 49K protein whose keratin-nature has not yet been established. The variable presence of 67K and 58K keratins is probably due to suprabasal contamination of the basal fraction. Similar results have recently been described for human epidermis [48,49, 58, 691 which indicate a high degree of interspecies homology in this respect. The 67K and 58K keratins become progressively more abundant in the higher living layers and can, as previously suggested [69], be considered as specific markers to terminal differentiation. It has not yet been clearly established whether a positionally controlled switch in keratin gene expression occurs, but similar observations have been made for several mammalian systems, both by immunofluorescence [4, 42, 67, 691 and biochemical studies [21, 47, 58, 691. However, according to a recent report, this is apparently not a postmitotic event, because there is no direct link to cessation of DNA synthesis and replication [6q. It is also interesting to note that small amounts of the “basal-type”- keratins (60K, 53K) do persist throughout the epidermis, due to the inherent stability of these proteins. Recent work with peptide mapping (P.E. Bowden et al., in preparation) has shown that the major stratum corneum keratins (64K,62K, 58.5K, 57.5K) are structurally related to the keratins abundant in the granular layer (67K, 58K). Furthermore, the major stratum corneum keratins are not encoded by their own mRNA [20, 541 and cannot be detected after short-term incubation of epidermis (2-5 h) in medium containing labelled amino acids (P.E. Bowden and D. Breitkreutz, unpublished observations). Keratinocytes in primary culture contained two major keratins that were abundant in epidermal basal cells (60K, 53K) and once established in culture, expressed two other major keratins (59K, 49K). While a close relationship between the corresponding 60K and 53K keratins (in vivo and in vitro) is strongly suggested by our recent data (in preparation) from two-dimensional gels and peptide mapping, the relationship between the 49K keratin of cultured cells and the 49K protein found in vivo in small amounts is still under investigation. It also remains a possibility that the basal cells in vivo express small amounts of the 59K keratin abundant in culture. Keratin composition undergoes subtle changes during epidermal differentiation and isolates invariably contain spinous and granular cells which possess large amounts of the differentiation-specifickeratins (67K, 58K). These differentiated cells adhere to the monolayer, especially when keratinocytes are plated at high cell density. However, plating at low density and vigorous washing every 24 h for the first few days results in the rapid loss of these keratins from culture extracts. These results are at variance with previous work on mouse epidermal keratins in culture [60], but are generally consistent with more recent keratin mRNA studies [S]. Labelling experiments further substantiated that these in vivo keratins are carried-over from the isolate and not made in the cultured keratinocytes, while expression of the 59K and 49K keratins occurred within 24 h of plating (see also [24]). Thus, there appear to be two alterations of keratin gene expression related to differentiation. In the majority of cases, basal cells synthesise two keratins (60K,53K), but may contain low levels of the other four keratins (67K, 59K, 58K, 49K). Once committed to terminal differentiation in vivo, there is no synthesis of 59K and 49K keratins,

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a decrease of 60K and 53K keratins and a concomitant increase of 67K and 58K keratins. When cells are placed in primary culture, the opposite is found. An increase of 59K and 49K keratins occurs, while the 60K and 53K keratins are maintained and the 67K and 58K keratins rapidly disappear. At this point, it should be noted that the apparent molecular weights used for the keratins have been determined by the calculation of their relative mobility compared to several standard proteins and to the mouse epidermal keratin that has recently been sequenced [61]. The latter has a molecular weight of 58,350 including the serine-bound phosphate, and is referred to as the “58K keratin”. It is analogous to “component VI”, an acidic mouse epidermal keratin [19], and to mouse “keratin 12” as defined in a later paper [55]. In general, our estimates of molecular weight for the other keratins also compare