Progressive renal diseases lead to prolonged
glomerular hypertension, which induces the proliferation of mesangial
cells. This proliferation is thought to be involved in the development of renal injury. Here we investigate mitogen-activated protein kinase
(MAPK) activation and cell proliferation in mesangial cells under
conditions of high pressure. After pressure-load, the phosphorylation level of MAPK (at Tyr-204) increases rapidly with a peak at 1 min,
although the amount of MAPK remains almost constant during pressure-load. To confirm the activation of MAPK, we carried out an
immunoprecipitation-kinase assay. MAPK activity during pressure-load shows kinetics similar to that of the tyrosine phosphorylation. In
contrast, c-Jun N-terminal kinase 1 (JNK1) phosphorylation falls below
basal levels in response to high pressure. Immunocytochemical observations show phosphorylated MAPK in the nucleus at 10 min. The
expression of c-Fos, a nuclear transcription factor, is induced by high
pressure, and the induction is significantly inhibited by PD98059 (50 µM), an upstream MAPK/extracellular
signal-regulated kinase kinase (MEK) inhibitor of MAPK. The expression
of the c-Jun that is induced by JNK1 activation remains unchanged
during pressure-load. MAPK phosphorylation and cell proliferation by
applied pressure are significantly inhibited by genistein, a tyrosine
kinase inhibitor in a dose-dependent manner, but not by
protein kinase C inhibitors, chelerythrine and GF109203X. Genistein
also blocks pressure-induced tyrosine phosphorylation of proteins with
molecular masses of 35, 53, and 180 kDa. To clarify the physiological
role in MAPK activation under high pressure conditions, we transfected
antisense MAPK DNA into mesangial cells. The antisense DNA (2 µM) inhibited MAPK expression by 80% compared with
expression in the presence of sense or scrambled DNA, and significantly
blocked pressure-induced cell proliferation. Treatment of cells with
MEK inhibitor also produced a similar result. MEK inhibitor strongly
suppresses DNA synthesis induced by pressure-load. Cyclin D1 expression
is significantly increased under high pressure conditions, and the
increase is blocked by treatment with MEK inhibitor. These findings
show that pressure-load, a novel activator of MAPK, induces the
activation of tyrosine kinases, and enhances the proliferation of
mesangial cells, probably through cyclin D1 expression.
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INTRODUCTION |
Mesangial cells in glomeruli are located under a fenestrated
capillary endothelium and are exposed to hydrostatic pressure necessary
to sustain normal filtration (1-3). Progressive renal diseases, such
as diabetic nephropathy, remnant kidney, and hypertensive nephropathy,
lead to prolonged glomerular hypertension, which is involved in the
mesangial cell proliferation that is considered to be the most
important factor mediating glomerular sclerosis (4-11). However, the
mechanism of these changes under glomerular hypertension remains
largely unknown. Hishikawa et al. (12) have reported that,
in vascular smooth muscle cells, pressure promotes DNA synthesis and
cell growth probably via protein kinase C
(PKC),1 although the detailed
mechanism between pressure as an extracellular stimulus and
proliferation is unknown.
Various growth factors and mitogenic stimuli are known to induce the
activation of mitogen-activated protein kinase (MAPK), a
serine/threonine kinase (13, 14). This kinase activity is up-regulated
through phosphorylation on tyrosine and threonine residues by
MAPK/extracellular signal-regulated kinase kinases (MEKs) (15, 16).
MEKs are substrates for Raf-1 (17, 18), which has been reported to be
activated either through receptors involved in Ras or a
PKC-dependent pathway (19, 20). These MAPK activators cause
the translocation of MAPK from the cytosol to the nucleus (21-23),
where transcription factors such as Elk-1 (24) and c-Ets (25, 26) are
substrates for MAPK. This indicates that MAPK serves as an important
regulator of transcriptional activity related to proliferation.
