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Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School and Shriners Burns Hospital, Boston, Massachusetts 02114, USA
2Correspondence: Shriners Hospitals for Children, 51 Blossom St., Boston, MA 02114, USA.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5ß1 and delayed
expression of keratin 10 and transglutaminase. However, barrier
formation of the epidermis was not disrupted. These results demonstrate
for the first time that a single growth factor can alter the
3-dimensional organization and proliferative function of an in
vitro epidermis. In addition to new strategies for tissue
engineering, such a well-defined system will be useful for analyzing
growth factor effects on the complex links between cell proliferation,
cell movement and differentiation within a stratified
tissue.—Andreadis, S. T., Hamoen, K. E., Yarmush, M. L., Morgan, J. R. Keratinocyte growth factor induces
hyperproliferation and delays differentiation in a skin equivalent
model system.
Key Words: KGF • fibroblast growth factor • cell proliferation • rete ridges
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Although these skin equivalents have been used
clinically to repair burns and wounds (3
, 4)
and are being
considered for gene therapy strategies (5
6
7
8
9
10
11)
, there has
been little use of these skin equivalents to study the factors that
control the proliferation, differentiation, and 3-dimensional
organization of the epidermis. Such information is important to
understand the factors that control epidermal morphogenesis, pathways
to diseases of the epidermis, and the design of improved tissue
engineering strategies for the repair of defective skin.
Diploid human keratinocytes are readily cultured in vitro;
when seeded on the surface of analogs of the dermis and exposed to the
air/liquid interface, the cells will stratify, differentiate, and form
an epidermis complete with basal, spinous, granular, and cornified
layers (12)
. We used this system to study the effects of
keratinocyte growth factor (KGF). KGF, a member of the fibroblast
growth factor family (FGF-7), is a paracrine mediator of epithelial
cell growth (13)
that is expressed by stromal cells in a
variety of tissues including lung, prostate, mammary gland, stomach,
bladder, and skin (14)
. Expression of KGF is restricted to
cells of mesenchymal origin, whereas its mitogenic activity is
restricted to epithelial cells (14)
and is mediated
through the KGF receptor (KGFR), a splice variant of FGF-2 receptor
encoded by the gene fgfr-2 (15
, 16)
. KGF is thought to
play an important role in tissue development and response to cutaneous
injury and has been implicated in tissue morphogenesis, especially in
those tissues whose development is dependent on mesenchymal–epithelial
interactions (17
, 18)
. Targeting KGF expression to the
basal keratinocytes of a developing mouse epidermis caused epidermal
hyperthickening accompanied by alterations in epidermal growth and
differentiation (19)
. Expression of KGF is stimulated
(20)
during normal wound healing, and this up-regulation
is significantly reduced and delayed in diabetic (21)
and
glucocorticoid-treated mice (22)
.
