Differential regulation of Clminus transport proteins by PKC in Calu-3 cells

Carole M. Liedtke, Derek Cody, and Thomas S. Cole

The Cystic Fibrosis Center, Departments of Pediatrics and Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4948


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cl- transport proteins expressed in a Calu-3 airway epithelial cell line were differentiated by function and regulation by protein kinase C (PKC) isotypes. mRNA expression of Cl- transporters was semiquantitated by RT-PCR after transfection with a sense or antisense oligonucleotide to the PKC isotypes that modulate the activity of the cystic fibrosis transmembrane conductance regulator [CFTR (PKC-epsilon )] or of the Na/K/2Cl (NKCC1) cotransporter (PKC-delta ). Expression of NKCC1 and CFTR mRNAs and proteins was independent of antisense oligonucleotide treatment. Transport function was measured in cell monolayers grown on a plastic surface or on filter inserts. With both culture methods, the antisense oligonucleotide to PKC-epsilon decreased the amount of PKC-epsilon and reduced cAMP-dependent activation of CFTR but not alpha 1-adrenergic activation of NKCC1. The antisense oligonucleotide to PKC-delta did not affect CFTR function but did block alpha 1-adrenergic activation of NKCC1 and reduce PKC-delta mass. These results provide the first evidence for mRNA and protein expression of NKCC1 in Calu-3 cells and establish the differential regulation of CFTR and NKCC1 function by specific PKC isotypes at a site distal to mRNA expression and translation in airway epithelial cells.

sodium-potassium-2chloride cotransport; antisense oligonucleotide; permeabilized monolayer; reverse transcriptase-polymerase chain reaction; actin; alpha -adrenergic activation; methoxamine; phorbol ester; cystic fibrosis transmembrane conductance regulator; protein kinase C


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TWO CHLORIDE TRANSPORT PROTEINS, basolateral Na/K/2Cl (NKCC1) cotransporter and apical Cl- channel-designated cystic fibrosis transmembrane conductance regulator (CFTR), act in a coordinated manner to mediate salt and water secretion in the epithelial cells that line the airways, sweat glands, and salivary glands. Regulation of NKCC1 appears to occur at a posttranslational level, with increased activity linked to the phosphorylation of existing transport molecules (25). In previous studies (15, 19, 20), our laboratory demonstrated that protein kinase (PK) C is a key effector enzyme in the hormonal activation of NKCC1 in airway epithelial cells. More recent studies (16, 18) that used an antisense approach identified PKC-delta as the PKC isotype that is required for activation by alpha 1-adrenergic stimulation.

Although CFTR is an apical Cl- channel regulated primarily by PKA, it is stimulated to a modest extent by PKC. In addition, PKC appears to act synergistically with PKA to maximize CFTR Cl- channel activity. For example, in membrane patches excised from cells expressing CFTR, the addition of exogenous PKC caused a modest increase in CFTR channel activity (3, 33) and enhanced the rate and magnitude of subsequent PKA stimulation of open probability (33). New evidence from this laboratory and others provides more direct evidence for PKC regulation of CFTR function. Our laboratory (17) found that inhibition of PKC with chelerythrine blocked efflux of 36Cl from Calu-3 cells stimulated by cAMP-generating agents and reduced cAMP-dependent CFTR activity, suggesting that constitutive PKC activity in unstimulated cells regulates maximal activation of CFTR. Findings similar to ours were also reported by Jia et al. (13) from patch-clamp studies of CHO and BHK cells that express wild-type CFTR and by Middleton and Harvey (23) from whole cell patch-clamp studies of guinea pig ventricular myocytes. Our laboratory extended its studies to identify a PKC isotype involved in this response by using an antisense approach and demonstrated that the antisense oligonucleotide to PKC-epsilon reduced PKC-epsilon mass and activity and prevented cAMP-dependent activation of CFTR (17).

PKC has, however, been found to have other functions in epithelial cells. In colonic cells, PKC modulates the level of CFTR mRNA expression (1, 14, 35) and NKCC1 mRNA expression (8). In T84 and HT-29 colonic epithelial cells, treatment with phorbol 12-myristate 13-acetate (PMA) for 12 h downregulated CFTR mRNA transcript numbers in a dose- and time-dependent manner. More importantly, PMA-treated cells were unable to respond to forskolin treatment with activation of Cl- secretion. PMA also downregulated CFTR in a human liver epithelial BC1 cell line, with a concomitant inhibition of stimulated Cl- efflux, and activated PKC-alpha and -epsilon as indicated by a cytosol-to-membrane translocation of both PKC isotypes (14). In T84 cells, NKCC1 mRNA and protein expression were reduced after 24 h of treatment with high concentrations of PMA (8). NKCC1 mRNA expression can also be regulated by hormonal stimuli. In vascular endothelial cells, NKCC1 mRNA expression was selectively regulated by inflammatory cytokines after 6 h of treatment and by fluid mechanical stimuli after 24 h of treatment (34). There is no evidence that PKC is necessary for these responses.

