Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
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ABSTRACT |
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Upregulation of the platelet-derived
growth factor (PDGF) receptor- (PDGFR-
) is a
mechanism of myofibroblast hyperplasia during pulmonary fibrosis. We
previously identified interleukin (IL)-1
as a major inducer of the
PDGFR-
in rat pulmonary myofibroblasts in vitro. In this study, we
report that staurosporine, a broad-spectrum kinase inhibitor,
upregulates PDGFR-
gene expression and protein. A variety of other
kinase inhibitors did not induce PDGFR-
expression. Staurosporine
did not act via an IL-1
autocrine loop because the IL-1 receptor
antagonist protein did not block staurosporine-induced PDGFR-
expression. Furthermore, staurosporine did not activate a variety of
signaling molecules that were activated by IL-1
, including nuclear
factor-
B, extracellular signal-regulated kinase, and c-Jun
NH2-terminal kinase. However, both staurosporine- and IL-1
-induced phosphorylation of p38 mitogen-activated protein kinase
and upregulation of PDGFR-
by these two agents was inhibited by the
p38 inhibitor SB-203580. Finally, staurosporine inhibited basal and
PDGF-stimulated mitogenesis over the same concentration range that
induced PDGFR-
expression. Collectively, these data demonstrate that
staurosporine is a useful tool for elucidating the signaling mechanisms
that regulate PDGFR expression in lung connective tissue cells and
possibly for evaluating the role of the PDGFR-
as a growth
arrest-specific gene.
pulmonary fibrosis; protein kinase C; p38 mitogen-activated protein
kinase; interleukin-1; platelet-derived growth factor
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INTRODUCTION |
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PLATELET-DERIVED GROWTH
FACTOR (PDGF) is a potent mitogen for connective tissue cells
that plays a critical role in lung development (6, 34) and
has been implicated in the etiology of pulmonary fibrosis (25,
26). PDGF exists as a disulfide-linked dimer of two polypeptide
chains, A and B, that form PDGF-AA, PDGF-BB, or PDGF-AB
(9). Two PDGF receptor (PDGFR) subtypes ( and
) have different affinities for the three isoforms. PDGFR-
can only interact with B chain-containing isoforms, whereas PDGFR-
can
bind all three isoforms (32). Dimerization of these
receptor subtypes occurs in response to interaction with PDGF ligands
followed by receptor tyrosine kinase phosphorylation and interaction
with a variety of signal transduction molecules (for a review, see Ref.
9).
Recent studies (5, 20) have shown that the PDGFR- is
inducible in rat models of pulmonary fibrosis, and this represents a
potential mechanism of mesenchymal cell hyperplasia that could contribute to the progression of the disease. Upregulation of the
PDGFR-
in vitro in rat pulmonary myofibroblasts and rat osteoblasts is stimulated by interleukin (IL)-1
(7, 22, 27, 35), and induction of PDGFR-
renders these cell types hyperresponsive to
the mitogenic and chemotactic effects of PDGF. These observations are
consistent with several studies (27, 30, 33) that showed that maximal responses to PDGF-AB or -BB require coexpression of
PDGFR-
and PDGFR-
. Wang et al. (36) recently
reported that IL-1
-induced upregulation of the PDGFR-
gene
requires the activation of p38 mitogen-activated protein (MAP) kinase.
The microbial alkaloid staurosporine is a broad-spectrum protein kinase
inhibitor that has been documented as a useful tool for the study of
apoptosis and growth arrest (4, 11, 14, 41).
