Institut National de la Santé et de la Recherche Médicale Unité 467, Faculté de Médecine Necker-Enfants Malades, 75015 Paris, France
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ABSTRACT |
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Hypertonicity has pleiotropic effects on cell function,
including activation of transporters and regulation of gene expression. It is important to investigate the action of hypertonicity on cystic
fibrosis gene expression because cystic fibrosis transmembrane conductance regulator (CFTR), the cAMP-regulated
Cl channel, regulates ion
transport across the secretory epithelia, which are often in a
hypertonic environment. We found that adding >150 mosmol/l NaCl,
urea, or mannitol to the culture medium reduced the amount of CFTR mRNA
in colon-derived HT-29 cells in a time-dependent manner. Studies with
inhibitors of various kinases [H-89 (protein kinase A inhibitor),
bisindolylmaleimide (protein kinase C inhibitor), staurosporine
(serine/threonine kinase inhibitor) and herbimycin A (tyrosine kinase
inhibitor), SB-203580 and PD-098059 (mitogen-activated protein kinase
inhibitors)] showed that CFTR gene expression and its decrease by
added NaCl required p38 kinase cascade activity. The CFTR gene activity
is regulated at the transcriptional level, since adding NaCl diminished
the luciferase activity of HeLa cells transiently transfected with the
CFTR promoter. This regulation requires protein synthesis. The
complexity of the reactions involved in blocking CFTR gene
transcription by NaCl strongly suggests that the decrease in CFTR mRNA
is part of a general cell response to hyperosmolar stress.
cystic fibrosis transmembrane conductance regulator gene expression; extracellular sodium chloride; mitogen-activated protein kinases; p38 mitogen-activated protein kinase
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INTRODUCTION |
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THE CYSTIC FIBROSIS transmembrane conductance regulator
(CFTR) protein is an epithelial transport protein encoded by a large gene in which >800 mutations have been identified. The CFTR protein is a cAMP-stimulated Cl
channel, and mutation-induced alterations in it lead to cystic fibrosis
(CF), the most frequent and severe autosomic recessive disease in the
Caucasian population. A large amount of data have been obtained on the
properties of the CFTR gene and protein (14), but several questions
about the function of CFTR protein in integrated epithelial physiology
and the regulation of CFTR gene expression remain unanswered.
CFTR protein homologues, which have the same properties as the CFTR
protein, are overexpressed in euryhaline fishes and sea birds during a
chronic high-salt load and are involved in salt excretion (12, 29).
Many ionic disturbances in patients suffering from CF have been
attributed to the dysfunction of CFTR. The development of the
inflammatory and infectious syndromes that dominate the disease may be
due to the increased epithelial secretion viscosity caused by the
defective water excretion provoked by the abnormal regulation of ion
transport, which is normally assumed or controlled by CFTR activation
(28). However, the exact mechanism of the dysregulation is not known,
and contradictory data have been reported concerning the ionic
composition and the osmolarity of the secretions (33). One study
described an elevated NaCl concentration in the pulmonary secretions of
CF patients (30) that was shown to prevent the defensive effect of the
endogenous antibiotic -defensin (17). Other studies found isosmolar
surfactant (20). Inhalation of hypertonic saline has been shown
recently to improve the medical status of CF patients (2). A better
knowledge of the relationship between extracellular osmolarity and CFTR
might help to clarify CF physiopathology. The present work was
therefore undertaken to determine whether CFTR gene expression is
upregulated by extracellular hypersalinity in humans as it is in sea
birds and fishes.
Extracellular hyperosmolarity enhances the expression of genes coding for the transporters of the organic molecules (betaine, taurine, inositol) involved in adaptation of the cell medium and also those of ions (Na+, K+) in mammals (7). The cellular processes involved include many cytosolic reactions and the activation of several early activated genes coding for transcription factors (c-jun, Hsf, Egr1, Elk-1, CHOP-1, etc.) that are implicated in the response of cells to stress. The major transduction pathway is the reactivating kinase cascade, homologous to the murine p38 kinase and to the yeast protein Hog1 devoted to defense against hyperosmolarity (6, 18). Increased extracellular NaCl activates the p38 kinase cascade in several types of cells (9). However, hyperosmolarity may also activate other mitogen-activated protein (MAP) kinase cascades (23), and recent data indicate that there are many interactions between the various transduction pathways (19).
