1 Canadian Institutes of Health Research Group in Lung Development, Programme in Lung Biology, Research Institute, and 4 Department of Pediatrics, The Hospital for Sick Children, Toronto M5G 1X8; and Departments of 3 Physiology and 2 Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada M5S 1A1
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ABSTRACT |
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Herein, we
investigated the activity of mitogen-activated protein kinase (MAPK), a
key component of downstream signaling events, which is activated
subsequent to platelet-derived growth factor (PDGF)-BB stimulation.
Specifically, p42MAPK activity peaked 60 min after addition
of PDGF-BB, declined thereafter, and was determined not to be a
direct or necessary component of glycosaminoglycan (GAG)
synthesis. PDGF-BB also activated MAPK kinase 2 (MAPKK2) but had no
effect on MAPKK1 and Raf-1 activity. Chemical inhibition of Janus
kinase, phosphatidylinositol 3-kinase, Src kinase, or tyrosine
phosphorylation inhibition of the PDGF -receptor (PDGFR-
) did not
abrogate PDGF-BB-induced p42MAPK activation or its
threonine or tyrosine phosphorylation. A dominant negative cytoplasmic
receptor for hyaluronan-mediated motility variant 4 (RHAMMv4), a
regulator of MAPKK-MAPK interaction and activation, did not inhibit
PDGF-BB-induced p42MAPK activation nor did a construct
expressing PDGFR-
with cytoplasmic tyrosines mutated to
phenylalanine. However, overexpression of a dominant negative PDGFR-
lacking the cytoplasmic signaling domain abrogated p42MAPK
activity. These results suggest that PDGF-BB-mediated activation of
p42MAPK requires the PDGFR-
but is independent of its
tyrosine phosphorylation.
mitogen-activated protein kinase; platelet-derived growth factor receptor; fetal development; lung fibroblasts; tyrosine phosphorylation
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INTRODUCTION |
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PLATELET-DERIVED GROWTH
FACTOR (PDGF) is a dimeric peptide composed of two closely
related, but not identical, chains, denoted A and B, which are linked
by disulfide bonds (18). PDGF regulates its biological
functions though its binding to specific high-affinity receptors on
cell surface. The PDGF -receptor binds PDGF-AA and PDGF-BB, whereas
the
-receptor (PDGFR-
) binds PDGF-BB only. Binding of PDGF to its
receptors induces receptor dimerization (17) and initiates
activation of intrinsic kinase activity of the receptor
(27). Upon activation, the receptor associates with a
number of signaling molecules, including phospholipase C-
(PLC-
) (30), phosphatidylinositol 3-kinase
(PI3K) (27), Ras GTPase-activating
protein (RasGAP) (25), and tyrosine
phosphatase Syp/SH-PTP2 (39). Downstream signaling
components subsequent to PDGF stimulation include
mitogen-activated protein kinases (MAPKs) or extracellular
signal-regulated kinases (ERKs), which are central elements of growth
factor-induced signal transduction cascades (9). The
activation of MAPK results from phosphorylation of adjacent
tyrosine and serine/threonine residues within the MAPK by its upstream
component, termed MAPK kinase (MAPKK) (9, 12). A
family of MAPK isoforms has been identified recently (3).
ERK1 (p44MAPK) and ERK2 (p42MAPK) were the
first two members of the MAPK family to be purified and cloned. Both of
these enzymes are substantially homologous, with ERK1 and ERK2 sharing
~90% amino acid identity in the catalytic core and 80% identity
overall (2). Some of the other, less-understood members of
the family include the p62 ERK3 (3, 16) and the p45 ERK4
(2). MAPKs mediate the phosphorylation and activation of
nuclear transcription factors that regulate cell growth
(3). MAPK activation may result from stimulation of either
tyrosine kinase activity or G protein-coupled receptors
(21). Receptor tyrosine kinase (RPTK)-mediated MAPK
activation involves a series of SH2- and SH3-dependent protein-protein
interactions between the tyrosine-phosphorylated receptor, Shc, Grb2,
and Sos, resulting in Ras-dependent MAPK activation (39).
The Janus family of kinases (JAK) (10, 38), initially
characterized as transmitting signals from cytokine receptors, includes
four members: JAK1, JAK2, JAK3, and Tyk2. The JAKs can catalyze the
activation of two distinct signaling pathways. Growth factors or
cytokines can activate either the MAPK cascade or a family of
gene-regulating transcription factors known as signal transducers
and activators of transcription (STATs) (38). Recently,
JAK2 activation has been demonstrated to provide a convergent signaling
element for both pathways (35); moreover, MAPK has been
shown to regulate the activation of early response genes by modifying
the JAK-STAT signaling cascade (11). Protein kinase C
(PKC) and PI3K have been found to be involved in the activation of MAPK
(24, 39). Protein kinase A (PKA) has been reported to
negatively regulate MAPK (48). Furthermore, a novel
extracellular matrix binding protein, receptor for hyaluronan-mediated motility (RHAMM), which was originally characterized for its ability to
regulate cell mobility (16), has been demonstrated to
regulate the MAPK signaling pathway at the level of MAPKK
(52). An isoform of RHAMM, RHAMM variant 4 (RHAMMv4),
occurs only in the cytoplasm and, in conjunction with cell surface
RHAMM isoforms, can regulate activation of MAPK by growth factors such
as PDGF (52). Moreover, nonclassical means of signal
transduction, which exclude phosphorylation-dependent mechanisms, may
also be considered when addressing MAPK activation. The
Drosophila Numb, a protein involved in the development of the nervous system, does not absolutely require ligand phosphorylation for binding (31).
In previous studies, we have found that PDGF-BB stimulated glycosaminoglycan (GAG) synthesis, but not proliferation, of fetal lung fibroblasts (6) and that PI3K mediates PDGF-BB-induced GAG synthesis (32). To further explore the downstream mechanisms by which PDGF-BB signaling is relayed, we studied the effects of PDGF-BB on activation of MAPK.
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METHODS |
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Materials.
