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INTRODUCTION |
During water deprivation, the extracellular osmolality in the
mammalian renal medulla can exceed 3000 mosmol/kg of H2O.
While such an osmolality is incompatible with the survival of cells from other organs, kidney cells tolerate it well. The survival of
kidney medullary cells in hypertonic environment is essential for the
generation of the concentrating gradient, which is key for maintenance
of body solute and water homeostasis.
Many organisms, including bacteria, yeast, plants, and animals, adapt
to sustained hyperosmotic stress by accumulating osmotically active
organic solutes (compatible organic osmolytes) (1). These compounds do
not perturb cellular macromolecules and are preferentially accumulated
over inorganic salts (1). Extracellular hyperosmolality exerted by a
non-permeable solute (e.g. NaCl or raffinose, but not urea)
has a hypertonic effect, i.e. it shrinks cells and increases
intracellular potassium and sodium concentrations (ionic strength).
There is evidence that increased intracellular ionic strength is among
the initial signals for the induction of genes responsible for organic
osmolytes accumulation (osmoprotective genes) (2, 3). How the increased
intracellular ionic strength ultimately induces transcription of these
genes remains to be determined. The molecular mechanisms involved in
this adaptive process in mammalian cells have been extensively studied
in two renal epithelial cell lines, Madin-Darby canine kidney
(MDCK)1 and PAP-HT25 (rabbit
renal papillary epithelium) (4). In these cells, hypertonicity induces
the transcription of genes that encode proteins (specific enzymes and
transporters) directly involved in the metabolism and transport of
organic osmolytes such as sorbitol (2, 5, 6), betaine (7), and inositol
(8). The induction of these genes by hypertonic stress appears to be
relatively specific, since it is not produced by other stresses such as
heat shock (9). Hypertonicity also induces mRNA for heat shock
protein 70 (HSP70), and it is proposed that the induction of heat shock proteins during osmotic stress protects intracellular macromolecules from the harmful effects of elevated intracellular ionic strength until
the cell accumulates the appropriate level of organic osmolytes (9,
10).
Recently, it was reported that the mitogen-activated protein kinase
(MAPK) HOG1 and its activator (MAPK kinase), PBS2, are involved in
osmosensing signal transduction pathway in yeast, and that cells that
carry mutations in these genes fail to grow in medium supplemented with
NaCl (11). In mammalian cells, activation by osmotic stress of three
MAPK pathways has been shown so far, JNK (also known as
stress-activated protein kinase (SAPK)) (12, 13), ERK (13, 14) and p38,
a lipopolysaccharide-induced MAPK in mammalian cells (HOG1 homologue),
which complements HOG1 gene mutant yeast cells that fail to grow
otherwise in hypertonic medium (15).
While demonstration of MAPKK and ERK activation by osmotic stress in
MDCK cells has been shown (14), ERK activity does not appear to be
essential for either transcriptional regulation of the betaine
transporter gene (16) or the stimulation of inositol uptake (17) during
osmotic stress. Furthermore, despite evidence linking N-terminal Jun
phosphorylation to osmosensing transduction pathway proximal to gene
activation (12), it is unclear which MAPK transduction pathway is
essential for osmotically driven gene transcription in mammalian
cells.
Here we show that the p38 kinase inhibitor SB203580 blocks the
osmotically driven induction of mRNAs for HSP70 and BGT1, the betaine transporter. The effect is dose-dependent and
correlates with in situ p38 kinase activity. Consistent with
previous reports (18, 19), ERK-1 and -2 activities were not inhibited
by SB203580, but surprisingly, inhibition of p38 kinase in
hypertonically stressed cells correlates with marked up-regulation of
JNK-1 activity. In addition, we find that the thermal induction of
HSP70 mRNA is not dependent on the activity of p38 kinase,
suggesting divergent pathways for induction of HSP70 gene by different
stresses.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Confluent MDCK cells (American Type Culture
Collection, Rockville, MD), grown on polystyrene (Corning, NY) dishes
were used at passages 65-78. Cells were grown in serum-free,
previously defined medium (20, 21) (315 mosmol/kg of H2O),
containing 120 µM myoinositol (inositol) and no betaine
or taurine. In some experiments, the media were made hyperosmotic by
the addition of 200 mosmol of NaCl/kg of H2O (final
osmolality of 515-525 mosmol/kg of H2O). All cultures were
maintained in 5% CO2, 95% air at 37 °C. Heat shock was
induced by placing cells in 42 °C incubator for predetermined
periods of time.
