(Received for publication, September 14, 1995; and in revised form, October 25, 1995)
From the
We have investigated the role of transforming growth
factor-1 (TGF-
1) in suspension-induced programmed cell death
of cultured human keratinocytes. Suspension of keratinocytes in
semisolid medium induces TGF-
1 mRNA levels and synthesis of
bioactive TGF-
1 protein. Concomitant with the suspension-induced
increase in secreted TGF-
1 levels, steady state mRNA levels for
c-myc are decreased. Both exogenously added and endogenously
produced TGF-
1 attenuate suspension-induced nucleosomal
fragmentation in keratinocytes. We propose that TGF-
1 may function
to protect keratinocytes from DNA fragmentation following loss of
cell-substratum and/or cell-cell contact. Taken together, our findings
suggest that loss of cell-substratum and/or cell-cell adhesion is an
important component of an apoptotic signal transduction cascade
regulated by TGF-
1 in normal human stratified squamous epithelia.
We have described the morphological and biochemical features of
apoptosis in cultured normal human keratinocytes and identified loss of
cell-cell and/or cell-substratum contact as an important signal for
keratinocyte cell death in vitro. ()The pleiotropic
molecule transforming growth factor
1 (TGF-
1) (
)has been shown to be a potent modulator of the apoptotic
process in a variety of cell types. In certain simple epithelial cell
types and hematopoietic cells, TGF-
1 has been reported to induce
programmed cell death (Oberhammer et al., 1992; Rotello et
al., 1991; Lotem and Sachs, 1992). TGF-
1 also inhibits the
growth of normal human keratinocytes in vitro (Pietenpol et al., 1990). Based on studies using normal human
keratinocytes and the murine keratinocyte cell line, BALB/MK, it is
known that TGF-
1 inhibits keratinocytes in the G
phase
of the cell cycle (Shipley et al., 1986) through a mechanism
that most likely involves transcriptional down-regulation of c-myc expression (Pietenpol et al., 1990; Munger et
al., 1992). In the present study, we identify TGF-
1 as an
important inhibitor of apoptosis in cultured normal human
keratinocytes. We propose that TGF-
1 may function as a signal to
forestall apoptosis as well as replication in the basal layer of human
skin.
Cells were suspended following removal
from tissue culture plates with 0.5 mM EDTA, 0.1% trypsin,
washed with serum-containing medium to inactivate residual trypsin,
then with serum-free medium, and suspended in the desired semisolid
medium at a density of 1 10
cells/ml in sterile
50-ml polypropylene tubes. Suspended cells were incubated at 37 °C
in a humidified 5% CO
atmosphere. Cells were recovered from
suspension at various times by repeated dilution of the semisolid
medium with serum-free medium followed by centrifugation at 440
g. Adherent control cultures were treated with medium alone
for the indicated times.
Intact and fragmented DNA was
3`-end-labeled with [-
P]ddATP using
terminal dideoxynucleotidylexotransferase as described by Tilly and
Hsueh (1993). Briefly, each reaction mixture contained 1
reaction buffer (0.2 M potassium cacodylate, 25 mM Tris-HCl, 0.25 mg/ml bovine serum albumin, pH 6.6), 2.5 mM CoCl
, 25 units of terminal
dideoxynucleotidyltransferase (Boehringer Mannheim), and
[
-
P]ddATP (specific activity of 3000
Ci/mmol, Amersham Corp.) diluted with cold ddATP to a final
concentration of 340 pM ddATP or 12.5 µCi/reaction. Each
reaction was initiated by the addition of 360 ng of sample DNA,
incubated for 60 min at 37 °C, and terminated by the addition of 25
mM EDTA, pH 8.0, final concentration. Labeled DNA was
separated from unincorporated [
-
P]ddATP by
precipitation with ice-cold 100% ethanol in the presence of 2 M ammonium acetate and 50 µg of yeast tRNA (Sigma) followed by a
second precipitation with ice-cold 100% ethanol. The pellet was
collected by centrifugation, rinsed in ice-cold 80% ethanol, dissolved
in 50 µl of TE buffer, pH 8.0, and stored at -20 °C. One
half of each labeled sample was loaded onto a 1.5% agarose gel and
electrophoresed. Gels were dried without heat using a vacuum dryer
(Bio-Rad) or with heat using a SE 1200 Easy Breeze (Hoeffer Scientific,
San Francisco, CA) and exposed to X-Omat photographic film.
