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INTRODUCTION |
The cystic fibrosis transmembrane conductance regulator
(CFTR)1 is a member of the
ATP binding cassette family and functions as a cAMP-activated chloride
channel that is polarized to the apical plasma membrane in a number of
epithelial cells such as those of the airways, intestine, and kidney
(1, 2). CFTR apical localization is critical for the vectorial
transport of chloride and is crucial for the normal functioning of
epithelial cells (3). Mutation of CFTR (
F508) prevents the export of CFTR from the endoplasmic reticulum to the apical plasma membrane and
thus causes cystic fibrosis (CF) disease (4). The COOH terminus of CFTR
interacts with PDZ domain-containing proteins, such as EBP50 or E3KARP
(5, 6), and this interaction plays a key role in the polarization of
CFTR to the apical membrane (7, 8). The PDZ domains, which are named
for the three proteins in which these domains were originally
characterized (namely, PSD-95, Dlg, ZO-1), play an essential role in
determining the polarity of several proteins (7-9). EBP50 contains two
PDZ domains that mediate the assembly of transmembrane and cytosolic
proteins into functional signal transduction complexes. It has been
shown that the EBP50 PDZ1 domain interacts specifically with C terminus motifs present at the carboxyl terminus of the beta (2) adrenergic receptor (
2AR), the platelet-derived growth factor
receptor (PDGFR), and the CFTR, and plays a central role in the
physiological regulation of these proteins (7, 10-12). CFTR and
2AR bind equally well to the PDZ1 domain of NHERF1 (10).
The PDZ domains form a complex that plays a major role in stabilizing
CFTR at the apical membrane region of airway epithelial cells (6) and
may be involved in the regulation of membrane trafficking events. The
sorting of
2AR between recycling endosomes and lysosomal
compartments is controlled by their association with EBP50 (13, 14).
Moreover,
2ARs are linked to the actin cytoskeleton via
EBP50-ezrin interactions, since ezrin contains actin-binding sites at
its C-terminal domain. It has been recently shown that PDZ proteins
regulate the endocytic re-cycling of CFTR in polarized Madin-Darby
canine kidney cells (15). This suggests that
2AR-EBP50-ezrin and -actin interactions may enhance the
maturation of CFTR and its expression at the apical membrane of
epithelial cells. The
2AR belongs to the class of seven-transmembrane domain receptors for hormones and
neurotransmitters, the stimulation of which leads to alterations in the
metabolism, excitability, differentiation, and growth of many cell
types. Signal transduction by
2AR occurs upon ligand
binding via the activation of adenylate cyclase, which increases the
concentration of intracellular cAMP and produces a variety of
cell-specific physiological responses. In epithelial cells, activation
of PKA by cAMP elevation, which in turn activates
CFTR-dependent chloride transport, is among the myriad of
cell processes that are regulated by cAMP signals. It has been reported
that, in addition to the activation of CFTR channel gating, cAMP
increases CFTR apical membrane expression by inducing the apical
membrane insertion of the protein in CFTR-transfected Madin-Darby
canine kidney cells. However, this effect was shown to be dependent
upon the level of protein expressed (16). In Calu3 cells, a human
secretory airway epithelial cell line, cAMP, induces the stimulation of CFTR channels present in the apical plasma membrane but not the recruitment of CFTR from an intracellular pool to the apical plasma membrane (17).
Although CFTR-PDZ interactions have been analyzed in several epithelial
cell types, CFTR-PDZ interactions and the effect of agonists of
2AR on CFTR apical expression in human well
differentiated primary cultures of surface epithelial cells remains
unknown. In the present study, cultures of primary human airway surface epithelial cells were used to analyze whether a long term
2AR agonist may modulate the expression of membrane CFTR
via the cAMP/PKA pathway. We report that exposure of primary airway
epithelial cells to
2AR agonist induces a
post-transcriptional, time-dependent increase in CFTR
expression. This effect is mediated by
2AR but does not
involve the cAMP/PKA pathway.
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EXPERIMENTAL PROCEDURES |
Materials--
The primary antibodies used for both
immunohistochemistry and Western blots were as follows: 24-1 mouse
monoclonal anti-human CFTR (R&D Systems), rabbit polyclonal
anti-human
2AR and goat polyclonal anti-human ezrin
(Santa Cruz Biotechnology, Inc., Santa Cruz), mouse monoclonal
anti-human EBP50 (Transduction Laboratories), and mouse monoclonal
anti-human occludin (Zymed Laboratories Inc., San
Francisco). The appropriate biotinylated secondary antibodies were all
purchased from Amersham Biosciences.
Salmeterol was provided by GlaxoSmithKline. Rolipram
(Rp)-adenosine 3',5'-cyclic monophosphate triethylamine and
8-bromoadenosine-3',5'-cyclic monophosphate were purchased from Sigma.
