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Institut du Thorax, Institut National de la Sante et de la Recherche Medicale U533, Facultee de Meedecine, Nantes, France (A.R., G.T., C.G.)
Laboratoire de Pharmacologie de la Facultee de Pharmacie, Institut National de la Sante et de la Recherche Medicale EMI-0356, Universitee Victor Segalen Bordeaux 2, Bordeaux, France (V.L.)
Facultee des Sciences et Techniques, Nantes, France (C.G.)
Abstract
We have shown previously that in a heterologous mammalian expression system A549 cells, 3-adrenoceptor (3-AR) stimulation regulates the activity of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The present investigation was carried out to determine the signaling pathway involved in this regulation. A549 cells were intranuclearly injected with plasmids encoding human CFTR and 3-AR. CFTR activity was functionally assessed by microcytofluorimetry. The application of 1 e 4-[3-t-butylamino-2-hydroxypropoxy]benzimidazol-2-1 hydrochloride (CGP-12177), a 3-AR agonist, produced a CFTR activation that was not abolished by protein kinase A inhibitors. In pertussis toxin-pretreated cells, the CFTR activation induced by CGP-12177 was abolished. The overexpression of -adrenoceptor receptor kinase, an inhibitor of subunits, abolished the CGP-12177eCinduced CFTR activation, suggesting the involvement of subunits of Gi/o proteins. The pretreatment of A549 cells with selective inhibitors of either phosphoinositide 3-kinase (PI3K), wortmannin, and 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), or extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein kinase (MAPK), 2'-amino-3'-methoxyflavone (PD98059), and 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene (U0126), abolished the effects of CGP-12177 on the CFTR activity. Immunohistochemical assays showed that only the cells expressing 3-AR exhibited MAPK activation in response to CGP-12177. Furthermore, CFTR activity increased in cells pretreated with 10% fetal bovine serum both in A549 cells injected only with CFTR and in T84 cells, which endogenously express CFTR, indicating that CFTR activity can be regulated by the MAPK independently of the 3-AR stimulation. In conclusion, we have demonstrated that CFTR is regulated through a Gi/o/PI3K/ERK1/2 MAPK signaling cascade dependently or not on an activation of 3-ARs. This pathway represents a new regulation for CFTR.
Cystic fibrosis (CF), the most common lethal recessive genetic disorder among white persons, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (Collins, 1992). The gene encodes the CFTR protein, a cAMP-regulated CleC channel that controls salt and water transport across the epithelium in many tissues. CFTR proteins are expressed in airway epithelia and submucosal glands, sweat glands, gastrointestinal tract, pancreatic bile, and reproductive ducts. Gating of the channel is tightly regulated by phosphorylation of the regulatory domain. The regulatory domain contains multiple phosphorylation sites for cAMP-dependent protein kinase A (PKA) (Cheng et al., 1991). Phosphorylation at most of these sites stimulates CleC channel activity (Wilkinson et al., 1997). CFTR also contains protein kinase C (PKC) phosphorylation sites. There is evidence that prior phosphorylation by PKC primes CFTR for subsequent phosphorylation and activation by PKA (Jia et al., 1997; Liedtke and Cole, 1998). The mechanism for this priming effect of PKC is unknown (Kirk, 2000). CFTR can also be activated by cGMP via cGMP-dependent protein kinase (PKG)-mediated phosphorylation in cells expressing the PKGII isoform but not in airway epithelium (Berger et al., 1993) that lack PKGII isoforms (French et al., 1995). Finally, several studies suggest that CFTR might be regulated either directly or indirectly by tyrosine phosphorylation (Dahan et al., 2001).
