Division of Differentiation and Carcinogenesis (B0600), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
* Author for correspondence (e-mail: n.fusenig{at}dkfz.de)
Accepted 11 March 2003
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Summary |
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Key words: HaCaT, Keratinocytes, Skin equivalent, Epithelial-mesenchymal interaction, Cytokines
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Introduction |
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These regulatory mechanisms are also operative in the more in-vivo-like
organotypic co-culture system representing an in vitro skin equivalent model
consisting of a differentiated stratified epithelium on top of a dermal
equivalent formed by fibroblasts in a collagen type I gel (for reviews, see
Fusenig, 1994;
Maas-Szabowski et al., 2002
;
Bell et al., 1981
). Thus, in
vitro regeneration of a structured epidermis is regulated by the same double
paracrine mechanisms involving keratinocyte-released IL-1 as inducer, and
KGF/FGF-7 as well as GMCSF as fibroblast-produced effector molecules to
stimulate keratinocyte growth and differentiation
(Maas-Szabowski et al., 2000
;
Maas-Szabowski et al., 2001
;
Szabowski et al., 2000
).
Obviously, these signaling molecules are only part of a more complex
regulatory mechanism of epithelial-stromal interactions controlling tissue
regeneration and homeostasis. In the absence of stromal cells both KGF and
GM-CSF are unable to fully support epidermal reconstitution in organotypic
co-cultures (Maas-Szabowski et al.,
2001
).
The study of the identity and mechanism of action of further players in
this mechanism is often complicated by inter-individual variations of early
passage keratinocytes as well as of fibroblasts when comparing cell strains
isolated from different donors (Watt et
al., 1987; Boukamp et al.,
1990
). Whether these differences are due to variations in the
isolation technique, to variable contaminations of fibroblasts in the
keratinocyte populations, varying composition of the dermis-derived cell
populations or a combination of all three determinants, is difficult to
discriminate when using cultures of early passaged cells. These variables may
also obscur donor-age or body-site related differences in the regenerative
capacity of keratinocytes and the supportive behaviour of fibroblasts. By
contrast, keratinocytes of passages higher than three often exhibit reduced
regenerative capacities (Gilchrest et al.,
1983
; Stanulis-Praeger and Gilchrest, 1986).
Thus, for studying in detail the complex basic mechanisms of
epidermal-dermal cell interactions, a standardized and reproducible in vitro
skin equivalent model would be advantageous. Obviously, established cell lines
with maintained functional capacities would be the most suitable candidates to
replace freshly isolated cells. Established cell lines, however, generally
exhibit altered functional properties, although fibroblast cell lines, such as
mouse 3T3 or comparably immortalized embryonic cell lines, seem to function
similarly to adult primary fibroblasts when combined with normal human
keratinocytes in organotypic co-culture
(Kaur and Carter, 1992;
Choi and Fuchs, 1994
;
Szabowski et al., 2000
;
Maas-Szabowski et al., 2001
).
Although these studies have demonstrated functioning of the
epithelial-fibroblast interactions across species barriers, excessive growth
of established fibroblasts in the collagen gel and/or other functional
variations may lead to enhanced and atypical growth behaviour of keratinocytes
(Kaur and Carter, 1992
;
Choi and Fuchs, 1994
). Varying
proliferative activities of fibroblasts in the collagen gel with the
consequence of variable stromal cell numbers are another disturbing problem.
However, this could, for the most part, be eliminated by using permanently
postmitotic cells following
-irradiation comparable with feeder layer
fibroblasts. These irradiated fibroblasts maintained fairly constant cell
numbers and functioned as well as proliferating cells to support keratinocyte
growth and differentiation into 2D and 3D co-cultures
(Maas-Szabowski and Fusenig,
1996
; Maas-Szabowski et al.,
2000
).
