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Hypertonicity-Induced Expression of
Monocyte Chemoattractant Protein-1
through a Novel Cis-Acting Element and
MAPK Signaling Pathways
Ryoji Kojima, Hajime Taniguchi, Aya Tsuzuki, Kanako
Nakamura, Yumi Sakakura and Mikio Ito
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2010 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2010; 184:5253-5262; Prepublished online 5
April 2010;
doi: 10.4049/jimmunol.0901298
http://www.jimmunol.org/content/184/9/5253
The Journal of Immunology
Hypertonicity-Induced Expression of Monocyte
Chemoattractant Protein-1 through a Novel Cis-Acting
Element and MAPK Signaling Pathways
Ryoji Kojima, Hajime Taniguchi, Aya Tsuzuki, Kanako Nakamura, Yumi Sakakura,
and Mikio Ito
M
CP1, a C-C chemokine, activates monocytes/macrophages and CD8-positive T cells, which then infiltrate
locally into the inflammatory region. The action of MCP1
as a chemoattractant for these cells suggests that it is involved in the
pathophysiologic processes of progressive diseases such as glomerulonephritis (1, 2) and diabetic nephropathy (3), which are characterized by massive urinary albumin excretion and the marked
recruitment of monocytes/macrophages and T cells in glomeruli and
tubulointerstitum. Recent studies have suggested that MCP1 is involved in the development of renal fibrosis, including glomerulosclerosis and tubulointerstitial fibrosis (4). In addition to renal fibrosis,
MCP1 is also implicated in the pathogenesis of peritoneal fibrosis. In
peritoneal fibrosis, it has been suggested that high glucose concentration in conventional peritoneal dialysis solutions, which are used
for continuous ambulatory peritoneal dialysis, plays a role in fibrotic
lesions by increasing MCP1 gene expression (5, 6).
In regard to the molecular mechanisms underling MCP1 gene expression in these pathophysiologic conditions, recent studies have
Laboratory of Analytical Pharmacology, Faculty of Pharmacy, Meijo University,
Nagoya, Japan
Received for publication April 28, 2009. Accepted for publication February 22, 2010.
This work was supported by grants from the Ministry of Education, Science, Sports
and Culture of Japan (12672132), and the Meijo University Research Institute (Grantin-Aid for Specially Promoted Research).
Address correspondence and reprint requests to Dr. Ryoji Kojima, Laboratory of Analytical Pharmacology, Faculty of Pharmacy, Meijo University, Nagoya 468-8503,
Japan. E-mail address: [email protected]
Abbreviations used in this paper: CHX, cycloheximide; DAB, diaminobenzidine; DFO,
deferoxamine; DPI, diphely iodonium; Iso, isotonic medium; Man, Mannitol; N, normal; Na, NaCl; PDTC, pyrolidine dithiocarbanate; PD, PD98059; PKC, protein kinase
C; SB, SB203580; SP, SP600125; U, U0126.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901298
demonstrated a relationship between intracellular pathways responsible
for MCP1 gene expression and exogenous stimuli. High glucose, for
instance, stimulates MCP1 gene expression by activating AP1 (5), or
protein kinase C (PKC) and NF-kB (6), in peritoneal mesothelial cells.
High glucose also increases MCP1 mRNA levels in renal mesangial
cells through NF-kB activation (7), which provides insight into understanding the development of diabetic nephropathy. In renal proximal
tubular cells, albumin induces MCP1 expression by producing reactive
oxygen species and, in turn, activating NF-kB when the cells are treated
with an excess of albumin (8). In addition, LPS, which activates
proinflammatory cascades, has been found to induce MCP1 expression
through the activation of NF-kB (9) and/or C/E binding protein (BP)
regulatory element in the promoter region of the MCP1 gene (10).
In comparison with these stimuli, the molecular mechanisms of the
expression of MCP1 in response to hyperosmolality remain poorly
understood. Recently, Matsuo et al. (6) reported that in peritoneal
mesothelial cells, mannitol at a dose of 140 mM induced expression of
MCP1 mRNA and protein through PKC activation and a NF-kB cisacting element localized in the distal region of the MCP1 gene.
However, previous studies on the molecular mechanisms of protective
adaptation to hyperosmolality identified a hypertonicity-sensitive cisacting element, TonE/ORE, localized in the BGT1 (11) and aldose
reductase (AR) genes (12). Investigations further identified the TonEBP (13)/OREBP (14)—also known as NFAT5 (15)—as a transcription factor that binds to the TonE/ORE enhancer element. It has
been also reported that the consensus sequence of the TonE/ORE
is the 59-(C/T)GGAANNN(C/T)N(C/T)-39 and/or 59-NGGAAA
(A/T)(T/A/G)(C/A/T)(A/C)C-39 in the canine BGT1 gene (16) and/
or AR gene (17, 18), respectively. In addition to the BGT1 and AR
genes, the TonE/ORE has been identified in the proximal region of
the 59-flanking region of stress protein genes, such as Hsp70 (19)
and Osp94 (20), which protect cells from the deleterious effects of
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
MCP1 is upregulated by various stimuli, including LPS, high glucose, and hyperosmolality. However, the molecular mechanisms
of transcriptional regulation of the MCP1 gene under hyperosmolar conditions are poorly understood. Treatment of NRK52E
cells with NaCl or mannitol resulted in significant elevation of MCP1 mRNA and protein in a time- and dose-dependent
manner. Treatment with a p38MAPK inhibitor (SB203580), an ERK inhibitor (PD98059), or an MEK inhibitor (U0126),
suppressed the increase in MCP1 expression caused by hypertonic NaCl, whereas a JNK inhibitor (SP600125) and an AP1
inhibitor (curcumin) failed to attenuate MCP1 mRNA expression by NaCl. In the 59-flanking region of the MCP1 gene, there is
a sequence motif similar to the consensus TonE/ORE as well as the consensus C/E binding protein (BP), NF-kB, and AP1/Sp1
sites. Luciferase activity in cells transfected with reporter constructs containing a putative TonE/ORE element (MCP1-TonE/
ORE) enhanced reporter gene expression under hypertonic stress. Results of electrophoretic gel mobility shift assay showed
a slow migration of the MCP1-TonE/ORE probe, representing the binding of TonEBP/OREBP/NFAT5 to this enhancer element. These results indicate that the 59-flanking region of MCP1 contains a hypertonicity-sensitive cis-acting element, MCP1TonE/ORE, as a novel element in the MCP1 gene. Furthermore, p38MAPK and MEK–ERK pathways appear to be, at least in
part, involved in hypertonic stress-mediated regulation of MCP1 expression through the MCP1-TonE/ORE. The Journal of
Immunology, 2010, 184: 5253–5262.
5254
Materials and Methods
RT-PCR analysis
Normal rat kidney (NRK52E) cells (ATCC#; CRL-1571) were exposed to
hypertonic stress by addition of NaCl (100 mM) or mannitol (200 mM). At
3, 6, 9, 12 and 24 h after treatment, total RNA was isolated using the TRizol
reagent (Invitrogen, Carlsbad, CA), in accordance with the manufacturer’s
instructions. In addition, NRK52E cells were exposed to glucose (100
mM) for 3 d, or 10 mg/ml albumin or 1 mg/ml LPS for 24 h. Furthermore,
NaCl (50, 100 or 150 mM), mannitol (100, 200, or 300 mM), glycerol (200
mM) or urea (200 mM) were also applied to cells for 3 h. RT-PCR was
performed using the following primers: for MCP1, sense, 59-TGTTCACAGTTGCTGCCTGT-39, and antisense, 59-CTACAGAAGTGCTTGAGGTG-39; and for GAPDH, sense, 59-AATGCATCCTGCACCACCAA-39,
and antisense, 59-GTAGCCATATTCATTGTCATA-39.