well with published values. Mouse epidermal keratins in vivo (67K, 60K, 58K, 53K) correspond to murine keratins 1,6,12,16 respectively (nomenclature of [55]) and to the values determined by Roop et al. [54]. This is also true of the culture-type mouse keratins (WK, 59K, 53K, 49K) equivalent to murine keratins 6, 7, 16, 18 [55]. It is also interesting that the size relationships that exist amongst these mouse keratins appear analogous to those found for human keratins in vivo, in cell fractions and in culture (D. Breitkreutz et al., in preparation; P.E. Bowden et al., in preparation; see also [50, 65, 691). Molecular weight determination is dependent on the gel system and standards used (e.g. [46]; personal observations) and comparisons with data from various laboratories should be made with care until further sequence data and a unified nomenclature is available. Keratin profiles showed little, if any, variation under the different culture conditions examined, even when alterations in morphology and filament organisation were observed, for example at low Ca2+ (cf. [31, 32, 50]), after treatment with retinoic acid (cf. [47]) or the tumour promoter TPA (cf. [30]). However, a minor change did occur after continuous treatment with retinoic acid and a protein (40K) appeared just below actin, which may be analogous to the 40K keratin described by others [22,48, 721. Changes in keratin expression have been described in cultures grown under reduced vitamin A levels [22], with partial re-expression of the 67K keratin occurring. A similar observation was made in the cells shed from the monolayer under low CazC-conditions,where low levels of the 67K and 58K keratins were noted, but this observation requires further substantiation including labelling experiments. Therefore, the keratin profile found in cells in primary culture appeared very stable over extended periods of time and may represent a basic differentiation programme of keratinocytes, which is commonly executed in vitro [50]. Earlier findings with developing or established mouse keratinocytes in vitro and carcinoma-derived cell lines [9, 11, 18, 24, 741 showed that this long-term stability of keratin profile often remains even after transformation. Although the keratin profile of cultured cells remained stable, the relative proportions of non-crosslinked and crosslinked (disulphide-bridged) keratins did change under various conditions. In general, both in the living epidermis and cultured cells, the keratins are poor in disulphide crosslinks. Changes in cell membrane permeability occur as differentiation proceeds [29, 53,621 and the reducing potential of the cells is lost. This has the effect of oxidising SH-groups

already in close proximity, which occurs “naturally” above the upper granular layer [45, 591 and artificially during certain extraction procedures or storage of keratin preparations (cf. [63]). Thus, very careful attention must be paid towards methodology in order to assess the fraction of crosslinked keratins. In vivo, large quantities of keratin are soluble in SDS without a reducing agent (see Fig. 5), but after prior extraction with Triton X-100 and low/high salt buffers and extensive homogenisation, most of the keratins are no longer soluble in the absence of a reducing agent. The ability of keratins to become crosslinked under oxidising conditions was also observed in early primary cultures where the rounded-up cells found in the medium or cells washed from the monolayer contained keratins specific for living cells in vivo, which were now disulphide-crosslinked. The same was true of the abundant cells floating above low CaZ+-cultures. Generally, in primary culture, there were only small amounts of crosslinked keratins which were of similar electrophoretic profile to the uncrosslinked keratins. However, in older primary cultures (1-2 weeks) and long-term cultures (e.g., on 3T3 feeder layers, 2 months), the amount of crosslinked keratin increased in proportion to the thickness of the horny layer (cf. [63]) and evidence of proteolytic processing was apparent. This was analogous to the situation for epidermis in vivo, where disulphide crosslinking is accompanied by a change in keratin profile [7], presumably due to proteolytic modification [8]. Thus, certain aspects of the differentiation programme appear to be present in primary culture but, as demonstrated here, individual biochemical markers are often incorrectly linked with characteristic