Expression of markers of differentiation in a transformed human keratinocyte line induced by coculture with a fibroblast line

Experimental

Expression of Markers Human Keratinocyte

T. KAMALATI,’

Cell Research

185 (1989) 453463

of Differentiation in a Transformed Line Induced by Coculture with a Fi broblast Line

Z. McIVOR, M. HOWARD, and R. F. BROOKS

M. R. GREEN,*

Department of Anatomy and Human Biology, Kings College London, Strand, London, WC2R 2LS, United Kingdom, and *Unilever Research, Colworth Laboratory, Colworth House, Sharnbrook, Bedford MK44 lLQ, United Kingdom

SVK14, an SV40-transformed human keratinocyte line, has previously been reported to be almost completely unable to differentiate, and indeed, to express a set of keratins characteristic of simple epithelia rather than the stratifying epithelium from which they were derived. We have recently shown that IGF I stimulation of SVK14 results in expression of keratin 14, a marker of stratifying epithelia, as well as expression of markers which are characteristic of differentiation in normal human keratinocytes such as involucrin and keratin 10. To study further the capacity of SVK14 to differentiate, we have cocultured SVK14 with a variety of fibroblastic cell lines with a view to examining whether the cocultured partner can promote or interfere with their differentiation. We have observed that SVK14, when cocultured with Swiss 3T3, form organized structures through specific cell-cell interactions in which SVK14 express keratins 14 and 5 and involucrin, while maintaining T-antigen expression. These results are interesting since they show coculturing of a transformed human keratinocyte cell line and a particular tibroblast line can result in induction of characteristics of stratifying epithelia in a cell line with characteristics of simple epithelia. This may be analogous to the epithelial-mesenchymal interactions seen during epithelial development in the very early embryo. 0 1989 Academic PKSS, IIK.

The specific functions of skin depend largely on the properties of the epidermis which is locally specialized to form the various skin appendages: hair, glands, and nails. Keratinocytes, the epithelial cells of the epidermis, constitute a major part of the epidermis and are successfully cultured using a feeder layer of 3T3 cells [I]. The growth of normal human keratinocytes in culture leads to formation of terminally differentiating, stratified colonies, which eventually fuse into a continuous epithelium and form cornified envelopes [2, 31, a process reminiscent of the defined program of differentiation in skin. These cultures, however, do not form structures resembling hair follicles or other skin appendages. Transformed human keratinocyte cell lines grow in culture without the aid of feeder cells but show a limited capacity to differentiate. The human keratinocyte cell line, SVK14 [4] has been reported previously to be almost completely unable to differentiate [5, 61, as marked by lack of expression of markers of differentiation and expression of a set of keratins characteristic of simple epithelia (keratins 7, 8, 18, and 19) [4,5]. SVK14 cells were originally isolated using 3T3 feeders but have become feeder-independent as a consequence of transformation. To determine whether the impaired ability of SVK14 cells to differentiate might have ’ To whom reprint requests should be addressed. 453

Copyright 0 1989 by Academic Press, Inc. All ri&ts of reproduction in any form reserved 0014-4827/89 $03 .@I

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been influenced in any way by the subsequent absence of mesodermally derived cells (such as 3T3), we have reexamined the behavior of SVK14 cells in the presence of a variety of fibroblastic cell types. In this paper we report that SVK14, when cocultured with 3T3 cells, form organized structures, most likely due to specific cell-cell interactions, in which SVK14 express keratins 14 and 5 (markers of stratifying epithelia [7]) and involucrin (a marker of differentiating stratifying epithelia). The possibility exists that the cell-cell interactions between SVK14 and 3T3 demonstrated here, are analogous to the epithelial-mesenchymal interactions seen during epithelial development in the early embryo [8]. These results are interesting in that they show that coculturing of a transformed human keratinocyte and a specific fibroblast cell line can result in a degree of normalization of the differentiation process in keratinocytes while the cells maintain T-antigen expression. This suggests that transformation can be compatible with preservation of some level of differentiation potential and retention of sensitivity to appropriate environmental signals.

