From the Department of Dermatology and Cutaneous Biology and the
Kimmel Cancer Center, Thomas Jefferson University,
Philadelphia, Pennsylvania 19107 and the Department of
Microbiology and Immunology and the Sylvester Comprehensive Cancer
Center, University of Miami School of Medicine,
Miami, Florida 33101
Received for publication, September 7, 2000, and in revised form, November 21, 2000
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ABSTRACT |
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Previous work has shown that the epidermal growth
factor receptor (EGFR) tyrosine kinase moiety provides protection to
normal human keratinocytes against apoptosis. This protection is, at least in part, due to EGFR-dependent expression of the
antiapoptotic Bcl-2 family member, Bcl-xL. Here we
focused on intracellular signaling pathways relevant to keratinocyte
survival and/or Bcl-xL expression. By using pharmacological
inhibitors and dominant negative expression constructs, we observed
that phosphatidylinositol 3-kinase/AKT and phospholipase C Extracellular signals are essential to the survival of normal
cells in multicellular organisms. Appropriate signals are transmitted in cell type-specific fashion by growth factor/cytokine, cell-matrix adhesion, or cell-cell adhesion receptors. Removal of essential survival signals triggers physiological cell death by default. Recent
studies have implicated many cell surface receptors previously known to
affect cell cycle progression and proliferation also in cellular
survival. For example, normal human keratinocytes require activation of
the epidermal growth factor receptor
(EGFR)1 by exogenous or
endogenous signals to survive (1-4). Specifically, EGFR blockade using
a ligand-antagonistic monoclonal antibody results in enhanced
susceptibility to cell death provoked by cellular stressors such as
passaging (1) or UVB
irradiation.2 Several lines
of evidence highlight a prominent role of Bcl-xL, an
anti-apoptotic Bcl-2 family member, in EGFR-dependent
keratinocyte survival. First, activation of the EGFR on keratinocytes
by exogenous or endogenous ligands is associated with up-regulation of
Bcl-xL expression (2). Second, induction of cell death by
EGFR blockade is a slow process requiring at least several days of EGFR
blockade and is preceded by down-regulation of Bcl-xL (1).
Third, forced expression of Bcl-xL in immortalized
keratinocytes rescues these cells from cell death associated with EGFR
blockade (4). EGFR-dependent tyrosine phosphorylation
appears to be required for protection against cell death because
EGFR-selective tyrosine kinase inhibitors of the tyrphostin class cause
Bcl-xL down-regulation and cell death in a manner similar
to the EGFR-antagonistic monoclonal antibody (2). Taken together, these
earlier results implicate signal transduction pathways activated by
EGFR-dependent phosphorylation events in keratinocyte survival.
The multiplicity of cell surface receptors activated by exogenous
signals is contrasted by the relative uniformity of intracellular signaling pathways triggered by these receptors. For example, EGFR
activation leads to activation of parallel signal transduction pathways
including SRC, RAS, phosphatidylinositol (PI)-3 kinase, signal
transducer and activator of transcription (STAT) 3, and phospholipase
(PLC) In this study, we attempted to distinguish signaling pathways
obligatory to keratinocyte survival, which depend on EGFR activation from those that are required independently of EGFR activation. By using
a combination of small molecular weight inhibitors and dominant
negative expression constructs, we probed the contribution of several
signaling cascades including the SRC, PI 3-kinase/AKT, PLC Materials and Cells--
Properties of the EGFR antagonistic mAb
425 have been described earlier (6-9). The MEK inhibitor PD98059, PI
3-kinase inhibitor LY294002, and tyrphostins AG1478 and AG1295 were
purchased from Calbiochem-Novabiochem. SRC inhibitors PP1 and PP2, the
control compound PP3, PLC
Normal foreskin keratinocytes and HaCaT cells were maintained in
culture as described earlier (10). For treatment with inhibitors cells
were seeded at subconfluency in complete MCDB medium (4). After
attachment medium was replaced with base MCDB medium (4), supplemented
with insulin as the only proteinaceous growth factor. Inhibitors were
diluted from Me2SO stocks directly into the culture medium. The Me2SO concentration was adjusted to 0.5% in
all conditions including controls. After 48 h cells were harvested
for analysis as described below.
