Institut National de la Santé et de la Recherche Médicale U. 467, Faculté de Médecine Necker, 75015 Paris, France
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ABSTRACT |
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We have previously shown that
ouabain, which changes the electrochemical properties of cell membranes
by inhibiting Na+,K+-ATPase, induces the
expression of multidrug resistance (MDR-1) gene in several human cell
lines. Because the expressions of the MDR-1 and CFTR (which encodes the
cAMP-activated Cl channel associated with cystic
fibrosis) genes are physiologically regulated in opposing directions,
we wanted to determine whether ouabain also decreases CFTR transcripts
and subsequently to analyze its mechanism of action. We found that the
submicromolar concentrations of ouabain that increase MDR-1 mRNAs
decrease the CFTR transcripts with analogous time-dependency in human
pulmonary Calu-3 cells. By altering or reproducing the ouabain-induced
changes in intracellular ionic activities (decreasing in external
Na+ or K+ or using Na+ ionophore),
we show that the ouabain-induced regulations of both CFTR and MDR-1
transcripts depend on the Na+/K+ pump
inhibition but that the decrease in CFTR mRNAs also proceeds from
cytoplasm reactions simultaneously activated by ouabain. These data,
which emphasize the complex mechanism of action of ouabain, suggest
that changes in intracellular ionic activities modulate CFTR/MDR-1 gene expressions.
cystic fibrosis transmembrane conductance regulator; Na+,K+-ATPase inhibition; gene expression; transduction mechanisms
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INTRODUCTION |
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OUABAIN CONTROLS THE ACTIVITY of most ion transporters either directly or indirectly (3) by inhibiting Na+,K+-ATPase, which supports the ionic concentration gradients between the intra- and the extracellular fluids and the electrical potential of the cell membrane (5). Recent studies have shown that it has other effects that are not clearly linked to the inhibition of transmembrane ion transport, such as altering cell growth and differentiation and triggering apoptosis (4, 11, 14, 24). The expression of several genes is modulated in these processes, both early activated genes (c-fos, c-jun, etc.) (14, 21, 26) and late-response genes. The late-response genes encode cytosolic and secreted proteins (11) or membrane proteins (10, 15, 20, 30), including the Na+,K+-ATPase subunits, whose synthesis is controlled by ouabain in cardiomyocytes (10, 15, 30) and kidney cells (20).
Several cytosolic pathways transduce the actions of ouabain. Ouabain
activates the early-response genes and the gene encoding -skeletal
actin by increasing the cytosolic Ca2+ concentration
(Cai) and activating protein kinase C in rat cardiomyocytes (10, 26). However, it stimulates c-fos and
Egr-1 gene transcription in human monocytic leukemia cells with no
increase in Cai and no stimulation of protein kinase C
(22). Ouabain stimulates the synthesis of the
1 and
1
Na+,K+-ATPase subunits in kidney epithelial
cells via the increased intracellular Na+ concentration
([Nai]) produced by inhibiting
Na+,K+-ATPase (20), and high
[Na]i triggers the activation of MAP kinase cascades in
several cell types (16). On the other hand, the
stimulation of p42/44 MAP kinase in rat cardiomyocytes is independent
of increased [Na]i (14). This increase is
triggered, together with the production of reactive oxygen species
(ROS), by the activation of Ras produced by the interaction of
Na+,K+-ATPase and the other membrane proteins
Src and EGFR (epidermal growth factor receptor) (7, 8, 18, 19,
31).
We have previously shown that ouabain stimulates the expression of the
MDR-1 (multidrug resistance) gene and the synthesis of an active
P-glycoprotein (P-gp) in some human epithelial cells (4),
particularly pulmonary Calu-3 cells, which have the properties of the
serous cells of the pulmonary submucosal glands (9). These
cells are often used to study the expression of the CFTR (cystic
fibrosis transmembrane conductance regulator) gene. This gene undergoes
numerous mutations, causing cystic fibrosis. It encodes a transmembrane
protein acting as a cAMP-activated Cl channel
(27), and its expression is regulated, transcriptionally or posttranscriptionally, by several cytoplasm transduction processes, among which are the activation of protein kinase C and MAP kinase cascades (1, 35). Like P-gp, CFTR is one of the ATP
binding cassette (ABC) proteins. The proteins are also linked by the
fact that the CFTR and MDR-1 genes are frequently regulated in opposite directions in various epithelial cells by hormones and during cell
differentiation (33). The origin of the balance is still unknown. The present study was undertaken to determine whether stimulating Calu-3 cells with ouabain produced this opposing regulation of the CFTR and MDR-1 genes and whether the phenomenon was directly linked to altered ion transports.