ICI 118,551 hydrochloride was purchased from Fisher.
HAEC Isolation and Culture--
Nasal polyps were collected from
non-cystic fibrosis patients and immediately immersed in Hanks'-Hepes
culture medium containing 200 units/ml penicillin, 200 µg/ml
streptomycin. After repeated washings with cold Hanks'-Hepes medium,
the polyps were digested with 0.1% Pronase (Sigma) at 4 °C
overnight under continuous rotation. The cell suspension obtained after
digestion was centrifuged, and the pellet was shaken in 20 ml of fresh
Hanks'-Hepes medium. The cell suspension obtained was then
centrifuged, and the pellet was resuspended in RPMI 1640 (Invitrogen)
containing insulin (1 µg/ml; Sigma), apo-transferrin (1 µg/ml;
Serva, Heidelberg, Germany), epidermal growth factor (10 ng/ml; Serva),
retinoic acid (10 ng/ml; Sigma), hydroxycortisone (0.5 µg/ml; Sigma),
amphotericin B (2.5 µg/ml; Sigma), streptomycin (100g/ml), and
penicillin (100 units/ml). The cells were plated on 5-mm-thick collagen
type I gels in 12-well plastic dishes and incubated at 37 °C, 5%
CO2. The culture medium was changed daily.
Transmission Electron Microscopy--
Confluent monolayers of
HAEC were grown on Transwells and fixed in 2.5% glutaraldehyde in
phosphate-buffer saline (0.1 M), pH 7.4, for 1-2 h at room
temperature. Cells were then washed 3 times with PBS and post-fixed for
1 h in OsO4 (2% in distilled water) at pH 7.2-7.4 before being
dehydrated through a graded series of ethanol.
The specimens were then embedded in Epon and cut with an
ultramicrotome, and ultra-thin sections (0.08 µm) were mounted on copper grids, contrasted with uranyl acetate and lead citrate, and
examined with a transmission electron microscope (Hitachi 300, Elexience, Verrieres-leBuisson, France) at 75 kV.
Transepithelial Resistance Measurements--
The degree of
tightness of the cells in culture was evaluated by measuring the
transepithelial electrical resistance (TER) of HAEC grown on collagen
I-coated filters (0.4 µm). TER was measured every 2 days using an ERS
electrical resistance system (Millipore). After a 1-h equilibration of
sterile electrodes in culture medium, resistance values were measured
and normalized by subtracting the contribution of the collagen-coated
filter and bathing solution. To obtain TER values that were independent
of the area of membrane used, we calculated the product of the measured
resistance and the area of effective membrane on the Transwells. The
value obtained was expressed in ohm·cm2.
Immunofluorescence Microscopy--
Expression and localization
of
2AR, CFTR, and CFTR-associated proteins (EBP50 and
ezrin) were assessed by immunohistochemistry. Cells were seeded onto
12-well plates, which were previously covered by 5-mm-thick collagen
type I gels, at 50% confluence. Five to 6 days later confluent cells
and their gels were embedded in optimum cutting temperature compound
(Tissue Tek), cryofixed in liquid nitrogen, and stored at
80 °C. Transverse frozen sections (5-µm thick) were placed on
gelatin-coated glass slides and fixed in cooled methanol (
20 °C)
for 10 min. After washing 2 times with PBS, cells were incubated in PBS
containing 1% bovine serum albumin to block nonspecific sites. Primary
antibodies (dilution, 1:40 each in PBS) were added to the cells for
1 h at room temperature. Cells were washed 3 times with PBS, 1%
bovine serum albumin and incubated with the biotinylated secondary
antibody for 1 h at room temperature. After washing the cells 3 times in PBS, streptavidin-coupled fluorescein isothiocyanate (1:50 in
PBS) was added. Nuclei were counterstained with Harris hematoxylin
solution (Sigma), mounted in Citifluor antifading solution (Agar
Scientific), and observed with an Axiophot microscope (Zeiss, Le Pecq,
France) at a magnification of ×40.
Quantitation of CFTR Transcripts--
HAEC total RNA was
extracted using a High Pure RNA Isolation kit (Roche Applied
Science). RT-PCR was performed with 10 ng of total RNA by using
the GeneAmp Thermostable RNA PCR kit (PerkinElmer Life Sciences) with
pairs of primers for CFTR and for 28 S control amplification
(Eurogentec, Seraing, Belgium). Forward and reverse primers for human
CFTR and 28 S were designed as follows: CFTR primers (forward
5'-GCTTCCTATGACCCGGATAACAAG-3'; reverse 5'-GTGCCAATGCAAGTCCTTCATCAA-3') and 28 S primers (forward 5'-GTTCACCCACTAATAGGGAACGTGA-3'; reverse 5'-GGATTCTGACTTAGAGGCGT TCAGT-3'). Reverse transcription was
performed at 70 °C for 15 min followed by a 2-min incubation at
95 °C to melt RNA-DNA heteroduplexes. For PCR amplification, we used
27 cycles for CFTR and 17 cycles for 28 S; each cycle consisted of 15 s at 94 °C, 20 s at 68 °C, 10 s at
72 °C.
