Epidermal Tissue Regeneration

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Introduction

Regeneration and maintenance of epithelial tissue homeostasisrequires a complex interplay with neighbouring cells andextracellular matrix of the adjacent stroma. This is wellunderstood in skin based on data from wound healing,transplantation and cell culture studies, which stronglyindicate that epidermal tissue regeneration is regulated by anetwork of cytokines and growth factors controlling functionalbehaviour of keratinocytes and dermal fibroblasts (Lugerand Schwarz, 1990; Fusenig, 1994; Mackenzie et al., 1993;Werner et al., 1994; Breitkreutz et al., 1997; Boukamp etal., 1990; Stark et al., 2001). Comparably, keratinocyte

proliferation in conventional cell culture is strongly enhancedby stromal cell interactions, a process routinely used in2D feeder layer co-cultures with postmitotic fibroblasts(Rheinwald and Green, 1975; Limat et al., 1989).Accumulating data strongly indicate that this stromal cellsupport of epithelial cell proliferation is, for the most part,mediated by diffusible factors (Waelti et al., 1992; Smolaet al., 1993; Maas-Szabowski et al., 1999). Importantly, thesupport of keratinocyte proliferation is not based on theirpassive utilization of growth factors constitutively producedby fibroblasts but represents an active interplay between bothcell types in a double paracrine mechanism. Hereby,keratinocytes actively control the production of their growth

factors in fibroblasts by release of interleukin-1 (IL-1) and, which induce the enhanced expression of growth factors instromal cells such as keratinocyte growth factor/fibroblastgrowth factor 7 (KGF/FGF-7) and granulocyte-macrophagecolony-stimulating factor (GM-CSF). Both factors stronglystimulate keratinocyte proliferation in vitro and areupregulated in wound healing (Maas-Szabowski et al., 2001;Werner, 1998; Mann et al., 2001; Groves and Schmidt-Lucke,2000).

These regulatory mechanisms are also operative in the morein-vivo-like organotypic co-culture system representing an invitro skin equivalent model consisting of a differentiatedstratified epithelium on top of a dermal equivalent formed byfibroblasts in a collagen type I gel (for reviews, see Fusenig,1994; Maas-Szabowski et al., 2002; Bell et al., 1981). Thus, invitro regeneration of a structured epidermis is regulated by thesame double paracrine mechanisms involving keratinocyte-released IL-1 as inducer, and KGF/FGF-7 as well as GM-CSF as fibroblast-produced effector molecules to stimulatekeratinocyte growth and differentiation (Maas-Szabowski etal., 2000; Maas-Szabowski et al., 2001; Szabowski et al.,2000). Obviously, these signaling molecules are only part of amore complex regulatory mechanism of epithelial-stromalinteractions controlling tissue regeneration and homeostasis. Inthe absence of stromal cells both KGF and GM-CSF are unable

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The human keratinocyte cell line HaCaT expressesessentially all epidermal differentiation markers butexhibits deficiencies in tissue organization as surfacetransplants in nude mice and even more so in organotypicco-cultures with fibroblasts. Whereas tissue differentiation

by normal keratinocytes (NEKs) is regulated by stromalinteractions, this mechanism is impaired in HaCaT cells.This regulatory process is initiated by interleukin-1 (IL-1)release in keratinocytes, which induces expression ofkeratinocyte growth factor (KGF/FGF-7) and granulocytemacrophage-colony stimulating factor (GM-CSF) infibroblasts. Production and release of IL-1 is very low and,consequently, expression of the fibroblast-derived growthfactors KGF/FGF-7 and GM-CSF is absent in HaCaT-fibroblast co-cultures. However, addition of KGF and GM-CSF, respectively, is inefficient to improve stratification anddifferentiation by HaCaT cells due to the low expression oftheir cognate receptors. More importantly, expression and

release of the autocrine keratinocyte growth factor TGF-is dramatically decreased in HaCaT cells. Addition of TGF- or EGF stimulated HaCaT cell proliferation but, evenmore effectively, suppressed apoptosis, thus facilitatingthe formation of a regularly stratified epithelium.

Furthermore, TGF- enhanced the expression of thereceptors for KGF and GM-CSF so that addition of thesegrowth factors, or of their inducer IL-1, further improvedepidermal tissue differentiation leading to in vitro skinequivalents comparable with cultures of NEKs. Thus,supplementing TGF- normalized epidermal tissueregeneration by immortal HaCaT keratinocytes and theirinteraction with stromal cells so that regular skinequivalents are produced as standardized in vitro models.

Key words: HaCaT, Keratinocytes, Skin equivalent, Epithelial-mesenchymal interaction, Cytokines

Summary

Epidermal tissue regeneration and stromal interactionin HaCaT cells is initiated by TGF-

Nicole Maas-Szabowski, Anja Strker and Norbert E. Fusenig*

Division of Differentiation and Carcinogenesis (B0600), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg,Germany*Author for correspondence (e-mail: [email protected])

Accepted 11 March 2003Journal of Cell Science 116, 2937-2948 2003 The Company of Biologists Ltddoi:10.1242/jcs.00474

Research Article


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to fully support epidermal reconstitution in organotypic co-cultures (Maas-Szabowski et al., 2001).

The study of the identity and mechanism of action of furtherplayers in this mechanism is often complicated by inter-individual variations of early passage keratinocytes as well asof fibroblasts when comparing cell strains isolated fromdifferent donors (Watt et al., 1987; Boukamp et al., 1990).

Whether these differences are due to variations in the isolationtechnique, to variable contaminations of fibroblasts in thekeratinocyte populations, varying composition of the dermis-derived cell populations or a combination of all threedeterminants, is difficult to discriminate when using culturesof early passaged cells. These variables may also obscur donor-age or body-site related differences in the regenerative capacityof keratinocytes and the supportive behaviour of fibroblasts. Bycontrast, keratinocytes of passages higher than three oftenexhibit reduced regenerative capacities (Gilchrest et al., 1983;Stanulis-Praeger and Gilchrest, 1986).

