From the Department of Molecular Genetics,
Biochemistry, and Microbiology, University of Cincinnati College of
Medicine, Cincinnati, Ohio 45267-0524 and the ¶ Division of
Pulmonary Biology, Children's Hospital Medical Center,
Cincinnati, Ohio 45229
Received for publication, January 12, 2001, and in revised form, February 6, 2001
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ABSTRACT |
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Aquaporin 5 (AQP5), the major water
channel expressed in alveolar, tracheal, and upper bronchial
epithelium, is significantly down-regulated during pulmonary
inflammation and edema. The mechanisms that underlie this decrease in
AQP5 levels are therefore of considerable interest. Here we show that
AQP5 expression in cultured lung epithelial cells is decreased 2-fold
at the mRNA level and 10-fold at the protein level by the
proinflammatory cytokine tumor necrosis factor Aquaporins (AQPs)1are
water channel proteins that function to increase plasma membrane water
permeability in secretory and absorptive cells in response to osmotic
gradients (1). Aquaporins are found in tissues where the rapid and
regulated transport of fluid is necessary such as in the kidney,
salivary glands, and lung. Deficiency of AQPs results in human diseases
such as nephrogenic diabetes insipidus due to mutations in AQP2 and
cataract formation from AQP0 mutations (2, 3). AQP5 is a mammalian
water channel expressed on alveolar type I and II cells, tracheal and
bronchial epithelium in the lung, in salivary and lacrimal gland
epithelia, and in corneal epithelium (4,
5).2 Mice that are deficient
in AQP5 have decreased production of saliva as well as altered saliva
composition (6).3 In
addition, AQP5 knockout mice have a 90% decrease in airspace-capillary water permeability (7). These studies demonstrate the importance of
AQP5 under normal conditions in both the salivary gland and lung.
Several AQPs have recently been demonstrated to undergo complex
regulation; for instance, AQP2 is regulated in response to vasopressin
both at the transcriptional and post-translational levels as well as
through shuttling of the protein to the membrane (8). In addition,
multiple AQPs are regulated under pathophysiological conditions such as
altered expression of AQP1, AQP2, AQP3, and AQP4 in the kidney in a
number of water balance disorders (9). Recently, through intratracheal
infection of mice with adenovirus (10, 11), Towne et al.
(12) showed that AQP5 mRNA and protein expression are decreased in
a mouse model of pulmonary inflammation and edema. The decreased
expression of AQP5 was found uniformly throughout the lung and was not
restricted to regions of overt inflammation, suggesting local effects
of a diffusible factor released in response to adenoviral infection.
AQP5 was decreased both 7 and 14 days after adenoviral infection;
however, the mechanism responsible for the regulation of AQP5 in
inflammation is unknown.
Proinflammatory cytokines, such as tumor necrosis factor alpha
(TNF- This study was therefore designed to assess directly the potential
effects of TNF- Experimental Reagents--
Murine TNF- Cell Culture and Drug Treatments--
Murine lung epithelial
cells (MLE-12) were a gift from Dr. Jeffrey Whitsett (Children's
Hospital Medical Center, Cincinnati, OH) (18). MLE-12 cells were
propagated at 37 °C with 5% CO2 in RPMI 1640 medium
(Life Technologies, Inc.) supplemented with L-glutamine,
100 units/ml penicillin, 100 µg/ml streptomycin, and 3% fetal bovine
serum (Life Technologies, Inc.). Approximately 48 h before study,
cells were seeded onto six-well tissue culture dishes at 5 × 105 cells/well and were serum starved 24 h before
study by replacing the medium with RPMI medium containing 1% fetal
bovine serum. In the MAP kinase and NF- Cell Viability--
Effects of TNF- Northern Analysis--
Following the specified treatment, medium
was aspirated, MLE cells were washed with phosphate-buffered saline,
and 1 ml of TriReagent (Molecular Research Center Inc., Cincinnati, OH)
was added for the isolation of total RNA as per the manufacturer's instructions. RNA was solubilized in Formazol (Molecular Research Center, Inc.), and RNA concentrations were determined via
spectrophotometry and confirmed with agarose gel electrophoresis. Total
RNA, 5 µg/sample, was size fractionated by gel electrophoresis as
described (12). The AQP5 cDNA probe was generated as described
previously (12). As controls for loading of total RNA, the 28 S and
18 S ribosomal RNA bands were examined with ultraviolet exposure of
the ethidium bromide-stained gel, and subsequent to probing with AQP5,
blots were stripped in boiling 0.5% SDS for 30 min and reprobed with a
mouse L32 ribosomal protein mRNA probe as described (12, 19). Northern blots probed with either the AQP5 or L32 were quantified by
exposure of a phosphor screen, scanned by means of a Storm 840 scanner,
and analyzed using ImageQuant software (all from Molecular Dynamics,
Sunnyvale, CA). RNA values are reported as the AQP5/L32 ratio for each
sample. Phosphorimaging results are reported as volume-integrated
values and are expressed in percentages compared with the mean values
in controls (vehicle-treated) (100%).
