(Received for publication, October 19, 1995; and in revised form, January 12, 1996)
From the
Keratinocyte growth factor (KGF) exhibits paracrine action on numerous epithelia, including skin. We have found that cultures of normal human keratinocytes must attain confluence before KGF promotes an increase in cell number relative to untreated controls. In postconfluent cultures, treatment with KGF promoted tight packing of keratinocytes with a small basal cell morphology. Based on these observations, we hypothesized that KGF increased cell number in postconfluent cultures by affecting the ability of normal keratinocytes to undergo terminal differentiation and/or programmed cell death. In support of this hypothesis, keratinocytes treated with KGF produced fewer cross-linked envelopes and exhibited reduced membrane-associated transglutaminase activity relative to cells treated with epidermal growth factor or untreated controls. We also found that nucleosomal fragmentation was reduced in postconfluent cultures of KGF-treated keratinocytes. Furthermore, KGF-treated keratinocytes were more resistant to suspension-induced nucleosomal fragmentation than control or epidermal growth factor-treated cultures. Therefore, it appears that KGF modulates aspects of keratinocyte terminal differentiation which share features with programmed cell death. We propose that stromally-derived KGF may act as a paracrine survival factor in skin and perhaps other renewal tissues.
Keratinocyte growth factor (KGF or FGF-7), ()synthesized and secreted by stromally-derived cells, is a
member of the fibroblast growth factor (FGF) superfamily (Rubin et
al., 1989). The FGFs are a family of structurally related
polypeptide growth factors found to modulate proliferation and
differentiation in a variety of cell types (reviewed by Basilico and
Moscatelli(1992) and Mason(1994)). Early experiments with KGF
demonstrated that it differs from other members of the FGF family by
virtue of its production in cells of mesenchymal origin and its target
cell specificity for epithelial cells (Finch et al., 1989).
The 2.4-kilobase KGF transcript has been detected in the dermal layer
of rodent skin as well as in several fibroblast cell lines; however,
transcripts were not detected in epithelial cell lines, endothelial
cells, or melanocytes (Finch et al., 1989). The KGF receptor
(KGFR) is a splice variant of FGF receptor-2 (FGFR2) encoded by the fgfr-2 gene (Miki et al., 1991, 1992). In skin, KGFR
is localized to the spinous cells directly adjacent to the basal cell
layer (LaRochelle et al., 1995). The expression pattern of KGF
and KGFR, as well as the known biological activities of KGF, suggest
that it functions as a paracrine mediator of epithelial cell growth in
numerous tissues (reviewed by Rubin et al.(1995)).
KGF-mediated mesenchymal-epithelial communication has been shown to
play a role in regeneration of epidermis during wound healing (Werner et al., 1992; Tsuboi et al., 1993; Brauchle et
al., 1994; Staiano-Coico et al., 1993; Pierce et
al., 1994) and appears to be critical for the establishment of
normal skin architecture during development (Guo et al., 1993;
Werner et al., 1994; Finch et al., 1995). In
developing mouse skin, KGF expression targeted to the basal layer by
the keratin 14 promoter results in hyperthickening of the epidermis and
a pronounced increase in cell density of inner cell layers (Guo et
al., 1993). Conversely, blocking KGFR function by targeted
expression of a dominant-negative KGFR to basal keratinocytes of
transgenic mice results in severe atrophy of the epidermis, with basal
cells becoming flattened and exhibiting pyknotic nuclei (Werner et
al., 1994).