Inhibition of protein synthesis, PKC, NF-kB, NADPH oxidase
and MAPKs, and iron chelation
NRK52E cells were pretreated with 10 mg/ml cycloheximide (CHX; SigmaAldrich, St. Louis, MO), a protein synthesis inhibitor; 500 nM calphostin C
(Calbiochem, La Jolla, CA), a protein kinase C (PKC) inhibitor; 20 mM pyrolidine dithiocarbanate (PDTC; Sigma-Aldrich, St Louis, MO), an NF-kB
inhibitor; 10 mM diphely iodonium (DPI; Sigma-Aldrich), an NADPH oxidase
inhibitor; or 100 mM deferoxamine (DFO; Sigma-Aldrich), an iron chelator. At
1 h after treatment with inhibitors, cells were exposed to NaCl (100 mM, 3 h).
For MAPKs inhibition studies, cells were pretreated with SB20385 (5, 10, or 20
mM; Calbiochem), a p38 MAPK inhibitor; PD98059 (5, 10 or 20 mM, Calbiochem), an ERK inhibitor, U0126 (5, 10, or 20 mM; Calbiochem), an MEK
inhibitor; SP600125 (10, 20, or 30 mM; Calbiochem), a JNK inhibitor; or
curcumin (5 or 15 mM; Sigma-Aldrich), an AP1 inhibitor, at the indicated
doses for 1 h, followed by exposure to NaCl (100 mM, 3 h). RT-PCR analysis
was performed as described above.
Western blot analysis
For analysis of MCP1 expression induced by hyperosmolality, cells were exposed to NaCl (100 mM) or mannitol (200 mM) for 6, 9, 12, 15, and 24 h. In
addition, NaCl (50, 100, 150, or 200 mM) or mannitol (100, 200, 300, or 400
mM) was applied to cells for 12 h. For analysis of changes in MAPK activity in
response to hypertonicity, cells were treated with NaCl (100 mM) for 15, 30, 60,
and 180 min. Moreover, cells were pretreated with MAPK inhibitors for 1 h,
followedbyexposuretoNaCl(100mM,30min).Pelletedcellsweretreatedwith
lysis buffer containing 25 mM HEPES/KOH (pH 7.9), 150 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 50 mM NaF, 1 mM Na3VO3, 0.5% Triton X-100, 20
mM b-glycerophosphate, 1 mM PMSF, 5 mM DTT, and protease inhibitor
(Roche Applied Science, Mannheim, Germany), followed by centrifugation at
4000 3 g for 10 min. The resulting supernatant was subjected to SDS-PAGE
and Western blot analysis using anti-rat MCP1 (1:1,500, Chemicon, Temecula,
CA), and anti-phospho-p38MAPK, phospho-ERK or phospho-JNK (1:1000;
Cell Signaling Technology, Danvers, MA). Total MAPK (1:1000; Cell Signaling Technology), b-actin (1:500; Sigma-Aldrich), and GAPDH (1:3000;
StressGen, Ann Arbor, MI) leves were also detected.
Measurement of MCP1 by ELISA
At the indicated time points after treatment of NRK52E cells with NaCl
(100 mM) or mannitol (200 mM), aliquots of medium were centrifuged at
14,000 3 g for 5 min at 4˚C, and MCP1 levels in the supernatant were
measured using an MCP1 Rat ELISA Biotrak system (GE Heathcare,
Buckinghamshire, England). MCP1 levels were normalized against cell
protein contents.
Luciferase reporter gene construction
On the basis of sequence data of the rat MCP1 gene (GenBank accession no.
AF079313), the promoter region of the MCP1 gene (2939 to +59) was
amplified using rat genomic DNA prepared from NRK52E cells and the
following primers: sense; 59-TCTCTCCTCATAGTCCTTGG-39 (2939 to
2920) and antisense; 59-AGAGATCTGGCTTCAGTGAG-39 (+40 to +59).
The promoter region (WT, 2939 to +59) of the MCP1 gene isolated above
and its deletion mutant constructs (D1, D2, D3, D4, and D5) were prepared
using each combination of the antisense primer (59-AGAGATCTGGCTTCAGTGAG-39, +40 to +59) and the following sense primers: for D1; 59CAGGGAATCTTAGGGCAATT-39 (2749 to 2730), for D2; 59-CCAGGTCTGGAATTGTACAA-39 (2559 to 2540), for D3; 59-AGGTATCTTCTCCCTTAGGA-39 (2359 to 2340), for D4; 59-CATTTGCTCCCAGTAGTGGC39 (2219 to 2200), for D5; 59-ATCCGCGGTTTCTCCCTTCT-39 (2179 to
2160). Each PCR fragment was inserted into pGL3-basic vector (Promega,
Madison, WI).
In separate experiments, a 40-bp promoter region (D440) from 2219
to 2180, including a putative MCP1 tonicity response element (59-TGGAAAAACACCAA-39, MCP1-TonE/ORE), which spans from 2186 to 2199
of the MCP1 gene, was generated by annealing two complementary oligonucleotides and inserted into the pGL3-SV40-promoter vector (Promega).
Several deletion mutants (D440M1, D440M2, D440M3) of D440 were also
analyzed to further characterize a tonicity response element in the MCP1 gene.
In further experiments, the D440M2 fragment (59-TGGAAAAACACCAAATTCCA-39) was deleted from its 59- or 39-end to identify a minimum
essential element responsive to hypertonicity, and reporter constructs with
multiple concatenated copies of the minimum element were also synthesized and analyzed.
DNA transfection and luciferase assay
NRK52E cells were cotransfected with each firefly luciferase reporter construct
and pRL (Renilla luciferase)-SV40 reporter vector (Promega) as a control of
transfection efficacy. Cells were incubated in isotonic medium containing 5%
FBS for 18 h, and were then treated with either fresh isotonic or hypertonic (200
mM mannitol) medium for 6 h (20). Cell lysates prepared with lysis buffer
(Promega) were analyzed for firefly and Renilla luciferase activities using the
Dual-Luciferase Reporter Assay System (Promega) in accordance with the
manufacturer’s instructions, and the light emitted was measured by a MiniLumat LB 9506 luminometer (Berthold, Nashua, NH). The firefly luciferase
activity in relative light units was normalized against Renilla luciferase activity
as a control, and osmotic response was calculated as the hypertonic-to-isotonic
ratio. The average activity from three wells of six-well plates was used, and at
least three independent experiments were performed. For statistical analysis,
the mean 6 SD was calculated.
EMSA
Nuclear extracts were prepared as described previously (20). After treatment with isotonic medium or NaCl (100 mM, 6 h), NRK52E cells were
lysed in buffer composed of 10 mM HEPES/KOH (pH 7.9), 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5% Nonidet P-40, and 0.5 mM
PMSF, and were centrifuged at 14,000 3 g for 5 min. The nuclear pellet
was then resuspended in buffer containing 20 mM HEPES/KOH (pH 7.9),
0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM
PMSF, and 25% glycerol; incubated for 45 min at 4˚C; and centrifuged at
14,000 3 g for 30 min at 4˚C. The nuclear extract was stored at 280˚C
until use for EMSA.