In past studies, our laboratory (16, 17) downregulated PKC isotypes using antisense oligonucleotides to identify a PKC isotype linked to regulation of each Cl- transporter. Because the turnover time for PKC is 24 h, the cells were treated for 48 h to downregulate the amount of PKC. However, this time frame is sufficient to downregulate CFTR if CFTR mRNA expression is also dependent on PKC-epsilon . Likewise, for NKCC1 mRNA expression, the effects of the antisense oligonucleotide to PKC-delta could be attributed to the downregulation of NKCC1 mRNA.

The purpose of the present study was twofold. First, we wanted to resolve the question of potential downstream effects of antisense oligonucleotides to PKC isotypes on NKCC1 and CFTR mRNA expression. Second, we wanted to analyze and characterize the function and regulation of the Cl- transporters that are required for Cl- secretion in a single cell line. We selected a Calu-3 epithelial cell line that has been shown to functionally express CFTR (17, 21, 24, 31). The Calu-3 cell line is a human lung carcinoma cell line that displays electrophysiological properties. Calu-3 cells express high levels of CFTR and respond to cAMP- and Ca2+-mediating agents with changes in net transepithelial ion transport, indicative of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Cl- secretion, respectively (7, 31). Reports (7, 32) of NKCC1 in Calu-3 cells are limited to activity, which is measured as bumetanide-sensitive short-circuit current (Isc). For the present studies, Calu-3 cells were treated with sense or antisense oligonucleotides to PKC-delta or -epsilon and analyzed for NKCC1 and CFTR mRNA expression by semiquantitative RT-PCR; for the expression of NKCC1, CFTR, and PKC isotypes by Western blot analysis; and for the function of NKCC1 and CFTR. We found that the antisense oligonucleotide to PKC-delta or -epsilon downregulated the respective PKC isotype but did not alter the expression of NKCC1 and CFTR mRNA or protein. Moreover, the antisense oligonucleotide to a specific PKC isotype selectively blocked hormone-stimulated NKCC1 or CFTR function.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture and oligonucleotide treatment. Calu-3 cells were purchased from the American Type Culture Collection and were grown on 60-mm plastic tissue culture dishes in Eagle's medium with Earle's balanced salt solution supplemented with 10% fetal bovine serum in a humidified CO2 incubator at 37°C. For treatment with PMA, cells were incubated for 48 h in serum-free medium in the absence of an oligonucleotide-LIPOFECTIN mixture; PMA was administered during the final 18-h period. Oligonucleotide treatments were performed as previously described (16-18). Briefly, oligonucleotide-LIPOFECTIN mixtures in serum-free medium were added to cell cultures every 12 h for 2 days. The antisense oligonucleotides were complementary to the translation initiation region of mRNA specific for mouse PKC-delta (AGGGTGCCATGATGGA) (27) and human PKC-epsilon (antisense 1, GGCTGGTACCATCACAAG; antisense 2, GAACACTACCATGGTCGG). Sense oligonucleotides to PKC-delta (TCGATCATGGCACCCT) and to PKC-epsilon (CCGACCATGGTAGTGTTC) were used as controls. Oligonucleotides were dissolved in sterile deionized water to a final concentration of 1 mM, divided into aliquots, and stored at -20°C until ready for use. The effect of oligonucleotide treatment on the amount of PKC-delta and -epsilon was analyzed by laser densitometry of Western blots of the PKC isotypes as previously described (16, 17). In an earlier study (16), our laboratory found that the antisense oligonucleotide to PKC-delta decreased the amount of PKC-delta in Calu-3 cells by 73.7% and that the antisense oligonucleotide to PKC-epsilon decreased the amount of PKC-epsilon by 76.1%. For the current study, we repeated these measurements and found that the antisense oligonucleotide to PKC-epsilon decreased PKC-epsilon mass to 28.7 ± 9% (P < 0.02; n = 3 cultures) of levels in untreated cells; this was a loss of 71.9% of PKC-epsilon . The amounts of PKC-delta and -zeta did not change. The antisense oligonucleotide to PKC-delta reduced the amount of PKC-delta to 28.5 ± 11% (P < 0.01; n = 3) of the PKC-delta mass in untreated cells but did not affect the amount of PKC-epsilon or -zeta . Sense oligonucleotides to PKC-delta and -epsilon did not significantly alter the expression of PKC-delta , -epsilon , and -zeta .

For measurement of electrolyte transport function, cells were passaged onto 4.52-cm2 filter inserts at a seeding density of 2.0 × 106 cells/filter, onto 0.6-cm2 Transwell Snapwell clear filter inserts at a seeding density of 0.5 × 106 cells/filter, or onto six-well tissue culture dishes. The culture medium was changed at 48-h intervals until confluence was reached. Confluence was assessed by microscopic examination of the cell monolayer and, for the cells grown on filter inserts, by measurement of the electrical resistance across the monolayer with chopstick electrodes and an epithelial voltohmmeter (EVOM; World Precision Instruments). Values were corrected for background resistance of the filter alone bathed in medium. Filters were used for experiments when the resistance exceeded 250 Omega /cm2 6-9 days after seeding. The resistance of untreated and oligonucleotide-treated cells was matched for resistance before use.