Interestingly, the PDGFR- gene has been identified as a growth
arrest-specific gene, and accumulation of the PDGFR-
gene product
has been suggested to facilitate the exiting of cells from growth
arrest after stimulation with PDGF (21). In this study, we
report that staurosporine upregulates the PDGFR-
gene over the same
concentration range (1-100 nM) that causes growth arrest in rat
pulmonary myofibroblasts. The mechanism of staurosporine-induced PDGFR-
upregulation was not related to inhibition of protein kinase
(PK) C activity or inhibition of receptor tyrosine kinase activity. In
contrast to IL-1
, staurosporine did not cause activation of nuclear
factor-
B (NF-
B) or the MAP kinases c-Jun NH2-terminal kinase (JNK) or extracellular signal-regulated kinase (ERK). However, staurosporine and IL-1
caused phosphorylation of p38 MAP kinase, and
upregulation of the PDGFR-
was inhibited by the p38 MAP kinase inhibitor SB-203580. These data are consistent with the idea that the
PDGFR-
is a growth arrest-specific gene that is regulated via the
activation of p38 MAP kinase.
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METHODS |
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Reagents.
The protein kinase inhibitors staurosporine, genistein, tyrphostin
AG-1296, bisindolylmaleimide I, GF-109203X, Ro -31-8220, and H-89 were
purchased from Calbiochem (La Jolla, CA). The rat cDNA probe for the
PDGFR- was the generous gift of Dr. Yutaka Kitami (Ehime University,
Ehime, Japan). PDGF isoforms (AA, AB, and BB), IL-1
, and the
IL-1 receptor antagonist protein (IRAP) were purchased from R&D Systems
(Minneapolis, MN). 125I-PDGF-AA (specific activity of
125 µCi/µg) was from Biomedical Technologies (Stoughton, MA).
Antibodies to mouse PDGFR-
and human PDGFR-
were from Upstate
Biotechnology (Lake Placid, NY); the swine anti-rabbit horseradish
peroxidase (HRP)-conjugated secondary antibody was from DAKO (Santa
Barbara, CA). The phosphotyrosine monoclonal PY20 and the goat
anti-mouse HRP-conjugated secondary antibodies were from Transduction
Laboratories (Lexington, KY). An antibody specific for the
phosphorylated form of p38 MAP kinase was from New England Biolabs.
[
-32P]ATP was from Amersham (Arlington Heights, IL).
JNK and ERK kits were purchased from Stratagene (La Jolla, CA). NF-
B
consensus double-strand oligonucleotide and
poly(dI-dC) · poly(dI-dC) were purchased from Promega
(Madison, WI). Fischer 344 rat pulmonary myofibroblasts were isolated
and characterized as described previously (10). TRI
Reagent was from Molecular Research Center (Cincinnati, OH).
Immobilon-S membranes were purchased from Millipore (Bedford, MA). The
Prime-It II random-primer labeling kit was from Stratagene.
125I-PDGF-AA binding assay. Myofibroblasts in 24-well plates were grown to confluence in 10% fetal bovine serum-Dulbecco's modified Eagle's medium (FBS-DMEM) and then rendered quiescent for 24 h in serum-free defined medium (SFDM; Ham's F-12 medium with HEPES, CaCl2, and 0.25% BSA supplemented with an insulin-transferrin-selenium mixture from Boehringer Mannheim, Indianapolis, IN) for 24 h. Cultures were chilled to 4°C, rinsed in ice-cold binding buffer (Ham's F-12 with HEPES, CaCl2, and 0.25% BSA), and exposed to 1 ng/ml of 125I-PDGF-AA for 3-4 h at 4°C on an oscillating platform in the absence and presence of an excess of cold PDGF-AA. Cells were then rinsed three times in ice-cold binding buffer, solubilized in 1% Triton X-100, 0.1% BSA, and 0.1 M NaOH, and cell-associated radioactivity was measured with a gamma counter. Total binding was measured with 125I-PDGF-AA alone, and nonspecific binding was measured in parallel wells with 125I-PDGF-AA plus a 500-fold excess of nonradioactive PDGF-AA. Specific binding was defined as the difference between total and nonspecific binding.
Northern blot analysis.