A probe for the putative tonicity enhancer element (59-TGGAAAAACACCAA-39, MCP1-TonE/ORE) in the promoter region of the MCP1 gene
was prepared by annealing two complementary oligonucleotides and endlabeled with (g-[32P])-ATP using T4 polynucleotide kinase. The probe
(30,000 cpm) was incubated with 5 mg of the nuclear extract prepared above
in reaction buffer containing 10 mM Tris/HCl (pH 7.5), 1 mM MgCl2, 0.5
mM EDTA, 0.5 mM DTT, 50 mM NaCl, 2 mg poly (dI-dC), and 4% glycerol.
After incubation for 20 min at room temperature, the reaction products were
subjected to 4% PAGE and visualized by autoradiography. For competition
experiments, 100-fold excess of unlabeled oligomer was added to the reaction mixture prior to the addition of labeled probe.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
hyperosmolity. Recent studies on intracellular signaling pathways
responsive to hypertonicity have suggested that MAPKs pathways
play a role in the cellular mechanisms underling the regulation of
gene expression in response to hypertonicity (21–24).
Taken together, we hypothesized that MCP1 expression induced by
hyperosmolality may be regulated through a hypertonicity response
element, such as TonE/ORE or NF-kB, in the MCP1 gene. Interestingly,
in a homologic search of the MCP1 gene sequence and TonE/ORE,
there is a sequence motif similar to TonE/ORE in the proximal region of
the 59-flanking region of the MCP1 gene. Therefore, to investigate the
molecular basis of transcriptional regulation of MCP1 in response to
hypertonicity, we sought to identify a functionally specific enhancer
element in response to hypertonic stress in the MCP1 gene, and we
examined the intracellular signaling pathways participating in the regulation of MCP1 expression. In this study, we report that in NRK52E
cells, MCP1 is upregulated through a novel hypertonicity-sensitive cisacting element localized in the proximal region of the MCP1 gene, and
p38 MAPK and MEK–ERK are, at least in part, implicated in cellular
signaling pathways leading to significant expression of MCP1 mRNA
and protein under NaCl- and mannitol-induced hyperosmolality.
HYPERTONICITY-INDUCED MCP1 EXPRESSION
The Journal of Immunology
5255
Western blot and immunocytochemical analyses of nuclear
distribution of TonEBP/OREBP/NFAT5
Results
Male Sprague-Dawley rats (n = 4) weighing 160 g (6 wk old; SLC, Shizuoka,
Japan) were deprived of water for 3 d and then sacrificed under deep anesthesia in
accordance with the Animal Rights Association of Meijo University. Kidneys
were fixed with PBS containing 4% paraformaldehyde and embedded in paraffin. Kidney sections were treated in a microwave oven (500 W, 12 min) in
citrate buffer (pH 6.0), blocked wtih 10% goat serum in PBS for 1 h, and incubated with anti-rat MCP1 (1:200; Chemicon) and/or anti-NFAT5 (1:500;
Calbiochem) at 4˚C overnight. Sections were then subjected to diaminobenzidine (DAB) staining using the Vectastain Elite ABC kit (Vector
Laboratories, Burlingame, CA) and the DAB substrate kit (Vector Laboratories),
according to the manufacturer’s instructions.
Statistical analysis
Data were analyzed by one-way ANOVA and Tukey’s multiple-comparison
test or nonparametric statistics. Data are expressed as mean 6 SD, and
significance was set at p , 0.05.
H2O Iso
Glucose H2O
In the first experiment, we used glucose (5–7), albumin (8), and LPS (9,
10) as positive controls that induce MCP1 mRNA expression. As shown
in Fig. 1A, treatment of NRK52E cells with glucose (200 mM for 3 d),
albumin (10 mg/ml for 24 h), or LPS (1 mg/ml for 24 h) resulted in
significant elevation of MCP1 mRNA. Similarly, when NRK52E cells
were treated with NaCl (100 mM) or mannitol (200 mM), MCP1
mRNA levels were markedly increased compared with treatment with
isotonic medium. Treatment with NaCl or mannitol revealed that MCP1
was upregulated in a time-dependent manner by the NaCl- or mannitolinduced hyperosmotic stress response (Fig. 1B). NaCl (150 mM) or
mannitol (300 mM) treatment at more than 600 mOsm/l resulted in
decreased induction of MCP1 mRNA, compared with lower doses of
NaCl and mannitol (Fig. 1C). It has also been reported that urea and
glycerol elicit a hyperosmotic response in mouse renal medullary cells
(23) and yeast Saccharomyces cerevisiae (25). However, in NRK52E
cells, treatment with glycerol (200 mM) or urea (200 mM) failed to
induce MCP1 gene expression (Fig. 1D).
Western blot analysis of MCP1 expression induced by NaCl
and mannitol-induced hyperosmolality, and ELISA analysis of
MCP1 released from cells under hyperosmolality
Effect of dehydration on MCP1 expression in the kidney
in vivo
A
Induction of MCP1 gene expression by NaCl and
mannitol-induced hyperosmolality
To examine whether MCP1 protein synthesis is promoted by NaCl or
mannitol-induced hyperosmolality, Western blot analysis was performed. In NRK52E cells treated with NaCl (100 mM; Fig. 2A), MCP1
protein was significantly increased, and higher levels remained at 24 h
after treatment. Cells treated with mannitol (200 mM) also showed
a marked elevation of MCP1 protein, which peaked at 12 h before
decreasing (Fig. 2A). When NaCl or mannitol were applied to the cells
at different doses, Western blot analysis showed no detectable expression of MCP1 protein at doses of more than 150 or 300 mM,
respectively, suggesting that extreme hyperosmotic stress (.600
mOsm/l) suppresses MCP1 protein expression, possibly through
translational inhibition (Fig. 2B). ELISA analysis of MCP1 in the
culture medium revealed that MCP1 protein was released into the
C
Iso Albumin
MCP-1
300
H2O Iso
NaCl
400
500
+ 50 + 100
600
+ 150
MCP-1
GAPDH
H2O Iso
LPS
mOsm/l
mM
GAPDH
H2OIso NaCl Iso Mannitol
MCP-1
H2O
GAPDH
300
Iso
Mannitol
400
500
+ 100 + 200
600
+ 300
mOsm/l
mM
MCP-1
B
Iso
H2O 0
NaCl (+ 100 mM)
3
6
9
12
Iso
24
24
GAPDH
hr
MCP-1
GAPDH
D
H2O
Iso
NaCl Glycerol Urea
MCP-1
Iso
H2O 0
Mannitol (+ 200 mM)
3
6
9
12
Iso
24
24
hr
GAPDH
MCP-1
GAPDH
FIGURE 1. RT-PCR analysis of MCP1 gene expression. A, Effect of glucose, albumin, LPS, NaCl, and mannitol on MCP1 expression in NRK52E cells.
NRK52E cells were exposed to glucose (100 mM) for 3 d or albumin (10 mg/ml), LPS (1 mg/ml), NaCl (100 mM), or mannitol (200 mM) for 24 h. B, Time
course of MCP1 expression under NaCl- or mannitol-induced hypertonic stress. NRK52E cells were treated with NaCl (100 mM) or mannitol (200 mM) for
24 h. Total RNA was prepared at the indicated time points and was subjected to RT-PCR. C, Effect of osmolality on NaCl- or mannitol-induced MCP1
expression. NRK52E cells were exposed to hypertonic medium at 400, 500, and 600 mOsm/l by addition of NaCl (50, 100, or 150 mM) or mannitol (100,
200, or 300 mM) for 3 h. D, Effect of glycerol and urea on MCP1 expression. NRK52E cells were exposed to NaCl (100 mM), glycerol (200 mM), or urea
(200 mM) for 3 h. For each case, three independent experiments were performed. Iso, isotonic medium.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
NRK52E cells were pretreated with SB203580 (5, 10, or 20 mM), PD98059 (5,
10, or 20 mM), U0125 (5, 10, or 20 mM), or SP600125 (10 or 20 mM), followed
by exposure to hypertonic stress (100 mM NaCl or 200 mM mannitol for 6 h).