Female (200-250 g) and male (250-300 g) Wistar rats were
purchased from Charles River (St. Constant, QC, Canada) and bred in our
animal facility. The sources of all cell culture material have been
described elsewhere (7). [-32P]ATP was
from ICN Biomedicals (St. Laurent, QC, Canada). Human recombinant
PDGF-BB, PDGF-AA, epidermal growth factor (EGF), insulin-like growth
factor I (IGF-I), myelin basic protein (MBP), inactive p44MAPK, and MAPKK-glutathione S-transferase
(GST) fusion proteins and antibodies to PDGFR-
, phosphotyrosine,
Raf-1, MAPKK1, MAPKK2, JAK1, JAK2, and Tyk2 were purchased from Upstate
Biotechnology (Lake Placid, NY). Antibodies to p44MAPK and
p42MAPK were from Santa Cruz Biotechnology (Santa Cruz,
CA). Antibodies against p44MAPK/p42MAPK
(panMAPK) were from New England Biolabs (Beverly, MA). Tyrphostin 9 and perillic acid were from BIOMOL (Plymouth Meeting, PA). Wortmannin was purchased from Calbiochem (La Jolla, CA). AG-490 was purchased from
Calbiochem (San Diego, CA).
N-[2-(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H-8) and
N-(2-quanidinoethyl)-5-isoquinoline sulfonamide hydrochloride (HA-1004) were obtained from Seikagaku America (Rockville, MD). Enhanced chemiluminescence detection reagent
was from Amersham (Oakville, ON, Canada). Herbimycin A, guanosine
5'-O-(2-thiodiphosphate (GDP
S), genistein, phorbol 12-myristate 13-acetate (PMA), NiCl2, nifedipine, and
3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) were from Sigma (St.
Louis, MO). NIH/3T3 cells were from American Type Culture Collection
(Manassas, VA). 4,6-Diamidino-2-phenylindole (DAPI) was from Vector
Laboratories, Burlingame, CA. The enhanced green fluorescent protein
vector (pEGFP-C1) was from Clontech (Palo Alto, CA). TLC plates
(20 × 20-cm, 0.10-mm cellulose) were from Alltech (Deersfield,
IL). Polyvinylidene difluoride (PVDF) membrane (0.2-µm pore size) was
from Helix Technologies (Scarborough, ON, Canada). Nitrocellulose
mebrane (0.2-µm pore size) was from Bio-Rad Laboratories (Missisauga,
ON, Canada).
Cell culture.
Timed-gestation (day 19) Wistar rats (term = 22 days)
were killed by diethylether excess, and the fetuses were aseptically removed. The fetal lungs were dissected out, placed in cold Hank's balanced salt solution without calcium and magnesium [HBSS()] and
cleared of major airways and vessels. The lungs were washed twice in
HBSS(
), minced, and suspended with HBSS(
). Fibroblasts were
isolated from the fetal lungs as previously described (7).
Stimulation of fetal lung fibroblasts with PDGF-BB. Subconfluent fetal lung fibroblasts cultured in 75-cm2 culture flasks were washed three times with serum-free MEM and serum starved for 24 h at 37°C. The cells were then washed again once with serum-free MEM and incubated with either MEM alone or MEM supplemented with 20 ng/ml of either PDGF-BB, PDGF-AA, EGF, or IGF-I for 30 min at 37°C. Cells were also incubated in MEM supplemented with PDGF-BB ranging in concentration from 0 to 20 ng/ml. The incubation was stopped by removing the medium and washing cells with ice-cold PBS three times. The cells were then lysed in (in mM) 50 HEPES, pH 7.4, 150 NaCl, 1.5 MgCl2, 1 EGTA, 100 sodium fluoride, 10 pyrophosphate, and 1 phenylmethylsulfonyl fluoride, 10% (vol/vol) glycerol, 200 µM Na3VO4, 10 µg/ml aprotinin, 1% (vol/vol) Triton X-100, and 10 µg/ml leupeptin, sonicated, and centrifuged for 15 min at 10,000 g at 4°C. The protein content was determined according to the method of Bradford (4). For time-course and dose-response studies, the cells were cultured in 6-well plates, serum starved for 24 h, and stimulated with 20 ng/ml PDGF-BB for various time periods or with different concentrations of PDGF-BB for 30 min at 37°C.
Immunoprecipitation of MAPK, MAPKK, Raf-1, PDGFR- and
tyrosine-phosphorylated p42MAPK and immunoblotting.
Aliquots containing 300 mg of lysate proteins were precleaned by
incubation with nonimmune rabbit mouse IgG for 30 min at 4°C,
followed by incubation with 10% (vol/vol) formalin-fixed Staphylococcus aureus Cowan strain A (Zysorbin) or protein
G-Sepharose in PBS for another 30 min at 4°C. Polyclonal antibodies
against p44MAPK, p42MAPK, MAPKK1, MAPKK2, and
PDGFR-
as well as monoclonal antibodies to Raf-1 were added to the
samples and incubated overnight on an end-to-end rotator at 4°C.
Zysorbin or protein G-Sepharose was used to collect immune complexes
(28). After a 60-min incubation at 4°C, the pellet was
washed three times with lysis buffer. Immunoprecipitates of PDGFR-
were boiled for 5 min in sample buffer and subjected to 5% (wt/vol)
SDS-PAGE and subsequently assayed by Western blot for detection of
PDGFR-
. Immunoprecipitates of p44MAPK and
p42MAPK were boiled for 5 min in sample buffer, subjected
to 10% (wt/vol) SDS-PAGE, and then assayed for activity by gel
renaturation. Immunoprecipitates of MAPKK and Raf-1 were assayed for
MAPKK and Raf-1 activity. To determine p42MAPK tyrosine
phosphorylation on PDGF-BB stimulation, monoclonal phosphotyrosine antibody (PY-99) was cross-linked to protein G-Sepharose by incubation with 20 mM dimethylpimelimidate in 0.2 M sodium borate (pH. 9) for 30 min, followed by three washes and incubation with 0.2 M ethanolamine
for 2 h. After extensive washing with PBS, the protein G-Sepharose-PY-99 complex was added to the samples to immunoprecipitate tyrosine-phosphorylated proteins. Immunoprecipitates were subjected to
10% (wt/vol) SDS-PAGE and transferred to nitrocellulose membrane. Nonspecific binding was blocked with 3% (wt/vol) dry milk powder in
PBS, and anti-p42MAPK antibody was added. After
overnight incubation at 4°C, the membrane was washed three
times with PBS, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit (1:30,000). After
several washes with PBS, blots were developed with an enhanced
chemiluminescence detection kit (Amersham, Oakville, ON, Canada).