Total RNA was isolated with TRI Reagent. Twenty micrograms of each
sample were electrophoresed in 1% agarose-2 M formaldehyde gels and
capillary transferred onto Immobilon-S membranes. A rat cDNA probe for
the PDGFR- was labeled with [
-32P]dCTP with a
Prime-It II random-primer labeling kit. The autoradiographic signal was
visualized with a phosphorimager (Molecular Dynamics, Sunnyvale, CA).
Western blot analysis.
Cells were washed with PBS and 250 µl of lysis buffer [50 mM
Tris · HCl; 1% Triton X-100; 150 mM NaCl; 1 mM EGTA; 1 mM
phenylmethylsulfonyl fluoride (PMSF); 0.25% sodium deoxycholate; 1 µg/ml each of aprotinin, leupeptin, and pepstatin; 1 mM
Na3VO4; and 1 mM NaF] were added to cover the
surface of the attached cells for 20 min at 0-4°C. Extracts were
collected without scraping and stored at 70°C. Twenty microliters
of each sample were mixed with 5 µl of reducing sample buffer and
boiled for 5 min before electrophoresis in a 2-15% Tris-glycine
SDS-polyacrylamide gel for 2 h at 130 V and 30 mA. The protein on
the gel was transferred to a nitrocellulose membrane (Hybond,
Amersham). The membrane was blocked with 3% milk-PBS for 1 h
before the addition of a rabbit anti-mouse PDGFR-
antibody or a
rabbit anti-human PDGFR-
antibody (at a dilution of 1:500)
overnight. After being washed three times with PBS-Tween, a secondary
HRP-conjugated swine anti-rabbit antibody was added for 1.5 h at a
dilution of 1:2,000. For measurement of autophosphorylation on
tyrosine, cell lysates were collected as described above for PDGFR
Western blotting. The membranes were blocked with 3% milk for 1 h
and then incubated for 24 h at 4°C with a 1:500 dilution of
anti-phosphotyrosine (PY20) monoclonal antibody. The membranes were
washed three times with PBS-Tween and then incubated with a 1:2,000
dilution of goat anti-mouse IgG-HRP for 90 min. For measurement of
inhibitor of NF-
B (I
B-
), cell lysates were collected with
scraping, and electrophoresed on 12% Tris-glycine-EDTA (TGE) gels, and membranes were incubated with a rabbit anti-human I
B-
antibody. For measurement of p38 MAP kinase activation, an
anti-phospho-p38 antibody (New England Biolabs) was used at a dilution
of 1:1,000. The secondary antibody for I
B-
or p38 MAP kinase
Western blots was a 1:2,000 dilution of HRP-swine anti-rabbit IgG
(DakoPatts, Carpinteria, CA). After a thorough washing with PBS-Tween,
all Western blots were developed with an enhanced chemiluminescence luminol kit (Amersham).
Electrophoretic mobility shift assay.
Nuclear extracts were prepared as follows. Cells were washed with PBS,
trypsinized, and centrifuged at 1,500 rpm for 10 min at 4°C. Cell
pellets were resuspended in 400 µl of buffer A [10 mM
HEPES, pH 7.9; 2 mM MgCl2; 10 mM KCl; 1.0 mM dithiothreitol (DTT); 1.0 mM PMSF; 5 µg/ml each of aprotinin, pepstatin, and leupeptin; and 0.1% Triton X-100], incubated for 15 min on ice, vortexed for 15 s, and centrifuged for 10 min at 14,000 rpm.