Nuclear extact, prepared as described for EMSA, was subjected to Western blot
analysis using anti-NFAT5 (1:750; Calbiochem).
For immunocytochemicalobservation of thenuclear distribution of TonEBP/
OREBP/NFAT5, NRK52E cells were grown in a chamber slide (Lab-Tek II;
Nalgen Nunc, Naperville, IL) and exposed to NaCl (100 mM) or mannitol
(200mM) for6 h,after 1h treatment with SB20385 (20mM),PD98059(20mM),
U0126 (20 mM) or SP600125 (20 mM). Cells were fixed with cold-methanol
(220˚C) for 2 min, incubated with anti-NFAT5 (1:500; Calbiochem) at 4˚C
overnight, and treated with Alexa 488-conjugated anti-rabbit IgG (1:1000;
Molecular Probes, Eugene, OR) for 3 h at room temperature. Cells were then
viewed on a Zeiss Meta 510 microscope (Carl Zeiss, Jena, Germany) equipped
with a confocal imaging system.
In addition, NRK52E cells grown on a 96-well plate were treated as
described above, except that the cells were stained with Hoechist 33446
(Dojindo, Kumamoto, Japan) for 20 min at room temperature. Fluorescence
images of nuclear TonEBP/OREBP/NFAT5 and the nuclei (Hoechist 33446)
from separate cell views (∼500 cells in each view, and ∼2000 cells in total)
were subjected to quantitative analysis using an ImageXpress High Content Screening System (Molecular Devices, Sunnyvale, CA) and were
expressed as fluorescence intensity per cell.
5256
HYPERTONICITY-INDUCED MCP1 EXPRESSION
A
NaCl (+ 100 mM)
Iso
B
6
9
12
15
Mannitol (+ 200 mM)
24
hr
Iso
6
9
12
15
24
hr
MCP-1
MCP-1
GAPDH
GAPDH
NaCl
Mannitol
400 500 600 700 mOsm/l
Iso +50 +100 +150 +200 mM
400 500 600 700 mOsm/l
Iso +100 +200 +300 +400 mM
MCP-1
GAPDH
6
24 6 9 12 15 24
Iso NaCl (+ 100 mM)
hr
***
***
***
45
40
35
30
25
20
15
10
5
0
***
***
***
45
40
35
30
25
20
15
10
5
0
***
ng/ml/mg
C
ng/ml/mg
***
MCP-1
GAPDH
6 24 6 9 12 15 24 hr
Iso Mannitol (+ 200 mM)
culture medium in a time-dependent manner after NaCl or mannitol
stimulation (Fig. 2C).
Signaling pathways participating in induction of MCP1
expression under hypertonic stress
It has been shown that inhibition of protein synthesis with CHX blocks
hyperosmolality-induced gene expression in some cases (26), implicating de novo synthesized protein in gene expression regulation. In this
study, MCP1 mRNA elevated by NaCl was suppressed by treatment
with CHX (Fig. 3A). PKC and NF-kB signaling pathways have been
demonstrated to be activated in cells cultured in medium containing high
glucose (6, 7), high albumin (8), or high urea (27). However, calphostin
A
FIGURE 3. Effects of protein synthesis inhibition
(A), PKC inhibition (B), NF-kB inhibition (C), NADPH
oxidase inhibition (D), and iron chelation (E) on MCP1
gene expression under hyperosmolar NaCl. NRK52E
cells were pretreated with CHX (10 mg/ml), calphostin
C (500 nM), PDTC (20 mM), DPI (10 mM) ,or DFO
(100 mM) and were then exposed to NaCl (100 mM) for
3 h. MCP1 mRNA levels were evaluated by RT-PCR.
Pretreatment with CHX resulted in the suppression of
MCP1 mRNA expression.
C
E
H2O
Iso
H2O Iso
H2O
Iso
B
NaCl +
NaCl CHX CHX
Iso
NaCl +
NaCl calpho calpho
MCP-1
MCP-1
GAPDH
GAPDH
D
NaCl +
NaCl PDTC PDTC
NaCl
H2O
H2O
Iso
NaCl +
DPI
NaCl DPI
MCP-1
MCP-1
GAPDH
GAPDH
NaCl +
DFO DFO
MCP-1
GAPDH
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 2. Western blot analysis of time course (A) and dose-dependency
(B) of MCP1 protein expression induced by NaCl and mannitol hyperosmolality,
and time course of MCP1 protein released into culture medium under hyperosmolality (C). A, NRK52E cells were treated with NaCl (100 mM) or mannitol
(200 mM) for 6, 9, 12, 15, and 24 h, and cell lysates were subjected to Western
blot analysis. In NRK52E cells treated with NaCl (100 mM), MCP1 protein was
significantly elevated, and the levels remained higher at 24 h after treatment.
Mannitol (200 mM) also induced a marked elevation in MCP1 protein reaching,
which peaked at 12 h. B, NaCl (50, 100, 150, or 200 mM) or mannitol (100, 200,
300, or 400 mM) were applied to cells for 12 h. NaCl and mannitol induced
marked MCP1 protein expression at doses of 100 and 200 mM, respectively. C,
NRK52E cells were treated with NaCl (100 mM) or mannitol (200 mM) at the
indicated time points, and MCP1 protein released into culture medium was
measured by ELISA. pppp , 0.01 versus Normal (6 or 24 h).
C (a PKC inhibitor) and PDTC (an NF-kB inhibitor) showed no apparent inhibition of NaCl-induced MCP1 mRNA expression in
NRK52E cells (Fig. 3B, 3C). Moreover, although other NF-kB inhibitors, such as DPI (NADPH oxidase inhibitor) and DFO (iron
chelator), which indirectly block NF-kB by inhibiting the production
of reactive oxygen species, such as O22 and H2O2, were used, they
also had no effect on the suppression of MCP1 mRNA by NaCl (Fig.
3D, 3E). These results suggest that PKC and NF-kB cell signaling
pathways are not involved in the regulation of the MCP1 gene under
hyperosmotic stress conditions induced by NaCl in NRK52E cells.
Several lines of evidence have demonstrated that MAPKs, such as
p38MAPK (21, 22, 24), ERK (21–24) and JNK (21, 22, 24), are activated by hypertonic stress associated with NaCl. Thus, to further characterize the cell signaling pathways stimulated by hypertosmolality,
involvement of MAPKs in the regulation of MCP1 expression was
analyzed. Fig. 4A–C shows the time courses of activation of MAPKs
after NaCl-hyperosmotic stimuli. NaCl-treated NRK52E cells exhibited
time-dependent activation of MAPKs, namely p38MAPK, ERK, and
JNK with their highest activation at 30 min after the treatment. This
finding indicates that MAPK signaling pathways participate in the cellular response to hypertonic stress in NRK52E cells. To determine the
inhibitory effects of each MAPK inhibitor on the activity of each
MAPK, SB203580, PD98059, U0126, and SP600125 were used. Each
inhibitor at the indicated doses markedly blocked MAPK activity
(Fig. 4D–G).
