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Wnt/β-catenin signaling modulates human airway sensitization induced by β2-adrenoceptor stimulation.

Faisy C, Grassin-Delyle S, Blouquit-Laye S, Brollo M, Naline E, Chapelier A, Devillier P - PLoS ONE (2014)

Bottom Line: Compared to paired controls, fenoterol-sensitization was abolished by inhibition/blockage of the Wnt/β-catenin signaling, especially the cell-surface LRP5/6 co-receptors or Fzd receptors (1 µM SFRP1 or 1 µM DKK1) and the nuclear recruitment of TCF/LEF transcriptions factors (0.3 µM FH535).Wnt proteins secretion did not seem to be involved in the fenoterol-induced sensitization since the mRNA expression of Wnt remained low after fenoterol exposure and the inactivator of Wnt secretion (1 µM IWP2) had no effect on the fenoterol-sensitization.Collectively, our pharmacological investigations indicate that fenoterol-sensitization is modulated by the inhibition/blockage of canonical Wnt/β-catenin pathway, suggesting a phenomenon of biased agonism in connection with the β2-adrenoceptor stimulation.

View Article: PubMed Central - PubMed

Affiliation: Unité Propre de Recherche de l'Enseignement Supérieur, Equipe d'Accueil 220, Université Versailles Saint-Quentin, Hôpital Foch, Suresnes, France; Medical Intensive Care Unit, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, Paris, France.

ABSTRACT

Background: Regular use of β2-agonists may enhance non-specific airway responsiveness. The wingless/integrated (Wnt) signaling pathways are responsible for several cellular processes, including airway inflammation and remodeling while cAMP-PKA cascade can activate the Wnt signaling. We aimed to investigate whether the Wnt signaling pathways are involved in the bronchial hyperresponsiveness induced by prolonged exposure to β2-adrenoceptor agonists in human isolated airways.

Methods: Bronchi were surgically removed from 44 thoracic surgery patients. After preparation, bronchial rings and primary cultures of bronchial epithelial cells were incubated with fenoterol (0.1 µM, 15 hours, 37 °C), a β2-agonist with high intrinsic efficacy. The effects of inhibitors/blockers of Wnt signaling on the fenoterol-induced airway sensitization were examined and the impact of fenoterol exposure on the mRNA expression of genes interacting with Wnt signaling or cAMP-PKA cascade was assessed in complete bronchi and in cultured epithelial cells.

Results: Compared to paired controls, fenoterol-sensitization was abolished by inhibition/blockage of the Wnt/β-catenin signaling, especially the cell-surface LRP5/6 co-receptors or Fzd receptors (1 µM SFRP1 or 1 µM DKK1) and the nuclear recruitment of TCF/LEF transcriptions factors (0.3 µM FH535). Wnt proteins secretion did not seem to be involved in the fenoterol-induced sensitization since the mRNA expression of Wnt remained low after fenoterol exposure and the inactivator of Wnt secretion (1 µM IWP2) had no effect on the fenoterol-sensitization. Fenoterol exposure did not change the mRNA expression of genes regulating Wnt signaling or cAMP-PKA cascade.

Conclusions: Collectively, our pharmacological investigations indicate that fenoterol-sensitization is modulated by the inhibition/blockage of canonical Wnt/β-catenin pathway, suggesting a phenomenon of biased agonism in connection with the β2-adrenoceptor stimulation. Future experiments based on the results of the present study will be needed to determine the impact of prolonged fenoterol exposure on the extra- and intracellular Wnt signaling pathways at the protein expression level.