Northern Blot Analysis--
Total RNA was isolated using RNAzol
(22), and poly(A)+ RNA was isolated with oligo(dT) columns
(Collaborative Biomedical Products), as described previously (6).
Electrophoresis was performed by loading equal amounts of
poly(A)+ RNA per lane on 1% agarose, 2.2 M
formaldehyde gel, followed by transfer to GeenScreen membrane (NEN Life
Science Products) (6). Dog betaine transporter cDNA was obtained by
NotI, MluI digestion of pBGT1 (23) (a generous
gift from Drs. Kwon and Handler, Johns Hopkins School of Medicine,
Baltimore, MD). Human HSP70 insert (2.3 kb) was obtained by
BamHI, HindIII digestion of pAT153 (American Type
Culture Collection). Human (2 kb) full-length
-actin cDNA was
purchased from CLONTECH. Inserts were labeled with
[
-32P]dCTP (Random Primed DNA labeling kit, Boehringer
Mannheim) for use as probes. Probes are hybridized to the blots
overnight at 42 °C in a solution containing 40% formamide, 5 × SSC (0.75 M NaCl, 75 mM trisodium citrate,
pH 7), 5 × Denhardt's solution (0.5% (w/v)
polyvinylpyrrolidone, 0.5% (w/v) Ficoll 400), 0.5% SDS, 250 µg/ml
salmon sperm DNA, 10 mM Tris, pH 7.5, and 10% dextran sulfate. The blots were then washed under high stringency at 65 °C as follows: for 30 min twice in 3 × SSC, 0.5% SDS; 1 h in
3 × SSC, 0.5% SDS; 30 min twice in 0.3 × SSC, 0.5% SDS. A
35-nucleotide p38-specific antisense oligonucleotide probe was
designed, based on published data and used as probe. The sequence of
the p38-specific probe is 5
-TGG TCT GTA CCA GGA AAC AAT GTT CTT CCA
GTC AA-3
and corresponds to bases 857-891 of human p38 kinase (19)
(GenBank accession no. L35264). Sequence specificity of the probe was determined using BLAST search of the National Center for Biotechnology Information (NCBI) data bases, aided by Baylor College of Medicine Molecular Biology Computational Resource Center (MBCR) services. End-labeled oligonucleotide was hybridized to blots overnight at
42 °C in a solution as above but containing 100 mg/ml salmon sperm
DNA and no formamide. These blots were then washed under high
stringency at 42 °C as follows: for 30 min in 5 × SSC, 0.5% SDS; 1 h in 2 × SSC, 0.5% SDS; 30 min in 0.5 × SSC,
0.5% SDS; and 30 min in 0.1 × SSC, 0.5% SDS. The BGT1 probe
hybridizes in MDCK cells to 2.8- and 3.4-kb bands that behave similarly
under hypertonic conditions. HSP70 and
-actin probes hybridize to
2.8- and 2.2-kb bands, respectively. Autoradiographs were prepared using X-Omat AR film (Eastman Kodak Co.) with an intensifying screen.
Relative band intensities were determined by scanning laser
densitometry (Ultroscan, LKB Biotechnology). mRNA abundance was
determined relative to that of cells maintained in isotonic medium,
after normalization of band intensities to
-actin bands for each
respective time point. For consistency of BGT1 data analysis, the
2.8-kb bands were compared. mRNA abundance for HSP70 was determined relative to that of non-heat-stressed cells.