Thus, for studying in detail the complex basic mechanismsof epidermal-dermal cell interactions, a standardized andreproducible in vitro skin equivalent model would be

advantageous. Obviously, established cell lines withmaintained functional capacities would be the most suitablecandidates to replace freshly isolated cells. Established celllines, however, generally exhibit altered functional properties,although fibroblast cell lines, such as mouse 3T3 orcomparably immortalized embryonic cell lines, seem tofunction similarly to adult primary fibroblasts when combinedwith normal human keratinocytes in organotypic co-culture(Kaur and Carter, 1992; Choi and Fuchs, 1994; Szabowski etal., 2000; Maas-Szabowski et al., 2001). Although thesestudies have demonstrated functioning of the epithelial-fibroblast interactions across species barriers, excessive growthof established fibroblasts in the collagen gel and/or otherfunctional variations may lead to enhanced and atypical growth

behaviour of keratinocytes (Kaur and Carter, 1992; Choi andFuchs, 1994). Varying proliferative activities of fibroblasts inthe collagen gel with the consequence of variable stromal cellnumbers are another disturbing problem. However, this could,for the most part, be eliminated by using permanentlypostmitotic cells following -irradiation comparable withfeeder layer fibroblasts. These irradiated fibroblasts maintainedfairly constant cell numbers and functioned as well asproliferating cells to support keratinocyte growth anddifferentiation into 2D and 3D co-cultures (Maas-Szabowskiand Fusenig, 1996; Maas-Szabowski et al., 2000).

However, the use of human keratinocyte cell lines inorganotypic cultures was less successful. They have beenestablished mostly by immortalization with viral oncogenesand exhibited minor or major variations in their differentiationcapacity (Blanton et al., 1991; Durst et al., 1989; Oda et al.,1996; Tsunenanga et al., 1994; Lechner and Laimins, 1991).Otherwise, spontaneously immortalized human keratinocytelines generally have maintained a rather high degree ofdifferentiation potential (Boukamp et al., 1988; Baden et al.,1987; Allen-Hoffmann et al., 2000; Rice et al., 1993). This isbest demonstrated with the HaCaT cell line, a non-tumorigenickeratinocyte population derived from adult trunk skinexhibiting a rather normal differentiation capacity despitemultiple chromosomal alterations (Boukamp et al., 1988;Boukamp et al., 1997; Breitkreutz et al., 1997; Breitkreutz et

al., 1998; Ryle et al., 1989). However, organotypic co-culturesof HaCaT cells with skin fibroblasts exhibit some stratificationbut usually lack typical criteria of an ordered structure andregular keratinization (Haake and Polakowska, 1993; Syrjnenet al., 1996; Steinstrsser et al., 1997; Boelsma et al., 1999).This deficiency, however, was not due to the permanent loss ofessential differentiation functions. We have recently

demonstrated that HaCaT cells under optimal environmentalconditions (i.e. in surface transplants on nude mice) were ableto reform a regularly structured differentiated epidermis,although with some delay and minor deficiencies comparedwith normal keratinocytes (Breitkreutz et al., 1997; Breitkreutzet al., 1998). Furthermore, we documented that improvedorganotypic co-culture conditions with increased numbers ofsupporting fibroblasts in the collagen gel, significantlyenhanced stratification and differentiation of HaCaT epithelia(Schoop et al., 1999). Nevertheless, growth and differentiationof the squamous epithelia was delayed and incomplete,suggesting deficient signal transduction.

Here, we demonstrate that HaCaT cells exhibit distinctfunctional deficiencies that substantially reduce their

interaction with stromal cells as well as their response tofibroblast-produced growth factors. The expression and releaseof the inducer molecule IL-1 is very low in HaCaT cells, withthe consequence of a minimal induction of KGF and GM-CSFin fibroblasts. More importantly, expression of the receptors forboth growth factors is strongly decreased on HaCaT cells sothat signal transduction is significantly impaired. Finally, thelevel of the autocrine acting keratinocyte growth factor TGF- is drastically reduced in HaCaT cells. By addition of TGF-, deficiencies in growth and differentiation of HaCaTorganotypic cultures can be completely rescued. This ismediated by the direct stimulatory effect of TGF- on HaCaTcell proliferation and survival, and, moreover, by the enhancedexpression of IL-1 and the receptors of KGF and GM-CSF

resulting in normalized interaction with stromal cells. Thus,following supplementation of one growth factor (TGF-) skinequivalents with rather normal epidermal structures could bereproducibly obtained to serve as a standardized skinequivalent model system.

Materials and MethodsCell culture

Normal human skin keratinocytes (NEKs) and dermal fibroblasts(HDFs) were derived from adult skin obtained from surgery, aspreviously described (Smola et al., 1993; Stark et al., 1999). HaCaTcells (passages 35-40) (Boukamp et al., 1988) and fibroblasts(passages 5 to 9) were grown in DMEM (Bio Whittaker, Serva,

Heidelberg, Germany) supplemented with 10% FCS. For preparingfibroblast feeder cells (HDFis), trypsinized fibroblast suspensions(0.05% trypsin/0.025% EDTA, v/v) were -irradiated with 70 Gray.NEKs were expanded on X-irradiated feeder cells (5000 cells/cm2) inFAD medium (DMEM:Hams F12, 3:1) with 100 U/ml penicillin, 50g/ml streptomycin and supplemented with 5% FCS, 5 g/ml insulin,1 ng/ml recombinant human EGF, 1010 M cholera toxin, 104 Madenine and 0.4 g/ml hydrocortisone (Sigma, Deisenhofen,Germany), as described (Smola et al., 1993); for RNA expressionstudies EGF was omitted. HaCaT cells were plated at 6000 cells/cm2

in DMEM with 10% FCS as monocultures; in 2D co-cultures 5000cells/cm2 were plated on feeder cells (5000/cm2) in DMEM with 10%FCS. NEKs were grown at 7000 cells/cm2 on feeder cells (5000/cm2)in FAD medium without EGF. To determine the proliferation response

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to growth factors, HaCaT monocultures were grown for 24 hours inthe respective serum-containing medium. Thereafter, medium wasreplaced by serum-free DMEM additionally containing one of thefollowing additives: 10 ng/ml KGF (BTS, St Leon, Germany), 2 ng/mlEGF (Sigma, Deisenhofen, Germany), 2 ng/ml TGF-, 100 ng/mlGM-CSF, 5 ng/ml IL-1 (all R&D Systems, Wiesbaden, Germany),and 5% FCS, respectively, as control. For cell number basedcalculations of cytokine concentration in supernatants, cells weretrypsinized and counted at day 3, 5 and 7. Data are given as means standard deviation of duplicate measurements performed in 2-3independent experiments.

