Stimulation of beta 2-Adrenergic Receptor Increases Cystic Fibrosis Transmembrane Conductance Regulator Expression in Human Airway Epithelial Cells through a cAMP/Protein Kinase A-independent Pathway*

Karima TaouilDagger , Jocelyne HinnraskyDagger , Coralie HologneDagger , Pascal Corlieu§, Jean-Michel Klossek, and Edith PuchelleDagger ||

From the Dagger  INSERM 514, IFR 53, Centre Hospitalier Universitaire Maison Blanche, Reims, 51092 Cedex, France, § Hopital Tenon, 4 rue de la Chine, 75020 Paris, France, and  Hopital Jean Bernard, BP 577, 86021 Poitiers, France

Received for publication, December 2, 2002, and in revised form, February 28, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PSD-95/Dlg-A/ZO-1 (PDZ) domains play an essential role in determining cell polarity. The Na+/H+ exchanger regulatory factor (NHERF), also known as EBP50, contains two PDZ domains that mediate the assembly of transmembrane and cytosolic proteins into functional signal transduction complexes. Moreover, it has been shown that cystic fibrosis transmembrane conductance regulator (CFTR) and beta 2-adrenergic receptor (beta 2AR) bind equally well to the PDZ1 domain of EBP50. We hypothesized that beta 2AR activation may regulate CFTR protein expression. To verify this, we evaluated the effects of a pharmacologically relevant concentration of salmeterol (2.10-7 M), a long acting beta 2AR agonist, on CFTR expression in primary human airway epithelial cells (HAEC). beta 2AR stimulation induced a time-dependent increase in apical CFTR protein expression, with a maximal response reached after treatment for 24 h. This effect was post-transcriptional, dependent upon the beta 2AR agonist binding to beta 2AR and independent of the known beta 2AR agonist-mediated cAMP/PKA pathway. We demonstrated by immunohistochemistry that CFTR, beta 2AR, and EBP50 localize to the apical membrane of HAEC. Analyses of anti-EBP50 protein immunoprecipitate showed that salmeterol induced an increase in the amount of CFTR that binds to EBP50. These data suggest that beta 2AR activation regulates the association of CFTR with EBP50 in polarized HAEC.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (Delta 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 (beta 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 beta 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 beta 2AR between recycling endosomes and lysosomal compartments is controlled by their association with EBP50 (13, 14). Moreover, beta 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 beta 2AR-EBP50-ezrin and -actin interactions may enhance the maturation of CFTR and its expression at the apical membrane of epithelial cells. The beta 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 beta 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 beta 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 beta 2AR agonist may modulate the expression of membrane CFTR via the cAMP/PKA pathway. We report that exposure of primary airway epithelial cells to beta 2AR agonist induces a post-transcriptional, time-dependent increase in CFTR expression. This effect is mediated by beta 2AR but does not involve the cAMP/PKA pathway.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

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 beta 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 beta 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta 1-integrin, a basal plasma membrane marker. The staining of beta 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 beta 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 beta 2AR, mouse monoclonal antibodies specific for human CFTR, EBP50, beta 1-integrin, and occludin, or rat polyclonal antibody specific for the EBP50-associated protein ezrin. Immunoreactivity was visualized using streptavidin-coupled fluorescein isothiocyanate staining.

beta Agonist Stimulation Increases CFTR Expression in HAEC-- The effect of beta 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 beta 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 beta 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 beta 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 beta 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 beta 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.

To test whether the increase in CFTR after beta 2AR agonist administration was due to effects on steady-state mRNA levels, we performed a semiquantitative RT-PCR analysis from total cellular RNA. The beta 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.

The Effect of beta 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 beta 2AR down-regulation and desensitization, we performed Western blots to analyze the expression of membrane beta 2AR in HAEC after salmeterol treatment (Fig. 6A). In contrast to its effect on CFTR, salmeterol induced a significant decrease of membrane beta 2AR levels after 4 and 24 h of cell treatment (Fig. 6B). beta 2AR corresponded to a band of 54 kDa that disappeared when blots were treated with the anti-human beta 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 beta 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. beta 2AR was detected after incubation with rabbit polyclonal anti-human beta 2AR and via ECL protocols. For the identification of beta 2AR bands, similarly treated blots were incubated with rabbit polyclonal anti-human beta 2AR and its blocking peptide (C). In B, histograms represent the relative changes in membrane beta 2AR expression upon salmeterol treatment. Data are arbitrary densitometry units, where control values have been normalized to 1.

The Effect of beta 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 beta 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 beta 2AR agonist-mediated increase of CFTR expression was investigated using 8-bromo cAMP (10-6 M) to mimic the cAMP signal induced by beta 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 beta 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.

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 beta 2AR agonist), suggesting that the PKA/cAMP pathway is not involved in the regulation of CFTR expression under basal conditions.

Co-immunoprecipitation of beta 2AR and CFTR with an Anti-human EBP50 Antibody-- To verify whether beta 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 beta 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 beta 2AR antibody, a 54-kDa band that most likely corresponds to beta 2AR was revealed.