Due to the complexity of the in vivo setting and the
presence of numerous cell types, it is often difficult to establish
direct cause-and-effect relationships between a growth factor and a
change to tissue organization. Therefore, we took advantage of the skin
equivalent system to study KGF’s effects on a 3-dimensional human
epidermis reconstituted by a single cell type. KGF either was added
exogenously as a recombinant protein or was produced by keratinocytes
genetically modified to express the KGF gene. KGF induced significant
changes to the cellular organization of the in vitro
epidermis including an increase in epidermal thickness, crowding and
elongation of the cells of the basal layer, and an induction of
suprabasal cell proliferation. Although terminal differentiation was
delayed, normal granular and cornified layers were produced. Most of
KGF’s effects appear to target the proliferative compartment of the
epidermis and possibly are mediated by its induction of 5ß1, an
integrin that might increase the adhesiveness of the basal cells. These
results support the importance of KGF in controlling the proliferation
of the epidermis and demonstrate the usefulness of using skin
equivalents to investigate the effects of growth factors on the
3-dimensional organization and function of the epidermis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Primary cultures of keratinocytes were established
by cocultivation with 3T3-J2 mouse fibroblasts (originally provided by
H. Green, Harvard Medical School, Boston, Mass.) pretreated with 15
µg/ml mitomycin C (Boehringer Mannheim, Indianapolis, Ind.). The
keratinocyte culture medium was a 3:1 mixture of Dulbecco’s modified
Eagle’s medium (DMEM, high glucose) (Gibco/BRL, Gaithersburg, Md.) and
Ham’s F12 medium (Gibco./BRL) supplemented with adenine, 1.8 x
10-4 M (Sigma Chemical, St. Louis, Mo.); cholera
toxin, 10-10 M (Vibrio cholerae, Type
Inaba 569 B; Calbiochem, La Jolla, Calif.); hydrocortisone, 0.4 µg/ml
(Calbiochem); insulin, 5 µg/ml (Novo Nordisk, Princeton, N.J.);
transferrin, 5 µg/ml (Boehringer Mannheim);
triiodo-L-thyronine, 2 x
10-9 M (Sigma); and penicillin-streptomycin, 100
IU/ml-100 µg/ml (Boehringer Mannheim). Epidermal growth factor
(Collaborative Biomedical Products, Bedford, Mass.) was added at 10
ng/ml with the first medium change 3 days after cell isolation.
Cultures were incubated in a humidified 10% CO2
atmosphere at 37°C and medium was changed every 3–4 days.
Keratinocytes were subcultured by first removing the feeder layer cells
with a trypsin-EDTA wash (1 min), then treating keratinocytes with
trypsin-EDTA for 10–15 min and reseeding them on mitomycin-C 3T3-J2
mouse fibroblasts. Swiss mouse 3T3-J2 and virus producer cell lines were passaged twice a week in DMEM (high glucose) supplemented with 10% bovine calf serum (HyClone, Logan, Utah) and penicillin-streptomycin, (100 IU/ml to 100 µg/ml) and incubated in a humidified 10% CO2 atmosphere at 37°C.
Keratinocytes were seeded onto the papillary side of acellular dermis
using methods similar to those previously described (24)
with slight modifications of media described by Ponec et al.
(12)
. Acellular dermis was placed into 35 mm tissue
culture dishes; cells in keratinocyte seeding medium (described below)
were seeded onto the surface (5x105
cells/cm2). After 
2 h, the skin equivalents
were submerged in keratinocyte seeding medium for 24 h.
Keratinocyte seeding medium was a 3:1 mixture of DMEM (high glucose)
(GIBCO-BRL) and Ham’s F-12 medium (GIBCO-BRL) supplemented with 1%
FBS, 10-10 M cholera toxin (Vibrio
cholerae, Type Inaba 569 B; Calbiochem), 0.2 µg/ml
hydrocortisone (Calbiochem), 5 µg/ml insulin (Novo Nordisk,
Princeton, N.J.), 50 µg/ml ascorbic acid (Sigma), and 100 IU/ml and
100 µg/ml penicillin-streptomycin (Boehringer Mannheim). After
24 h, keratinocyte seeding medium was removed and the skin
equivalents were submerged for an additional 48 h in a
keratinocyte priming medium. Keratinocyte priming medium was composed
of keratinocyte seeding medium supplemented with 24 µM bovine serum
albumin (BSA; Sigma), 1.0 mM L-serine (Sigma), 10
µM L-carnitine (Sigma), and a mixture of fatty
acids: 25 µM oleic acid, (Sigma), 15 µM linoleic acid (Sigma), 7
µM arachidonic acid (Sigma), and 25 µM palmitic acid (Sigma)
(25)
. After 48 h in priming medium, skin equivalents
were placed on stainless steel screens, raised to the air-liquid
interface, and cultured with an air-liquid interface medium composed of
serum-free keratinocyte priming medium supplemented with 1.0 ng/ml
epidermal growth factor (Collaborative Biomedical Products).