Kinase Assays--
p38 kinase activity was determined as per
Pombo et al. (24) and Moriguchi et al. (12) with
slight modifications. Briefly, experimental medium was removed and
cells were washed using ice-cold phosphate-buffered saline of equal
osmolality. Dishes were kept on ice, and cells were lysed in buffer
containing: 20 mM Tris, pH 7.4, 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 25 mM
-glycerophosphate, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, and 1 µg/ml leupeptin.Cell lysate was
cleared of large particles by 10-min centrifugation at 15,000 × g. After immobilizing anti-p38 kinase antibody (p38 antibodies from Santa Cruz Biotechnology, Santa Cruz, CA; p38b antibodies were a generous gift from J. Han, Scripps Institute, La
Jolla, CA) with Pansorbin (Calbiochem, La Jolla, CA), cell lysate (100 µg of protein) was added, and p38 kinase was immunoprecipitated. The
immunoprecipitate was washed five times in lysis buffer, followed by
five additional washes in kinase buffer. p38 kinase activity was
determined by in vitro labeling of activating transcription factor 2 (Santa Cruz Biotechnology) with 32P, in a buffer
containing: 25 mM Hepes, pH 7.4, 25 mM
-glycerophosphate, 25 mM MgCl2, 2 mM dithiothreitol, 0.1 mM sodium vanadate, 25 µM ATP. Following 20 min of incubation at 30 °C,
kinase reaction was stopped by 2-min centrifugation at 12,000 × g. Supernatant was suspended in Laemmli sample buffer (25),
and resolved on a 10% SDS-PAGE (polyacrylamide gel electrophoresis).
Gels were dried and autoradiographs taken. JNK-1 activity was
determined using 1-79 N-terminal amino acids of c-Jun (Santa Cruz
Biotechnology) as substrate and anti-JNK-1 antibodies (26) (Santa Cruz
Biotechnology) for immunoprecipitation. For ERK-1 and -2 assay, the
above procedure was used except for the following. Anti-ERK antibodies
(Upstate Biotechnology Inc.; the antibody cross-reacts with ERK-1 and
ERK-2) were used for immunoprecipitation, protein A/G-agarose was used to immobilize the antibodies, myelin basic protein (Sigma) was used as
substrate, and the reaction supernatant was resolved on 15% SDS-PAGE.
MAPKAP kinase-2 activity was determined as per Krump et al.
(27) with slight modification; cell lysate was obtained as described above. MAPKAP kinase-2 was immunoprecipitated using anti-MAPKAP kinase
2 antibodies (Stress Gen. Biotechnologies Corp., Victoria, British
Columbia, Canada), and protein A/G-agarose (Santa Cruz Biotechnology)
was used to immobilize the antibody. Immunoprecipitate was suspended in
25 µl of kinase buffer (20 mM MgCl2, 1 mM sodium vanadate, 5 mM NaF, 20 mM
-glycerophosphate, 20 mM
p-nitrophenylphosphate, 2 mM dithiothreitol, 20 mM ATP, 5 µCi of [
-32P]ATP, 50 mM HEPES, pH 7.4), and kinase reaction was started by the
addition of 3 µg of HSP27 substrate (Stress Gen.). Following 20 min
of incubation at 30 °C, the reaction was stopped by 2-min centrifugation at 12,000 × g. Supernatant was suspended in
Laemmli sample buffer, boiled for 2 min, and analyzed on 15% SDS-PAGE. The pellet was processed for Western blotting as described below.
Protein Quantitation--
p38 protein was quantified using a
modification of the method of Laemmli et al. (25). Briefly,
equal amounts of protein were loaded and run on 12% reducing SDS-PAGE.
Proteins were transferred at room temperature, 250 mA, onto
HybondTM-ECL membrane (Amersham Life Sciences) in a buffer containing
10% methanol, 0.03% SDS, 25 mM Tris, 52 mM
glycine, pH 8.3. Blots were blocked for 15 min with 5% dried milk in
TBST (20 mM Tris, pH 7.6, 137 mM NaCl, 0.05% Tween 20), and incubated for 45 min at room temperature, with 1:100 (1 µg/ml) dilution of polyclonal rabbit anti-p38 antibody (Santa Cruz
Biotechnology), in TBST containing 5% milk. After a 15-min wash in
TBST, blots were incubated with 1:1000 dilution of horseradish
peroxidase-conjugated secondary antibody in TBST buffer containing 5%
milk. Protein was visualized using ECL detection system (Amersham Life
Sciences) and quantified by densitometric analysis of the resultant
bands.