To quantify CFTR mRNA levels, a synthetic RNA standard
(pCTR) was introduced to each sample before PCR. To produce this
standard, a PCR fragment corresponding to the CFTR RT-PCR amplification products with a 16-bp deletion was generated by PCR and cloned in the
pCR®4-TOPO®-cloning (Invitrogen). A sequence-verified construct was
then linearized, purified, and used as the template for RNA synthesis
(SP6/T7 transcription kit, Roche Applied Science). RT-PCR amplification
of this synthetic RNA with the pairs of primers designed for CFTR
RT-PCR generated amplification products shorter than those obtained
from the cellular RNA, enabling them to be discriminated by electrophoresis.
RT-PCR products were separated by electrophoresis, stained with
SYBR gold (Molecular Probes), and quantified by fluorometric scanning (LAS-1000, Fuji). The products from pCTR endogenous CFTR and
endogenous 28 S were 260, 276, and 212 bp, respectively. Results are
expressed as a ratio of endogenous CFTR mRNA to its specific internal control pCTR after normalization with 28 S mRNA level.
Western Blot Analyses--
Whole HAEC proteins were extracted in
Tris buffer (50 mM Tris-HCl, pH 7.5) containing 1 mM phenylmethylsulfonyl fluoride and precipitated at
4 °C overnight with 4% (v/v) trichloroacetic acid. After
centrifugation (10,000 × g for 5 min at 4 °C), the
pellet was dissolved in SDS-PAGE disaggregation buffer (50 mM Tris-HCl, pH 6.8, 2% SDS (w/v), 15% glycerol (w/v), 1 mM EDTA, and 0.02% bromphenol blue (w/v)) at a protein
concentration of 3 mg/ml. Equal amounts of protein extracts were
separated by electrophoresis on 7.5% SDS-polyacrylamide gels
containing SDS and electroblotted to nitrocellulose membranes using 20 V overnight at 4 °C in a 25 mM Tris-HCl, 150 mM glycine buffer. Membranes were first incubated for
1 h in a blocking buffer containing 5% nonfat dried milk in PBS,
then for 2 h with the relevant primary monoclonal antibody, and
finally with secondary fluorescein isothiocyanate-conjugated IgG. Blots
were revealed by the enhanced ECL method using an ECL kit (Amersham
Biosciences). For membrane extracts, confluent cells were disrupted
mechanically in cold Tris buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA with complete protease inhibitor mixture (Roche Applied Science)) for 15-20 min on ice. After homogenization, and
centrifugation (14,000 × g for 3 min), the
supernatants were centrifuged at 100,000 × g for
1 h. The pellet, corresponding to the membrane protein fraction,
was dissolved in sample buffer and analyzed as above.
Co-immunoprecipitation--
Confluent cells from 3 wells (12 wells/plate) were disrupted mechanically and dissolved in Tris buffer
(50 mM Tris-HCl, pH 7.5) containing complete protease
inhibitor mixture for 15-20 min on ice. Proteins were precipitated at
4 °C with 4% trichloroacetic acid (v/v) overnight. After
centrifugation (10,000 × g for 5 min at 4 °C),
pellets were dissolved in 100 µl of disaggregating buffer (containing
1% Nonidet P-40, 1 mM EDTA, 10% glycerol, in PBS), and
the concentration of protein was assessed using BCA protein assay
reagent (Pierce). Proteins (400 µg) were incubated with 4 µg of
human EBP50 antibody for 2 h on a rotating wheel at 4 °C.
Washed protein G-Sepharose beads (25 µl) were then added to each
sample and incubated for 1 h at 4 °C. After centrifugation, immunoprecipitates were washed four times with PBS buffer and resuspended in SDS sample buffer before immunoblotting analyses.
Cellular cAMP Measurements--
We used the cAMP enzyme
immunoassay (EIA) system Biotrak (Amersham Biosciences) to assess
cellular cAMP levels in both control and salmeterol-treated cells
according to standard manufacturer's protocols. Each experiment was
performed in triplicate.