We have used human colonic HT-29 cells, which strongly express the CFTR gene and have been widely used in the studies on the regulation of CFTR gene expression, to analyze by Northern blotting the effect of increasing extracellular NaCl concentration on CFTR gene expression. We then explored the transduction mechanisms involved.
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MATERIALS AND METHODS |
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Cell culture and drug treatment. Most experiments were performed on human HT-29 carcinoma cells obtained from the American Type Culture Collection (Rockville, MD) and propagated in DMEM plus 10% FCS. T84 and Calu-3 cells (from the ATCC) were also used. The cells were cultured in DMEM-F-12 medium buffered with 15 mmol/l HEPES plus 10% FCS (T84 cells) and in MEM-Eagle salt solution supplemented with 10% FCS, 5 mmol/l sodium pyruvate, and 0.1 mmol/l nonessential amino acid (Calu-3 cells). HeLa cells (from the ATCC) were used for transfection experiments. The HeLa cells were cultured in DMEM plus 10% FCS. All of the culture products were obtained from Life Technologies (Les Ulis, France).
The cells were seeded at 104 per square centimeter and were grown for 2 days. The subconfluent cultures were then placed in serum-free medium for 24 h. NaCl was added to the culture medium (from a 2 mol/l stock solution) for the indicated time. Urea was added in the same way as NaCl, whereas mannitol was dissolved directly in DMEM (400 mmol/l), and this solution was mixed (1:1) with the cell culture medium. Permeant 8-(4-chlorophenylthio)cAMP (CPT-cAMP) was used to stimulate protein kinase A (PKA), and phorbol 12-myristate 13-acetate (PMA) was used to stimulate protein kinase C (PKC). The inhibitors used were H-89 (PKA inhibitor), bisindolylmaleimide (BIM, PKC inhibitor), staurosporine (broad serine/threonine protein kinase inhibitor), herbimycin A (tyrosine phosphorylation inhibitor), and inhibitors of the MAP kinase cascades, the pyridinil imidazole SB-203580 and the methoxyflavone PD-098059, inhibitors of p38 and of p42/p44 kinases (1, 11). Actinomycin D and cycloheximide were used to inhibit gene transcription and protein synthesis. The drugs were dissolved in the minimum volume of water or ethanol, and the control cells were always incubated in medium containing the same volume of solvent (<0.1%). The various compounds were added to the medium 15 min before NaCl, except for cycloheximide, which was added 2 h before NaCl. SB-203580 and PD-098059 were from Alexis (France), and all the other drugs were from Sigma (France).
Cell viability. Control cell cultures and those incubated with NaCl (+100 mmol/l) for 24 h were washed with PBS, trypsinized, suspended in serum-free medium, and centrifuged. Cells were washed two more times with PBS and suspended in PBS. This suspension was used to test cell viability with trypan blue, for cell counting (in a Malassez cell), and to measure protein concentration (22).
RNA extraction and analysis. Total RNA
was isolated by the phenol/chloroform method (8) using the Trizol
reagent (Life Technologies). The RNA was then fractionated on 0.9%
agarose gels (15 µg/well), transferred to nylon membranes
(Stratagene), and fixed by heating. The filters were hybridized with
32P-labeled cDNA probes (specific
activity >109
counts · min1 · µg
1)
with the Quik Hyb protocol provided by Stratagene, washed under stringent conditions (0.1 saline sodium citrate, 0.1% SDS at 52°C for 20 min), and autoradiographed at
80°C. The CFTR probe
was the 1.5-kb EcoR
I-EcoR I fragment of human CFTR-cDNA
labeled by random priming. The membranes were rehybridized with a human
-actin cDNA probe from Oncogene Science. The mRNAs were quantified
by densitometric scanning of the autoradiograms on an ImageMaster VSD
(Pharmacia-Biotech-Amersham, Orsay, France), and the amounts of CFTR
mRNA were normalized to those of
-actin.
Functional studies. The CFTR-related
cAMP-dependent Cl fluxes
were analyzed on Calu-3 cells incubated with either normal medium or in
the presence of +100 mmol/l NaCl for 48 h, using the video-imaging technology previously described (13). Briefly, the cells were grown on
glass coverslips and loaded with the fluorescent halide indicator
6-methoxy-N-ethylquinolinium (MEQ).
They were equilibrated in physiological saline in which
Cl
was replaced by
I
, which gives a minimal
intracellular fluorescence, since
I
reacts with MEQ and
quenches its fluorescence. Replacement of I
by nitrate ions in the
solution increases the MEQ fluorescence if there are anion conductive
pathways in the cell membrane, since the nitrate ions, which thus
replace the intracellular
I
, do not react with MEQ.