cDNA Constructs and Transfections--
A plasmid containing
dominant negative MEK1 cDNA (pMCL-MKK1-8E (11)) was kindly provided
by Dr. Natalie Ahn. The MKK1-8E mutant carries a methionine
substitution in the active center of the kinase domain (Lys-79) and is
HA-tagged at the N terminus. MKK1-8E was cloned into pCEPTetP to create
pCEPTetP-MKK1-8E. Constructs encoding dominant negative STAT3 mutants
pCAGGS-STAT3D and pCAGGS-STAT3F were a generous gift from Dr. T. Hirano
(12). In STAT3F, the C-terminal tyrosine (Tyr-705) has been changed to
phenylalanine, which prevents phosphorylation of this mutant. The
replacement of two glutamic acids (Glu-34 and Glu-35) in the DNA
binding domain of the STAT3D mutant renders this protein unable to bind
DNA. The Stat3D and Stat3F fragments are HA-tagged and were cloned as
NheI/BamHI fragments into pCEPTetP to create
pCEPTetP-Stat3D and pCEPTetP-Stat3F. A dominant negative AKT-encoding
plasmid was obtained from Dr. Thomas F. Franke (pCMV-HA-AKT(K179M))
(13). By replacing a lysine (Lys-179) with methionine in the kinase domain, it has been rendered inactive. HA-AKT(K179M) was subcloned into pCEPTetP.
Transfections were performed using Fugene 6 (Roche Molecular
Biochemicals). Briefly, cells were seeded at a density of ~3 × 103/cm2 in complete MCDB medium and allowed to
attach overnight. Transfections were performed 1 day after seeding
using 0.5 µg of DNA and 1.7-2.0 µl of Fugene 6 per 105
cells following the manufacturer's protocol. Selection was started 48-72 h after transfection using 0.2 mg/ml G419 (Mediatech, Herndon, VA), 0.1 mg/ml hygromycin B (Roche Molecular Biochemicals), and 2 µg/ml tetracycline (Sigma).
Reporter Gene Assays--
Transient transfections using a
1.1-kilobase pair Bcl-xL promoter fragment cloned into pGL3
(Promega) driving expression of luciferase were performed as described
(14). Luciferase and Immunoblot Analyses--
For Western blot analyses, attached
cells were washed once in cold PBS and then lysed in 1× nonreducing
Laemmli buffer followed by boiling for 3-5 min. Protein content was
determined using the BCA method (Pierce). Equal amounts of protein were
separated by SDS-polyacrylamide gel electrophoresis and blotted onto
polyvinylidene difluoride membranes (Millipore). Membranes were treated
with blocking buffer (5% dry milk in PBS or 5% dry milk, 0.05% Tween 20 (Sigma) in TBS). Primary antibodies were diluted in PBS or in TBS
containing 5% BSA, 0.05% Tween 20 and incubated with membranes for
12 h at 4 °C followed by incubation (1 h at room temperature) in dilutions of horseradish peroxidase-conjugated secondary antibodies in the same buffers. Following antibody incubations membranes were
washed in 0.5% Tween 20 in PBS or TBS. Signals were visualized by
chemiluminescence using reagents from Pierce according to the manufacturer's instructions. After detection, blots were washed and
inactivated by staining with SG substrate (Vector Labs) and used for
further antibody incubations.
Kinase Assays--
AKT and MAPK assays were performed using
nonradioactive kits manufactured by New England Biolabs. Briefly, cells
were treated for 48 h and lysed in 1× lysis buffer. Equal amounts
of proteins were immunoprecipitated with either an immobilized
phospho-MAPK antibody that only cross-reacts with the phosphorylated,
i.e. active, form of MAPK or with an immobilized AKT
antibody that recognizes both active and inactive AKT. The immobilized
precipitated enzymes were then used for kinase assays using either
ELK-1 (for MAPK) or GSK-3 Apoptosis Detection Assays--
To detect DNA nicks and breaks
as markers of apoptotic cell death, TUNEL assays were performed.
Briefly, floating and attached cells were collected and fixed in 70%
ethanol at 4 °C. Prior to staining with Biotin-16-dUTP (Roche
Molecular Biochemicals), cells were washed in PBS containing 1% BSA.
Staining was performed using TdT enzyme and buffers purchased from
Roche Molecular Biochemicals for 1 h at 37 °C. After the TUNEL
reaction cells were stained with 2.5 µg/ml fluorescein
isothiocyanate/Avidin (Vector Laboratories, Burlingame, CA) in 4× SSC
(1× SSC is 0.15 M NaCl, 0.015 sodium citrate), 0.1%
Triton X-100, 5% (w/v) nonfat dry milk followed by a wash with PBS
containing 1% BSA and 0.1% Triton X-100. Cells were analyzed without
delay using a FACScan cytometer (Coulter, Fullerton, CA).