Statistical Analyses--
For statistical analyses, the
Student's t test and analysis of variance analysis were
performed. A value of p < 0.05 was considered as significant.
 |
RESULTS |
Characterization of Polarized HAEC in Primary Culture--
Because
protein trafficking and synthesis and certain drug effects depend on
cell polarity and tightness, we therefore first characterized the
polarity and tightness of HAEC under our culture conditions. A
distinguishing characteristic of the polarized epithelial cells is the
presence of tight junctions. Transmission electron microscopy revealed
the presence of structures corresponding to tight junctions (Fig.
1A), along with which the
exclusion of lanthanum nitrate precipitate by these structures
suggested that they were functional (Fig. 1B). To further
evaluate the functionality of the tight junctions observed, we measured
TER, a commonly used method to assess tight junction function. As shown
in Fig. 1C, the cultured cells showed an increase in their
TER over time, with a maximal value of 1500 ohm·cm2
reached 2 days after confluence. The presence of tight junctions correlated with the polarized distribution of apical and basolateral plasma membrane markers in these HAEC. Transverse sections of the cells
and culture support (collagen I gel) enabled clear visualization of the
apical and basolateral regions of the cells. Consistent with the TEM
data and TER measurements, the immunofluorescence staining (Fig.
2) for occludin, a tight junction
protein, revealed an apical staining pattern located at the point of
cell-cell contact. Moreover, these primary airway epithelial cells also
showed distinct apical localization of CFTR, ezrin, and CFTR-associated
protein EBP50 and a basal localization of
1-integrin, a
basal plasma membrane marker. The staining of
2AR was
apical and patchy in the cytoplasm (Fig. 2).

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Fig. 1.
Transmission electron micrographs of primary
HAEC. After 6 days of culture, HAEC appeared well differentiated
and polarized with basal nuclei and numerous microvilli located at the
apical membrane (A). Cells were connected by well developed
tight junctions (TJ) that were functional as shown by
lanthanum nitrate (La) exclusion (B).
Bars in A and B, 0.6 µm. The TER of
HAEC in culture was measured, and the value obtained was expressed in
ohm·cm2. The cultured cells showed an increase in their
TER over time, with a maximal value of 1500 ohm·cm2
reached 2 days after confluence (C).
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Fig. 2.
Expression and localization of
2 adrenergic receptors, CFTR, and
CFTR-associated protein EBP50 in primary HAEC. HAEC isolated from
human nasal polyps were cultivated on collagen I gels until confluent.
Cryofixed longitudinal sections of the cells (5 µm thick) were
incubated with rabbit polyclonal antibody specific for human
2AR, mouse monoclonal antibodies specific for human
CFTR, EBP50, 1-integrin, and occludin, or rat polyclonal
antibody specific for the EBP50-associated protein ezrin.
Immunoreactivity was visualized using streptavidin-coupled fluorescein
isothiocyanate staining.
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Agonist Stimulation Increases CFTR Expression in HAEC--
The
effect of
2AR agonist on endogenous CFTR expression by
airway epithelial cells was assessed by Western blotting analyses using
a mouse monoclonal anti-human CFTR antibody (C24-1). As shown in Fig.
3, A and B, cell
incubation with 2.10
7 M salmeterol, a long
acting
2AR agonist, resulted in a significant increase
in mature CFTR levels after 4 and 24 h of agonist administration. CFTR bands observed in Western blots were the N-glycosylated
B and C forms, since a shift in the molecular weights was observed after N-glycanase treatment (data not shown). Cell treatment
with salmeterol (24 h) produced a 2.2-fold increase over base line for
the fully mature band C (p < 0.05). To
verify that the effect of the agonist on CFTR expression was mediated
by
2AR and not as a result of a direct effect of the
agonist on CFTR expression, cells were exposed to ICI 118,551 (100 nM), a highly specific
2AR antagonist, for
20 min before treatment with salmeterol. Because this maneuver
prevented the effect of the salmeterol, we concluded that the effect of
salmeterol on CFTR expression was dependent upon its interaction with
2AR. Moreover, cells treated with ICI 118,551 (100 nM) alone did not show any change in CFTR levels,
suggesting that this receptor is not involved in the regulation of CFTR
expression under resting conditions.

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Fig. 3.
Western blot analysis of salmeterol effect on
CFTR protein expression in primary HAEC. In A, primary
HAEC were grown onto collagen I gels until confluent (5-7 days of
culture) and then treated with salmeterol (2.10 7
M) for 0.5, 4, or 24 h. Protein extracts were
prepared, and equal aliquots were separated by electrophoresis on 7%
SDS-polyacrylamide gel and transferred onto nitrocellulose membranes.