changes in morphology and ultrastructure (and vice versa). However, care should be exercised in the use of stratification, keratin crosslinking and cornified envelope formation [29, 53,621 as “differentiation markers” in cultured keratinocytes. These processes apparently mimic epidermal differentiation in vivo although they can occur in vitro independent of differentiation or in the wrong time sequence triggered by events other than a true differentiation programme. For example, cornified envelope formation has been induced in cultured keratinocytes by such “unphysiological” conditions as treatment with TPA [30] or suspension in methylcellulose [29, 53, 621. Virtually complete restoration of epidermal differentiation was found in transplanted keratinocytes and normalisation of morphology and ultrastructure was accompanied by a significant alteration in keratin composition. Establishment of in vivo tissue architecture, as previously demonstrated by histology and immunofluorescence in mouse and human transplants [3, 25, 26, 33, 70, 71, 731, was further substantiated by electron microscopy, revealing typical, welldeveloped cell strata. A continuous basal lamina and abundant hemidesmosomes are not observed in primary culture on plastic substrata or in organotypic cultures on collagen (A. Bohnert et al., in preparation; [261), but are a consistent feature of transplants. A defined stratum granulosum with abundant keratohyalin granules was present and the stratum corneum exhibited an electron-dense “ keratin pattern” closely resembling that found in newborn mouse epidermis. Direct contact with host tissue was apparently not required for this normalisation, since epithelia developed similarly when separated from the graft bed by a layer of collagen. The most striking feature of the transplants was that the keratin profile changed towards that

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characteristic of in vivo epidermis, the extent paralleling the development of epidermal morphology. In the first few weeks, expression of 67K and 58K keratins was apparent [lo], but persistence of the culture-type keratins was a common feature, analogous to the situation when cells are first plated in primary culture. The use of labelling here may distinguish which keratins are residual and which are newly synthesised. Once the transplants were established, the typical stratum corneum keratins (64K,62K), which are never found in culture, were apparent. The keratin profiles were not completely normalised to the adult type, as might be expected, but this is not surprising as inflammatory responses in the graft bed cause hyperplasia, which has been shown to effect keratin profiles [57]. A similar environmental influence on keratin synthesis, as well as ultrastructural features, has been previously demonstrated in regressing keratinising cysts formed after the injection of keratinocytes into nude mice [15, 391. So far, comparable changes in keratin expression have not been observed after transplanting malignant epidermal cell lines, although keratinising features have been apparent [I 1,641. Therefore, in conclusion, it is apparent that keratinocytes do not lose their intrinsic potential for epidermalspecific differentiation while growing in primary culture. Transplantation to an ‘in vivo’ environment is sufficient to normalise, almost completely, the tissue morphology, ultrastructure and keratin expression. These features are temporarily lost in culture and our experiments suggest that various diffusible factors are absolute requirements for normal epidermal differentiation. Identification of these substances would allow normal epidermal differentiation to occur in culture and would also provide a valuable in vitro system for studying changes in differentiation at different stages of transformation, in a manner more relevant to the situation in vivo. The crucial role of diffusible factors or the conditioning effects of the mesenchyme is implicated by our recent experiments with “recombinants” in vitro, where viable dermis is placed underneath “ organotypic” cultures (Mackenzie et al., in preparation; [43]). Under these culture conditions, epidermal differentiation is essentially improved as judged by morphology, ultrastructure and by preliminary results on keratins. Acknowledgements. The skilful technical assistance of Birgit Kahl, Angelika Krischke, Christine Marston and Jutta PBhlmann and the help of Monika Matejka and Angela Scherer in preparing the manuscript is gratefully acknowledged. We thank Dr. Werner W. Franke for the keratin antisera and Monika Gross for advice in cell separation on Percoll gradients. This work was supported by the Deutsche Forschungsgemeinschaft(Fu 91/2-1).