Isolation of RNA, reverse transcription and PCR

Mono- and co-cultures of HDFs, NEKs and HaCaT cells were grownin the respective medium to subconfluence. Cells were lysed inguanidinium-isothiocyanate solution and total RNA was extracted(Chomczynski and Sacchi, 1987). Furthermore, RNA was extractedfrom subconfluent HaCaT monolayer cultures, as controls or treatedfor 10 hours and 24 hours with 2 ng/ml TGF- (in serum-freemedium). RT-PCR was performed according to an established methodwith some modifications, as reported (Maas-Szabowski and Fusenig,1996). In brief, cDNA was transcribed at 42C for 60 minutes in 50

l final volume containing 5 g total RNA, 5 l 10 PCR-buffer, 10l MgCl2 (25 mM), 3 l of each dNTP (10 mM), 0.5 l RNasin (0.5U/l), 0.5 l reverse transcriptase (50 U/l), 1 l oligo dT15 (50 M)and 1 l random hexamers (50 M) (GeneAmp-PCR-Kit, PerkinElmer, Weiterstadt, Germany). Four microliters of first-strand cDNAwere added to the PCR-mixture up to a volume of 50 l following theproduct description. The mixture was amplified in a thermal cycler(Biometra, Gttingen, Germany) at the indicated annealingtemperature performing 24-30 cycles, such that the product yield wasin the exponential range. Twenty-mer primers from separate exonsflanking the regions of the following locations on cDNA were chosen:GAPDH 69-308 (62C); IL-1 84-504 (62C); IL-1 (174-564;64C); TGF- (240-448; 64C); EGF (3800-4393; 63C); KGF (208-474; 60C); KGF-R (870-1022; 64C); GM-CSF (66-388; 60C);GM-CSF-R (83-524; 56.5C); and GM-CSF-R (981-1318;

60.5C). PCR fragments were separated on 1.5% agarose gels(Seakem; Biozym, Oldendorf, Germany), ethidiumbromide-stained,and identified by their running positions on the gel and by restrictionmapping with two different enzymes. The mRNA amount of thehouse-keeping gene GAPDH was used as internal standard.

Protein determination by ELISA

Selected cytokines were quantified by enzyme-linked immuno-sorbent assays (ELISA) in aliquots of culture medium of feeder-layerco-cultures. Medium was collected at day 3, 5 and 7, always 48 hoursafter medium change. ELISA kits for IL-1 were purchased fromEndogen (Eching, Germany), for TGF- from Calbiochem (BadSoden, Germany), and for EGF, KGF and GM-CSF from R&DSystems (Wiesbaden, Germany). Protein values were calculted as

pg/105

cells and mean values standard deviation of data derivedfrom duplicate measurements from 2-3 independent experiments aregiven.

Organotypic co-cultures (OTCs)

NEKs (passage 2) and HaCaT cells (passage 35-40) were seeded(1106/insert) onto collagen type I gels (rat tail tendon) containing3105/ml postmitotic fibroblasts (HDFi) cast in cell culture filterinserts (pore size 3.0 m, polycarbonat; Falcon, Becton Dickinson,Heidelberg, Germany) as described in detail previously (Stark et al.,1999). In HaCaT cultures medium was replaced after 24 hours byDME medium [10% FCS and 50 g/ml L-ascorbic acid (Sigma)] andcultures were raised to the air-liquid interface by lowering the upper

medium level to the lower part of the collagen gel. Medium wasreplaced every 2 days with or without any of the following additives:10 ng/ml KGF (BTS), 2 ng/ml EGF (Sigma), 2 ng/ml TGF-, 100ng/ml GM-CSF, 5 ng/ml IL-1 (all R&D Systems) and 1 g/ml mousemonoclonal anti-human EGF-receptor antibody (C225, Imclone, NewYork, NY). NEK OTCs were cultivated in rFAD medium (FAD with10% FCS, 5 g/ml insulin, 0.4 g/ml hydrocortisone, 50 g/ml L-ascorbic acid) as previously described (Stark et al., 1999). Beforefixation organotypic cultures were incubated for 16 hours with 63 Mbromodeoxyuridine (BrdU) to label proliferating cells. Cultures wereeither fixed according to a standardized protocol in 3.7% phosphate-buffered formaldehyde for routine histology and staining inhematoxylin and eosin (H and E), or embedded in Tissue Tec OCTCompound (Medim, Gieen, Germany) and frozen in liquid nitrogenvapor for cryosectioning.

Indirect immunofluorescence microscopy

Cryosections mounted on glass slides (Histobond, Medim, Gieen,Germany) were fixed for 5 minutes in 80% methanol at 4C followedby 2 minutes in acetone at 20C, rehydrated in PBS, and blocked for15 minutes in PBS with 1% BSA. First, antibodies were incubatedovernight at 4C in a moist chamber; after three washes in PBS the

sections were incubated for 1 hour at room temperature with species-specific, fluorochrome-conjugated secondary antibodies (Dianova,Hamburg, Germany), as well as 0.5 g/ml bisbenzimide (Hoechst No.33258) DNA dye for nuclear counter staining. After three final washeswith PBS, specimens were mounted with Mowiol (Medim, Gieen,Germany) under a coverslip and examined and photographed using aLeica microscope (Leitz DMRBE, Bensheim, Germany) equippedwith epifluorescence optics. To visualize proliferating cells, amonoclonal mouse anti-BrdU antibody (Progen, Heidelberg,Germany) was used and proliferation was quantitated by counting theratio of labelled to total nuclei within the basal keratinocyte layer ofthe epithelium.