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EXPERIMENTAL PROCEDURES |
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Cell culture and treatment. Calu-3 cells were obtained from the ATCC and cultured on plastic in DMEM containing 1 mM sodium pyruvate, nonessential amino acids, and 10% fetal calf serum (FCS) at 37°C in a 5% CO2-95% air atmosphere, except when otherwise noted. They were incubated with freshly prepared ouabain (Sigma-Aldrich) for the indicated time (generally 24 h). Ouabain toxicity was assessed by treating the subconfluent cultures with ouabain for 24 and 48 h and then counting the living cells (trypan blue exclusion). Some experiments were performed in buffered saline (BS) (basal composition in mM: 140 NaCl, 5 KCl, 1.1 MgSO4, 1 CaCl2, 0.34 Na2HPO4, 0.44 NaH2PO4, 10 Na acetate, 5 glucose, and 25 HEPES, pH 7.4), which allows ionic substitutions (140 mM NaCl was replaced by 140 mM choline chloride, while 5 mM KCl was replaced by 5 mM choline chloride). The DMEM was replaced by these warmed solutions 30 min before adding ouabain, and the cells were incubated for 24 h at 37°C in normal air.
RNA extraction and analysis.
Confluent cells were placed in serum-free medium for 24 h and then
incubated with ouabain in the absence of FCS. Total RNAs were isolated
with phenol/chloroform using the Trizol reagent (Invitrogen) according
to the manufacturer's instructions, fractionated on 0.9% agarose gels
(15 µg/well), and transferred to nylon membranes (Stratagene). The
membranes were hybridized with 32P-labeled cDNA probes
(specific activity >109 cpm/µg) with the Quik Hyb
solution provided by Stratagene. The CFTR probe was the 1.5-kb
EcoR1-EcoR1 fragment of human CFTR cDNA (a gift
from Dr. Pascale Fanen, Institut de la Santé et de la Recherche
Médicale U.468, Hôpital Henri Mondor, Créteil,
France). The human MDR-1 probe, kindly provided by Dr. Jean-Pierre
Marie (Service d'hématologie, Hôtel-Dieu, Paris, France),
was a 1.5-kb EcoR1-EcoR1 fragment of human MDR-1
cDNA. The -actin cDNA probe was purchased from Oncogene Science. The
mRNAs were quantified by densitometry using an ImageMaster VSD
(Pharmacia-Biotech-Amersham, Orsay, France), and the amounts of CFTR
mRNA were normalized to those of
-actin. All experiments were
repeated at least six times.
Statistical analysis. When appropriate, the statistical significance of the results was checked using ANOVA or Student's paired or unpaired t-test.
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RESULTS |
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Influence of ouabain on CFTR gene expression.
Serum-deprived confluent Calu-3 cells were treated with ouabain for
24 h, and the absence of toxicity of the treatment was checked
under microscope and by trypan blue exclusion. There were 603 ± 48 thousand living cells per cm2 in control
cultures, and 623 ± 46 thousand living cells per cm2
after incubation in 107 M ouabain, 601 ± 50 after
incubation in 2 × 10
7 M ouabain, and 490 ± 49 after incubation with 5 × 10
7 M ouabain
(n = 6). Cells incubated with ouabain (5 × 10
8 to 5 × 10
7 M) exhibited a
dose-dependent decrease in CFTR transcripts (Fig. 1, left). The typical Northern
blot shown at the top of the figure shows that ouabain enhanced MDR-1
gene expression in the same cells. The curves representing the changes
in expression of the CFTR and MDR-1 genes quantified as CFTR and
MDR-1/
actin ratios show that about 1.5 × 10
7 M
ouabain produced half inhibition of the CFTR message; this concentration also caused half-maximal stimulation of the MDR-1 gene
expression.
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Implication of
Na+,K+-ATPase
inhibition.
The cells were then treated with digoxin, another agent that binds to
the pump specifically and also stimulates MDR-1 gene expression
(4), to determine whether inhibiting
Na+,K+-ATPase reduced CFTR gene activity.
Digoxin also dose-dependently decreased CFTR mRNA (Fig.
3), with a time course (Fig.
3A) and an activity (Fig. 3B) very similar to
those of ouabain.
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Involvement of ionic factors in the regulation of CFTR and MDR-1
gene expressions.
Blocking the ion pump may be involved in modulating the gene
expressions via alternating ion concentrations, particularly by
increasing intracellular Na+ concentration. We checked this
by replacing most of the extracellular Na+ by choline
(leaving only 20 mM Na+) to prevent the increase in
[Na+]i caused by inhibiting
Na+,K+- ATPase (23). Cells
incubated in low-Na+ medium for 24 h showed little
change in CFTR gene expression, but the ouabain-induced decrease in
CFTR mRNA was blocked (Fig. 5).
