From the Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262
Received for publication, September 30, 2002, and in revised form, January 15, 2003
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ABSTRACT |
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Aquaporin-1 (AQP1) is a water channel
that is induced by hypertonicity. The present study was undertaken
to clarify the osmoregulation mechanism of AQP1 in renal medullary
cells. In cultured mouse medullary (mIMCD-3) cells, AQP1 expression was
significantly induced by hypertonic treatment with impermeable solutes,
whereas urea had no effect on AQP1 expression. This result indicates
the requirement of a hypertonic gradient. Hypertonicity activated ERK,
p38 kinase, and JNK in mIMCD-3 cells. Furthermore, all three MAPKs were
phosphorylated by the upstream activation of MEK1/2, MKK3/6, and MKK4,
respectively. The treatments with MEK inhibitor U0126, p38 kinase
inhibitor SB203580, and JNK inhibitor SP600125 significantly attenuated hypertonicity-induced AQP1 expression in mIMCD-3 cells. In addition, hypertonicity-induced AQP1 expression was significantly reduced by both
the dominant-negative mutants of JNK1- and JNK2-expressing mIMCD-3
cells. NaCl-inducible activity of AQP1 promoter, which contains a
hypertonicity response element, was attenuated in the presence of
U0126, SB203580, and SP600125 in a dose-dependent manner
and was also significantly reduced by the dominant-negative mutants of
JNK1 and JNK2. These data demonstrate that the activation of ERK, p38
kinase, and JNK pathways and the hypertonicity response element in the
AQP1 promoter are involved in hypertonicity-induced AQP1 expression in
mIMCD-3 cells.
Aquaporins (AQPs),1 a
family of water channels, function as a water-selective transporting
protein in cell membranes (1). Aquaporin-1 (AQP1) was first discovered
in human erythrocytes as a water channel for high osmotic water
permeability (2, 3). In addition to erythrocytes, AQP1 is abundantly
present in the epithelium of kidney proximal tubules and descending
thin limbs and endothelium of the descending vasa recta (4). AQP1 has
been suggested to be important in constitutive water reabsorption, especially in the epithelial cells of the renal medulla.
The AQP1-expressing vasa recta of the renal medulla are critical in
generating and maintaining an axial osmotic gradient through the
medulla (5). Sodium chloride (NaCl), urea, and water transporters in
the inner medullary collecting duct (IMCD) all play an important role
in the regulation of solute-free water excretion in the kidney. Although most cells in mammals are not normally stressed by
hypertonicity, epithelial cells of the renal medulla are constantly
subjected to a hypertonic condition. Specifically, as a consequence of
the urinary concentrating mechanism, cells in the renal inner medulla are normally exposed to a variety of high concentrations of NaCl and
urea. Hypertonicity, which results from a high concentration of salt
and urea, provides a mechanical stress to shrink medullary cells.
However, medullary cells adapt to hypertonicity by a variety of
responses through an acute influx of NaCl and water (6), chronic
accumulation of organic osmolytes (7), and acute activation of
immediate early and heat shock genes (8, 9). Although the amounts of
total RNA transcription, DNA synthesis, and protein synthesis are
significantly decreased by hypertonicity, a limited number of genes are
up-regulated by hypertonicity-induced transcription (10, 11).
The cellular and molecular mechanisms of aquaporin-2 (AQP2) regulation
via vasopressin in the collecting duct of the kidney are well
understood (12-14). However, the AQP1 regulation in the kidney is
still unknown. Studies performed in the human primary proximal tubule
epithelial cell (15) and mouse medullary cell line (mIMCD-3) (16)
demonstrate that AQP1 mRNA and protein expressions were
up-regulated by hypertonicity. It was also reported that the AQP1
transcript was up-regulated by a hypertonicity response element (HRE)
in the promoter region of AQP1 gene that was located at The objective of the present study was to elicit the cellular and
molecular mechanisms of hypertonicity-induced AQP1 expression in renal
medullary cells. We show here that hypertonicity-induced AQP1
expression is mediated by the activation of the ERK, p38 kinase, and
JNK pathways in mIMCD-3 cells. Functional analysis of the AQP1 promoter
containing the HRE sequence also demonstrated that the AQP1 promoter
activity was stimulated by hypertonicity and regulated by MAPK pathways.