Apoptotic cells were detected by TUNEL staining. Paraffin sectionsmounted on glass slides (Histobond, Medim) were dewaxed in xyloland rehydrated in a graded series of ethanol. Sections were incubated

with proteinase K as recommended in the protocol (In situ cell deathdetection kit TMR red; Roche, Mannheim, Germany). TUNELreaction mixture was applied for 45 minutes at 37C. Nuclei werecounterstained for 15 minutes at RT by 0.5 g/ml bisbenzimide(Hoechst No. 33258) DNA dye. Sections were washed twice inPBS and mounted with mowiol. Specimens were examined andphotographed using a microscope equipped with epifluorescenceoptics. The percentage of TUNEL-positive nuclei to the total cellnumber within all layers of the epithelium was calculated.

Differentiated keratinocytes were characterized by labeling withspecific antibodies to human transglutaminase and filaggrin (bothmouse monoclonal, Cell Systems, St Katharinen, Germany), EGF-receptor (mAb425, mouse monoclonal, kind gift of U. Rodeck,Philadelphia, PA) as well as loricrin (rabbit polyclonal, kind gift ofD. Hohl, Lausanne, Switzerland) and visualized by appropriate

fluorochrome-coupled secondary antibodies.

Results

Delayed tissue regeneration of HaCaT cells

In organotypic co-cultures with dermal fibroblasts embeddedin a collagen type I gel, HaCaT keratinocytes show drasticallyreduced growth and differentiation potential compared withepidermal keratinocytes (NEKs) (Fig. 1). This deficiency ispartially compensated by augmenting fibroblast numbers in thecollagen matrix (Schoop et al., 1999) although, withproliferating cells, their actual numbers at distinct time pointsare difficult to control (Coulomb et al., 1989). Postmitotic (X-


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irradiated) fibroblasts, commonly used asfeeder cells for keratinocytes (Rheinwaldand Green, 1975), also function inorganotypic co-cultures and maintain astable number for at least 2 weeks (Maas-Szabowski et al., 2000). Thus, thefibroblast number required for optimal

NEK and HaCaT cell growth and tissuerestoration has been more accuratelydetermined as 6105 irradiated cells/mlcollagen gel for HaCaT cell growth and3105 cells for NEKs [data not shown(Schoop et al., 1999; Maas-Szabowski etal., 2000)]. However, epidermal tissueformation by HaCaT cells wassignificantly delayed for 2 weeks and,even after 3 weeks, was incompletecompared with organotypic cultures ofNEKs.

To study the molecular defects ofHaCaT cells in their interaction with

stromal cells, the fibroblast number wasadjusted to be optimal for NEKs butsuboptimal for HaCaT cells. Thus, with2105 irradiated fibroblasts per mlcollagen gel, normal keratinocytesderived from adult skin grow to a typically structuredepidermal tissue within 1 week, followed by increaseddifferentiation and formation of a structured basementmembrane after 2 and 3 weeks [Fig. 1 (see also Stark et al.,1999)]. In contrast, HaCaT cells had formed only a 2-3 layereddisorganized epithelium under the same conditions within 1week, which increased in thickness after 2 weeks, but did notexhibit morphological features of differentiation. Thicknessand differentiation of the HaCaT epithelia increased further

after 3-4 weeks exhibiting a thin parakeratotic cell layer, butHaCaT epithelia were still less keratinized than NEK culturesafter 2 weeks [data not shown (Schoop et al., 1999)].

Lack of IL-1 impedes stromal interaction

This discrepancy in epidermal tissue regeneration of HaCaTcells, compared with that of NEKs, was possibly due to


Fig. 1. Deficient epithelial tissue formation of HaCaT cells in organotypic co-cultures.Epidermal tissue morphology of organotypic co-cultures of normal keratinocytes (NEK) orHaCaT cells grown on collagen type I gels containing 2105/ml postmitotic fibroblaststhroughout a 12-day-culture period (H and E staining; same magnification in all panels; bar,100 m).

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CFig. 2. (A) Schematic outline of the double paracrine regulation ofkeratinocyte growth and differentiation by fibroblast interactions(figure modified from Szabowski et al., 2000). (B) Reduced IL-1expression in HaCaT cultures. Expression levels of IL-1 and IL-1in monocultures of postmitotic (irradiated) fibroblasts (HDFi),keratinocytes (NEK) and HaCaT cells as well as in 2D co-culturesof NEK and HaCaT cells, both with HDFi, and in epithelia oforganotypic cultures of NEK and HaCaT cells (OTC). Steady-statemRNA levels were determined by semiquantitative RT-PCR usingGAPDH expression as internal standard. The RNA expressionprofile is representative of duplicate determinations of threeindependent experiments. (C) Lack of IL-1 production in HaCaTcells. Concentrations of IL-1 in supernatants of 3-, 5- and 7-day-old co-cultures with irradiated fibroblasts of NEK and HaCaT cellswere determined in aliquots of 2-day-conditioned media by ELISAand calculated as pg/105 cells. Bars represent means s.d. ofduplicate measurements performed in three independentexperiments.


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deficiencies in epithelial-stromal interaction mechanisms [Fig.2A (Maas-Szabowski et al., 2001)]. This double paracrineepithelial-stromal interaction is induced by keratinocyte-released IL-1 and -1 resulting in AP1-mediated enhancedexpression of KGF and GM-CSF, two strong stimulators ofkeratinocyte proliferation (Szabowski et al., 2000). To identifyany possible alterations in this interaction in HaCaT cells

compared with that in NEKs, expression of growth factors wasanalyzed at the RNA- and protein level in mono- and co-cultures. Thus, RNA levels were determined in both 2D and3D cultures with similar expression levels (Maas-Szabowskiet al., 1999; Maas-Szabowski et al., 2000), whereas proteinconcentrations were quantitated in supernatants of 2D cultures(to avoid absorption to the collagen gel).

The expression of the signalling factors IL-1 and IL-1,essential for the double paracrine mechanisms, was drasticallydecreased in HaCaT mono- and co-cultures when comparedwith that in NEKs (Fig. 2B). Comparably, the secretion of IL-1 into the culture supernatants remained extremely low overa 5-day-culture period and rose only slightly thereafter (Fig.2C). Moreover, in contrast to NEKs, HaCaT cells were unable

to store IL-1 intracellularly, since HaCaT cell lysates of 5-day cultures showed only minimal amounts (1.980.88 pg/105

cells) compared with a massive accumulation in NEKs(28124.9 pg/105 cells). Similar protein values were measuredin the culture supernatants for IL-1 (data not shown). Asexpected, and due to the lack of both inducer cytokines, RNAexpression (data not shown) and protein secretion of KGF andGM-CSF by co-cultured fibroblasts were strongly reducedthroughout the first week in culture in contrast to co-cultureswith NEK [Fig. 3A (Maas-Szabowski et al., 2000; Szabowskiet al., 2000)].