Measurements of surface electrical capacitance
Formation of epidermal barrier was measured with a dermal phase
meter (DPM 9003; NOVA Technology, Gloucester, Mass.) by placing the 6
mm sensor probe on the surface of the composite grafts. The dermal
phase meter measures surface capacitance, which is a measure of surface
skin hydration, by integrating measurements at different frequencies of
the applied alternating current (26)
. Ten serial readings
were recorded at 1 s intervals and were immediately displayed on
the LCD screen of the instrument and stored in a computer for further
analysis. Instrument readings were converted to pF by use of the
formula (27)
:
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Recombinant retrovirus
A cDNA encoding human KGF was kingly provided by William
LaRochelle of National Cancer Institute and was inserted into the
NcoI and BamHI site of the MFG retroviral vector
(28)
by a two-step procedure. MFG vector DNA was digested
with NcoI and BamHI and gel purified. Vector DNA
was ligated to two annealed oligonucleotides encoding the first few
amino acids of KGF (5'-CATGCACAAATGGATACTGACATG-3' and
5'-GATCCATGTCAGTATCCATTTGTG-3'). MFG, with this small oligo insert, was
isolated, digested with BamHI, treated with calf intestinal
phosphatase, and gel purified. The remainder of the KGF open reading
frame was prepared by PCR and ligated into this vector. The PCR product
encoding the rest of KGF was prepared using
5'-CATGCACAAATGGATACTGACATG-3' as a forward primer and
5'-AGTCCAGGATCCAGATCTTAAGTTATTGCCATAGGAAGAAAGTGG-3' as a reverse
primer. The PCR product was digested with BamHI and
gel purified before ligation into the final vector. The fidelity of the
entire insert in the final MFG-KGF vector was verified by DNA
sequencing. To generate a virus-producing cell line, MFG-KGF plasmid
DNA was transfected into individual 
-CRIP packaging cell lines as
described (5)
.
Measurement of KGF production
The time course of KGF secretion by genetically modified cells
was determined by an enzyme-linked immunosorbent assay (ELISA). ELISA
96-well plates (Fisher Scientific, Agawan, Mass.) were coated with 100
µl of a 1.0 µg/ml mouse antihuman KGF monoclonal antibody (R&D
Systems Minneapolis, Minn.) in PBS/0.1% BSA by overnight incubation at
room temperature. The next day, the antibody was removed; the plate
washed three times with buffer solution (PBS/0.05% Tween 20, pH 7.4)
and blocked with 300 µl of PBS with 1% BSA, 5% sucrose, and 0.05%
NaN3 for 1 h at room temperature. The plate
was washed three times with buffer solution and the conditioned medium
containing KGF along with KGF standards (R&D Systems) was added in
triplicate (100 µl per well) for 2 h at room temperature. The
plate was washed three times and 100 µl of biotinylated goat
polyclonal anti-hKGF detection antibody (R&D Systems) was added at 200
ng/ml in diluent (0.1% BSA, 0.05% Tween 20 in Tris-buffered saline,
pH 7.3) for 2 h at room temperature. After three washes, 100 µl
of a horseradish peroxidase (HRP) -conjugated avidin (Zymed
Laboratories, South San Francisco, Calif.) was added at a dilution of
1:5,000 in PBS/0.1% BSA for 1 h at room temperature. The
substrate (10 mg o-phenylenediamine dihydrochloride) and 10
µl of H2O2 in 25 ml of
substrate buffer (5.1 mg/ml citric acid mono-hydrate and 13.78 mg/ml
Na2HPO4·7H20
in dH2O) were added (100 µl per well) and the
reaction was allowed to proceed for 10 min before addition of 50 µl
per well of 8N H2SO4 (stop
solution). The optical density was read at 490–650 nm with an ELISA
plate reader (ThermoMax plate reader, Molecular Devices, Palo Alto,
Calif.).