However, the use of human keratinocyte cell lines in organotypic cultures
was less successful. They have been established mostly by immortalization with
viral oncogenes and exhibited minor or major variations in their
differentiation capacity (Blanton et al.,
1991; Durst et al.,
1989
; Oda et al.,
1996
; Tsunenanga et al.,
1994
; Lechner and Laimins,
1991
). Otherwise, spontaneously immortalized human keratinocyte
lines generally have maintained a rather high degree of differentiation
potential (Boukamp et al.,
1988
; Baden et al.,
1987
; Allen-Hoffmann et al.,
2000
; Rice et al.,
1993
). This is best demonstrated with the HaCaT cell line, a
non-tumorigenic keratinocyte population derived from adult trunk skin
exhibiting a rather normal differentiation capacity despite multiple
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-cultures of HaCaT cells with skin
fibroblasts exhibit some stratification but usually lack typical criteria of
an ordered structure and regular keratinization
(Haake and Polakowska, 1993
;
Syrjänen et al., 1996
;
Steinsträsser et al.,
1997
; Boelsma et al.,
1999
). This deficiency, however, was not due to the permanent loss
of essential differentiation functions. We have recently demonstrated that
HaCaT cells under optimal environmental conditions (i.e. in surface
transplants on nude mice) were able to reform a regularly structured
differentiated epidermis, although with some delay and minor deficiencies
compared with normal keratinocytes
(Breitkreutz et al., 1997
;
Breitkreutz et al., 1998
).
Furthermore, we documented that improved organotypic co-culture conditions
with increased numbers of supporting fibroblasts in the collagen gel,
significantly enhanced stratification and differentiation of HaCaT epithelia
(Schoop et al., 1999
).
Nevertheless, growth and differentiation of the squamous epithelia was delayed
and incomplete, suggesting deficient signal transduction.
Here, we demonstrate that HaCaT cells exhibit distinct functional
deficiencies that substantially reduce their interaction with stromal cells as
well as their response to fibroblast-produced growth factors. The expression
and release of the inducer molecule IL-1 is very low in HaCaT cells, with the
consequence of a minimal induction of KGF and GM-CSF in fibroblasts. More
importantly, expression of the receptors for both growth factors is strongly
decreased on HaCaT cells so that signal transduction is significantly
impaired. Finally, the level of the autocrine acting keratinocyte growth
factor TGF- is drastically reduced in HaCaT cells. By addition of
TGF-
deficiencies in growth and differentiation of HaCaT organotypic
cultures can be completely rescued. This is mediated by the direct stimulatory
effect of TGF-
on HaCaT cell proliferation and survival, and, moreover,
by the enhanced expression 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-
) skin equivalents with
rather normal epidermal structures could be reproducibly obtained to serve as
a standardized skin equivalent model system.
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Materials and Methods |
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Isolation of RNA, reverse transcription and PCR
Mono- and co-cultures of HDFs, NEKs and HaCaT cells were grown in the
respective medium to subconfluence. Cells were lysed in
guanidinium-isothiocyanate solution and total RNA was extracted
(Chomczynski and Sacchi,
1987). Furthermore, RNA was extracted from subconfluent HaCaT
monolayer cultures, as controls or treated for 10 hours and 24 hours with 2
ng/ml TGF-
(in serum-free medium). RT-PCR was performed according to an
established method with some modifications, as reported
(Maas-Szabowski and Fusenig,
1996
). In brief, cDNA was transcribed at 42°C for 60 minutes
in 50 µl final volume containing 5 µg total RNA, 5 µl 10x
PCR-buffer, 10 µl MgCl2 (25 mM), 3 µl of each dNTP (10 mM),
0.5 µl RNasin (0.5 U/µl), 0.5 µl reverse transcriptase (50 U/µl),
1 µl oligo dT15 (50 µM) and 1 µl random hexamers (50 µM)
(GeneAmp-PCR-Kit, Perkin Elmer, Weiterstadt, Germany). Four microliters of
first-strand cDNA were added to the PCR-mixture up to a volume of 50 µl
following the product description. The mixture was amplified in a thermal
cycler (Biometra, Göttingen, Germany) at the indicated annealing
temperature performing 24-30 cycles, such that the product yield was in the
exponential range. Twenty-mer primers from separate exons flanking the regions
of the following locations on cDNA were chosen: GAPDH 69-308 (62°C);
IL-1
84-504 (62°C); IL-1ß (174-564; 64°C); TGF-
(240-448; 64°C); EGF (3800-4393; 63°C); KGF (208-474; 60°C); KGF-R
(870-1022; 64°C); GM-CSF (66-388; 60°C); GM-CSF-R
(83-524;
56.5°C); and GM-CSF-Rß (981-1318; 60.5°C). 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 restriction mapping with two different enzymes. The mRNA amount of the
house-keeping gene GAPDH was used as internal standard.