MATERIALS

AND

METHODS

Antibodies Primary anribodies. A mouse monoclonal antibody directed against HLA (monotypic), W6/32 191, and a rat monoclonal antibody against mouse H-2 (monotypic) [lo] from Hybritech (Nottingham, UK) were used at a dilution of 1 : 100. Mouse monoclonal antibody against involucrin, 3Al [ll], a gift from Dr. T. Rupniak (Glaxo, Greenford), and the mouse monoclonal antibody specific for the SV40 Tantigen, PAb416 [12], a gift from Dr. L. Crawford (ICRF, Lincolns Inn Fields), were used undiluted. Mouse monoclonal antibodies, LP34, a general antikeratin antibody [13], and LLOOl, monospecific for K14 [14], gifts from Dr. B. Lane (ICRF), were used undiluted. Mouse monoclonal antibody RCK102 directed against keratins 5 and 8 [15] from Bio-Nuclear Services (UK) was used at a dilution of 1 : 1. A mouse monoclonal antibody directed against vimentin, VIM 13.2 [16], from Sigma chemical Co., Ltd., was used at a dilution of 1: 10. All dilutions were made in a solution of PBS containing 1% BSA and 0.1% sodium azide. Secondary antibodies. Fluorescein-conjugated goat anti-mouse immunoglobulin (Cappel Laboratories) was used for immunofluorescence at a dilution of 1: 50 in PBS containing 1% BSA and 0.1% azide. Horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin (Dakopatt) was used for immunoblotting at a dilution of 1 : 50 in PBS containing 1% BSA.

Cells Swiss 3T3 cells used here (passage 10-25) were clone 4AiC5 described previously [17]. Human fetal tracheal tibroblasts, passage 12, were a gift from Dr. R. Littleton (Kings College Hospital, London). Human embryonic lung tibroblasts, MRC.5, passage 19, were a gift from Dr. E. Gherardi (Department of Pathology, University of Cambridge, UK). Several strains of human newborn dermal fibroblasts, passage 6-9, derived from newborn foreskin, were gifts from Miss C. Linge and Mr. A. Wallace (Kings College, London). Human fetal dermal fibroblasts, passage 8, derived from ventral skin of a 24-week fetus, were a gift from Dr. G. Jones (Kings College, London). Human dermal papillae cells, passage 4, derived from skin from the temporal region of a 47-year old donor, were a gift from Dr. R. Dover (ICRF, Lincolns Inn Fields). Mouse dermal fibroblasts, passage 2, were a gift from Ms. S. Murphy (Kings College, London). SVK14 (SV40-transformed human epidermal keratinocytes) were a gift from Dr. J. Taylor-Papadimitriou (ICRF, Lincolns Inn Fields). Cos [18], HeLa [19], and A431 1201 cells were gifts from Dr. L. Buluwela (LMB, Cambridge).

Cell Culture SVKII. Stock cultures of SVK14 were grown in RPM1 1640 supplemented with 10% fetal calf serum (FCS), 50 rig/ml cholera toxin, 5 yglml hydrocortisone, and sodium hydrogen carbonate to give a final concentration of 3.7 g per liter.

Coculture

of SVKl4

cells with Swiss 3T3fibroblasts

455

Other cells. Stock cultures of all other cell types used were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS. All cells were grown at 37°C in a humidified 12% CO2 atmosphere.

Cocultures Cocultures were initiated by seeding exponentially growing SVKl4 at 1.5~ 10’ cells together with 1.5X l@ cells of the cell type to be cocultured in 2 ml of medium in 3-cm dishes. The medium used for the coculture was the same as that used for SVK14 stock cultures. The cocultures and their controls were assayed after 4-5 days.

Conditioned

Medium

Conditioned medium was obtained by replacing the DMEM on exponentially growing 3T3 cells with the supplemented RPM1 medium (as above) for 2-3 days. The conditioned medium was centrifuged at 350g for 5 min using a bench top centrifuge to remove cell debris and stored at 4°C until use.

Mitomycin

C Treatment

Exponentially growing 3T3 were treated with 4 ug of mitomycin C per milliliter of medium and incubated at 37°C for 2 b. The cells were then washed, harvested, and used immediately.

Preparation

of Collagen

Gel

A solution of 1 g collagen from rat tail tendon per 300 ml of 1: 1000 (0.017 M) acetic acid was prepared according to the procedure of Michalopoulos and Pitot [21] (rats aged 3-4 months). The collagen gel was prepared using the following recipe: 500 ul collagen solution, 97 ul fetal calf serum, 323 ul 3X RPMI, and 50 ul 7.5% sodium bicarbonate. The components were kept and mixed at 4°C. One milliliter of the collagen mixture was pipetted into each 3-cm dish, spread evenly by rocking, and left to gel at room temperature.