Effects of EGFR Blockade on Major Signal Transduction Pathways in
Human Keratinocytes--
To identify signal transduction pathways
relevant to EGFR-dependent Bcl-xL expression or
survival of human keratinocytes, we first determined the effects of
EGFR blockade by EGFR-antagonistic mAb 425 on several pathways
previously shown to be affected by EGFR engagement in different cell
types. These included PI 3-kinase/AKT (15, 16), MEK/MAPK (17, 18),
STAT3 (19, 20), and PLC
STAT3 was expressed prominently in HaCaT keratinocytes but was not
phosphorylated under steady-state conditions (not shown). Similarly,
short term stimulation of HaCaT cells (Fig. 1C) or normal
primary keratinocytes (not shown) with EGF (10 ng/ml) failed to
detectably induce STAT3 phosphorylation although it was associated with
robust induction of MAPK phosphorylation that was inhibitable by the
EGFR-selective tyrphostin AG1478. This was in contrast to A431 squamous
carcinoma cells that contained comparatively high levels of
constitutively phosphorylated STAT3 and in which STAT3 phosphorylation
was further stimulated by exogenous EGF. Inhibition of the EGFR
tyrosine kinase moiety by use of AG1478 abrogated the effect of EGF on
STAT3 and MAPK phosphorylation in A431 cells but did not affect
base-line STAT3 phosphorylation in these cells. Previously, STAT3
phosphorylation was observed in primary mouse keratinocytes after
placing them into forced suspension culture (27). Consistent with these
earlier findings we also observed STAT3 phosphorylation in primary
keratinocytes placed in suspension culture (not shown). This result
demonstrates that STAT3 phosphorylation was inducible in human
keratinocytes albeit not through activation of the EGFR.
Similar to STAT3, we did not observe PLC Effects of Pharmacological Inhibitors of Signaling Pathways on
Keratinocyte Survival--
We next examined the effects of small
molecular weight compounds commonly used to inhibit signaling through
PI 3-kinase/AKT, PLC Effects of Pharmacological Inhibitors of Signaling Pathways on
Bcl-xL Protein Expression--
Our previous studies
demonstrated that EGFR blockade is associated with down-regulation of
Bcl-xL mRNA and protein expression prior to
manifestation of cell death and that Bcl-xL overexpression obviates the enhanced apoptosis susceptibility observed in
keratinocytes treated with EGFR inhibitors (2, 4). Therefore, we
determined the effects of signal transduction inhibitors on
Bcl-xL protein expression as determined by Western blot
analysis. These experiments were done after 48 h of incubation
with the respective compound. As shown in Fig.
2 and consistent with a very recent
report (28), inhibition of either SRC or SRC-like kinases by PP1 was
associated with down-regulated Bcl-xL protein expression in
HaCaT cells. Similarly, treatment with the MEK inhibitor PD98059 was
associated with consistently lower steady-state Bcl-xL
protein levels. By contrast, inhibition of PI 3-kinase, PLC Dominant Negative AKT Induces Keratinocyte Apoptosis Independently
of Bcl-xL Expression--
To obtain independent evidence
for the requirement of PI 3-kinase/AKT signaling for keratinocyte
survival, we generated HaCaT cells, which conditionally expressed a
dominant negative AKT construct under tetracycline control. This
construct encodes an AKT derivative rendered kinase-inactive by point
mutation within the AKT catalytic domain (13); upon overexpression in
HaCaT cells it was functional as demonstrated by its ability to
down-regulate GSK-3 Dominant Negative MEK Down-regulates Bcl-xL Expression
and Impairs Keratinocyte Survival--
To target the MEK/MAPK pathway
specifically, we used a dominant interfering MEK construct that has no
kinase activity (11). Upon transfection into HaCaT cells, we first
confirmed conditional expression of the MKK1-8E construct (Fig.
4A). When overexpressed, MKK1-8E induced spontaneous apoptosis in HaCaT cells (Fig.
4B) consistent with an important role of
MEK-dependent signaling in maintaining keratinocyte
survival. Furthermore, overexpression of MKK1-8E was accompanied by
moderately but consistently down-regulated Bcl-xL
expression in HaCaT keratinocytes (Fig. 4A); in different experiments Bcl-xL expression levels were reduced by
30-50% in MKK-8E overexpressing HaCaT keratinocytes when compared
with uninduced or mock-transfected HaCaT cells. This result was
consistent with the observation that the MEK inhibitor PD98059 reduced
Bcl-xL expression in HaCaT cells (see above). Next, we
assessed the effect of transiently transfected MKK1-8E on
Bcl-x promoter activity. To this end, we used a 1.1-kilobase
pair Bcl-x promoter fragment driving expression of the
luciferase gene in HaCaT keratinocytes (Fig.