Protein determination by ELISA
Selected cytokines were quantified by enzyme-linked immunosorbent assays
(ELISA) in aliquots of culture medium of feeder-layer co-cultures. Medium was
collected at day 3, 5 and 7, always 48 hours after medium change. ELISA kits
for IL-1 were purchased from Endogen (Eching, Germany), for TGF- from
Calbiochem (Bad Soden, Germany), and for EGF, KGF and GM-CSF from R&D
Systems (Wiesbaden, Germany). Protein values were calculted as
pg/105 cells and mean values ± standard deviation of data
derived from duplicate measurements from 2-3 independent experiments are
given.
Organotypic co-cultures (OTCs)
NEKs (passage 2) and HaCaT cells (passage 35-40) were seeded
(1x106/insert) onto collagen type I gels (rat tail tendon)
containing 3x105/ml postmitotic fibroblasts (HDFi) cast in
cell culture filter inserts (pore size 3.0 µm, polycarbonat; Falcon, Becton
Dickinson, Heidelberg, Germany) as described in detail previously
(Stark et al., 1999). In HaCaT
cultures medium was replaced after 24 hours by DME medium [10% FCS and 50
µg/ml L-ascorbic acid (Sigma)] and cultures were raised to the air-liquid
interface by lowering the upper medium level to the lower part of the collagen
gel. Medium was replaced 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-
, 100
ng/ml GM-CSF, 5 ng/ml IL-1
(all R&D Systems) and 1 µg/ml mouse
monoclonal anti-human EGF-receptor antibody (C225, Imclone, New York, NY). NEK
OTCs were cultivated in rFAD medium (FAD with 10% FCS, 5 µg/ml insulin, 0.4
µg/ml hydrocortisone, 50 µg/ml L-ascorbic acid) as previously described
(Stark et al., 1999
). Before
fixation organotypic cultures were incubated for 16 hours with 63 µM
bromodeoxyuridine (BrdU) to label proliferating cells. Cultures were either
fixed according to a standardized protocol in 3.7% phosphate-buffered
formaldehyde for routine histology and staining in hematoxylin and eosin (H
and E), or embedded in Tissue Tec OCT Compound (Medim, Gießen, Germany)
and frozen in liquid nitrogen vapor for cryosectioning.
Indirect immunofluorescence microscopy
Cryosections mounted on glass slides (Histobond, Medim, Gießen,
Germany) were fixed for 5 minutes in 80% methanol at 4°C followed by 2
minutes in acetone at -20°C, rehydrated in PBS, and blocked for 15 minutes
in PBS with 1% BSA. First, antibodies were incubated overnight at 4°C 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 µg/ml
bisbenzimide (Hoechst No. 33258) DNA dye for nuclear counter staining. After
three final washes with PBS, specimens were mounted with Mowiol (Medim,
Gießen, Germany) under a coverslip and examined and photographed using a
Leica microscope (Leitz DMRBE, Bensheim, Germany) equipped with
epifluorescence optics. To visualize proliferating cells, a monoclonal mouse
anti-BrdU antibody (Progen, Heidelberg, Germany) was used and proliferation
was quantitated by counting the ratio of labelled to total nuclei within the
basal keratinocyte layer of the epithelium.
Apoptotic cells were detected by TUNEL staining. Paraffin sections mounted on glass slides (Histobond, Medim) were dewaxed in xylol and rehydrated in a graded series of ethanol. Sections were incubated with proteinase K as recommended in the protocol (In situ cell death detection kit TMR red; Roche, Mannheim, Germany). TUNEL reaction mixture was applied for 45 minutes at 37°C. Nuclei were counterstained for 15 minutes at RT by 0.5 µg/ml bisbenzimide (Hoechst No. 33258) DNA dye. Sections were washed twice in PBS and mounted with mowiol. Specimens were examined and photographed using a microscope equipped with epifluorescence optics. The percentage of TUNEL-positive nuclei to the total cell number within all layers of the epithelium was calculated.
Differentiated keratinocytes were characterized by labeling with specific antibodies to human transglutaminase and filaggrin (both mouse 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 of D. Hohl, Lausanne, Switzerland) and visualized by appropriate fluorochrome-coupled secondary antibodies.