In an additional experiment to clarify which MAPKs are involved in
MCP1 regulation, the inhibitory effects of MAPK inhibitors on MCP1
mRNA expression were studied by RT-PCR analysis. Fig. 5 illustrates
the effects of MAPK inhibitors on MCP1 gene expression. Treatment
of NRK52E cells with SB203580 showed a marked decrease in MCP1
mRNA levels induced by hypertonic stress in a dose-dependent manner
(Fig. 5A). PD98059 suppressed the increase in MCP1 mRNA associated with hypertonic stress (Fig. 5B). Similarly, U0126 also resulted in
decreased MCP1 mRNA elevated by NaCl hypertonicity (Fig. 5B).
Interestingly, the JNK inhibitor SP600125 failed to suppress the
increase in expression of MCP1 by NaCl (Fig. 5C). In addition,
curcumin, which abolishes AP1 activity (28), showed no suppressive effects on MCP1 mRNA expression (Fig. 5C), although
curcumin at the same dose suppressed PMA-induced MCP1 expression, in which PMA stimulates the transcription of MCP1
through PKC and AP1 activation (29) (Fig. 5C). Hyperosmolar
mannitol showed similar results to NaCl treatment (data not shown).
Furthermore, the effects of MAPK inhibitors on MCP1 protein expression were analyzed. As illustrated in Fig. 6, MAPK inhibitors,
except for the JNK inhibitor, suppressed the increase in MCP1
protein levels by NaCl or mannitol in a dose-dependent manner, and
The Journal of Immunology
5257
A
FIGURE 4. Western blot analysis of time course
of activation of p38MAPK (A), MEK/ERK (B) and
JNK (C), and effects of MAPK inhibitors on
p38MAPK (D), MEK/ERK (E, G) and JNK (F) in
NaCl-treated NRK52E cells. NRK53E cells were
treated with NaCl (100 mM) for 15, 30, 60, and
180 min, and cell lysates were then subjected to
Western blot analysis to detect time-dependent activation of phosphorylated and nonphosphorylated
p38MAPK, ERK, and JNK. MAPK showed timedependent activation caused by hypertonicity, with
peak activity being seen at 30 min after treatment.
Furthermore, NRK52E cells were pretreated with
each MAPK inhibitor for 1 h, followed by exposure
to 100 mM NaCl for 30 min. Cell lysates were
then used for detecting phosphorylated and nonphosphorylated p38MAPK, ERK, and JNK to determine the effects of MAPK inhibitors. Each
MAPK inhibitor suppressed the elevated activation
of each kinase by hyperosmolality.
Iso
B
NaCl (+ 100 mM)
15
30
60
180
mim
P-p38
MAPK
p38
MAPK
D
10
E
μM
P-p38
MAPK
p38
MAPK
20
B
5
10
20
μM
NaCl
5
10
20
Iso NaCl
5
10
20
60
180
F
NaCl+PD98059
5
10
20
mim
- P-p54 JNK
- P-p46 JNK
- p54 JNK
- p46 JNK
μM
- P-p44 ERK
- P-p42 ERK
- p44 ERK
- p42 ERK
NaCl+SP600125
Iso NaCl 10 20
30
μM
- P-p54 JNK
- P-p46 JNK
- p54 JNK
- p46 JNK
β-actin
response element in the MCP1 promoter region similar to the TonE/
ORE identified previously.
Identification of TonE/ORE of MCP1
To elucidate the element responsive to hyperosmolality in the D4
construct, a synthesized oligonucleotide construct (D440) spanning
–219 to –180, including a putative tonicity response element (MCP1TonE/ORE), was inserted into the pGL3-SV40-promoter driven luciferase reporter construct. As shown in Fig. 8A, the deletion constructs
of D440, D440M1, and D440M2 showed equal reporter activity to that
in the D440 construct. However, the D440M3 deletion construct with
an incomplete MCP1-TonE/ORE sequence was insensitive to hyperosmolality, suggesting that the putative tonicity response element
MCP1-TonE/ORE is a functional element responsive to hypertonicity.
The capacity of MCP1-TonE/ORE to behave as a tonicity-responsive
element was further examined by subjecting the D440M2 construct to
additional deletions and insertion into an SV40 promoter-driven luciferase reporter construct. As shown in Fig. 8B, deletion of Tat the 59-end
resulted in a slightly weaker reporter activity than the D440M2 construct
(Fig. 8B; D440M2-D1). Further removal of 59-TGG at the 59-end
completely abolished the osmotic response of the D440M2 construct
(Fig. 8B; D440M2-D2). In addition, deletion of 59-AAATTCCA at the
39-end of D440M2, in which 59-AA at the 39-end of the MCP1-TonE/
ORE was removed, also diminished reporter activity to control levels
C
NaCl + SP600125
H2O Iso
20
NaCl 10
30
μM
MCP-1
GAPDH
GAPDH
NaCl +
curcumin
H2O Iso
NaCl
5
15
μM
MCP-1
MCP-1
GAPDH
GAPDH
PMA +
curcumin
μM
30
β-actin
NaCl + U0126
H2O
NaCl (+ 100 mM)
15
β-actin
β-actin
MCP-1
μM
Iso
mim
- P-p44 ERK
- P-p42 ERK
- p44 ERK
- p42 ERK
NaCl+U0126
Iso NaCl 5 10 20
μM
- P-p44 ERK
- P-p42 ERK
- p44 ERK
- p42 ERK
NaCl + PD98059
H2O Iso
180
H2O
Iso
PMA
5
15
μM
MCP-1
MCP-1
GAPDH
GAPDH
FIGURE 5. RT-PCR analysis of the effects of
SB203580 (A), PD98059 (B), U0126 (B), SP600125 (C)
and curcumin (C), on MCP1 gene expression under NaClinduced hypertonic stress. NRK52E cells were treated
with SB203580 (5, 10, or 20 mM), PD98059 (5, 10, or
20 mM), U0126 (5, 10, or 20 mM), SP600125 (10, 20, or
30 mM) or curcumin (5 or 15 mM) for 1 h, followed by
exposure to 100 mM NaCl for 3 h. In addition, NRK52E
cells were also pretreated with curcumin (5 or 15 mM) for
1 h and then stimulated with PMA (100 ng/ml) for 3 h.
Treatment of NRK52E cells with SB203580, PD98059, or
U0126, but not SP600125 or curcumin, produced a dosedependent decrease in MCP1 mRNA levels under NaClinduced hypertonic stress.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
NaCl + SB203580
60
G
Fig. 7A schematically illustrates the location of potential regulatory
elements of the 59-flanking region of the MCP1 gene. Previous studies
identified the location and functional activities of C/EBP (30), NF-kB
(9), AP1 (9) and Sp-1 (9) cis-elements at 23118/23107 and 22591/
22579, 22287/22278 and 22261/22252, and 254/244 and 251/
239, respectively, in the rat MCP1 gene. At position 2199 to 2186 bp
upstream of the transcription start site, there is a sequence (labeled
MCP1-ToE/ORE; 59-TGGAAAAACACCAA-39, 2199 to 2186)
similar to a uniform TonE/ORE consensus sequence (59-(C/T)GGAANNN(C/T)N(C/T)-39 (16) and/or 59-NGGAAA(A/T)(T/A/G)(C/A/
T)(A/C)C-39) (17, 18). Thus, to identify the cis-acting regulatory enhancer elements within the MCP1 gene, which is responsive to hypertonic stress, the promoter region (2939 to +59) of the MCP1 gene,
which contains the putative MCP1-TonE/ORE, was isolated, and
a series of deletion constructs were created for luciferase reporter assay.