Similarly, this ionic substitution did not significantly affect the
basal cell content of MDR-1 mRNAs, but it also inhibited the ouabain-induced stimulation of MDR-1 gene expression. These data confirm that MDR-1 gene expression depends on
Na+,K+-ATPase inhibition and point to increased
[Na+]i being involved in the inhibition of
CFTR gene expression by ouabain. Treating the Calu-3 cells with sodium
ionophore monensin (106 M), added 30 min before ouabain,
which corresponded to the optimal concentration, confirmed this
hypothesis. In preliminary experiments, we verified that treating the
cells with 10
6 M of monensin for the time of the
experiment was not toxic (we have compared the number of living cells
on one hand, and on the other, the total RNA content in the
monensin-treated and untreated cultures). Monensin decreased CFTR mRNAs
and stimulated MDR-1 gene expression. It also prevented any further
increase in MDR-1 mRNAs by ouabain but did not prevent any additional
inhibition by ouabain.
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Cytosolic reactions involved in the regulation of CFTR gene expression by ouabain. Several cytosolic reactions triggered by ouabain are involved in modulating the expression of several genes in rat cardiomyocytes. Among these are the activation of protein kinase C and p42/44 MAP kinase cascade (8, 14, 19) and the production of ROS (18, 34). The present research was not designed to analyze these ouabain-induced reactions, and we simply checked their involvement in the control of CFTR and MDR-1 gene expression by Northern blotting.
The modulation of both genes by ouabain expressions was first explored using inhibitors of the protein kinase C and p38 and p42/44 MAP kinase cascades. We used concentrations that did not alter the viability of the Calu-3 cells and found that only PD 98059, the p42/44 MAP kinase cascade blocker, inhibited the decrease of CFTR transcripts caused by ouabain (Table 1). The stimulation of MDR-1 gene expression by ouabain remained unchanged in all the experiments.
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DISCUSSION |
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Submicromolar concentrations of the specific Na+,K+-ATPase inhibitor, ouabain, which stimulate MDR-1 gene expression and P-gp synthesis, also decrease the concentrations of CFTR transcripts in the human Calu-3 cells. The pulmonary cells used in this study have the characteristics of the serous cells of the submucosal pulmonary glands devoted to mucous secretion (9).
We used ouabain concentrations in the same range as those that act on other human cells to decrease the synthesis of membrane proteins in T-84 colon cells (28), stimulate ROS production in HeLa cells (18), and modulate the expression of several genes in THP-1 (human monocytic) cells (22). The time course for the loss of CFTR mRNAs is also similar to that of the late changes caused by ouabain in the expression of genes encoding membrane or cytosolic proteins in cardiomyocytes or PC-12 cells (10, 30). Therefore, the CFTR gene, like the MDR-1 gene, is one of the genes whose expression is affected by ouabain.
Ouabain decreases the amount of CFTR mRNAs while stimulating MDR-1 gene
expression and P-glycoprotein (P-gp) synthesis in Calu-3 cells. This
replacement of CFTR by P-gp has been described in response to both
pharmacological stimulation and under physiological conditions,
particularly during cell differentiation (33). The expressions of many other genes are affected during cell
differentiation, and changes in Na+,K+-ATPase
-subunit isoforms and ouabain affinity are often observed (25). As there are several endogenous
Na+,K+-ATPase inhibitors whose physiology
remains unclear (29), our results raise the possibility of
a link between the modulation of the CFTR, MDR-1, and
Na+,K+-ATPase gene expressions. For example,
the
1-subunit is replaced by the
2-subunit with a higher affinity for ouabain in the
pulmonary tract at birth (13), together with decreased
CFTR activity (32).
The control of the expressions of CFTR and MDR-1 genes expression by ouabain raises two problems. The first concerns the control of gene expression by the altered electrochemical properties and transmembrane ion gradients caused by inhibiting Na+,K+-ATPase. The second problem is that of the existence and the nature of a common intracellular signal that affects the expressions of the CFTR and MDR-1 genes in opposite fashions.
Inhibition of Na+,K+-ATPase alone appears to
increase the cell content of MDR-1 transcripts, because incubating the
cells in K+-free medium or with ouabain or digoxin all had
the same effect of increasing MDR-1 mRNAs. Our studies on the
inhibition of RNA or protein synthesis indicate that the
ouabain-induced increase in MDR-1 transcripts results from the direct
stimulation of gene transcription. Similar direct stimulation of
early-activated gene transcription by ouabain also occurs in rat
cardiomyocytes (23, 26) and in human fibroblasts, in which
time course is the same as that for MDR-1 gene stimulation in Calu-3
cells (21). Like MDR-1, these early-activated genes are
not expressed in quiescent cells, and proteins they encode are directly
involved in the cell response to extracellular stresses. This
characteristic may be linked to the direct stimulation of their
expression when Na+,K+-ATPase is inhibited. In
contrast, the CFTR gene codes for a constitutive protein, and it may be
regulated via several mechanisms, coordinated to maintain correct cell
metabolism and function. The lack of effect of the K+-free
medium indicates that simply inhibiting
Na+,K+-ATPase cannot cause the inhibition of
the CFTR gene, as this process is complex and involves intermediate
protein(s). Nevertheless, if not sufficient,
Na+,K+-ATPase inhibition appears to be
necessary for inhibiting CFTR gene expression. This was suggested by
the effect of digoxin, another Na+,K+-ATPase
inhibitor, which reproduced the ouabain effect. Moreover, neither the
decrease of the CFTR transcripts by ouabain nor the stimulation of
MDR-1 gene expression occurred when cells were placed in
low-Na+ medium, because blockade of the pump by ouabain
cannot increase intracellular the Na+ concentration. An
increase in [Na+]i, caused by inhibiting
Na+,K+-ATPase or by monensin, can directly
stimulate the transcription of early-activated gene (22).