To compensate this deficiency of HaCaT cells in inducingtheir own growth factors in co-cultured fibroblasts, the additionof these factors or their inducer should restore their capacity to

form differentiated epithelia. However, neither the continuousaddition of IL-1, KGF, GM-CSF or their combination (notshown) significantly induced HaCaT cell growth or tissueformation in organotypic co-cultures (Fig. 3B). This lack ofresponse was most probably due to defects in signal receptionand transduction in HaCaT cells, as shown by the reducedexpression of the receptors for KGF and GM-CSF (Fig. 4A),

and paralleled at the protein level by immunofluorescentstaining with specific antibodies (data not shown). Theexpression of both receptors increased with culture timeand cell density: a possible explanation for the delayedstratification of organotypic HaCaT epithelia at 3 and 4 weeks(Schoop et al., 1999).

Abrogated TGF- expression impairs HaCaT cellproliferation and survial

More importantly, the expression of the transforming growthfactor (TGF-), a known autocrine acting keratinocytegrowth factor, was strongly reduced in HaCaT epithelia incontrast to that in NEKs (Fig. 4A). The reduced TGF-

production was more dramatically evident at the protein levelin supernatants of HaCaT mono- or co-cultures (Fig. 4B). Thiswas not due to a lack of cleavage of this factor from itsmembrane-bound form (Bosenberg et al., 1992), since HaCaTcell lysates showed similar low levels in the ELISA assay (datanot shown).

Supplementation of TGF- rapidly and efficiently restoredthe capacity of HaCaT cells to form structured epithelia inorganotypic co-culture (Fig. 5A). The same normalizing effectwas seen with EGF (data not shown), sharing the same receptorwith TGF-, the EGF receptor, which is constitutivelyexpressed in HaCaT cells (Game et al., 1992; Stoll et al., 1998).The essential role of the EGF-receptor-mediated signaltransduction for epidermal tissue formation was also

demonstrated in organotypic cultures of TGF-/EGF-substituted HaCaT cells as well as those of NEKs, by receptorblocking studies. Addition of the EGFR-blocking monoclonalantibody C225 drastically inhibited epithelial tissue formationby both cell types (Fig. 5A). EGF itself, although expressed atthe RNA level in NEK, HaCaT and fibroblast culture (Fig. 5B),was not detected in the culture supernatants of any mono- orco-cultures (data not shown). Thus, TGF- is the effectivegrowth factor in skin cells acting in an autocrine manner on

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Fig. 3. (A) Deficient production of KGF and GM-CSF infibroblasts co-cultured with HaCaT cells. Kinetics of KGF andGM-CSF production, measured in culture supernatants of co-

cultures with NEK and HaCaT cells, of 2-day-conditioned media,by ELISA and calculated as pg/105 cells. Bars represent means s.d. of duplicate measurements performed in three independentexperiments. (B) Ineffectiveness of KGF, GM-CSF or IL-1 on

epidermal tissue restoration by HaCaT cellsin organotypic co-cultures with postmitoticfibroblasts. Organotypic co-cultures ofHaCaT cells with postmitotic fibroblasts(3105/ml) in collagen type-I gel were grownfor 10 days in DMEM with 10% FCS(Control) and after addition of IL-1 (5ng/ml), KGF (10 ng/ml) or GM-CSF (100ng/ml); medium was changed every secondday (H and E staining; bar, 100 m).


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keratinocytes (Schulz et al., 1991; Carpenter, 1993; Comptonet al., 1995).

TGF- enhances HaCaT cell proliferation in mono-culturewith a sixfold higher cell number after 5-day treatmentin serum-free medium and in 2D co-cultures (data not

shown). Furthermore, TGF- treatment strongly stimulatesstratification in HaCaT mono-cultures on fibroblast-freecollagen gels, indicating its major effect as a direct actingfactor on HaCaT cells. Moreover, TGF- induces theexpression of IL-1 and, more importantly, the receptors ofKGF and GM-CSF (Fig. 5B). The elevated RNA levels arenot due to stabilization of mRNA, because the increase is

abrogated by actinomycin D treatment. The upregulation ofreceptor expression is most probably indirect, because itwas blocked by the addition of cycloheximide (data notshown). Although there is a basal expression of bothreceptors at the RNA level, the immunofluorescence dataexhibit only background staining in control co-cultures (datanot shown).

Based on the observed stimulation of HaCaT cellproliferation, we hypothesized that the effect of TGF- ontissue reconstruction was mainly due to this effect. However,in organotypic co-cultures, TGF- enhanced HaCaT cellproliferation only initially (at day 3), whereas at later culturetimepoints BrdU-labelled cells were even more frequent in thincontrol epithelia than in TGF--treated multilayered cultures

(Fig. 6A).The explanation for this discrepancy was found when

cultures were analyzed for the presence of apoptotic cells (Fig.6). During the first week, the rate of TUNEL-positive cellswas four- to five-times higher in control cultures comparedwith that in TGF--treated cultures (Fig. 6C). Whendifferentiation began (day 9), the rate of apoptotic cellsincreased in TGF--treated cultures and after 12 days washigher than in controls. Whereas TUNEL-positive nuclei wereseen throughout the whole epithelia in control cultures, inTGF--treated cultures they were exclusively localized inthe upper flattened cell layers representing terminallydifferentiating cells (Fig. 6B). Nearly all nuclei of theparakeratotic superficial cell layers stained positively in the

TUNEL assay, whereas the nuclei of the cuboidal cells of the4 to 6 lower cell layers were unstained.