For quantitation of MAPKAPK-2, the pelete from the kinase assay (as
described above) was suspended in Laemmli sample buffer (25), boiled
for 2 min, and run on 12% reducing SDS-PAGE. Proteins were transferred
at room temperature, 250 mA for 1 h, onto HybondTM-ECL membrane
(Amersham Life Sciences) in a buffer containing, 10% methanol, 0.1%
SDS, 25 mM Tris, 52 mM glycine, pH 8.3. Blots
were blocked for 1 h with 5% dried milk in PBST (100 mM NaPO4, pH 7.5, 300 mM NaCl,
0.1% Tween 20), and incubated overnight at 4 °C, with 2 µg/ml
polyclonal anti-rabbit MAPKAPK-2 (Upstate Biotechnology Inc.), in PBST
containing 5% milk. After a 15-min wash in PBST, blots were incubated
for 1 h at room temperature, with 1:1000 dilution of horseradish
peroxidase-conjugated secondary antibody in PBST buffer containing 5%
milk. Protein was visualized using an ECL detection system (Amersham
Life Sciences) and quantified by densitometric analysis of the
resultant bands.
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RESULTS |
SB203580 Inhibits Hypertonicity-induced Betaine Transporter (BGT1)
and HSP70 mRNAs--
As mentioned above, hypertonicity shrinks
cells and increases intracellular ionic strength. The rise in
intracellular ionic strength is believed to be among the initial
signals for induction of osmoprotective genes (2, 3). To investigate
the involvement of p38 kinase in mediating osmotically driven
transcription of these genes, MDCK cells were exposed for 16 h to
hypertonic medium, in the absence or presence of increasing
concentrations of SB203580, a potent inhibitor of p38 kinase, and the
abundance of BGT1 mRNA was measured. NaCl increases the abundance
of BGT1 mRNA 8-fold above that of cells in isotonic medium (Fig.
1). SB203580 attenuates the hypertonic
induction of the mRNA in a dose-dependent manner with
IC50 of 35-40 µM and complete inhibition at
100 µM.

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Fig. 1.
P38 kinase inhibitor, SB203580, abolishes
hypertonically induced betaine transporter mRNA. MDCK cells
were grown in isotonic medium (315 mosmol/kg of H2O). At
time zero, they were switched to same medium, or medium made hypertonic
(500 mosmol/kg of H2O) by addition of NaCl in the absence
or, presence of variable concentrations of SB203580. After 16 h,
they were harvested for measurement of betaine mRNA abundance.
Line represents the means of two or three independent
determinations. Betaine (BGT1) mRNA abundance is expressed relative
to that of cells maintained in isotonic medium (dotted line
at a value of 1-fold) after normalization of band intensities to
-actin bands. Inset, representative Northern blot is
shown (HT, hypertonicity). Lane 1, isotonic
medium; lane 2, NaCl and 10 µM SB203580;
lane 3, NaCl and 100 µM SB203580; lane
4, NaCl and 50 µM SB203580; lane 5, NaCl
alone.
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In addition to induction of osmoprotective genes, hypertonicity was
shown to induce heat shock proteins
B-crystallin and HSP70 (10, 28).
Therefore, we examined whether p38 kinase is involved in hypertonic
induction of HSP70. Hypertonicity increases the abundance of HSP70
mRNA 6-fold above that of cells in isotonic medium (Fig.
2). As seen with BGT1, hypertonic
induction of HSP70 mRNA is inhibited by SB203580 with similar
IC50. We conclude that p38 kinase activity is essential for
hypertonic induction of BGT1 mRNA, a representative gene involved
in the accumulation of compatible organic solute betaine in MDCK cells.
In addition, p38 kinase activity appears to be essential for hypertonic
induction of HSP70 mRNA as well. These findings suggest that p38
kinase is essential for the adaptation of kidney cells to osmotic
stress, either by induction of heat shock proteins or of osmoprotective
genes.

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Fig. 2.
P38 kinase inhibitor, SB203580, abolishes
hypertonically induced HSP70 mRNA. MDCK cells were grown in
isotonic medium (315 mosmol/kg of H2O). At time zero, they
were switched to the same medium or to medium made hypertonic (500 mosmol/kg of H2O) by addition of NaCl in the absence or
presence of variable concentrations of SB203580. After 16 h, they
were harvested for measurement of HSP70 mRNA abundance.