Histology and immunohistochemistry
To detect the presence of Ki67, keratin 10, and transglutaminase
the skin equivalents were fixed in 4% paraformaldehyde in PBS for
4 h at 4°C, followed by treatment with 0.1M ice-cold glycine for
1 h and overnight incubation in 0.6M sucrose solution at 4°C.
Tissues were embedded in OCT and placed in dry ice. For
immunohistochemistry, cryostat sections (8 µm) were washed with PBS
and stained with a staining kit (VECTASTAIN Elite ABC; Vector
Laboratories, Burlingame, Calif.) following the manufacturer’s
recommendations. Briefly, the slides were incubated with blocking
solution (10% horse serum in PBS) for 1 h at room temperature.
Sections were then incubated with 50 µl of mouse monoclonal
antibodies, anti-K10 (1:400 dilution in blocking solution; 30 min at
room temperature; Chemicon International, Temecula, Calif.), antihuman
keratinocyte transglutaminase (1:40 dilution in blocking solution;
1 h at 37°C; Biomedical Technologies, Stoughton, Mass.),
antihuman Ki67 (1:100 dilution in blocking solution; 30 min at room
temperature; PharMingen, Torrey Pines, Calif.), or antihuman 5ß1
(1:100 dilution in blocking solution; overnight at 4°C; Chemicon
International). Slides were then washed five times with PBS and
incubated with 50 µl horse antimouse biotinylated antibody (1:200 in
blocking solution) for 30 min at room temperature. The slides were then
incubated with avidin-HRP from 30 min and developed with a substrate
kit (Vector Laboratories) according to manufacturer’s recommendations.
Slides were washed five times with PBS, followed by a 5 min wash with
tap H2O and counterstained with hematoxylin (15 s
with Richard-Allan hematoxylin or 50 s with Harris hematoxylin).
The slides were washed with tap H2O for 10 min
and mounted with an aqueous mounting medium (Crystal/Mount; Biomeda,
Foster City, Calif.). Paraffin-embedded sections (5 µm) were stained
with hematoxylin and eosin.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Skin equivalents secreting KGF showed striking differences in tissue
morphology as compared to controls (Fig. 2
). After 1 wk at the air/liquid interface, the thickness of the
KGF-expressing epidermis was increased mainly because of an increase in
the suprabasal and spinous cell layers. There was a slight decrease in
the stratum corneum as well, probably due to a delay in
differentiation. The density of the cells in the basal layer was also
markedly increased and the cells were tightly packed and highly
elongated. Similar results were obtained when various concentrations of
recombinant KGF protein were added (25 and 100 ng/ml). Increased
thickness of KGF-treated and KGF-expressing skin equivalents was also
present after 2 wk at the air/liquid interface.
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Quantitation of the number of cells per unit length of basement
membrane showed that basal cell density was 50% higher in the KGF
epidermis vs. control epidermis at 1 wk (Fig. 2)
. Addition of exogenous
KGF also increased basal cell number, and this increase was independent
of the KGF concentrations tested (25–100 ng/ml). Basal cell density
was also increased at 2 wk
Another change to the epidermis in response to KGF was the flattening of the basal layer and the disappearance of the rete ridges. In controls, keratinocytes filled in and preserved the typical rete ridge pattern characteristic of the acellular dermis. In contrast, the epidermal/dermal interface was flattened and the rete ridges in KGF-expressing and KGF-treated skin equivalents were not as obvious.
The KGF-expressing epidermis also showed unusual ripple-like patterns at the junction of the granular and cornified layers, in contrast to controls, where this junction was mostly flat in appearance. These undulations were also apparent in skin equivalents treated with the highest dose of KGF (100 ng/ml).