RNA isolation. Calu-3 cells grown to confluence (7-8 days after subculture) were washed three times with ice-cold PBS, harvested, and collected by centrifugation at 1,200 rpm at 4°C for 10 min. Total RNA was isolated by hot phenol extraction. The pelleted cells were resuspended in ice-cold 10 mM sodium acetate (pH 4.5) and 1% SDS and incubated in water-saturated phenol at 60°C for 5 min. Samples were centrifuged at 3,000 rpm at 4°C for 10 min, and phenol extractions were repeated on the supernatants. RNA was precipitated by the addition of ice-cold 3 M sodium acetate (pH 5.2) and 2 volumes of 100% ethanol and by incubation at -80°C for 30 min. The precipitated RNA was collected by centrifugation at 7,000 rpm at 4°C for 10 min, air-dried, and dissolved in 200 µl of diethyl pyrocarbonate (DEPC) water. The ethanol precipitation was repeated once. The final RNA pellet was dried under vacuum and then dissolved in 50 µl of DEPC water and stored at -80°C.

RT-PCR. RT-PCR was performed with subunit-specific primers based on sequences of NKCC1 (26) and CFTR. beta -Actin was amplified as a control (12). Primers for beta -actin were chosen from exons 3 and 5 of beta -actin. Amplification of cDNA done with these primers is predicted to produce a 228-bp fragment, whereas amplification from genomic DNA is predicted to produce a 435-bp fragment. Total RNA (1 µg) was reverse transcribed at 42°C for 50 min with Superscript II Moloney murine leukemia virus RT and random hexamer primer. For cDNA synthesis, 1 µg of RNA was mixed with 1 µl of random hexamer primer and DEPC-treated water to a final volume of 12 µl in a 0.5-ml Eppendorf tube, incubated at 70°C for 10 min, and then cooled at 4°C for 1 min. A first-strand buffer consisting of 250 mM Tris-Cl, pH 8.3, 375 mM KCl, 15 mM MgCl2, 10 mM dithiothreitol, and 0.5 mM deoxynucleotide triphosphate mix was added to the reaction mixtures and incubated at 42°C for 2 min. Two hundred units of RT were added to the reaction mixtures, and the reaction was continued for 50 min at 42°C. The RT was heat inactivated at 70°C for 15 min, followed by the addition of 1 µl of RNAse H for 20 min at 37°C. Reactions were run without RT as a control for endogenous RT or without RNA.

PCR was performed with the Elongase enzyme amplification kit in combination with four primer sets (Table 1). Reactions in a final volume of 50 µl consisted of 2 µl of cDNA, 0.2 µM forward and reverse primers, 0.2 mM each deoxynucleotide triphosphate, and 2 mM MgCl2. Single-strand cDNA was amplified with a PTC-100 thermal cycler from MJ Research (Watertown, MA). For NKCC1 and actin primers, PCRs were denatured at 94°C for 3 min followed by 30 cycles at 94°C for 1 min, 62°C for 1 min, and 72°C for 1 min and a final extension at 72°C for 7 min. For CFTR primers, reactions were denatured for 3 min followed by 30 cycles at 94°C for 1 min, 58°C for 1 min, 72°C for 1 min, and a final extension at 72°C for 7 min. For controls, DEPC water replaced the cDNA. PCR products were analyzed by gel electrophoresis on 1% agarose gels and were stained with SYBR Green I. 

                              
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Table 1.   PCR primers

cDNA sequencing. Sense and antisense strands of cloned NKCC1, CFTR, and actin cDNAs were sequenced with the dideoxynucleotide chain termination methods (30). Automated sequencing reactions were performed with synthetic oligonucleotide primers and fluorescent dideoxynucleotide terminators on a DNA sequencer (model 377, Applied Biosystems, Foster City, CA). Sequence data were analyzed with a BLAST (National Institutes of Health) sequence similarity database.

Fluorescent imaging. PCR gels were analyzed on a FluorImager SI in conjunction with ImageQuant analysis software (Molecular Dynamics, Sunnyvale, CA) for quantitative measurements. A 228-kb PCR-amplified fragment of the beta -actin gene was used to normalize for total RNA.

Western blot analysis of PKC isotypes and NKCC1. Cell monolayers grown to confluence on filter inserts were untransfected or transfected for 48 h with an oligonucleotide- LIPOFECTIN combination. The culture medium was removed, and the cell monolayers were immediately washed twice with ice-cold PBS, harvested in lysis buffer (16), and assayed for protein. For PKC isotypes, aliquots were solubilized in SDS-Laemmli buffer and subjected to 8% SDS-PAGE. Protein bands were transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA) and immunoblotted with polyclonal antibodies to specific PKC isotypes. Immunoreactive protein bands were detected with enhanced chemiluminescence and were quantitated by laser densitometry. For NKCC1, aliquots of the lysate were incubated with 60 mM Tris, 10.0% (vol/vol) glycerol, 3.5% (wt/vol) SDS and 5% (vol/vol) beta -mercaptoethanol at 37°C for 30 min. The aliquots were applied to 6% SDS-PAGE gels and transferred to Immobilon-P paper for immunoblot analysis with the T4 monoclonal antibody (26). Because the T4 monoclonal antibody was raised against a peptide epitope of T84 NKCC1, lysates of T84 cells were analyzed as a control. Immunoreactive protein bands were detected with enhanced chemiluminescence.