Recently, Lavoie et al. (27) reported that cyclin D1
expression, which is one of the earliest cell cycle-related events to
occur during the G0/G1 to S phase transition,
is regulated positively by MAPK. Therefore, increasing interest has
been paid to the role of MAPK in the cell cycle (28-30). We recently
showed that pressure enhances G1/S progression and promotes
the rate of DNA synthesis in mesangial cells (31). However, MAPK
activation and its physiological effects in glomerular hypertension are
presently unknown. We investigated MAPK activation and cell
proliferation in mesangial cells using a pressure-loading apparatus. We
show here that applied pressure is a novel activator of MAPK.
Furthermore, we demonstrate that MAPK activation plays a role in
pressure-induced proliferation, probably via cyclin D1 expression.
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EXPERIMENTAL PROCEDURES |
Materials--
Anti-phospho-MAPK antibody (Tyr-204),
anti-phospho-Elk-1 (Ser-383) antibody, Elk-1 fusion protein, and
PD98059 (MEK inhibitor) were bought from New England Biolabs (Beverly,
MA). Anti-phospho-JNK1 antibody (Thr-183 and Tyr-185), anti-JNK1
antibody, anti-MAPK antibody, anti-c-Fos antibody, anti-cyclin D1
antibody, peptide substrate of myelin basic protein (APRTPGGRR), and
protein A/G-agarose were from Santa Cruz Biotechnology (Santa Cruz,
CA). Anti-c-Jun antibody and anti-phospho-tyrosine antibody were from
Transduction Laboratories (Lexington, KY). Anti-rabbit and
anti-mouse immunoglobulin G antibodies coupled to peroxidase were
obtained from Promega (Madison, WI). Anti-rabbit immunoglobulin G
antibody-linked fluorescein isothiocyanate was from Amersham
International plc (Buckinghamshire, UK). The enhanced chemiluminescence
reaction assay kit and PKC assay kit were from Pierce. Cell growth was
estimated with a cell counting kit (Dojindo Laboratories, Kumamoto,
Japan), which is a modified MTT assay kit using WST-1. Genistein,
chelerythrine, and GF109203X were from Calbiochem-Novabiochem
International (San Diego, CA). All other chemicals were commercially
available.
Preparation of Cells--
Rat mesangial cells were isolated as
described previously (31, 32). Mesangial cells were plated at a density
of 5 × 105 cells/dish in 100-mm culture dishes. After
incubation in serum-free RPMI 1640 medium for 72 h, the cells were
placed for the indicated times under high pressure conditions at
37 °C. High pressure conditions were applied using a
pressure-loading apparatus allowing for several levels of air pressure
under constant O2 and CO2 concentration and
temperature as described previously (31). For each time period, cells
were collected with a rubber policeman and used for biochemical
analyses.
Electrophoresis and Immunoblotting--
The collected cells were
lysed with lysis buffer (1% Triton X-100, 20 mM Tris (pH
7.5), 150 mM NaCl, 1 mM EDTA, 1 mM
EGTA, 2.5 mM sodium pyrophosphate, 1 mM
-glycerol phosphate, 1 mM sodium orthovanadate,
leupeptin (1 µg/ml), 1 mM phenylmethanesulfonyl fluoride). The cellular extracts and molecular mass standards were
electrophoresed in 12.5% (w/v) polyacrylamide gels in the presence of
SDS and transferred to polyvinylidene difluoride membranes (0.45 µm,
Millipore, Bedford, MA) in the case of phospho-MAPK and phospho-Elk-1,
or nitrocellulose membranes for other proteins. The blots were blocked
with 5% non-fat dry milk in Tris-buffered saline containing 0.05%
(w/v) Tween 20, and incubated with antibody. After the blots were
washed, the antigens were visualized by enhanced chemiluminescent
detection reagents. The levels of MAPK phosphorylation were determined
from the immunoblots by densitometric analysis and were corrected for
amounts of MAPK protein.