Western Analysis--
Following treatment of cells in the
specified medium for the indicated amount of time, the medium was
aspirated, and the cells were washed with ice-cold phosphate-buffered
saline. Ice-cold isolation solution (250 mM sucrose, 10 mM triethanolamine, 1 µg/ml leupeptin, and 0.1 mg/ml
phenylmethylsulfonyl fluoride, adjusted to pH 7.6) was added to each
well for scraping and collecting of the cells. Cells were then lysed
via three successive freezing and thawing cycles in dry ice and a
37 °C water bath, respectively. Total protein content was determined
by the BCA assay with bovine serum albumin as the standard. Cell
homogenates, 5 µg/sample, were solubilized in Laemmli sample buffer
and boiled for 5 min. SDS-polyacrylamide gel electrophoresis and
Western blotting were carried out as described previously (12).
Membranes were incubated overnight at 4 °C with anti-AQP5 antibody
at a dilution of 0.5 µg/ml and anti- Statistical Analysis--
Statistical analysis of AQP5/L32
density ratios for RNA expression and AQP5/ TNF-
To analyze the time course of TNF-
The decrease in AQP5 mRNA and protein expression was not the result
of apoptosis or the general inhibition of cell metabolism because the
number of live cells (trypan blue exclusion) and the protein content
after 24 and 48 h of incubation with 100 units/ml mTNF- Inhibition of AQP5 Expression Is Signaled through the p55 TNF- Decreased AQP5 Expression by TNF-
To examine the effect of ERK inhibition on AQP5 protein expression in
response to TNF-
Similar results were obtained with the use of SB203580, a drug that
specifically inhibits p38 kinase activity (30, 31). MLE-12 cells were
treated with 10 µM SB203580 for 1 h before the addition of TNF-
Several compounds have been demonstrated to inhibit activation of JNK
by TNF- Decreased AQP5 Expression in Response to TNF-
SN50 is a cell-permeable synthetic peptide that specifically competes
with the nuclear localization sequence of the p50 subunit of NF- Pulmonary inflammation is characterized by increased cytokine
expression, inflammatory cell infiltration, and excess fluid accumulation or pulmonary edema (42). Recently, aquaporins in the lung
were demonstrated to be down-regulated in a mouse model of pulmonary
inflammation (12). Through the use of knockout mice, AQP5 was shown to
be required for the majority of water transport in the lung (7).
Therefore, altered expression of AQP5 in inflammation may play a
significant role in the edema seen in pulmonary infection. Although the
regulation of AQP5 is of considerable interest, the mechanisms
regulating AQP expression remain poorly understood. Here we demonstrate
that AQP5 is down-regulated in a time- and dose-dependent
manner by TNF- TNF- The biological actions of TNF- Signaling through TNFR1 leads to alterations in gene expression via
activation of multiple signal transduction pathways including activation of the MAP kinase family, ERK1/2, p38, and JNK (22, 26). Two
principal transcription factors activated by TNF- TNF- Inflammatory lung diseases, such as chronic bronchitis, adult
respiratory distress syndrome, cystic fibrosis, and asthma, are
associated with elevated levels of TNF- In summary, TNF- (TNF-
). Treatment
of murine lung epithelial cells (MLE-12) with TNF-
results in a
concentration- and time-dependent decrease in AQP5 mRNA
and protein expression. Activation of the p55 TNF-
receptor (TNFR1)
with an agonist antibody is sufficient to cause decreased AQP5
expression, demonstrating that the TNF-
effect is mediated through
TNFR1. Inhibition of nuclear factor
B (NF-
B) translocation to the
nucleus blocks the effect of TNF-
on AQP5 expression, indicating
that activation of NF-
B is required, whereas inhibition of
extracellular signal-regulated or p38 mitogen-activated protein
kinases showed no effect. These data show that TNF-
decreases AQP5
mRNA and protein expression and that the molecular pathway for this
effect involves TNFR1 and activated NF-
B. The ability of
inflammatory cytokines to decrease aquaporin expression may help
explain the connection between inflammation and edema.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) and interferon gamma, are increased in expression
shortly after infection with adenovirus (10, 12) and may be potential mediators of the decrease in AQP5 expression. TNF-
is increased in
mouse lungs as early as 6 h postinfection and remains elevated both 7 and 14 days after infection (12, 13). The possibility that
cytokines may be responsible for the decrease of AQP5 in pulmonary
inflammation was therefore hypothesized. TNF-
has been implicated in
many biological conditions, most notably autoimmune diseases (such as
rheumatoid arthritis and inflammatory bowel disease), asthma, septic
shock, and human immunodeficiency virus infection (14). Extensive
studies show that TNF-
plays a pivotal role in inflammation
including modulating the expression of many genes such as other
proinflammatory cytokines, prostaglandins, major histocompatibility
complex antigens, oncogenes, and transcription factors (14, 15). The
ability of TNF-
to induce such a wide variety of effects is likely
because of its ability to activate multiple signal transduction
pathways including mitogen-activated protein (MAP) kinases and nuclear
factor
B (NF-
B) (16).
on AQP5 expression and to identify the signal
transduction pathways mediating the response. Both AQP5 mRNA and
protein expression are decreased in cultured murine lung epithelial
cells in a time- and dose-dependent manner in response to
TNF-
acting through the p55 type 1 TNF-
receptor 1 (TNFR1). This
decrease in expression requires the nuclear translocation of NF-
B.