Adding a permeant cAMP to the nitrate solution then stimulates the
cAMP-triggered anion efflux, which reflects the CFTR function. The rate
of change of the fluorescence (
F/
t) in basal
(
Fbasal) nitrate solution and
in the presence of cAMP
(
FcAMP) reflects the
importance of the various conductances, and the ratio
FcAMP to
Fbasal indicates CFTR activity,
which can be compared in control and NaCl-treated cells.
Plasmid construction and
transfections. The (2150/+52) CFTR-luc plasmid,
kindly provided by Dr. G. S. McKnight (University of Washington,
Seattle, WA; see Ref. 25), was digested with Sac I and
Hind III (partial digestion). The
2,000-bp fragment, representing the
2150/+52 region of the CFTR
gene, was isolated by agarose gel electrophoresis and was inserted in
the pGL3 basic plasmid polylinker containing the luciferase gene
(Promega) digested by Sac I and
Hind III. Plasmid DNA was purified
using the Quiagen Plasmid endofree Maxi Kit according to the
manufacturer's instructions and was checked by sequencing.
HeLa cells (from the ATCC) were transiently transfected using the
Lipofectamine Plus reagent (Life Technologies) as indicated by the
manufacturer. Cells (4 × 105
cells in 2 ml) were plated out in 3.5-cm dishes for 24 h. They were
incubated for 3 h with 0.7 µg of 2150/+52 of pGL3 reporter plasmid together with 0.3 µg of RSV
Gal (Promega) reference
plasmid used to correct for differences in transfection efficiency.
The transfected cells were allowed to recover for 20 h in DMEM-10% FCS
and then were transferred to fresh medium with or without extra NaCl
for 15 h. The cells were washed three times in cold PBS, harvested, and
lysed in 220 µl lysis buffer (Promega). Luciferase activity was
assayed by measuring the luminescent signals produced by mixing 20-µl
aliquots of cell lysate with 100 µl Luciferase Assay Reagent
(Promega) in a Lumat LB9507 luminometer (Berthold). The
-galactosidase activity was measured colorimetrically.
Statistical analysis. The data are presented as means ± SE and were analyzed by ANOVA or Student's paired or unpaired t-test, as indicated in RESULTS. Differences were considered significant at P < 0.05.
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RESULTS |
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Effect of increased extracellular NaCl on the cell
content of CFTR mRNA. Addition of 100 mmol/l NaCl to
the HT-29 cell culture medium (normal NaCl concentration: 110 mmol/l;
osmolarity: 329 ± 16 mosmol/l, n = 6) produced a time-dependent decrease in the cell content of CFTR mRNA
(Fig. 1). The decrease was measurable after
6 h, and the CFTR message was close to zero after 24 h. The decrease in
CFTR mRNA was not due to apoptosis or the general inhibition of cell
metabolism, since the number of live cells (trypan blue exclusion) and
the protein content after 24 h of incubation were the same in control
and hyperosmotic (+100 mmol/l NaCl) cells (Table
1). The cell -actin mRNA contents
visualized on the same blots as CFTR mRNA also remained unchanged.
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Cells incubated for 8 h in medium containing extra NaCl (+25 to +150
mmol/l) showed a dose-dependent decrease in CFTR mRNA between +75 and
+150 mmol/l NaCl so that CFTR mRNA was almost undetectable with +150
mmol/l NaCl (Fig. 2). The cell content of
CFTR mRNA was slightly increased by adding +25 or +50 mmol/l NaCl to
the medium, since the CFTR-to--actin mRNA ratio (expressed as
percentage of the control) was 129 ± 19% (+25 mmol/l NaCl) and 138 ± 18% (+50 mmol/l NaCl; n = 6;
0.02 < P < 0.05; Student's paired
t-test). This effect was not
investigated further.
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We checked the specificity of the reduced CFTR mRNA in response to high
extracellular NaCl by repeating the experiments with equivalent amounts
of mannitol and urea (+200 mmol/l, i.e., +200 mosmol/l). CFTR mRNA was
decreased in all cases, regardless of the solute added. Hyperosmolarity
thus appeared to be the trigger of the downregulation of CFTR gene
expression. Analogous results were obtained with T84 and Calu-3 cells
(Fig. 3, A
and B).