Aliquots containing 20 µg of lysate protein were also subjected to
10% (wt/vol) SDS-PAGE and transferred to nitrocellulose membrane for
the purpose of detecting panMAPK. Blots were processed and developed as
aforementioned, except that the primary antibody employed was the
anti-panMAPK antibody (1:500).
MAPK assay.
MAPK activity was measured by gel renaturation assay with the use of
MBP as the substrate. MBP (0.5 mg/ml) was mixed with polyacrylamide gel
solution before polymerization. After electrophoresis, the SDS was
removed by soaking the gel in 20% (vol/vol) isopropyl alcohol in 50 mM
Tris · HCl, pH 8.0, for 30 min with two changes. The gel was
then equilibrated with 50 mM Tris · HCl, pH 8.0, and 5 mM
-mercaptoethanol for 1 h, followed by incubation with 6 M
guanidine in 50 mM Tris · HCl, pH 8.0, and 5 mM
-mercaptoethanol, and subsequently renatured with five changes of
0.04% (vol/vol) Tween 40 in 50 mM Tris · HCl, pH 8.0, and 5 mM
-mercaptoethanol over 12-18 h at 4°C. The kinase activity was
assayed by incubation of the gel with (in mM) 10 HEPES, pH 8.0, 2 dithiothreitol (DTT), 0.1 EGTA, 5 MgCl2, and 20 ATP and 5 µCi/ml of [
-32P]ATP for 1 h at room
temperature. The gel was washed thoroughly with 5% (wt/vol)
trichloroacetic acid containing 1% (wt/vol) sodium pyrophosphate until
the radioactivity of washing solution was <100 cpm/ml. The gel was
dried and exposed to Kodak X-OMAT film.
MAPKK activity assay.
MAPKK was immunoprecipitated as described above, and 30 µl of the
immunoprecipitates were mixed with 20 µl of (in mM) 10 HEPES, pH 8.0, 2 DTT, 0.1 EGTA, 5 MgCl2, and 20 ATP and 10 µl of
inactive p44MAPK-GST fusion protein (35 µg/ml). The
reaction was initiated by adding 5 µCi of [-32P]ATP.
After 1 h of incubation at 30°C, the reaction was terminated by
boiling the samples in the SDS sample buffer for 3 min, and p44MAPK-GST phosphorylation was revealed by SDS-PAGE and autoradiography.
Raf-1 activity assay.
Quiescent fetal lung fibroblasts were incubated with and without 20 ng/ml PDGF-BB. Raf-1 proteins were immunoprecipitated, and the
immunoprecipitated Raf-1 complexes were resuspended and incubated for
20 min at 25°C in 40 µl of kinase buffer containing (in mM) 30 HEPES, pH 7.4, 7 MnCl2, 5 MgCl2, 1 DTT, 15 ATP,
and 20 µCi of [-32P]ATP (Amersham) and 10 µl of
inactive MAPKK-GST fusion protein (35 µg/ml). The reaction was
terminated by boiling for 5 min after the addition of 25 µl of sample
buffer containing 33% (vol/vol) glycerol, 0.3 M DTT, and 6.7%
(vol/vol) SDS. Samples were resolved on 7.5% (wt/vol) SDS-PAGE and
visualized by autoradiography (36).
Inhibition of PDGF-BB-induced-MAPK activation.
Fibroblasts, serum starved overnight, were preincubated for 30 min in
serum-free MEM with and without either 1 µM tyrphostin 9, 1 µM
tyrphostin 1, 1 µg/ml herbimycin A, 0.1 µM calphostin C, 0.5 µM
PMA, 1 µM perillic acid, 500 µM GDPS, 30 µM H-8, 30 µM
HA-1004, 500 nM wortmannin, 10 µM nifedipine, and 50 µM AG-490 or
50 µM NiCl2 and 25 µg/ml genistein for 1 h at
37°C. In preliminary studies, we had found that these
concentrations were not cytotoxic for fetal lung fibroblasts in 24-h
culture experiments (33). Cells were then stimulated with
20 ng/ml PDGF-BB for 30 min at 37°C, and MAPK activity was determined
as described above.
PDGF-BB-induced phosphorylation of p42MAPK.
Subconfluent fetal lung fibroblast cultures in 75-cm2
flasks were first serum starved overnight and then incubated in either MEM, MEM + 20 ng/ml PDGF-BB, MEM + 1 µM tyrphostin 9 + 20 ng/ml PDGF-BB, or MEM + 1 µg/ml herbimycin A + 20 ng/ml
PDGF-BB. Cells were first treated with the appropriate inhibitor for
2 h and then incubated with 1 mCi of
[32P]orthophosphate (Amersham) for 4 h before
stimulation with 20 ng/ml PDGF-BB for 30 min. Various time points for
the maximal activation of MAPK in lung fibroblasts have been examined,
and it was determined that 30 min of PDGF-BB stimulation is optimal. Cells were then washed with ice-cold PBS, lysed in lysis buffer, and
immunoprecipitated with anti-p42MAPK. Radiolabeled
p42MAPK was resolved by 10% (wt/vol) SDS-PAGE and
transferred onto PVDF membranes using CAPS buffer, and
p42MAPK was cut from individual lanes with a single-edged
razor blade. The pieces of membrane containing the p42MAPK
samples were soaked in 0.5% (wt/vol) polyvinylpyrrolidone (PVP-360) in
100 mM acetic acid for 30 min at 37°C. The liquid was aspirated, and
the membranes were washed extensively in deionized water and air-dried,
followed by the addition of 200 µl of 6 N HCl and incubation for 60 min at 110°C. The samples were centrifuged for 5 min in a
microcentrifuge, and the supernatant was transferred to a new microcentrifuge tube and evaporated in a lyophilizer. The phosphoamino acids were separated by two-dimensional TLC (47) in a HTLE
7000 TLC chamber (CBS Scientific, Del Mar, CA). Phosphoamino acid
marker mixtures containing 1 mg/ml each of phosphoserine,
phosphothreonine, and phosphotyrosine were added to the samples before
electrophoresis. After electrophoresis, the TLC plates were sprayed
with 0.25% (wt/vol) ninhydrin in acetone and baked for 15 min at
65°C to develop the stain of the three phosphoamino acid markers. The TLC plates were marked with fluorescent ink and exposed to X-ray film
at 70°C with an intensifier screen. The radioactive phosphoamino acids were identified by aligning the stained standards with the X-ray film.