Cell Line and Culture Condition--
mIMCD-3 cells used in this
study were obtained from the American Type Culture Collection. Cells
were cultured at 37 °C and 5% CO2 in Dulbecco's
modified Eagle's/F-12 medium supplemented with 10% fetal bovine serum.
Reverse Transcription-PCR--
Total RNA was isolated from
mIMCD-3 cells using a TRIzol reagent (Invitrogen). Two micrograms of
total RNA was reverse-transcribed and then directly amplified by PCR
using two sets of primers: AQP1 sense primer,
5'-CGGGCTGTCATGTACATCATCGCCCA-3' (nucleotides 276-301); AQP1
antisense primer, 5'-CCCAATGAACGGCCCCACCCAGAAA-3' (nucleotides
632-656); glyceraldehyde-3-phosphate dehydrogenase sense primer,
5'-ATGGGAAGCTTGTCATCAACGGGAA-3' (nucleotides 184-208); glyceraldehyde-3-phosphate dehydrogenase antisense primer,
5'-TGGCAGGTTTCTCCAGGCGGCACGT-3' (nucleotides 729-753). The PCR
amplification was performed for 30 cycles as follows: 94 °C for
30 s, 60 °C for 60 s, and 72 °C for 60 s. The PCR
products were analyzed on a 2% agarose gel and visualized by ethidium
bromide staining.
Immunoblot Analysis--
mIMCD-3 cells grown on 6-cm dishes were
washed with ice-cold phosphate-buffered saline and suspended with 10 mM Tris-HCl, pH 7.5, containing 200 mM sucrose
and homogenized by 10 passages through a 27-gauge needle. The
homogenate was centrifuged at 3000 × g for 10 min at
4 °C. The supernatant was collected, and protein concentration was
measured using the Bradford protein assay method (Bio-Rad protein assay
kit). Protein (10 µg) from the cell extract was resolved on a 12%
SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride
membrane. The membrane was incubated with a rabbit polyclonal
anti-human AQP1 antibody for 1 h. As a protein loading control,
the membrane was also incubated with anti- Plasmid Construction for Reporter Assay--
The plasmid
pCAT-basic (Promega, Madison, WI) was used to examine the promoter
activity of the 5'-flanking region in the human AQP1 gene (17, 20, 21).
The AQP1 promoter construct ( Transient Transfections and CAT Assay--
The transient
transfection experiment was performed by the modification of previous
reports (17, 20-22). mIMCD-3 cells were resuspended at 2 × 107 cells/ml in Dulbecco's modified Eagle's/F-12 medium
without serum and transfected by electroporation under 300 V at 500 microfarads in a 0.4-mm cuvette. Each transfection was performed with
10 µg of the AQP1 promoter-CAT construct (CP-54) and 5 µg of the
plasmid pSV- Hypertonicty-induced AQP1 Expression in mIMCD-3 Cells--
A
previous study demonstrated that AQP1 expression was induced by
hypertonic medium supplemented with NaCl in mouse renal medullary
(mIMCD-3) cells (16). To examine the hypertonic induction of AQP1 in
mIMCD-3 cells, cells were incubated in medium supplemented with
hyperosmotic agents (NaCl and urea) and impermeable osmolytes (raffinose, glucose, sucrose, and sorbitol). After the treatment with
hyperosmolar medium, cell lysates were immunoblotted by using anti-AQP1
antibody. AQP1 protein expression was increased by NaCl, raffinose,
glucose, sucrose, and sorbitol but not urea (Fig.
1A). These data indicate that
AQP1 induction by hypertonicity requires a hypertonic gradient. Next,
to investigate the time course for hypertonic induction of AQP1
protein, mIMCD-3 cells were incubated in hypertonic medium by the
addition of 100 mM NaCl. Samples were harvested at specific
times for immunoblot analysis. As shown in Fig. 1B, AQP1
protein significantly increased by 12 h after exposure to
hypertonic medium and peaked at 16-20 h.