CFTR bands B and C were detected after incubation with mouse monoclonal
anti-human CFTR following standard protocols for ECL Western detection
(Amersham Biosciences). In B, histograms represent the
relative changes in mature CFTR after salmeterol treatment. Data are
arbitrary densitometry units, where control values have been normalized
to 1. *, p < 0.05 versus control
values. In C, primary HAEC were pretreated for 20 min with
the highly specific 2AR antagonist ICI 118,551 (100 nM). After this period of time, salmeterol
(2.10 7 M) was added, and the cells were
incubated for the indicated times. In D, the relative
changes in CFTR (band C) upon ICI 118,551 (100 nM) and
salmeterol treatments are shown. Data are arbitrary densitometry units,
where control values have been normalized to 1. *, p
< 0.05 for salmeterol-treated cells compared with
nontreated cells.
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To test whether the increase in CFTR after
2AR agonist
administration was due to effects on steady-state mRNA levels, we performed a semiquantitative RT-PCR analysis from total cellular RNA.
The
2AR agonist did not appear to cause significant
changes in CFTR mRNA levels, suggesting that increased CFTR
mRNA did not account for the protein increase observed (Fig.
4).

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Fig. 4.
RT-PCR analysis of the salmeterol effect on
CFTR mRNA expression in primary HAEC. The total RNA from cells
was converted to first-strand DNA and then amplified with CFTR and 28 S-specific primers for 27 and 17 amplification cycles, respectively.
The amplified fragments were electrophoresed in polyacrylamide gels and
stained with SYBR gold. Endogenous CFTR and 28 S were detected at the
expected sizes (276 and 212 bp, respectively). The internal control RNA
template for CFTR (pCTR) was detected at 260 bp
(A). In B, CFTR was expressed as a ratio to its
specific internal control. The levels of mRNA were normalized with
28 S level.
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The Effect of
2AR Agonist on CFTR Expression Is
Specific--
To demonstrate that the effect of the agonist was
specific and not a result of a global alteration in cell protein
expression, we evaluated by Western blotting the levels of EBP50 and
ezrin (members of the protein network associated with CFTR) in both soluble and cytoskeletal fractions of untreated and treated cell lysates (Fig. 5, A and
B). In both fractions EBP50 and ezrin levels remained
unchanged after salmeterol treatment (Fig. 5, C and
D). Because agonist treatment has been previously reported
to cause
2AR down-regulation and desensitization, we
performed Western blots to analyze the expression of membrane
2AR in HAEC after salmeterol treatment (Fig.
6A). In contrast to its effect
on CFTR, salmeterol induced a significant decrease of membrane
2AR levels after 4 and 24 h of cell treatment (Fig.
6B).
2AR corresponded to a band of 54 kDa
that disappeared when blots were treated with the anti-human
2AR antibody blocking peptide (Fig. 6C).

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Fig. 5.
Western blot analysis of the effect of
salmeterol on expression of CFTR-associated protein EBP50 and
EBP50-associated protein ezrin expression in primary HAEC. Primary
HAEC were grown onto collagen I gels until confluent and then treated
(or not, control (C)) with salmeterol (2.10 7
M: S) for 24 h. Soluble and insoluble
protein extracts were prepared, and equal aliquots were separated by
electrophoresis on 12% SDS-polyacrylamide gels and electroblotted onto
nitrocellulose membranes. EBP50 was detected after incubation with a
mouse monoclonal anti-human EBP50 and using ECL protocols
(A). Similarly, ezrin was detected after incubation with rat
monoclonal anti-human ezrin (C). Histograms in B
and D represent the relative changes in EBP50 and ezrin
expression, respectively, after salmeterol treatment. Data show
arbitrary densitometry units, where control values have been normalized
to 1.
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Fig. 6.
Membrane
2AR down-regulation after salmeterol
treatment. Confluent primary epithelial cells cultured on collagen
I gels and treated for the indicated times with salmeterol were
incubated in lysis buffer and mechanically disrupted. Membrane protein
fractions were obtained by centrifugation of the post-nuclear
supernatants and resuspension of the pellets in sample buffer. In
A, equal aliquots of these protein extracts were separated
by electrophoresis on 12% SDS-polyacrylamide gels and electroblotted
onto nitrocellulose membranes. 2AR was detected after
incubation with rabbit polyclonal anti-human 2AR and via
ECL protocols. For the identification of 2AR bands,
similarly treated blots were incubated with rabbit polyclonal
anti-human 2AR and its blocking peptide (C).
In B, histograms represent the relative changes in membrane
2AR expression upon salmeterol treatment. Data are
arbitrary densitometry units, where control values have been normalized
to 1.
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The Effect of
2AR Agonist on CFTR Levels Is Not
Mediated by cAMP--
Treatment of cells with salmeterol also induced
an increase in cellular cAMP. This increase was detected after 2 min of
treatment, lasted for at least 30 min, and was partially restored to
near normal at 4 and 24 h (Fig.