Pelleted nuclei were resuspended in 40 µl of buffer C [20
mM HEPES, pH 7.9, 25% (vol/vol) glycerol, 0.42 M NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 1.0 mM DTT, 1.0 mM PMSF, and 5 µg/ml
each of aprotinin and leupeptin], incubated for 30 min on ice, and
centrifuged for 10 min at 14,000 rpm. Supernatants were diluted with 20 µl of buffer D [20 mM HEPES, pH 7.9, 20% (vol/vol)
glycerol, 50 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF] and
stored at 80°C. Protein concentrations were determined by the
Bradford assay. Three micrograms of nuclear extract were
incubated in binding buffer [4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-Cl, pH 7.5, and 0.5 mg/ml
of poly(dI-dC) · poly(dI-dC)] with
[
-32P]ATP-labeled NF-
B oligonucleotide in a total
reaction volume of 20 µl for 20 min at room temperature. Samples were
electrophoresed on 6% polyacrylamide gels (0.5× Tris-glycine) with
0.5× Tris-glycine as a running buffer. Gels were dried and exposed to
film at
80°C for 2-16 h.
Immunoprecipitation of ERK and phosphorylated heat- and acid-stable protein substrate-1 kinase assay. ERK activity in myofibroblast cell lysates was measured by the ability of these lysates to phosphorylate phosphorylated heat- and acid-stable protein substrate-1 (PHAS-1). Cells grown to confluence in 75-cm2 tissue culture flasks were rendered quiescent in SFDM for 24 h. After 30 min of treatment with the agent of interest, the cells were placed on ice, washed twice with PBS, and scraped off with 800 µl of lysate buffer consisting of 50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, and 20 µg/ml each of aprotinin, leupeptin, and pepstatin. Lysates were clarified by centrifugation at 13,000 rpm for 10 min, and protein concentrations were determined by Bradford assay. Immunoprecipitation was performed by incubating 200 µl of lysate with 2 µg of anti-ERK 2 (p42) antibody for 2 h and then adding 20 µl of protein A agarose (Santa Cruz Biotechnology). After an overnight incubation at 0-4°C with end-over-end mixing, the immune complex was recovered by centrifugation and washed three times with lysis buffer and one time with 250 mM HEPES (pH 7.4), 10 mM MgCl2, and 200 µM Na3VO4. Immune complex kinase assays were performed with a MAP kinase assay kit (Stratagene) according to the manufacturer's instructions; briefly, the ERK pellets were resuspended in Stratagene reaction buffer containing 120 µg of PHAS-1 substrate along with 3-5 µCi of [32P]ATP in a final volume of 180 µl. Kinase reactions took place for 30 min at room temperature and were stopped by the addition of 4× SDS-PAGE reducing sample buffer and by boiling for 10 min. ERK-PHAS samples were resolved on 4-20% PAGE gels, dried, and autoradiographed.
JNK assay.
Cell lysates were collected as described in Immunoprecipitation
of ERK and PHAS-1 kinase assay for the ERK assay. JNK was immunoprecipitated from 200 µl of lysate by first incubating with 2 µg of an anti-JNK-1 (p46) polyclonal IgG (Santa Cruz Biotechnology) for 3 h and then adding 20 µl of protein A agarose for an
overnight incubation at 0-4°C with end-over-end mixing. The
immune complex was recovered by centrifugation and washed three times
with lysis buffer (50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 1 mM
PMSF, and 20 µg/ml each of aprotinin, leupeptin, and pepstatin) and one time with JNK kinase buffer (20 mM HEPES, pH 7.9, 15 mM
MgCl2, 1 mM DTT, 100 µM Na3VO4,
and 25 mM -glycerophosphate). The pellet was resuspended in 180 µl
of kinase buffer containing 30 µg of glutathione
S-transferase-c-Jun(1-79) (Stratagene), 100 µM ATP, and 3-5 µCi of [32P]ATP. The reaction
was allowed to proceed for 30 min at room temperature and was
terminated by the addition of SDS loading buffer and boiling for 10 min. Phosphorylated glutathione
S-transferase-c-Jun(1-79) was resolved on a 12%
SDS-polyacrylamide gel and then autoradiographed.