MAPK Activity and PKC Activity Assay--
MAPK activity was
determined as described previously (23). Briefly, the cells were lysed
in lysis buffer and clarified by centrifugation at 15,000 × g for 10 min at 4 °C. Protein content was normalized, and
the lysates were incubated with anti-phospho-MAPK antibody followed by
protein A/G-agarose. The complexes were washed twice in lysis buffer
and twice in kinase buffer (25 mM Tris (pH 7.5), 5 mM
-glycerol phosphate, 2 mM dithiothreitol,
0.1 mM sodium orthovanadate, 10 mM
MgCl2). Kinase reactions were carried out by resuspending
the complexes in 50 µl of kinase buffer containing 100 µM ATP and 1 µg of Elk-1, and incubating for 30 min at
30 °C. The reaction products were electrophoresed in
SDS-polyacrylamide (12.5%) gels, transferred to polyvinylidene
difluoride membranes, and probed with anti-phospho-Elk-1 antibody. As
another method, after cell lysates were incubated with anti-MAPK
antibody, MAPK activity was measured by incubating cell extracts for 10 min at 30 °C with 1 mM peptide substrate containing the
sequence of myelin basic protein phosphorylated by MAPK (APRTPGGRR) in
buffer (25 mM Tris-HCl, pH 7.4, 10 mM
MgCl2, 1 mM dithiothreitol, 40 µM
ATP, 2 µCi of [
-32P]ATP, 2 mM protein
kinase inhibitor peptide, 0.5 mM EGTA). The reaction was
stopped by the addition of 0.6% HCl, 1 mM ATP, and 1%
bovine serum albumin, and the mixture was centrifuged at 3000 × g for 5 min. The supernatant was spotted onto 1.0 × 1.0-cm squares of P81 paper (Whatman), which was washed five times for
10 min each time with 0.5% phosphoric acid, rinsed once with ethanol, dried, and counted by the Cerenkov technique (33). PKC activity was
measured with a colorimetric PKC assay kit (Pierce) according to the
manufacturer's instructions.
Immunofluorescent Staining of Phospho-MAPK--
Mesangial cells
were seeded in a Chamber Slide (Lab-Tek, Nunc Inc., Naperville, IL) at
a density of 3 × 104 cells/well. The cells were
subjected to 70 mm Hg high pressure for 10 min and then fixed with 4%
paraformaldehyde in phosphate- buffered saline (PBS) for 15 min at
4 °C. Following fixation, the cells were permeabilized with 0.2%
Triton X-100 in PBS for 15 min and blocked with 10% fetal calf serum
(FCS) in PBS for 30 min. The cells were then incubated for 1 h at
room temperature with antibody against phospho-MAPK at 1:100 dilution
in PBS containing 0.1% sheep serum albumin, washed with PBS, and
incubated for an additional hour at room temperature with fluorescein
isothiocyanate-conjugated anti-rabbit immunoglobulin G antibody at
1:100 dilution in PBS. The cells were viewed with a fluorescence
microscope (Axioplan 2, Carl Zeiss Co., Heidelberg, Germany).
Transfection of Antisense MAPK into Mesangial
Cells--
Oligonucleotide transfection was determined as described
previously (34). Briefly, the phosphorothioate oligonucleotide with the
sequence 5'-GCC GCC GCC GCC GCC AT-3' was synthesized as an antisense
DNA. Control phosphorothioate oligonucleotides were synthesized with
the following sequences: 5'-ATG GCG GCG GCG GCG GC-3' (sense) and
5'-CGC GCG CTC GCG CAC CC-3' (scrambled). The cells (typically 80%
confluent in 24-well dishes) were washed three times with PBS.
Appropriate dilutions of oligonucleotides in 200 µl of serum-free
RPMI 1640 including liposomes (Tfx-50, Promega Co.) were preincubated
at room temperature for 15 min. The cells were incubated for 2 h
at 37 °C in the presence of 5% CO2. At the end of the
incubation period, 1 ml of medium containing 10% FCS was added. After
incubating for 48 h, the cells were reseeded in 96-well dishes,
and incubated with serum-free medium for 24 h after washing with
500 µl of PBS. After a further 24 h the medium was replaced with
medium containing 0.5% FCS. After incubation under high pressure
conditions, cell number was estimated by WST-1 assay. The densitometric
measurements were done with plate analyzer (ETY3A, Toyo Sokki Co.,
Kanagawa, Japan).