Electrolyte transport function. NKCC1 activity was measured as bumetanide-sensitive basolateral-to-apical flux of 86Rb, a congener of K, or as bumetanide-sensitive Isc (see Materials). Cell monolayers were preincubated for 10 min at 37°C with vehicle or with 50 µM bumetanide in the basolateral solution, which consisted of 10 mM HEPES (pH 7.5)-buffered Ringer solution (HBR) (15). Cells were permeabilized at the apical membrane with 175 U/ml of nystatin in an apical cytosolic medium containing (in mM) 110 KCl, 20 NaCl, 2.0 EGTA, 1.0 MgSO4 · 7H2O, and 10 HEPES, pH 7.5. To initiate transmonolayer flux, 1 µCi of 86Rb was added to the basolateral solution. The apical perfusion medium was collected for radioactive counts at 2.5-min intervals for 10 min and replaced with an equal volume of nystatin-supplemented cytosolic medium. At 10 min, methoxamine was added to the basolateral solution to a final concentration of 10 µM, and sampling was continued at 2.5-min intervals for 10 min. After the last sampling, cell monolayers were washed in 1% PBS and then extracted with 0.5 ml of 0.1 N NaOH. Aliquots of the cell extract were assayed for protein content. The accumulation of 86Rb in the apical compartment was calculated as nanomoles per milligram of protein over time.

The activity of CFTR was measured as the rate of 36Cl efflux as previously described (17). In brief, cell cultures were grown to confluence in six-well tissue culture dishes or on filter inserts and preincubated in serum-free medium for 24 h before use. Cells were preincubated for 1 h at 35°C with 3.5 µCi of 36Cl in HBR. The media with radioactive tracer were removed, and cells were washed four times with HBR to remove extracellular 36Cl. After the last wash, sequential 0.5-ml aliquots of isotope-free HBR were added and removed every 60 s for up to 11 min. The first three aliquots were used to establish a stable baseline in efflux buffer only. Agonists were added after the third aliquot was removed. Inhibitors were present in the bathing medium for the last 30 min of the 36Cl loading period and during the efflux period. Radioactive counts that remained in the cells were extracted with 0.1 N NaOH. The fraction of intracellular 36Cl remaining in the cell layer during each time point was calculated from the sample and extract counts. Time-dependent rates of 36Cl efflux were calculated as ln(36Clt=1/36Clt=2)/(t1-t2), where 36Cl is the percent intracellular Cl- at time t, and t1 and t2 are successive time points.

For electrophysiological studies, when cells grown on Transwell Snapwell clear filter inserts attained a conductance of <6 mS/cm2, cultures were inserted into an Ussing chamber (World Precision Instruments) and perfused on the apical and basolateral surfaces with Hanks' balanced salt solution (HBSS) at ambient room temperature (5, 20). The apical perfusion solution was changed to a nominal Cl--free medium that consisted of (in mM) 136.9 sodium gluconate, 5.4 potassium gluconate, 4.2 NaHCO3, 1.3 calcium gluconate, 0.4 KH2PO4, 0.9 MgSO4 · 7H2O, 0.3 Na2HPO4 · 7H2O, 10 HEPES, and 10 glucose. The pH was adjusted with NaOH to pH 7.4, and the solution was gassed with air. The serosal surface was perfused with HBSS in all subsequent experiments. Electrical measurements were made with conventional four-electrode circuits and were performed as previously described (5, 20). Cell monolayers were continuously short circuited with automatic voltage clamps (Iowa Bioengineering) except at 20-s intervals when a 2-mV bipolar pulse was imposed. Resistance was calculated by Ohm's law.

Materials. 86Rb (specific activity 154 Bq/g Rb, 4,200 Ci/g Rb) and an enhanced chemiluminescence kit were purchased from Amersham Life Sciences (Arlington Heights, IL). 36Cl (specific activity 260 MBq/g Cl-, 7.5 mCi/g Cl-) was purchased from ICN Radiochemical (Irvine, CA). Polyclonal anti-PKC isotype-specific antibodies and recombinant PKC isotypes were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and the T4 monoclonal antibody was from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, Iowa). Methoxamine HCl was supplied by Burroughs Wellcome (Research Triangle Park, NC). Chelerythrine chloride and PMA were purchased from Research Biochemicals International (Natick, MA), and bumetanide and nystatin were from Sigma (St. Louis, MO). LIPOFECTIN reagent, the Elongase enzyme amplification kit, Superscript II Moloney murine leukemia virus RT, RNAse H, PCR primers, custom sense and antisense oligonucleotides, and tissue culture supplies were purchased from GIBCO BRL (Life Technologies, Gaithersburg, MD). All other chemicals were of reagent grade.