[3H]thymidine incorporation assay. Cells were grown to confluence with 10% FBS-DMEM in 24-well tissue culture plates (2-cm2 wells) and then rendered quiescent for 24 h with SFDM containing 0.5% FBS. The cells were treated with fresh 0.5% FBS-SFDM containing staurosporine (1-100 nM) or DMSO vehicle for 24 h at 37°C. PDGF-AA, -AB, or -BB (50 ng/ml) or medium alone containing no growth factor was spiked into the medium along with 5 µCi/ml of [3H]thymidine (Amersham) for 24 h. The cells were washed with Ham's F-12 medium at 25°C, placed on ice, and incubated with 0.5 ml/well of 5% trichloroacetic acid for 10 min. After three washes with ice-cold distilled water, solubilization was performed with 0.5 ml/well of 0.2 N NaOH containing 0.1% SDS for 30 min on an oscillating platform. One hundred microliters of each sample were added to 1 ml of Ecolume (Costa Mesa, CA), and radioactivity was measured with a liquid scintillation counter.
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RESULTS |
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Staurosporine induction of PDGFR- expression.
Staurosporine caused a dose-dependent increase in PDGFR-
mRNA as
determined by Northern blot analysis (Fig.
1A) and upregulated PDGFR-
protein as determined by Western blot analysis (Fig. 1B). Moreover, staurosporine also caused upregulation of functional cell
surface PDGFR-
in a concentration-dependent manner as determined by
125I-PDGF-AA binding assays (Fig. 1C). The
concentration range of staurosporine used in these experiments
(1-100 nM) did not cause significant cytotoxicity as determined by
trypan blue exclusion staining or detachment of cells after 24 h
in culture (data not shown).
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Staurosporine-induced upregulation of PDGFR- is not due to PKC
or PKA inhibition.
The effect of staurosporine on PDGFR-
expression was not mimicked by
more specific inhibitors of PKC (Ro-31-8220, GF-109203X, or calphostin
C; Table 1). Furthermore, these
inhibitors did not block IL-1
-induced upregulation of PDGFR-
. To
further rule out the involvement of PKC isozymes, cells were treated
for 24 h with 0.1-10 µM phorbol 12-myristate 13-acetate to
downregulate PKC activity. However, phorbol 12-myristate 13-acetate
depletion of PKC activity had no effect on the increase in
125I-PDGF-AA binding caused by staurosporine or
IL-1
(data not shown). Staurosporine is also a known inhibitor of
PKA (10). Treatment of cells with the PKA inhibitor H-89
did not induce PDGFR-
expression (Table 1). Moreover, H-89 did not
affect IL-1
induction of PDGFR-
, further supporting the idea that
this kinase is not involved in PDGFR-
regulation.
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PDGFR- upregulation by staurosporine is not due to an IL-1
autocrine loop.
Because IL-1
is a potent inducer of PDGFR-
in rat pulmonary
myofibroblasts (19), we investigated whether staurosporine was inducing PDGFR-
via an IL-1
autocrine loop. Pretreatment of
myofibroblasts for 2 h with 2 µg/ml of IRAP before addition of
100 nM staurosporine had no effect on the increase in
125I-PDGF-AA binding (Fig.
2). As expected, IRAP completely blocked the upregulation caused by IL-1
. These data confirmed that
staurosporine was not causing upregulation of PDGFR-
by stimulating
the production of IL-1
by the myofibroblasts.
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Staurosporine-induced PDGFR- expression does not require NF-
B
activation.
The activation of the transcription factor NF-
B is involved in the
IL-1
-mediated induction of a number of genes (1, 2, 13, 17,
18). Moreover, a recent report (24) has suggested that both IL-1
and staurosporine activate NF-
B, thereby
increasing IL-2 production in EL4 thymoma cells. We sought to determine
if activation of NF-
B was a common mechanism whereby IL-1
and
staurosporine induce PDGFR-
. Using electrophoretic mobility shift
assays, we clearly showed that staurosporine did not activate NF-
B,
whereas IL-1
was a strong activator of NF-
B (Fig.