Generation of mutant PDGFR- lacking the intracellular
signaling domain.
Total RNA from rat lungs was isolated using the RNeasy total RNA kit
(QIAGEN, Chatham, CA), and RT-PCR was performed. The primer set chosen
for amplification of the extracellular and transmembranous portion of
the PDGFR-
was based on the mouse sequence (GenBank no. M84607) and
predicts a 1,725-nucleotide product. The sequence of the 5' primer was
5'-TCAAGCTTCCATCTGTA GCCCGGACAC-3', and that of the 3' primer was
5'-GATCTAGACTACTCATAGCGTGGCTTCTTCTGC-3'. The PCR products
were subcloned into the PCR vector (Invitrogen, San Diego, CA) and
transformed into Escherichia coli for propagation. The PCR
products were confirmed by sequencing. After sequence confirmation, the
mutant PDGFR-
cDNA was subcloned into the pcDNA3 vector (Invitrogen).
Transfection of fetal lung fibroblasts.
Fetal rat lung fibroblasts seeded onto 6-well plates were transfected
with pcDNA3-PDGFR- mutant plasmid (intracellular signaling domain
deleted) by use of cytofectin GS (Glen Research, Sterling, VA) in
MEM + 5% (vol/vol) fetal calf serum (FCS). Transfection was
allowed to proceed for 6 h before the medium was aspirated and
replaced with MEM + 10% (vol/vol) FCS without rinsing. The medium
was replaced 12 h later, and cultures were left undisturbed for
another 60 h. Cells were then incubated overnight in serum-free medium before stimulation with 20 ng/ml PDGF-BB. After a 30-min PDGF-BB
exposure, cells were lysed, and MAPK activity and tyrosine phosphorylation of PDGFR-
were measured. Transient transfections were also performed using a plasmid containing a mutated PDGFR-
construct, F5 (23), which has had
tyrosine-to-phenylalanine mutations at residues 740, 751, 771, 1009, and 1021 that are required for the recruitment of PI3K, GAP, SHP-2, and
PLC-
, respectively (gift from Dr. A. Kazlaukas, Harvard Medical
School, Boston, MA). A dominant negative form of RHAMMv4 that
is incapable of activating MAPK (51) was also employed in
transient transfections (gift from Dr. E. Turley, Hospital for Sick
Children, Toronto, ON). Transfection efficiency was determined by
transiently transfecting fibroblasts with a plasmid (pEGFP-C1)
expressing enhanced green fluorescent protein under the control of the
cytomegalovirus promoter (Clontech, Palo Alto, CA). The transfection
procedure was performed as aforementioned, except that after 24 h,
cells were fixed and nuclei were stained with mounting medium
containing DAPI. The cells were subsequently observed under a
fluorescence microscope (Leica Laborlux D), and the number of green
fluorescent cells were compared with the number of cell nuclei
stained with DAPI. The transfection efficiency was determined to be
75 ± 5%.
Western blotting of PDGFR-.
After incubation with and without PDGF-BB, fetal lung fibroblasts were
scraped in PBS, sonicated, and centrifugated at 12,000 g for
10 min. The membrane fraction was pelleted by centrifugation at 50,000 g for 60 min. After resuspension in lysis buffer, membrane protein content was determined according to the method of Bradford (4). Samples containing equal amounts of proteins were
subjected to 5% (wt/vol) SDS-PAGE and subsequently transferred onto
nitrocellulose membrane. Nonspecific binding was blocked by incubation
with 3% (wt/vol) dry milk powder in PBS at 4°C for 60 min. After
overnight incubation with anti-PDGFR-
antibody at 4°C, the
membrane was washed three times with PBS, followed by incubation with
horseradish peroxidase-conjugated goat anti-rabbit IgG (1:30,000).
After three washes with PBS, the blots were developed with an enhanced
chemiluminescence detection kit (Amersham). To assay for the presense
of RHAMMv4 in rat fetal lung fibroblasts, lysates were boiled for 5 min
in sample buffer and subjected to 5% (wt/vol) SDS-PAGE and
subsequently assayed by Western blot for detection of RHAMMv4. After
overnight incubation with primary antibody (gift from Dr. E. Turley),
the blot was processed as aforementioned.
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RESULTS |
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PDGF-BB induces MAPK tyrosine and threonine phosphorylation and
activation.
As a first step toward identifying the effect of PDGF-BB on MAPK
activity in fetal lung fibroblasts, we measured MAPK activity from the
cell lysates after stimulating cells with PDGF-BB. MAPK activity was
analyzed using MBP substrate gel renaturation. PDGF-BB stimulated a
phosphorylating activity of a 42-kDa protein, whereas weak
phosphorylating activity was noticed for a 44-kDa protein (Fig.