The epidermis is a multilayered renewal tissue
composed primarily of keratinocytes. Differentiated keratinocytes are
continuously lost from the surface and replaced by the proliferation of
basal keratinocytes. The rate at which a basal cell initiates and
completes its differentiation program appears to be tightly regulated,
although the molecular controls responsible for such regulation are
ill-defined (reviewed by Fuchs(1993)). In vivo, the final
stages of the terminal differentiation process are characterized by
numerous changes including filaggrin-mediated keratin intermediate
filament bundling (Dale et al., 1978), release of lipids from
membrane-coating granules into the intercellular space (reviewed by
Schurer et al.(1991)), and formation of the cornified envelope
which consists of several proteins that are covalently cross-linked by
isodipeptide bonds catalyzed by the action of calcium-dependent
transglutaminases found in keratinocytes (reviewed by Aeschlimann and
Paulsson(1994) and Reichert et al.(1993)). Ultimately,
keratinocytes lose intracellular organelles and enucleate in the upper
layers of the epidermis. Molecular mechanisms which govern keratinocyte
enucleation and terminal differentiation are poorly understood. Recent
studies in our laboratory ()and those of others (reviewed by
Fesus et al.(1991), Haake and Polakowska(1993), and Polakowska et al.(1994)) suggest that terminal differentiation in
keratinocytes may constitute a specialized form of apoptosis. Although
several studies have suggested a link between specific mediators of
apoptosis and inherited or induced dermatopathologies in humans
(Bianchi et al., 1994; Nakagawa et al., 1994;
reviewed by Haake and Polakowska(1993), Paus et al.(1993), and
Polakowska and Hake(1994)) it has only recently been demonstrated that
apoptosis is a naturally occurring process in normal human skin
(Polakowska et al., 1994).
In the present study, we have investigated the effects of KGF on terminal differentiation and features of apoptosis in cultured epidermal keratinocytes. We examined the effect of KGF on several aspects of terminal differentiation in vitro, including cross-linked envelope (CE) formation and membrane-associated transglutaminase activity, as well as nucleosomal DNA fragmentation, a classical marker of apoptosis. Our studies suggest that KGF can forestall the ability of normal human keratinocytes to both initiate terminal differentiation and undergo features of apoptosis in vitro.
For
suspension studies, preconfluent cultures from each treatment group
described above were removed from culture dishes with 0.5 mM EDTA, 0.1% trypsin and washed in serum-containing medium to
inactivate any residual trypsin. After a short centrifugation (440
g for 3 min), cells were resuspended at 1
10
cells/ml in serum-free and additive-free semi-solid
medium supplemented with BSA, 0.5 nM EGF, or 0.5 nM KGF as indicated and incubated for 18-20 h at 37 °C in
humidified 5% CO
incubators. Semi-solid medium was prepared
as described previously (Sadek and Allen-Hoffmann, 1994) and consisted
of 1.68% methylcellulose (4,000 centipoises, Fisher Scientific) in 3
parts Ham's F-12 plus 1 part Dulbecco's modified
Eagle's medium without serum or additives. Cells were recovered
from suspension by repeated dilution with serum-free medium and
centrifugation (440
g for 10 min). Following two
rinses with phosphate-buffered saline (0.137 M NaCl, 2.7
mM KCl, 8.1 mM Na
HPO
, 1.4
mM KH
PO
pH 7.2) (PBS), cells were
either resuspended in PBS (pH 7.2) to assay for CE formation or lysed
in SLS buffer (50 mM Tris, 10 mM EDTA, pH 8.0, 0.5%
(w/v) sodium lauryl sarcosine) to determine DNA fragmentation. Adherent
controls consisted of keratinocytes treated for similar times in serum-
and additive-free medium supplemented with the indicated growth factor,
rinsed twice with PBS (pH 7.2), and scraped into the appropriate
buffer.
For confluence experiments, keratinocytes were removed from
tissue culture plates with 0.5 mM EDTA, 0.1% trypsin at the
desired level of confluence. Cultures typically became confluent 5 days
following plating at a density of 1.5
10
cells/cm
. Preconfluent cultures were harvested for
assay
4 days postplating. Postconfluent cultures were maintained
in various treatment groups for the indicated times. Keratinocytes were
washed in serum-containing medium, rinsed twice with PBS (pH 7.2) to
remove residual culture medium, and resuspended in PBS (pH 7.2) or
lysed in SLS buffer to assay CE formation and DNA fragmentation,
respectively. For transglutaminase enzyme assays, treatment groups at
different stages of confluence were rinsed with PBS, scraped, and
enzyme activity was immediately assayed.