5). We observed consistent
down-regulation of promoter activity (40-60%) in cells cotransfected
with MKK1-8E when compared with cells that were transfected with a mock
construct or with HA-AKT(K179M).
No Effect of Dominant Negative STAT3 on Bcl-xL
Expression or Keratinocyte Survival--
A recent study identified
STAT3 as a critical mediator of EGFR-induced signals in support of
Bcl-xL expression in squamous carcinoma cells (29).
Consistent with this idea EGF treatment of A431 squamous carcinoma
cells induced STAT3 phosphorylation (see Fig. 1). However, in HaCaT
cells, no measurable STAT3 phosphorylation was observed in the absence
or the presence of exogenous EGF. This result suggested that STAT3
signaling downstream of the EGFR was not relevant to survival or
Bcl-xL expression in HaCaT cells. To test this contention
directly, HaCaT-derived cells were generated in which either one of the
two dominant negative STAT3 constructs (STAT3D and STAT3F) were
conditionally expressed using the tetracycline-regulated expression
system. As shown in Fig. 6A,
strong transgene expression was achieved in tetracycline regulatable
fashion. However, even after induction of DN STAT3 expression for 2-4
days no significant change in Bcl-xL protein expression was
evident (Fig. 6). Similarly, keratinocyte survival was not affected by
overexpression of either of the two dominant negative STAT3 constructs
for 2 days (Fig. 6B). These results were consistent with the
observation that, although expressed in HaCaT keratinocytes, STAT3
plays a minor role in EGFR-induced signal transduction in this cell
line (see Fig. 1).
The most salient findings of this study can be summarized as
follows. 1) Activation of multiple signaling pathways including SRC
kinases, MEK1, PI 3-kinase/AKT, and PLC/protein
kinase C
activation were required for keratinocyte survival
independently of EGFR activation or Bcl-xL expression. By
contrast, MEK activity required EGFR activation and, as shown by use of
the MEK inhibitor PD98059 and a dominant negative MEK construct, was
necessary for Bcl-xL expression and survival. Consistent
with an earlier study, blocking SRC kinase activities similarly led to
down-regulation of Bcl-xL protein expression and impaired
keratinocyte survival. In conclusion, our results demonstrate that
EGFR-dependent MEK activity contributes to both
Bcl-xL expression and survival of normal keratinocytes. Other signaling pathways (i.e. phosphatidylinositol
3-kinase/AKT and phospholipase C
/protein kinase C
) are obligatory
to keratinocyte survival but not to Bcl-xL expression, and
control of these pathways by EGFR activation is not rate-limiting to
normal keratinocyte survival.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in different cell types (reviewed in Ref. 5). The same
signaling components are also affected by engagement of various other
growth factor receptors and of adhesion receptors of the integrin
family. Thus, integration of multiple signals from diverse growth
factor and adhesion receptors must take place at a membrane-proximal
location to generate parallel second messengers. By implication, signal
strength for individual signaling pathways may be provided either by
activation of multiple receptors by diverse ligands or by strong
signaling by a single class of receptors. The former scenario is likely
to apply to normal cells, whereas the latter may operate in malignant
cells, which have become independent of certain extracellular signals
for survival. In view of this interpretation, it is important to define
the relative contribution of each single class of cell surface
receptors to signal transduction events required for cellular survival.
/PKC,
STAT, and MEK/MAPK pathways to keratinocyte survival and
Bcl-xL expression. The effects of abrogating the function of specific signal transducers were related to the effects of EGFR
blockade on keratinocyte survival and to Bcl-xL expression. We describe that PI3-kinase/AKT, PLC
, and PKC
activities were essential to keratinocyte survival. However, these pathways did not
depend on the EGFR tyrosine kinase activity to support keratinocyte survival and did not affect Bcl-xL expression. By contrast,
MEK activity was dependent on EGFR activation and was required for sustained Bcl-xL expression and keratinocyte survival.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
inhibitor U73122, and the control compound U73343 were purchased from Calbiochem or Biomol (Plymouth Meeting, PA).