Data analysis. Data are means ± SE for values obtained from four separate cell cultures. To determine the level of significance, Student's t-test was performed.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of NKCC1 and CFTR mRNA. CFTR was detected with primers that encode a 553-bp cDNA fragment starting at nucleotide 2131 and encompassing the regulatory (R) domain. PCR primers for NKCC1 were designed from transmembrane-spanning domains in human colonic basolateral NKCC1 (26). Amplification of cDNA reverse transcribed from the total RNA of Calu-3 cells with primers to NKCC1 and CFTR produced DNA fragments of the correct size (Fig. 1). beta -Actin was selected as a control to confirm that cDNA had been synthesized and to check for genomic DNA contamination. As shown in Fig. 1, RT-PCR with beta -actin primers yielded a 228-bp fragment, indicating the synthesis of cDNA and the absence of genomic DNA. The cDNA sequence of the PCR products was >99% homologous to the expected cDNA sequence of the corresponding protein (data not shown). The results were consistent with a report on CFTR and beta -actin (12) but represent the first report of human respiratory epithelial NKCC1, which apparently shares a high degree of homology in the transmembrane domains with other mammalian NKCC isoforms (25).


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Fig. 1.   SYBR green-stained RT-PCR products from RNA in Calu-3 cells demonstrating expression of Na/K/2Cl (NKCC1) and cystic fibrosis transmembrane conductance regulator (CFTR) mRNA relative to beta -actin. Cells were untreated (lane 1), treated with 10 nM phorbol 12-myristate 13-acetate (PMA) for 18 h (lane 2), or treated with 10 µg/ml of LIPOFECTIN for 48 h (lane 3). Oligonucleotide-treated cells were incubated with 10 µg/ml of LIPOFECTIN plus 1 µM sense oligonucleotide to protein kinase C (PKC)-delta (lane 4), 1 µM antisense oligonucleotide to PKC-delta (lane 5), 1 µM sense oligonucleotide to PKC-epsilon (lane 6), or 1 µM antisense oligonucleotide to PKC-epsilon (lane 7) for 48 h in serum-free cell culture medium. PCR products were detected by 1% agarose gel electrophoresis as a single band of expected size. Results are representative of 4 separate experiments.

To verify the sensitivity of the RT-PCR methodology, we treated cells with 10 nM PMA for 18 h. This treatment with PMA has been shown to downregulate CFTR mRNA expression (1, 35) but not beta -actin mRNA expression (35). Similar results were obtained in Calu-3 cells (Fig. 1, lanes 1 and 2). Arbitrary fluorescence ratios of CFTR mRNA relative to beta -actin were reduced from 0.50 to 0.23 (P < 0.05) after treatment with PMA (Table 2). This effect was specific for CFTR because the levels of NKCC1 mRNA expression were not decreased by PMA (Fig. 1, lanes 1 and 2; Table 2). These results are consistent with the reported effects of PMA on actin (35) and CFTR (1, 35) mRNA expression and, moreover, constitute the first direct observation of NKCC1 mRNA expression and its independence from PMA in Calu-3 cells.

                              
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Table 2.   Effect of long-term PMA treatment on relative mRNA expression

Effect of oligonucleotides on mRNA expression. To assess whether treatment with antisense or sense oligonucleotides altered mRNA expression of Cl- transporters, we semiquantitated the expression of mRNA for NKCC1 and CFTR relative to beta -actin in cells that were transfected with LIPOFECTIN alone or in combination with oligonucleotides. The results are presented in Fig. 2. Expression of mRNA for NKCC1, CFTR, or beta -actin was not affected by LIPOFECTIN (Fig. 1, lane 3). When expressed relative to beta -actin, mRNA expression of CFTR was 0.56 ± 0.13 (n = 4 cultures) and of NKCC1 was 0.44 ± 0.09 (n = 4 cultures). After treatment with LIPOFECTIN in combination with sense or antisense oligonucleotide to PKC-epsilon or PKC-delta , mRNA expression of NKCC1 varied over a range from 0.69 ± 0.04 to 0.73 ± 0.08 (n = 4 cultures). mRNA expression of CFTR varied from 0.41 ± 0.06 to 0.43 ± 0.08 (n = 4 cultures). None of the treatments significantly altered mRNA expression of NKCC1 and CFTR.


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Fig. 2.   Effect of oligonucleotides to PKC-delta and -epsilon on NKCC1 and CFTR mRNA expression. Calu-3 cells were incubated with LIPOFECTIN (LPF) alone or with LPF in combination with sense or antisense oligonucleotide to PKC-delta or PKC-epsilon . Total RNA was isolated, reverse transcribed, and analyzed by fluorescent imaging as described in METHODS. Arbitrary fluorescent values for NKCC1 and CFTR PCR products were divided by the value for beta -actin. Data are means ± SE for 4 separate cell cultures. There was no significant difference in mRNA expression of NKCC1 and CFTR among the treatment regimens.