1A). The minimal concentration
of PDGF-BB required to stimulate MAPK was determined by measuring MAPK
activity from cell lysates after fetal lung fibroblasts were stimulated
with various amounts of PDGF-BB. It is apparent that diminishing
concentrations of PDGF-BB leads to diminished MAPK activity. The
minimum threshold for MAPK activity was determined after stimulation
with 1 ng/ml of PDGF-BB (Fig. 1B). Consistent
activation of MAPK was determined to be at 3 ng/ml. PDGF-BB at 20 ng/ml, resulting in maximal MAPK activation, was used to stimulate
fibroblasts throughout the experiments contained herein. To further
confirm the specific MAPK isoforms activated by PDGF-BB, treated and
untreated samples were immunoprecipitated with antibodies against
p44MAPK or p42MAPK. Immunoprecipitates were
then assayed for MAPK activity by MBP substrate gel renaturation.
PDGF-BB significantly induced p42MAPK but not
p44MAPK activation (Fig. 1C). Immunoblotting
showed that anti-p44MAPK and anti-p42MAPK
antibodies specifically recognized the 44- and 42-kDa proteins, respectively (Fig. 1D). Immunoblotting using a
panMAPK-specific antibody determined that both p44MAPK
and p42MAPK were expressed similarly (Fig. 1E).
These findings are consistent with p42MAPK activation by
PDGF-BB.
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PDGF-BB activates MAPKK2, an upstream activator of MAPK, but not
Raf-1, an upstream element of MAPKK.
Phosphorylation of MAPK by a mixed-function kinase termed MAPKK results
in the activation of MAPK (1, 9). To explore the
possibility that PDGF-BB stimulates MAPK through its upstream activator, MAPKK, resting or PDGF-BB-stimulated cell lysates were immunoprecipitated with antibodies against either MAPKK1 or MAPKK2. The
immunoprecipites were assayed for their ability to phosphorylate a
kinase-defective recombinant MAPK. MAPKK1 activity was not detectible in either unstimulated or stimulated cells. However, PDGF-BB caused a
marked increase in MAPKK2 activity, judged by increased
32P incorporation into inactive recombinant MAPK (Fig.
2A). MAPKK has been reported
to be activated by autophosphorylation (39). In our
results, there were no detectable proteins at 45 kDa even with
treatment of PDGF-BB, indicating that MAPKK was not significantly autophosphorylated (data not shown). The specificity of antibodies used
to immunoprecipitate MAPKK1 and MAPKK2 was demonstrated by direct
Western blotting (Fig. 2B). In addition, we also determined whether PDGF-BB activated Raf-1, a further upstream kinase in the
signaling cascade (1, 3). The activity of Raf-1 was increased in 3T3 cells on stimulation with PDGF-BB, whereas exposure of
fetal lung fibroblasts with PDGF-BB did not stimulate Raf-1 activity
(Fig. 2C).
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PDGF-BB-induced MAPK tyrosine and threonine phosphorylation and
activation are independent of tyrosine phosphorylation of PDGFR-.
PDGFR-
is a ligand-activated tyrosine kinase that autophosphorylates
and subsequently binds downstream proteins linking the cell surface
message to the nucleus. To determine whether PDGFR-
kinase activity
is crucial for PDGF-BB-mediated MAPK activation, serum-starved cells
were preincubated with and without tyrphostin 9, a potent, reversible
inhibitor of intrinsic tyrosine kinase of PDGFR-
(3,
39), followed by stimulation of 20 ng/ml PDGF-BB. Samples were
analyzed by immunoprecipitation with anti-PY antibody, followed by
immunoblotting with anti-PDGFR-
antibody for PDGFR-
tyrosine
phosphorylation, and, in parallel, samples were assayed for MAPK
activity. Tyrphostin 9 had no effect on either basal or
PDGF-BB-stimulated MAPK activity (Fig.
3A). However,
tyrphostin 9 prevented PDGFR-
tyrosine phosphorylation on PDGF-BB
stimulation (Fig. 3B), suggesting that PDGF-BB-mediated MAPK
activation is not through tyrosine phosphorylation of PDGFR-
. Also,
herbimycin A, another tyrosine kinase inhibitor, had no influence on
PDGF-BB-induced-MAPK activation (Fig. 3A). To investigate
whether tyrphostin 9 and herbimycin A influenced tyrosine and threonine
phosphorylation of p42MAPK, cells pretreated or untreated
with tyrphostin 9 or herbimycin A followed by stimulation with 20 ng/ml
of PDGF-BB were either 1) immunoprecipitated with anti-PY
antibody and analyzed by immunoblotting with p42MAPK
antibody (Fig. 4A) or
2) labeled with 32P and immunoprecipitated
with antibody against p42MAPK, followed by two-dimensional
TLC for phosphoamino analysis (Fig. 4B). To address
potentially more subtle effects of tyrphostin 9 and herbimycin A on
tyrosine and threonine phosphorylation of p42MAPK, PDGF-BB
was also employed at a concentration of 3 ng/ml (Fig. 4C).
At either concentation of PDGF-BB, neither inhibitor influenced PDGF-BB-induced p42MAPK tyrosine and threonine
phosphorylation, suggesting that tyrosine and threonine
phosphorylation of p42MAPK after PDGF-BB exposure
does not require tyrosine phosphorylation of the PDGFR-
.
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PDGFR is required for PDGF-BB-induced-activation of MAPK.
To explore whether binding of PDGF-BB to PDGFR- is essential for
MAPK activation, serum-starved fibroblasts were preincubated with and
without 20 ng/ml PDGF-BB for 2-6 h for the purpose of downregulating the PDGF receptors and then restimulated with 20 ng/ml
PDGF-BB for 30 min. Membrane fractions were isolated, and cell surface
PDGFR-
was determined by immunoblotting with anti-PDGFR-
antibody. In parallel experiments, MAPK activity was assayed from fetal
lung fibroblasts stimulated with PDGF-BB. As a control, fetal lung
fibroblasts were also stimulated with EGF, IGF-1, and PDGF-AA; however,
none of these growth factors activated MAPK (Fig.