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Fig. 8.   Co-immunoprecipitation (IP) of CFTR and beta 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-beta 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.

Interestingly, this band intensity was decreased by 24 h of beta 2AR agonist treatment (Fig. 8D). These results are in accordance with our direct Western blot analyses of cell protein extracts. The immunoprecipitate contained beta 2AR, CFTR, and EBP50.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

beta 2AR agonists represent key therapeutic agents in the treatment of obstructive lung diseases. beta 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 beta 2AR in airway epithelial cells. To visualize beta 2AR in transverse sections of primary airway epithelial cells cultured on collagen I gels, we used a polyclonal antibody directed against human beta 2AR and its blocking peptide to identify the specificity of the staining observed. We found that the immunolabeling of beta 2AR in HAEC was not uniform and that the beta 2AR could be localized to the apical plasma membrane and be found in the cytoplasm. Importantly, this immunostaining study demonstrates that, besides a beta 2AR cytoplasmic localization, which probably corresponds to constitutively internalized receptors, beta 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 beta 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 beta 2AR agonist on CFTR protein expression was post-transcriptional. Because beta 2AR stimulation leads to alterations in the metabolism, excitability, differentiation, and growth of many cell types, we verified that the effect of the beta 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 beta 2AR agonist on CFTR expression. beta 2AR down-regulation in response to chronic exposure to beta 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, beta 2AR was down-regulated after prolonged agonist administration (18).

In human tracheal cells in primary culture, adrenergic stimulation affects cAMP levels only through beta 2AR (17). Other studies have shown that the beta  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 beta 2AR agonists or activators of protein kinase C (25). The beta 2AR down-regulation after exposure to beta 2AR agonist occurs in a dose- and time-dependent fashion in HAEC. The regulation of beta 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 beta 2AR density in response to either forskolin or dibutyryl-cAMP treatment (26). Moreover, a 24-h exposure of these cells to beta 2AR agonist had no effect on the steady-state levels of beta 2AR mRNA, suggesting that the process of beta 2AR down-regulation does not involve changes in beta 2AR gene transcription or stability of the beta 2AR message (26). According to these findings, our Western blot analyses of beta 2AR protein in primary HAEC showed that a 24-h exposure to the long-acting beta 2AR agonist induced a significant decrease in membrane beta 2AR and that this effect is cAMP-independent because cell treatment with 8-Br-cAMP did not change the levels of membrane beta 2AR (data not shown). Moreover, immunoprecipitation of beta 2AR with a monoclonal antibody directed against EBP50 demonstrated that 1) this receptor binds to EBP50 and 2) the level of beta 2AR bound was decreased after 24 h of salmeterol treatment, which indicated that the beta 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 beta 2AR. This result is confirmed by the absence of apical beta 2AR immunostaining after 24 h of salmeterol treatment.

Although it has been previously demonstrated that beta 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 beta 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 beta 2AR agonist on CFTR expression levels. However, when a specific beta 2AR blocker was administered to the cells before stimulation with the agonist, CFTR expression remained unchanged. This result demonstrates that the beta 2AR agonist effect on CFTR is dependent upon its binding to the receptors and rules out a potentially direct effect of beta 2AR agonist on CFTR.

The most interesting finding of this study is that both CFTR and beta 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 beta 2AR agonist increases the expression of membrane CFTR and down-regulates membrane beta 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 beta 2AR and CFTR compete for the same binding site on EBP50, for instance the PDZ domain, and thus the down-regulation of beta 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 beta 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 beta 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 beta 2AR agonist on protein expression or maturation.

Activation of the beta 2AR expressed on bronchial smooth muscle cells induces relaxation with a resultant increase in airway diameter, which is the most readily observable effect of beta  agonists on lung function. Other proposed actions of beta 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, beta 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 beta AR agonist, isoprenaline, on guinea-pig trachealis and the positive inotropic response produced by the beta 2AR agonist, zinterol, in adult ventricular myocytes are not mediated by cAMP-dependent PKA (28, 29). The increased CFTR expression associated with beta 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.

    ACKNOWLEDGEMENTS

We thank Myriam Polette for designing the CFTR primers and internal control. We thank Dr. Malcolm Johnson for helpful suggestions, support of our work, and critical reading of the manuscript.

    FOOTNOTES

* This work was supported by INSERM, GlaxoSmithKline, and by the Association Vaincre la Mucoviscidose.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.

|| To whom correspondence should be addressed: INSERM 514, Centre Hospitalier Universitaire Maison Blanche 45, rue Cognacq Jay, 51092 Reims Cedex, France. Tel.: 33-3-26-78-77-70; Fax: 33-3-26-06-58-01; E-mail: epuche@worldnet.fr.

Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M212227200

    ABBREVIATIONS

The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; NHERF, Na+/H+ exchanger regulatory factor; HAEC, human airway epithelial cells; beta 2AR, beta 2-adrenergic receptor; PKA, protein kinase A; PBS, phosphate-buffered saline; TER, transepithelial electrical resistance; RT, reverse transcription; 8-Br-cAMP, 5-bromo-cAMP.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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