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Results |
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To study the molecular defects of HaCaT cells in their interaction with
stromal cells, the fibroblast number was adjusted to be optimal for NEKs but
suboptimal for HaCaT cells. Thus, with 2x105 irradiated
fibroblasts per ml collagen gel, normal keratinocytes derived from adult skin
grow to a typically structured epidermal tissue within 1 week, followed by
increased differentiation and formation of a structured basement membrane
after 2 and 3 weeks [Fig. 1
(see also Stark et al.,
1999)]. In contrast, HaCaT cells had formed only a 2-3 layered
disorganized epithelium under the same conditions within 1 week, which
increased in thickness after 2 weeks, but did not exhibit morphological
features of differentiation. Thickness and differentiation of the HaCaT
epithelia increased further after 3-4 weeks exhibiting a thin parakeratotic
cell layer, but HaCaT epithelia were still less keratinized than NEK cultures
after 2 weeks [data not shown (Schoop et
al., 1999
)].
Lack of IL-1 impedes stromal interaction
This discrepancy in epidermal tissue regeneration of HaCaT cells, compared
with that of NEKs, was possibly due to deficiencies in epithelial-stromal
interaction mechanisms [Fig. 2A
(Maas-Szabowski et al.,
2001)]. This double paracrine epithelial-stromal interaction is
induced by keratinocyte-released IL-1
and -1ß resulting in
AP1-mediated enhanced expression of KGF and GM-CSF, two strong stimulators of
keratinocyte proliferation (Szabowski et
al., 2000
). To identify any possible alterations in this
interaction in HaCaT cells compared with that in NEKs, expression of growth
factors was analyzed at the RNA- and protein level in mono- and co-cultures.
Thus, RNA levels were determined in both 2D and 3D cultures with similar
expression levels (Maas-Szabowski et al.,
1999
; Maas-Szabowski et al.,
2000
), whereas protein concentrations were quantitated in
supernatants of 2D cultures (to avoid absorption to the collagen gel).
|
The expression of the signalling factors IL-1 and IL-1ß,
essential for the double paracrine mechanisms, was drastically decreased in
HaCaT mono- and co-cultures when compared with that in NEKs
(Fig. 2B). Comparably, the
secretion of IL-1
into the culture supernatants remained extremely low
over a 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 (1.98±0.88 pg/105 cells) compared with a
massive accumulation in NEKs (281±24.9 pg/105 cells).
Similar protein values were measured in the culture supernatants for
IL-1ß (data not shown). As expected, and due to the lack of both inducer
cytokines, RNA expression (data not shown) and protein secretion of KGF and
GM-CSF by co-cultured fibroblasts were strongly reduced throughout the first
week in culture in contrast to co-cultures with NEK
[Fig. 3A
(Maas-Szabowski et al., 2000
;
Szabowski et al., 2000
)].
|
To compensate this deficiency of HaCaT cells in inducing their own growth
factors in co-cultured fibroblasts, the addition of these factors or their
inducer should restore their capacity to form differentiated epithelia.
However, neither the continuous addition of IL-1, KGF, GM-CSF or their
combination (not shown) significantly induced HaCaT cell growth or tissue
formation in organotypic co-cultures (Fig.
3B). This lack of response was most probably due to defects in
signal reception and transduction in HaCaT cells, as shown by the reduced
expression of the receptors for KGF and GM-CSF
(Fig. 4A), and paralleled at
the protein level by immunofluorescent staining with specific antibodies (data
not shown). The expression of both receptors increased with culture time and
cell density: a possible explanation for the delayed stratification of
organotypic HaCaT epithelia at 3 and 4 weeks
(Schoop et al., 1999
).
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Abrogated TGF- expression impairs HaCaT cell proliferation and
survial
More importantly, the expression of the transforming growth factor
(TGF-
), a known autocrine acting keratinocyte growth factor, was
strongly reduced in HaCaT epithelia in contrast to that in NEKs
(Fig. 4A). The reduced
TGF-
production was more dramatically evident at the protein level in
supernatants of HaCaT mono- or co-cultures
(Fig. 4B). This was not due to
a lack of cleavage of this factor from its membrane-bound form
(Bosenberg et al., 1992
), since
HaCaT cell lysates showed similar low levels in the ELISA assay (data not
shown).