To test the AQP1 induction in medium osmolality, cells were incubated
in medium by the addition of NaCl, raffinose, or glucose with different
hyperosmolality. A minimum addition of 50 mM NaCl, 100 mM raffinose, or 100 mM glucose to the medium
was needed to increase AQP1 protein expression in mIMCD-3 cells, and
stronger induction of AQP1 was obtained by the exposure to higher
medium osmolality (Fig. 2). However, the
addition of 200 mM raffinose to the medium showed much less
AQP1 expression than that of 100 and 150 mM raffinose,
because the cell viability significantly decreased at that condition.
Taken together, these data indicate that AQP1 expression in mIMCD-3
cells is significantly increased in hypertonic condition in a time- and
dose-dependent fashion.
Transcriptional and Post-transcriptional Regulations of
Hypertonicity-induced AQP1 Expression in mIMCD-3
Cells--
Hypertonicity-induced AQP1 expression may be caused by
mRNA induction or stability or protein stability. To distinguish
the mechanism, cells were pretreated with the RNA synthesis inhibitor actinomycin D or protein synthesis inhibitor cycloheximide and then
incubated under isotonic or hypertonic condition. The expression levels
of AQP1 mRNA and protein were examined by reverse transcription-PCR and immunoblot analysis, respectively. As shown in Fig.
3, pretreatment with actinomycin D or
cycloheximide completely inhibited NaCl-induced AQP1 mRNA and
protein expressions. These results suggest that both transcriptional
and post-transcriptional regulations are required for AQP1 induction by
hypertonicity.
ERK, p38 Kinase, and JNK Activation by Hypertonicity in mIMCD-3
Cells--
Previous works show that all three MAPKs (ERK, p38 kinase,
and JNK) can be activated by hypertonicity (18, 19). To determine whether MAPK-mediating signaling was involved in hypertonic induction of AQP1 in mIMCD-3 cells, antibodies specific for either the
phosphorylated or total form of each of three MAPKs were used. Cells
were treated with hyperosmotic condition containing 100 mM
NaCl or 200 mM urea for 10 min, and then the
phosphorylation of all three MAPKs were investigated by immunoblot
analysis. NaCl significantly activated the ERK, p38 kinase, and JNK
pathways in mIMCD-3 cells, whereas urea activated only the ERK pathway
(Fig. 4). These data confirmed the
results reported previously (18, 23).
Next, activation of MAPKs is mediated by MEK or MKKs. ERK is activated
by MEK1 and MEK2, whereas p38 kinase is activated by MKK3 and MKK6, and
JNK is activated by MKK4 (24-28). To further investigate the
differential activation of MAPKs in mIMCD-3 cells in response to
hypertonicity, the phosphorylation for the upstream activation of MAPKs
was assessed using antibodies specific for the phosphorylated or total
form of MEK1/2, MKK3/6, or MKK4. As shown in Fig. 4, NaCl activated the
MEK1/2, MKK3/6, and MKK4 pathways, whereas urea activated only the
MEK1/2 pathway. Thus, NaCl significantly stimulated the phosphorylation
of all three subfamilies of MAPKs in mIMCD-3 cells, but urea only
the ERK phosphorylation.
Inhibition by MAPK Inhibitors on Hypertonicity-induced AQP1
Expression--
To determine whether the activation of ERK, p38
kinase, and JNK was involved in the regulation of hypertonicity-induced
AQP1 expression, a MEK inhibitor, U0126, a p38 kinase inhibitor,
SB203580, and a novel specific JNK inhibitor, SP600125 (29, 30), were used. To investigate the specificity of each MAPK inhibitor, mIMCD-3 cells were pretreated with the different concentrations of U0126, SB203580, or SP600125, and then NaCl-induced ERK, p38 kinase, and JNK
phosphorylations were analyzed by immunoblot. As shown in Fig.
5, all three MAPK inhibitors revealed the
specific selectivity for other MAPKs within the range of a given
concentration.