Intact and fragmented DNA were 3`-end labeled with
[-
P]ddATP using a terminal
dideoxytransferase procedure as described (Tilly and Hsueh, 1993). Each
reaction mixture was comprised of 1
reaction buffer (0.2 M potassium cacodylate, 0.25 mg/ml bovine serum albumin, 25 mM Tris-HCl, pH 6.6), 2.5 mM cobalt chloride, terminal
dideoxynucleotidyl transferase enzyme (from calf thymus, 25 units;
Boehringer Mannheim), 4.25 pmol of
[
-
P]ddATP (specific activity 3,000 Ci/mmol,
Amersham), and 12.75 pmol of unlabeled ddATP. Dideoxynucleotides were
utilized to ensure that only one labeled molecule was incorporated per
3` end. Briefly, DNA (
360 ng) was added to 29 µl of reaction
mixture to yield a final volume of 50 µl and incubated for 60 min
at 37 °C. The reaction was terminated by the addition of 0.25 M EDTA (pH 8.0). Using 50 µg of yeast tRNA (Sigma) as a carrier,
labeled DNA was separated from unincorporated nucleotides by two rounds
of precipitation with 3
volume of cold 100% ethanol in the
presence of 2 M ammonium acetate at -85 °C. DNA was
collected by centrifugation at 16,000
g for 30 min at
4 °C and dissolved in 40 µl of TE buffer. One quarter of each
sample was separated by electrophoresis on a 1.5% agarose gel at 60 V
for 4 h. Gels were dried without heat for 8 h (SE 1200 Eazy Breeze;
Hoefer Scientific, San Fransisco, CA) and visualized by
autoradiography.
Figure 1: KGF treatment maintains basal morphology in postconfluent keratinocytes cultures. Normal human keratinocytes were cultured in standard growth medium supplemented with BSA (A), 1.6 nM EGF (B), or 0.5 nM KGF (C) for 14 days. Bar = 50 µm.
Cultivation with KGF promoted tight packing of keratinocytes with a small basal cell morphology in postconfluent cultures. Based upon these observations, we asked if KGF promoted accumulation of keratinocytes at confluence by sustaining cell growth. To examine this possibility, control keratinocytes were plated at a preconfluent density in defined growth medium. Twenty-four h after plating, cells were treated with 0.5 nM KGF, 0.5 nM EGF, or BSA. Keratinocytes were counted for up to 12 days. Prior to confluence, keratinocyte cell numbers were similar, regardless of treatment, and all treatment groups achieved confluence 7 days after plating (Fig. 2). However, once confluence was attained, KGF-treated cultures accumulated over 2-fold more cells than did EGF-treated or control cultures. We have observed KGF-induced accumulation of keratinocytes following confluence in serum-containing standard medium (0.66 mM calcium) as well. EGF-treatment induced membrane ruffling in preconfluent keratinocyte cultures (data not shown), but did not promote accumulation of keratinocytes in postconfluent cultures. These findings suggest that KGF does not simply act as a mitogen but may affect the balance between proliferation and differentiation once cells have achieved confluence.
Figure 2:
KGF
induces accumulation of keratinocytes after cultures attain confluence.
Keratinocytes were plated at 1 10
cells/cm
in 6-well cluster dishes and treated with defined, serum-free KBM
growth medium supplemented with BSA (
), 0.5 nM EGF
(
), or 0.5 nM KGF (
). Cells were maintained in the
presence of growth factors for 11 days. Confluence is defined as the
point at which the entire surface of the dish is covered by cells.
Values represent the mean of four samples with standard deviations
indicated by the bar.
Figure 3: KGF and EGF attenuate cross-linked envelope formation in postconfluent cultures. Keratinocyte cultures were maintained in standard medium supplemented with BSA (open bar), 0.5 nM EGF (hatched bar), or 0.5 nM KGF (closed bar) for 3 days (preconfluent), 5 days (confluent), or 6-10 days (postconfluent). Detergent-resistant CEs were quantified by hemacytometer counting and data expressed as a percentage of input cell number. Values represent the mean of triplicate samples with standard deviations indicated by the bar.