Line represents the means of two or three independent
determinations. HSP70 mRNA abundance is expressed relative to that
of cells maintained in isotonic medium (dotted line at a
value of 1-fold) after normalization of band intensities to -actin
bands. Inset, representative Northern blot is shown (HT, hypertonicity). Lane 1, isotonic medium;
lane 2, NaCl alone; lane 3, NaCl and 50 µM SB203580; lane 4, NaCl and 100 µM SB203580; lane 5, NaCl and 10 µM SB203580.
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SB203580 Inhibits Native p38 Kinase Activity, but Not ERK-1 or -2 in Osmotically Stressed Cells--
This inhibition correlates with
up-regulation of native JNK-1 activity. The pyridinyl imidazole
compounds to which SB203580 belongs demonstrate a highly specific and
potent inhibitory activity against p38 kinase. In earlier studies,
these compounds showed no inhibitory activity against ERK-1 and -2, JNK, MAPK-activated protein kinase-2 (MAPKAPK-2), MAPKK, protein kinase
C, calmodulin-dependent protein kinase, or
cAMP-dependent protein kinase (18, 19). In fact, the
binding specificity of these compounds was utilized to identify and
affinity-purify p38 kinase (19). Recently, a second p38 kinase, p38b,
was identified (18). Both kinases are equally inhibited by pyridinyl
imidazole and display near identical response to tumor necrosis factor,
interleukin-1, epidermal growth factor,
phorbol-12-myristate-13-acetate, and extracellular stresses such as UV
irradiation, H2O2, osmotic stress, and
arsenate. Since the inhibitory effect of the emidazol compounds on p38
kinase is reversible (29), in vitro examination of p38
kinase activity may not reflect its in situ activity. To
examine the specificity of SB203580 on the activity of p38 kinase in
our system, MDCK cells were placed for 16 h in hypertonic medium
in the presence of 0, 10, 25, 50, or 100 µM SB203580, and
cell lysates were prepared for determination of native MAPKAPK-2
activity. p38 kinase is the only known activator of MAPKAPK-2, and the
activity of MAPKAPK-2 is dependent on its phosphorylation by p38 kinase
(27). Therefore, the in vitro activity of immunoprecipitated
MAPKAPK-2 reflects the in situ activity of p38 kinase.
MAPKAPK-2 activity declines with increasing medium concentrations of
SB203580, with an IC50 of 35-40 µM (Fig.
3, A and B). The
kinetics of MAPKAPK-2 activity is similar to the kinetics of hypertonic
induction of HSP70 and BGT1 mRNAs in the presence of SB203580 (see
Figs. 1 and 2). To exclude the possibility of change in MAPKAPK-2 or
p38 kinase protein abundance, as a potential cause for the change in
p38 kinase activity, Western blot analysis was performed on cell
lysates. Cell treatment with SB203580 for 16 h does not affect the
abundance of MAPKAPK-2 or p38 kinase protein (Fig. 3C). It
is concluded that SB203580 exerts specific inhibitory effect on p38
kinase in MDCK cells. The decline in p38 kinase activity does not
result from diminished protein. Rather, it results from inhibition of
p38 kinase enzymatic activity. However, the concentration of SB203580
that is required for p38 kinase inhibition in situ in these
cells is higher than that reported for hematopoietic cells (18, 19,
30). These data are consistent with a role for p38 kinase in the
induction of BGT1 and HSP70 genes during osmotic stress.

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Fig. 3.
A, SB203580 inhibits p38 kinase in
situ as determined by examination of MAPKAPK-2 activity. The
in vitro activity of immunoprecipitated MAPKAP kinase-2
declines with increasing medium concentrations of SB203580. MDCK cells
were grown in isotonic medium (315 mosmol/kg of H2O). At
time zero, they were switched to the same medium or medium that was
made hypertonic (515 mosmol/kg of H2O) by the addition of
NaCl, in the presence of variable concentrations of SB203580. After
16 h, cells were harvested and cell lysates prepared for
determination of MAPKAPK-2 activity. Line represents the
means of four or five independent determinations. MAPKAPK-2 activity is
expressed relative to that of cells maintained in hypertonic medium.