3A). Also, I
B-
Western blot analysis showed that staurosporine did not cause degradation of
cytosolic I
B-
, whereas IL-1
caused complete degradation of
I
B-
within 30 min (Fig. 3B).
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PDGFR- increase by staurosporine is not due to inhibition of
PDGFR tyrosine kinase activity.
Because staurosporine is known to inhibit PDGFR tyrosine kinase
activity (31), we determined if staurosporine-induced
PDGFR-
expression was caused by inhibition of PDGFR
autophosphorylation (i.e., initiation of a feedback mechanism involving
synthesis of new PDGFR). Cells were pretreated with staurosporine or
two other receptor tyrosine kinase inhibitors, genistein and tyrphostin AG-1296, and then stimulated with PDGF isoforms for 5 min. PDGF-AB and
PDGF-BB induced strong autophosphorylation of PDGFR, whereas PDGF-AA
did not cause autophosphorylation because of the constitutively low
expression of this receptor. All three receptor tyrosine kinase inhibitors blocked autophosphorylation by >80% (Fig.
4A). However, genistein and tyrphostin AG-1296 did not upregulate
125I-PDGF-AA binding to intact myofibroblast monolayers
(Fig. 4B), indicating that inhibition of PDGFR tyrosine
kinase activity did not account for upregulation of PDGFR-
by
staurosporine.
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Staurosporine-induced upregulation of PDGFR- is due to
activation of p38 MAP kinase.
We previously reported (23) that IL-1
activates JNK-1
and ERK in rat pulmonary myofibroblasts. Therefore, IL-1
was used as
a known activator of these MAP kinases and was compared with staurosporine in kinase assays. Although IL-1
clearly activated JNK-induced phosphorylation of c-Jun- and ERK-induced phosphorylation of PHAS-1, staurosporine had no effect on these kinases (Fig. 5). However, Western blot analysis with a
phospho-p38 MAP kinase antibody demonstrated that staurosporine and
IL-1
activated p38 MAP kinase in a time-dependent manner (Fig.
6A). Moreover,
staurosporine-induced upregulation of 125I-PDGF-AA binding
was inhibited >70% by pretreatment with 20 µM p38 inhibitor
SB-203580 (Fig. 6B).
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Staurosporine inhibits PDGF-stimulated mitogenesis.
Lindroos et al. (22) previously reported that
upregulation of the PDGFR- by IL-1
renders myofibroblasts
hyperresponsive to the mitogenic effects of PDGF (22).
However, staurosporine inhibited basal thymidine incorporation (0.5%
FBS-DMEM with no PDGF) as well as mitogenesis stimulated by all three
PDGF isoforms (Fig. 7). Maximal
inhibition of basal mitogenesis was observed at 10 nM staurosporine,
whereas maximal inhibition of PDGF-stimulated mitogenesis was observed
at 50 nM staurosporine. The concentration range used to inhibit
mitogenesis (1-100 nM) in these experiments was the same as that
used to upregulate the PDGFR-
(Fig. 1). No significant cytotoxicity
was observed in these experiments by trypan blue nuclear exclusion with
as much as 100 nM staurosporine (data not shown).
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DISCUSSION |
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In this study, we report that staurosporine, a broad-spectrum
protein kinase inhibitor, strongly induces PDGFR-. Whereas staurosporine inhibits a variety of protein kinases, p38 MAP kinase was
phosphorylated in response to staurosporine treatment, and upregulation
of the PDGFR-
was due, at least in part, to activation of p38 MAP
kinase as determined by inhibition of staurosporine-induced PDGFR-
expression with SB-203580. Two members of the MAP kinase family, JNK
and ERK, were not activated by staurosporine. Wang et al.
(36) recently reported that IL-1
-induced upregulation of the PDGFR-
requires p38 MAP kinase activation. Thus p38 MAP kinase appears to be a common signaling intermediate required for
induction of the PDGFR-
gene by either staurosporine or IL-1
.