To confirm that KGF specifically affects CE formation, preconfluent cultures of keratinocytes cultured with or without equimolar concentrations of KGF or EGF were triggered to undergo differentiation by suspension for 18 h in medium made semi-solid with methylcellulose. Many aspects of terminal differentiation, including differential expression of keratins (Drozdoff and Pledger, 1993) and CE formation (Green, 1977), can be triggered in vitro by loss of keratinocyte cell-cell and cell-substratum adhesion. We found that regardless of treatment group, <5% of all adherent preconfluent keratinocytes produce a CE (Table 1). Following 18 h of suspension, control cultures readily produced CEs. However, keratinocytes cultured with KGF and to a lesser extent, with EGF, exhibited an attenuated ability to form CE following suspension. These findings are consistent with our observations that postconfluent keratinocytes cultured in the presence of KGF produced fewer CE when compared to control or EGF-treated postconfluent cultures.
Figure 4: KGF decreases activity of membrane-associated transglutaminase in postconfluent keratinocyte cultures. Normal human keratinocytes were treated with BSA (open bar), 0.5 nM EGF (hatched bar), or 0.5 nM KGF (closed bar) in standard medium for 3 days (preconfluent), 5 days (confluent), or 8 and 10 days (postconfluent). Samples were assayed for TGase activity as described. Values represent the mean of triplicate samples with standard deviations indicated by the bar.
Figure 5:
KGF diminishes fragmentation of
keratinocyte DNA in postconfluent cultures and following suspension. Panel A, DNA was isolated from keratinocyte cultures treated
with BSA, 0.5 nM EGF, or 0.5 mM KGF in standard
medium for 3 days (preconfluent), 5 days (confluent), and 10 days
(postconfluent). Panel B, keratinocyte cultures were treated
with BSA or 0.5 mM KGF in standard medium for 3 days
(preconfluent). Cells were then suspended in the corresponding
serum-free, additive-free semi-solid medium supplemented with BSA or
0.5 nM KGF for 20 h. Identical adherent cultures were also
incubated for 20 h in serum-free, additive-free medium containing BSA
or 0.05 mM KGF. Following treatments, genomic DNA was
isolated, 3` end-labeled with [-
P]ddATP,
displayed on a 1.5% agarose gel, and visualized by
autoradiography.
DNA fragmentation was also assayed by a quantitative sandwich enzyme immunoassay using mouse monoclonal antibodies directed against DNA and BrdUrd. Following BrdUrd incorporation, keratinocytes from treatment groups remained adherent or were suspended in serum-free, additive-free semi-solid medium containing 0.5 nM KGF, 0.5 nM EGF, or BSA for 18 h. Adherent cells were treated with serum-free medium containing KGF, EGF, or BSA for the identical time. Similar to the results obtained with 3` end labeling, adherent cultures exhibited little fragmented DNA regardless of the presence or absence of growth factors (Fig. 6). However, keratinocytes cultivated in KGF-containing medium and suspended in semi-solid medium exhibited substantially less DNA fragmentation compared to suspended control cells. Cultivation in an equimolar concentration of EGF resulted in a modest attenuation of DNA fragmentation in this assay. Taken together, these findings suggest that a novel paracrine effect of KGF is to forestall keratinocyte nucleosomal fragmentation following confluence or suspension-induced differentiation.
Figure 6: KGF decreases DNA fragmentation in suspended epidermal keratinocytes. Keratinocyte cultures were treated with standard medium supplemented with BSA, 0.5 nM EGF, or 0.5 nM KGF for 3 days (preconfluent). Cells in each treatment group were then labeled for 6 h with 10 µM BrdUrd. Labeled cells were suspended in their corresponding serum-free, additive-free semi-solid media supplemented with BSA (open bar), 0.5 nM EGF (hatched bar), or 0.5 nM KGF (closed bar) for 18 h. Identical adherent cultures were also labeled and incubated in serum-free, additive-free medium containing the indicated growth regulatory agents. Adherent and suspended normal human keratinocytes were lysed and supernatants containing labeled DNA fragments were quantified using the Cellular DNA Fragmentation ELISA. Values represent the mean of triplicate samples with standard deviations indicated by bar.