-adrenoceptor (-AR) stimulation is well known to regulate the intracellular cAMP levels. -AR agonists exert a variety of effects on airway epithelial cells, such as an increase in ciliary beat frequency (Sanderson and Dirksen, 1989) and an activation of ion transport by opening apical ion channels like CFTR (Bennett, 2002; Salathe, 2002). In epithelial cells, cAMP also activated CFTR (Gadsby and Nairn, 1999), but no data are available concerning an elevation of cAMP by 2-AR stimulation in those cells. However, in Xenopus laevis oocytes, the 2-AR stimulation produced an activation of adenylyl cyclase that increased the concentration of intracellular cAMP, leading to an activation of CFTR channel gating (Wotta et al., 1997). Our group has demonstrated in a recombinant system, a human lung epithelial-derived cell line (A549), that CFTR is regulated by 3-AR stimulation produced either by isoproterenol (a nonselective -AR agonist) in the presence of nadolol (a 1, 2-AR antagonist), SR 58611A (a preferential 3-AR agonist), or CGP-12177 (a partial 3-AR agonist) (Leblais et al., 1999). This CFTR activation induced by 3-AR stimulation was not prevented by PKA inhibitors, ruling out the involvement of a cAMP/PKA pathway in this response (Leblais et al., 1999). Thus, the present investigation was carried out to characterize the signaling pathway involved in the regulation of CFTR by 3-AR in A549 cells. Using pharmacological and biochemical approaches, we demonstrated the involvement of a new signaling pathway in the regulation of CFTR: Gi/o protein/phosphoinositide 3 kinase (PI3K)/ERK1/2 mitogen-activated protein kinase (MAPK).
Materials and Methods
Cell Culture. The human lung epithelial-derived cell line A549 and the human colonic carcinoma cell line T84 were provided by the American Type Culture Collection (Manassas, VA). A549 cells were cultured as reported previously (Mohammad-Panah et al., 1998). T84 cells were grown in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and antibiotics (100 IU/ml penicillin and 100 e/ml streptomycin) (Invitrogen, Paisley, UK) maintained in a humidified incubator (95% air/5% CO2) at 37°C and passaged weekly.
Plasmids. Transgene cDNAs were subcloned into pcDNA3 mammalian expression vector under the control of a cytomegalovirus promoter. The pcDNA3-CFTR plasmid (a gift from J. Ricardo, Lisbon, Portugal) encoded the wild-type human CFTR protein, and the pcDNA3-2 and pcDNA3-3A plasmids (gifts from D. Langin, Toulouse, France) encoded human 2-AR and A isoform of the human 3-AR, respectively. The pcDNA3--adrenoceptor receptor kinase (-ARK) plasmid (a gift from R. J. Lefkowitz, Durham, NC) encoded the C-terminal region of -ARK.
Intranuclear Injection of Plasmids. Cells were microinjected with plasmids (30 e/ml for human CFTR, 0.1 e/ml for human 3-AR, and/or 0.3 e/ml for human 2-AR) 1 day after plating on glass coverslips (Nunclon; NUNC A/S, Roskilde, Denmark). Our protocol to intranuclearly microinject individual cells using the Eppendorf ECET microinjector 5246 system and the ECET micromanipulator 5171 system has been reported in detail elsewhere (Mohammad-Panah et al., 1998). Plasmids were diluted in an injection buffer composed of 50 mM HEPES, 50 mM NaOH, and 40 mM NaCl, pH 7.4. Fluorescein isothiocyanate-labeled dextran (0.5%) was added to the injection medium to visualize successfully microinjected cells.
SPQ Fluorescence Assay. The CleC channel activity of CFTR was assessed using the halide-sensitive fluorescent probe 6-methoxy-N-(3-sulfopropyl)quinolium (SPQ; Molecular Probes, Leiden, Netherlands) as described previously (Mohammad-Panah et al., 1998). Twenty-four hours after injection, cells were loaded with intracellular SPQ dye by incubation in Ca2+-free hypotonic (50% dilution) medium containing 10 mM SPQ at 37°C for 15 min. The coverslips were mounted on the stage of an inverted microscope (Diaphot; Nikon, Tokyo, Japan) equipped for fluorescence and illuminated at 360 nm. The emitted light was collected at 456 ± 33 nm by a high-resolution image intensifier coupled to a video camera (Extended ISIS Camera System; Photonic Science, Roberts-Bridge, UK) connected to a digital-image processing board controlled by FLUO software (Imstar, Paris, France). Cells were maintained at 37°C and continuously superfused with an extracellular solution containing 145 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES, and 5 mM glucose, pH 7.4. A microperfusion system allowed local application and rapid change of the different experimental media. IeC- and media were identical with the extracellular solution except that IeC and replaced CleC as the dominant extracellular anions. All extracellular media also contained 10 e bumetamide to inhibit the CleC/Na+/K+ cotransporter.
