Anchored PDE4 regulates chloride conductance in wild‐type and ΔF508‐CFTR human airway epithelia

Blanchard, Elise; Zlock, Lorna; Lao, Anna; Mika, Delphine; Namkung, W.; Xie, Moses; Scheitrum, Colleen; Gruenert, Dieter C.; Finkbeiner, Walter E.; Conti, Marco; Richter, Wito

doi:10.1096/fj.13-240861

Cited by 34 publications

(36 citation statements)

References 65 publications

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“…Taken together, these data suggest that both correctors modulate the balance between the macromolecular complexes regulating the cAMP signaling machinery in the vicinity of CFTR that either attenuate or enhance its activation (Blanchard et al, 2014;Li and Naren, 2005) and, in this way, restore the cAMP and PKA compartmentalization in the membrane region and improve the cellular environment that has been perturbed by the lack of a functional CFTR on the cell membrane.…”

Section: Discussionmentioning

confidence: 80%

Correctors of mutant CFTR enhance subcortical cAMP–PKA signaling through modulating ezrin phosphorylation and cytoskeleton organization

Abbattiscianni

Favia

Mancini

et al. 2016

Journal of Cell Science

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The most common mutation of the cystic fibrosis transmembrane regulator (CFTR) gene, F508del, produces a misfolded protein resulting in its defective trafficking to the cell surface and an impaired chloride secretion. Pharmacological treatments partially rescue F508del CFTR activity either directly by interacting with the mutant protein and/or indirectly by altering the cellular protein homeostasis. Here, we show that the phosphorylation of ezrin together with its binding to phosphatidylinositol-4,5-bisphosphate (PIP 2 ) tethers the F508del CFTR to the actin cytoskeleton, stabilizing it on the apical membrane and rescuing the sub-membrane compartmentalization of cAMP and activated PKA. Both the small molecules trimethylangelicin (TMA) and VX-809, which act as 'correctors' for F508del CFTR by rescuing F508del-CFTRdependent chloride secretion, also restore the apical expression of phosphorylated ezrin and actin organization and increase cAMP and activated PKA submembrane compartmentalization in both primary and secondary cystic fibrosis airway cells. Latrunculin B treatment or expression of the inactive ezrin mutant T567A reverse the TMA and VX-809-induced effects highlighting the role of corrector-dependent ezrin activation and actin re-organization in creating the conditions to generate a sub-cortical cAMP pool of adequate amplitude to activate the F508del-CFTR-dependent chloride secretion.

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Section: Discussionmentioning

confidence: 80%

Correctors of mutant CFTR enhance subcortical cAMP–PKA signaling through modulating ezrin phosphorylation and cytoskeleton organization

Abbattiscianni

Favia

Mancini

et al. 2016

Journal of Cell Science

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“…Sodium current was blocked with amiloride. Cells incubated with 10 mM VX-809 alone (Blanchard et al, 2014) showed increased short-circuit current in response to forskolin and genistein, which was inhibited by CFTR inh -172 (Fig. 7A).…”

Section: Cfbe41omentioning

confidence: 98%

Synergy-Based Small-Molecule Screen Using a Human Lung Epithelial Cell Line Yields ΔF508-CFTR Correctors That Augment VX-809 Maximal Efficacy