PKC
/
inhibitor Gö6976 and PKC
inhibitor Rottlerin were purchased from Biomol. Antibodies to Bcl-xL were purchased
from Transduction Laboratories (Lexington, KY), to
-actin from
Amersham Pharmacia Biotech, and to
-tubulin from Oncogene Research
Products (Boston, MA). Antibodies to signal transduction components
(STAT3, phospho-STAT3, p42/44-MAPK, phospho-MAPK, AKT, phospho-AKT,
phospho-PKC
/
, PKC
, and PLC
) were purchased from Cell
Signaling Technology (Beverly, MA) or Santa Cruz Biotechnology (Santa
Cruz, CA). HA tag antibodies were from Covance (Richmond, CA).
-galactosidase assays were performed using
reagents from PharMingen (San Diego, CA) and Tropix Inc. (Bedford, MA)
as described earlier (14).
(for AKT) followed by Western blot
analysis with antibodies that allow detection and quantitation of
phosphorylated substrates.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(21). These experiments were performed using
immortalized keratinocytes (HaCaT) in a serum-free medium containing
insulin as the sole exogenous growth factor to account for
EGFR-dependent signaling events induced by autocrine EGFR
ligands, several of which are known to be produced by cultured
keratinocytes (22-25). In addition, these experiments were performed
using steady-state conditions by maintaining cells for 24-48 h under
the same media conditions. In select experiments EGF was added short
term (5-10 min) to the medium to provide a strong receptor activation
signal. As shown in Fig. 1A,
EGFR blockade by mAb 425 used at 66 nM markedly reduced steady-state phosphorylation of the MAPK substrate ELK-1 in normal human keratinocytes. By contrast, EGFR blockade using mAb 425 had
little effect on GSK-3
phosphorylation (Fig.
1B); GSK-3
is a major target of the serine/threonine
kinase AKT (26). As controls we used the MEK-1 inhibitor PD98059 to
inhibit ELK-1 phosphorylation and the PI 3-kinase inhibitor LY294002 to
inhibit GSK-3
phosphorylation.
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Fig. 1.
Effects of EGFR blockade on ELK-1,
GSK-3 , and STAT3 phosphorylation in
immortalized HaCaT keratinocytes. A, in
vitro phosphorylation of ELK-1 after treatment with mAb 425 (10 µg/ml) for 48 h; as a positive control the effects of the MEK
inhibitor PD98059 at different concentrations are shown. B,
in vitro phosphorylation of GSK-3
after treatment with
mAb 425 for 48 h; as a positive control the effects of the PI
3-kinase inhibitor LY294002 at different concentrations are shown.
C, expression and phosphorylation levels of STAT3 in HaCaT
keratinocytes induced by short term EGF treatment (10 ng/ml; 5 min) in
the presence and absence of EGFR inhibitor AG1478 (10 µM); STAT3 phosphorylation patterns of A431 cells exposed
to the identical culture conditions are also shown. Control cells were
HaCaT keratinocytes overexpressing STAT3D. The bottom panel
shows EGF-dependent phosphorylation of p42/44 MAPK in HaCaT
and A431 cells as determined by use of an antibody to phosphorylated
MAPK.
phosphorylation in normal
and immortalized keratinocytes in the presence or absence of EGF (not
shown), although short term EGF treatment induced readily detectable
PLC
phosphorylation in A431 cells. In summary, these results
indicated that EGFR activation in primary and HaCaT keratinocytes
contributed substantially to MEK/MAPK but not to AKT or STAT3 activation.
/PKC, and the RAS/RAF/MEK cascade on
keratinocyte survival (Table I). We used
LY294002 to inhibit PI 3-kinase activity, PD98059 to inhibit
MEK-dependent phosphorylation events, the amino steroid
U73122 to inhibit PLC-dependent signaling, and Rottlerin and Gö6976 to inhibit PKC
and PKC
activities, respectively (Table I). In addition, we determined by use of the SRC inhibitors PP1
or PP2 whether SRC family members were required for keratinocyte survival. The inhibitors were used at concentrations needed to inhibit
functional activity of the respective signaling pathways. In these
assays, the fraction of apoptotic keratinocytes after 24-48-h
treatments was determined by TUNEL staining combined with FACS
analysis. This assay is highly quantitative as it measures the
frequency of cells with DNA nicks within at least 2 × 104 cells/sample. We have reported previously (1, 2) that
EGFR inhibition by mAb 425 does not induce cell death within the first 24-48 h of treatment but rather sensitizes these cells to cellular stress. To provide a direct comparison between the effects attributable to EGFR blockade and the effects of inhibitors of signal transduction pathways, mAb 425 was included in all assays. As expected, treatment with mAb 425 did not affect survival of either normal keratinocytes or
HaCaT cells under steady-state conditions in the absence of cellular
stress. Similarly, pharmacological inhibition of MEK or PKC
activity
had only marginable effects on cell survival in this setting. By
contrast, inhibition of PI 3-kinase, SRC, PLC
, and PKC
activities
induced significant increases in the fraction of TUNEL-positive cells
within 48 h of treatment. The PLC
inhibitor U73122 and the
PKC
inhibitor Gö6976 induced cell death most effectively as
judged by the fraction of TUNEL-positive cells after 48 h. Both
inhibitors also caused significant cell death as early as 12 h
after addition (not shown). These results are consistent with the view
that activation of PI 3-kinase, SRC, and PLC
/PKC
is obligatory to
keratinocyte survival but that EGFR activation is not essential to
maintain levels of activation of these signaling components required
for survival of this cell type.