Single-cell fluorescence intensity was measured from digital-image processing and displayed against time. Fluorescence intensity was standardized according to the equation F = (F eC Fo)/Fo x 100, where F is the relative fluorescence and Fo is the fluorescence intensity measured in the presence of IeC. The membrane permeability to halides was determined as the rate of SPQ dequenching upon perfusion with nitrates. At least three successive data points were collected immediately after the medium application and then fitted using a linear regression analysis. The slope of the straight line reflected the membrane permeability to halides and was used as an index of CFTR activity.
Immunohistochemical Assays. After injection, cells were placed for 12 h in a medium containing 10% fetal bovine serum (FBS). Then, cells were placed in a medium without FBS for an additional 12 h to reduce basal levels of phosphorylation. After aspiration of the culture medium, cells were treated by adding fresh medium without FBS, containing or not 1 e CGP-12177 for 5 min at 37°C. Cells were washed with phosphate-buffered saline and fixed successively in 3% paraformaldehyde for 30 min at 4°C and 100% methanol for 10 min at eC20°C. After washing with phosphate-buffered saline, the fixed cells were incubated with primary monoclonal antibody raised against human 3-AR (a gift from GlaxoSmithKline, Welwyn Garden City, Hertfordshire, UK) for 3 h at room temperature. This antiserum was revealed with peroxidase-conjugated second serum. After the control of the efficiency of the 3-AR staining procedure under microscope, a second staining was performed with the phospho-p44/42 MAPK antiserum (Thr202/Tyr204; Cell Signaling Technology Inc., Beverly, MA) overnight at 4°C and revealed with alkaline phosphatase-conjugated second serum.
Drugs. Intracellular cAMP was increased with a mixture containing 10 e forskolin plus 100 e 3-isobutyl-1-methylxanthine (both from Sigma-Aldrich, Saint Quentin Fallavier, France). CGP-12177 was purchased from Sigma-RBI (Clermont-Ferrand, France); salbutamol, (eC)-isoproterenol, wortmannin, and bumetamide were from Sigma-Aldrich; and pertussis toxin (PTX), PD98059, LY294002, and U0126 were from Calbiochem (Meudon, France). The two PKA inhibitors, Rp-8-bromoadenosine-3',5'-cyclic monophosphorothiorate (Rp-8-Br-cAMPS) and Rp-8-(chlorophenyl-thio)adenosine-3',5'-cyclic monophosphorothiorate (Rp-8-CPT-cAMPS) were obtained from Biolog (Bremen, Germany). For SPQ experiments, drugs were dissolved in dimethyl sulfoxide (Sigma-Aldrich) so that final concentration of the solvent was less than 0.1%.
Statistical Analysis. Data are expressed as the means ± S.E. of n experiments. The statistical significance of a drug effect versus baseline was assessed using a one-way analysis of variance completed when appropriate by a Bonferroni test. The significant influence of a pretreatment on a drug effect was assessed by two-way analysis of variance.
Results
CFTR Activation by 3-AR Stimulation. In A549 cells injected only with the plasmid encoding human CFTR, the application of 10 e forskolin, a direct activator of adenylate cyclase, increased the p value by 8-fold, whereas the application of 1 e CGP-12177 did not modify basal CFTR activity (Fig. 1A). In A549 cells coexpressing CFTR and 3-AR, the CFTR basal permeability to halides (p) was 0.15 ± 0.01 mineC1. The application of increasing concentrations (0.01eC10 e) of CGP-12177 (a 3-AR agonist) produced an increase of p values in a concentration-dependent manner. The maximal effect was obtained at 1 e. At this concentration, CGP-12177 increased p values by approximately 6-fold after 15 min of perfusion (Fig. 1B). Then, for the following experiments, CGP-12177 was used at 1 e.