Fig. 4. Decreasedcytokine receptor andTGF- expression inHaCaT cells.(A) Expression of KGFand GM-CSF receptorsas well as of TGF-were determined inorganotypic fibroblast-co-cultures ofkeratinocytes (NEK)and HaCaT cells bysemiquantitative RT-PCR using GAPDHexpression as internalstandard. The RNAexpression profile isrepresentative ofduplicate determinationsof three independentexperiments. (B) TGF-concentrations in culturesupernatants of 3-7-day-

old co-cultures of NEKand HaCaT cells withpostmitotic fibroblastswere determined inaliquots of 2-day-conditioned media byELISA and calculatedas pg/105cells. Barsrepresent means s.d. of duplicate measurements performed in threeindependent experiments.

Fig. 5. EGF-receptor signalling is essential fororganotypic growth and differentiation of HaCaTcells. (A) Organotypic co-cultures of HaCaT cellswith postmitotic fibroblast (3105/ml) in collagentype-1 gel were grown for 10 days in DMEM as acontrol (HaCaT), and after addition (every secondday) of TGF- (2 ng/ml) or EGF (2 ng/ml) as wellas both factors (1 ng each), together with the EGF-receptor blocking antibody C225 (+Ab). Forcomparison, organotypic co-cultures of normalepidermal keratinocytes were grown for 10 dayson collagen gels with 3105/ml postmitoticfibroblasts (NEK) and with EGF-receptor blockingantibodies (+Ab). (H and E staining; bar, 100 m).(B) Cytokine and receptor expression in HaCaTepithelia of organotypic co-cultures stimulated for16 hours with TGF- (2 ng/ml) and EGF,respectively. Expression levels of IL-1, IL-1,KGF-R, GM-CSF-R, GM-CSF-R, EGF-R andTGF- were determined by semiquantitative RT-PCR using GAPDH expression as an internalstandard. The RNA expression profile isrepresentative for duplicate determinations ofthree independent experiments.

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*

C

Fig. 6. Effects of TGF- on proliferation and apoptosisof HaCaT cells in organotypic co-culture.(A) Proliferation of HaCaT cells in organotypic co-

cultures with postmitotic fibroblasts (3105/ml) grownin DMEM + 10% FCS (control) and after addition ofTGF- (2 ng/ml, every second day) was evaluated bycounting nuclei stained with an anti-BrdU-specificantibody. Bars represent the percentage of BrdU-positive basal keratinocytes identified and counted oncross-sections. Three vision fields per experiment werecounted in three independent experiments.(B) Fragmentation of DNA was detected in organotypicco-cultures grown for 12 days in DMEM with 10%FCS (control) and after addition of TGF- (2 ng/ml,every second day) by TUNEL labeling (red). Nucleiwere counterstained with Hoechst DNA dye (blue). InTGF--treated epithelia at day 12, TUNEL-positivecells are localized in the newly formed stratum

granulosum and corneum. Bar, 100 m. (C) Percentageof HaCaT cells with DNA fragmentation in organotypicco-cultures grown for 12 days in DMEM with 10%FCS (control) and after addition of TGF- (2 ng/ml,every second day) was evaluated on cross-sections bycounting TUNEL-positive and total number of(bisbenzimide-stained) nuclei. Bars representpercentage of TUNEL-positive cells per epithelium(means s.d.). Three vision fields per experiment werecounted in three independent experiments. *TUNEL-positive cells at 9 and 12 days are localized in thenewly formed stratum granulosum and corneum.

Fig. 7. Normalized tissue architecture ofHaCaT organotypic epithelia by TGF-.Morphologic features (H and E staining) ofHaCaT organotypic co-cultures withpostmitotic fibroblasts (3105/ml) in collagentype-I gel grown for 10 days in DMEM with10% FCS (HaCaT) and with TGF- (+ TGF).For comparison, NEK organotypic epitheliawere grown and stained under the sameconditions. By indirect immunofluorescencemicroscopy the EGF receptor (EGF-R, red), theearly epidermal differentiation markertransglutaminase (TG, red), and the late markerloricrin (Lor, red) have been labeled. Nucleiwere counterstained with bisbenzimide (blue).Bar, 100 m.


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TGF- normalizes HaCaT cell growth and differentiation

Replenishment of TGF- in the deficient HaCaT organotypiccultures not only enabled these immortal keratinocytes to reforma structured squamous epithelium but also allowed a rathernormal differentiation (Fig. 7). This is clearly visible in H-and-E-stained sections of 2-week-old cultures, and sections stainedwith antibodies to markers of early [e.g. keratin 1 and 10 (notshown) and transglutaminase] or later stages of keratinization,such as loricrin. The components are typically localized in theupper layers of the stratified epithelium comparable with, thoughstill less regular than, those in cultures of NEKs. Furthermore,the stratum granulosum formation is rather poor, the stratumcorneum is thin and mostly parakeratotic, as indicated by theremnant nuclei in the uppermost flattened cells.

This deficit in differentiation of HaCaT epithelia, however,is repaired to the most part by further supplementing theculture media with KGF, GM-CSF and IL-1, respectively(Fig. 8). This resulted not only in improved morphologicorganization of the reconstituted stratified epithelia but also ina more regular localization of the late differentiation productsin the uppermost cell layers very similar to NEKs. Remarkably,as already seen earlier (Szabowski et al., 2000), GM-CSFnot only exerts effects on keratinocyte proliferation but alsoenhances keratinization, demonstrated by the intensifiedstaining with the late differentiation marker loricrin. This issimilarly seen after addition of IL-1, which induces GM-CSFin fibroblasts (Maas-Szabowski et al., 2001).

Thus, by compensating for the missing expression of a single

autocrine acting growth and survival factor, i.e. TGF-, thedelayed and deficient growth and differentiation capacity of theimmortal HaCaT keratinocytes is restored. TGF- inducesproliferation with enhanced survival as well as the expressionof IL-1 and the receptors of KGF and GM-CSF so thatthe HaCaT cells are able to respond typically to stromalinteractions regulating keratinocyte growth and differentiation.This skin equivalent reconstructed by the immortal HaCaTkeratinocytes represents a better standardized in vitro modelto study further regulatory mechanisms in skin physiologyand may serve as a highly reproducible test system forpharmakotoxicology.