5A). Pretreatment of
fibroblasts with PDGF-BB downregulated cell surface-associated
PDGFR-
, whereas restimulation with 20 ng/ml PDGF-BB after
downregulation of PDGFR-
did not increase MAPK activity in fetal rat
lung fibroblasts (Fig. 5, B and C). To further
confirm that PDGF-BB-induced MAPK activation is solely through the
PDGFR-
, cells were transiently transfected with a dominant negative
construct of the PDGFR-
and stimulated with PDGF-BB. Scatchard
analysis with 125I-labeled PDGF-BB has demonstrated that
fetal rat lung fibroblasts have a binding capacity of 4.0 × 10
20 mol/cell (5), whereas an esophageal
carcinoma cell line has a 100-fold greater binding capacity for PDGF-BB
(34). Because fetal rat lung fibroblasts have a very low
number of PDGFR-
s, transient transfection of a dominant negative
construct under the control of a strong viral promoter is predicted to
overwhelm the endogenous system. PDGF-BB stimulation of cells
transfected with dominant negative PDGFR-
lacking the cytoplasmic
portion resulted in the abolishment of tyrosine phosphorylation of
PDGFR-
compared with untransfected cells (Fig.
6A). Moreover, insignificant activation of MAPK was observed upon stimulation with PDGF-BB compared
with control (Fig. 6B). We also stimulated cells with EGF,
IGF-I, and PDGF-AA. None of these growth factors activated MAPK in
fetal lung fibroblasts (Fig. 5A). These observations
substantiate the premise that binding of PDGF-BB to PDGFR-
is
required for activation of MAPK. Furthermore, PDGF-BB stimulation of
cells transfected with a construct expressing a PDGFR-
(F5), in
which tyrosines were converted to phenylalanine, abolished
PDGFR-
phosphorylation (Fig. 6C) without inhibiting MAPK
activation (Fig. 6D). These data indicate that the receptor
is crucial for mediating the signal-tranducing event toward MAPK
activation but that the process is independent of phosphorylation.
Transfection with the PDGFR F5 construct, however, inhibited
PDGF-BB-induced GAG synthesis (data not shown), which we have shown to
be dependent on PDGFR tyrosine phosphorylation (32).
|
|
Lack of effects of inhibitors on PDGF-BB-induced
p42MAPK activation.
In an effort to dissect possible signaling pathways that relay the
PDGFR- signal to MAPK, a series of specific blockers was used to
identify the role of individual pathways in the MAPK cascade. Wortmannin, which we recently employed to block PI3K signaling in fetal
lung fibroblasts (32), did not alter PDGF-BB-induced p42MAPK activation. Calphostin C (a PKC inhibitor), H-8,
and HA-1004 (both PKA inhibitors), GDP
S (p21 Ras inhibitor), and
perillic acid (small G protein inhibitor) did not block PDGF-BB-induced p42MAPK activation. Neither nifedipine nor
NiCl2 (Ca2+ channel blockers) modulated
PDGF-BB-induced p42MAPK activation in fetal lung
fibroblasts (Fig. 7, A and
B). Although JAK2 kinase, but not JAK1 and Tyk kinases, was
found in fetal lung fibroblasts (Fig.
8A), treatment of cells with
AG-490, a specific inhibitor of JAK2 kinase, alone or in combination
with tyrphostin 9, did not inhibit PDGF-BB-induced
p42MAPK activation (Fig. 8B). These
results imply that PDGF-BB activation of p42MAPK is
not through the regular growth factor-induced transduction pathways.
|
|
Dominant negative RHAMMv4 does not inhibit PDGF-BB-induced
p42MAPK activation.
The molecular mechanism by which RHAMM modifies protein tyrosine
phosphorylation is not yet clear. Although it has been demonstrated that RHAMM acts at modifying MAPKK-MAPK interactions, it is not known
whether these interactions occur directly or through a modular protein
complex (52). RHAMMv4 complexes p44MAPK more
than p42MAPK; nevertheless, it is predicted that there
should be some inhibition of PDGF-BB-induced p42MAPK
activation subsequent to transfection with dominant negative RHAMMv4.
However, although RHAMMv4 is present in fetal lung fibroblasts (Fig.
9A), transient transfection of
a dominant negative RHAMMv4 into fibroblasts does not inhibit
PDGF-BB-induced p42MAPK activation (Fig. 9B).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although MAP kinases have been widely studied in transformed
cells, little is known in primary cells at the physiological level. Our
study revealed that PDGF-BB stimulated predominantly p42MAPK. There was a minor increase in p44MAPK
activity from cells stimulated with PDGF-BB that coincided with p42MAPK activity in gel renaturation assays but not in
kinase assays from isoform-specific p44MAPK
immunoprecipitations. The upstream regulator of p42MAPK,
MAPKK2, but not MAPKK1, was activated by PDGF-BB, demonstrating the
selective action of PDGF-BB in triggering MAPK signaling in primary
fetal lung fibroblasts. RPTKs have been shown to transmit intracellular
signals that are essential in regulating a wide array of cellular
processes, which include proliferation and differentiation (15). Although tyrosine phosphorylation is an integral and
early component of many signal transduction pathways, the majority of proteins downstream of RPTK activation are phosphorylated by
serine/threonine kinases (40). The MAP kinases are a
highly conserved family of serine/threonine kinases that appear to be
the focus of a variety of signal transduction pathways that are
initiated by RPTK and at which point mitogenic and nonmitogenic
extracellular signals converge (13). In the present study,
we found that neither tyrphostin 9, an inhibitor of PDGFR tyrosine
phosphorylation (32), nor the tyrosine kinase blocker
herbimycin A abolished tyrosine and threonine phosphorylation of
p42MAPK or the activation of MAPKK2. We have previously
demonstrated that the PDGFR- remains phosphorylated at 3 ng/ml of
PDGF-BB (32). We now demonstrate that MAPK is activated at
this lower concentration of PDGF-BB and that a direct correlation
exists between diminishing levels of PDGF-BB stimulation and
diminishing levels of MAPK activity. Prevention of PDGF-BB binding to
functional PDGFR-
s by overexpression of a dominant negative
-receptor, in which the cytoplasmic portion of the receptor
containing the signal transduction related sites was eliminated,
abolished p42MAPK activation subsequent to PDGF-BB
stimulation of fibroblasts at concentrations as high as 20 ng/ml.