On the basis of the result in Fig. 5, we proceeded to assess the effect
of MAPK inhibitors on NaCl-induced AQP1 protein expression. Cells were
pretreated with each MAPK inhibitor and stimulated with NaCl. Cell
lysates were analyzed by immunoblot. Results were normalized to
expression of Inhibition by Dominant-negative Mutants of JNK Isoforms on
Hypertonicity-induced AQP1 Expression--
As an alternative approach
to the involvement of JNK pathway on hypertonicity-induced AQP1
expression, mIMCD-3 cells were transiently cotransfected with the
dominant-negative mutants of JNK1 (JNK1-DN) or JNK2 (JNK2-DN) and
pSV- Effect of Potent ERK Stimulators on AQP1 Expression--
Our
results demonstrated that both NaCl and urea activated the ERK pathway.
To determine whether the ERK activation is sufficient to induce AQP1
expression, arginine vasopressin, phorbol-12-myristate 13-acetate, or
calcium ionophore (ionomycin), which are known as potent stimulators of
ERK pathway, were tested. As shown in Fig.
8A, all three potent
stimulators indeed activated the ERK pathway. However, they did not
induce AQP1 protein expression under isotonic or hypertonic condition
(Fig. 8B). Therefore, the ERK activation is necessary but
not sufficient to induce AQP1 expression by hypertonicity.
Inhibition of AQP1 Promoter Activity by U0126, SB203580, SP600125,
or Dominant-negative Mutants of JNK1 or JNK2--
Our previous study
demonstrates that a novel HRE is present in the AQP1 promoter region,
which is located in the region from In the book From Fish to Philosopher, Smith suggests
that the capacity to conserve solute-free water excretion by activating a urinary concentrating mechanism was an important factor in the evolution from fresh water fishes to land-dwelling mammals (31). Much
has been learned about the mammalian urinary-concentrating mechanism,
including the osmotic and non-osmotic release of the antidiuretic
hormone, vasopressin (32), the countercurrent concentrating and
exchange mechanisms (33), the cloning of the collecting duct
vasopressin V2 receptor (34), and most recently, the cloning of the
renal water channels, termed aquaporins (35). Although the
up-regulation and trafficking to the collecting apical membrane of AQP2
is a critical factor in the urinary-concentrating mechanism (36), there
is recent evidence implicating an important role of AQP1. AQP1 resides
in the proximal tubule, descending thin limb of Henle's loop and
endothelial cells of the vasa recta (4). Mutations of the AQP1 gene
both in knockout mice (37) and humans (38) have been shown to exhibit a
defect in maximal urinary concentrating capacity. A recent study shows
that a hypertonic NaCl environment in mouse medullary cells is
associated with an up-regulation of AQP1 (16). The mechanism for this
effect has, however, not been elucidated. Such an up-regulation of AQP1
protein expression would likely enhance the urinary concentrating
mechanism, since mutations of AQP1 impair urinary concentration. The
present study was undertaken to examine the cellular and molecular
mechanisms on the effect of hypertonic stress to up-regulate AQP1
protein expression.
The initial study was performed with mouse medullary (mIMCD-3) cells
incubated in medium supplemented with hyperosmotic NaCl or urea as well
as with medium containing impermeable osmolytes including raffinose,
glucose, sucrose, and sorbitol. AQP1 protein expression was increased
in medium containing all of these substrates except urea. Because urea
crosses cell membrane freely and, thus, does not create an osmotic
gradient, these results implicate a hypertonic gradient in this
induction of AQP1 protein expression. Further studies demonstrated the
time- and dose-dependent effects of the hypertonic gradient
using NaCl, raffinose, or glucose to induce AQP1 protein expression.
The actinomycin D and cycloheximide treatment experiments suggested the
respective involvement of transcriptional and post-transcriptional
regulations in hypertonicity-induced AQP1 expression. A recent study
demonstrated that AQP1 protein stability was crucial in AQP1 induction
by hypertonicity in mouse BALB/c fibroblasts, which was mediated by the
proteasome degradation pathway (39). Therefore, we conclude that AQP1
induction by hypertonicity occurs at both the transcriptional and
posttranscriptional levels.