Immunofluorescence Cells were fixed in methanol: acetone (1:l) for 10 min, air-dried for 20 mitt, and rehydrated PBS for 10 min. Antibody binding to fixed cells was performed at 37°C in a humidified chamber, an incubation period of 60 min for both primary and secondary antibodies. The preparations mounted in glycerol containing the “antifade” agent 1,4-diazabicyclo[2.2.2]octane (DABCO) and visualized using a Leitz Orthoplan microscope equipped with epi-illumination.

with using were [22],

Immunoblots Keratins were extracted according to the procedure of Rosenberg et al. 1231. Keratin extract from SVK14 monocultures (100 ug), 3T3 monocultures (50 ug), and SVK14/3T3 cocultures (50 ug) were separated by discontinuous SDS-polyacrylamide gel electrophoresis (8.5 %) [24], electrophoretically transferred to nitrocellulose filter [25], and probed with monoclonal antibodies. After electrophoretic transfer, the nitrocellulose filters were blocked with PBS containing 0.5% Tween 20 and incubated with primary antibody. Filters were then washed in PBS containing 0.5% Tween 20 for 30 min at room temperature and incubated with horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin. Filters were again washed with PBS containing 0.5% Tween 20 for 30 min at room temperature and peroxidase activity was visualized by using 4-chlor-1-naphthol [26]. Blocking and antibody incubation steps were each performed by gentle shaking overnight at room temperature.

RESULTS Coculture of SVK14 with Swiss 3T3 cells results in the formation of threedimensional, nodular structures composed of fibroblast aggregates dispersed among a monolayer of SVK14 cells. Here we describe in detail the series of events which leads to the formation of these structures when SVK14 are cocultured with 3T3. In coculturing SVK14 with 3T3, where the cultures are initiated 30-898342

Fig. I. Morphology of SVK14 cells after coculture with 3T3, demonstrating the three-dimensional, nodular structures. (A) Low-power view, and (B) a higher magnification of one of the nodules. Bar, 1000 urn for A and 500 urn for B.

as single-cell suspensions at high densities, the cells reassort such that individual cells of each cell type segregrate out of the mixed culture and migrate into discrete groups of like cells (visible within 36 h). Subsequently the cells assemble into marked nodular structures (Fig. 1) which on immunofluorescent staining with anti-HLA and anti-H-2 antibodies are seen to be composed of a core of 3T3 surrounded by a collar of SVK14 cells (Fig. 2; some nodules appear to be completely covered by SVK14 cells, data not shown). Thus it appears as though the SVK14 cells have “herded” together the 3T3 cells into small aggregates TABLE

1

Ability of a variety of cell types to form three-dimensional, coculture with SVK14

Coculture systems Fibroblastic cell lines SVKlUSwiss 3T3 SVKlUmitomycin C-treated Swiss 3T3 SVK14/dermal papilla tibroblasts SVK14Ifoetal dermal tibroblasts SVKlrllnewbom dermal flbroblasts” SVKlWembryonic lung fibroblasts (MRCS) SVK14Ifetal tracheal fibroblasts SVK14lmouse dermal fibroblasts Epithelial cell lines SVKlUCOS SVKIUHELA SVK14/A431 Others HELA/3T3 A43 ll3T3 COS/3T3 a Several strains tested.

nodular structures on Formation of nodular structures

Positive Positive Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative

Coculture

of SW14

cells with Swiss 3T3fibroblasts

457

Fig. 2. Immunofluorescent staining of an SVK14/3T3 coculture to demonstrate the distribution of the fibroblasts in relation to the epithelial cells. (A, C, and E) Phase contrast; (B, D, and F) fluorescence. Immunostaining with W6/32, a monoclonal antibody directed against HLA (monotypic) (C and D), and LP34, a general anti-keratin monoclonal antibody (E and fl, shows that the cells on the outside of the three-dimensional structures are composed of epithelial SVK14 cells while the core is composed of 3T3 fibroblasts, as demonstrated by staining with a monoclonal antibody against H-2 (monotypic, A and B). Bar, 500 urn.

(clearly visible by 72 h). Formation of these structures is not dependant on simultaneous seeding of the two cell types, since addition of SVK14 cells to a culture dish containing attached and growing 3T3 cells (or vice versa) results in appearance of the structures shown in Figs. 1 and 2. Further, the density of 3T3 cells affects only the time course and not the development of these structures. Furthermore, mitomycin C treatment of 3T3 such that the cells are rendered incapable of cell division, yet retain full metabolic activity, does not appear to interfere with the formation of these structures.

Fig. 3. Immunostaining of a coculture of SVK14 with human dermal fibroblasts using a monoclonal antibody against vimentin, Vim 13.2, which decorates dermal tibroblasts only. (A and C) Phase contrast; (B and D) fluorescence. (A and B) After 5 days; (C and 0) after 12 days. Unlike the cocultures of SVK14 with 3T3 cells, dermal tibroblasts and SVK14 cells do not re-sort to form threedimensional structures. The dermal tibroblasts appear as single cells dispersed uniformly among SVK14 cells. Bar; 500 pm.