7A). To test whether the
effect of salmeterol on CFTR expression levels was mediated by this
cAMP signal, we treated HAEC for different periods of time with
8-Br-cAMP (a cell-permeable analogue of cAMP), or with 8-Br-cAMP along
with salmeterol, and then analyzed CFTR expression by Western blotting (Fig. 7, B and C). As shown in Fig.
7B', cellular CFTR levels were not significantly modified by
exposure of cells to 8-Br-cAMP alone, whereas when the
2AR agonist was added, CFTR expression was significantly
increased (Fig. 7C').

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Fig. 7.
The cAMP pathway is not involved in the
effect of salmeterol on CFTR expression. In A, cAMP
levels in lysates from cells treated with salmeterol for the indicated
times are reported. The role of cAMP in the 2AR
agonist-mediated increase of CFTR expression was investigated using
8-bromo cAMP (10 6 M) to mimic the cAMP signal
induced by 2AR agonist. In B, cells incubated
with 8-bromo cAMP (10 6 M) for the indicated
times did not show any significant changes in CFTR expression, whereas
cells treated with 8-bromo cAMP (10 6 M) and
salmeterol (2 ×10 7 M) for the indicated
times showed an increase in CFTR expression comparable with the effect
of salmeterol alone (C). Incubation of cells with protein
kinase A-inhibiting peptide (3 × 10 6 M)
for the indicated times did not prevent the effect of
2AR agonist on CFTR expression (D). B', C',
and D' histograms represent the relative changes in CFTR expression
after treatments with 8-bromo cAMP alone, 8-bromo cAMP in combination
with salmeterol, and PKA-inhibiting peptide, respectively. Data show
arbitrary densitometry units, where control values have been normalized
to 1.
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Similarly, treatment with the protein kinase A inhibitor Rp-cAMP, which
is known to inhibit cAMP-dependent PKA-mediated effects in
several cell types, did not prevent the increase in CFTR expression after salmeterol treatment. This suggests that PKA activity is not
required for the agonist to increase CFTR levels. The cAMP analogue and
the PKA inhibitor did not affect CFTR levels in control cells (not
treated with
2AR agonist), suggesting that the PKA/cAMP pathway is not involved in the regulation of CFTR expression under basal conditions.
Co-immunoprecipitation of
2AR and CFTR with an
Anti-human EBP50 Antibody--
To verify whether
2AR
stimulation increases levels of CFTR that bind to EBP50 in HAEC, we
performed co-immunoprecipitation experiments using an anti-human EBP50
followed by Western blotting analysis of the resulting
immunoprecipitates with anti-human CFTR or anti-human EBP50. As shown
in Fig. 8C, the treatment of
cells for 24 h with
2AR agonist induced a
detectable increase in the intensity of the band recognized by
anti-human CFTR without any associated EBP50 alteration. When the
immunoprecipitate blots were incubated with the anti-human
2AR antibody, a 54-kDa band that most likely corresponds
to
2AR was revealed.

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Fig. 8.
Co-immunoprecipitation (IP)
of CFTR and 2AR with an anti-EBP50
in primary HAEC. Salmeterol-treated cell lysates were incubated
with a monoclonal anti-human EBP50 antibody and then precipitated using
protein G beads at 4 °C. The precipitates were resolved via
SDS-PAGE, and proteins were electroblotted (WB) to
nitrocellulose membranes. Blots were then incubated with an anti-EBP50
antibody (B), an anti-CFTR (C), or an
anti- 2AR (D) followed by incubation with the
relevant secondary antibody. For controls, similarly treated blots were
incubated with anti-mouse IgG (A). Blots were visualized
using an ECL protocol.
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Interestingly, this band intensity was decreased by 24 h of
2AR agonist treatment (Fig. 8D). These
results are in accordance with our direct Western blot analyses of cell
protein extracts. The immunoprecipitate contained
2AR,
CFTR, and EBP50.
 |
DISCUSSION |
2AR agonists represent key therapeutic agents in
the treatment of obstructive lung diseases.
2ARs are
expressed within the lung on multiple cell types including bronchial
smooth muscle cells, tracheal epithelial cells, and presynaptic
cholinergic nerve terminals and on multiple immune cells present
therein, including macrophages (17-19). To our knowledge, no data are
available concerning the localization of
2AR in airway
epithelial cells. To visualize
2AR in transverse
sections of primary airway epithelial cells cultured on collagen I
gels, we used a polyclonal antibody directed against human
2AR and its blocking peptide to identify the specificity
of the staining observed. We found that the immunolabeling of
2AR in HAEC was not uniform and that the
2AR could be localized to the apical plasma membrane and
be found in the cytoplasm. Importantly, this immunostaining study
demonstrates that, besides a
2AR cytoplasmic localization, which probably corresponds to constitutively internalized receptors,
2AR could localize to the apical domain of
cultured cells, where CFTR resides normally. As such, this finding is
of major interest.