Phuan¹,

Veit²,

Tan³

et al. 2014

Mol Pharmacol

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The most prevalent cystic fibrosis transmembrane conductance regulator (CFTR) mutation causing cystic fibrosis, DF508, impairs folding of nucleotide binding domain (NBD) 1 and stability of the interface between NBD1 and the membranespanning domains. The interfacial stability defect can be partially corrected by the investigational drug Second-generation DF508-CFTR correctors are needed to improve on the modest efficacy of existing cystic fibrosis correctors. We postulated that a second corrector targeting a distinct folding/interfacial defect might act in synergy with VX-809 or the R1070W suppressor mutation. A biochemical screen for DF508-CFTR cell surface expression was developed in a human lung epithelium-derived cell line (CFBE41o 2 ) by expressing chimeric CFTRs with a horseradish peroxidase (HRP) in the fourth exofacial loop in either the presence or absence of R1070W. Using a luminescence readout of HRP activity, screening of approximately 110,000 small molecules produced nine novel corrector scaffolds that increased cell surface ΔF508-CFTR expression by up to 200% in the presence versus absence of maximal VX-809. Further screening of 1006 analogs of compounds identified from the primary screen produced 15 correctors with an EC 50 , 5 mM. Eight chemical scaffolds showed synergy with VX-809 in restoring chloride permeability in ΔF508-expressing A549 cells. An aminothiazole increased chloride conductance in human bronchial epithelial cells from a DF508 homozygous subject beyond that of maximal VX-809. Mechanistic studies suggested that NBD2 is required for the aminothiazole rescue. Our results provide proof of concept for synergy screening to identify second-generation correctors, which, when used in combination, may overcome the "therapeutic ceiling" of first-generation correctors.

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“…PDE4 family, the primary cAMP-specific enzyme, comprises four genes (PDE4A-D), each with multiple variants that encode more than 20 different protein products (8). The therapeutic potential of PDE4 inhibition in airway inflammatory diseases such as chronic obstructive pulmonary disease (COPD), asthma, and cystic fibrosis (CF) has long been explored (9)(10)(11)(12). Recently, roflumilast has been approved as the first PDE4 inhibitor for oral, once-daily treatment of severe COPD with symptoms of chronic bronchitis and a history of exacerbations (12)(13)(14).…”

mentioning

confidence: 99%

Cross-talk between PKA-Cβ and p65 mediates synergistic induction of PDE4B by roflumilast and NTHi

Susuki-Miyata

Miyata

Lee

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Phosphodiesterase 4B (PDE4B) plays a key role in regulating inflammation. Roflumilast, a phosphodiesterase (PDE)4-selective inhibitor, has recently been approved for treating severe chronic obstructive pulmonary disease (COPD) patients with exacerbation. However, there is also clinical evidence suggesting the development of tachyphylaxis or tolerance on repeated dosing of roflumilast and the possible contribution of PDE4B up-regulation, which could be counterproductive for suppressing inflammation. Thus, understanding how PDE4B is up-regulated in the context of the complex pathogenesis and medications of COPD may help improve the efficacy and possibly ameliorate the tolerance of roflumilast. Here we show that roflumilast synergizes with nontypeable Haemophilus influenzae (NTHi), a major bacterial cause of COPD exacerbation, to up-regulate PDE4B2 expression in human airway epithelial cells in vitro and in vivo. Up-regulated PDE4B2 contributes to the induction of certain important chemokines in both enzymatic activity-dependent and activity-independent manners. We also found that protein kinase A catalytic subunit β (PKA-Cβ) and nuclear factor-κB (NF-κB) p65 subunit were required for the synergistic induction of PDE4B2. PKA-Cβ phosphorylates p65 in a cAMP-dependent manner. Moreover, Ser276 of p65 is critical for mediating the PKA-Cβ-induced p65 phosphorylation and the synergistic induction of PDE4B2. Collectively, our data unveil a previously unidentified mechanism underlying synergistic up-regulation of PDE4B2 via a cross-talk between PKA-Cβ and p65 and may help develop new therapeutic strategies to improve the efficacy of PDE4 inhibitor.PDE4B | Roflumilast | nontypeable Haemophilus influenzae | PKA-Cβ | p65

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Anchored PDE4 regulates chloride conductance in wild‐type and ΔF508‐CFTR human airway epithelia

Cited by 34 publications

References 65 publications

Correctors of mutant CFTR enhance subcortical cAMP–PKA signaling through modulating ezrin phosphorylation and cytoskeleton organization

Correctors of mutant CFTR enhance subcortical cAMP–PKA signaling through modulating ezrin phosphorylation and cytoskeleton organization

Synergy-Based Small-Molecule Screen Using a Human Lung Epithelial Cell Line Yields ΔF508-CFTR Correctors That Augment VX-809 Maximal Efficacy

Cross-talk between PKA-Cβ and p65 mediates synergistic induction of PDE4B by roflumilast and NTHi

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