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The presence of the CFTR protein in the membrane of the Calu-3 cells
allowed us to look for a functional effect of incubating them for 48 h
in hyperosmolar medium. Preincubation of the Calu-3 cells for 48 h in
+100 mmol/l (+200 mosmol/l) NaCl partly inhibited the cAMP-triggered
anion-induced increase in MEQ fluorescence measured by video imaging
(Fig. 3C), as shown by the decrease in the
FcAMP-to-
Fbasal
ratio brought about by incubation in NaCl-supplemented medium
([
FcAMP/
Fbasal]
for NaCl = 1.75 ± 0.24 vs.
[
FcAMP/
Fbasal]
for control = 3.24 ± 0.27; n = 24).
Action of added extracellular NaCl.
Because PKA and PKC control both CFTR gene expression and the activity
of many ionic transporters, their involvement in the action of the
added NaCl was checked by stimulating and inhibiting them (Table
2). Neither a permeant cAMP (CPT-cAMP,
104 mol/l) nor PMA (100 ng/ml) altered the decrease in CFTR mRNA caused by incubating cells in
medium supplemented with 200 mosmol/l NaCl for 8 h. Similarly,
inhibition of PKA or PKC activities with 10
6 mol/l H-89 or
10
5 mol/l BIM did not
modify the effect of +200 mosmol/l NaCl on CFTR mRNA. Thus regulation
of CFTR gene expression is not due to simple altered transduction.
Inhibition of tyrosine kinase activity with herbimycin A
(10
6 mol/l) did not alter
the effect of NaCl supplementation, and only staurosporine
(10
7 mol/l), the broad
spectrum inhibitor of serine/threonine kinases, significantly decreased
it (Table 2). However, when the two kinase inhibitors (herbimycin A and
staurosporine) were added together at the same concentrations, they did
not alter the cell response to extra NaCl. This suggests that the
effect of NaCl requires serial phosphorylations.
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Cells were then placed in medium with +200 mosmol/l NaCl plus
105 mol/l SB-203580, which
specifically inhibits p38 kinase activation, and
10
5 mol/l PD-98059, the
specific inhibitor of p42/p44 [extracellular signal-regulated
kinase (ERK1/ERK2)] MAP kinases. SB-203580 prevented the decrease
in CFTR mRNA caused by added extracellular NaCl, whereas PD-98059 had
no action (Fig. 4,
A and
B). SB-203580 dose dependently
decreased the inhibition of CFTR mRNA caused by added NaCl (+200
mosmol/l) between 5 × 10
6 and 2 × 10
5 mol/l (Fig.
5B), but
it also downregulated the CFTR transcripts by itself, at
10
5 and 2 × 10
5 mol/l, under control
conditions (Fig. 5A). Both basal
CFTR gene expression and its inhibition by added NaCl thus appear to be controlled by the activity of the p38 kinase cascade.
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Transcription and protein synthesis in the
downregulation of CFTR mRNA by increased extracellular
NaCl. The decrease in CFTR mRNA was not directly linked
to the cytosolic reactions triggered by extracellular hyperosmolarity,
since it did not occur when NaCl was added to the culture medium after
adding actinomycin D (5 µg/ml) or cycloheximide (6 × 106 mol/l; Fig.
6, A and
B). Therefore, the process requires
both gene transcription and protein synthesis. The decrease in CFTR mRNA caused by adding 10
5
mol/l SB-203580 also disappeared with actinomycin D. Addition of
actinomycin D to the cultures unmasked another effect of increased extracellular saline. In the presence of actinomycin D (Fig.
6B, center), CFTR gene expression was
higher in cells incubated for 8 h with +200 mosmol/l NaCl than in
controls (+69 ± 22%, P < 0.01, n = 6). This suggests that added
extracellular NaCl stabilizes CFTR mRNA when transcription is blocked.
Because SB-203580 does not alter the CFTR mRNA under the same
conditions, the stabilizing effect of added NaCl does not involve p38
kinase activation.
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No significant effect of adding NaCl or SB-203580 was found on cells
treated with cycloheximide (6 × 106 mol/l; Fig.
6B,
right), indicating that the
hyperosmolarity- and p38 kinase-controlled regulation of CFTR mRNA
requires protein synthesis.
We used HeLa cells transfected with the CFTR promoter (2150 to
+52) coupled to the luciferase gene to determine whether NaCl-induced downregulation of CFTR gene expression occurred at the transcriptional level. Incubation of the transfected cells in medium containing added
NaCl from +50 to +300 mosmol/l for 9 h dose dependently decreased the
luciferase activity (Fig.