To our knowledge these studies provide the first example of regulation
of an aquaporin by TNF-
and also provide the first evidence that
aquaporin expression can be regulated by NF-
B.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(mTNF-
) and human
TNF-
(hTNF-
) were obtained from Roche Biochemicals
(Indianapolis). Monoclonal hamster anti-mouse TNFR1 (55R539) antibody
was purchased from R & D Systems (Minneapolis). PD98059 was obtained
from New England BioLabs (Beverly, MA) and was dissolved in dimethyl
sulfoxide. U0126, SB205380, curcumin, MG-132,
pyrrolidinedithiocarbonate (PDTC), and NF-
B SN50 inhibitor peptide were purchased from Calbiochem. U0126, SB203580,
curcumin, and MG-132 were dissolved in dimethyl sulfoxide. PDTC and
SN50 were both dissolved in RPMI medium with 1% fetal bovine serum. Quercetin and dicoumarol were purchased from Sigma. Quercetin was
dissolved in dimethyl sulfoxide. Dicoumarol was dissolved in ethanol.
Electrophoresis reagents were from Bio-Rad. Reagents for enhanced
chemiluminescence (SuperSignal) and the BCA protein assay kit were from
Pierce. The antibody to
-actin was an anti-mouse monoclonal antibody
purchased from Sigma. The rabbit, anti-mouse AQP5 antibody (LL639;
Lofstrand Laboratories, Gaithersburg, MD) was generated against a
synthetic peptide corresponding to the mouse AQP5 carboxyl terminus and
affinity purified on a SulfoLink column (Pierce) conjugated with the
immunizing peptide (17). The horseradish peroxidase-labeled anti-mouse
and anti-rabbit IgG secondary antibodies were from Roche.
B inhibition studies,
inhibitors were added in the specified concentrations for 1 h
before the addition of 100 units/ml (1.5 ng/ml) mTNF-
as determined
by a dose-response curve (see Fig. 1). Cells were then maintained in
the medium for the duration of the experiment. Each experiment was
replicated in its entirety at least twice with at least three
independent wells/experimental group.
and the inhibitors
PD98059, SB203580, and SN50 on cell viability were measured with a
standard trypan blue uptake assay after treatment with 100 units/ml
mTNF-
for 8 and 24 h. Cell cultures were also examined
morphologically via light microscopy.
-actin antibody at a dilution
of 1:50,000 in 0.5% blocking solution (Roche). After washing, the
membranes were incubated with 100 milliunits/ml horseradish
peroxidase-labeled anti-rabbit secondary antibody and 50 milliunits/ml
peroxidase-labeled anti-mouse secondary antibody for 1 h at room
temperature, washed again, and visualized via enhanced
chemiluminescence with variable film exposures. For quantification,
films of Western blots were scanned using a Hewlett-Packard scanner and
Adobe Photoshop. Scanning was performed using chemiluminescence
exposures that gave control bands in the lower gray scale. The labeling
density was quantified using ImageQuant software. Values for AQP5 were
corrected by quantification of the
-actin values and were expressed
as an AQP5/
-actin ratio. Densitometry results are reported as
volume-integrated values and expressed in percentages compared with the
mean values in controls (vehicle-treated) (100%).
-actin density ratios for
protein expression were performed using unpaired Student's
t test with equal variance. Results are expressed as
means ± S.E. A p value of <0.05 was considered statistically significant.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Reduces AQP5 mRNA and Protein Expression in MLE-12
Cells--
MLE-12 cells were utilized to examine the effects of
TNF-
on AQP5 mRNA and protein expression. MLE-12 cells were
treated with various concentrations of mTNF-
for various time points to determine whether the response to TNF-
was dose- and
time-dependent. Cells were incubated in media supplemented
with 10, 50, 100, 500, or 1,000 units/ml mTNF-
for 8 h before
isolation of total RNA for Northern blot analysis. AQP5 mRNA levels
were decreased significantly with 50, 100, and 500 units/ml mTNF-
treatment (Fig. 1, A and B). AQP5 protein levels were decreased significantly after
24 h of treatment with 50, 100, 500, and 1,000 units/ml mTNF-
(Fig. 1, C and D) in a dose-dependent
manner. AQP5 mRNA and protein were decreased maximally after
treatment with 100 units/ml mTNF-
, therefore this concentration was
used for subsequent experiments.
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Fig. 1.