We have found that cultivation with KGF sustains proliferation of human keratinocytes after cultures have achieved confluence. Following confluence, KGF-treated cultures exhibit both a decreased capacity to form CEs and reduced membrane-associated TGase activity compared to control or EGF-treated cultures. Furthermore, internucleosomal DNA fragmentation of keratinocyte DNA, a hallmark of apoptosis, is reduced by cultivation with KGF. Our findings suggest that KGF plays an important role in modulating features of terminal differentiation that may be related to programmed cell death.
Little is known about the specific biological activities of KGF in human keratinocytes. It is generally accepted that KGF, which is produced by dermal fibroblasts, acts in a paracrine manner on keratinocytes in the overlying epidermis (Finch et al., 1989). It has been previously shown that KGF is a potent mitogen for differentiation-defective murine keratinocytes, BALB/MK, inducing up to a 500-1000-fold increase in thymidine incorporation (Rubin et al., 1989). In normal murine keratinocytes, KGF has been shown to increase cell number approximately 2-fold following 7 days of treatment (Dlugosz et al., 1994). Marchese and co-workers(1990) also reported that KGF produced increases in human keratinocyte proliferation as measured by cumulative cell number. In both reports, the cell growth experiments were plated at high cell densities which likely resulted in cultures reaching confluence early during the treatment periods. We found that neither KGF nor EGF enhanced cell proliferation in low density cultures. Prior to confluence, there was no difference in cumulative cell numbers between control, EGF-treated, and KGF-treated keratinocytes. Following confluence, only KGF-treated cultures continued to accumulate cells while cell numbers in EGF-treated or control cultures appeared to plateau. Our findings with cultured human keratinocytes are consistent with a recent report on skin abnormalities exhibited by transgenic mice overexpressing KGF targeted to basal cells by the human keratin 14 promoter (Guo et al., 1993). Specifically, KGF expression resulted in an unusually high density of tightly packed keratinocytes in the epidermis. These mice had a pronounced thickening of the epidermis, primarily the result of an increase of cells in the spinous and granular layers. Taken together, these data suggest that KGF promotes accumulation of keratinocytes that have retained proliferative potential.
We
observed that EGF did not affect the rate of cell growth or increase
the accumulation of keratinocytes into tightly packed cell monolayers
after confluence. Early experiments by Rheinwald and Green(1977) and
subsequent studies by Barrandon and Green(1987) also found that
multiplication of small colonies of keratinocytes undergoing
exponential growth was unaffected by EGF or TGF-. They concluded
that the effects of EGF and TGF-
on multiplication are dependent
upon stimulation of migration. More recently, Dlugosz and co-workers
(1994) demonstrated that KGF increases steady state TGF-
mRNA
levels and secretion of TGF-
in primary cultures of keratinocytes
isolated from newborn BALB/c mice. Given these data, it is reasonable
to postulate that KGF-induced TGF-
expression may contribute to
the accumulation of keratinocytes we have observed in postconfluent
cultures by activating EGF receptor signaling. However, our studies
show that EGF treatment does not promote tightly packed monolayers in
postconfluent cultures as does KGF treatment. Furthermore, mice
overexpressing TGF-
driven by the keratin 14 promoter also do not
exhibit the increase in cell density or cell packing observed in KGF
transgenic mice (Vassar and Fuchs, 1991; Guo et al., 1993).
Given the divergent patterns of cell density and cell packing exhibited
by KGF and TGF-
transgenic mice and our similar findings with KGF
and EGF-treated human keratinocyte cultures, it is unlikely that KGF
action is mediated solely through increased production of keratinocyte
TGF-
.