7A),
demonstrating that extra NaCl depressed the activity of the CFTR
promoter. SB-203580 (10
5
mol/l) did not significantly inhibit the decrease in luciferase activity caused by adding +200 mosmol/l NaCl (decrease without SB-203580:
51 ± 6%; with SB-203580:
54 ± 5%;
n = 6; Fig.
7B). However, SB-203580 alone did
not depress, but slightly enhanced, the CFTR promoter activity. These
data show that the inhibition of CFTR gene transcription caused by
added NaCl does not depend on p38 kinase activation. They also indicate
that the inhibition of the NaCl-induced decrease in CFTR mRNA by
SB-203580 is linked to its ability to depress basal CFTR gene
expression.
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DISCUSSION |
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Our main finding is that increasing the extracellular NaCl
concentration modulates CFTR gene expression in HT-29 cells. Small additions of extracellular NaCl (+25 and +50 mmol/l) produce a slight
increase in CFTR transcripts, too small to be further investigated, but
increasing the NaCl concentration by >75 mmol/l dose dependently downregulates CFTR mRNA in HT-29 cells. This NaCl-induced decrease in
CFTR mRNA also occurs in T84 and Calu-3 cells and appears to be
triggered by extracellular hyperosmolarity more than by
Na+ or
Cl, since urea and mannitol
have the same effect as NaCl. The changes in CFTR gene expression in
these human cells incubated with high extracellular NaCl thus conflict
with the upregulation of the homologous CFTR proteins found in fishes
and birds (29, 12). Also, the effect of urea, which passes through the
membrane and does not affect cell volume, shows that the decrease in
CFTR mRNA is not directly linked to the control of cell volume or
intracellular ionic strength. Most of the published data relate that
hyperosmolar NaCl or mannitol, but not urea, increases expression of
ion transporter proteins (see Ref. 4) or expression of proteins
involved in the synthesis of osmolytes (see Ref. 15). The decrease in
CFTR mRNA by high extracellular NaCl thus does not evoke an adaptative process to hyperosmolarity. It rather looks like a part of the cell's
response to the stress of a disturbed environment. A similar stress-induced downregulation of CFTR gene expression has been used as
an argument for the "housekeeping" nature of the CFTR gene (34).
Analysis of the cAMP-stimulated anionic efflux from Calu-3 cells shows
that incubation in NaCl-enriched medium also decreases the CFTR protein
function, and this indicates the physiological relevance of the changes
in mRNA. However, whether the decrease in the anion efflux is due only
to the decreased mRNA or not cannot be ascertained, as many cytoplasmic
components influence the intracellular maturation and activation of the
CFTR protein (for review, see Refs. 21 and 16).
The reduced amount of CFTR transcripts caused by adding NaCl is not directly due to the activation of PKA or PKC, which are important in controlling electrolyte-water secretion in epithelial cells and which control CFTR gene expression (3, 5, 32, 34). On the other hand, since it is inhibited by staurosporine, the downregulation of CFTR expression induced by NaCl requires some other serine/threonine phosphorylation. The lack of action of staurosporine and herbimycin A combined suggests that this serine/threonine phosphorylation occurs together with a tyrosine phosphorylation.
The pharmacological study of the role of p38 MAP kinase also unmasks a
complex regulation of CFTR mRNA. Because the decrease in CFTR message
caused by added NaCl was offset by adding SB-203580, a specific
inhibitor of p38 kinase (11), p38 kinase activation seems to be
required for the hyperosmolarity-triggered modulation of CFTR gene
expression. However, addition of SB-203580 to the isosmotic culture
medium also caused a dose-dependent decrease in CFTR mRNA. This may
reflect a "basal" activation of the p38 kinase cascade in our
experimental model. Cytokine secreted by HT-29 cells (27) may be
responsible for this, since substances like interleukin-8 or tumor
necrosis factor- can activate p38 (9). The effect of SB-203580
suggests that p38 kinase activation enhances CFTR gene expression and
thus cannot alone cause the reduction in CFTR mRNA triggered by added
NaCl. The analogous downregulation of CFTR gene expression observed
with both added NaCl and SB-203580 may thus be due to some cytosolic
cross-talk between the various MAP kinase cascades (31) and
particularly that between p38 kinase and Jun
NH2-terminal kinase
cascades, which is favored by SB-203580 itself (19), or from different effects of hyperosmolarity and SB-203580 acting at various levels on
CFTR mRNA production or degradation.
