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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 selectively reduces the GTP-bound form of Cdc42.Before lysis, cells were submitted to 10 μM ML141 for 30 min and 1% DMSO (v/v) was used as the control condition. After enrichment of the activated forms of GTPases in the clarified lysates, GST-pull-down was performed. Cdc42, Rac1 or RhoA protein amounts were then assessed in the resulting samples. (A) Representative Western blot images are shown. Densitometric quantification of bands was normalized to DMSO condition. (B) Histogram displays relative activated GTPase amounts, expressed as the percentage of control. Data represent means ± SEM of 3 independent experiments each performed in duplicate. *: p<0.05, ns: non significant.
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pone.0118943.g001: ML141 selectively reduces the GTP-bound form of Cdc42.Before lysis, cells were submitted to 10 μM ML141 for 30 min and 1% DMSO (v/v) was used as the control condition. After enrichment of the activated forms of GTPases in the clarified lysates, GST-pull-down was performed. Cdc42, Rac1 or RhoA protein amounts were then assessed in the resulting samples. (A) Representative Western blot images are shown. Densitometric quantification of bands was normalized to DMSO condition. (B) Histogram displays relative activated GTPase amounts, expressed as the percentage of control. Data represent means ± SEM of 3 independent experiments each performed in duplicate. *: p<0.05, ns: non significant.

Mentions: Few compounds specifically interfering with Cdc42 pathway are available. To inhibit Cdc42, we used ML141, a new lead molecule that was identified by the Kansas University Specialized Chemistry Center [20]. First, we tested its inhibition selectivity amongst several GTPases, before using it to study CFTR trafficking. We performed an in vitro assay based on the binding of GTPases to an interaction domain of their effectors, to estimate the activation state of RhoA, Rac1 and Cdc42. CFBE-wtCFTR cells were treated with 10 μM ML141 for 30 min, according to the manufacturer’s suggestion, and the ratios of activated to total GTPases in the cells were determined (Fig. 1). As expected, when the cells were exposed to ML141 the proportion of activated Cdc42 was reduced to 50%, compared to the DMSO control. In the cases of Rac1 and RhoA, we found no impact of ML141. As its inhibitory effect appeared specific in CFBE-wtCFTR cells, in the subsequent steps of our study we exposed the cells for 30 min to 10 μM ML141.


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 selectively reduces the GTP-bound form of Cdc42.Before lysis, cells were submitted to 10 μM ML141 for 30 min and 1% DMSO (v/v) was used as the control condition. After enrichment of the activated forms of GTPases in the clarified lysates, GST-pull-down was performed. Cdc42, Rac1 or RhoA protein amounts were then assessed in the resulting samples. (A) Representative Western blot images are shown. Densitometric quantification of bands was normalized to DMSO condition. (B) Histogram displays relative activated GTPase amounts, expressed as the percentage of control. Data represent means ± SEM of 3 independent experiments each performed in duplicate. *: p<0.05, ns: non significant.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118943.g001: ML141 selectively reduces the GTP-bound form of Cdc42.Before lysis, cells were submitted to 10 μM ML141 for 30 min and 1% DMSO (v/v) was used as the control condition. After enrichment of the activated forms of GTPases in the clarified lysates, GST-pull-down was performed. Cdc42, Rac1 or RhoA protein amounts were then assessed in the resulting samples. (A) Representative Western blot images are shown. Densitometric quantification of bands was normalized to DMSO condition. (B) Histogram displays relative activated GTPase amounts, expressed as the percentage of control. Data represent means ± SEM of 3 independent experiments each performed in duplicate. *: p<0.05, ns: non significant.
Mentions: Few compounds specifically interfering with Cdc42 pathway are available. To inhibit Cdc42, we used ML141, a new lead molecule that was identified by the Kansas University Specialized Chemistry Center [20]. First, we tested its inhibition selectivity amongst several GTPases, before using it to study CFTR trafficking. We performed an in vitro assay based on the binding of GTPases to an interaction domain of their effectors, to estimate the activation state of RhoA, Rac1 and Cdc42. CFBE-wtCFTR cells were treated with 10 μM ML141 for 30 min, according to the manufacturer’s suggestion, and the ratios of activated to total GTPases in the cells were determined (Fig. 1). As expected, when the cells were exposed to ML141 the proportion of activated Cdc42 was reduced to 50%, compared to the DMSO control. In the cases of Rac1 and RhoA, we found no impact of ML141. As its inhibitory effect appeared specific in CFBE-wtCFTR cells, in the subsequent steps of our study we exposed the cells for 30 min to 10 μM ML141.

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