Coculturing SVK14 with a variety of cell types shows that of the cells tested only 3T3 promote the formation of these organized three-dimensional structures (Table 1). Cocultures with all other cell types tested failed to show re-assorting or segregation of the mixed cultures as described above, and hence were devoid of three-dimensional structures. In these the cocultured partner of SVK14 either remained as individual cells, evenly dispersed among the SVKl4 cells for the entire culture life span (up to 12-15 days), as seen with dermal tibroblasts (Fig. 3), or formed small colonies dispersed among SVK14 cells [as seen with HeLa, Cos, and A431 cells (data not shown)]. Formation of the three-dimensional nodular structures described above does not appear to be induced when the two cell types are separated by a collagen gel matrix, i.e., by seeding the 3T3 cells underneath the collagen gel or by embedding them in the gel matrix while SVK14 cells are seeded on its surface. Further, 3T3-conditioned medium does not appear to induce formation of these structures in a pure SVK14 culture. This suggests that direct cell-cell contact or short-range interactions are involved. Immunofluorescent staining with anti-involucrin and anti-T-antigen antibodies showed that the SVK14 cells within the structures express involucrin, a marker of differentiation in stratifying epithelia, while maintaining T-antigen expression

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of SVK14 cells with Swiss 3T3fibroblasts

459

Fig. 4. Immunofluorescent staining of an SVK14/3T3 coculture with a monoclonal antibody against involucrin, 3Al (A and B), and a monoclonal antibody against the SV40 T-antigen, PAb416 (C and D), demonstrating that the SVK14 cells in the three-dimensional structures formed as a direct result of coculture express involucrin while maintaining T-antigen expression. (A and B) Phase contrast; (B and D) fluorescence. Bar; 500 urn.

(Fig. 4). Examination of the keratin profile of the cocultures by SDS-polyacrylamide gel electrophoresis and immunoblotting revealed the expression of two new keratins, keratin 5 and keratin 14 (markers of stratifying epithelia [7]), which are absent in pure SVKl4 and 3T3 cultures. Thus immunoblotting with LLOOI, an antibody monospecific to K14 revealed a single band of MW 50 kDa, compatible with K14, while the presence in the cocultures of a new band of MW 58 kDa compatible with K5 was demonstrated using the antibody RCK102, specific for keratins 5 and 8 (Fig. 5).

DISCUSSION We have previously shown that IGF I/insulin treatment of SVK14 results in expression of markers of differentiation (involucrin, keratin 14, and keratin 10) typical of stratifying human keratinocytes at levels detectable by immunofluorescence, while the cells apparently maintain T-antigen expression [27]. Induction of markers of epithelial differentiation in SVK14 in the presence of T-antigen suggests transformation can be compatible with retention of some defferentiation potential and sensitivity to appropriate environmental signals. Here we present

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I 94-

67-

43-

1 A0

II

2 AB

94-

67-

-K14 43-

30-

Fig. 5. Induction of expression of keratins 5 and 14 as a result of coculture of SVK14 with 3T3. (Z) Electrophoretic separation of a keratin extract from pure SVK14 cultures (lane I, 100 up), pure 3T3 cultures (lane 2, 50 ug), and SVK14/3T3 cocultures (lane 3, 50 ug) (Coomassie-stained gel). Appearance of two new bands at 58 and 50 kDa (weak) compatible with the molecular weights for keratins 5 and 14, respectively, is demonstrated in the cocultures (lane 3). The weak band of MW 59 kDa seen in the SVK14 track (lane 1) is not immunoreactive with the antikeratin 5 antibody (see II, IA) and is therefore not keratin 5. The left-hand lane shows the molecular weight protein markers (Pharmacia) phosphorylase B (94 kDa), albumin (67 kDa), ovalbumin (43 kDa), and carbonic anhydrase (30 kDa). (SZZ)Immunoblotting with RCKlO2, a monoclonal antibody against keratins 5 and 8 reveals only that keratin 5 is present in the coculture (II?) but is absent in the pure SVK14 cultures (1 A). Similarly, immunoblotting with LLOOl, a monoclonal antibody against keratin 14, reveals the presence of keratin 14 in the coculture (2B) and its absence in the pure SVK14 culture (2A).