NKCC1 protein expression and function. Reports (7, 24, 31) of Cl- secretion in Calu-3 cells have provided variable insight into the source of Cl- for secretion. NKCC1 is a highly regulated Cl- transporter in airway epithelial cells (15, 16, 18) and might serve as a major source of Cl- for secretion in Calu-3 cells. The goal of these experiments was to demonstrate the protein expression and function of NKCC1 in Calu-3 cells. Western blot analysis of cell lysates with T4 monoclonal antibody raised against human colonic NKCC1 (22) revealed immunoreactivity to two protein bands that were also detected in the lysates of T84 cells (Fig. 3A). Both cell types expressed a lower molecular mass band that might be indicative of incomplete glycosylation of the expressed protein (26). The higher molecular mass band corresponded to a molecular mass of ~170 kDa and is thought to represent a fully glycosylated cotransporter delivered to the plasma membrane (26). Treatment with sense or antisense oligonucleotide to PKC-delta or -epsilon did not affect the amount of NKCC1 (Fig. 3B) or the amount of CFTR expressed in Calu-3 cells (data not shown).


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Fig. 3.   Western blot analysis of NKCC1 expression in Calu-3 cells. Aliquots of Calu-3 cell lysates were separated on 6% SDS-PAGE gels and probed with T4 monoclonal antibody at 1:500 dilution as described in METHODS. A: expression of NKCC1 in Calu-3 cells. Lysate protein (15 µg) from Calu-3 and T84 cells was analyzed. NKCC1 was detected as a doublet, with a major band at ~170 kDa. B: effect of oligonucleotides on NKCC1 expression. Calu-3 cells were untransfected or transfected with oligonucleotide for 48 h. Lysate protein (20 µg) was applied to 6% SDS-PAGE gels for analysis. Lane 1, untransfected cells; lane 2, sense oligonucleotide to PKC-delta ; lane 3, antisense oligonucleotide to PKC-delta ; lane 4, sense oligonucleotide to PKC-epsilon ; lane 5, antisense oligonucleotide to PKC-epsilon ; lane 6, T84 cell lysate. T84 lysate (40 µg) served as a positive control for the T4 antibody. Nos. at left, molecular mass markers in kDa. Blots are representative of 3 separate experiments.

Functional activity of NKCC1 was measured as bumetanide-sensitive basolateral-to-apical 86Rb flux in cell monolayers grown on Costar Transwell filter inserts and treated on the apical surface with the pore-forming antibiotic nystatin (16). Nystatin permeabilizes the apical membrane and thus isolates NKCC1 activity and its regulation by intracellular effector enzymes from CFTR at the apical plasma membrane. In untransfected cells, stimulation with methoxamine, an alpha 1-adrenergic agonist, increased bumetanide-sensitive NKCC1 activity 2.9-fold (Table 3). Treatment with the antisense oligonucleotide to PKC-delta , but not to PKC-epsilon , prevented alpha 1-adrenergic stimulation of NKCC1 (Table 3). As a control for the antisense sequence, the sense oligonucleotide to PKC-delta was also tested but was found to have no effect on NKCC1 activation by methoxamine. Thus NKCC1 expressed in Calu-3 cells shares with human tracheal epithelial cells and CF/T43 cells (16, 18) a requirement for PKC-delta for hormonal activation.

                              
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Table 3.   Antisense oligonucleotides to PKC isotypes modulate distinct epithelial electrolyte transporters

CFTR function. CFTR function was assessed as the efflux of 36Cl or by the traditional electrophysiology of cell monolayers grown on a filter insert and mounted in a modified Ussing chamber. The data in Table 3 show that the antisense oligonucleotide to PKC-delta did not affect cAMP-dependent 36Cl efflux. However, CFTR function remained sensitive to the antisense oligonucleotide to PKC-epsilon (Table 3). Two antisense oligonucleotides markedly attenuated the cAMP-dependent efflux of 36Cl, with a similar reduction of 86% compared with cells transfected with the sense oligonucleotide.

Untransfected cell monolayers displayed a basal Isc of 2.2 ± 0.5 µA/cm2 (range 0.6-3.7; n = 8 cultures) and a transmonolayer resistance of 290.0 ± 15.4 Omega  · cm2 (range 168-468). Basal Isc was not significantly altered by transfection with sense or antisense oligonucleotides (Table 4). Switching the apical perfusion medium to a Cl--free medium increased Isc by 21.5 ± 2.4 µA/cm2 (n = 10 cultures) in untransfected cell monolayers, by 16.8 ± 2.2 µA/cm2 (n = 6 cultures) in cell monolayers transfected with sense oligonucleotide to PKC-epsilon , and by 18.1 ± 1.8 µA/cm2 (n = 5 cultures) in cell monolayers transfected with antisense oligonucleotide to PKC-epsilon . There was no significant difference among these responses. The antisense oligonucleotide to PKC-epsilon significantly reduced the response to basolateral application of L-isoproterenol from 21.3 to 12.6 µA/cm2 (P < 0.02; Table 4). Bumetanide inhibited 53.9% of the L-isoproterenol-stimulated Isc in untransfected cells and 45.3% in antisense oligonucleotide-transfected cells.