Hypertonicity activated all three MAPK signaling pathways (ERK, p38
kinase, and JNK) in mIMCD-3 cells. These signaling pathways were shown
to mediate hypertonicity-induced AQP1 expression. Specifically, it was
demonstrated that the blockade of any one of ERK, p38 kinase, or JNK
signaling pathway by a specific inhibitor significantly reduced
hypertonicity-induced AQP1 expression. U0126, SB203580, or SP600125
selectively inhibited the ERK, p38 kinase, or JNK pathway in a
dose-dependent fashion, respectively. Interestingly, the
partial ERK inhibition by SB203580 was observed at 5 µM
but not 2.5 µM (data not shown). The maximal dose used in
the present study was 2.5 µM. The similar observation was
previously reported by Yang et al. (23) and suggested a
potential interrelationship between ERK and p38 kinase activation.
Although SP600125, which was recently identified as a specific JNK
inhibitor, blocked the JNK activation in a dose-dependent
fashion, it has been demonstrated that this inhibitor also had a
partial p38 inhibition at a concentration higher than the maximal dose
used in the present study (29). It should be noted that mIMCD-3 cells
are highly sensitive to pharmacological inhibition of MAPK signaling
pathway. Furthermore, we showed that the blockade of JNK pathway by
dominant-negative mutants of JNK1 and JNK2 as an alternative approach
significantly reduced hypertonicity-induced AQP1 expression. Taken
together, our findings suggest that the activation of all three MAPK is indispensable for AQP1 induction by hypertonicity.
In mIMCD-3 cells, although NaCl activated the ERK, p38 kinase, and JNK,
urea activated only the ERK. Although both NaCl and urea activated the
ERK, only NaCl induced AQP1 expression. Furthermore, other ERK
stimulators such as arginine vasopressin, phorbol-12-myristate 13-acetate, and ionomycin did not induce AQP1 expression under isotonic
or hypertonic condition. These results indicate that the ERK activation
is necessary but not sufficient to induce AQP1 expression by
hypertonicity. A similar study also demonstrates that
hypertonicity-induced AQP5 expression in mouse lung epithelial cells
was mediated by the ERK-dependent pathway, although the ERK
activation was not sufficient for AQP5 induction by hypertonicity (40).
The detailed regulatory mechanism via MAPK pathways in hypertonicity-induced AQP1 expression is still unclear. Our data suggest that the three MAPKs may be necessary to phosphorylate transcriptional factors that bind to hypertonicity-responsive elements
in the AQP1 gene.
The induction of AQP1 gene by hypertonicity results from a sequential
MAPK-signaling pathway that directly affects the promoter region of
AQP1 gene. Previous studies demonstrate that the genes of the
sodium/myo-inositol cotransporter (41),
sodium/chloride/betaine cotransporter (42), and aldose reductase (43)
are up-regulated in response to hypertonicity. The tonicity responsive
enhancer (TonE) consensus sequence present in these genes mediates the transcriptional stimulation. It seems that AQP1 induction by
hypertonicity is mediated by an osmotic response element such as TonE.
However, no TonE consensus sequence is present in a 1.8-kilobase AQP1
promoter region (20). We recently characterized a novel HRE in the AQP1 gene that is different from TonE consensus sequence (17). Using the
AQP1 promoter-chloramphenicol acetyltransferase construct that contains
the HRE sequence, we showed that any of three MAPK specific inhibitors
and dominant-negative mutants of JNK1 or JNK2 significantly reduced
NaCl-induced AQP1 promoter activity in a dose-response manner. In
contrast, urea did not induce the promoter activity. In addition, the
promoter activity was not affected by U0126, SB203580, or SP600125
under isotonic conditions.2
Thus, the novel HRE, which only responds to hypertonicity, is essential
for the transcriptional regulation of hypertonicity-induced AQP1
expression. Identification of a trans-acting HRE-binding protein that binds to the HRE should further elucidate the
transcriptional regulation of hypertonicity-induced AQP1 expression.