DiscussionThe immortalized human keratinocyte line HaCaT exhibitsdistinct genetic features of cell transformation and representsan early stage in the skin carcinogenesis process (Boukamp etal., 1988; Boukamp et al., 1997; Fusenig and Boukamp, 1998).However, under in vivo conditions, HaCaT cells are non-tumorigenic but still respond typically to environmental controlmechanims by reconstituting a rather normal stratifiedepithelium in surface transplants on nude mice (Breitkreutz etal., 1998). Although epidermal tissue reconstitution in vivo wasdelayed and showed some deficiencies, these data clearlydemonstrated that HaCaT cells, although carrying severe


Fig. 8. Normalization of epidermal differentiation of HaCaT organotypic co-cultures by cytokines. Epidermal tissue morphology of HaCaTorganotypic co-cultures with postmitotic fibroblasts (3105) in collagen type-1 gel grown for 12 days in DMEM with 10% FCS (control) andtreated with 2 ng/ml TGF-. Stimulated cultures were additionally supplemented with KGF (10 ng/ml), GM-CSF (100 ng/ml) or IL-1 (5ng/ml). For comparison, organotypic co-cultures of normal epidermal keratinocytes (NEK) grown for 12 days on collagen gels with 3 105/mlpostmitotic fibroblasts are included (top row: H and E staining). By indirect immunofluorescence the late epidermal differentiation markersfilaggrin (red), and loricrin (red) have been labelled. Nuclei were counterstained with bisbenzimide (blue). Bar, 100 m.


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chromosomal abnormalities, had not permanently lost theirdifferentiation capacities. Owing to the well-maintaineddifferentiation properties, also shown by the expression ofmany different keratins as well as other biochemical markersof differentiation in monolayer cultures (Ryle et al., 1989;Breitkreutz et al., 1997), HaCaT cells have become a paradigmfor skin keratinocyte cultures.

In organotypic co-cultures with fibroblasts, conditions thatinduce normal keratinocytes to form stratified epithelia with allfeatures of a normal epidermis (Smola et al., 1998; Stark et al.,1999), HaCaT cells have been described to be deficient inthis complex function of tissue regeneration (Haake andPolakowska, 1993; Syrjnen et al., 1996; Steinstrsser et al.,1997; Boelsma et al., 1999). We have been able to improvetheir epidermal tissue formation and differentiation in thismodel by intensifying stromal influences by elevating thenumber of supporting fibroblasts (Schoop et al., 1999).However, tissue architecture and keratinization was delayedand stayed deficient, indicating major defects in transductionof the major signals mediating epithelial-stromal interplay.

Here we demonstrate that the deficiency of HaCaT cell

interaction with fibroblasts is based on their very lowconstitutive expression of the prime signalling cytokineinterleukin-1 (IL-1) by which normal epidermal keratinocytes(NEKs) induce expression of their growth factors in fibroblasts(Maas-Szabowski et al., 2000). Moreover, the response ofHaCaT cells to these stromal-cell produced factors KGF andGM-CSF is abolished due to the low level of expression of therespective receptors. More importantly, production oftransforming growth factor (TGF-), known as a potentautocrine acting stimulator of keratinocyte proliferation, isbarely detectable in HaCaT cells. Since its cognate receptor,the EGF receptor, is expressed, addition of TGF- or EGFrapidly re-established the capacity of HaCaT cells to grow toa stratified epithelium and to exhibit typical differentiation

markers of the epidermis. Furthermore, with substitution ofTGF-, the effect of stromal cell-derived factors, in particularof GM-CSF, on keratinocyte differentiation is restored,resulting in improved differentiation in HaCaT organotypiccultures comparable with skin equivalents of NEKs (Maas-Szabowski et al., 2001).

This remarkable effect of one single growth factor on thecomplex mechanism of tissue regeneration was mediated inHaCaT cells by a coordinated function: TGF--upregulated theexpression of IL-1, and receptors for KGF and GM-CSF; itexhibited an autocrine stimulation of cell proliferation; and,last but not least, it suppressed cell apoptosis. As shown earlier(Maas-Szabowski et al., 1999; Maas-Szabowski et al., 2001),both IL-1 and IL-1 are released by keratinocytes in culturefollowing stress comparable with conditions of injury to theskin in vivo (e.g. Kupper and Groves, 1995; Wood et al., 1996).Whereas in skin, IL-1 signalling is mostly understood as partof a proinflammatory cytokine cascade to induce multipleeffects on fibroblasts, endothelial cells and inflammatory cells(for a review, see Dinarello, 1997), we have demonstrated thatit has additional major signaling functions in epidermal tissueregeneration.

In keratinocyte co-cultures with fibroblasts, IL-1 upregulatesof growth factors in stromal cells to stimulate keratinocyteproliferation, such as KGF and GM-CSF (Chedid et al., 1994;Maas-Szabowski and Fusenig, 1996; Maas-Szabowski et al.,

1999; Maas-Szabowski et al., 2000). Thus, deficiency in theconstitutive production of IL-1 in HaCaT cells, as alreadynoticed earlier (Ruhland and de Villiers, 2001), with theconsequence of inefficient induction of KGF and GM-CSF infibroblasts, could to some extent be overcome by increasing thenumber of producer cells in the collagen gel (Schoop et al.,1999). However, the improvement in tissue organization was

best noticed in later culture periods of 2-3 weeks. Upregulationof IL-1 by EGF and TGF-, as already noticed earlier both innormal keratinocytes (Lee et al., 1991) and in HaCaT cells(Philips et al., 1995), was also demonstrated here in HaCaT co-cultures. In addition to the proliferation stimulating effectof KGF and GM-CSF on keratinocytes, GM-CSF alsoenhanced epidermal differentiation in organotypic co-cultures(Szabowski et al., 2000). This differentiation-stimulating effectof GM-CSF was also noticed in HaCaT organotypic co-cultures; however, only after enhanced receptor expressionby TGF-. The same tendency to a morphologically betterdifferentiated HaCaT epithelium was noticed after addition ofIL-1, the inducer of both KGF and GM-CSF in the co-culturedfibroblasts.