AQP5 mRNA and protein are decreased in
MLE-12 cells after treatment with various concentrations of
TNF- . A, representative
Northern blot analysis of RNA (5 µg/lane) isolated from MLE-12 cells
incubated in medium containing 10, 50, 100, 500, or 1,000 units/ml
mouse TNF-
for 8 h. Upper panel, AQP5; lower
panel, L32. The L32 ribosomal protein mRNA probe was used to
control for equal loading. B, Northern blots were quantified
by phosphorimaging, and the AQP5/L32 ratio was calculated for each
sample. Data are plotted as a percentage of control, vehicle-treated,
samples (n = 6, mean ± S.E.; * = p < 0.01, ** = p < 0.05 versus control). C, Western blot analysis of cell
homogenates (5 µg/lane) isolated after treatment as in A
for 24 h. Immunoblotting was performed with an affinity-purified
anti-AQP5 antibody, and a
-actin-specific antibody was utilized to
control for equal loading. D, immunoblots were quantified by
densitometry and expressed as the AQP5/
-actin ratio for each sample.
Data are plotted as a percentage of control (n = 6, mean ± S.E.; * = p < 0.01, ** = p < 0.05 versus control).
-mediated inhibition of AQP5
expression, MLE-12 cells were treated with mTNF-
for various time
points before isolation of total RNA. AQP5 mRNA was decreased significantly to about 50% of control (medium alone) levels after 4, 8, and 24 h of treatment with mTNF-
(Fig.
2, A and B).
However, after 48 h of treatment with TNF-
, AQP5 mRNA
returned to control levels (Fig. 2, A and B).
AQP5 protein was decreased dramatically to about 10% of control levels
after 24, 48, and 72 h of treatment with mTNF-
(Fig. 2,
C and D). Therefore, although AQP5 mRNA
returns to base-line levels by 48 h of treatment with mTNF-
,
AQP5 protein levels do not return to base line even by 72 h,
suggesting that the regulation of this gene is accomplished at both the
transcriptional and the post-transcriptional levels.
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Fig. 2.
TNF- decreases AQP5
mRNA and protein expression in a time-dependent
manner. A, Northern blot analysis of RNA (5 µg/lane)
isolated from MLE-12 cells treated with 100 units/ml mTNF-
for 1, 2, 4, 8, 24, or 48 h. The L32 ribosomal protein mRNA probe was
used as a loading control. B, quantification of AQP5 and L32
signals by phosphorimaging is expressed as the AQP5/L32 ratio for each
sample and is plotted as a percentage of control, vehicle alone,
treated samples (n = 6, mean ± S.E., * = p < 0.01, ** = p < 0.05 versus control). C, MLE-12 cells were treated
with 100 units/ml mTNF-
for 24, 48, or 72 h before the
isolation of cell homogenates. Cell homogenates (5 µg/lane) were
subjected to Western blot analysis with AQP5 and
-actin-specific
antibodies. D, signals on immunoblots were quantified using
densitometry and expressed as the AQP5/
-actin ratio for each sample.
Data are plotted as a percentage of control, vehicle alone, treated
samples (n = 6, mean ± S.E., * = p < 0.05 compared with control).
were
the same in control and TNF-
-treated cells (data not shown).
Examination of cells by light microscopy demonstrated that cells
treated with mTNF-
remained adherent and morphologically similar to
control cells. The quality and quantity of total RNA recovered and L32
mRNA contents visualized on the same blots as AQP5 mRNA were
not influenced by TNF-
. These data demonstrate that AQP5 mRNA
and protein are decreased in MLE-12 cells after treatment with TNF-
in a time- and dose-dependent manner.
Receptor--
The first step in TNF action is binding to specific
receptors that are expressed on the plasma membrane of virtually all
cells except erythrocytes (20, 21). Two distinct receptors for TNF-
have been identified, the p55 type 1 receptor (TNFR1) and the p75 type
2 receptor (TNFR2) (21, 22). These receptors share structural homology
in the extracellular TNF-
binding domains and exhibit similar
binding affinities for TNF-
, but they induce separate cytoplasmic
signaling pathways after receptor-ligand binding (21, 22). Mouse
TNF-
, which was utilized in all previous experiments, is capable of
binding to and signaling through both TNF-
receptors on mouse cells
(23). The monoclonal TNFR1 agonist antibody 55R539 specifically binds
to and activates signaling through TNFR1 and does not cross-react with
TNFR2 (22, 24). To determine which TNF-
receptor signals the
decrease in AQP5, we treated MLE-12 cells with the TNFR1 agonist
antibody 55R539 and measured AQP5 mRNA and protein levels. MLE-12
cells were treated with the TNFR1 agonist antibody at 0.33, 1, or 3 µg/ml or with mTNF-
for 8 h, and AQP5 mRNA expression was
evaluated. Northern blot analysis demonstrated that activation of TNFR1
by treatment of MLE-12 cells with all three concentrations of the TNFR1
agonist antibody reduced AQP5 mRNA expression to an extent similar
to that obtained by the simultaneous triggering of both receptors by
mouse TNF-
(Fig. 3, A and
B). In addition, human TNF-
, which specifically binds and
activates TNFR1 on murine cells (20, 25), also promotes decreased AQP5
expression (data not shown). Treatment of MLE-12 cells with either
mouse TNF-
or 1 or 3 µg/ml agonist TNFR1 antibody for 24 h
also resulted in a similar decrease in AQP5 protein (Fig. 3,
C and D). Therefore, stimulation of TNFR1 alone
decreases both AQP5 mRNA and protein expression to a level equal to
that seen with mouse TNF-
, suggesting that decreased AQP5 expression
in response to TNF-
is mediated principally through TNFR1.