B, representative gel of a MAPKAPK-2 assay is shown.
Lanes 1 and 2, isotonic medium without SB203580;
lanes 3 and 4, hypertonic medium with zero
SB203580; lanes 5 and 6, hypertonic medium with 10 µM SB203580; lanes 7 and 8,
hypertonic medium with 25 µM SB203580; lanes 9 and 10, hypertonic medium with 50 µM SB203580;
lanes 11 and 12, hypertonic medium with 100 µM SB203580. Panel represents HSP27 phosphorylation
in vitro by immunoprecipitated MAPKAPK-2. C,
SB203580 does not affect p38 kinase or MAPKAPK-2 protein abundance. MDCK cells were treated as in A. Cell lysates (for p38
kinase) or immunoprecipitates (for MAPKAPK-2) were run on SDS-PAGE, and blots were reacted with anti-p38 kinase (upper panel) or
anti-MAPKAPK-2 antibodies (lower panel). Upper
panel, lane 1, isotonic medium without SB203580;
lane 2, hypertonic medium without SB203580; lane
3, hypertonic medium with 50 µM SB203580; lane
4, hypertonic medium with 100 µM SB203580.
Lower panel, lane 1, isotonic medium without
SB203580; lane 2, hypertonic medium without SB203580; lane 3, hypertonic medium with 10 µM SB203580;
lane 4, hypertonic medium with 25 µM SB203580;
lane 5, hypertonic medium with 50 µM SB203580;
lane 6, hypertonic medium with 100 µM
SB203580.
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Of interest, JNK-1 activity in the hypertonically stressed cells is
equal to that of non-stressed cells. Inhibition of native p38 kinase
activity in the hypertonically stressed cells correlates with
8-10-fold increase in native JNK-1 activity (Fig.
4). No change is seen in the activities
of ERK-1 and -2 with medium SB203580 concentrations up to 100 µM (Fig. 4). The data suggest that, in hypertonically
stressed cells, p38 kinase may have an inhibitory effect on JNK-1,
either directly or indirectly.

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Fig. 4.
In vitro activity of native ERK-1 and -2, JNK-1, p38, p38b, and MAPKAPK-2 from SB203580-treated and
hypertonically stressed MDCK cells. p38 kinase inhibition in
situ correlates with up-regulation of JNK-1 activity. The
activities of ERK-1 and -2 are not affected by SB203580. MDCK cells
were grown in isotonic medium (315 mosmol/kg of H2O). At
time zero they were switched to the same medium or medium that was made
hypertonic (515 mosmol/kg of H2O) by the addition of NaCl,
in the absence or presence of SB203580 (50 or 100 µM).
After 16 h, cells were harvested and cell lysates prepared for
determination of native ERK-1 and -2, p38, p38b, JNK-1, and MAPKAPK-2
activities. Lane 1, isotonic medium without SB203580;
lane 2, hypertonic medium without SB203580; lane
3, hypertonic medium with 50 µM SB203580; lane
4, hypertonic medium with 100 µM SB203580.
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SB203580 Does Not Affect Thermal Induction of HSP70
mRNA--
It was previously reported that HSP70 is induced by the
stress of hypertonicity, and accumulation of organic osmolytes by the
cell attenuates the thermal and hypertonic induction of HSP70 mRNA
(9). While heat shock proteins are induced in response to general
stresses (ischemia, arsenate, heat stress, osmotic stress, heavy
metals, alcohol, and amino acid analogues) (9, 31, 32), the
osmoprotective genes, such as the betaine transporter, are specifically
induced by osmotic stress (9). The inhibition of hypertonic induction
of HSP70 and betaine transporter mRNAs by SB203580 presents the
following question. Is the SB203580-sensitive pathway specific for
induction of genes following osmotic stress, or does it mediate gene
expression during exposure of kidney cells to general stresses?
SB203580-treated (0, 50, and 100 µM) MDCK cells were
heat-shocked (42 °C for up to 3 h), and HSP70 mRNA abundance was examined. As seen in Fig.