DNA Synthesis--
DNA synthesis was estimated with an
immunocytochemical assay kit using monoclonal anti-bromodeoxyuridine
(BrdUrd) antibody to detect BrdUrd incorporation into cellular DNA (RPN
20, Amersham International). Briefly, growing cells were seeded in
chamber slides at a density of 3 × 104/well (0.81 cm2/well). The cells were incubated with serum-free medium
for 24 h and then incubated with 0.5% FCS, RPMI 1640 medium under
high pressure conditions. After 24 or 48 h, BrdUrd was added to
each sample for 30 min, and the cells were then fixed with 4%
paraformaldehyde after washing with PBS. After incubation with
anti-BrdUrd antibody for 60 min and horseradish peroxidase-conjugated
anti-mouse immunoglobulin G antibody for 30 min, BrdUrd incorporation
was visualized with 3,3'-diaminobenzidine tetrahydrochloride as a
substrate according to the manufacturer's instructions.
BrdUrd-positive cells and total cells (200-300 cells) in 10 fields
that were selected randomly in each sample were counted, and the
proportion of positive cells to total cells was calculated.
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RESULTS |
MAPK Activation by High Pressure--
MAPK is activated through
the phosphorylation of Thr-202 and Tyr-204 by MEK (13, 35-37). We
initially examined the phosphorylation of MAPK in mesangial cells under
high pressure conditions by immunoblotting with an antibody that
recognizes phosphorylation at Tyr-204 in MAPK. In pressure-treated
cells, their level of MAPK phosphorylation was significantly increased
at 1 min after pressure-load as compared with untreated cells (Fig.
1, A and C). The
amount of MAPK remained almost constant throughout pressure-load (Fig.
1B). To confirm MAPK activation by high pressure, we
examined MAPK activity by an immunoprecipitation kinase assay using
Elk-1 as a substrate. MAPK activity rose rapidly after pressure-load
with almost the same kinetics as MAPK phosphorylation (Fig.
2, A and B). To
further confirm MAPK activation by high pressure, we carried out
another kinase assay using myelin basic protein as described under
"Experimental Procedures." MAPK activity with MAPK
immunoprecipitates and myelin basic protein peptide, significantly
increased (6-fold) with similar kinetics to MAPK phosphorylation and
MAPK activity using Elk-1 as a substrate (data not shown). When
mesangial cells were exposed to high pressure (30, 50, 70, or 90 mm Hg)
or to atmospheric pressure (0 mm Hg), the level of MAPK phosphorylation
increased in a pressure-dependent manner (Fig.
3, A and C). The
amount of MAPK remained almost constant in each lane (Fig.
3B). These findings show that MAPK is activated by tyrosine
phosphorylation in mesangial cells under high pressure conditions. It
is known that JNK1, a member of the MAPK superfamily, is activated by
phosphorylation by SEK1 in response to various stresses (38-41). We
measured the level of JNK1 phosphorylation necessary for activation by
immunoblotting with an anti-phospho-JNK1 antibody. As shown in Fig.
4A, JNK1 was already
phosphorylated under the control conditions, and the level of
phosphorylation was rather lower during pressure-load, although the
amount of JNK1 remained almost unchanged during pressure-load (Fig.
4B). These results indicate that JNK1 phosphorylation
decreases during pressure-load.

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Fig. 1.
Phosphorylation of MAPK in rat mesangial
cells under high pressure conditions. Cell extracts (40 µg of
protein) were prepared from mesangial cells exposed to 70 mm Hg high
pressure for the indicated times and subjected to immunoblotting with
anti-phospho-MAPK antibody (A) or anti-MAPK antibody
(B). A representative immunoblot from three independent
experiments is shown. The levels of MAPK phosphorylation were
determined from the immunoblots by densitometric analysis (mean ± S.E., n = 3) (C) as described under
"Experimental Procedures."
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Fig. 2.
MAPK activity in rat mesangial cells under
high pressure conditions. Cell extracts (600 µg of protein) were
prepared from mesangial cells exposed to 70 mm Hg high pressure for the
indicated times and subjected to immunoprecipitation with
anti-phospho-MAPK antibody followed by protein A/G-agarose. The
complexes were incubated with 100 µM ATP and 1.0 µg of
Elk-1, and subjected to immunoblotting with anti-phospho-Elk-1 antibody
(A). A representative immunoblot from three independent
experiments is shown. The MAPK activities were determined from the
immunoblots by densitometric analysis (mean ± S.E.,
n = 3) (B).