                              
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Table 4.   Effect of oligonucleotides on L-isoproterenol-stimulated Isc in Calu-3 cells


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The differential effects of antisense oligonucleotide to PKC-delta and PKC-epsilon on NKCC1 and CFTR in Calu-3 cells, respectively, demonstrate that downregulation of specific PKC isotypes blocks the function of distinct Cl- transport proteins. This study of Cl- transport proteins and their function in a single airway epithelial cell line establishes that the site of action of PKC isotypes is independent of mRNA expression (Fig. 1; Table 2) and NKCC1 (Fig. 3B) and CFTR protein expression. The results suggest that steps in an intracellular signaling mechanism proximal to each Cl- transporter are involved. One likely site is posttranslational modification by phosphorylation. NKCC1 and CFTR each have multiple consensus sites for PKC phosphorylation (6, 9). The deduced primary structure of mammalian NKCC1 displays one consensus site for PKA phosphorylation (26) and at least eight consensus sites for PKC phosphorylation (6, 26, 37). In addition, human colonic NKCC1 contains two threonines, Thr217 and Thr1135, which correspond to known phosphoacceptors in shark rectal gland NKCC1 (26). CFTR has 29 consensus sites for PKC phosphorylation; only 7 are located in the R domain (9). The role of PKC phosphorylation and specific sites of phosphorylation in the CFTR and NKCC1 function are not yet clear. PKC phosphorylates the R domain at CFTR in vitro, predominantly at Ser686, Ser700, and Ser790, and in vivo at sites Ser686, Ser737, Ser786, and Ser795 (28, 38). However, phosphorylation of these sites by PKC might not adequately explain the modulatory role of PKC. Rather, constitutive phosphorylation of CFTR might be a prerequisite for subsequent phosphorylation by PKA. Indeed, another possibility is that PKC isotype-dependent regulation might be localized to a multiprotein complex that interacts with and modulates the function of CFTR or NKCC1. Intracellular Cl- concentrations have also been proposed to regulate the activity of NKCC1 (4, 29) or to coordinate the activity of CFTR and NKCC1 (11). PKC could link regulatory proteins and intracellular Cl- in an isotype-specific manner and thus explain an apparent Cl--dependent NKCC1 activation (6).

PKC has been implicated in the regulation of expression of CFTR and NKCC1 in other cell lines. One study (36) of native mouse colonocytes highlighted a role for PKC-beta , a Ca2+-dependent PKC isotype, in CFTR expression. Increases in PKC-beta abundance and nuclear localization during proliferation apparently parallel alterations in CFTR expression at the mRNA and protein levels. Our study here provides the first evidence for the independence of NKCC1 mRNA expression from PMA and from the downregulation of PKC-delta and PKC-epsilon (Fig. 1; Table 2). Thus, in Calu-3 cells, these two PKC isotypes do not regulate the transcription of NKCC1 DNA. Nevertheless, our results with PMA differ markedly from a recent report (8) on the effect of PMA on NKCC1 mRNA and protein expression in colonic epithelial T84 cells. There are several key differences in the two studies that might account for the different results. First, the T84 cells were treated with a 10-fold higher concentration of PMA than was used with the Calu-3 cells (100 vs. 10 nM). Second, in T84 cells, 24 h of treatment with PMA downregulated NKCC1 mRNA and protein. However, after extended treatment for 72 h, NKCC1 mRNA and protein levels were comparable to those in untreated control cells, indicating recovery of NKCC1 mass. With Calu-3 cells, PMA treatment for 18 h did not affect NKCC1 mRNA expression (Figs. 1 and 2). The concentration of PMA used in our study was sufficient to induce a rapid increase in NKCC1 function (15) but did not downregulate PKC mass or activity in native tracheal and cystic fibrosis airway epithelial cells (18, 19). Third, long-term treatment with PMA caused a disassembly of T84 cells that could have adversely affected the expression and retention of NKCC1 mRNA and protein (8). We found that the viability and morphology of Calu-3 cells were not affected by PMA treatment for 18 h (data not shown). Thus the independence of Calu-3 NKCC1 mRNA expression from PMA may be a cell type-specific response that differentiates airway epithelial cells from colonic epithelial cells. This suggests that PMA could produce its effects in a PKC-dependent or PKC-independent manner depending on the time of exposure.