Effects of pharmacological inhibitors of signal transduction pathways
on spontaneous apoptosis of normal human keratinocytes (NHK) and
immortalized HaCaT keratinocytes
, or
PKC
had no detectable effect on Bcl-xL expression.
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Fig. 2.
Effects of pharmacological inhibitors of
signaling pathways on Bcl-xL protein expression in human
keratinocytes. A, effects of MEK inhibitor PD98059 (50 µM) and PI 3-kinase inhibitor LY294002 (20 µM) on Bcl-xL expression in HaCaT
keratinocytes. The blots were reprobed with an -actin antibody to
demonstrate equal loading. B, effect of PLC
inhibitor
U73122 (10 µM) on Bcl-xL expression in normal
keratinocytes. Controls consisted of the inactive compound U73343 as
well as PD98059. C, effect of the SRC inhibitor PP1 (20 µM) on Bcl-xL expression in HaCaT
keratinocytes. Controls consisted of the related but inactive compound
PP3 (20 µM). In this experiment Gö6976 (5 µM) and Rottlerin (3 µM) were also used to
inhibit PKC
and -
, respectively. Relative signal intensities
represent the ratio of the densitometrically measured
Bcl-xL signal to the
-actin signal in each sample
relative to controls shown as 1. Reduction of this ratio >20% was
considered significant.
phosphorylation (Fig.
3A). Similar to the PI
3-kinase inhibitor LY294002, overexpressed inactive AKT induced
spontaneous keratinocyte apoptosis over a 48-h period (Fig.
3C) but had no effect on Bcl-xL expression (Fig. 3B) during this time.
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Fig. 3.
Induction of apoptosis in HaCaT keratinocytes
by conditional expression of a dominant negative AKT construct.
A, transgene expression in the presence (+) and absence ( )
of tetracycline (Tet) and inhibition of GSK-3
phosphorylation in cells overexpressing dominant negative AKT (AKT(K179
M)); cells were maintained in the experimental conditions
indicated for 48 h. Mock-transfected HaCaT cells served as
controls for tetracycline-induced effects. B, expression of
Bcl-xL in HaCaT cells overexpressing dominant negative AKT.
C, TUNEL staining of HaCaT cells overexpressing dominant
negative AKT for 48 h as compared with uninduced cells and
mock-transfected controls. The proportion of TUNEL-positive cells in
each experimental condition is shown in the respective panels.
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Fig. 4.
Induction of apoptosis in HaCaT keratinocytes
by conditional expression of a dominant negative MEK1 construct.
A, MKK1-8E transgene expression in the presence (+) and
absence ( ) of tetracycline after 48 h and inhibition of MAPK
phosphorylation in HaCaT cells overexpressing dominant negative MEK.
MKK1-8E transgene expression was assessed using an antibody to the
hemagglutinin tag. In addition the expression of Bcl-xL in
MKK1-8E-overexpressing cells is shown relative to mock-transfected
control cells and to the uninduced state; the blot was reprobed with an
antibody to
-tubulin to account for differences in loading between
samples. B, TUNEL staining of HaCaT cells overexpressing
dominant negative MEK as compared with uninduced cells and
mock-transfected controls.
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Fig. 5.
Effects of dominant negative MEK on
Bcl-x promoter activity. HaCaT cells were
transiently cotransfected with MKK1-8E and a Bcl-x-luc
reporter construct schematically shown at the bottom of the
figure. Controls included mock-transfected HaCaT cells and cells
transfected with the dominant negative AKT construct AKT(K179M).
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Fig. 6.
Effects of dominant negative STAT3 constructs
on Bcl-xL expression and survival of HaCaT cells.
