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Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.

Ferru-Clément R, Fresquet F, Norez C, Métayé T, Becq F, Kitzis A, Thoreau V - PLoS ONE (2015)

Bottom Line: When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis.Total and PM CFTR amounts were increased, resulting in greater activation of CFTR.In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Génétique des Maladies Rares, Université de Poitiers, Poitiers, France.

ABSTRACT
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed on the apical plasma membrane (PM) of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD) and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o-) expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i) Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii) it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii) it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.

No MeSH data available.


Related in: MedlinePlus

ML141 or wiskostatin treatments stimulate CFTR endocytosis.(A) Representative Western blots and (B and C) histograms summarizing the data are presented. First, cells were submitted to 10 μM ML141 for 30 min or 10 μM wiskostatin for 120 min. Treatment with 1% DMSO (v/v) was used as a negative control. Surface proteins were then biotinylated. Total CFTR protein amount was assessed by immunoblot in denatured samples obtained from 20 μg of clarified lysates (α1 Na+/K+ ATPase was used as a normalization control). Biotinylated proteins were purified from 100 μg of clarified lysates and the amount of labelled CFTR was assessed in the resulting samples. (B) The amount of plasma membrane CFTR, expressed as the percentage of DMSO treatment condition, decreased following both pharmacological treatments. Alternatively, the biotinylated PM proteins were allowed to enter the inner cell compartment through 5 min incubation of cell cultures at 37°C. Surface-exposed biotin moieties were then stripped by MESNA reduction. Biotinylated (internalized) proteins were purified from 600 μg clarified lysates and analyzed by Western blot. The ratio of the densitometric quantification of bands to the relative initial PM-CFTR amounts was then calculated. (C) Relative CFTR internalization, expressed as the percentage of DMSO control condition, appeared to have increased following ML141 or wiskostatin treatments. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001, **: p<0.01, *: p<0.05.
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pone.0118943.g002: ML141 or wiskostatin treatments stimulate CFTR endocytosis.(A) Representative Western blots and (B and C) histograms summarizing the data are presented. First, cells were submitted to 10 μM ML141 for 30 min or 10 μM wiskostatin for 120 min. Treatment with 1% DMSO (v/v) was used as a negative control. Surface proteins were then biotinylated. Total CFTR protein amount was assessed by immunoblot in denatured samples obtained from 20 μg of clarified lysates (α1 Na+/K+ ATPase was used as a normalization control). Biotinylated proteins were purified from 100 μg of clarified lysates and the amount of labelled CFTR was assessed in the resulting samples. (B) The amount of plasma membrane CFTR, expressed as the percentage of DMSO treatment condition, decreased following both pharmacological treatments. Alternatively, the biotinylated PM proteins were allowed to enter the inner cell compartment through 5 min incubation of cell cultures at 37°C. Surface-exposed biotin moieties were then stripped by MESNA reduction. Biotinylated (internalized) proteins were purified from 600 μg clarified lysates and analyzed by Western blot. The ratio of the densitometric quantification of bands to the relative initial PM-CFTR amounts was then calculated. (C) Relative CFTR internalization, expressed as the percentage of DMSO control condition, appeared to have increased following ML141 or wiskostatin treatments. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001, **: p<0.01, *: p<0.05.

Mentions: Cells were exposed to either chemical, in the aforementioned conditions, and by means of a surface biotinylation assay we estimated the amounts of plasma membrane CFTR (PM-CFTR); both chemicals elicited a significant decrease of labelled PM-CFTR (Fig. 2A and 2B). To address the functional impact of this effect upon CFTR trafficking, we performed iodide efflux experiments after pharmacological inhibition of Cdc42 or of N-WASP in CFBE-wtCFTR cells; both ML141 and wiskostatin treatments led to a significant decrease of CFTR activation (Fig. 3) in accordance with our PM-CFTR quantification data.


Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.

Ferru-Clément R, Fresquet F, Norez C, Métayé T, Becq F, Kitzis A, Thoreau V - PLoS ONE (2015)

ML141 or wiskostatin treatments stimulate CFTR endocytosis.(A) Representative Western blots and (B and C) histograms summarizing the data are presented. First, cells were submitted to 10 μM ML141 for 30 min or 10 μM wiskostatin for 120 min. Treatment with 1% DMSO (v/v) was used as a negative control. Surface proteins were then biotinylated. Total CFTR protein amount was assessed by immunoblot in denatured samples obtained from 20 μg of clarified lysates (α1 Na+/K+ ATPase was used as a normalization control). Biotinylated proteins were purified from 100 μg of clarified lysates and the amount of labelled CFTR was assessed in the resulting samples. (B) The amount of plasma membrane CFTR, expressed as the percentage of DMSO treatment condition, decreased following both pharmacological treatments. Alternatively, the biotinylated PM proteins were allowed to enter the inner cell compartment through 5 min incubation of cell cultures at 37°C. Surface-exposed biotin moieties were then stripped by MESNA reduction. Biotinylated (internalized) proteins were purified from 600 μg clarified lysates and analyzed by Western blot. The ratio of the densitometric quantification of bands to the relative initial PM-CFTR amounts was then calculated. (C) Relative CFTR internalization, expressed as the percentage of DMSO control condition, appeared to have increased following ML141 or wiskostatin treatments. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001, **: p<0.01, *: p<0.05.
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pone.0118943.g002: ML141 or wiskostatin treatments stimulate CFTR endocytosis.(A) Representative Western blots and (B and C) histograms summarizing the data are presented. First, cells were submitted to 10 μM ML141 for 30 min or 10 μM wiskostatin for 120 min. Treatment with 1% DMSO (v/v) was used as a negative control. Surface proteins were then biotinylated. Total CFTR protein amount was assessed by immunoblot in denatured samples obtained from 20 μg of clarified lysates (α1 Na+/K+ ATPase was used as a normalization control). Biotinylated proteins were purified from 100 μg of clarified lysates and the amount of labelled CFTR was assessed in the resulting samples. (B) The amount of plasma membrane CFTR, expressed as the percentage of DMSO treatment condition, decreased following both pharmacological treatments. Alternatively, the biotinylated PM proteins were allowed to enter the inner cell compartment through 5 min incubation of cell cultures at 37°C. Surface-exposed biotin moieties were then stripped by MESNA reduction. Biotinylated (internalized) proteins were purified from 600 μg clarified lysates and analyzed by Western blot. The ratio of the densitometric quantification of bands to the relative initial PM-CFTR amounts was then calculated. (C) Relative CFTR internalization, expressed as the percentage of DMSO control condition, appeared to have increased following ML141 or wiskostatin treatments. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001, **: p<0.01, *: p<0.05.
Mentions: Cells were exposed to either chemical, in the aforementioned conditions, and by means of a surface biotinylation assay we estimated the amounts of plasma membrane CFTR (PM-CFTR); both chemicals elicited a significant decrease of labelled PM-CFTR (Fig. 2A and 2B). To address the functional impact of this effect upon CFTR trafficking, we performed iodide efflux experiments after pharmacological inhibition of Cdc42 or of N-WASP in CFBE-wtCFTR cells; both ML141 and wiskostatin treatments led to a significant decrease of CFTR activation (Fig. 3) in accordance with our PM-CFTR quantification data.

Bottom Line: When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis.Total and PM CFTR amounts were increased, resulting in greater activation of CFTR.In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Génétique des Maladies Rares, Université de Poitiers, Poitiers, France.

ABSTRACT
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed on the apical plasma membrane (PM) of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD) and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o-) expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i) Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii) it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii) it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.

No MeSH data available.


Related in: MedlinePlus