Involvement of Gi/o Protein in CFTR Activation Induced by 3-AR Stimulation. To determine the putative involvement of PKA in the CFTR regulation by 3-AR stimulation, cells were incubated for 20 min with a mixture of two PKA inhibitors, Rp-8-Br-cAMPS (100 e) and Rp-8-CPT-cAMPS (100 e). In such conditions, the effect of CGP-12177 on CFTR activity was not modified (Fig. 2A). To check the efficiency of PKA inhibitors, the effects of isoproterenol, a nonselective -AR agonist, and salbutamol, a 2-AR agonist, were investigated in the absence and presence of PKA inhibitors (Rp-8-Br-cAMPS and Rp-8-CPT-cAMPS). In A549 cells injected with CFTR alone, isoproterenol through stimulation of the endogenous 1- and/or 2-AR stimulation increased significantly the activity of CFTR. This effect was abolished in cells pretreated with PKA inhibitors (Fig. 2B). In cells injected with CFTR and 2-AR, 10 e salbutamol increased significantly the CFTR activity. This effect was fully abolished in cells pretreated with PKA inhibitors (Fig. 2C).
The pretreatment with PTX (500 ng/ml) during 36 h did not modify the activation of CFTR induced by forskolin in cells expressing CFTR alone (Fig. 3A). In contrast, in cells coexpressing CFTR and 3-AR, the response to CGP-12177 was fully abolished after pretreatment with PTX (Fig. 3B), suggesting that CFTR activation by 3-AR stimulation involved Gi/o proteins. To determine the Gi/o subunits (i/o and/or ) involved in this effect, we coinjected A549 cells with the plasmid encoding for the C-terminal tail of -ARK (5 e/ml), an inhibitor of the complex (Koch et al., 1994), in addition to plasmids encoding for CFTR and 3-AR. In such conditions, the CFTR activation induced by CGP-12177 was fully inhibited. In the other hand, the forskolin response was not modified (Fig. 4).
Involvement of the PI3K-MAPK Pathway in CFTR Activation Induced by 3-AR Stimulation. In cells pretreated with 50 e wortmannin, a PI3K inhibitor (Gerhardt et al., 1999), for 25 min, CGP-12177 only increased p values by approximately 2-fold (Fig. 5A). After 25-min pretreatment with 10 e LY294002, a highly selective PI3K inhibitor (Gerhardt et al., 1999), the response to CGP-12177 was fully abolished (Fig. 5B). The effects of CGP-12177 were also abolished in cells deprived of FBS, an activator of MAPK pathway, and pretreated for 24 h with either 20 e PD98059 (Fig. 6A) or 10 e U0126 (Fig. 6B), two highly selective ERK1/2 MAPK inhibitors (Gerhardt et al., 1999).
To strengthen the MAPK involvement in CFTR regulation suggested by the present pharmacological studies, a sequential dual immunolabeling method was used. To avoid the activation of MAPK by the serum, the A549 culture medium was replaced by a fresh medium without FBS. Successfully injected cells with 3-AR were identified by fluorescein isothiocyanate under an incident light fluorescence microscope (Fig. 7). Then, cells were incubated in the absence or the presence of 1 e CGP-12177 during 5 min. In the first step, incubation with the antibody raised against human 3-AR was performed. We verified that the noninjected cells did not present any staining, whereas cells injected with 3-AR were labeled after this procedure (Fig. 7, B and C). In the second step, a second incubation was performed with the phospho-p44/42-MAPK antiserum. No labeling was obtained in wild-type or microinjected cells untreated by CGP-12177. On the other hand, a strong nuclear and perinuclear labeling was obtained with the phospho-p44/42 MAPK antibody only in cells injected with 3-AR and pretreated with CGP-12177 (Fig. 7, B and C).