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Fig. 3.
Effect of pressure-load on MAPK
phosphorylation in rat mesangial cells. Cell extracts (40 µg of
protein) were prepared from mesangial cells exposed to the indicated
pressures for 3 min and subjected to immunoblotting with
anti-phospho-MAPK antibody (A) or anti-MAPK antibody
(B). A representative immunoblot from three independent
experiments is shown. The levels of MAPK phosphorylation were
determined from the immunoblots by densitometric analysis (mean ± S.E., n = 3, *p < 0.05 versus control) (C).
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Fig. 4.
Phosphorylation of JNK1 in rat mesangial
cells under high pressure conditions. Cell extracts (40 µg of
protein) were prepared from mesangial cells exposed to 70 mm Hg high
pressure for the indicated times, and subjected to immunoblotting with
anti-phospho-JNK1 antibody (A) or anti-JNK1 antibody
(B). A representative immunoblot from three independent
experiments is shown.
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To investigate upstream kinases in the pressure-induced MAPK
activation, we examined the MAPK phosphorylation in the presence of
inhibitors for PKC and tyrosine kinases. Genistein, a tyrosine kinase
inhibitor, inhibited pressure-induced MAPK phosphorylation in a
dose-dependent manner (Fig.
5, A and C).
However, chelerythrine and GF109203X, PKC inhibitors, did not affect
MAPK phosphorylation (Fig. 5, A and C). The
amount of MAPK was almost constant in each lane (Fig. 5B).
Furthermore, to confirm the effect of genistein, we examined tyrosine
phosphorylation with immunoblotting using anti-phospho-tyrosine
antibody. Applied pressure induces the tyrosine phosphorylation of
proteins with molecular masses of 35, 53, and 180 kDa, and the tyrosine
phosphorylation was inhibited by genistein (Fig. 5D) in a
dose-dependent manner (0.1-10 µM). PKC
inhibitors had no effects on the pressure-induced tyrosine
phosphorylation (Fig. 5D). Genistein also inhibited
pressure-induced cell proliferation, whereas PKC inhibitors had no
effect (data not shown). PKC activity was weakly increased by applied
pressure, the activity of which was blocked by PKC inhibitors, but not
by 10 µM genistein (data not shown). These findings
suggest that tyrosine kinases are involved in the pressure-induced MAPK
activation.

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Fig. 5.
Effect of various inhibitors on
pressure-induced MAPK phosphorylation and tyrosine
phosphorylation. Cell extracts (40 µg protein) were prepared
from mesangial cells exposed to 70 mm Hg high pressure for 3 min in the
presence of each inhibitor and subjected to immunoblotting with
anti-phospho-MAPK antibody (A), anti-MAPK antibody
(B), or anti-phopho-tyrosine antibody (D). A
representative immunoblot from three independent experiments is shown.
The levels of MAPK phosphorylation were determined from the immunoblots
by densitometric analysis (mean ± S.E., n = 3, *p < 0.05) (C).
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MAPK substrates are present in various subcellular fractions (21, 22).
To examine the physiological effect of MAPK under high pressure
conditions, we observed the subcellular localization of MAPK by
immunocytochemical staining using an anti-phospho-MAPK antibody.
Immunocytochemical observations showed the phosphorylated MAPK to be
present mainly in the cytoplasm 1 min after pressure-load (data
not shown), and the nuclear staining was significantly increased after
10 min of pressure-load (Fig. 6,
C and D). The staining was not observed under
control conditions (atmospheric pressure) (Fig. 6, A and
B) and was inhibited by preincubating the primary antibody
with activated MAPK (data not shown). These findings suggest that MAPK
has a physiological effect on the nucleus.

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Fig. 6.