We initially speculated that staurosporine-induced upregulation of the
PDGFR- could be due to PKC inhibition because other investigators
(12) have reported that overexpression of PKC-
caused
suppression of PDGFR-
in Swiss/3T3 fibroblasts. However, other more
specific inhibitors of PKC (i.e., Ro-31-8220, GF-109203X, and
calphostin C) did not mimic the staurosporine effect on PDGFR-
expression. PKA inhibition by staurosporine was also excluded as a
possible mediator because the more specific PKA inhibitor H-89 had no
effect on PDGFR-
expression. Moreover, the ability of staurosporine
to block tyrosine phosphorylation of the PDGFR and thereby activate a
possible feedback loop for increased PDGFR-
synthesis was ruled out
because other receptor tyrosine kinase inhibitors (genistein and
tyrphostin AG-1296) did not upregulate PDGFR-
.
Other investigators have shown that staurosporine can induce various
biological responses via the activation of MAP kinases. A recent study
by Xiao et al. (38) showed that staurosporine-induced production of macrophage inflammatory protein-2 in rat peritoneal neutrophils is dependent on the activation of p38 MAP kinase and ERK.
Yao et al. (39) found that staurosporine activated a novel JNK isoform, but not JNK-1 or ERK, in rat PC-12 cells, and this contributed to neurite outgrowth in these cells. Although we observed that p38 MAP kinase is involved in staurosporine-induced PDGFR- expression, we did not detect activation of either ERK or JNK after
treatment of rat pulmonary myofibroblasts with staurosporine. In
contrast, IL-1
was a strong activator of JNK and ERK in rat pulmonary myofibroblasts.
Both staurosporine and IL-1 activated p38 MAP kinase in rat
pulmonary myofibroblasts, and upregulation of 125I-PDGF-AA
binding by either of these agents was significantly inhibited by
SB-203580. This observation suggests that staurosporine and IL-1
act
through a similar mechanism to induce PDGFR-
gene expression.
IL-1
activates a diversity of signaling intermediates including
NF-
B, PKC isozymes, and all three classes of MAP kinases (ERKs,
JNKs, and p38 MAP kinase). The broad spectrum of intracellular mediators activated by IL-1
has complicated the search for signaling pathways that control PDGFR-
expression. To our knowledge, p38 MAP
kinase is the only signaling intermediate that is strongly activated by
staurosporine in rat pulmonary myofibroblasts, and all other
IL-1-activated pathways (NF-
B, ERK, JNK, and PKC) are not affected
by staurosporine. IL-1
-induced upregulation of the PDGFR-
also
requires activation of p38 MAP kinase, which serves to stabilize the
mRNA that encodes the PDGFR-
(38). In contrast to
staurosporine, IL-1
activates ERK, which leads to suppression of
PDGFR-
expression (23). Thus the suppression or
induction of PDGFR-
by IL-1
appears to involve activation of both
ERK and p38 MAP kinases, respectively. In future studies, staurosporine will serve as a useful tool to better understand the mechanisms through
which p38 MAP kinase regulates PDGFR-
expression.
Other investigators have shown that staurosporine upregulates the
epidermal growth factor (EGF) receptor in PC-12 cells
(28), the tumor necrosis factor- (TNF-
) receptor in
myeloid and epithelial cells (40), and the human serotonin
receptor in choriocarcinoma cells (29). In all of these
cases, receptor upregulation was associated with enhanced biological
responses. Raffioni and Bradshaw (28) showed that
staurosporine increased EGF receptor expression in PC-12 cells and that
staurosporine enhanced EGF-induced receptor tyrosine phosphorylation.
In contrast, we found that staurosporine inhibited PDGF-induced
receptor tyrosine phosphorylation, even though it upregulated
PDGFR-
. These results are in agreement with those of Secrist et
al. (31) wherein staurosporine was reported as
a potent inhibitor of PDGFR phosphorylation. We demonstrated in this
report that inhibition of PDGFR tyrosine phosphorylation was not a
mechanism that contributes to PDGFR-
upregulation because genistein
and tyrphostin both blocked PDGFR phosphorylation but did not induce
PDGFR-
expression. Similar results were obtained by Zhang et al.