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Fig. 3.
Decreased AQP5 mRNA and protein
expression in response to TNF- occurs, at
least primarily, by signaling through the TNFR1 receptor.
A, Northern blot analysis of RNA (5 µg/lane) isolated from
MLE-12 cells treated with 100 units/ml mTNF-
or 0.33, 1, or 3 µg/ml monoclonal TNFR1 agonist antibody 55R539 for 8 h.
B, quantification of AQP5 signals by phosphorimaging is
expressed as the AQP5/L32 ratio for each sample. Data are plotted as a
percentage of control, vehicle alone, treated samples
(n = 6, mean ± S.E., * = p < 0.01 compared with control). C, MLE-12 cells were treated
with mTNF-
or 1 or 3 µg/ml TNFR1 agonist antibody for 24 h
before isolation of cell homogenates and subsequent Western blot
analysis (5 µg/lane). D, immunoblotting was quantified by
densitometry and is expressed as the AQP5/
-actin ratio for each
sample. Data are plotted as a percentage of control, vehicle alone,
treated samples (n = 6, mean ± S.E., * = p < 0.01 compared with control).
Does Not Require Activation of
the ERK or p38 MAP Kinase Pathway--
Signaling through TNFR1 leads
to distinct effector functions, including MAP kinase activation and the
activation of NF-
B (14, 26). TNF-
activates signaling through
three MAP kinase pathways: extracellular signal-regulated kinase (ERK),
Jun N-terminal kinase (JNK), and p38 (16, 22), and all three pathways
are activated by mTNF-
treatment of MLE-12 cells (27). To
investigate the possible involvement of the ERK cascade in decreased
AQP5 expression in response to TNF-
, the effects of the MEK1/2 (the upstream kinase of ERK1/2) inhibitor PD98059 (28, 29) were examined.
PD98059 inhibits MEK1/2 by blocking activation of MEK1/2 by Raf kinase
(28). 20 µM PD98059 was added to MLE cells for 1 h
before the addition of mTNF-
, and cells were collected for RNA
isolation 8 h after the addition of TNF-
. Northern blot
analysis demonstrated that the addition of PD98059 alone did not alter AQP5 mRNA expression significantly. AQP5 mRNA expression was
decreased to the same extent with the addition of PD98059 and TNF-
as with TNF-
alone (Fig. 4,
A and B). Therefore, decreased AQP5 mRNA expression in response to TNF-
does not require signaling through ERK MAP kinase.
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Fig. 4.
Decreased expression of AQP5 in response to
TNF- does not require activation of ERK MAP
kinase. A, Northern blot analysis of RNA (5 µg/lane)
isolated from MLE-12 cells treated with mTNF-
, the ERK inhibitor, 20 µM PD98059 alone, PD98059 plus TNF-
, or vehicle alone
for 8 h. B, quantification of AQP5 mRNA levels by
phosphorimaging is expressed as the AQP5/L32 ratio for each sample and
is plotted as a percentage of control, vehicle alone, treated cells
(n = 6, mean ± S.E., * = p < 0.05 compared with control). C, Western blot analysis of
cell homogenates (5 µg/lane) isolated after treatment with vehicle
alone, mTNF-
, 20 µM PD98059, or PD98059 plus TNF-
for 24 h probed with AQP5 and
-actin specific antibodies.
D, immunoblotting was quantified by densitometry and is
expressed as the AQP5/
-actin ratio for each sample. Data are plotted
as a percentage of control, vehicle alone, treated cells
(n = 6, mean ± S.E., * = p < 0.01 compared with control).
, MLE-12 cells were treated with 20 µM
PD98059 for 1 h before the addition of TNF-
, and cell
homogenates were collected 24 h after the addition of TNF-
.
Western blot analysis demonstrated that AQP5 protein was unaltered with
the addition of PD98059 alone; when PD98059 was incubated in
combination with TNF-
, AQP5 expression was decreased to the same
extent as with TNF-
alone (Fig. 4, C and D).
Together these data demonstrate that the MEK inhibitor PD98059 has no
effect on either the decrease in AQP5 mRNA or protein expression
seen in response to TNF-
. Therefore, decreased AQP5 expression in
response to TNF-
does not require activation of the ERK MAP kinase pathway.
. Cells were then isolated 8 and 24 h after the addition of TNF-
for RNA and protein isolation, respectively. SB203580 alone did not alter AQP5 mRNA or protein expression
significantly (Fig. 5, A-D),
and addition of the inhibitor plus TNF-
resulted in a decrease in
AQP5 expression which was similar to that with TNF-
alone (Fig. 5,
A-D). These data show that the p38 inhibitor SB203580 has
no effect on the decrease in AQP5 mRNA and protein expression seen
in response to TNF-
, suggesting that decreased AQP5 expression in
response to TNF-
does not require activation of the p38 MAP kinase
pathway.