The detection of NKCC1 mRNA and protein in Calu-3 cells is the first demonstration at the molecular level of a Na/K/2Cl cotransporter that accounts for bumetanide-sensitive Isc in unstimulated Calu-3 cells and in cells treated with agents that induce Cl- secretion (7, 31, 32). Models of basal Isc in Calu-3 cells stressed an important role of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Cl- exchange in concentrating Cl- above the equilibrium potential for Cl-. However, another study (7) showed that, in stimulated cells, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Cl- exchange played only a minor role. Instead, bumetanide-sensitive Isc preferentially increased with thapsigargin, a Ca2+-mobilizing agent, cAMP-generating agents, or the K+-channel activator 1-ethyl-2-benzimidazolinone (1-EBIO). The study reported here provides the first direct evidence for the expression and function of NKCC1 in Calu-3 cells. In addition, several features of NKCC1 function emerge from our studies. First, basolateral-to-apical 86Rb flux is stimulated by the alpha 1-adrenergic agonist and is inhibited by bumetanide when these agents are applied to the basolateral perfusion solution (Table 3). These findings support the designation of NKCC1, a basolateral isoform of Na/K/2Cl cotransport (6), as the bumetanide-sensitive transporter in Calu-3 cells. Second, an increase in 86Rb flux was detected after basolateral application of an alpha 1-adrenergic agent, methoxamine, which has been shown to selectively activate NKCC1 in native tracheal epithelial cells (15, 16). The Calu-3 cell line is a cancer cell line that has been functionally classified as tracheal serous gland cells. Tracheal serous gland cells also express numerous alpha 1-adrenergic receptors in situ (2). The stimulatory effect of the alpha 1-adrenergic agonist methoxamine in Calu-3 cells indicates that NKCC1 expressed in Calu-3 cells and in native tracheal epithelial cells share similar hormonal regulation. Third, the antisense oligonucleotide to PKC-delta , but not to PKC-epsilon , abolished alpha 1-adrenergic stimulation of NKCC1, indicating that PKC-delta is a key effector enzyme in the activation of NKCC1 (Table 3). This result provides additional evidence to support the conclusion that the NKCC1 expressed in Calu-3 cells functionally resembles the NKCC1 expressed in non-cystic fibrosis (CF) airway epithelial cells (17) and in CF/T43 cells (18).

The NKCC1 activity that we detected as bumetanide-sensitive Isc was observed in Calu-3 cell monolayers that were treated with the beta -adrenergic agonist L-isoproterenol, a cAMP-generating agent (Table 4). We used L-isoproterenol instead of forskolin, a cAMP-generating agent that bypasses beta -adrenergic receptors, because of conflicting reports on the ability of forskolin to increase Cl- secretion and bumetanide-sensitive Isc in Calu-3 cells (7, 24). The baseline electrophysiological properties of Calu-3 cell monolayers that we used for these studies resembles the Isc (2.2 vs. 13 µA/cm2) and transepithelial resistance (290 vs. 353 Omega  · cm2) of cells used in a study of 1-EBIO, a drug that increases Isc by bypassing hormone receptors (7). In that study, 1-EBIO stimulated a basolateral Ca2+-activated K conductance and activated an apical membrane Cl- channel that was thought to be CFTR (7, 10). Bumetanide blocked ~50% of the response to 1-EBIO (7), a value similar to the 53.9% bumetanide-sensitive Isc observed in the L-isoproterenol-stimulated cells used in our study. The antisense oligonucleotide to PKC-epsilon blocked the response to L-isoproterenol, indicating loss of cAMP-dependent CFTR function (Table 4). Transfection with the antisense oligonucleotide to PKC-epsilon did not affect NKCC1 activity, which was measured in cells grown on filter inserts and quantitated as radiolabeled flux (Table 3) or as bumetanide-sensitive Isc (Table 4). These results demonstrate that NKCC1 activity in Calu-3 cells is detected with selected stimulatory agents. Our study does not explain the results with forskolin stimulation that have been obtained by different laboratories. One possibility is that the Calu-3 cell line is heterogeneous with respect to cell population and that cell culture conditions are selective for cell types that are responsive to specific hormones or drugs.

In summary, this study demonstrates the independence of CFTR and mRNA expression from PKC-delta and PKC-epsilon and provides evidence for PKC isotype-specific regulation of CFTR and NKCC1 in Calu-3 cells. The cell line, therefore, is a reasonable model for native airway epithelial cells and will prove useful in further studies to determine an intracellular signaling mechanism that explains how NKCC1 cotransport is regulated to account for its increased activity during Cl- secretion and how constitutive activity of PKC-epsilon regulates CFTR. Overall, the studies from this laboratory indicate that PKC isotypes differentially target unique electrolyte transporters localized to opposing plasma membranes of epithelial cells in an isotype-specific manner.


    ACKNOWLEDGEMENTS

We thank Dr. D. Kube for assistance in designing the primers to the cystic fibrosis transmembrane conductance regulator and Anthony Skalak and Robert Moore for technical assistance.


    FOOTNOTES

The research was supported by National Heart, Lung, and Blood Institute Grants HL-50160 and HL-58598 and Cystic Fibrosis Foundation Grant LIEDTK98G0.

Address for reprint requests and other correspondence: C. M. Liedtke, Pediatric Pulmonology, Case Western Reserve Univ., BRB, Rm. 824, 2109 Adelbert Rd., Cleveland, OH 44106-4948 (E-mail: cxl7{at}po.cwru.edu).

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.

Received 16 June 2000; accepted in final form 30 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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