KGF increases basal cell proliferation and induces suprabasal cell
proliferation
To determine KGF’s influence on cell proliferation, skin
equivalents were stained for the nuclear proliferation antigen Ki67
(brown) and counterstained with hematoxylin (blue)(Fig. 3
). In control skin equivalents, Ki67-positive cells were largely
confined to the basal cell layer. In KGF-expressing and KGF-treated
skin equivalents, not only was the number of Ki67-positive basal cells
increased by twofold (from 50 to 100%; data not shown), but a
significant proportion of Ki67-positive cells was also found in the
suprabasal cell layers. At low KGF concentrations (25 ng/ml), there was
approximately a fourfold increase in the number of Ki67-positive cells,
but only a twofold increase at higher KGF concentrations (50 and 100
ng/ml). The number of Ki67-positive cells of KGF-expressing and
KGF-treated skin equivalents subsided to the levels of control grafts
by 2 wk at the air/liquid interface and there was still evidence of
suprabasal cell proliferation.
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KGF delays differentiation
To investigate the effect of KGF on differentiation, sections were
stained for keratin-10 (K10) and transglutaminase (TGase I) (19
, 29
, 30)
. In control epidermis (days 7 and 14), K10 and TGase I
were expressed in all cell layers except for the basal cells
(Fig. 4
and Fig. 5
). In KGF-expressing and KGF-treated epidermis, not only was the basal
cell layer negative for these differentiation markers, but an
additional 3–4 suprabasal cell layers were also negative. These K10
and TGase I negative cell layers coincided with the Ki67-positive
cells.
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KGF stimulates 5ß1 integrin expression
Since KGF had induced major changes to the cellular organization
of the epidermis, we stained skin equivalents for alterations to
integrin expression. When stained for 5ß1, controls had little if
any expression, whereas KGF-expressing and KGF-treated skin equivalents
had strong staining for 5ß1 throughout the basal and suprabasal
layers (Fig. 6
). A preponderance of staining for 5ß1 in the basal cell layer was
localized to the basal domain of the basal cells.
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KGF does not alter the barrier function
To determine whether KGF’s perturbation of epidermal structure
also disrupted barrier formation, we measured the surface electrical
capacitance (SEC) of control, KGF-expressing and KGF-treated skin
equivalents. SEC is a measure of the surface hydration of the skin and
has been used to monitor epidermal barrier function (27
, 31
32
33)
. SEC levels for control and KGF skin equivalents were
high at days 1 and 3 after lifting to the air-liquid interface. During
this transition time, the cells are stratifying, differentiating and
forming a barrier in response to the air (Fig. 7
). By day 4 and beyond, SEC declined to a low and steady level and there
was no significant difference between control and KGF skin equivalents.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 20
, 34
, 35)
.
However, it is difficult to reach conclusions about the direct actions
of KGF on the epidermis in vivo, because skin has several
other cell types in addition to keratinocytes that could be influenced
by transgene expression and, during mouse development, unknown
compensatory mechanisms may operate in response to transgene
expression. Moreover, species differences could exist between mouse and
human keratinocytes.
Using an in vitro differentiated skin equivalent system, we
show that KGF induces dramatic changes to the proliferation and
3-dimensional organization of the human epidermis. These changes
include hyperthickening of the epidermis, an increase in proliferating
cells; an elongation of basal cell morphology and an increase in basal
cell density, an induction of suprabasal cell proliferation; a delay in
differentiation, an induction of 5ß1 expression; the loss of rete
ridges and the formation of undulations in the junction of the granular
and cornified layers. Since this skin equivalent system has only one
cell type (human keratinocytes), we can conclude that KGF is able to
influence not only cell proliferation, but the 3-dimensional
organization of the epidermis as well, and that no other cell types are
needed to induce these hyperproliferative changes to the epidermis.
These changes were manifest when either recombinant KGF protein was
added or if the cells were genetically modified to produce their own
KGF.
In the normal epidermis, cell proliferation and discrete steps of
differentiation occur at distinct spatial locations in the tissue.