Most astonishingly, however, was the crucial role of theEGF receptor and its ligands TGF- and EGF in regulatingepidermal tissue regeneration by HaCaT cells. A very lowrelease and expression of TGF- by HaCaT clones, comparedwith ras-transfected tumorigenic HaCaT cells, had alreadybeen observed earlier (Game et al., 1992), although this facthad not been associated with their reduced tissue regeneration.Here we clearly demonstrate that the low production rate ofTGF- is due to reduced RNA expression and not a failure ofrelease of the active compound from its membrane-boundprecursor. In transplants in vivo, the deficiency of HaCaT cellsin TGF- expression may be compensated by EGF providedby the circulation (Derynck, 1988). As shown earlier, both highand low affinity EGF receptors are functional on HaCaT cells

(Game et al., 1992) and this has been confirmed recently(Kaufmann and Thiel, 2002). Thus, EGF and TGF- stimulateHaCaT cell proliferation and migration similar to that observedin normal keratinocytes in culture (Pittelkow et al., 1989; Ju etal., 1993), whereas EGF receptor inhibition induces growtharrest (Peus et al., 1997) and consequently blocks stratificationin skin equivalents.

Interestingly, in organotypic cultures of HaCaT cells, thestimulating effect of TGF- on cell proliferation wasprominent only at early culture time points, whereas at laterstages the rate of DNA synthesis was comparable withuntreated control cultures. By contrast, the percentage ofapoptotic cells in the non-differentiated epidermal layers wasdrastically decreased in TGF--treated cultures from thebeginning. This indicated that the enhancement of survival ofHaCaT cells was a major function of TGF- in the developingepidermal tissue and, consequently, a major contribution tothe increased number of cell layers. With the beginning ofkeratinization in the TGF--treated HaCaT epithelia, thenumber of TUNEL-positive cells also increased here; however,apoptotic cells were located in the upper parakeratotic celllayers. As shown earlier (Schoop et al., 1999), the remnantnuclei of the incompletely differentiated cells stain positivewith the TUNEL reaction owing to incomplete DNAdegradation (Bernerd and Asselineau, 1997).

An important role of EGF receptor signalling for cell


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survival in epithelial cells has been postulated earlier. EGFRactivation in keratinocytes increases the expression of bcl-XL,a member of the anti-apoptotic bcl-2 family of proteins, andhuman keratinocytes exhibit enhanced apoptosis when EGFR-signalling is inhibited (Rodeck et al., 1997a; Rodeck et al.,1997b; Stoll et al., 1998; Sibilia et al., 2000). Similarly,blockade of EGFR decreased bcl-XL expression and enhanced

apoptosis in HaCaT cells following UV-B irradiation (Jostet al., 2001). Thus, EGFR-signalling is important for bothkeratinocyte proliferation and survival and the latter functionseems to play a major role in organotypic cultures when cellshave to detach from their specific extracellular matrix, thebasement membrane, and migrate upwards to form amultilayered tissue.

On the other hand, stable transfection and overexpression ofbcl-2 into HaCaT cells reduced their apoptotic rate inorganotypic cultures but did not show noticeable effects ontheir tissue organization and epidermal differentiation features(Delehedde et al., 2001). Thus, the combined effect of TGF-on cell proliferation and survival is required for regularstratification and differentiation of HaCaT cells.

Furthermore, a major component of the TGF- effect onHaCaT cells is the enhanced expression of IL-1 and of thereceptors for KGF and GM-CSF, rendering HaCaT cellsreactive and responsive for stromal growth regulatory signals.In particular, the induction of the KGF receptor seems to be anessential step to gaining sustained keratinocyte proliferationboth in wound healing in vivo as well as in cell culture (for areview, see Werner, 1998; Marchese et al., 1997; Werner et al.,1992). It had been noticed that expression of the KGF receptoris deficient in sparse HaCaT cultures but upregulated uponconfluence and the onset of differentiation (Capone et al., 2000).Moreover, the induction of KGFR in keratinocyte culturesgrown at high calcium concentration has been interpreted in thatits signalling plays a possible role in keratinization control

(Marchese et al., 1997). We have shown recently, however, thataddition of KGF effects mainly proliferation in organotypiccultures of normal keratinocytes co-cultured with AP-1-defective fibroblasts, whereas differentiation was not affected(Szabowski et al., 2000). Keratinization and formation ofspecific differentiated strata (i.e. stratum granulosum) wasenhanced by GM-CSF and a regular tissue structure requiredthe combined action of both growth factors.

Clearly, the complex process of epidermal tissueregeneration, differentiation and homeostasis is unlikely to beregulated only by the four factors identified so far, i.e. TGF-,IL-1, KGF and GM-CSF. Further regulating factors have to beidentified and their molecular mechanisms of action elucidated.For these investigations, in-vivo-like but reproducible andsimple-to-handle skin equivalent in vitro models are required.Owing to the rather faithful mimicry of the basic functionsof epidermal proliferation, differentiation and tissuereorganization by the immortal HaCaT cells in TGF--supplemented organotypic co-culture with fibroblasts, thisreproducible skin equivalent represents a biologically relevantmodel for such studies. Furthermore, as an immortalizedcell line, HaCaT cells can be genetically modified byoverexpression and/or blockade of specific genes so that theirconsequences can be studied in a tissue context. Finally, withfurther improvement of the quality of structural organization,differentiation and barrier functions of the stratified epithelia

formed by HaCaT cells, such skin equivalents may becomehighly standardized in vitro tissue models for routine testing inpharmacology and toxicology.

Critical discussion and helpful comments of Hans-Jrgen Stark,Dirk Breitkreutz, Axel Szabowski and Petra Boukamp, as well as thetechnical assistance of Iris Martin, Eva Goedecke, Silke Haid andAngelika Krischke, are gratefully acknowledged. We thank DanielHohl (Lausanne) for kindly providing the loricrin-specific antibody,Peter Bohlen (ImClone, New York, NY) for the gift of the EGFRblocking antibody C225 and Ulrich Rodeck (Philadelphia, PA) for theEGF receptor antibody for immunostaining (mAB 425). Work wassupported by grants from the Deutsche ForschungsgemeinschaftDFG-Schwerpunkt Epitheliale Differenzierung (Fu 94-5), the Israel-DKFZ Cooperation Program by the German Ministry of Research (Ca94) and Beiersdorf AG, Hamburg.

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Epidermal Tissue Regeneration

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