References 1. Baden HP, Kubilus J, Argyris TS (1980) Modification of polypeptide composition in keratinocyte fibrous protein. J Invest Dermatol75 :383-387 2. Balmain A, Loehren D, Fischer J, Alonso A (1977) Protein synthesis during fetal development of mouse epidermis. Dev Biol60:442452 3. Banks Schlegel SP, Green H (1980) Formation of epidermis by serially cultivated human epidermal cells transplanted as an epithelium to athymic mice. Transplantation 29: 308-313 4. Banks-Schlegel SP, Schlegel R, Pinkus GS (1981) Keratin pro-

tein domains within the human epidermis. Exp Cell Res 136:465-469 5. Bladon PT, Bowden PE, Cunliffe WJ, Wood EJ (1982) Prekeratin biosynthesis in human scalp epidermis. Biochem J 208:179-187 6. Bonner WM, Laskey RA (1974) A film detection method for tritium labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem 46:83-88 7. Bowden PE, Cunliffe WJ (1981) Modification of human prekeratin during epidermal differentiation. Biochem J 199:145-1 54 8. Bowden PE, Cunliffe WJ (1983) Isolation and characterisation of different keratin types from human epidermis. In: Marks R, Plewig G (eds) Stratum corneum. Springer, Berlin Heidelberg New York, pp 9-18 9. Breitkreutz D, Boukamp P, Lueder M, Fusenig NE (1981 a) Morphological and biochemical criteria for keratinization in primary and permanent mouse epidermal cell cultures. In: Prunieras M (ed) Frontiers of matrix biology 9. Karger, Basel, pp 57-82 10. Breitkreutz D, Hornung J, Bohnert A, Herzmann E, Fusenig NE (1981b) Re-expression of epidermis-specificdifferentiation in transplants of cultured keratinocytes. J Invest Dermatol 76 :429 11. Breitkreutz D, Tilgen W, Boukamp P, Fusenig NE (1981~) Correlation of prekratin peptides and ultrastructure in epithelial cells of human skin tumors in vivo and in vitro. Anticancer Res 1:323-328 12. Briggaman RA (1982) Epidermaldermal interactions in adult skin. J Invest Dermatol79:21~-24s 13. Brysk MM, Snider JM, Smith EB (1981) Separation of newborn rat epidermal cells on discontinuous isokinetic gradients of percoll. J Invest Dermatol77:205-209 14. Delescluse C, Prunieras M (1978) Homeostatic regulation of epidermal cells in culture. Bull Cancer (Paris) 65: 223-226 15. Doran TI, Vidrich A, Sun TT (1980) Intrinsic and .extrinsic regulation of the differentiation of skin, corncal and esophageal epithelial cells. Cell 22: 17-25 16. Eisinger M, So0 Lee JI, Hefton JM, Danynkiewicz Z, Chiao JW, de Harven E (1979) Human epidermal cell cultures: Growth and differentiation in the absence of dermal components or medium supplements. Proc Natl Acad Sci USA 76:5340-5344 17. Franke WW, Weber K, Osborn M, Schmid E, Freudenstein C (1978) Antibody to prekeratin. Exp Cell Res 116:42%445 18. Franke WW, Schmid E, Breitkreutz D, Lueder M, Boukamp P, Fusenig NE, Osborn M, Weber K (1979) Simultaneous expression of two different types of intermediate-sized filaments in mouse keratinocytes proliferating in vitro. Differentiation 14:35-50 19. Franke WW, Schiller DL, Moll R, Winter S, Schmid E, Engelbrecht I (1981) Diversity of cytokeratins. Differentiation specific expression of cytokeratin polypeptides in epithelial cells and tissues. J Mol Biol153:933-959 20. Fuchs E, Green H (1979) Multiple keratins of cultured human epidermal cells are translated from different mRNA molecules. Cell 17:573-582 21. Fuchs E, Green H (1980) Changes in keratin gene expression during terminal differentiation of the kerdtinocyte. Cell 19~1033-1042 22. Fuchs E, Green H (1981) Regulation of terminal differentiation of cultured human keratinocytes by vitamin A. Cell 25:617-625 23. Fusenig NE, Worst PKM (1975) Mouse epidermal cell cultures. 11. Isolation, characterization and cultivation of epidermal cells from perinatal mouse skin. Exp Cell Res 93:443-457 24. Fusenig NE, Breitkreutz D, Boukamp P, Lueder M, Irmscher G, Worst PKM (1979) Chemical carcinogenesis in mouse epidermal cell cultures : Altered expression of tissue specific functions accompanying cell transformation. In : Franks LM, Wigley CB (eds) Neoplastic transformation in differentiated epithelial cell systems in vitro. Academic Press, New York London, pp 37-98