Subcellular localization of phospho-MAPK in
mesangial cells under high pressure conditions. The mesangial
cells were incubated for 0 min (A and B) or 10 min (C and D) at 37 °C under the condition of
70 mm Hg high pressure, fixed in 4% paraformaldehyde, and stained with
anti-phospho-MAPK antibody as described under "Experimental
Procedures." Phase-contrast microscopic photographs were shown in
A and C. A representative photograph from three
independent experiments is shown. Final magnification, × 400.
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It is known that MAPK in the nucleus can phosphorylate Elk-1 (24), a
transcription factor, and that this phosphorylation subsequently leads
to an increase in the expression of c-Fos (42). We examined the
expression of c-Fos under high pressure conditions by immunoblotting
and found it to be significantly increased for 12 h after
pressure-load (Fig. 7, A and
B). This increase was inhibited by treatment with 50 µM MEK inhibitor (Fig. 8,
A and B). In addition, antisense DNA against
MAPK, as described below, also inhibits pressure-induced c-Fos
expression (data not shown). On the other hand, the expression of
c-Jun, a nuclear transcription factor that is induced by JNK
activation, remained almost unchanged under 70 mm Hg pressure for up to
48 h (Fig. 9). These observations demonstrate that applied pressure induces the expression of c-Fos via
the activation of MAPK.

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Fig. 7.
Expression of c-Fos in rat mesangial cells
under high pressure conditions. Cell extracts (40 µg of protein)
were prepared from mesangial cells exposed to 70 mm Hg high pressure
for the indicated times, and subjected to immunoblotting with
anti-c-Fos antibody (A). A representative immunoblot from
three independent experiments is shown. The amount of c-Fos was
determined from the immunoblots by densitometric analysis (mean ± S.E., n = 3, *p < 0.05 versus control) (B).
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Fig. 8.
Effect of MEK inhibitor on c-Fos expression
in mesangial cells under high pressure conditions. Cell extracts
(40 µg of protein) were prepared from the mesangial cells exposed to
70 mm Hg high pressure for 24 h in the presence or absence of MEK
inhibitor (50 µM PD98059) and subjected to immunoblotting
with anti-c-Fos antibody (A). A representative immunoblot
from three independent experiments is shown. The amount of c-Fos was
determined from the immunoblots by densitometric analysis (mean ± S.E., n = 3, *p < 0.05)
(B).
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Fig. 9.
Expression of c-Jun in rat mesangial cells
under high pressure conditions. Cell extracts (40 µg of protein)
were prepared from mesangial cells exposed to 70 mm Hg high pressure
for the indicated times and subjected to immunoblotting with anti-c-Jun
antibody. A representative immunoblot from three independent
experiments is shown.
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Cell Proliferation through MAPK Activation by High
Pressure--
We recently showed that high pressure enhances the rates
of DNA synthesis and cell proliferation (31). To investigate the role
of MAPK in pressure-induced proliferation, we transfected an antisense
oligonucleotide against MAPK into mesangial cells, and found MAPK
expression to be inhibited in a concentration-dependent manner (0.1-2.0 µM) (Fig.
10, A and B).
Incubation of cells with 2 µM sense or scrambled DNA for
MAPK did not significantly affect MAPK expression for up to 4 days.
JNK1 expression remained almost unchanged in cells transfected with
each MAPK DNA as compared with untreated cells. We examined cell
proliferation using the WST-1 assay. Mesangial cells whose MAPK protein
was depleted by antisense DNA, completely inhibited pressure-induced
proliferation, but sense DNA or scrambled DNA-treated cells enhanced
proliferation to the same extent as untreated cells for up to 48 h
under high pressure conditions (Fig.
11). Cell treatment with MEK inhibitor produced similar results in terms of cell numbers (data not shown). DNA
synthesis in mesangial cells was enhanced under 70 mm Hg high pressure
condition as described previously (31). This enhancement was strongly
blocked by 50 µM MEK inhibitor (Fig.
12). These findings show that MAPK
activation participates in pressure-induced proliferation. In
addition, the applied pressure for 30 min weakly induced the cell
proliferation at 24 h under the condition of atmospheric pressure,
but not at 48 h (data not shown), which suggests that transient
pressure can proliferate mesangial cells.

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Fig. 10.