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Fig. 5.
Decreased expression of AQP5 in response to
TNF- does not require activation of p38 MAP
kinase. A, Northern blot analysis of 5 µg/lane RNA
isolated from MLE-12 cells treated with mTNF-
, 10 µM
p38-specific inhibitor SB203580 alone, SB203580 plus TNF-
, or
vehicle alone for 8 h. B, quantification of AQP5
mRNA levels by phosphorimaging is expressed as the AQP5/L32 ratio
for each sample and plotted as a percentage of control, vehicle alone,
treated cells (n = 6, mean ± S.E., * = p < 0.01 compared with control). C, cells
were treated with vehicle alone, mTNF-
, 10 µM
SB203580, or SB203580 plus TNF-
for 24 h before the isolation
of cell homogenates and subsequent Western blot analysis with AQP5 and
-actin-specific antibodies. D, immunoblotting was
quantified by densitometry and is expressed as the AQP5/
-actin ratio
for each sample. Data are plotted as a percentage of control, vehicle
alone, treated cells (n = 6, mean ± S.E., * = p < 0.01 compared with control).
; however, these inhibitors are fairly nonspecific for JNK
and affect other processes in the cell. Dicoumarol is a quinone
reductase inhibitor (32) that has been demonstrated to inhibit JNK
activation; however, use of this compound in combination with TNF-
resulted in rapid cell death, thus the effect of dicoumarol on AQP5
expression could not be assessed. Quercetin and curcumin are a plant
flavanoid and pigment, respectively, and previously they have been
demonstrated to inhibit JNK activation (33-35). Use of both inhibitors
individually resulted in decreased AQP5 mRNA expression with
inhibitor alone (data not shown). Expression was not decreased further
with inhibitor plus TNF-
. However, curcumin has been demonstrated to
decrease NF-
B activation as well as JNK activation (34), so the
effects of this inhibitor could be the result of inhibition of either
pathway. These results could suggest that JNK and/or NF-
B activation
is necessary for both basal and decreased AQP5 expression in response
to TNF-
.
May Require the
Nuclear Translocation of NF-
B--
NF-
B is a ubiquitous
transcription factor that is activated by proinflammatory cytokines
such as TNF-
and interleukin-1 (14, 26, 36). In addition, studies
have demonstrated that TNF-
activates NF-
B by signaling through
TNFR1 (36, 37). NF-
B is a homo- or heterodimer of DNA binding
subunits whose activity is regulated by the I
B proteins. Under
unstimulated conditions, NF-
B dimers are retained in an inactive
form in the cytoplasm through association with one of the I
B
proteins. Upon stimulation, I
B molecules are rapidly phosphorylated
and degraded through a ubiquitin/proteasome pathway, thus unmasking a
nuclear localization signal in NF-
B. Free NF-
B then translocates
from the cytoplasm to the nucleus where it can regulate transcription of genes with a
B site (16, 36, 37). To examine the possible involvement of NF-
B activation in the TNF-
-mediated
down-regulation of AQP5, several inhibitors of NF-
B were utilized.
Multiple compounds have been reported to inhibit NF-
B activity
through a variety of mechanisms, the majority of which are not specific
for NF-
B. Inhibitors of the proteasome, such as MG-132, inhibit
activation of NF-
B by blocking degradation of I
B (38). PDTC
inhibits NF-
B by suppressing the release of the inhibitor subunit
I
B from the latent cytoplasmic form of NF-
B (39) and thus blocks the activation of NF-
B; however, PDTC is also a potent antioxidant and affects many other processes in the cell. Treatment of MLE-12 cells
with 10 µM MG-132 or 100 mM PDTC,
concentrations that were shown previously to inhibit NF-
B activity
in response to TNF-
(38, 39), resulted in decreased AQP5 expression
with inhibitor alone (data not shown). No difference was seen between
cells treated with inhibitor or inhibitor plus TNF-
; however, AQP5
expression was already decreased to the same extent with inhibitor
alone as with TNF-
alone (data not shown). Therefore, no direct
conclusions can be drawn about the importance of NF-
B signaling on
AQP5 expression with the use of these inhibitors.