Supplementation of TGF- rapidly and efficiently restored the
capacity of HaCaT cells to form structured epithelia in organotypic co-culture
(Fig. 5A). The same normalizing
effect was seen with EGF (data not shown), sharing the same receptor with
TGF-
, the EGF receptor, which is constitutively expressed in HaCaT
cells (Game et al., 1992
;
Stoll et al., 1998
). The
essential role of the EGF-receptor-mediated signal transduction for epidermal
tissue formation was also demonstrated in organotypic cultures of
TGF-
/EGF-substituted HaCaT cells as well as those of NEKs, by receptor
blocking studies. Addition of the EGFR-blocking monoclonal antibody C225
drastically inhibited epithelial tissue formation by both cell types
(Fig. 5A). EGF itself, although
expressed at the RNA level in NEK, HaCaT and fibroblast culture
(Fig. 5B), was not detected in
the culture supernatants of any mono- or co-cultures (data not shown). Thus,
TGF-
is the effective growth factor in skin cells acting in an
autocrine manner on keratinocytes (Schulz
et al., 1991
; Carpenter,
1993
; Compton et al.,
1995
).
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TGF- enhances HaCaT cell proliferation in mono-culture with a
sixfold higher cell number after 5-day treatment in serum-free medium and in
2D co-cultures (data not shown). Furthermore, TGF-
treatment strongly
stimulates stratification in HaCaT mono-cultures on fibroblast-free collagen
gels, indicating its major effect as a direct acting factor on HaCaT cells.
Moreover, TGF-
induces the expression of IL-1
and, more
importantly, the receptors of KGF and GM-CSF
(Fig. 5B). The elevated RNA
levels are not due to stabilization of mRNA, because the increase is abrogated
by actinomycin D treatment. The upregulation of receptor expression is most
probably indirect, because it was blocked by the addition of cycloheximide
(data not shown). Although there is a basal expression of both receptors at
the RNA level, the immunofluorescence data exhibit only background staining in
control co-cultures (data not shown).
Based on the observed stimulation of HaCaT cell proliferation, we
hypothesized that the effect of TGF- on tissue reconstruction was
mainly due to this effect. However, in organotypic co-cultures, TGF-
enhanced HaCaT cell proliferation only initially (at day 3), whereas at later
culture timepoints BrdU-labelled cells were even more frequent in thin control
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 cells was
four- to five-times higher in control cultures compared with that in
TGF--treated cultures (Fig.
6C). When differentiation began (day 9), the rate of apoptotic
cells increased in TGF-
-treated cultures and after 12 days was higher
than in controls. Whereas TUNEL-positive nuclei were seen throughout the whole
epithelia in control cultures, in TGF-
-treated cultures they were
exclusively localized in the upper flattened cell layers representing
terminally differentiating cells (Fig.
6B). Nearly all nuclei of the parakeratotic superficial cell
layers stained positively in the TUNEL assay, whereas the nuclei of the
cuboidal cells of the 4 to 6 lower cell layers were unstained.
TGF- normalizes HaCaT cell growth and differentiation
Replenishment of TGF- in the deficient HaCaT organotypic cultures
not only enabled these immortal keratinocytes to reform a structured squamous
epithelium but also allowed a rather normal differentiation
(Fig. 7). This is clearly
visible in H-and-E-stained sections of 2-week-old cultures, and sections
stained with antibodies to markers of early [e.g. keratin 1 and 10 (not shown)
and transglutaminase] or later stages of keratinization, such as loricrin. The
components are typically localized in the upper layers of the stratified
epithelium comparable with, though still less regular than, those in cultures
of NEKs. Furthermore, the stratum granulosum formation is rather poor, the
stratum corneum is thin and mostly parakeratotic, as indicated by the remnant
nuclei in the uppermost flattened cells.
|
This deficit in differentiation of HaCaT epithelia, however, is repaired to
the most part by further supplementing the culture media with KGF, GM-CSF and
IL-1, respectively (Fig.
8). This resulted not only in improved morphologic organization of
the reconstituted stratified epithelia but also in a more regular localization
of the late differentiation products in the uppermost cell layers very similar
to NEKs. Remarkably, as already seen earlier
(Szabowski et al., 2000
),
GM-CSF not only exerts effects on keratinocyte proliferation but also enhances
keratinization, demonstrated by the intensified staining with the late
differentiation marker loricrin. This is similarly seen after addition of
IL-1, which induces GM-CSF in 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-, the delayed and deficient
growth and differentiation capacity of the immortal HaCaT keratinocytes is
restored. TGF-
induces proliferation with enhanced survival as well as
the expression of IL-1 and the receptors of KGF and GM-CSF so that the HaCaT
cells are able to respond typically to stromal interactions regulating
keratinocyte growth and differentiation. This skin equivalent reconstructed by
the immortal HaCaT keratinocytes represents a better standardized in vitro
model to study further regulatory mechanisms in skin physiology and may serve
as a highly reproducible test system for pharmakotoxicology.