Effect of MAPK antisense DNA on MAPK
expression in rat mesangial cells under high pressure conditions.
Cell extracts (20 µg of protein) were prepared from mesangial cells
transfected with antisense DNA or sense DNA and subjected to
immunoblotting with anti-MAPK antibody (A). A representative
immunoblot from two independent experiments is shown. The amount of
MAPK was determined from the immunoblots by densitometric analysis
(B).
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Fig. 11.
Effect of MAPK antisense DNA on the
proliferation of mesangial cells under high pressure conditions.
Mesangial cells were pretreated with 2 µM of scrambled
DNA (open column), sense DNA (closed column), or
antisense DNA (hatched column) for 2 h in RPMI 1640 containing liposomes (Tfx-50). Cell numbers under the conditions of 0 mm Hg (atmospheric pressure) or 70 mm Hg (high pressure) for the
indicated times were determined by WST-1 assay (mean ± S.E., n = 4, *p < 0.05) as
described under "Experimental Procedures."
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Fig. 12.
Effect of MEK inhibitor on DNA synthesis in
mesangial cells under high pressure conditions. Mesangial cells
were incubated in the presence or absence of MEK inhibitor (50 µM; PD98059) under the conditions of 0 mm Hg (atmospheric
pressure) or 70 mm Hg (high pressure) for the indicated times. DNA
synthesis was determined by immunocytochemical assay using anti-BrdUrd
(BrdU) antibody to detect BrdUrd incorporation into cellular
DNA as described under "Experimental Procedures." Ten fields were
examined in a sample, BrdUrd-positive cells in total cells (200-300
cells) were counted in each field, and the proportion of
BrdUrd-positive cells to total cells was calculated (mean ± S.E.,
n = 4, *p < 0.05).
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It was recently reported that cyclin D1 expression, which is required
to pass the G1 restriction point, is positively regulated by MAPK (27). We examined cyclin D1 expression under high pressure conditions by immunoblotting using an anti-cyclin D1 antibody. The
expression of cyclin D1 was induced after 12 h of pressure-load (Fig. 13, A and
B), and the expression was significantly suppressed to basal
levels by MEK inhibitor (Fig. 14,
A and B). The results suggest that cyclin D1,
induced by MAPK activation, may be involved in cell proliferation under
high pressure conditions.

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Fig. 13.
Expression of cyclin D1 in rat mesangial
cells under high pressure conditions. Cell extracts (40 µg of
protein) were prepared from mesangial cells exposed to 0 mm Hg
(atmospheric pressure) or 70 mm Hg (high pressure) for the indicated
times and subjected to immunoblotting with anti-cyclin D1 antibody
(A). A representative immunoblot from three independent
experiments is shown. The amount of cyclin D1 was determined from the
immunoblots by densitometric analysis (mean ± S.E.,
n = 3, *p < 0.05)
(B).
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Fig. 14.
Effect of MEK inhibitor on cyclin D1
expression in mesangial cells under high pressure conditions. Cell
extracts (40 µg of protein) were prepared from the mesangial cells
exposed to 0 mm Hg (atmospheric pressure) or 70 mm Hg (high pressure)
for 24 h in the presence or absence of MEK inhibitor (50 µM; PD98059), and subjected to immunoblotting with
anti-cyclin D1 antibody (A). A representative immunoblot
from three independent experiments is shown. The amount of cyclin D1
was determined from the immunoblots by densitometric analysis
(mean ± S.E., n = 3, *p < 0.05)
(B).
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DISCUSSION |
We have investigated MAPK activation in mesangial cells exposed to
high pressure. During the study we demonstrated that a few minutes of
pressure-load induces the activation and nuclear translocation of MAPK,
which leads to an increase in the expression of c-Fos during the
pressure-load. MAPK activation under high pressure conditions increases
cyclin D1 expression and DNA synthesis, and finally enhances cell cycle
progression. In addition, pressure-induced MAPK activation and cell
proliferation might be involved in tyrosine kinases.