As shown in Fig. 7B, reporter assay with truncation constructs showed
a significant difference in luciferase activity between D4 and D5 deletion constructs, thus suggesting that there is a putative hypertonicity
NaCl
Iso NaCl
β-actin
Response of 59-flanking region of MCP1 gene to hypertonic stress
H2O Iso
30
β-actin
NaCl+SB203580
Iso NaCl 5
C
NaCl (+ 100 mM)
15
β-actin
this was consistent with the results shown in Fig. 5. This finding
indicates that the p38MAPK and MEK–ERK pathways, but not the
JNK-AP1 pathway, participate in cell signaling for MCP1 gene
expression under hypertonic stimulation by NaCl or mannitol.
A
Iso
5258
HYPERTONICITY-INDUCED MCP1 EXPRESSION
A
Iso
NaCl
SB203580
5
10
20
B
μM
Man
SB203580
5
10
20
Iso
Man
PD98059
5
10
20
μM
Iso
Man
5
U0126
10
20
μM
Iso
Man 10
SP600125
20
30
μM
Iso
μM
MCP-1
MCP-1
Western blot and immunocytochemical analyses of nuclear
translocation of TonEBP/OREBP/NFAT5
GAPDH
GAPDH
Iso
NaCl
PD98059
5
10
20
μM
MCP-1
MCP-1
GAPDH
GAPDH
Iso
NaCl
U0126
5
10
20
μM
MCP-1
MCP-1
GAPDH
GAPDH
Iso
NaCl
SP600125
10
20
30
μM
MCP-1
MCP-1
GAPDH
GAPDH
osmolality. In addition, EMSA showed a slow migrating band in the
cells exposed to hypertonic NaCl, with a stronger intensity as compared
with isotonic exposure (Fig. 9).
(Fig. 8B; D440M2-D4). However, deletion of 59-ATTCCA at the 39-end
of D440M2, thus creating the MCP1-TonE/ORE, maintained equal
reporter activity as the D440M2 construct (Fig. 8B; D440M2-D3).
Furthermore, reporter constructs with multiple concatenated copies
(MCP1-TonE/ORE 32 and 33) of MCP1-TonE/ORE showed a higher
transcriptional activity than that in constructs with a single copy (MCP1TonE/ORE 31; Fig. 8C). These results indicate that the 59-TGG and 59AAA sequences at the 59-end and 39-end, respectively, are essential to
elicit functional activity in response to hyperosmolality, and MCP1TonE/ORE is the minimum essential sequence sensitive to hyper-
Effect of dehydration on MCP1 expression and nuclear
distribution of TonEBP/OREBP/NFAT5 in rat kidney in vivo
After treatment of rats with dehydration, immunoreactivity for MCP1
in normal kidney was not apparent, whereas MCP1 immunostaining in
dehydrated rats was detected in tubular cells in the outer strip of the
renal outer medulla (Fig. 11A). In addition, immunostaining of TonEBP/OREBP/NFAT5 revealed that nuclear abundance of TonEBP/
OREBP/NFAT5 was increased in dehydrated rats, as compared with
normal rats (Fig. 11B).
Discussion
In the inflammatory process, chemokines and cytokines play an important role in the pathophysiologic features of progressive diseases.
A
B
FIGURE 7. Schematic presentation of location of potential regulatory elements within MCP1 59-flanking region (A) and its promoter activity in response
to hyperosmorality (B). A, The MCP1 promoter region from 2939 to +59 was isolated by PCR-based genome cloning; cis-acting elements in the MCP1
gene, such as C/EBP, NF-kB and AP1/Sp-1, are illustrated on the bases of the published data. In addition, a putative element responsive to hyperosmolality,
which has a consensus sequence with TonE/ORE, is localized at 2199 to 2186 of the MCP1 promoter region. B, Schematic presentation of luciferase
reporter constructs used for transfection and relative luciferase activity of the six reporter constructs in the cells exposed to hypertonic stress. Each deletion
fragment was directionally inserted into pGL3-basic vectors at the KpnI and XhoI sites. Cells transfected with the reporter constracts were treated with
isotonic or hypertonic (200 mM mannitol) medium. Data are expressed as firefly luciferase activity relative to Renilla luciferase and were normalized
against basal control levels (isotonic medium). Data in each panel represent mean 6 SD, and the number of independent transfections for each construct
was $ 3. ppp , 0.01 versus pGL3basic.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 6. Western blot analysis of the effects of MAPK inhibitors on
MCP1 protein expression under NaCl-induced (A) and mannitol-induced (B)
hypertonic stress. NRK52E cells were treated with SB203580 (5, 10, or
20 mM), PD98059 (5, 10, or 20 mM), U0126 (5, 10, or 20 mM) or SP600125
(10, 20, or 30 mM) for 1 h, followed by exposure to 100 mM NaCl or 200 mM
mannitol for 12 h. Treatment of NRK52E cells with SB203580, PD98059, or
U0126, but not SP600125, led to a dose-dependent decrease in the MCP1
protein production induced by NaCl or mannitol.
Western blot analysis showed that in NRK52E cells cultured in hypertonic medium with NaCl and/or mannitol, TonEBP/OREBP/NFAT5
was localized in nuclei in response to hypertonicity. Exposure to the
MAPK inhibitors SB203580, PD98059, and U0126 resulted in a reduction in the nuclear TonEBP/OREBP/NFAT5 abundance caused by
hypertonic stress (Fig. 10A).
Immunocytochemical observation also confirmed that the hypertonicity-induced nuclear translocation of TonEBP/OREBP/NFAT5 was
suppressed by MAPK inhibitors, except for SP6001225 (Fig. 10B).
Similarly, quantitative analysis based on immunocytochemical observation demonstrated reduced nuclear translocation of TonEBP/OREBP/
NFAT5 in cells pretreated with SB203580, PD98059, and/or U0126.
These results indicate that the p38MAPK and MEK–ERK pathways
participate in the activation of TonEBP/OREBP/NFAT5 in NRK52E
cells stimulated with NaCl or mannitol hyperosmolality.
The Journal of Immunology
5259
A
B
C
Competitor
Hypertonic
Isotonic
Hypertonic
Isotonic
scriptional activity (25, 38) and/or mRNA stability (39). In this study,
MCP1 expression induced by NaCl was also inhibited by pretreatment
with CHX, a translational inhibitor (Fig. 3A), thus suggesting that
newly synthesized, hypertonicity-induced factors might be involved
in the transcriptional activation of MCP1, although additional
Probe
Amongchemokines,MCP1is apotentchemoattractantand activatorfor
circulating monocytes/macrophages and T lymphocytes, and thus it has
been implicated in the development of fibrotic diseases, such as liver
fibrosis (31) and pulmonary fibrosis (32), as well as renal fibrosis and
peritoneal fibrosis. For the prevention of organ dysfunction owing to
fibrotic lesions, the molecular mechanisms underlying MCP1 regulation need to be understood at the molecular level. Previous studies have
reported numerous stimuli that lead to expression of MCP1, including
insulin (30), hypoxia/reoxygenation (33), mechanical stress (34), and
advanced glycation end-product (35), as well as glucose, albumin and
LPS. However, the regulation of MCP1 induction by hypertonicity
alone remains poorly understood. In this study, we investigated the
molecular regulation of MCP1 in response to hypertonicity.
