The Cystic Fibrosis Center, Departments of Pediatrics and Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4948
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ABSTRACT |
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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-
)] or of the
Na/K/2Cl (NKCC1) cotransporter (PKC-
). 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-
decreased the
amount of PKC-
and reduced cAMP-dependent activation of CFTR but not
1-adrenergic activation of NKCC1. The antisense
oligonucleotide to PKC-
did not affect CFTR function but did block
1-adrenergic activation of NKCC1 and reduce PKC-
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; -adrenergic activation; methoxamine; phorbol ester; cystic fibrosis transmembrane conductance regulator; protein kinase C
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INTRODUCTION |
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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-
as the PKC isotype that is required for activation by
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-
reduced PKC-
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-
and -
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-
. Likewise, for NKCC1 mRNA expression, the effects of the antisense oligonucleotide to PKC-
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
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-
or
-
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-
or -
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.
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METHODS |
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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-
(AGGGTGCCATGATGGA) (27) and human PKC-
(antisense
1, GGCTGGTACCATCACAAG; antisense 2, GAACACTACCATGGTCGG). Sense oligonucleotides to PKC-
(TCGATCATGGCACCCT) and to PKC-
(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-
and -
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-
decreased the
amount of PKC-
in Calu-3 cells by 73.7% and that the antisense
oligonucleotide to PKC-
decreased the amount of PKC-
by 76.1%.
For the current study, we repeated these measurements and found that
the antisense oligonucleotide to PKC-
decreased PKC-
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-
. The amounts of PKC-
and -
did not change. The antisense
oligonucleotide to PKC-
reduced the amount of PKC-
to 28.5 ± 11% (P < 0.01; n = 3) of the
PKC-
mass in untreated cells but did not affect the amount of
PKC-
or -
. Sense oligonucleotides to PKC-
and -
did not
significantly alter the expression of PKC-
, -
, and -
.
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. -Actin was amplified as a control (12). Primers for
-actin were chosen from exons
3 and 5 of
-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.
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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
-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)
-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)/(t1Materials.
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.
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RESULTS |
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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). -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
-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
-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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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
-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
-actin was not affected by LIPOFECTIN (Fig. 1, lane 3).
When expressed relative to
-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-
or PKC-
, 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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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-
or -
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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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- did not affect cAMP-dependent 36Cl efflux. However, CFTR function remained
sensitive to the antisense oligonucleotide to PKC-
(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.
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DISCUSSION |
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The differential effects of antisense oligonucleotide to PKC-
and PKC-
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-, a
Ca2+-dependent PKC isotype, in CFTR expression. Increases
in PKC-
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-
and PKC-
(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
exchange in concentrating
Cl
above the equilibrium potential for
Cl
. However, another study (7)
showed that, in stimulated cells, HCO
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
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
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
1-adrenergic receptors in situ (2). The
stimulatory effect of the
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-
, but not to
PKC-
, abolished
1-adrenergic stimulation of NKCC1,
indicating that PKC-
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 -adrenergic agonist
L-isoproterenol, a cAMP-generating agent (Table 4). We used
L-isoproterenol instead of forskolin, a cAMP-generating agent that bypasses
-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
· 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-
blocked the response to
L-isoproterenol, indicating loss of cAMP-dependent CFTR
function (Table 4). Transfection with the antisense oligonucleotide to
PKC-
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- and PKC-
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-
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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