Figure 1:
Suspension increases steady state
TGF-1 mRNA levels in cultured normal human keratinocytes. Adherent
keratinocytes (0 h), strains SK-2-Ep and NS-2-Ep, were
suspended for 6, 12, 24, and 72 h in serum-free, semisolid growth
medium without EGF. Poly(A)
RNA was prepared from each
time point, and 3 µg of RNA was loaded per lane. Northern analysis
was performed using random primer-labeled
[
P]cDNA probes for TGF-
1 and
c-myc. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was used as a loading control. Kb,
kilobases.
We next determined if suspended keratinocytes produced increased
levels of biologically active TGF-1. Keratinocytes were suspended
in serum-free and additive-free semisolid medium for 0, 12, 24, and 48
h. Following suspension, keratinocytes were removed by centrifugation
to generate keratinocyte-conditioned semisolid medium. To control for
lysis of keratinocytes following centrifugation, keratinocytes were
suspended in semisolid medium and immediately removed (t = 0). Anchorage-independent growth of NRK-49F cells was
used as a bioassay to determine the presence of biologically active
TGF-
1 in keratinocyte-conditioned medium. Neutralizing
anti-TGF-
1 antibody was added to the keratinocyte-conditioned
medium to confirm that TGF-
1 alone was inducing
anchorage-independent growth. As seen in Fig. 2,
keratinocyte-conditioned medium supports the anchorage-independent
growth of the NRK-49F indicator cells. Surprisingly, non-acidified
keratinocyte-conditioned medium supported NRK-49F colony formation as
well as acidified medium (data not shown). These results demonstrate
that the number of anchorage-independent colonies is proportional to
the amount of time the keratinocytes conditioned the semisolid medium.
Suspended keratinocytes typically produced the equivalent of between 50
and 100 pg of recombinant human TGF-
1/ml of semisolid medium.
Inhibition of TGF-
1 with neutralizing anti-TGF-
1 antibody
decreased NRK-49F colony formation to background levels. Twice as much
antibody was required to neutralize the activity in 48-h
keratinocyte-conditioned medium. Therefore, loss of keratinocyte
cell-cell and/or cell-substratum interaction is an inducer of
TGF-
1 gene expression and ultimately promotes increased synthesis
and perhaps activation of this epithelial growth inhibitor.
Figure 2:
Conditioned medium from suspended
keratinocytes contains active TGF-1. Cultured keratinocytes were
suspended in serum-free, additive-free, semisolid growth medium for 12,
24, and 48 h to generate conditioned semisolid medium (CM).
Keratinocytes were removed from the conditioned medium by
centrifugation without dilution of the semisolid medium. To control for
possible cell lysis during centrifugation, keratinocytes were suspended
in semisolid medium and immediately removed by centrifugation (0 h
conditioned semisolid medium). Indicator cells, NRK-49F, were assayed
for anchorage-independent growth in the keratinocyte-conditioned media.
Conditioned medium was incubated with or without a neutralizing
anti-human TGF-
1 antibody (2.5 µg/ml) for 1 h prior to the
addition of NRK-49F cells. As a control for background levels of
anchorage-independent growth, NRK-49F cells were suspended in semisolid
medium supplemented with EGF alone. A standard curve for
TGF-
1-dependent anchorage-independent NRK-49F cell growth was
generated for each experiment using the following concentrations of
recombinant TGF-
1: 0.05, 0.1, 0.5, 1.0, and 2.0 ng/ml semisolid
medium. After 7 days, anchorage-independent colonies of NRK-49F cells
were scored. Data represent the average number (mean ± S.D. of
three replicates per condition) of colonies (>60 µm) per
well.
Figure 3:
TGF-1 inhibits internucleosomal
cleavage in suspended keratinocytes. Keratinocytes (NS-2-Ep) were
suspended for 24 h in serum-free, semisolid growth medium containing 5
ng/ml TGF-
1 or an equal volume of diluent (4 mM HCl, 1
mg/ml bovine serum albumin). Adherent control cells were refed with
serum-free growth medium containing 5 ng/ml TGF-
1 or diluent at
the beginning of the treatment period. A, keratinocytes were
plated onto mitomycin C-treated 3T3 feeder layers to determine the
effect of treatments on replicative potential in surface culture. After
14 days, cultures were fixed with formaldehyde, stained with methylene
blue, and counted. Data represent the average colony-forming efficiency (CFE, mean ± S.D. of six replicates per treatment) for
each condition. B, following treatment, genomic DNA was
isolated, labeled on 3`-ends with
[
-
P]ddATP, displayed on a 1.5% agarose gel,
and subjected to autoradiography.
