Date Published: October 31, 2014
Publisher: Public Library of Science
Author(s): Christophe Faisy, Stanislas Grassin-Delyle, Sabine Blouquit-Laye, Marion Brollo, Emmanuel Naline, Alain Chapelier, Philippe Devillier, Zhuo Zhang.
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.
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.
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.
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.
Wnt (wingless/integrated) is a large family of secreted glycoproteins with highly conserved cysteine residues involved in lung development and diseases . The WNT gene family includes 19 members encoding Wnts, which can activate three distinct signaling pathways. The best characterized canonical Wnt/β-catenin pathway implicated the inhibition of glycogen synthase kinase GSK-3β, resulting a cytoplasmic accumulation of β-catenin and its nuclear translocation –. The two non-canonical Wnt pathways do not require β-catenin as a co-transcription factor , . Therefore, the Wnt/Ca2+ signaling pathway is mediated by protein kinase C (PKC) and the Wnt/planar cell polarity (PCP) pathway activates the small G proteins Rho and the mitogen-activated proteins kinases (MAPK) cascade or alternatively triggers activation of the c-Jun-N-terminal kinase (JNK) leading to the transcription of target genes through the activator protein-1 (AP-1) stimulation , , . Wnts are expressed in the distal mesenchyme and in airway epithelium and act via the seven membrane-spanning Fzd cell-surface receptors , , , . The Fzd family includes 10 distinct members , , , most of which can activate β-catenin signaling when combined with the lipoprotein-related co-receptors LRP5/6 , . The Wnt-induced phosphorylation of LRP is critical for Fzd-LRP association , . Unlike the canonical pathways, the Wnt/Ca2+ and the Wnt/PCP signaling pathways are independent from LRP5/6 , . The Wnt signaling pathways are responsible for several cellular processes, including cell movement and polarity, proliferation and differentiation of the airway epithelium, airway epithelial repair and cytoskeletal reorganization after airway stretching , , , –. Wnts also exert autocrine-signaling activity on airway epithelial cells , . Depending on the cellular context, Wnts stimulate the canonical signaling pathway, thereby up-regulating inflammatory genes such as cyclo-oxygenase 2 (COX-2), interleukine-8 (IL-8), and matrix metalloproteinases (MMPs) , , , . Conversely, the inflammatory mediator nuclear factor κ-B (NF-κB) modulates Fzd mRNA expression and GSK-3β suppression can induce NF-κB-mediated transcription , . It has been suggested that modulating the β-catenin pathway in the airway epithelium could have promising impact on airway inflammation and remodeling , , . However, the involvement of the Wnt/β-catenin signaling pathway in human airway responsiveness remains scarce.
This study shows that fenoterol, a β2-agonist with high intrinsic efficacy, sensitized human bronchi at least in part via the Wnt/β-catenin signaling, notably the LRP5/6 co-receptor and the nuclear recruitment of TCF/LEF. Conversely, fenoterol exposure did not change the Wnt secretion and the mRNA expression of genes interacting with the regulation of Wnt proteins, Fzd-receptors, LRP5/6 co-receptor, PKC, p38/JNK or Rho/MAPK, and cAMP–PKA cascade. Moreover, the present study confirms the implication of p38/JNK and Rho/MAPK signaling pathways in the fenoterol-induced sensitizing effect  which could also be explained by the involvement of the non-canonical Wnt signaling. Taken together, our observations suggest that 15 hours of fenoterol exposure provokes the recruitment of Wnt/β-catenin signaling via the LRP5/6 co-receptor and/or Fzd-receptors phosphorylation, regardless of the Wnt secretion. In this way, the involvement of Wnt/β-catenin signaling in the fenoterol-sensitization suggests a mechanism of functional selectivity or biased agonism in connection with the β2-adrenoceptor stimulation –. However, the stimulation of Wnt/β-catenin signaling pathway by fenoterol does not fully account for the fenoterol-induced sensitizing effect. Indeed, previous reports have shown that untoward effect was mediated by various pro-inflammatory mediators activated via the prolonged stimulation of the β2-receptor –. Pertinently, co-incubation with anti-inflammatory drugs or proinflammatory mediator receptor antagonist such as corticosteroids or non-steroidal anti-inflammatory drugs did not fully inhibit the fenoterol-sensitization , . The involvement of the Wnt/β-catenin signaling pathway, which is not directly influenced by these drugs, brings a better understanding of previous published data.
Collectively, our pharmacological investigations indicated that fenoterol may activate, at least in part, the Wnt/β-catenin signaling pathway in human isolated bronchi, suggesting a mechanism of biased agonism. To a clinical point of view, these findings highlight the interest of Wnt signaling pathways in inflammatory airway diseases. 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.