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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

Alteration of dynamin 2-dependent mechanisms impairs the stimulation of CFTR endocytosis by ML141 or wiskostatin treatments.CFBE-wtCFTR cells were transfected with the corresponding siRNA to deplete dynamin 2 or caveolin 1 proteins 48 h before performing quantification of PM-CFTR or of internalized CFTR, as described in Fig. 2 legend. Histograms summarizing analyzed data are displayed. Results were compared with negative control siRNA transfection conditions. (A) Dynamin 2 depletion elicited an increase of PM-CFTR amount, (B) correlated to lower CFTR internalization, (A and B) whereas caveolin 1 depletion had no impact upon PM- and internalized CFTR amounts. Then, 48 h after transfection with indicated siRNA, cells were treated with 10 μM ML141 for 30 min or 10 μM wiskostatin for 120 min, before labelling and during the 5 min internalization period. Results were compared to vehicle treatment conditions. (C) When dynamin 2 is depleted, CFTR internalization increase upon pharmacological treatments is abolished. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001, **: p<0.01, *: p<0.05, ns: non significant.
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pone.0118943.g004: Alteration of dynamin 2-dependent mechanisms impairs the stimulation of CFTR endocytosis by ML141 or wiskostatin treatments.CFBE-wtCFTR cells were transfected with the corresponding siRNA to deplete dynamin 2 or caveolin 1 proteins 48 h before performing quantification of PM-CFTR or of internalized CFTR, as described in Fig. 2 legend. Histograms summarizing analyzed data are displayed. Results were compared with negative control siRNA transfection conditions. (A) Dynamin 2 depletion elicited an increase of PM-CFTR amount, (B) correlated to lower CFTR internalization, (A and B) whereas caveolin 1 depletion had no impact upon PM- and internalized CFTR amounts. Then, 48 h after transfection with indicated siRNA, cells were treated with 10 μM ML141 for 30 min or 10 μM wiskostatin for 120 min, before labelling and during the 5 min internalization period. Results were compared to vehicle treatment conditions. (C) When dynamin 2 is depleted, CFTR internalization increase upon pharmacological treatments is abolished. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001, **: p<0.01, *: p<0.05, ns: non significant.

Mentions: We then tried to determine which endocytosis pathway is involved during increased internalization. As dynamin is involved in clathrin- and caveolin-mediated endocytosis [24], we used siRNA-mediated depletion of dynamin 2 or caveolin 1 to interfere with the corresponding pathways. Knock-down efficiency was assessed by Western blot (S2 Fig.). As expected, while dynamin 2 depletion had no effect upon total CFTR amount (not shown), it elicited an increase in PM-CFTR abundance (Fig. 4A), correlated to a decrease in CFTR internalization (Fig. 4B). This finding highlights dynamin-dependent CFTR endocytosis in CFBE-wtCFTR cells. However, caveolin 1 depletion exhibited no impact upon either PM-CFTR amount (Fig. 4A), or CFTR internalization (Fig. 4B). This result suggests that caveolae do not mediate PM-CFTR uptake in these cells, and that dynamin-dependent endocytosis should occur mainly via clathrin-coated vesicles. When we treated dynamin 2-depleted cells with ML141 or wiskostatin, as described above, PM-CFTR amounts (not shown) and CFTR internalization were found to be unchanged compared to DMSO control (Fig. 4C). We concluded that treatments with these chemicals trigger increased clathrin-mediated internalization of CFTR.


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)

Alteration of dynamin 2-dependent mechanisms impairs the stimulation of CFTR endocytosis by ML141 or wiskostatin treatments.CFBE-wtCFTR cells were transfected with the corresponding siRNA to deplete dynamin 2 or caveolin 1 proteins 48 h before performing quantification of PM-CFTR or of internalized CFTR, as described in Fig. 2 legend. Histograms summarizing analyzed data are displayed. Results were compared with negative control siRNA transfection conditions. (A) Dynamin 2 depletion elicited an increase of PM-CFTR amount, (B) correlated to lower CFTR internalization, (A and B) whereas caveolin 1 depletion had no impact upon PM- and internalized CFTR amounts. Then, 48 h after transfection with indicated siRNA, cells were treated with 10 μM ML141 for 30 min or 10 μM wiskostatin for 120 min, before labelling and during the 5 min internalization period. Results were compared to vehicle treatment conditions. (C) When dynamin 2 is depleted, CFTR internalization increase upon pharmacological treatments is abolished. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001, **: p<0.01, *: p<0.05, ns: non significant.
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pone.0118943.g004: Alteration of dynamin 2-dependent mechanisms impairs the stimulation of CFTR endocytosis by ML141 or wiskostatin treatments.CFBE-wtCFTR cells were transfected with the corresponding siRNA to deplete dynamin 2 or caveolin 1 proteins 48 h before performing quantification of PM-CFTR or of internalized CFTR, as described in Fig. 2 legend. Histograms summarizing analyzed data are displayed. Results were compared with negative control siRNA transfection conditions. (A) Dynamin 2 depletion elicited an increase of PM-CFTR amount, (B) correlated to lower CFTR internalization, (A and B) whereas caveolin 1 depletion had no impact upon PM- and internalized CFTR amounts. Then, 48 h after transfection with indicated siRNA, cells were treated with 10 μM ML141 for 30 min or 10 μM wiskostatin for 120 min, before labelling and during the 5 min internalization period. Results were compared to vehicle treatment conditions. (C) When dynamin 2 is depleted, CFTR internalization increase upon pharmacological treatments is abolished. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001, **: p<0.01, *: p<0.05, ns: non significant.
Mentions: We then tried to determine which endocytosis pathway is involved during increased internalization. As dynamin is involved in clathrin- and caveolin-mediated endocytosis [24], we used siRNA-mediated depletion of dynamin 2 or caveolin 1 to interfere with the corresponding pathways. Knock-down efficiency was assessed by Western blot (S2 Fig.). As expected, while dynamin 2 depletion had no effect upon total CFTR amount (not shown), it elicited an increase in PM-CFTR abundance (Fig. 4A), correlated to a decrease in CFTR internalization (Fig. 4B). This finding highlights dynamin-dependent CFTR endocytosis in CFBE-wtCFTR cells. However, caveolin 1 depletion exhibited no impact upon either PM-CFTR amount (Fig. 4A), or CFTR internalization (Fig. 4B). This result suggests that caveolae do not mediate PM-CFTR uptake in these cells, and that dynamin-dependent endocytosis should occur mainly via clathrin-coated vesicles. When we treated dynamin 2-depleted cells with ML141 or wiskostatin, as described above, PM-CFTR amounts (not shown) and CFTR internalization were found to be unchanged compared to DMSO control (Fig. 4C). We concluded that treatments with these chemicals trigger increased clathrin-mediated internalization of CFTR.

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