It has been reported that, regardless of CFTR gene mutations, CFTR
apical targeting in vivo can be affected by the epithelium integrity, maturation, polarity, and differentiation (20-22).
Moreover, HAEC can be cultured under different conditions in
vitro with variable degrees of differentiation (23,
24). Under our culture conditions, primary HAEC showed tight junctions,
elevated transepithelial resistance, and a polarized distribution of
CFTR.
It has been reported that in human bronchial sections, EBP50 is
concentrated in the apical compartment of airway epithelial cells (5).
We therefore determined if EBP50 was similarly distributed in cultured
airway epithelial cells. Immunohistochemical analysis of these cells
using a monoclonal anti-human EBP50 demonstrated staining in the apical
region of these cells, suggesting that the process of cell isolation
and culture did not alter the distribution of EBP50.
As demonstrated by the Western blot analyses of airway epithelial cell
protein extracts, treatment with
2AR agonist increased mature CFTR protein expression in a time-dependent fashion.
The maximal effect was reached after 24 h of exposure to the
agonist. This stimulation did not affect the steady-state levels of
CFTR mRNA, suggesting that the effect of the
2AR
agonist on CFTR protein expression was post-transcriptional. Because
2AR stimulation leads to alterations in the metabolism,
excitability, differentiation, and growth of many cell types, we
verified that the effect of the
2AR agonist on CFTR
protein expression was specific and not simply a result of a global
alteration in airway epithelial cell protein expression. For this
purpose, we tested the effect of the stimulation on the CFTR-associated
proteins EBP50 and ezrin. The levels of these proteins were unaltered
by agonist treatment, suggesting a specific action of
2AR agonist on CFTR expression.
2AR
down-regulation in response to chronic exposure to
2AR
agonists is a virtually universal finding in all cell systems. However, the mechanisms involved in this down-regulation are usually
cell-specific and involve transcriptional, post-transcriptional, and
post-translational mechanisms that regulate receptor mRNA levels
and the rates of receptor protein synthesis and degradation. In
vivo studies demonstrated that in both human bronchial epithelial
cells and alveolar macrophages,
2AR was down-regulated
after prolonged agonist administration (18).
In human tracheal cells in primary culture, adrenergic stimulation
affects cAMP levels only through
2AR (17). Other studies have shown that the
receptor-coupled-adenylyl cyclase system is
highly expressed and functional on acutely dissociated HAEC. This
system has been shown to be rapidly desensitized by exposure to
2AR agonists or activators of protein kinase C (25). The
2AR down-regulation after exposure to
2AR
agonist occurs in a dose- and time-dependent fashion in
HAEC. The regulation of
2AR density in the human airway
epithelial cell line BEAS-2B has been shown to be largely
cAMP-independent. This is on account of the lack of changes in
2AR density in response to either forskolin or
dibutyryl-cAMP treatment (26). Moreover, a 24-h exposure of these cells
to
2AR agonist had no effect on the steady-state levels
of
2AR mRNA, suggesting that the process of
2AR down-regulation does not involve changes in
2AR gene transcription or stability of the
2AR message (26). According to these findings, our
Western blot analyses of
2AR protein in primary HAEC
showed that a 24-h exposure to the long-acting
2AR
agonist induced a significant decrease in membrane
2AR
and that this effect is cAMP-independent because cell treatment with
8-Br-cAMP did not change the levels of membrane
2AR
(data not shown). Moreover, immunoprecipitation of
2AR
with a monoclonal antibody directed against EBP50 demonstrated that 1)
this receptor binds to EBP50 and 2) the level of
2AR bound was decreased after 24 h of salmeterol treatment, which indicated that the
2AR bound to EBP50 was affected by
the down-regulation process. Because immunohistochemical studies showed
that EBP50 is localized exclusively to the apical plasma membrane,
results obtained after immunoprecipitation with an anti-EBP50 antibody suggest that salmeterol decreased the level of apical
2AR. This result is confirmed by the absence of apical
2AR immunostaining after 24 h of salmeterol treatment.