Overexpression of another mutated PDGFR-
, F5, which has
tyrosine-to-phenyalanine mutations at residues 740, 751, 771, 1009, and
1021 and are required for the recruitment of PI3K, GAP, SHP-2, and
PLC-
, respectively, did not inhibit MAPK activation subsequent to
PDGF-BB treatment. A mutation in the PLC-
binding site on the
PDGFR-
only diminished, but did not abrogate, PLC-
phosphorylation subsequent to PDGF-BB stimulation in vitro
(45). In vivo, loss of binding of PLC-
to the PDGFR-
does not disrupt receptor function (45). The remaining
tyrosines in the F5 PDGFR-
, Tyr579 and
Tyr581 (Src kinase binding site) and Tyr716
(Grb2 binding site), are not a concern in the activation of MAPK in
fetal lung fibroblasts. We have previously demonstrated that Grb2 does
not bind to the activated PDGFR-
nor does Src kinase become
activated subsequent to PDGF-BB stimulation (32). These findings suggest that binding of PDGF-BB with PDGFR-
is essential for activation of p42MAPK but that ligand-induced PDGFR-
tyrosine phosphorylation is not required to activate
p42MAPK in fetal rat lung fibroblasts. This result is
distinct from what is typically observed in transformed cell lines
where the activation of MAPK is through the signal-transducing pathway
originating from the phosphorylation of the intrinsic receptor tyrosine
kinase (27). However, PDGF-BB activation of the
transfected F5-mutated PDGFR-
in the nonneoplastic epithelial cell
line T51B leads to a MAPK activation that was of similar magnitude
compared with MAPK activation in nontransfected T51B cells
(20).
Reduction of PDGFR- content on the cell surface by preincubation
with PDGF-BB blocked activation of p42MAPK on subsequent
restimulation with PDGF-BB, corroborating that binding of PDGF-BB to
PDGFR-
is essential for the p42MAPK activation. The
finding that stimulation of fetal lung fibroblasts with PDGF-AA, which
only binds to the PDGFR-
, did not induce p42MAPK
activation further supported this concept. Surprisingly, EGF and IGF-I
treatment of fetal lung fibroblasts, which express the EGF receptor
(44) and both IGF receptors (41), also did
not lead to activation of MAP kinases. Despite differences in their physiological actions, many growth factors engage similar intracellular signaling pathways initiated by the autophosphorylation of specific transmembrane receptors. The autophosphorylation of receptors recruits
multiple signaling molecules including PLC-
, RasGAP, and PI3K into
the membrane-associated complex. The activation of their downstream
components elicits a cascade of phosphorylation and activation of
protein kinases, one of which is MAPK (1). In our previous
studies, we found that PDGF-BB activated PDGFR-
, followed by
association of PDGFR-
with PLC-
, RasGAP, and PI3K in fetal rat
lung fibroblasts (32). Remarkably, PDGF-BB stimulation augmented only PI3K activity. PDGF-BB-induced PI3K activation was
abolished by the PI3K inhibitor wortmannin or LY-294002
(32). We employed inhibitors to examine the possible
signal transduction pathways that mediate the activation of
p42MAPK in fetal lung fibroblasts. The activation of MAPK
was not abolished either by the PI3K inhibitor wortmannin or by Ras and
PKC inhibitors GDP
S and calphostin C, respectively. Also, AG-490, a
JAK2 kinase inhibitor, did not affect MAPK activation. In
addition, the role of PKA and Ca2+-dependent channels on
PDGF-BB-induced-MAPK activation was investigated by employing PKA
inhibitors H-8 and HA-1004 and Ca2+ channel blockers
NiCl2 and nifedipine; however, none influenced PDGF-BB-induced p42MAPK activation. Transient transfection
with dominant negative RHAMMv4, which has been shown to activate MAPK
(52), also did not affect PDGF-BB-induced MAPK activation.
MAPK plays pivotal roles in the regulation of cellular proliferation and differentiation in response to extracellular signals (1). Fetal rat lung fibroblasts respond to PDGF-BB with increased GAG synthesis but not mitogenesis (6). PDGF-BB-induced GAG synthesis is mediated via PI3K (32). The PI3K inhibitors wortmannin and LY-294002 did not block PDGF-BB-induced p42MAPK activation, suggesting that PDGF-BB-induced GAG synthesis is not mediated through this MAPK. This finding is consistent with PDGF-BB-induced GAG synthesis not being inhibited by the MAPKK inhibitor PD-098095 (32). The growth factor activation of MAPK is a prerequisite for fibroblast proliferation (39). Such a critical role of MAPK in the control of cell growth may be cell-type specific. Therefore, the role of each pathway and its signaling molecules will have to be established for each cell type. Recent studies suggest that the duration of MAPK activation may dictate proliferation and differentiation responses in PC12 cells (46). In addition, it has been shown that sustained activation of MAPK by nerve growth factor (NGF) in these cells allows for the nuclear translocation of MAPK (37). This may initiate a program of differentiation and growth arrest, presumably through the action of the nuclear substrates of MAPK or associated kinases. It has been reported that in mesangial cells, PDGF-BB triggers not only activation of the MAPK signaling cascade but also de novo synthesis of p42MAPK and p44MAPK and the upstream MAPKK, thus providing mesangial cells with an increased capacity to respond to mitogenic stimulation by PDGF and other growth factors (22). In our studies, we detected only a transient activation of p42MAPK during 30-60 min of PDGF-BB stimulation, and de novo synthesis of either p42MAPK or p44MAPK was not observed during the 24-h period after exposure to PDGF-BB. It has been demonstrated that PDGF induces sustained activation of p44MAPK in Chinese hamster embryo (IIC9) fibroblasts that results in the continued expression of cyclin D1, a protein known to positively regulate G1 progression (49). Moreover, it has been found that MAPKK1 is required for PDGF-induced p44MAPK activation in bovine tracheal myocytes and that MAPKK1 and MAPKs are required for PDGF-induced DNA synthesis (26). Thus it can be reasoned that a lack of persistent activation of MAPK in response to PDGF-BB in fetal lung fibroblasts may result in failure to elicit a mitogenic function in fetal lung fibroblasts.