Both added NaCl and SB-203580 act on gene transcription, since their effects disappear in HT-29 cells pretreated with actinomycin D, but the results obtained on the HeLa cells transiently transfected with the CFTR promoter indicate that there are two different mechanisms of action on CFTR gene. The dose-dependent decrease in luciferase activity found in these cells incubated in NaCl-enriched medium demonstrates that the CFTR promoter responds to a change in extracellular medium. Additionally, the regulation appears to occur in response to slight hyperosmolarity, since isolation of the gene transcription unmasked the effect of small additions of NaCl. On the other hand, SB-203580 neither inhibited the transfected CFTR promoter basal activity nor significantly decreased the effect of added NaCl in the same cells. This indicates that SB-203580 decreases CFTR mRNA by acting posttranscriptionally and shows that addition of extracellular NaCl inhibits CFTR gene transcription independently of p38 kinase activation. Hence, the SB-203580-induced inhibition of the effect of added NaCl on CFTR gene expression in HT-29 cells may be due to distal inhibition of the product of transcription, and the reaction would set aside the effect of previous transcriptional regulation caused by hyperosmolarity via another transduction mechanism. Because the NaCl-induced decrease in CFTR mRNA is blocked by cycloheximide in HT-29, CFTR gene transcription is not directly controlled by hyperosmolarity via cytosolic activation but requires protein synthesis. Various transcription factors, which bind to the CFTR promoter, are rapidly produced (early activated genes) after activation of the MAP kinase cascades (10). Such is the case for the c-jun and c-fos transcription factors, which are induced by the activated MAP kinase via the phosphorylation of transcription factors such as ternary complex factor, c-Jun, and ATF-2 (9). This is also true for CHOP, which is both induced and phosphorylated by MAP kinases and which, through the formation of CHOP-C/EBP heterodimers, modulates the DNA binding and activity of the C/EBP transcription factor (26), which is important in the regulation of CFTR gene expression (24). The downregulation of CFTR transcripts may involve one or several of these factors.
The modulation of CFTR gene expression by a high extracellular NaCl concentration is not due to the regulation of gene transcription alone but also includes posttranscriptional reactions. Supplementing the culture medium with NaCl increased CFTR transcripts in HT-29 cells that had been treated with actinomycin D. In the absence of gene transcription, this increase must have been due to increased stability of CFTR transcripts. An inhibition of CFTR mRNA degradation induced by adding extracellular NaCl may explain the small increase in CFTR transcripts caused by adding 50 or 100 mosmol/l NaCl to HT-29 cells. The idea of a dual (transcriptional and posttranscriptional) effect of hyperosmolarity is supported by the results obtained with the transfected HeLa cells, since adding small amounts of NaCl (+50 and +100 mosmol/l) does not increase but decreases the CFTR promoter activity in these cells. The CFTR mRNA is directly stabilized by hyperosmolarity, since the process occurs in HT-29 cells in the absence of both transcription and protein synthesis (blocked by actinomycin D and cycloheximide, respectively). Therefore, this effect of added NaCl does not appear to be related to the mRNA stabilizing action of p38 kinase activation (unmasked by the opposite effect of the inhibitor SB-203580), which disappears in the presence of either actinomycin D or cycloheximide. Further studies are now required to better characterize these mechanisms of CFTR mRNA stabilization and to determine whether the large amount of CFTR mRNA found in HT-29 cells is linked to the basal activation of p38 kinase present in these cells or not.
In conclusion, extracellular hyperosmolarity, which decreases CFTR mRNA in several cell lines, appears to affect several molecular targets that can modulate CFTR gene expression via transcriptional and posttranscriptional processes. These data demonstrate the existence of a large number of molecular reactions involved in the control of CFTR mRNA and confirm the housekeeping character of the CFTR gene. The regulation of CFTR mRNA by p38 MAP kinase activity suggests that CFTR gene expression is modulated by many extracellular stimuli.
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ACKNOWLEDGEMENTS |
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We thank Owen Parkes for correcting the manuscript.
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FOOTNOTES |
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These studies were supported by the Institut National de la Santé de la Recherche Médicale and by a grant from the Association Française de Lutte contre la Mucoviscidose.
Parts of this study were presented at the Annual Meeting of the American Cystic Fibrosis Foundation in Montreal (Canada), in October 1998, and appeared in abstract form (Pediatr. Pulmonol. S17: 215, 1998).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Baudouin-Legros, INSERM U. 467, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard, 75015 Paris, France (E-mail: legros{at}necker.fr).