Although it has been previously demonstrated that
2AR
interacts with NHERF, the rabbit homologue of EBP50, and that this interaction is mediated via the binding of the last few amino acids of
the
2AR tail to the first PDZ domain of NHERF (10, 11),
we demonstrate in this study for the first time that such an
interaction is also present in primary human airway epithelial cells
most likely at their apical domain. Similarly, the C terminus of CFTR
corresponds to a PDZ binding motif, which interacts with the first PDZ
domain of NHERF to bring ezrin into proximity with CFTR. Ezrin can
anchor protein kinase A at a position physiologically appropriate for
CFTR phosphorylation, and ezrin interactions with the cytoskeleton may
permit retention of CFTR at the apical membrane as a mechanism to
establish its polarity in these epithelial cells. In recent years,
protein interactions at several CFTR domains have been implicated in
channel function and trafficking. Thus, it has been demonstrated that
cAMP/protein kinase A-mediated activation of the CFTR results in
increased open channel probability in membrane patches and lipid
bilayers. Experiments on intact cells suggest that cAMP mediates
insertion of a submembranous pool of CFTR into the apical membrane
(27). However, it has also been reported that cAMP activation of
CFTR-mediated Cl
secretion does not involve the
recruitment of CFTR from an intracellular pool to the apical plasma
membrane in Calu3 cells (16). The involvement of protein kinase A in
the regulation of channel gating or membrane insertion is
controversial. More recent studies demonstrated that forskolin
stimulation increased insertion of virally expressed epitope-tagged
CFTR into the apical membrane of Madin-Darby canine kidney cells, but
this was only observed at a low multiplicity of infection (15). This
latter result suggests that the effect of channel stimulation on CFTR
insertion in the apical membrane of epithelial cells depends on protein
expression levels and is, therefore, a cell-specific effect. In our
study, cell treatment with 8-Br-cAMP, a cAMP mimetic, did not affect
the membrane expression level of CFTR, indicating that, as for Calu3
and unlike CFTR-transfected Madin-Darby canine kidney type I cells and
other cell types, cAMP has no significant effect on endogenous apical
expression of CFTR in primary HAEC. Furthermore, in our study the
inhibition of PKA with the PKA-inhibiting peptide Rp-cAMP did not
modify the effect of salmeterol on CFTR expression, suggesting that the
cAMP/PKA pathway is not involved in the effect of
2AR
agonist on CFTR expression levels. However, when a specific
2AR blocker was administered to the cells before
stimulation with the agonist, CFTR expression remained unchanged. This
result demonstrates that the
2AR agonist effect on CFTR
is dependent upon its binding to the receptors and rules out a
potentially direct effect of
2AR agonist on CFTR.
The most interesting finding of this study is that both CFTR and
2AR bind to EBP50 in polarized primary HAEC. EBP50 is
highly expressed in these cells and is exclusively localized to the
apical membrane. Exposure of cells to a
2AR agonist
increases the expression of membrane CFTR and down-regulates membrane
2AR in a time-dependent manner, whereas
EBP50 is unmodified. Moreover, neither effect is mediated by the
cAMP/PKA pathway. Taken together, these data may suggest that
2AR and CFTR compete for the same binding site on EBP50,
for instance the PDZ domain, and thus the down-regulation of
2AR liberates these binding sites for CFTR at the apical
plasma membrane. In fact PDZ domain-containing proteins as well as
PDZ-interacting domains play a key role in the apical polarization of
ion channels in epithelial cells (7).
Thus, the pharmacological treatment reveals an existent
cross-regulation between these two proteins, and the CFTR apical
targeting to the apical plasma membrane is improved by
2AR internalization. We cannot, however, exclude the
fact that these two modifications are completely independent and that
the augmentation of CFTR expression could be due to modifications in
total post-transcriptional production of this protein because the total
amount of CFTR (band B + band C) is increased in salmeterol-treated
cells. This implies that
2AR stimulation by salmeterol
enhance CFTR translation and/or maturation. Very little if anything is
known about the cAMP-independent effects of salmeterol or any other
2AR agonist on protein expression or maturation.
Activation of the
2AR expressed on bronchial smooth
muscle cells induces relaxation with a resultant increase in airway
diameter, which is the most readily observable effect of
agonists
on lung function. Other proposed actions of
2 agonists
in treating asthma and chronic obstructive lung disease include
improved ciliary function, modulation of immune cell functions, changes
in vascular permeability, and a decrease in acetylcholine release. All
these effects are mediated by the cAMP/PKA pathway. Several reports indicate that, besides these well known effects,
2AR
activation induces other effects on cell ion transport, protein
expression, proliferation, and differentiation. The potential clinical
benefits of these effects are still unclear. Some recent studies
demonstrated that the anti-spasmogenic effect of the
AR agonist,
isoprenaline, on guinea-pig trachealis and the positive inotropic
response produced by the
2AR agonist, zinterol,
in adult ventricular myocytes are not mediated by
cAMP-dependent PKA (28, 29). The increased CFTR
expression associated with
2AR stimulation may be of
major importance in a number of pathological situations such as cystic fibrosis and non cystic fibrosis diseases where remodeling and inflammation may decrease the apical CFTR expression of airway epithelial cells.