In summary, the present results demonstrate that hypertonicity-induced
AQP1 expression is regulated by ERK, p38 kinase, and JNK activation and
the HRE in the AQP1 promoter. Inhibition of each MAPK pathway results
in significant reduction of hypertonicity-induced AQP1 expression,
indicating that all three MAPK signaling pathways are indispensable for
AQP1 induction.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
54 to
46,
which responded to hypertonic stimulation (17). This HRE sequence is
different from previously reported tonicity-responsive enhancer (TonE)
consensus sequence, which is also responsible for hypertonicity.
Additional studies in mIMCD-3 cells showed that all three MAPKs (ERK,
p38 kinase, and JNK) were activated by hypertonicity (18, 19). Thus, it
was possible that the osmoregulation of AQP1 may be mediated by MAPK
pathways, which are induced by hypertonicity. However, the role of
MAPKs in hypertonicity-induced AQP1 expression remains unclear.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin antibody (Sigma).
For the analysis of MAPK signaling pathways, cells were lysed with
ice-cold lysis buffer (50 mM
-glycerophosphate, pH 7.2, 0.5% Triton X-100, 0.1 mM sodium vanadate, 2 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol). The lysates were resolved on a 12%
SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride
membrane. The membranes were incubated with antibodies to phospho-ERK,
ERK, phospho-p38, p38, phospho-JNK, JNK, phospho-mitogen-activated ERK
kinase (MEK) 1/2, MEK1/2, phospho-MAPK kinase (MKK) 3/6, MKK3, phospho-MKK4 (Cell Signaling Technology Inc., Beverly, MA), or MKK4
(StressGen, Victoria, British Columbia, Canada). After washing, the
membrane was incubated with anti-rabbit IgG horseradish peroxidase secondary antibody (Amersham Biosciences). The immunoreactive bands
were visualized by enhanced chemiluminescence method (PerkinElmer Life Sciences).
54/+23; CP-54), which contains the HRE
sequence, was generated by PCR and ligated into Hind III and
XbaI sites of pCAT-basic vector. The sense primer
corresponded to nucleotides
54 to
33 and contained an engineered
Hind III restriction site. The antisense primer for
amplification corresponded to nucleotides +23 to +4 and contained an
engineered XbaI restriction site. The CAT construct was
confirmed by sequence analysis.
-galactosidase (Promega) on a total volume of 300 µl.
After overnight incubation, cells were incubated with or without 100 mM NaCl. After a 48-h incubation, the cells were harvested,
and cell extracts were isolated using the reporter lysis buffer
(Promega).
-Galactosidase activity was measured in every experiment
for normalization. CAT activity was measured using
D-threo[dichloroacetyl-1-14C]chloramphenicol
(Amersham Biosciences) and the phase extraction method.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Hypertonicity-induced AQP1 expression in
mIMCD-3 cells. A, hypertonic induction of AQP1 by
different solutes in mIMCD-3 cells. Cells were incubated in isotonic or
hypertonic medium containing NaCl (100 mM), raffinose (100 mM), glucose (200 mM), sucrose (200 mM), sorbitol (200 mM), or urea (200 mM) for 16 h. Cells were harvested, and total protein
was analyzed by immunoblot with anti-AQP1 antibody. B, time
course for hypertonic induction of AQP1 protein. Cells were incubated
in hypertonic medium containing 100 mM NaCl. Samples were
harvested at specific times for immunoblot analysis.
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Fig. 2.
Dose-dependent induction on
hypertonicity-induced AQP1 expression in mIMCD-3 cells. Cells were
incubated in medium with different hyperosmolality for 16 h. Cells
were harvested, and total protein was analyzed by immunoblot.
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Fig. 3.
Effects of RNA and protein synthesis
inhibitors on hypertonicity-induced AQP1 expression in mIMCD-3
cells. Cells were incubated without or with 100 mM
NaCl for 16 h in the presence or absence of actinomycin D (5 µg/ml) or cycloheximide (2 µg/ml). A, reverse
transcription-PCR analysis was performed for the detection of AQP1 and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs.