(40) who found that upregulation of the TNF-
receptor
by staurosporine in a human erythroblastoid leukemic cell line was not
due to inhibition of tyrosine kinases.
In contrast to IL-1- or lipopolysaccharide-induced upregulation of
PDGFR-
(10, 22), staurosporine-induced upregulation of
PDGFR-
did not result in an enhanced mitogenic response to PDGF
isoforms but, instead, inhibited PDGF-stimulated
[3H]thymidine uptake in myofibroblasts (Fig. 7). This is
likely due to the fact that staurosporine inhibited PDGFR tyrosine
kinase activity (Fig. 4). Basal [3H]thymidine uptake in
the presence of 0.5% FBS-DMEM with no PDGF was also suppressed in a
concentration-dependent manner by staurosporine, and this is consistent
with the well-known activity of staurosporine in the nanomolar range to
cause growth arrest via the inhibition of cell cycle kinases (14,
41). Higher concentrations of staurosporine (i.e.,
100-1,000 µM) have been reported to cause apoptosis in a variety
of cell types (4, 11). However, we did not observe significant cytotoxicity in rat pulmonary myofibroblasts within the
concentration range used in this study (1-100 nM).
We investigated the possibility that the signal transduction pathways
mediating PDGFR- upregulation by IL-1
and staurosporine could
converge at the level of transcriptional activation by NF-
B. Activation of NF-
B is necessary for the induction of gene expression by IL-1
in a number of cells (1, 2, 13, 17, 18), and a
B site exists in the PDGFR-
promoter region (19).
Furthermore, activation of NF-
B by staurosporine and IL-1
in EL4
thymoma cells has been reported as a common mechanism that leads to
increased IL-2 production (24), and Chabot and Breton
(8) reported that staurosporine activated NF-
B in human
keratinocytes. However, we clearly demonstrated that staurosporine did
not activate NF-
B in rat pulmonary myofibroblasts, nor did
staurosporine cause degradation of cytosolic I
B-
in these cells.
These findings are in agreement with some other investigators (3,
15, 16) who reported no effect of staurosporine on NF-
B
activation. Also, a report by Warshamana et al. (37)
demonstrated that dexamethasone upregulated PDGFR-
mRNA and protein,
although dexamethasone suppresses NF-
B activity. In further support
of this idea, we have reported that TNF-
strongly activates NF-
B
in pulmonary myofibroblasts but does not upregulate PDGFR-
(23). Thus it appears that NF-
B activation is not
related to upregulation of the PDGFR-
, even though some inducers of
PDGFR-
expression (IL-1
and lipopolysaccharide) are NF-
B activators.
In summary, we report that the PK inhibitor staurosporine upregulates
PDGFR- in rat pulmonary myofibroblasts via a p38 MAP kinase-dependent pathway. Induction of PDGFR-
by staurosporine was
not due to inhibition of PKC, PKA, or receptor tyrosine kinase activity. Moreover, the effect of staurosporine on PDGFR-
expression was not due to the production of IL-1
, the major endogenous inducer of PDGFR-
in the lung. Although staurosporine upregulated
PDGFR-
expression, it also caused growth arrest of rat pulmonary
myofibroblasts and inhibited PDGF-stimulated mitogenesis. Collectively,
these data support the idea that the PDGFR-
is a growth
arrest-specific gene and demonstrate that staurosporine is a useful
tool for elucidating the signaling mechanisms that regulate PDGFR
expression in lung connective tissue cells.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. C. Bonnner, NIEHS, PO Box 12233, Research Triangle Park, NC 27709 (E-mail: bonnerj{at}niehs.nih.gov).
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.
Received 2 February 2000; accepted in final form 14 August 2000.
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