5, thermal induction of HSP70 mRNA is
not affected by SB203580 at concentrations sufficient to block the
hypertonic induction of HSP70 completely. These results suggest that
HSP70 (and perhaps other heat shock proteins) induction is mediated
through more than one pathway; hypertonic induction of HSP70 is
mediated by an SB203580-sensitive pathway, similar to the pathway that
mediates hypertonic induction of betaine transporter mRNA, while
thermal induction of HSP70 mRNA is not SB203580-sensitive.

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Fig. 5.
SB203580 does not inhibit thermal induction
of HSP70 mRNA. Control or SB203580-treated MDCK cells were
subjected to 42 °C heat shock for 0, 1, or 3 h. Cells were
harvested for determination of HSP70 (upper panel) and
-actin (lower panel) mRNA abundance. Lanes
1 and 8, without heat stress and without SB203580;
lanes 2, 4, and 6 correspond to 1-h
heat stress with 0, 50, and 100 µM SB203580,
respectively; lanes 3, 5, and 7 correspond to 3-h heat stress with 0, 50, and 100 µM
SB203580, respectively.
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p38 Kinase mRNA Is Induced by Hypertonic Stress; This Induction
Is SB203580-sensitive--
Regulatory proteins can modulate the
abundance of their own mRNAs directly or indirectly (33). If a
positive loop existed between p38 kinase activity and the regulation of
its own mRNA, it may be possible to demonstrate down-regulation of
p38 kinase mRNA with inhibition of kinase activity. MDCK cells were
exposed for 16 h to hypertonic medium, in the absence or presence
of increasing concentrations of SB203580. As seen in Fig.
6, a 4.2-kb band is detected with a
p38-specific oligonucleotide probe. The mRNA is induced 1.5-2-fold
by hypertonic stress. This induction is markedly attenuated (2.5-fold,
relative to hypertonically stressed cells) in the presence of SB203580
at concentrations greater than 50 µM. A 5-kb mRNA is
detected with p38b-specific antisense oligonucleotide probe that
behaves similarly (data not shown). We conclude that p38 is
up-regulated by hypertonicity at the mRNA level and that p38 kinase
positively regulates the abundance of its own mRNA. The decline in
mRNA abundance without concomitant change in p38 kinase protein
after 16 h of SB203580 treatment (as shown in Fig. 3C),
suggests that the half-life of the protein may exceed 16 h.

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Fig. 6.
P38 kinase mRNA is induced by
hypertonicity; inhibition of p38 kinase activity, using SB203580,
attenuates the induction of p38 kinase mRNA by hypertonicity.
MDCK cells were grown in isotonic medium (315 mosmol/kg of
H2O). At time zero, they were switched to the same medium
or medium made hypertonic (500 mosmol/kg of H2O) by
addition of NaCl in the absence or presence of variable concentrations
of SB203580. After 16 h, they were harvested for measurement of
p38 (upper panel) and -actin (lower panel)
mRNA abundance. Representative Northern blot is shown. Lane
1, isotonic medium; lane 2, NaCl alone; lane
3, NaCl and 50 µM SB203580; lane 4, NaCl
and 100 µM SB203580; lane 5, NaCl and 10 µM SB203580.
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DISCUSSION |
Our findings demonstrate that p38 kinase activity is essential for
the hypertonic induction of mRNAs for HSP70 and betaine transporter
BGT1, but not for thermal induction of HSP70 mRNA. In addition,
JNK-1 activity may not be required for HSP70 and BGT1 mRNAs
induction under hypertonic conditions. These findings represent the
first direct evidence linking p38 kinase to regulation of genes
involved in the adaptation to osmotic stress in mammalian cells.