Figure 4:
Neutralization of keratinocyte-derived
TGF-1 promotes internucleosomal cleavage in suspended
keratinocytes. Keratinocytes (NS-2-Ep) were suspended for 24 h in
serum-free semisolid growth medium in the presence or absence of a
neutralizing antibody (Ab) to human TGF-
1. Adherent
control cells received serum-free growth medium with or without
anti-TGF-
1 antibody at the beginning of the 24-h treatment period.
Following treatments, genomic DNA was isolated, labeled on 3`-ends with
[
-
P]ddATP, displayed on a 1.5% agarose gel,
and subjected to autoradiography.
We found that suspended keratinocytes contained increased
levels of both TGF-1 mRNA and activated protein. TGF-
1 is an
autocrine growth factor that inhibits the growth of adherent human
keratinocytes in vitro (Shipley et al., 1986;
Pietenpol et al., 1990). Suspension of keratinocytes also
results in a decrease in the steady state mRNA levels of
c-myc. This finding is consistent with the known effects of
suspension and TGF-
1 on the growth of keratinocytes. We suspect
that the suspension-induced decrease in c-myc mRNA levels may
be mediated, in part, through increased production of TGF-
1. The
role of TGF-
1 in terminal differentiation or programmed cell death
in stratified squamous epithelia is unclear. In vivo,
TGF-
1 plays a role in the maintenance of normal tissue
architecture in rodent skin. For example, transgenic mice homozygous
for a targeted disruption of the TGF-
1 locus exhibit epidermal
hyperproliferation as measured by an increase in the labeling index of
basal cell nuclei following administration of 5-bromo-2`-deoxyuridine
(Glick et al., 1993). However, these TGF-
1 null mice
express the differentiation-specific keratins, K1 and K10, normally
(Glick et al., 1993). Based on these reports and our findings
it is reasonable to postulate that TGF-
1 may exert its effect on
epidermal homeostasis by affecting the number of cells present in the
tissue as opposed to directly affecting the synthesis and/or
organization of differentiation-specific proteins.
We have recently
found that suspension of normal human keratinocytes induces both
morphological and biochemical features of programmed cell death, such
as membrane blebbing and DNA fragmentation. Because
TGF-
1 has been implicated in the regulation of programmed cell
death in a variety of epithelial cell types (Rotello et al.,
1991; Oberhammer et al., 1991; Bursch et al., 1993),
we determined if endogenous production of TGF-
1 played a role in
suspension-mediated apoptotic signaling in keratinocytes. In contrast
to findings in hepatocytes and hematopoietic and lymphoid cells, we
found that TGF-
1 protected against suspension-induced DNA
fragmentation in keratinocytes. Treatment of suspended keratinocytes
with exogenously added TGF-
1 dramatically attenuated
suspension-induced nucleosomal fragmentation. Furthermore, neutralizing
endogenously produced TGF-
1 activity in suspended keratinocytes
increased DNA fragmentation. Neither DNA fragmentation nor
colony-forming efficiency was affected by treatment of adherent cells
with TGF-
1. It is possible that TGF-
1 delays the onset of
suspension-induced DNA fragmentation rather than blocking the apoptotic
response altogether. Nevertheless, it is clear that TGF-
1 does not
promote apoptosis in adherent or suspended human keratinocytes in
vitro. Based on these findings, we propose that TGF-
1
functions to protect cultured keratinocytes from suspension-induced DNA
fragmentation. Consistent with this interpretation, TGF-
1 is
present exclusively in the basal layer of normal mouse epidermis (Glick et al., 1993) where it may serve to protect against apoptosis
in the replicative compartment of skin. TGF-
1 also increases
expression of cell adhesion molecules such as fibronectin, collagens,
and their cognate cell surface receptors (reviewed by Massague(1990)),
thereby potentially increasing cellular adhesiveness. Our findings may
have important implications for the current understanding of normal
epidermal wound healing (reviewed by Gailit and Clark(1994)). For
example, increased endogenous levels of TGF-
1, as well as that
contributed by platelets, may serve to forestall injury-induced
apoptosis in a wound while increasing the production of extracellular
matrix and keratinocyte adhesiveness. TGF-
1, although a potent
keratinocyte growth inhibitor, may serve to forestall apoptosis in this
renewal tissue during times of tissue regeneration.