It may also, either directly or indirectly, control the expression of
late response genes, such as those encoding angiotensin II receptor 2 (30), IL-1 (22), and
Na+,K+-ATPase subunits (20). In
the Calu-3 cells, this increased [Na+]i thus
appears to be the common trigger of the opposing responses of the MDR-1
and CFTR gene expressions to ouabain, which could also be seen with
monensin. However, the results obtained with monensin also show that
increased [Na+]i alone cannot support the
decrease in CFTR transcripts caused by ouabain, which remains active in
the presence of the ionophore. Little is known about how high
[Na+]i may directly modulate gene expression,
but the effects of this parameter have been linked to a secondary
increase in [Ca2+]i. Our results obtained
with BAPTA and thapsigargin are consistent with such a relationship in
the Calu-3 cells, because BAPTA prevents the effects of ouabain on the
expression of the CFTR and MDR-1 genes and thapsigargin enhances it.
Thapsigargin alone does not modify the "basal" amount of MDR-1
mRNAs, although it decreases that of CFTR transcripts. This points to a
complex regulation of CFTR gene expression involving some
calcium-sensitive cellular pathway(s), which could also be activated by
ouabain independently of Na+,K+-ATPase
inhibition (8).
Indeed, the absence of any change in CFTR mRNAs when extracellular K+ is removed and the conservation of the ouabain effect under these conditions clearly show that other reaction(s), besides the inhibition of Na+,K+-ATPase, are required to inhibit CFTR gene expression. Modulating gene expression by ouabain involves both increased [Ca2+]i and the ion-independent activation of protein kinase C, of p42/44 MAP kinase cascade, and of ROS production in rat cardiomyocytes (31). Pharmacological inhibition of protein kinase C and the p38 MAP kinase cascade did not alter the effects of ouabain on either gene, but blocking the p42/44 MAP kinase cascade decreased the inhibition of CFTR gene expression by ouabain without affecting MDR-1 gene transcription. This MAP kinase cascade is activated by ouabain, in a Na+-dependent fashion, in several cells (17). Its activation by ouabain in Calu-3 cells was very slight and short-lived (<30 min, result not shown) and thus difficult to link to the decrease in CFTR transcripts.
However, inhibiting ROS production, which did not affect MDR-1 mRNAs, blocked the ouabain-induced regulation of CFTR gene expression. ROS formation, which occurs in response to ouabain independently of Na+,K+-ATPase inhibition (18), may thus be the second stimulus for decreasing CFTR transcripts. ROS are now considered to be a second messenger in several pathways involved in the control of gene expression (6). They may also be implicated in the induction of IL-1 gene expression (12), which is also stimulated by ouabain (22). Little is known about their precise molecular actions, but they are usually part of the overall cell response to extracellular stresses. This supports their implication in the inhibition of CFTR gene expression, which commonly occurs in response to extracellular stimulation (1, 2).
We conclude that ouabain and other Na+,K+-ATPase inhibitors regulate the expression of CFTR and MDR-1 genes in opposing senses. Inhibiting Na+,K+-ATPase can induce MDR-1 gene expression by itself and is also necessary to decrease CFTR mRNAs. However, the downregulation of CFTR gene expression by ouabain is a complex phenomenon that also involves transduction pathways triggered by the interaction of ouabain with the Na+/K+ pump. This illustrates both the control of gene expression by Na+,K+-ATPase activity and [Na+]i and the wide spectrum of action of the ouabain-like drugs.
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ACKNOWLEDGEMENTS |
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This work was supported by Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Association Vaincre la Mucoviscidose, and Association ABCF protéines. F. Brouillard and A. Hinzpeter were supported by fellowships from the Association Vaincre la Mucoviscidose.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. Baudouin-Legros, 156 rue de Vaugirard, 75015 Paris, France (E-mail: legros{at}necker.fr).
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.
10.1152/ajpcell.00457.2002
Received 30 September 2002; accepted in final form 28 October 2002.
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