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Related in: MedlinePlus

Schematic representation of the canonical (Wnt/β-catenin) and non-canonical (Wnt/Ca2+ and Wnt/PCP) signaling pathways.Drugs used in the experiments and their action on Wnt signaling pathways are figured in black. Activation and inhibition are symbolized by  and , respectively. In the presence of Wnt ligand, Fzd and LRP5/6 form a receptor complex leading to the recruitment of cytosolic proteins Dsh and LRP5/6 phosphorylation. The phosphorylation and partial internalization of LRP5/6 initiate the disruption of the destruction complex (axin, APC, CK-1, and GSK-3β), allowing cytosolic β-catenin accumulation and then translocation into the nucleus, where β-catenin serves as a coactivator of TCF/LEF transcription factors to control gene transcription. Depending on the cellular context, the non-canonical Wnt signaling pathway is stimulated by the binding of Fzd and ROR2/RYK coreceptors. The Wnt/Ca2+ pathway induces PKC activation via the intracellular Ca2+ increase. The Wnt/PCP pathway activates the small G proteins Rho and the MAPK cascade via the recruitment of the cytosolic proteins Dsh and DAAM1, or, alternatively, triggers activation of JNK leading to the transcription of target genes through AP-1 activation.
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pone-0111350-g001: Schematic representation of the canonical (Wnt/β-catenin) and non-canonical (Wnt/Ca2+ and Wnt/PCP) signaling pathways.Drugs used in the experiments and their action on Wnt signaling pathways are figured in black. Activation and inhibition are symbolized by and , respectively. In the presence of Wnt ligand, Fzd and LRP5/6 form a receptor complex leading to the recruitment of cytosolic proteins Dsh and LRP5/6 phosphorylation. The phosphorylation and partial internalization of LRP5/6 initiate the disruption of the destruction complex (axin, APC, CK-1, and GSK-3β), allowing cytosolic β-catenin accumulation and then translocation into the nucleus, where β-catenin serves as a coactivator of TCF/LEF transcription factors to control gene transcription. Depending on the cellular context, the non-canonical Wnt signaling pathway is stimulated by the binding of Fzd and ROR2/RYK coreceptors. The Wnt/Ca2+ pathway induces PKC activation via the intracellular Ca2+ increase. The Wnt/PCP pathway activates the small G proteins Rho and the MAPK cascade via the recruitment of the cytosolic proteins Dsh and DAAM1, or, alternatively, triggers activation of JNK leading to the transcription of target genes through AP-1 activation.

Mentions: Tissues were dissected and washed in oxygenated Krebs-Henseleit solution (composition in mM: 119 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 25 NaHCO3 and 11.7 glucose). For function, rings of similar length from the same bronchi were randomly distributed in paired groups. Half of the rings were placed in oxygenated Krebs-Henseleit solution, in parallel the other half was placed in oxygenated Krebs-Henseleit solution with 0.1 µM fenoterol (Boehringer-Ingelheim, Germany) and both were incubated (37°C) for 15 hours as previously described [22]. To investigate the implication of the Wnt-signaling pathways in the fenoterol-induced bronchial hyperresponsiveness, experiments were run in parallel (control and pretreated groups) in the absence or presence of (Fig. 1): 1) two extracellular Wnt pathway modulators, which inhibit Wnt signaling by directly sequestering Wnt ligands and inhibiting both canonical and non-canonical Wnt signaling [3], [4], [8], [13], [25]: secreted fizzled-related protein 1 (SFRP1, 1 µM) or WNT inhibitory factor 1 (WIF1, 1 µM); 2) an extracellular Wnt antagonist, which inhibits Wnt/β-catenin signaling by binding LRP5/6 [1], [2], [4], [28], [29]: Dickkopf protein family 1 (DKK1, 1 µM); 3) an intracellular inhibitor of Wnt processing and secretion, which inactivates a regulator of Wnt secretion (porcupine) [4], [30]: IWP2 (1 µM); 4) a selective Rho-kinase (ROCK1/2) blocker, which inhibits the Wnt-planar cell polarity (PCP) pathway [12]: Y27632 (1 µM); 5) a blocker of phosphoinositide 3-kinases (PI3Ks), which inhibits the Wnt-Ca2+ pathway (4, 15): wortmaninn (1 µM); 6) a selective peroxysome proliferator activated receptor-γ (PPAR-γ) agonist, which inhibits both noncanonical Wnt/JNK and canonical Wnt/β-catenin signaling [31], [32]: pioglitazone (1 µM); 7) a modulator of Wnt signaling in the nucleus, which inhibits the recruitment of the co-activators β-catenin (TCF/LEF) [4]: FH535 (0.3 µM). Because of its very short half-life, wortmaninn was added only after 15 hours of incubation at 37°C, immediately to the organ bath after the last change of fresh Krebs−Henseleit solution during the equilibration period. SFRP1, WIF1 and DKK1 were purchased from Abcam (Cambridge, MA). IWP2 came from Tocris Biosciences (Bristol, United Kingdom). Y27632 was purchased from Cayman Chemicals (Ann Arbor, MI). Pioglitazone came from Adipogen (San Diego, CA). Wortmannin and FH535 were purchased from Enzo Life Sciences (Farmingdale, NY). All drugs were dissolved in distilled water except for wortmannin, FH535 and pioglitazone which were dissolved in pure ethanol and DMSO and then diluted in Krebs-Henseleit solution. The final ethanol or DMSO concentration (0.03%) did not alter airway tone or contractility [19]. For RNA isolation, paired bronchial rings were immediately (H0) immersed in RNAlater (Sigma, St. Louis, MO) or were first incubated without or with 0.1 µM fenoterol in oxygenated Krebs-Henseleit solution (37°C) for 15 hours (H15) before immersion in RNAlater. Bronchi were then stored at −80°C until use.