A, expression of the dominant negative STAT3D and STAT3F
constructs in HaCaT cells as indicated; transgene expression was probed
using an antibody to the hemagglutinin tag. Also shown is
Bcl-xL expression in the presence (+) and absence ( ) of
tetracycline after 48 h. The blot was reprobed with an antibody to
-tubulin to account for differences in loading between samples.
B, TUNEL staining of HaCaT cells expressing STAT3D and
STAT3F. All experiments were performed 48 h after removal of
tetracycline (Tet) to induce transgene expression.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/PKC
but not STAT3 were
required for keratinocyte survival in vitro. 2) EGFR
activation in keratinocytes was not necessary to maintain PI 3-kinase
or PLC
/PKC
signaling sufficient to support survival of
keratinocytes. 3) By contrast, MEK activity in human keratinocytes was
largely dependent on EGFR activation. 4) Inhibition of either the EGFR tyrosine kinase or of MEK enzymatic activity was associated with down-regulation of Bcl-xL expression and enhanced
susceptibility to cell death induction. Based on these results we
suggest that EGFR activation supports keratinocyte survival and
Bcl-xL expression in part through activation of the
MEK/MAPK cascade (Fig. 7).
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Fig. 7.
Schematic view of signaling pathways required
for keratinocyte survival in vitro. Multiple
signals depending on MEK, PI 3-kinase, and PLC activities appear
necessary to sustain survival of this cell type. To maintain MEK
activity in cultured keratinocytes EGFR activation is indispensable. By
contrast, PI 3-K/AKT and PLC
/PKC
activation levels required for
normal keratinocyte survival can be maintained independently of EGFR
activation. MEK-dependent signals regulate expression of
Bcl-xL and, possibly, additional targets relevant to cell
survival.
Previous work strongly implicates PI 3-kinase in nerve growth factor-
and IGF-1-dependent survival of several cell types, including fibroblasts and neuronal cells (13, 30-33) and some hemopoietic cells (34, 35). We extend these observations to human
keratinocytes, which similarly required PI 3-kinase/AKT activity for
survival. However, our findings also indicate that, in keratinocytes,
EGFR activation was not necessary for PI
3-kinase/AKT-dependent protection against apoptosis under
steady-state culture conditions. In support of this view, inhibition of
the EGFR by use of mAb 425 did not substantially affect AKT-mediated
phosphorylation of GSK-3. This result is consistent with previous
observations that the EGFR is relatively ineffective in stimulating PI
3-kinase activity (18, 36). We described earlier that activation of the
IGF1R by insulin used at supraphysiological concentrations supports
keratinocyte survival under the culture condition used here (4). It
remains to be determined whether activation of the IGF-1R contributes
to the PI 3-kinase activity necessary for keratinocyte survival as
suggested recently (37).
Activation of PLC is required for survival of endothelial cells (38)
but promotes apoptotic death of myoblasts (39) and thymocytes (40). In
hemopoietic precursor cells suppression of PLC
activity by the
inhibitor D609 has been associated with down-regulation of Bcl-2
expression followed by cell death (41). These findings led us to
investigate the role of PLC
in keratinocyte survival and
Bcl-xL expression. We demonstrate that the PLC
inhibitor U73122 did not affect Bcl-xL expression in keratinocytes
although it effectively induced keratinocyte death. Similar to the PI
3-kinase/AKT pathway, EGFR activation was also not required to sustain
basal PLC
activity. Furthermore, keratinocytes required activation of conventional PKCs (either PKC
, -
, or -
) but not the
Ca2+-independent PKC
for survival. This conclusion is
supported by the observations that the PKC
inhibitor Rottlerin had
no measurable effects on keratinocyte survival, whereas the PKC
inhibitor Gö6976 potently induced keratinocyte apoptosis. These
results are consistent with several recent reports in the literature.
Specifically, overexpression of PKC
has been described to induce
cell death in keratinocytes (42), and PKC
participates in
caspase-mediated apoptosis in various cell types (43) including human
keratinocytes (44). By contrast, PKC
is essential for cell survival
in B lymphocytes, glioma (45), and hepatocellular carcinoma cells (46,
47). Furthermore, PKC
overexpression was observed to stimulate AKT activity in a PI 3-kinase-independent manner and suppress apoptosis of
32D myeloid progenitor cells (48). Taken together, these results assign
important roles to both PLC
and PKC
in survival of normal
keratinocytes. However, EGFR activation is not required to maintain
PLC
/PKC
activities sufficient for survival of this cell type in
the steady-state culture conditions chosen by us.