B, immunoblot analysis was performed for the detection of
AQP1 protein.
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Fig. 4.
Activation of MAPK and MAPK kinase pathways
by NaCl and urea in mIMCD-3 cells. For the MAPK immunoblotting,
cells were incubated in isotonic or hypertonic medium containing 100 mM NaCl or 200 mM urea for 10 min. Cells were
harvested, and cell lysates were prepared using lysis buffer.
Immunoblot analysis was performed by using antibodies to phospho-ERK,
ERK, phospho-p38 kinase, p38 kinase, phospho-JNK, JNK, phospho-MEK1/2,
MEK1/2, phospho-MKK3/6, MKK3, phospho-MKK4, or MKK4.
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Fig. 5.
Effect of MAP kinase inhibitors on MAPK
phosphorylations in mIMCD-3 cells. After pretreated with the MEK
inhibitor U0126 (0.5, 1, or 2.5 µM), the p38 kinase
inhibitor SB203580 (0.5, 1, or 2.5 µM), or the JNK
inhibitor SP600125 (2, 5, or 10 µM) for 30 min, cells
were incubated in hypertonic medium containing 100 mM NaCl
for 10 min. Cells were harvested, and cell lysates were prepared using
lysis buffer. Immunoblot analysis was performed by using antibodies to
phospho-ERK, ERK, phospho-p38 kinase, p38 kinase, phospho-JNK, or
JNK.
-actin by densitometry of immunoblot (Fig.
6). NaCl-induced AQP1 expression was
significantly blocked by U0126 (0.5-2.5 µM), SB203580
(0.5-2.5 µM), and SP600125 (2-10 µM). The
treatment of a higher concentration with each MAPK inhibitor almost
completely abolished NaCl-induced AQP1 expression (data not shown).
Therefore, this result provides that AQP1 induction by hypertonicity
requires the activation of ERK, p38 kinase, and JNK pathways.
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Fig. 6.
Effect of MAPK inhibitors on NaCl-induced
AQP1 expression in mIMCD-3 cells. After pretreated with U0126
(0.5, 1, or 2.5 µM), SB203580 (0.5, 1, or 2.5 µM), or SP600125 (2, 5, or 10 µM) for 30 min, cells were incubated in hypertonic medium containing 100 mM NaCl for 16 h. Cells were harvested, and total
protein was analyzed by immunoblot. The membranes were also incubated
with anti- -actin antibody as a protein loading control in
immunoblot. Each protein blot was analyzed by densitometry. AQP1
expression in 100 mM NaCl was assigned as 100%. Values
represent the mean ± S.E. of five independent sets of
experiments.
-galactosidase plasmids. After 16 h of incubation without
or with 100 mM NaCl, AQP1 protein expression was examined
by immunoblot analysis.
-Galactosidase activity was measured in each
experiment for correction of transfection efficiency. Results were
normalized to expression of
-actin by densitometry of immunoblot. As
shown in Fig. 7, NaCl-induced AQP1 expression was significantly reduced by both the JNK1-DN- and JNK2-DN-transfected mIMCD-3 cells. Taken together, the activation of
ERK, p38 kinase, and JNK pathways is necessary for AQP1 up-regulation in mIMCD-3 cells that is responsible for hypertonicity.
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Fig. 7.
Effect of dominant-negative mutants of JNK
isoforms on NaCl-induced AQP1 expression in mIMCD-3 cells. Cells
were cotransfected with the dominant-negative mutant of JNK1 (JNK1-DN)
or JNK2 (JNK2-DN) and pSV- -galactosidase plasmids followed by
treatment without or with 100 mM NaCl for 16 h. Cells
were harvested, and total protein was analyzed by immunoblot. The
membranes were also incubated with anti-
-actin antibody as a protein
loading control in immunoblot. Transfection efficiency to each
experiment was determined by
-galactosidase activity. Each protein
blot was analyzed by densitometry. AQP1 expression in 100 mM NaCl was assigned as 100%. Values represent the
mean ± S.E. of three independent sets of experiments.