Proliferation is confined to the basal layer and there is a steady
upward migration and progressive differentiation of cells leading to
the formation of the granular and cornified layers. Our data
demonstrate that KGF alters this spatial control of proliferation and
gives rise to suprabasal cell proliferation. Under normal conditions,
keratinocytes that detach from the basal lamina lose their growth
potential, and this process is linked to the onset of terminal
differentiation (36
, 37)
. Thus, KGF is able to circumvent
this process, possibly by substituting for those signals from the basal
lamina that preserve growth potential. Even though the epidermis of the
mice expressing KGF was hyperproliferative, there was no evidence of
suprabasal cell proliferation (19)
.
We also show for the first time that KGF induces 5ß1 expression.
5ß1 binds fibronectin, and loss of its activity has been linked to
migration of cells from the basal layer and terminal differentiation
(37
, 38)
. By stimulating 5ß1 expression, KGF may
enhance the adhesive properties of basal keratinocytes. In fact, a
preponderance of 5ß1 protein in KGF-treated skin equivalents is
localized to the basal domain of the basal cells. This increase in
adhesion may slow the upward migration of basal cells, which in
combination with an enhanced rate of proliferation could explain our
observation that KGF induces crowding of the basal layer.
Another 3-dimensional change mediated by KGF in our experiments is the
loss of rete ridges. The acellular dermis used as a substrate for the
formation of an epidermis has a complex surface topography because it
retains the papillary projections (24)
. When keratinocytes
are seeded on the surface of this material, they settle in between the
papillary projections and reform rete ridges. In the presence of KGF,
these rete ridges were not formed and we observed a flattening of the
epidermal-dermal junction. One explanation might be that the increased
proliferation and the resulting crowding of the basal layer forced a
stretching or flattening of the epidermal-dermal junction. Consistent
with this interpretation is the increased expression of 5ß1 and
its location to the basal domain of basal cells. Another explanation
might be that KGF induces remodeling of the papillary projections by
induction of various metalloproteases (39
40
41)
.
An interesting observation was KGF’s effect on the junction between the granular and cornified layers. In contrast to the relatively flat interface in controls, high-dose KGF and cells expressing KGF produced undulations in this interface. The cause of this change is unclear, but it may be linked to the increased basal cell density. As this excess of cells moves upward and differentiates, there may be too many cells to be accommodated by a simple flat interface and so an undulating interface of increased surface area is formed. Alternatively, these undulations could arise if the rate of upward migration of cells was not homogenous throughout the skin equivalent. Localized areas where the rate of migration was increased would also have increased numbers of cells arriving at the granular/cornified interface, and undulations might form to accommodate these cells.
Changes to the 3-dimensional organization of the epidermis also occur
in the hyperproliferative disease psoriasis. Clearly the immune system
plays a critical role in this disease (42)
; however, the
nature of the molecular signals that activates the epidermis to undergo
such significant changes to proliferation and cell–cell organization
is largely unknown. Our results demonstrate that in the absence of
immune cells, KGF mediates several but not all of the changes to the
epidermis characteristic of psoriasis such as hyperproliferation,
suprabasal proliferation, and induction of 5ß1 localized to the
basal domain of the basal cells (43)
. Our data are also
consistent with a previous report demonstrating that KGF and its
receptor were up-regulated in psoriasis and that this subsided in
response to an antiproliferative agent (44)
.
In summary, KGF induces dramatic changes to the organization of the
proliferative cells of the epidermis possibly by its induction of
proliferation and an increase in adhesiveness through 5ß1. These
data demonstrate for the first time that a growth factor such as KGF
can induce significant changes to the 3-dimensional organization and
function of a skin equivalent in vitro and provide a model
system for further understanding of KGF’s direct effects on the
epidermis. Moreover, KGF-treated or KGF-expressing grafts may be useful
as part of a tissue engineering/gene therapy approach to repairing
defects of the skin.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication August 22, 2000.
Revision received November 10, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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