Activation of CFTR through the MAPK Pathway Independently of 3-AR Stimulation. Another set of experiments was performed in A549 cells injected with CFTR alone to determine whether CFTR activation in response to the MAPK pathway recruitment might be observed in the absence of 3-AR stimulation. A 15-min application of FBS, which is known to be an activator of MAPK pathway (Schramek et al., 1996), increased p values by 5-fold (Fig. 8A). The pretreatment of cells with PD98059 significantly decreased the CFTR activation induced by FBS without modifying the response to forskolin (Fig. 8A). In T84 cells, which endogenously express the CFTR protein (Cohn et al., 1992), the application of FBS increased p values by 3-fold. CFTR activation induced by FBS was also abolished by the pretreatment of T84 cells with PD98059 (Fig. 8B).
Discussion
We have demonstrated previously that 3-ARs were functionally coupled to the CFTR protein (Leblais et al., 1999). In the present study, using pharmacological and biochemical approaches, we have demonstrated that the regulation of CFTR by 3-ARs in A549 cells is independent of the cAMP/PKA pathway but involves a Gi/o protein/PI3K/ERK1/2 MAPK pathway. This last pathway could also regulate CFTR activity independently of the 3-AR stimulation.
3-AR stimulation induced by CGP-12177 increased CFTR activity in a concentration-dependent manner, with a maximal effect at 1 e. This increase was not observed in A549 cells only injected with the plasmid encoding CFTR alone, demonstrating that the partial 3-AR agonist, CGP-12177, activated only 3-ARs in our recombinant system. This increase of the CFTR activity induced by 3-AR stimulation was not modified by the pretreatment of A549 cells with PKA inhibitors. In contrast, in cells expressing only CFTR, the increased CFTR activity induced by the stimulation of endogenous 1- and 2-ARs by isoproterenol was abolished in PKA inhibitor-pretreated cells. Furthermore, in cells coexpressing CFTR and 2-AR, salbutamol also produced an activation of CFTR that was abolished by PKA inhibitors. All together, these results demonstrated that 1) the blockade of PKA was efficient in our model, 2) the GeC-adenylyl cyclase/cAMP/PKA pathway was functional in A549 cells, and 3) the overexpression of the 2-AR did not disturb its coupling to cAMP/PKA pathway. Thus, our results demonstrated that 3-AR did not link to the cAMP/PKA pathway in A549 cells, although this pathway was present and functional.
CFTR activation by the 3-AR stimulation was abolished by pretreatment with PTX, indicating that PTX-sensitive G proteins, Gi/0, were involved in this effect. Several studies performed in different cellular types or tissues have already reported that 3-ARs could be linked to Gs and/or Gi/0 proteins. In CHO cells transfected with rat 3-ARs (Granneman et al., 1991) and murine 3T3-F442A preadipocytes (Feve et al., 1991), 3-ARs seemed to be coupled to Gs proteins, leading to an increase in intracellular cAMP level. In rat and mice adipocytes, in addition to their coupling to Gs, 3-ARs coupled to Gi/0 proteins, which inhibited adenylyl cyclase (Chaudhry et al., 1994; Begin-Heick, 1995). Finally, in some tissues such as human endomyocardial biopsies, 3-ARs were shown to be exclusively linked to Gi/0 proteins (Gauthier et al., 1996). The coupling of 3-ARs to several types of G proteins is not specific to this receptor. Such apparently "promiscuous" coupling has been actually observed with several G protein-coupled receptors, even in native cells (Gudermann et al., 1997). Thus, the 2-AR has been shown to activate both Gs and Gi/0 proteins in rat adult cardiac myocytes (Xiao et al., 1995).
