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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

Cdc42 depletion does not alter fibrillar actin content.RNAi-mediated depletions of Cdc42 or N-WASP were performed for 48 h and F-actin content was quantified as described in Fig. 5 legend. As displayed in histograms, N-WASP depletion alone elicited a decrease of polymerized actin content, compared with negative control RNAi condition. Data represent means ± SEM of 3–8 independent experiments, each performed in triplicate. **: p<0.01, ns: non-significant.
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pone.0118943.g006: Cdc42 depletion does not alter fibrillar actin content.RNAi-mediated depletions of Cdc42 or N-WASP were performed for 48 h and F-actin content was quantified as described in Fig. 5 legend. As displayed in histograms, N-WASP depletion alone elicited a decrease of polymerized actin content, compared with negative control RNAi condition. Data represent means ± SEM of 3–8 independent experiments, each performed in triplicate. **: p<0.01, ns: non-significant.

Mentions: To interfere with Cdc42 pathway in CFBE-wtCFTR cells, we applied a siRNA knock-down approach in order to deplete cells in the proteins that we had pharmacologically inhibited in the first part of our study. The efficiencies of these depletions were assessed by real-time RT-PCR and by Western blot (S3 Fig.). We first investigated the impact of these depletions upon polymerized actin content. As expected, N-WASP-depleted cells exhibited a significant decrease of F-actin amount (Fig. 6), and the consequences appear similar to those observed with wiskostatin inhibition of N-WASP. By contrast, when Cdc42 was depleted, no significant difference in F-actin content was observed when compared with cells transfected with negative control siRNA (Fig. 6), whereas ML141 inhibition of Cdc42 led to a decrease in polymerized actin content. Whatever may account for this difference, these findings suggest that alterations of CFTR trafficking resulting from Cdc42 depletion would not be triggered by actin scaffold-dependent mechanisms.


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)

Cdc42 depletion does not alter fibrillar actin content.RNAi-mediated depletions of Cdc42 or N-WASP were performed for 48 h and F-actin content was quantified as described in Fig. 5 legend. As displayed in histograms, N-WASP depletion alone elicited a decrease of polymerized actin content, compared with negative control RNAi condition. Data represent means ± SEM of 3–8 independent experiments, each performed in triplicate. **: p<0.01, ns: non-significant.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359135&req=5

pone.0118943.g006: Cdc42 depletion does not alter fibrillar actin content.RNAi-mediated depletions of Cdc42 or N-WASP were performed for 48 h and F-actin content was quantified as described in Fig. 5 legend. As displayed in histograms, N-WASP depletion alone elicited a decrease of polymerized actin content, compared with negative control RNAi condition. Data represent means ± SEM of 3–8 independent experiments, each performed in triplicate. **: p<0.01, ns: non-significant.
Mentions: To interfere with Cdc42 pathway in CFBE-wtCFTR cells, we applied a siRNA knock-down approach in order to deplete cells in the proteins that we had pharmacologically inhibited in the first part of our study. The efficiencies of these depletions were assessed by real-time RT-PCR and by Western blot (S3 Fig.). We first investigated the impact of these depletions upon polymerized actin content. As expected, N-WASP-depleted cells exhibited a significant decrease of F-actin amount (Fig. 6), and the consequences appear similar to those observed with wiskostatin inhibition of N-WASP. By contrast, when Cdc42 was depleted, no significant difference in F-actin content was observed when compared with cells transfected with negative control siRNA (Fig. 6), whereas ML141 inhibition of Cdc42 led to a decrease in polymerized actin content. Whatever may account for this difference, these findings suggest that alterations of CFTR trafficking resulting from Cdc42 depletion would not be triggered by actin scaffold-dependent mechanisms.

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