Coculture

of SVK14 cells with Swiss 3T3Jibroblasts

461

data showing formation of organized structures and expression of involucrin and keratins 5 and 14 (markers of differentiating stratifying epithelia) as a direct result of coculture of SVK14 with 3T3 cells in submerged cultures grown on plastic dishes. Out of the 10 coculture partners examined, only 3T3 gave rise to the structures described above on coculture with SVK14. The formation of these structures is therefore not a general response to mesenchymally derived cells but rather depends on some particular property of ,3T3 cells. Furthermore, since the effect could not be reproduced when cell-cell contact was reduced (in collagen gels) or conditioned medium used, it is likely to be mediated directly by cell-cell contact or shortrange inter-actions. Superficially, the reassortment of 3T3 and SVK14 cells described here resembles the sorting out of cell types reported by Steinberg for mixed suspensions of embryonic cells [28]. The latter phenomenon is believed to reflect differential adhesion between cell types such that the less cohesive cell population tends to envelope the more cohesive one. Whether a similar mechanism applies here to the nodules formed in the SVK14 and 3T3 cell cocultures remains to be determined. As SVK14 can be induced to express involucrin and keratin 14 by IGF I, it is possible that the keratin and involucrin expression of SVK14 in these cocultures is induced by IGF I-like molecules secreted by 3T3. Although 3T3 require added IGF I (or high insulin levels) for growth in culture in the absence of serum growth factors [29; Brooks et al., manuscript in preparation], suggesting an inability to produce a substitute for IGF I, it is conceivable that the production of such molecules may be induced directly as a result of coculture. However, the secretion of IGF I-like molecules would not account for a sorting out of cells into discrete cell types. Furthermore the nodular structures are not observed on coculturing SVK14 with dermal fibroblasts, the “natural” fibroblastic partner of keratinocytes, despite the fact that dermal fibroblasts are known to be capable of releasing IGF I [30]. Keratinocytes play a major role in skin morphogenesis and are successfully cultured into sheets of cells which can be used as autografts in the treatment of burns and ulcers [31, 321. However, the morphology of cultured keratinocytes differs markedly from a normal epidermis. When grown on plastic substrates submerged in medium, the basal cells are flattened rather than cuboidal, stratification is relatively poor, and keratinization is incomplete. On artificial collagen substrates at a gaseous interface in the presence of vital dermis, the architecture is more normal, with rounded basal cells and a thicker stratum corneum [33-361. Bell et al. [37] have reported what they describe as “crypts” or “follicular-like” structures when keratinocytes are cultured on a collagen gel impregnated with dermal tibroblasts at a gaseous interface for a long period of time. However, development or regeneration of recognizable, fully organized hair follicles does not occur in vitro. Cutaneous appendages such as hair follicles and glands are derived from invagination of epidermal cells during fetal development, although they differ completely in structure and function from the mature interfollicular epidermis. During the first few weeks of development, the embryo is covered by a transient

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layer of cells, the periderm, which is pushed outward by the developing epidermis as it begins to stratify, until it is eventually lost. Peridermal cells display ultrastructural features of simple epithelia and express keratins 8, 18, and 19, characteristic of simple epithelia [ 131 as well as keratins 4 and 13, characteristic of noncornifying stratifying epithelia [38, 391. SVK14 cells derived by transformation of normal human keratinocytes with SV40 virus [4] also exhibit characteristics of simple epithelia in that they express keratins 7, 8, 18, and 19 and show monolayer growth. They thus resemble the fetal periderm more closely than the stratifying epithelia from which they were derived. The organization of SVK14 and 3T3 cells in the nodular structures seen here is somewhat reminiscent of condensation of the anlagen of dermal papilla underneath the epidermis during development of primary hair germs in the early embryo [40, 81. Interactions between epithelium and mesenchyme are essential to the normal development and histogenesis of epithelia during embryogenesis [8] and important in maintenance of normal epithelial structure and function. In this context the induction of keratins 14 and 5 and involucrin expression in SVK14 by 3T3 is intriguing. Formation of three-dimensional structures, together with the induction of expression of markers of stratifying epithelia in SVK14 as a direct result of coculture, raises the possibility that this model is analogous to the epithelial mesenchymal interactions seen during epithelial development in the early embryo, where epithelial cells under some as yet unknown external signal(s), switch from a simple to a stratifying epithelial cell type [8]. The authors thank Dr. T. Rupniak, Dr. B. Lane, Dr. I. Leigh, and Dr. L. Crawford for gifts of antibodies, Dr. J. Taylor-Papadimitriou, Dr. R. Littleton, Dr. E. Gherardi, Dr. L. Buluwela, Dr. R. Dover, Dr. G. Jones, Ms. S. Murphy, Miss C. Linge, and Mr. A. Wallace for gifts of cells, Dr. N. Holder for electrophoresis apparatus and Dr. L. Buluwela for critical discussion. We also thank Cancer Research Campaign and Unilever for supporting this work.

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