Wnt/β-catenin signaling modulates human airway sensitization induced by β2-adrenoceptor stimulation.

Faisy C, Grassin-Delyle S, Blouquit-Laye S, Brollo M, Naline E, Chapelier A, Devillier P - PLoS ONE (2014)

Schematic representation of the canonical (Wnt/β-catenin) and non-canonical (Wnt/Ca2+ and Wnt/PCP) signaling pathways.Drugs used in the experiments and their action on Wnt signaling pathways are figured in black. Activation and inhibition are symbolized by  and , respectively. In the presence of Wnt ligand, Fzd and LRP5/6 form a receptor complex leading to the recruitment of cytosolic proteins Dsh and LRP5/6 phosphorylation. The phosphorylation and partial internalization of LRP5/6 initiate the disruption of the destruction complex (axin, APC, CK-1, and GSK-3β), allowing cytosolic β-catenin accumulation and then translocation into the nucleus, where β-catenin serves as a coactivator of TCF/LEF transcription factors to control gene transcription. Depending on the cellular context, the non-canonical Wnt signaling pathway is stimulated by the binding of Fzd and ROR2/RYK coreceptors. The Wnt/Ca2+ pathway induces PKC activation via the intracellular Ca2+ increase. The Wnt/PCP pathway activates the small G proteins Rho and the MAPK cascade via the recruitment of the cytosolic proteins Dsh and DAAM1, or, alternatively, triggers activation of JNK leading to the transcription of target genes through AP-1 activation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4216012&req=5

pone-0111350-g001: Schematic representation of the canonical (Wnt/β-catenin) and non-canonical (Wnt/Ca2+ and Wnt/PCP) signaling pathways.Drugs used in the experiments and their action on Wnt signaling pathways are figured in black. Activation and inhibition are symbolized by and , respectively. In the presence of Wnt ligand, Fzd and LRP5/6 form a receptor complex leading to the recruitment of cytosolic proteins Dsh and LRP5/6 phosphorylation. The phosphorylation and partial internalization of LRP5/6 initiate the disruption of the destruction complex (axin, APC, CK-1, and GSK-3β), allowing cytosolic β-catenin accumulation and then translocation into the nucleus, where β-catenin serves as a coactivator of TCF/LEF transcription factors to control gene transcription. Depending on the cellular context, the non-canonical Wnt signaling pathway is stimulated by the binding of Fzd and ROR2/RYK coreceptors. The Wnt/Ca2+ pathway induces PKC activation via the intracellular Ca2+ increase. The Wnt/PCP pathway activates the small G proteins Rho and the MAPK cascade via the recruitment of the cytosolic proteins Dsh and DAAM1, or, alternatively, triggers activation of JNK leading to the transcription of target genes through AP-1 activation.
Mentions: Tissues were dissected and washed in oxygenated Krebs-Henseleit solution (composition in mM: 119 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 25 NaHCO3 and 11.7 glucose). For function, rings of similar length from the same bronchi were randomly distributed in paired groups. Half of the rings were placed in oxygenated Krebs-Henseleit solution, in parallel the other half was placed in oxygenated Krebs-Henseleit solution with 0.1 µM fenoterol (Boehringer-Ingelheim, Germany) and both were incubated (37°C) for 15 hours as previously described [22]. To investigate the implication of the Wnt-signaling pathways in the fenoterol-induced bronchial hyperresponsiveness, experiments were run in parallel (control and pretreated groups) in the absence or presence of (Fig. 1): 1) two extracellular Wnt pathway modulators, which inhibit Wnt signaling by directly sequestering Wnt ligands and inhibiting both canonical and non-canonical Wnt signaling [3], [4], [8], [13], [25]: secreted fizzled-related protein 1 (SFRP1, 1 µM) or WNT inhibitory factor 1 (WIF1, 1 µM); 2) an extracellular Wnt antagonist, which inhibits Wnt/β-catenin signaling by binding LRP5/6 [1], [2], [4], [28], [29]: Dickkopf protein family 1 (DKK1, 1 µM); 3) an intracellular inhibitor of Wnt processing and secretion, which inactivates a regulator of Wnt secretion (porcupine) [4], [30]: IWP2 (1 µM); 4) a selective Rho-kinase (ROCK1/2) blocker, which inhibits the Wnt-planar cell polarity (PCP) pathway [12]: Y27632 (1 µM); 5) a blocker of phosphoinositide 3-kinases (PI3Ks), which inhibits the Wnt-Ca2+ pathway (4, 15): wortmaninn (1 µM); 6) a selective peroxysome proliferator activated receptor-γ (PPAR-γ) agonist, which inhibits both noncanonical Wnt/JNK and canonical Wnt/β-catenin signaling [31], [32]: pioglitazone (1 µM); 7) a modulator of Wnt signaling in the nucleus, which inhibits the recruitment of the co-activators β-catenin (TCF/LEF) [4]: FH535 (0.3 µM). Because of its very short half-life, wortmaninn was added only after 15 hours of incubation at 37°C, immediately to the organ bath after the last change of fresh Krebs−Henseleit solution during the equilibration period. SFRP1, WIF1 and DKK1 were purchased from Abcam (Cambridge, MA). IWP2 came from Tocris Biosciences (Bristol, United Kingdom). Y27632 was purchased from Cayman Chemicals (Ann Arbor, MI). Pioglitazone came from Adipogen (San Diego, CA). Wortmannin and FH535 were purchased from Enzo Life Sciences (Farmingdale, NY). All drugs were dissolved in distilled water except for wortmannin, FH535 and pioglitazone which were dissolved in pure ethanol and DMSO and then diluted in Krebs-Henseleit solution. The final ethanol or DMSO concentration (0.03%) did not alter airway tone or contractility [19]. For RNA isolation, paired bronchial rings were immediately (H0) immersed in RNAlater (Sigma, St. Louis, MO) or were first incubated without or with 0.1 µM fenoterol in oxygenated Krebs-Henseleit solution (37°C) for 15 hours (H15) before immersion in RNAlater. Bronchi were then stored at −80°C until use.

