Modulation of CFTR gene expression in HT-29 cells by extracellular hyperosmolarity

Maryvonne Baudouin-Legros, Franck Brouillard, Marc Cougnon, Danielle Tondelier, Tony Leclerc, and Aleksander Edelman

Institut National de la Santé et de la Recherche Médicale Unité 467, Faculté de Médecine Necker-Enfants Malades, 75015 Paris, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 · min-1 · 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 beta -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 beta -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 (Delta F/Delta t) in basal (Delta Fbasal) nitrate solution and in the presence of cAMP (Delta FcAMP) reflects the importance of the various conductances, and the ratio Delta FcAMP to Delta 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 beta 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 beta -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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -actin mRNA contents visualized on the same blots as CFTR mRNA also remained unchanged.


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Fig. 1.   Time course of the NaCl-induced decrease in cystic fibrosis transmembrane regulator (CFTR) mRNA. Serum-deprived HT-29 cells were incubated with serum-free medium plus 100 mmol/l NaCl, and total RNAs were extracted and analyzed by Northern blotting. A: typical Northern blot of 15 µg total RNAs extracted at the end of the indicated incubation periods. B: autoradiograms were quantified, and the intensity of the CFTR message was normalized to that of beta -actin. Each value is the mean ± SE of 6 determinations. [NaCl], NaCl concentration.


                              
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Table 1.   Cell viability

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-beta -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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Fig. 2.   Concentration dependence of the NaCl effect. Serum-deprived HT-29 cells were incubated in serum-free culture medium plus various amounts of NaCl for 8 h. A: typical Northern blot obtained with 15 µg total RNAs extracted from cells incubated with the indicated amounts of added NaCl. B: Northern blot data expressed by the CFTR-to-beta -actin mRNA ratios. Each value is the mean ± SE of 6 experiments.

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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Fig. 3.   Decrease in CFTR mRNA caused by adding various compounds to the medium. Serum-deprived HT-29, T84, and Calu-3 cells were incubated for 8 h in appropriate serum-free culture medium supplemented with 1) control medium, 2) NaCl (100 mmol/l; 200 mosmol/l), 3) urea (200 mmol/l; 200 mosmol/l), and 4) mannitol (200 mmol/l; 200 mosmol/l). A: Northern blots. B: results expressed as a percentage of the CFTR-to-beta -actin mRNA ratio, with 100% taken as cells incubated in control medium. Each result is the mean ± SE of 5 determinations. C: functional analysis. Fluorescence of intracellular 6-methoxy-N-ethylquinolinium was recorded in control Calu-3 cells () and in cells preincubated for 48 h in the presence of +200 mosmol/l added NaCl () under sequential anionic substitutions of the superfusing medium and addition of permeant 8-(4-chlorophenylthio)cAMP (10-4 mol/l) as indicated.

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 Delta FcAMP-to-Delta Fbasal ratio brought about by incubation in NaCl-supplemented medium ([Delta FcAMP/Delta Fbasal] for NaCl = 1.75 ± 0.24 vs. [Delta FcAMP/Delta 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, 10-4 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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Table 2.   Effect of kinase activators and inhibitors on CFTR transcripts in cells incubated in NaCl-supplemented medium

Cells were then placed in medium with +200 mosmol/l NaCl plus 10-5 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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Fig. 4.   Effect of SB-203580, inhibitor of p38 mitogen-activated protein (MAP) kinase, and PD-98059, inhibitor of p42/p44 MAP kinase. Serum-deprived HT-29 cells were incubated for 8 h in culture medium alone (1) or in medium supplemented with 200 mosmol/l NaCl (2) without (control) or with SB-203580 (10-5 mol/l) or PD-98059 (10-5 mol/l). A: typical Northern blot. B: quantification of the Northern blot data by the CFTR-to-beta -actin mRNA ratios. Each value is the mean ± SE of 6 experiments.



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Fig. 5.   Double effect of SB-203580. Serum-deprived HT-29 cells were incubated for 8 h with various concentrations of SB-203580 in control culture medium or with +200 mosmol/l NaCl added 15 min after the p38 kinase inhibitor. Amount of CFTR mRNA is given by the CFTR-to-beta -actin mRNA ratio calculated from the Northern blot data. A: Northern blot data, expressed as the CFTR-to-beta -actin ratio, for cells incubated with various concentrations of SB-203580 in unsupplemented medium or plus 200 mosmol/l added NaCl. Each value is the mean ± SE of 6 experiments. * P < 0.02 (Student's t-test using the control CFTR mRNA as reference). B: decrease in CFTR mRNA caused by adding +200 mosmol/l NaCl plus various concentrations of SB-203580. Each value is the mean ± SE of 6 determinations. * P < 0.02 (Student's t-test realized using the effect of added NaCl under control conditions as the reference).

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 × 10-6 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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Fig. 6.   Effect of actinomycin D and cycloheximide. Serum-deprived HT-29 cells were incubated for 8 h in isotonic (1) or NaCl-enriched (+200 mosmol/l) medium (2) without or with SB-203580 (10-5 mol/l), under basal conditions, with actinomycin D (5 µg/ml), or with cycloheximide (6 × 10-6 mol/l). A: typical Northern blots. B: CFTR-to-beta -actin mRNA ratios. Each value is the mean ± SE of 6 experiments.

No significant effect of adding NaCl or SB-203580 was found on cells treated with cycloheximide (6 × 10-6 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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Fig. 7.   Effect of high extracellular NaCl concentration on CFTR promoter activity. Top: construction transfected into HeLa cells. CFTR promoter (25) was subcloned in pGL3 basic plasmid. A: effect of added NaCl in the culture medium of HeLa cells. Results, expressed as the ratio of luciferase and beta -galactosidase activities, are means ± SE of 6 determinations. Ratios of luciferase and beta -galactosidase activities measured in mock controls (no CFTR promoter in the transfected plasmid) are shown on right. B: functional response of transfected CFTR gene promoter to SB-203580 (5 × 10-6 and 10-5 mol/l) added alone or together with +200 mosmol/l NaCl. Results, expressed as the ratio of luciferase and beta -galactosidase activities, are the means ± SE of 6 determinations. [SB-203580], SB-203580 concentration.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha 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.


    ACKNOWLEDGEMENTS

We thank Owen Parkes for correcting the manuscript.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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