PC12 cells have provided researchers with the best-studied example of
dual regulation within a single cell through the distinctive actions of NGF and EGF. Interestingly, both EGF and NGF activate a
receptor tyrosine kinase to phosphorylate and activate similar intracellular substrates, including Ras and MAPK. However, stimulation of PC12 cells with EGF or NGF results in distinct and disparate physiological responses. Stimulation with EGF results in proliferation and stimulation with NGF results in differentiation
(8). Agents have been tested that stimulate MAPK
without inducing differentiation but that can act combinatorially when
employed with other agents to trigger differentiation. The application
of forskolin and EGF to PC12 cells altered the physiological action of
both agents by inducing differentiation (51). Therefore,
it was proposed that cellular responses to growth factor action are
dependent not only on the activation of growth factor receptors by
specific growth factors but also on synchronous signals that elevate
intracellular signals like cAMP, which can activate MAPK within the
same cells. Thus a possible explanation for the failure of a mitogenic
response of fetal lung fibroblasts to PDGF-BB is that there is a
deficiency of other mitogen(s) necessary to trigger the mitogenic
pathway. It has recently been found that PDGF-AA is not mitogenic in
connective tissue cells (3T3) unless tissue growth factor-, which is
not itself mitogenic, was added simultaneously to the culture medium (43). Thus it is possible that fetal rat lung fibroblasts
in vitro lack the synchronous signals required for activating MAP kinases that can trigger a mitogenic effect in combination with PDGF-BB.
Another possible level of control for the activation of MAP kinases are scaffolding proteins. A recently identified scaffolding protein, MEK (MAPKK) partner 1 (MP1), enhances the activation of MAPKK1 by B-Raf and that of p44MAPK by MAPKK1 (42). MP1 selectively associates with MAPKK1 and p44MAPK but not with MAPKK2 or p42MAPK. Because p44MAPK and MAPKK1 were not activated by PDGF-BB treatment in fetal lung fibroblasts, perhaps the appropriate scaffolding proteins were not activated. Conversely, because p42MAPK and MAPKK2 were activated subsequent to PDGF-BB stimulation, it suggests that particular scaffolding proteins were activated and recruited in such a manner as to permit enzymatic activity.
Ligand-independent activation of PDGFR- by lysophosphatidic acid and
downstream MAPK activation has been reported (19, 21),
suggesting that, like PDGF-BB-mediated activation of
p42MAPK, associative proteins, such as G proteins, may
synergize to activate receptors and/or downstream signaling components.
A novel recognition motif on the fibroblast growth factor receptor-1 (FGFR-1) has been determined to mediate direct association and activation of suc-associated neurotrophic factor target (SNT) adapter proteins (50). A juxtamembrane segment of FGFR-1 (residues 401-434) and the phosphotyrosine binding (PTB) domain (31) of SNTs are necessary and sufficient for FGFR-1-mediated SNT tyrosine phosphorylation. In the common case, the PTB domain recognizes the phosphotyrosine in the sequence context NPXpY, where X can be any amino acid. There now exist exceptions to this rule. The Drosophila Numb, a protein involved in the develoment of the nervous system, interacts with a GPpY motif rather than with NPXpY and does not absolutely require ligand phosphorylation for binding (31). However, the SNT binding motif of FGFR-1 is distinct from other PTB domain recognition motifs, lacking both tyrosine and asparagine residues. Myristoylation of SNT is required for plasma membrane localization and subsequent tyrosine phosphorylation. The phosphotyrosine independence of SNT interaction with the FGFR-1 receptor has been linked to MAPK activation (29), and the underlying mechanism may be comparable to the processes leading to activation of the MAPK pathway in fetal lung fibroblasts.
The data contained herein demonstrate that PDGF-BB-mediated activation
of p42MAPK requires PDGFR- but is independent of its
tyrosine phosphorylation. It was determined that p42MAPK is
not a direct signaling component in the PDGF-BB-mediated GAG pathway.
However, MAPK activation may lead to differentiation of fetal lung
fibroblasts because PDGF-BB-stimulation limits cell proliferation
(6) and initiates an increase in GAG synthesis via a PI3K
signaling pathway (32). Furthermore, activation of p42MAPK has been demonstrated to phosphorylate
Ser112 in the proapoptotic molecule BAD and thereby to
contribute to its inactivation with respect to apoptosis
(14). Therefore, the role of p42MAPK in fetal
lung cells may be to initiate an alteration in cell function that
eventuates in cell differentiation, cell protection, and lung
maturation. These processes are now subject to further investigation.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Dr. E. Turley from the Hospital for Sick
Children, Toronto, Ontario, for gifts of anti-RHAMMv4 antibody and a
vector containing a dominant negative form of RHAMMv4. We further
gratefully acknowledgement Dr. A. Kazlauskas from Harvard University,
Boston, MA, for the gift of the mutated PDGF -receptor construct F5.
![]() |
FOOTNOTES |
---|
* Nicholas J. Cartel and Jason Liu contributed equally to this work.
This study was supported by a grant from the Canadian Institutes of Health Research (CIHR) and an equipment grant from the Ontario Thoracic Society. N. Cartel is a recipient of a doctoral research award from the CIHR. M. Post is the recipient of a Canadian Research Chair in Respiration.
Address for reprint requests and other correspondence: M. Post, Programme in Lung Biology, Research Institute, The Hospital for Sick Children, 555 University Ave., Toronto, ON, Canada, M5G 1X8 (E-mail: mppm{at}sickkids.on.ca).
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 1 February 2001; accepted in final form 20 April 2001.
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