Betaine transporter is a representative osmoprotective gene. HSP70 is a
representative heat shock protein, one of the major heat shock proteins
expressed in the kidney, and has been shown to play a major role in
early stages of adaptation of kidney cells to osmotic stress (9). Both
adaptive processes, the induction of heat shock proteins and the
accumulation of compatible organic solutes, are essential for the
survival of kidney cells in hyperosmotic environment. Hence, these
findings not only are important for the understanding of the molecular
physiology of the adaptation to osmotic stress in kidney cells, but
also may offer clues to disease states involving solute and water
handling by the kidney. In addition, these findings may provide insight
into osmotic stress adaptation in the brain, since brain cells behave
similar to kidney cells under hyperosmotic conditions (34). The low
JNK-1 activity we observe after 16 h of exposure to hypertonic
medium when p38 kinase is not inhibited is consistent with the low
JNK-1 activity that was found in outer medulla slices of rat kidney
after osmotic stress (35). The activation of JNK-1 concomitant with p38
inhibition is interesting and suggests that p38 kinase may have an
inhibitory effect on JNK-1 in hypertonically stressed MDCK cells,
either directly or indirectly. The significance of these findings
remains to be determined.
Recent reports suggest involvement of JNK in apoptotic signals
(36-39); ERK inhibits, whereas JNK mediates, cytokine-induced apoptosis (36). Hypertonic stress induces cell cycle arrest (40, 41),
inhibits general mRNA and protein synthesis (40, 41), and induces
DNase I-hypersensitive sites (42), consistent with the existence of
apoptotic signals during such stress. As kidney medulla cells are
exposed to variable extracellular tonicity, the existence of an
un-opposed apoptotic signal would obviously lead to cell death
immediately upon exposure of the medulla to the first cycle of
hypertonicity. To avoid uniform cell death, apoptosis must be
regulated. Since ERK, JNK, and p38 kinases are induced during osmotic
stress (12-15), and ERK may not be involved in the induction of
osmoprotective genes (16), it is proposed that the ERK pathway may
mediate growth signals and opposes the apoptotic signals that are
induced during osmotic stress. Whether JNK-1 mediates these
apoptotic signals remains to be determined.
At least three p38 kinase isoforms have been described thus far: p38
(19), p38b (18), and p38
(43) (known also as ERK6 or SAPK3) (44,
45). p38 and p38b (
) are expressed in many tissues including the
kidneys (43). They are equally inhibited by pyridinyl imidazole
compounds and display near identical responses to tumor necrosis
factor, interleukin-1, epidermal growth factor, phorbol-12-myristate-13-acetate, UV irradiation,
H2O2, osmotic stress, and arsenate (18).
p38
, on the other hand, is expressed exclusively in muscle, and may
have a unique role in myocyte differentiation (43, 44). Therefore, of
the known p38 kinases, p38 and p38b are the only p38 kinases relevant
to our studies in kidney cells.
A recent report, based on inhibition of MKK3 (one of the upstream
activators of p38 kinase) in rabbit papillary epithelial cells,
suggested that p38 kinase may not be required for induction of
osmoprotective genes in mammalian cells (46). Kidney cells express at
least p38 and p38b (18). Both kinases are activated equally by stimuli
such as cytokines and environmental stresses including osmotic stress,
yet differ in their upstream activators; p38 kinase is activated in
parallel by MKK3, MKK4, and MKK6, while p38
is activated
predominantly by MKK6 (18). Therefore, inhibition of MKK3, may not
affect the function of p38
kinase. As these pathways might be
redundant in function, if both kinases are not inhibited concomitantly,
one might erroneously conclude that elements of p38 kinase pathway are
not necessary for induction of osmoprotective genes in mammalian cells
(see Scheme 1). Hence, the availability of a specific inhibitor of p38 kinases offers a screening tool for
identification of relevant functions. The identity of the exact p38
kinase(s) responsible for osmoprotective genes induction remains to be
determined.
The regulation of heat shock response is complex, and our results
provide a hint to even greater complexity in its regulation. Heat shock
is a well known activator of p38 kinase, which mediates phosphorylation
of HSP25/27 (30). As p38 is not required for induction of HSP70 during
heat stress, it is possible that thermal activation of p38 kinase
serves distinct functions that may not be related to induction of
HSP70. The finding of divergent pathways mediating thermal and osmotic
induction of HSP70 is intriguing but not surprising. It is amply
reasonable that induction of proteins that are required for the
adaptive response to osmotic stress be mediated by a specific pathway
if specificity is important. Since some of the proteins that are
induced during osmotic stress, such as heat shock proteins, may also be
required for other cellular functions, their regulation may require
separate, function-specific pathways.