B
(40). SN50 has been demonstrated to penetrate cells rapidly (within 15 min) and inhibit NF-
B translocation to the nucleus, thereby
inhibiting NF-
B DNA binding (41). In addition, SN50 is specific for
the nuclear localization signal of NF-
B and therefore is a more
specific inhibitor of NF-
B activation. MLE-12 cells were treated
with 50 µg/ml SN50 for 1 h before the addition of TNF-
for
8 h and the subsequent isolation of RNA. Treatment with SN50 alone
caused a modest decrease in AQP5 mRNA; however, pretreatment with
SN50 prevented further inhibition of AQP5 mRNA expression by
TNF-
(Fig. 6, A and
B). AQP5 mRNA expression with inhibitor or inhibitor
plus TNF-
was significantly greater than with TNF-
alone, showing
that inhibition of NF-
B blocked the decrease in AQP5 expression in
response to TNF-
. Similar results were obtained when cells were
treated with SN50 for 1 h and then treated with TNF-
for
24 h followed by protein isolation and Western blot analysis. The
decrease in AQP5 protein expression was inhibited by pretreatment with
SN50 (Fig. 6, C and D). Therefore, NF-
B
translocation to the nucleus is likely necessary for decreased AQP5
expression in response to TNF-
.
View larger version (16K):
[in a new window]
Fig. 6.
Decreased AQP5 mRNA and protein
expression in response to TNF- may require the
nuclear translocation of NF-
B.
A, Northern blot analysis of 5 µg/lane RNA isolated from
MLE-12 cells treated with mTNF-
, the NF-
B-specific inhibitor, 50 µg/ml SN50 alone, SN50 plus TNF-
, or vehicle alone for 8 h.
B, AQP5 mRNA levels were quantified by phosphorimaging
and expressed as the AQP5/L32 ratio for each sample. Data are plotted
as a percentage of control, vehicle alone, treated cells
(n = 6, mean ± S.E., * = p < 0.05 compared with control, § = p < 0.01 compared
with cells treated with TNF-
alone). C, cells were
treated with vehicle alone, mTNF-
, 50 µg/ml SN50, or SN50 plus
TNF-
for 24 h before the isolation of cell homogenates and
subsequent Western blot analysis (5 µg/lane) with AQP5 and
-actin-specific antibodies. D, densitometry of Western
blot results expressed as the AQP5/
-actin ratio for each sample.
Data are plotted as a percentage of control, vehicle alone, treated
cells (n = 6, mean ± S.E., * = p < 0.05 compared with control, § = p < 0.01 compared
with cells treated with TNF-
alone).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
signaling through TNFR1. Inhibition of the activation
of the MAP kinases, ERK and p38, demonstrated activation of ERK, and
p38 is not necessary for the effect of TNF-
on AQP5 expression.
However, inhibition of the nuclear translocation of NF-
B showed that
the decrease of AQP5 mRNA and protein expression in response to
TNF-
is dependent upon the activation of NF-
B. To our knowledge,
these studies provide the first example of down-regulation of an
aquaporin by a proinflammatory cytokine as well as the first
demonstration that aquaporin expression can be regulated by
NF-
B.
is a pivotal mediator of inflammation and has been demonstrated
to regulate numerous genes essential to the inflammatory process,
including other cytokines and cell adhesion molecules (21, 22, 43).
Pulmonary inflammation is accompanied by edema, and administration of
TNF-
alone has been demonstrated to result in pulmonary edema (21).
Given the connection among TNF-
, inflammation, and edema, we
explored whether AQP expression might also be regulated by TNF-
.
AQP5 mRNA and protein were reduced by TNF-
in MLE-12 cells. AQP5
mRNA was decreased maximally within 4 h of treatment with
TNF-
and returned to normal levels by 48 h. AQP5 protein, on
the other hand, was decreased to a much greater extent than AQP5
mRNA and did not return to normal levels by 72 h of TNF-
treatment. The expression of several proteins, such as
CTP:phosphocholine cytidylyltransferase and I
B, is decreased in
response to TNF-
through increased protein degradation irrespective
of changes in mRNA levels (36, 44). TNF-
also inhibits skeletal
muscle protein synthesis and thus affects translational efficiency in a
number of genes (45). The observed inhibition of AQP5 protein expression is consistent with regulation at both pre- and
post-translational levels. Further experiments are required to
determine whether mechanisms that regulate protein level such as
translational efficiency, protein degradation, or other intracellular
signaling pathway(s) are involved in the TNF-
-induced
down-regulation of AQP5 protein.
are initiated by its binding to a
55-kDa receptor (TNFR1) and/or to a 75-kDa receptor (TNFR2) (22, 43).
Although these two receptors induce both distinct and overlapping
responses, the majority of effects of TNF-
studied occur through
signaling through TNFR1 (36, 37). However, some responses initiated by
TNF-
require the combined activation of both receptors or occur by
signaling through TNFR2 alone (22, 37). Through the use of a TNFR1
agonist antibody we demonstrated that activation of TNFR1 alone reduced
AQP5 mRNA and protein expression to the same extent as with mouse
TNF-
, which signals through both receptors. Thus, selective
signaling through TNFR1 results in the reduction of AQP5 gene
expression. Our results do not preclude the possibility that signaling
through TNFR2 might also decrease AQP5 expression but that signaling
through this receptor is not required.
are NF-
B and
activating protein 1 (14, 22). The AQP5 5'-flanking region contains
consensus binding sites for both activating protein 1 and NF-
B (17).