Bottom Line: Compared to paired controls, fenoterol-sensitization was abolished by inhibition/blockage of the Wnt/β-catenin signaling, especially the cell-surface LRP5/6 co-receptors or Fzd receptors (1 µM SFRP1 or 1 µM DKK1) and the nuclear recruitment of TCF/LEF transcriptions factors (0.3 µM FH535).Wnt proteins secretion did not seem to be involved in the fenoterol-induced sensitization since the mRNA expression of Wnt remained low after fenoterol exposure and the inactivator of Wnt secretion (1 µM IWP2) had no effect on the fenoterol-sensitization.Collectively, our pharmacological investigations indicate that fenoterol-sensitization is modulated by the inhibition/blockage of canonical Wnt/β-catenin pathway, suggesting a phenomenon of biased agonism in connection with the β2-adrenoceptor stimulation.

View Article: PubMed Central - PubMed

Affiliation: Unité Propre de Recherche de l'Enseignement Supérieur, Equipe d'Accueil 220, Université Versailles Saint-Quentin, Hôpital Foch, Suresnes, France; Medical Intensive Care Unit, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, Paris, France.

ABSTRACT

Background: Regular use of β2-agonists may enhance non-specific airway responsiveness. The wingless/integrated (Wnt) signaling pathways are responsible for several cellular processes, including airway inflammation and remodeling while cAMP-PKA cascade can activate the Wnt signaling. We aimed to investigate whether the Wnt signaling pathways are involved in the bronchial hyperresponsiveness induced by prolonged exposure to β2-adrenoceptor agonists in human isolated airways.

Methods: Bronchi were surgically removed from 44 thoracic surgery patients. After preparation, bronchial rings and primary cultures of bronchial epithelial cells were incubated with fenoterol (0.1 µM, 15 hours, 37 °C), a β2-agonist with high intrinsic efficacy. The effects of inhibitors/blockers of Wnt signaling on the fenoterol-induced airway sensitization were examined and the impact of fenoterol exposure on the mRNA expression of genes interacting with Wnt signaling or cAMP-PKA cascade was assessed in complete bronchi and in cultured epithelial cells.

Results: Compared to paired controls, fenoterol-sensitization was abolished by inhibition/blockage of the Wnt/β-catenin signaling, especially the cell-surface LRP5/6 co-receptors or Fzd receptors (1 µM SFRP1 or 1 µM DKK1) and the nuclear recruitment of TCF/LEF transcriptions factors (0.3 µM FH535). Wnt proteins secretion did not seem to be involved in the fenoterol-induced sensitization since the mRNA expression of Wnt remained low after fenoterol exposure and the inactivator of Wnt secretion (1 µM IWP2) had no effect on the fenoterol-sensitization. Fenoterol exposure did not change the mRNA expression of genes regulating Wnt signaling or cAMP-PKA cascade.

Conclusions: Collectively, our pharmacological investigations indicate that fenoterol-sensitization is modulated by the inhibition/blockage of canonical Wnt/β-catenin pathway, suggesting a phenomenon of biased agonism in connection with the β2-adrenoceptor stimulation. Future experiments based on the results of the present study will be needed to determine the impact of prolonged fenoterol exposure on the extra- and intracellular Wnt signaling pathways at the protein expression level.

Show MeSH
Related in: MedlinePlus