In contrast to the PI 3-kinase/AKT and the PLC/PKC
pathways, EGFR
activation was obligatory to phosphorylation of the MAPK target ELK-1
in keratinocytes. This conclusion is based on the observation that EGFR
blockade by use of mAb 425 reduced ELK-1 phosphorylation similar to the
MEK inhibitor PD98059. Inhibition of MEK activity by either PD98059 or
by use of a dominant interfering MEK construct also down-regulated
Bcl-xL promoter activity and protein expression in a manner
similar to EGFR blockade by mAb 425. Induction of the dominant negative
MEK construct for 48 h also led to spontaneous apoptosis in the
absence of obvious cellular stress indicating that EGFR-independent MEK
activity may provide survival signals other than supporting
Bcl-xL expression. Consistent with this view MEK has
recently been shown to contribute to phosphorylation of the
pro-apoptotic Bcl-2 family member Bad (34, 49). Interestingly, overexpression of the dominant negative MEK1 construct MKK1-8E led to
spontaneous apoptosis of HaCaT keratinocytes, whereas pharmacological inhibition of MEK activity did not. At present, this apparent discrepancy is unresolved. It is possible that the dominant negative MEK construct exerts effects on other targets in addition to MEK1 resulting in enhanced cell death.
The results presented here are consistent with an earlier study that implicated both the mitogen-activated protein kinase (MAPK) signaling pathway and SRC family kinases in survival of BaF/3 cells engineered to express the EGFR (50) and establish that the same pathways contribute to Bcl-xL protein expression in human keratinocytes. Furthermore, our results highlight the requirement for SRC family kinase activity also for keratinocyte survival. SRC can be activated by EGFR engagement in various cell types (51-53) and potentiates the ability of the EGFR to transform murine fibroblasts (54). It remains to be determined whether SRC kinases contribute to Bc-xL expression in keratinocytes primarily through the MEK/MAPK pathway or through STAT3 activation as described for NIH3T3 cells overexpressing the EGFR (55).
Interestingly, despite robust STAT3 expression we found no
evidence for STAT3 phosphorylation in HaCaT cells either in
steady-state conditions or when pulsed short term with exogenous EGF.
That STAT3 could be phosphorylated by EGFR activation in epithelial cells was shown in A431 squamous carcinoma cells which, however, express very high levels of EGFR. Consistent with a negligible role of
STAT3 in regulation of HaCaT cell survival stable overexpression of two
different dominant interfering STAT3 constructs in a
tetracycline-regulatable manner had no measurable effect on either
survival or Bcl-xL protein expression in these cells. We
conclude that, in contrast to AKT, PLC, and MEK activation,
STAT3-mediated events were not essential to survival of HaCaT
keratinocytes. Presently, we are investigating the possibility that
STAT3 signaling downstream of the EGFR is a characteristic of advanced
epithelial malignancies and does not extend to either normal
keratinocytes or early stages of malignant transformation.
In summary, this study provides evidence that interference with several
major signal transduction pathways channeled through either AKT, or
MEK, or PLC/PKC
provoked spontaneous apoptosis of normal and
immortalized keratinocytes in culture. These results suggest that
keratinocyte survival rests on the coordinate activation of a network
of signal transducers and that disruption of any of these pathways will
result in death by default. Conversely, disruption of EGF receptor
signaling impacted only one, i.e. the MEK/MAPK pathway, to
the extent that keratinocytes became prone to apoptosis.
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ACKNOWLEDGEMENTS |
---|
We are indebted to Drs. N. Ahn, T. Hirano, and T. F. Franke for providing cDNAs and plasmids.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants CA81008 (to U. R.) and CA77837 (to L. H. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Dermatology, Thomas Jefferson University, 319 BLSB, 233 S. 10th St., Philadelphia, PA 19107. Tel./Fax: 215-503-5622; E-mail: Ulrich.Rodeck@mail.tju.edu.
Published, JBC Papers in Press, November 29, 2000, DOI 10.1074/jbc.M008210200
2 M. Jost, F. P. Gasparro, and U. Rodeck, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; PI 3-kinase, phosphatidylinositol 3-kinase; mAb, monoclonal antibody; PKC, protein kinase C; PLC, phospholipase C; FACS, fluorescence-activated cell sorter; MAPK, mitogen-activated protein kinase; MEK, MAPK/extracellular signal-regulated kinase kinase; HA, hemagglutinin; PBS, phosphate-buffered saline; BSA, bovine serum albumin; TBS, Tris-buffered saline; TUNEL, terminal dUTP nick-end labeling; STAT, signal transducer and activator of transcription; IGR, insulin-like growth factor.
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