MAPK is strongly activated by growth factors and growth-promoting
hormones (13, 43-47), in contrast to JNK, which is preferentially activated by environmental stresses and pro-inflammatory cytokines (14,
38, 48, 49). In this study, applied pressure, which is a physical force
generated by hydrostatic pressure, promoted the activation of MAPK but
not JNK1. MAPK has been reported to phosphorylate Elk-1, a nuclear
transcription factor (50, 51). Elk-1 binds to a serum response element
within the c-fos promoter region together with a serum
response factor, inducing c-Fos expression (42, 52, 53). High pressure
induces c-Fos expression, and the induction is inhibited by both MAPK
antisense oligonucleotide and MEK inhibitor. These observations
demonstrate that MAPK activation is involved in the expression of c-Fos
under high pressure conditions. A transcription factor, c-Ets, is also
a substrate for MAPK (54, 55). The promoter region of cyclin D1 has an
Ets-like binding domain that regulates cyclin D1 expression (25, 54).
Recently, Lavoie et al. (27) reported that MAPK plays a
positive regulatory role in cyclin D1 expression. Consistent with their
report, our present data show that pressure-load activates MAPK, which
increases cyclin D1 expression and an enhancement of DNA synthesis and
cell growth. Cyclin D1 plays an important role in the entry of cells into S phase and cell cycle progression (56-59). Therefore, cyclin D1
expression may participate in pressure-induced proliferation since
pressure-load contributes to cell cycling by enhancing G1/S progression and promoting the rate of DNA synthesis in mesangial cells
as described previously (31).
The mechanism of MAPK activation under high pressure conditions is
poorly elucidated. It does not appear that mesangial cells secrete
growth factors that would activate MAPK during pressure-load for the
following two reasons. First, we added a supernatant from pressure-treated cells to untreated cells and observed no cell proliferation under the experimental conditions employed (data not
shown). This result is consistent with studies on smooth muscle cells
as described previously (12). Second, MAPK is rapidly activated,
reaching a peak at 1 min after pressure-load; there seems to be no time
delay due to the secretion of growth factors after pressure-load.
Hishikawa et al. (12) have reported that pressure promotes
the activation of PKC with Ca2+ mobilization in cultured
smooth muscle cells. It is well known that various stresses activate
distinct PKC isoforms (60-63). In mesangial cells as well as smooth
muscle cells, it is possible that pressure-load activates PKC isoforms.
In our study, PKC activity was weakly increased by applied pressure.
However, pressure-induced MAPK phosphorylation was inhibited by
tyrosine kinase inhibitor, but not by PKC inhibitors. In addition,
applied pressure induces tyrosine phosphorylation of proteins with
molecular masses of 180, 53, and 35 kDa, and tyrosine phosphorylation
was inhibited by genistein. Thus, it is strongly suggested that
tyrosine kinases are involved in the pressure-induced MAPK
activation.
MAPK activation in mesangial cells is thought to play an important role
in the development of renal injury. Pressure-load itself activates
MAPK, which contributes to the mesangial cell proliferation that is a
central feature of numerous glomerular diseases (8-11). In addition,
Bokemeyer et al. (64) have recently reported that MAPK is
significantly activated by proliferative glomerulonephritis in response
to immune injury. The proliferation induced by renal diseases is an
important aspect of the pathogenic process of glomerular sclerosis
(64). In these diseases, this is often accompanied not only by the
proliferation of mesangial cells but also by expansion of the
extracellular matrix (65-68). Transforming growth factor-
(TGF-
)
is reported to be a factor in regulating the extracellular matrix and
inducing glomerular sclerosis (67-69). It is known that the promoter
region of TGF-
contains an AP1 element (70-73) and that c-Fos
expression regulates TGF-
expression. In our study, we found that
pressure-load significantly increases the expression of TGF-
in
mesangial cells for 24 h.2 Pressure-load may
increase TGF-
expression through c-Fos induced by MAPK activation.
It is possible that pressure-load contributes to the development
of renal injury by both mesangial proliferation and matrix expansion
through MAPK activation. This MAPK activation of mesangial cells in
response to pressure-load might be an underlying mechanism in the
development of renal injury with glomerular hypertension. The present
study provides new insights into the role of MAPK in glomerular
hypertension.