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Discussion |
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In organotypic co-cultures with fibroblasts, conditions that induce normal
keratinocytes to form stratified epithelia with all features of a normal
epidermis (Smola et al., 1998;
Stark et al., 1999
), HaCaT
cells have been described to be deficient in this complex function of tissue
regeneration (Haake and Polakowska,
1993
; Syrjänen et al.,
1996
; Steinsträsser et
al., 1997
; Boelsma et al.,
1999
). We have been able to improve their epidermal tissue
formation and differentiation in this model by intensifying stromal influences
by elevating the number of supporting fibroblasts
(Schoop et al., 1999
).
However, tissue architecture and keratinization was delayed and stayed
deficient, indicating major defects in transduction of the major signals
mediating epithelial-stromal interplay.
Here we demonstrate that the deficiency of HaCaT cell interaction with
fibroblasts is based on their very low constitutive expression of the prime
signalling cytokine interleukin-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 of HaCaT cells to these stromal-cell produced factors
KGF and GM-CSF is abolished due to the low level of expression of the
respective receptors. More importantly, production of transforming growth
factor
(TGF-
), known as a potent autocrine acting stimulator of
keratinocyte proliferation, is barely detectable in HaCaT cells. Since its
cognate receptor, the EGF receptor, is expressed, addition of TGF-
or
EGF rapidly re-established the capacity of HaCaT cells to grow to a stratified
epithelium and to exhibit typical differentiation markers of the epidermis.
Furthermore, with substitution of TGF-
, the effect of stromal
cell-derived factors, in particular of GM-CSF, on keratinocyte differentiation
is restored, resulting in improved differentiation in HaCaT organotypic
cultures comparable with skin equivalents of NEKs
(Maas-Szabowski et al.,
2001
).
This remarkable effect of one single growth factor on the complex mechanism
of tissue regeneration was mediated in HaCaT cells by a coordinated function:
TGF--upregulated the expression of IL-1, and receptors for KGF and
GM-CSF; it exhibited 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-1ß are released by keratinocytes in culture
following stress comparable with conditions of injury to the skin in vivo
(e.g. Kupper and Groves, 1995
;
Wood et al., 1996
). Whereas in
skin, IL-1 signalling is mostly understood as part of a proinflammatory
cytokine cascade to induce multiple effects on fibroblasts, endothelial cells
and inflammatory cells (for a review, see
Dinarello, 1997
), we have
demonstrated that it has additional major signaling functions in epidermal
tissue regeneration.
In keratinocyte co-cultures with fibroblasts, IL-1 upregulates of growth
factors in stromal cells to stimulate keratinocyte proliferation, 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 the constitutive production of
IL-1 in HaCaT cells, as already noticed earlier
(Ruhland and de Villiers,
2001
), with the consequence of inefficient induction of KGF and
GM-CSF in fibroblasts, could to some extent be overcome by increasing the
number 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. Upregulation of IL-1 by EGF and TGF-
as
already noticed earlier both in normal 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 effect of KGF and
GM-CSF on keratinocytes, GM-CSF also enhanced epidermal differentiation in
organotypic co-cultures (Szabowski et al.,
2000
). This differentiation-stimulating effect of GM-CSF was also
noticed in HaCaT organotypic co-cultures; however, only after enhanced
receptor expression by TGF-
. The same tendency to a morphologically
better differentiated HaCaT epithelium was noticed after addition of IL-1, the
inducer of both KGF and GM-CSF in the co-cultured fibroblasts.
Most astonishingly, however, was the crucial role of the EGF receptor and
its ligands TGF- and EGF in regulating epidermal tissue regeneration by
HaCaT cells. A very low release and expression of TGF-
by HaCaT clones,
compared with ras-transfected tumorigenic HaCaT cells, had already been
observed earlier (Game et al.,
1992
), although this fact had not been associated with their
reduced tissue regeneration. Here we clearly demonstrate that the low
production rate of TGF-
is due to reduced RNA expression and not a
failure of release of the active compound from its membrane-bound precursor.
In transplants in vivo, the deficiency of HaCaT cells in TGF-
expression may be compensated by EGF provided by the circulation
(Derynck, 1988
). As shown
earlier, both high and 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-
stimulate HaCaT cell
proliferation and migration similar to that observed in normal keratinocytes
in culture (Pittelkow et al.,
1989
; Ju et al.,
1993
), whereas EGF receptor inhibition induces growth arrest
(Peus et al., 1997
) and
consequently blocks stratification in skin equivalents.