The present study shows that KGF treatment reduces nucleosomal fragmentation in postconfluent cultures of keratinocytes. Furthermore, we found that KGF-treated preconfluent cultures exhibit decreased nucleosomal fragmentation following loss of cell-cell and cell-substratum adhesion. It has been well documented that keratinocytes from all species studied to date can undergo apoptosis both in vivo and in vitro (Marthinuss et al., 1995a, 1995b; McCall and Cohen, 1991; Bianchi et al., 1994; Frisch and Francis, 1994; Sayama et al., 1994; Schwarz et al., 1995). Epidermal keratinocytes are destined to enucleate and lose metabolic activity as part of their differentiation pathway. As basal keratinocytes of intact skin proliferate, cells which leave the basal compartment progress upwards through a program of terminal differentiation. Fully differentiatied cells are eventually shed from the skin surface. A balance between basal cell proliferation and cell loss is required for the maintenance of normal epidermal architecture. Budtz and Spies(1989) studied the natural deletion of skin cells in the African toad, Bufo bufo, and demonstrated that apoptosis was one of the mechanisms by which surplus cells were lost. Using TUNEL analysis, Polakowska and co-workers(1994) demonstrated that intact tissue from developing fetal and adult human skin contains cells with DNA breaks in their nuclei. Keratinocytes that exhibited nuclear changes associated with apoptosis were located primarily in the upper granular layer and only occasionally in the spinous layer of epidermis. Because KGF-treated postconfluent cultures exhibited reduced nucleosomal fragmentation, it is reasonable to postulate that KGF promotes accumulation of cells by inhibiting basal cell loss through cell death pathways, such as apoptosis.
Many
parallels exist between keratinocyte terminal differentiation and
apoptosis. For example, mature cornified envelopes and apoptotic bodies
are transglutaminase cross-linked end-products of terminal
differentiation and programmed cell death pathways, respectively. The
amino acid composition of cornified envelopes from human skin and
apoptotic bodies from liver are very similar and show almost identical
peptide mapping patterns (Tarcsa et al., 1992). It has been
suggested that terminal differentiation of keratinocytes may actually
be a specialized form of apoptosis (Polakowska and Hake, 1994;
Polakowska et al., 1994; Fesus et al., 1991; Paus et al., 1993; Alison and Sarraf, 1992). We found that KGF
reduced CE and membrane-associated TGase activity in postconfluent but
not subconfluent cultures. These finding may, in part, explain the
basal-like, tightly packed morphology of KGF-treated postconfluent
cultures in contrast to the large, flattened cells observed in control
and EGF-treated cultures. Equimolar concentrations of EGF were not as
effective as KGF in reducing CE formation and membrane-associated TGase
activity in postconfluent keratinocyte cultures. Marchese and
co-workers(1990) reported that EGF, but not KGF, forestalls expression
of the differentiation-specific keratin K1 as well as filaggrin in
response to calcium-induced differentiation. This report, in
conjunction with the phenotypes exhibited by TGF- and KGF
transgenic mice suggest that these two growth regulatory factors most
likely have differing biological activities in keratinocytes. Werner
and co-workers (1994) recently reported that mice expressing a
dominant-negative KGF receptor transgene targeted to basal epidermal
keratinocytes exhibited severe atrophy of the epidermis. Specifically,
the transgenic basal keratinocytes contained pyknotic nuclei and
premature onset of cornification. It was concluded that addition of the
dominant-negative KGF receptor disturbed the balance between
proliferation and differentiation. Our findings of reduced nucleosomal
fragmentation, CE formation, and membrane-associated TGase activity in
postconfluent or suspended keratinocyte cultures suggest that KGF, and
to a lesser extent EGF, promotes survival of keratinocytes signalled to
differentiate. We propose that KGF may be functioning, in part, as a
survival factor, thus affecting those aspects of keratinocyte
differentiation which share features with cell death, such as
enucleation and formation of CE (cornification).