In the first set of experiments, we examined the capacity of hypertonic
NaCl or mannitol to induce MCP1 gene expression, in comparison with
LPS, and glucose and albumin at high doses, as positive controls.
Hyperosmolar NaCl and mannitol increased MCP1 mRNA levels in
a time-dependent manner (Fig. 1). Furthermore, MCP1 protein synthesis and its release into the culture medium were elevated in a timedependent manner by exposure to NaCl and mannitol (Fig. 2A, 2C).
However, MCP1 protein production was significantly abolished by
NaCl and mannitol at .600 mOsm/l (Fig. 2B). Previous studies have
revealed that hypertonic NaCl at .600 mOsm/l induces apoptosis (36)
through mitochondrial dysfunction (37). Thus, the observed suppression of MCP1 protein synthesis might have resulted from such cellular
damage when cells were exposed to the extremely high concentrations
of NaCl and mannitol.
This study further investigated the intracellular signaling pathways
participating in MCP1 expression in response to hypertonicity. In some
genes, de novo protein synthesis has been found to regulate tran-
100 X 100 X
Free probe
FIGURE 9. EMSA of MCP1-TonE/ORE in MCP1 gene. A [32P]-labeled
MCP1-TonE/ORE 14-bp oligonucleotide fragment was incubated with 5 mg
of nuclear extracts prepared from NRK52E cells maintained in isotonic or
hypertonic medium, in the absence (2) or presence of 100-fold excesses of
unlabeled 14-bp oligonucleotide, as described in Materials and Methods.
Probe, labeled 14-bp probe only; Isotonic, isotonic medium; Hypertonic,
hypertonic medium supplemented with 100 mM NaCl; Competitor, unlabeled oligonucleotide fragment. The arrow indicates a slowly migrating
band of MCP1-TonE/ORE and protein complex.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 8. Functional activity of MCP1-TonE/ORE under hypertonic stress. A, To confirm the location and functional activity of the osmotic responsive
element in the promoter region of the MCP1 gene, a 40-bp fragment (D440) from 2219 to 2180 containing the TonE/ORE-like element 59-TGGAAAAACACCAA-39 (MCP1-TonE/ORE) was prepared based on the D4 construct, and its deletion mutant constructs (D440M1, D440M2, D440M3) were also inserted
into the pGL3-SV40 promoter vector. There was a significant difference in luciferase activity between D440M2 and D440M3, suggesting that the D440M2
sequence is involved in the response to hyperosmolality. Underlining represents the consensus sequence to TonE/ORE. B, To characterize the functional activity
of the D440M2 fragment, D440M2 was deleted. All deletion mutant constructs, except for D4M2-D3, failed to respond to hyperosmolality, suggesting that the
D4M2-D3 sequence 59-TGGAAAAACACCAA-39 (MCP1-TonE/ORE) is a potential osmotic responsive element in the MCP1 gene. Underlining represents
the consensus sequence to TonE/ORE. C, To further examine the activity of MCP1-TonE/ORE, constructs with multiple copies of MCP1-TonE/ORE were
analyzed. Constructs having two (MCP1-TonE/ORE 32) or three (MCP1-TonE/ORE 33) copies of MCP1-TonE/ORE showed increased luciferase activity, as
compared with the construct with a single copy of MCP1-TonE/ORE. Data in each panel represent mean 6 SD, and the number of independent transfections for
each construct was $3. pp , 0.05; ppp , 0.01 versus SV40 promoter.
5260
HYPERTONICITY-INDUCED MCP1 EXPRESSION
A
NaCl
5
SB203580
10
20
μM
Iso
Man
5
SB203580
10
20
μM
Iso
NaCl
5
PD98059
10
20
μM
Iso
Man
5
PD98059
10
20
μM
Iso
NaCl
5
μM
Iso
Man
5
U0126
10
20
Iso
SP600125
NaCl 10
20
Iso
Man
SP600125
10
20
μM
U0126
10
20
μM
μM
B
NaCl
NaCl + SB203580
NaCl + U0125
NaCl + SP600125
No Ab
NaCl + PD98059
600
**
500
##
##
##
400
300
200
100
0
experiments are required to confirm whether this includes translation of
TonEBP/OREBP/NFAT5.
Matsuo et al. (6) reported that hyperosmolar mannitol induced
MCP1 mRNA and protein through the activation of PKC and NF-kB in
peritoneal mesothelial cells. PKC activates NADPH-oxidase, leading
to the production of reactive oxygen species, and in turn the reactive
oxygen species activate NF-kB transcription factor through the
phosphorylation of Ik-B, followed by proteasome degradation, which
induces expression of NF-kB–regulated genes (8, 40). However, our
results showed that the PKC inhibitor calphostin C, which broadly
inhibits the activity of PKC subtypes, including a, b, g, d, and ε, does
not suppress the increase in MCP1 mRNA caused by hyperosmotic
NaCl (Fig. 3B). Moreover, the NF-kB inhibitor PDTC and the NADPH
oxidase inhibitor DPI were also ineffective in attenuating the MCP1
expression induced by NaCl (Fig. 3C, 3D). Furthermore, the iron
chelator DFO, which blocks the Fenton reaction leading to the production of H2O2 that activates NF-kB (40), also showed no suppressive
effects on MCP1 mRNA levels (Fig. 3E). These results thus indicate
that, PKC, NADPH oxidase, and H2O2, which are related to NF-kB
activation, may not be involved in the signaling pathways underlying
MCP1 induction in response to hyperosmolar NaCl or mannitol in
NRK52 E cells. This finding differs from the previous findings of
Matsuo et al. (6) using peritoneal mesothelial cells, and it might be due
to the difference in cell types used in this study, although further study
is needed for clarification.
Recent studies on the adaptive mechanisms for hypertonicity have
substantially improved our knowledge of hypertonicity-mediated
intracellular signaling pathways and transcriptional regulation of to-
N
Na
SB
PD
U
SP
Arbitrary intensity units
(Fluorescence intensity/cell)
NaCl
Arbitrary intensity units
(Fluorescence intensity/cell)
C
Isotonic
Mannitol
500
**
##
400
##
##
300
200
100
0
N
Man SB
PD
U
SP
nicity-sensitive genes. In kidney tubular cells, such as Madin-Darby
canine kidney cells (21), mouse inner medullary collecting duct cells
(20, 22, 23), and rabbit kidney papillary epithelial (PAP-HT25) cells
(24), p38MAPK, ERK, and JNK have been found to be involved in
hypertonicity-mediated cell signaling cascades. The present study
also demonstrated that hypertonic NaCl treatment results in the activation of p38MAPK, ERK, and JNK in NRK52E cells (Fig. 4), and
MAPK inhibitors, except for the JNK inhibitor, abolished MCP1
mRNA (Fig. 5) and protein (Fig. 6) expression induced by NaCl.