Interestingly, in organotypic cultures of HaCaT cells, the stimulating
effect of TGF- on cell proliferation was prominent only at early
culture time points, whereas at later stages the rate of DNA synthesis was
comparable with untreated control cultures. By contrast, the percentage of
apoptotic cells in the non-differentiated epidermal layers was drastically
decreased in TGF-
-treated cultures from the beginning. This indicated
that the enhancement of survival of HaCaT cells was a major function of
TGF-
in the developing epidermal tissue and, consequently, a major
contribution to the increased number of cell layers. With the beginning of
keratinization in the TGF-
-treated HaCaT epithelia, the number of
TUNEL-positive cells also increased here; however, apoptotic cells were
located in the upper parakeratotic cell layers. As shown earlier
(Schoop et al., 1999
), the
remnant nuclei of the incompletely differentiated cells stain positive with
the TUNEL reaction owing to incomplete DNA degradation
(Bernerd and Asselineau,
1997
).
An important role of EGF receptor signalling for cell survival in
epithelial cells has been postulated earlier. EGFR activation in keratinocytes
increases the expression of bcl-XL, a member of the anti-apoptotic bcl-2
family of proteins, and human 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
(Jost et al., 2001
). Thus,
EGFR-signalling is important for both keratinocyte proliferation and survival
and the latter function seems to play a major role in organotypic cultures
when cells have to detach from their specific extracellular matrix, the
basement membrane, and migrate upwards to form a multilayered tissue.
On the other hand, stable transfection and overexpression of bcl-2 into
HaCaT cells reduced their apoptotic rate in organotypic cultures but did not
show noticeable effects on their tissue organization and epidermal
differentiation features (Delehedde et
al., 2001). Thus, the combined effect of TGF-
on cell
proliferation and survival is required for regular stratification and
differentiation of HaCaT cells.
Furthermore, a major component of the TGF- effect on HaCaT cells is
the enhanced expression of IL-1 and of the receptors for KGF and GM-CSF,
rendering HaCaT cells reactive and responsive for stromal growth regulatory
signals. In particular, the induction of the KGF receptor seems to be an
essential step to gaining sustained keratinocyte proliferation both in wound
healing in vivo as well as in cell culture (for a review, see
Werner, 1998
;
Marchese et al., 1997
;
Werner et al., 1992
). It had
been noticed that expression of the KGF receptor is deficient in sparse HaCaT
cultures but upregulated upon confluence and the onset of differentiation
(Capone et al., 2000
).
Moreover, the induction of KGFR in keratinocyte cultures grown at high calcium
concentration has been interpreted in that its signalling plays a possible
role in keratinization control (Marchese
et al., 1997
). We have shown recently, however, that addition of
KGF effects mainly proliferation in organotypic cultures of normal
keratinocytes co-cultured with AP-1-defective fibroblasts, whereas
differentiation was not affected
(Szabowski et al., 2000
).
Keratinization and formation of specific differentiated strata (i.e. stratum
granulosum) was enhanced by GM-CSF and a regular tissue structure required the
combined action of both growth factors.
Clearly, the complex process of epidermal tissue regeneration,
differentiation and homeostasis is unlikely to be regulated only by the four
factors identified so far, i.e. TGF-, IL-1, KGF and GM-CSF. Further
regulating factors have to be identified and their molecular mechanisms of
action elucidated. For these investigations, in-vivo-like but reproducible and
simple-to-handle skin equivalent in vitro models are required. Owing to the
rather faithful mimicry of the basic functions of epidermal proliferation,
differentiation and tissue reorganization by the immortal HaCaT cells in
TGF-
-supplemented organotypic co-culture with fibroblasts, this
reproducible skin equivalent represents a biologically relevant model for such
studies. Furthermore, as an immortalized cell line, HaCaT cells can be
genetically modified by overexpression and/or blockade of specific genes so
that their consequences can be studied in a tissue context. Finally, with
further improvement of the quality of structural organization, differentiation
and barrier functions of the stratified epithelia formed by HaCaT cells, such
skin equivalents may become highly standardized in vitro tissue models for
routine testing in pharmacology and toxicology.
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Acknowledgments |
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References |
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