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Fig. 8.
Effect of potent ERK stimulators on AQP1
expression in mIMCD-3 cells. A, ERK activation. Cells
were incubated with arginine vasopressin (AVP; 10 or 100 nM), phorbol-12-myristate 13-acetate (PMA) (1 or
10 nM), or ionomycin (10 or 100 nM) for 10 min.
Cells were harvested, and cell lysates were prepared using lysis
buffer. Immunoblot analysis was performed by using antibody specific
for the phospho-ERK or ERK. B, AQP1 expression. Cells were
incubated without or with 100 mM NaCl for 16 h after
pretreated with arginine vasopressin (10 or 100 nM),
phorbol-12-myristate 13-acetate (1 or 10 nM), or ionomycin
(10 or 100 nM) for 30 min. Cells were harvested, and total
protein was analyzed by immunoblot. The data are representative of
three independent experiments. Similar results were obtained in the
other two experiments.
54 to
46 (17). This critical
element responds to hypertonicity and is required for basal and
hypertonicity-induced expression of the AQP1 gene. To investigate the
involvement of ERK, p38 kinase, and JNK pathways in
hypertonicity-induced AQP1 expression at the transcriptional level,
transient transfection experiments were performed using the AQP1
promoter-chloramphenicol acetyltransferase construct CP-54 (
54/+23),
which contains the HRE sequence. These results are showed in Fig.
9. When cells were transfected with the
CP-54, the treatment with NaCl led to an ~3.4-fold increase in the
promoter activity, whereas the treatment with urea did not.
NaCl-induced promoter activity was attenuated in the presence of U0126,
SB203580, and SP600125 in a dose-dependent manner. In cotransfection experiments, NaCl-induced promoter activity was significantly inhibited by the dominant-negative mutants of JNK1 (JNK1-DN) or JNK2 (JNK2-DN). These results indicate that the HRE in
AQP1 promoter is essential for the hypertonicity-induced regulatory effects by ERK, p38 kinase, and JNK pathways on AQP1 expression.
View larger version (42K):
[in a new window]
Fig. 9.
Effect of MAPK inhibitors or
dominant-negative mutants of JNK1 and JNK2 on NaCl-induced AQP1
promoter activity in mIMCD-3 cells. Cells were transfected with
the AQP1 promoter-chloramphenicol acetyltransferase
(CAT) construct (CP-54) as described under "Experimental
Procedures." After pretreated with U0126 (0.5, 1, 2.5, or 25 µM), SB203580 (0.5, 1, 2.5, or 25 µM), or
SP600125 (2, 5, 10, or 20 µM) for 30 min, cells were
incubated in hypertonic medium containing 100 mM NaCl for
48 h. Also, the transfected cells were incubated with 200 mM urea for 48 h. The dominant-negative mutant of JNK1
(JNK1-DN) or JNK2 (JNK2-DN) was cotransfected
with the CP-54 followed by incubating without or with 100 mM NaCl for 48 h. The results were normalized using
-galactosidase assay. Each value is an average (±S.E.) of three to
five independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Erin M. Stone for helpful assistance and Lynn E. Heasley for kindly providing the dominant-negative mutants of JNK1 and JNK2 cDNAs.
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FOOTNOTES |
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* This work was supported by the National Institutes of Health Grant DK19928.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Division of Renal
Diseases and Hypertension, Dept. of Medicine, University of Colorado
Health Sciences Center, 4200 East Ninth Ave., Box C281, Denver, CO
80262. Tel.: 303-315-6715; Fax: 303-315-4852; E-mail: Fuminori.Umenishi@UCHSC.edu.
Published, JBC Papers in Press, February 21, 2003, DOI 10.1074/jbc.M209980200
2 F. Umenishi and R. W. Schrier, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: AQP1, aquaporin-1; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated extracellular signal-regulated kinase kinase; MKK, mitogen-activated protein kinase kinase; IMCD, inner medullary collecting duct; m-, mouse; HRE, hypertonicity response element; TonE, tonicity-responsive enhancer; DN, dominant negative.
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REFERENCES |
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