A
a
b
c
d
B
FIGURE 11. Effect of dehydration on MCP1 expression (A) and nuclear
distribution of TonEBP/OREBP/NFAT5 (B) in rat kidney in vivo. a and c,
Normal. b and d, Dehydration. Arrows indicate cytosolic MCP1 (a, b) and
nuclear TonEBP/OREBP/NFAT5 staining (c, d). Dehydration resulted in
elevated MCP1 protein expression and nuclear translocation of TonEBP/
OREBP/NFAT5.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 10. Western blot (A) and immunocytochemical (B, C) analyses of nuclear translocation of
TonEBP/OREBP/NFAT5. Nuclear distribution of TonEBP/OREBP/NFAT5 was suppressed by SB203580,
PD98059 and U0126, but not SP600125. A, NRK52E
cells were pretreated with SB203580 (5, 10, or 20
mM), PD98059 (5, 10, or 20 mM), U0126 (5, 10, or 20
mM) or SP600125 (10 or 20 mM) for 1 h, followed by
exposure to 100 mM NaCl and/or 200 mM mannitol
for 6 h. Cell lysates were then subjected to Western
blot analysis. B, NRK52E cells were pretreated with
SB203580 (20 mM), PD98059 (20 mM), U0126 (20
mM), or SP600125 (20 mM) for 1 h, and after hypertonic stress, were subjected to immunocytochemical
observation. C, Quantitative analysis of immunocytochemical observation using ImageXpress High Content
Screening System (Molecular Devices, Sunnyvale,
CA). N, normal; Na, NaCl (100 mM); Man, Mannitol
(200 mM); SB, SB203580 (20 mM); PD, PD98059
(20 mM); U, U0126 (20 mM); SP, SP600125 (20 mM).
ppp , 0.01 versus Normal; ##p , 0.01 versus NaCl or
Mannitol.
Iso
The Journal of Immunology
limbs and the S3 segment (pars recta) of proximal tubules in the rat
kidney (50). In the current study, dehydrated rats showed increased
MCP1 protein expression in the kidney, which is known to experience
an increase in tissue osmolality under such conditions. There was also
an increase in nuclear translocation of TonEBP/OREBP/NFAT5 in rat
kidneys during dehydration (Fig. 11), suggesting that MCP1 is induced by hypertonicity in the in vivo kidney, as well as in cultured
cells, through the nuclear distribution of TonEBP/OREBP/NFAT5.
Previous studies reported that hypertonicity induces expression of
cytokines, such as IL-8 (51) and TNF-a (52, 53), in PBMCs, T cells,
and fibroblasts; however, the biologic significance of their accelerated
expression is poorly understood. Sugiura et al. (54) showed that hyperosmotic stress activated the processing of latent TGF-b to the biologically active form in cultured fibroblast cells, leading to the stimulation of collagen synthesis that mediates fibrotic lesions. The results
of this study suggest that hypertonicity can activate proinflammatory
cascades in the kidney by promoting cytokine production. In contrast,
TNF-a has been found to stimulate the transcriptional activation of an
osmoprotective gene, the AR gene, through the TonE/ORE enhancer
element (55), suggesting that cytokine induction by hypertonicity can
contribute to osmoprotective adaptation. Further study is needed to
investigate the biologic significance of MCP1 expression in response
to hypertonicity in regard to osmoprotection or pathogenesis associated with tissue fibrosis.
The results of this study indicate that in NRK52E cells, NaCl, and
mannitol significantly induce MCP1 mRNA and protein production,
and the p38MAPK and MEK–ERK signaling pathways are involved
in the regulation of MCP1 gene expression. This study further identified a hypertonicity sensitive cis-acting element, MCP1-TonE/ORE,
in the 59-flanking region of the MCP1 gene, and MCP1-TonE/ORE is
transactivated through the TonEBP/OREBP/NFAT5, which is activated by the p38MAPK and MEK–ERK signaling pathways under
hypertonic stress. In addition, in vivo dehydration induces expression
of MCP1 in the rat kidney.
Disclosures
The authors have no financial conflicts of interest.
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These results indicate that p38 MAPK and MEK–ERK, but not JNK,
are involved in the tonicity-induced MCP1 expression in NRK52E
cells. As shown in Fig. 6, although SB203580 inhibited equally MCP1
protein level induced by NaCl and mannitol, PD98059 and U0126
moderately suppressed the increased MCP1 protein by mannitol, as
compared with the result with NaCl. A previous study has reported the
endothelin synthesis by a differential activation of p38MAPK and
ERK induced by hyperosmolar urea or betaine in Madin-Darby canine kidney cells (41), suggesting a differential participation of urea
and betain via p38MAPK and ERK activation in the cellular event.
Our result also suggests that in NRK52E cells NaCl and mannitol
might differentially participate in MCP1 expression through p38MAPK
and MEK–ERK signaling pathways with a different degree, although
transcriptional and translational analyzes are further needed to demonstrate the molecular mechanisms of the regulation of MCP1 synthesis.
The tonicity-responsiveelementTonE/ORE has been identified in the
59-flanking region of tonicity-sensitive genes (11, 12). Luciferase reporter assay using the MCP1 promoter region showed significant activity in luciferase reporter constructs containing the TonE/ORE
consensus sequence from the MCP1 gene (Fig. 7B). Moreover, based
on experiments with deletion mutant constructs (Fig. 8), 59-TGGAAAAACACCAA-39 is an essential sequence for functional activity in
response to hypertonicity and falls within the uniform consensus sequence of TonE/ORE, 59-(C/T)GGAANNN(C/T)N(C/T)-39 (16), or
59-NGGAAA(A/T)(T/A/G)(C/A/T)(A/C)C-39 (17, 18). When compared with the transcriptional activity between MCP1-TonE/ORE 32
and 33, the activities were almost equivalent (Fig. 8). This finding
might show that two copies of the elements are suffient to drive
a maximum transcriptional activity or that the observed equivalent
activity is due to the impaired efficiency of protein synthesis by hypertonic stress, although the molecular mechanism by which hypertinicity inhibits translation is not well understood (42). In addition, the
AP1/Sp-1 element of the MCP1 gene was insensitive to hyperosmolality because the D5 deletion construct containing the AP1/Sp-1
element failed to increase reporter activity (Fig. 7B). This observation
was consistent with the results shown in Fig. 5C, in which inhibition of
JNK failed to block the elevation of MCP1 mRNA by NaCl-hypertoniciy, indicating that the JNK-AP1 signaling pathway is not involved
in hypertonicity-mediated MCP1 regulation in NRK52E cells. Our
EMSA experiment showed a slowly migrating band for the MCP1TonE/ORE probe in cells exposed to hypertonic NaCl (Fig. 9), indicating that MCP1-TonE/ORE is a functional enhancer in response to
hypertonicity.
TonEBP/OREBP/NFAT5, a rel/NF-kB family member, has been
identified as a transcription factor that activates transcription of osmosensing genes in response to hypertonicity (13–15). TonEBP/
OREBPP/NFAT5 has been also found to be widely distributed in
various tissues, including the kidney (13), and is translocated into the
cellular nucleus (13, 14) by a nuclear localization signal (43) under
hypertonic stimulation. As shown in Fig. 10, p38MAPK and MEK–
ERK inhibitors, but not the JNK inhibitor, blocked the nuclear
translocation of TonEBP/OREBP/NFAT5 induced by NaCl or mannitol, suggesting that p38MAPK and MEK–ERK signaling pathways
(44, 45) are associated with the nuclear distribution of TonEBP/
OREBP/NFAT5 mediated by hypertonicity. This result is also consistent with the effects of MAPK inhibitors on MCP1 mRNA and
protein levels, as shown in Figs. 5 and 6, which suggests that MCP1
gene expression caused by hypertonicity with NaCl and mannitol is
driven by TonEBP/OREBP/NFAT5.
It has been reported that dehydration upregulates the expression
of tonicity-sensitive genes, such as an ion channel (46), a solute cotransporter (47), a metabolic enzyme (48), and heat shock proteins
(26, 49) in the kidney in vivo. In vivo dehydration also induces the
nuclear distribution of TonEBP/OREBP/NFAT5 in thick ascending
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HYPERTONICITY-INDUCED MCP1 EXPRESSION