It has been demonstrated recently that AQP5 expression is increased in
MLE cells in response to hypertonic stress through an
ERK-dependent mechanism (29). In the present study, the
decrease in AQP5 mRNA and protein expression in response to TNF-
was not affected by the MEK inhibitor PD98059, suggesting that
signaling through ERK MAP kinase was not required for the effect.
Therefore, although TNF-
activates ERK in MLE cells, AQP5 expression
was decreased, and this response was independent of ERK activation.
This result does not conflict with the previous study because although
ERK activation was necessary for the hypertonic induction of AQP5, it
was not sufficient as activation of ERK by TPA did not induce AQP5
(29). Likewise, inhibition of the activation of p38 MAP kinase by
SB203580 demonstrated that p38 does not likely have a role in the
reduction of AQP5 in response to TNF-
.
has both stimulatory and inhibitory effects on gene expression,
and its effects are often mediated through the activation of the
transcription factor NF-
B (26, 36, 43). This study provides the
first link between aquaporin expression and NF-
B activation. SN50,
which specifically inhibits entry of NF-
B into cell nuclei,
inhibited the decrease of AQP5 mRNA and protein in response to
TNF-
, suggesting that the TNF-
effect requires the nuclear
translocation of NF-
B. The mechanism by which NF-
B activation regulates AQP5 expression is not clear, but it could involve several mechanisms including direct interaction of NF-
B with one of the putative
B binding sequences present in the mouse AQP5 gene (17). Studies have shown that when NF-
B binds to a consensus NF-
B binding site as a heterodimer of p50/p65, it acts as a transcription activator; yet when the NF-
B p50/p50 homodimer binds to promoters, it functions as a transcriptional repressor (46, 47). In addition, both
p50 and p65 subunits have been demonstrated to decrease expression of
genes, such as the
1(I) collagen gene, through direct interaction with other transcription factors such as SP1 and
CCAAT/enhancer-binding protein (48, 49). Alternatively, NF-
B could
serve to activate a repressor that inhibits transcription of AQP5.
Further studies are required to dissect the mechanism behind NF-
B
regulation of AQP5 expression.
in lung fluids (43). Therapeutic approaches that inhibit the action of TNF-
are a focus
of intense research and have recently been employed in the treatment of
rheumatoid arthritis and inflammatory bowel disease (14, 36). In this
regard it is essential to understand the downstream effectors of
TNF-
to predict the efficacy of therapy as well as potential side
effects. TNF-mediated inhibition of AQP5 may help explain why pulmonary
inflammation is accompanied by pulmonary edema and could implicate
potential therapy aimed at TNF and/or AQP5.
decreased the level of AQP5 mRNA and protein in
MLE-12 cells. Decreased AQP5 expression in response to TNF-
occurs
through activation of TNFR1 and does not require activation of ERK or
p38 MAP kinases. However, translocation of NF-
B to the nucleus is
likely required for regulation of AQP5 by TNF-
. This is the first
report of a proinflammatory cytokine decreasing the expression of an
aquaporin and provides information that may begin to explain the
relationship between inflammation and edema in the lung.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Gary E. Shull for careful review of the manuscript and Dr. Guanhu Yang for helpful technical discussions.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grants RO1 DE138283 (to A. G. M.) and RO1 HL60907 from the National Institutes of Health (to C. J. B.), Grant HL61781 from the NHLBI Program of Excellence in Molecular Biology of Heart and Lung, National Institutes of Health (to A. G. M.), and a new investigator grant from this program (to C. M. K.), and by the Caroline Halfter-Spahn Trust.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.
§ Supported in part by a predoctoral fellowship from the University of Cincinnati and an Albert J. Ryan Foundation fellowship.
To whom correspondence should be addressed: Dept. of Molecular
Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, 3110 MSB, P. O. Box 670524, Cincinnati, OH 45267-0524. Tel.: 513-558-5534; Fax:
513-558-1885; E-mail: Anil.Menon@UC.edu.
Published, JBC Papers in Press, February 9, 2001, DOI 10.1074/jbc.M100322200
2 C. M. Krane, C. N. Fortner, A. R. Hand, D. W. McGraw, J. N. Lorenz, S. E. Wert, J. E. Towne, R. J. Paul, J. A. Whitsett, and A. G. Menon, submitted for publication.
3 Krane, C. M., Melvin, J. E., Nguyen, H. V., Richardson, L., Towne, J. E., Doetschman, T., and Menon, A. G. (2001) J. Biol. Chem. 276, in press.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
AQP(s), aquaporin(s);
TNF-, tumor necrosis factor alpha;
MAP, mitogen-activated protein;
NF-
B, nuclear factor
B;
TNFR1, p55
type 1 TNF-
receptor;
TNFR2, p75 type 2 TNF-
receptor;
mTNF-
, murine TNF-
;
hTNF-
, human TNF-
;
PDTC, pyrrolidinedithiocarbonate;
MLE cells, murine lung epithelial cells;
ERK, extracellular signal-regulated kinase;
JNK, Jun N-terminal kinase;
MEK, mitogen-activated protein kinase/extracellular signal-regulated
kinase kinase.
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