168 25. Fusenig NE, Breitkreutz D, Lueder M, Boukamp P, Worst PKM (1981)Keratinization and structural organization in epidermal cell cultures. In: Schweiger HG (ed) International cell biology. Springer, Berlin Heidelberg New York, pp 1004-1014 26. Fusenig NE, Breitkreutz D, Dzarlieva RT, Boukamp P, Herzmann E, Bohnert A, Piihlmann J, Rausch C, Schiitz S, Hornung J (1982) Epidermal cell differentiation and malignant transformation in culture. In: Smith GJ, Stewart BW ( 4 s ) Cancer forum 6. Australian Cancer Society, Sidney, pp 209-240 27. Fusenig NE, Breitkreutz D, Dzarlieva RT, Boukamp P, Bohnert A, Tilgen W (1983)Growth and differentiation characteristics of transformed keratinocytes from mouse and human skin in vitro and in vivo. J Invest Dermatol81: 168s-175s 28.Gibbs PEM, Freedberg IM (1980) Mammalian epidermal mRNA; identification and characterization of the keratin messengers. J Invest Dermatol74: 382-388 29.Green H (1977) Terminal differentiation of cultured human epidermal cells. Cell 11 :405416 30.Hawley-Nelson P, Stanley JR, Schmidt J, Gullino M, Yuspa SH (1982)The tumor promoter, 12-O-tetradecanoylphorbol-13acetate accelerates keratinocyte differentiation and stimulates growth of an unidentified cell type in cultured human epidermis. Exp Cell Res 137:155-167 31. Hennings H, Holbrook KA (1983)Calcium regulation of cellcell contact and differentiation of epidermal cells in culture. Exp Cell Res 143:127-142 32.Hennings H, Michael D, Cheng C, Steinert P, Holbrook KA, Yuspa SH (1980)Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell 19:245-254 33. Karasek MA (1968)Growth and differentiation of transplanted epithelial cell cultures. J Invest Dermatol 51 :247-252 34.Kitano Y,Endo H (1977)Differentiation of human keratinocytes in cell culture. In: Seiji M, Bemstein JA (eds) Biochemistry of cutaneous epidermal differentiation. University of Tokyo Press, Tokyo, pp 319-335 35. Kubilus J, Rand R, Baden HP (1981) Effects of retinoic acid and other retinoids on the growth and differentiation of 3T3 supported human keratinocytes. In Vitro 17:786-795 36.Kulesz-Martin MF, Koehler B, Hennings H, Yuspa SH (1980) Quantitative assay for carcinogen altered differentiation in mouse epidermal cells. Carcinogenesis I :995-1006 37. Laemmli U K (1970)Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:6 8 M 8 5 38. Laerum OD, Boyum A (1970)The separation and cultivation of basal and differentiating cells from hairless mouse epidermis. J Invest Dermatol54: 279-287 39.Lavker RM, Sun T-T (1983)Rapid modulation of keratinocyte differentiation by the external environment. J Invest Dermatol 80:228-237 40 Lee LD, Baden HP, Kubilus J, Fleming BF (1976)Immunology of epidermal fibrous proteins. J Invest Dermatol67: 521-525 41.Lillie JH, MacCallum DK, Jepsen A (1980) Fine structure of subcultivated stratified squamous epithelium grown on collagen rafts. Exp Cell Res 125:153-165 42. Loning T, Staquet MJ, Schmitt D, Thivolet J (1982)Immunocytochemical and ultrastructural localization of keratin polypeptides in normal epidermal and mucosal cells and tissues. J Invest Dermatol 78:4447 43.Mackenzie IC, Fusenig NE (1983) Regeneration of organized epithelial structure. J Invest Dermatol 81 : 189s-194s 44.Marcelo CL, Kim YG,Kaine JL, Voorhees JJ (1978)Stratification, specialization and proliferation of primary keratinocyte cultures. J Cell Biol 79:356-370 45.Matoltsy AG (1976) Keratinization. J Invest Dermatol 67:20-25 46.Matoltsy AG, Matoltsy M, Cliffel PJ (1981)Characterization of polypeptide chains of epidermal prekeratin and reconstituted filaments. Biochim Biophys Acta 668:160-166 47.Milstone LM, McGuire J, LaVigne J F (1982) Retinoic acid muses premature desquamation of cells from confluent cultures of stratified squamous epithelia. J Invest Dermatol79:253-260

48. Moll R, Franke WW, Schiller DL, Geiger B, Krepler R (1982a) The catalog of human cytokeratin polypeptides: patterns of expression of specific cytokeratins in normal epithelia, tumors and cultured cells. Cell 31 :I 1-24 49.Moll R, Franke WW, Volc-Platzer B, Krepler R (1982b)Different keratin polypeptides in epidermis and other epithelia of human skin: a specific cytokeratin of molecular weight 46,000 in epithelia of the pilosebaceous tract and basal cell epitheliomas. J Cell Biol95:28S295 50.Nelson WG, Sun T-T (1983)The 50-and 58-Kdalton keratin classes as molecular markers for stratified squamous epithelia : cell culture studies. J Cell Biol97:244-251 51.Prunikras M,Delescluse C, Regnier M (1976)Growth and differentiation of postembryonic mammalian epidermal keratinocytes in culture. In: Robert L (ed) Frontiers of matrix biology 3. Karger, Basel, pp 52-76 52. Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331-343 53. Rice RH, Green H (1978) Relationship of protein synthesis and transglutaminase activity to formation of the cross-linked envelope during terminal differentiation of the cultured human epidermal keratinocyte. J Cell Biol76 :70S711 54.Roop DR, Hawley-Nelson P, Cheng CK, Yuspa SH (1983) Keratin gene expression in mouse epidermis and cultured epidermal cells. Proc Natl Acad Sci USA 80: 716-720 55. Schiller DL, Franke WW, Geiger B (1982)A subfamily of relatively large and basic cytokeratin polypeptides as defined by peptide mapping is represented by one or several polypeptides in epithelial cells. EMBO J 1 :761-769 56. Schweizer J, Goerttler K (1980) Synthesis in vitro of keratin polypeptides directed by mRNA isolated from newborn and adult mouse epidermis. Eur J Biochem 112:243-249 57. Schweizer J, Winter H (1982)Changes in regional keratin polypeptide patterns during phorbol ester-mediated reversible and permanently sustained hyperplasia of mouse epidermis. Cancer Res 42:1517-1 529 58. Skerrow D, Skerrow CJ (1983) Tonofilament differentiation in human epidermis. Isolation and polypeptide chain composition of keratinocyte subpopulations. Exp Cell Res 143:27-35 59. Steinert PM, Idler WW (1979)Postsynthetic modifications of mammalian epidermal keratin. Biochemistry 18:5664-5669 60.Steinert PM, Yuspa SH (1978)Biochemical evidence for keratinization by mouse epidermal cells in culture. Science 200:1491-1493 61.Steinert PM, Rice RH, Roop DR, Trus BL, Steven AC (1983) Complete amino acid sequence of a mouse epidermal subunit and implications for the structure of intermediate filaments. Nature 302 :794-800 62.Sun TT,Green H (1976)Differentiation of the epidermal keratinocyte in cell culture: formation of the c o d i e d envelope. Cell 9:511-521 63.Sun TT,Green H (1978)Keratin filaments of cultured human epidermal cells; formation of intermolecular disulphide bonds during terminal differentiation. J Biol Chem 253 :2053-2060 64.Tilgen W, Boukamp P, Breitkreutz D, Dzarlieva RT, Engstner M, Haag D, Fusenig NE (1983)Preservation of morphological, functional and karyotypic traits during longterm culture and in vivo passage of two human skin squamous cell carcinomas. Cancer Res 43 : 5995-601 1 65.Tseng SCG, Jarvinen MJ, Nelson WG, Huang J-W, Woodcock-Mitchell J, Sun T-T (1982)Correlation of specific keratins with different types of epithelial differentiation: monoclonal antibody studies. Cell 30: 361-372 66.Van Neste D, Staquet MJ, Viac J, Lachapelle JM, Thivolet J (1983)A new way to evaluate the germinative compartment in human epidermis, using [3H]thymidine incorporation and immunoperoxidase staining of 67K polypeptide. Br J Dermatol 108:433-439 67.Viac J, Schmitt D, Staquet MJ, Thivolet J, Ortonne JP, Bustamante R (1980)Binding specificity of guinea pig anti-a-keratin

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polypeptide sera on human keratinocytes: comparison of their receptors with those of human epidermal cytoplasmic antibodies. Acta Derm Venereol (Stockh) 60:18%196 68. Voigt WH, Fusenig NE (1979) Organotypic difTerentiation of mouse keratinocytes in cell culture : a light and electronmicroscopic study. Biol Cell 34: 13-20 69. Woodcock-Mitchel J, Eichner R, Nelson WG, Sun TT (1982) Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies. J Cell Biol95: 580-588 70. Worst PKM, Valentine EA, Fusenig NE (1974) Formation of epidermis after reimplantation of pure primary epidermal cell cultures from perinatal mouse skin. J Natl Cancer Inst 53: 1061-1064 71. Worst PKM, Mackenzie IC, Fusenig NE (1982) Reformation

of organized epidermal structure by transplantation of suspen-

sions and cultures of epidermal and dermal cells. Cell Tissue Res 225 :65-77 72. Wu YJ, Rheinwald JG (1981) A new small (40 kd) keratin filament protein made by some cultured human squamous cell carcinomas. Cell 25 :627-635 73. Yuspa SH, Morgan DL, Walker RJ, Bates R R (1970) The growth of fetal mouse skin in cell culture and transplantation to F,-mice. J Invest Dermatol 55: 379-389 74. Yuspa SH, Hawley-Nelson P, Koehler B, Stanley JR (1980) A survey of transformation markers in differentiating epidermal cell lines in culture. Cancer Res 40:4694-4703 Received September 1983 / Accepted in revised form December 1983