Received 5 April 1999; accepted in final form 23 August 1999.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alessi, D. R.,
A. Cuenda,
P. Cohen,
D. T. Dudley,
and
A. R. Saltiel.
PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo.
J. Biol. Chem.
270:
27489-27494,
1995
2.
Ballman, M.,
and
H. Hardt.
Hypertonic saline and recombinant human DNASE: a randomised cross-over pilot study in patients with cystic fibrosis (Abstract).
Pediatr. Pulmonol.
S17:
343,
1998.
3.
Bargon, J.,
B. C. Trapnell,
C-S. Chu,
E. R. Rosenthal,
K. Yoshimura,
W. B. Guggino,
W. Dalemans,
A. Pavirani,
J. P. Lecocq,
and
R. G. Crystal.
Down-regulation of cystic fibrosis transmembrane conductance regulator gene expression by agents that modulate intracellular divalent cations.
Mol. Cell. Biol.
12:
1872-1878,
1992[Abstract].
4.
Bowen, J.
Regulation of Na+-K+-ATPase expression in cultured renal cells by incubation in hypertonic medium.
Am. J. Physiol. Cell Physiol.
262:
C845-C853,
1992
5.
Breuer, W.,
N. Kartner,
J. R. Riordan,
and
I. Cabantchik.
Induction of expression of the cystic fibrosis transmembrane conductance regulator.
J. Biol. Chem.
267:
10464-10469,
1992.
6.
Brewster, J. L.,
T. de Valoir,
N. D. Dwyer,
E. Winter,
and
M. C. Gustin.
An osmosensing signal transduction pathway in yeast.
Science
259:
1760-1763,
1993[ISI][Medline].
7.
Burg, M. B.
Molecular basis of osmotic regulation.
Am. J. Physiol. Renal Fluid Electrolyte Physiol.
268:
F983-F996,
1995
8.
Chomczynski, P.,
and
N. Sacchi.
Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[ISI][Medline].
9.
Cohen, P.
Dissection of protein kinase cascades that mediate cellular response to cytokines and cellular stress.
In: Intracellular Signal Transduction, edited by H. Hidaka,
and A. C. Nairn. San Diego, CA: Academic, 1996, vol. 36, p. 15-27.
10.
Cuenda, A.,
G. Alonso,
N. Morrice,
M. Jones,
R. Meir,
P. Cohen,
and
A. R. Nebreda.
Purification and cDNA cloning of SAPKK3, the major activator of RK/p38 in stress- and cytokine-stimulated monocytes and epithelial cells.
EMBO J.
15:
4293-4301,
1996.
11.
Cuenda, A.,
J. Rouse,
Y. N. Doza,
R. Meier,
P. Cohen,
T. F. Gallaher,
P. R. Young,
and
J. C. Lee.
SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1.
FEBS Lett.
364:
229-233,
1995[ISI][Medline].
12.
Ernst, S. A.,
K. M. Crawford,
M. A. Post,
and
J. A. Cohn.
Salt stress increases abundance and glycosylation of CFTR localized at apical surfaces of salt gland secretory cells.
Am. J. Physiol. Cell Physiol.
267:
C990-C1001,
1994
13.
Fanen, P.,
R. Labarthe,
F. Garnier,
M. Benharouga,
M. Goossens,
and
A. Edelman.
Cystic fibrosis phenotype associated with pancreatic insufficiency does not always reflect the cAMP-dependent chloride conductive pathway defect.
J. Biol. Chem.
272:
30563-30566,
1997
14.
Fuller, C. M.,
and
D. J. Benos.
CFTR!
Am. J. Physiol. Cell Physiol.
263:
C267-C286,
1992
15.
Garcia-Perez, A.,
B. Martin,
H. R. Murphy,
S. Uchida,
H. Murer,
B. D. Cowley,
J. S. Handler,
and
M. B. Burg.
Molecular cloning of cDNA coding for kidney aldose reductase: regulation of specific mRNA accumulation by NaCl-mediated osmotic stress.
J. Biol. Chem.
264:
16815-16821,
1989
16.
Gasby, D. C.,
and
A. C. Nairn.
Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis.
Physiol. Rev.
79:
S77-S107,
1999[Medline].
17.
Goldman, M. J.,
G. M. Anderson,
E. D. Stolzenberg,
U. P. Kari,
M. Zasloff,
and
J. M. Wilson.
Human -defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis.
Cell
88:
553-560,
1997[ISI][Medline].
18.