Then, to determine whether i/0 subunit and/or complex of the Gi/o protein were involved in CFTR activation induced by the 3-AR stimulation, A549 cells were coinjected with the plasmid encoding the C-terminal region of -ARK, an inhibitor of the complex, in addition to the plasmids encoding CFTR and 3-ARs. In such conditions, the CFTR activation induced by the 3-AR stimulation was fully abolished, indicating a total regulation of CFTR by the complex in our model. It should be noted that several studies reported that CFTR activity could be regulated by the i subunit. In cultured human airway epithelial cells, the purified i subunit produced an inhibition of CFTR-induced current, probably through an impairment of the CFTR trafficking (Schwiebert et al., 1994; Schreiber et al., 2001). In the presence of exogenous complex, the inhibition of CFTR-induced current was abolished, probably because of the assembly of the different subunits in an inactive complex (Stow et al., 1991).
In the present study, the pretreatment of A549 cells by selective inhibitors of PI3K and ERK1/2 MAPK abolished the CFTR activation induced by 3-AR stimulation. These pharmacological data were strengthened by the immunohistochemical study, showing a strong nuclear and perinuclear labeling with the phospho-p44/42 MAPK antibody in A549 cells incubated with the 3-AR agonist. This labeling was absent in wild-type or microinjected cells untreated with the 3-AR agonist. All together, these results demonstrate that 3-AR regulates CFTR activity through activation of a Gi/o protein/PI3K/MAPK pathway. Our results are in agreement with those of previous studies reporting that 3-ARs stimulated the MAPK pathway via the activation of Gi/0 proteins. In CHO/K1 cells overexpressing the human 3-AR, the 3-AR was simultaneously coupled to Gs and Gi/0 proteins, leading to the activation of adenylyl cyclase and MAPK, respectively (Gerhardt et al., 1999). In human embryonic kidney 293 cells expressing human 3-AR as well as in intact 3T3-F442A adipocytes, CL 316243, a 3-AR agonist, stimulated the MAPK pathway in a PTX-sensitive Gi-dependent manner rather than in a Gs-dependent manner (Soeder et al., 1999). In contrast, in 3T3-L1 adipocytes, Mizuno and colleagues (2000) proposed that several 3-AR agonists (BRL 37344, CGP-12177, and SR 58611A) produced an ERK1/2 phosphorylation via a pathway involving Gs protein and PKA but not via a Gi protein-dependent pathway. The different coupling of 3-ARs to Gs and/or Gi according to the studies could result from the diversity of the pharmacological agents used and/or the cell types.
In the present work, we have demonstrated that CFTR regulation by MAPK also occurred independently of the 3-AR stimulation. This response has been observed both in a recombinant system, A549 cells injected with the plasmid encoding for CFTR, and in native cells, T84, which endogenously express CFTR. To our knowledge, this is the first report relating the regulation of CFTR activity through the MAPK, although an endogenous activation of MAPK has been reported in CF (Ratner et al., 2001). Through the binding on their epithelial receptor sites, adherent bacteria produced increases in intracellular Ca2+ concentration, leading to MAPK activation and initiated interleukin-8 production (Ratner et al., 2001). This latter effect contributed to the general proinflammatory medium in CF airways (Heeckeren et al., 1997). However, the consequences of the MAPK activation were not identified.
The present work has been carried out in experimental cell systems and has highlighted a new regulation pathway for CFTR protein, the Gi/o/PI3K/ERK1/2 MAPK pathway. Further investigations are clearly necessary to determine whether MAPK could modulate CFTR activity in epithelial cells from healthy patients and those with CF. Until now, it has been widely believed that cAMP-dependent phosphorylation of CFTR is the predominant physiological mechanism for activating CFTR in a number of epithelial cells from airways, pancreas, intestines, and sweat glands. However, our results demonstrate that an alternative pathway of CFTR regulation by -AR stimulation, involving the 3-AR, exists. These findings might play an important role in airway pathologies in which 1- and 2-ARs are decreased, such as in CF (Sharma and Jeffery, 1990).
Acknowledgements
We thank Dr Jacques Noireaud for reading and criticizing the manuscript and Karine Laurent and Morteeza Erfanian for their expert technical assistance with cell cultures.
doi:10.1124/mol.104.002097.
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