Han, J.,
J-D. Lee,
L. Bibbs,
and
R. J. Ulevitch.
A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells.
Science
265:
808-810,
1994[ISI][Medline].
19.
Hazzalin, C. A.,
A. Cuenda,
E. Cano,
P. Cohen,
and
L. C. Mahadevan.
Effects of the inhibition of p38/RK MAP kinase on induction of five fos and jun genes by diverse stimuli.
Oncogene
15:
2323-2331,
1997.
20.
Knowles, M. R.,
J. M. Robinson,
R. E. Wood,
C. A. Pue,
W. M. Mentz,
G. C. Wager,
J. T. Gatzy,
and
R. C. Boucher.
Ion composition of airway surface liquid of patients with cystic fibrosis as compared with normal and disease-control subjects.
J. Clin. Invest.
100:
2588-2595,
1997
21.
Kopito, R. R.
Biosynthesis and degradation of CFTR.
Physiol. Rev.
79:
S167-S173,
1999[Medline].
22.
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr,
and
R. J. Randall.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951
23.
Matsuda, S.,
H. Kawasaki,
T. Moriguchi,
Y. Gotoh,
and
E. Nishida.
Activation of protein kinase cascades by osmotic shock.
J. Biol. Chem.
270:
12781-12786,
1995
24.
Matthews, R. P.,
and
G. S. McKnight.
Characterization of the cAMP response element of the cystic fibrosis transmembrane conductance regulator gene promoter.
J. Biol. Chem.
271:
31869-31877,
1996
25.
McDonald, R. A.,
R. P. Matthews,
R. L. Idzerda,
and
G. S. McKnight.
Basal expression of the cystic fibrosis transmembrane conductance regulator gene is dependent on protein kinase A activity.
Proc. Natl. Acad. Sci. USA
92:
7560-7564,
1995[Abstract].
26.
Ron, D.,
and
J. F. Habener.
CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription.
Genes Dev.
6:
439-453,
1992[Abstract].
27.
Saitoh, O.,
R. Matsuse,
K. Sugi,
K. Nakagawa,
K. Uchida,
K. Maemura,
K. Kojimata,
I. Hirata,
and
K. Katsu.
Cyclosporine A inhibits interleukin-8 production in a human colon epithelial cell line (HT-29).
J. Gastroenterol.
32:
605-610,
1997[ISI][Medline].
28.
Schwierbert, E. M.,
D. J. Benos,
M. E. Egan,
M. J. Stutts,
and
W. B. Guggino.
CFTR is a conductance regulator as well as a chloride channel.
Physiol. Rev.
79:
S145-S166,
1999[Medline].
29.
Singer, T. D.,
S. J. Tucker,
W. S. Marshall,
and
C. F. Higgins.
A divergent CFTR homologue: highly regulated salt transport in the euryhaline teleost F. heteroclitus.
Am. J. Physiol. Cell Physiol.
274:
C715-C723,
1998
30.
Smith, J. J.,
S. M. Travis,
E. P. Greenberg,
and
M. J. Welsh.
Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid.
Cell
85:
229-236,
1996[ISI][Medline].
31.
Tan, Y.,
J. R. Rouse,
A. Zhang,
S. Cariati,
C. Boccia,
P. Cohen,
and
M. J. Comb.
FGF and stress regulated CREB via a pathway involving the RK/p38 MAP kinase homologue and MAPKAP-2.
EMBO J.
15:
4629-4642,
1996[Abstract].
32.
Trapnell, B. C.,
P. L. Zeitlin,
C.-S. Chu,
K. Yoshimura,
H. Nakamura,
W. B. Guggino,
J. Bargon,
T. C. Banks,
W. Dalemans,
A. Pavirani,
J.-P. Lecocq,
and
R. G. Crystal.
Down-regulation of cystic fibrosis gene mRNA transcript levels and induction of the cystic fibrosis chloride secretory phenotype in epithelial cells by phorbol ester.
J. Biol. Chem.
266:
10319-10323,
1991
33.
Wine, J. J.
The genesis of cystic fibrosis lung disease.
J. Clin. Invest.
103:
309-312,
1999
34.
Yoshimura, K.,
H. Nakamura,
B. C. Trapnell,
W. Dalemans,
A. Pavirani,
J. P. Lecocq,
and
R. G. Crystal.
The cystic fibrosis gene has a "housekeeping"-type promoter and is expressed at low levels in cells of epithelial origin.
J. Biol. Chem.
266:
9140-9144,
1991