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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 increases CFTR channel activation.(A) In non-stimulated CFBE-wtCFTR cells (basal) or upon stimulation of CFTR activity by forskolin (Fsk, 10 μM) + genistein (Gst, 30 μM), iodide efflux curves were obtained after 48 h of siRNA-mediated depletions (negative control, Cdc42 or N-WASP), n = 4 in each condition. (B) Histograms show the mean relative rates of CFTR activity. Results obtained after Cdc42 and N-WASP depletions were compared to control RNAi condition. Means ± SEM are indicated. **: p<0.01, *: p<0.05.
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pone.0118943.g007: Cdc42 depletion increases CFTR channel activation.(A) In non-stimulated CFBE-wtCFTR cells (basal) or upon stimulation of CFTR activity by forskolin (Fsk, 10 μM) + genistein (Gst, 30 μM), iodide efflux curves were obtained after 48 h of siRNA-mediated depletions (negative control, Cdc42 or N-WASP), n = 4 in each condition. (B) Histograms show the mean relative rates of CFTR activity. Results obtained after Cdc42 and N-WASP depletions were compared to control RNAi condition. Means ± SEM are indicated. **: p<0.01, *: p<0.05.

Mentions: We first investigated the impact of Cdc42 or N-WASP depletions upon CFTR channel function. We performed iodide efflux experiments 48 h after siRNA transfections: in N-WASP-depleted cells, CFTR activity decreased, whereas in Cdc42-depleted cells it increased (Fig. 7). In the first case, there are pronounced similarities to the consequences of wiskostatin treatment. As for Cdc42 depletion impact, it contrasts with the previously observed ML141inhibition of CFTR channel activation.


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 increases CFTR channel activation.(A) In non-stimulated CFBE-wtCFTR cells (basal) or upon stimulation of CFTR activity by forskolin (Fsk, 10 μM) + genistein (Gst, 30 μM), iodide efflux curves were obtained after 48 h of siRNA-mediated depletions (negative control, Cdc42 or N-WASP), n = 4 in each condition. (B) Histograms show the mean relative rates of CFTR activity. Results obtained after Cdc42 and N-WASP depletions were compared to control RNAi condition. Means ± SEM are indicated. **: p<0.01, *: p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118943.g007: Cdc42 depletion increases CFTR channel activation.(A) In non-stimulated CFBE-wtCFTR cells (basal) or upon stimulation of CFTR activity by forskolin (Fsk, 10 μM) + genistein (Gst, 30 μM), iodide efflux curves were obtained after 48 h of siRNA-mediated depletions (negative control, Cdc42 or N-WASP), n = 4 in each condition. (B) Histograms show the mean relative rates of CFTR activity. Results obtained after Cdc42 and N-WASP depletions were compared to control RNAi condition. Means ± SEM are indicated. **: p<0.01, *: p<0.05.
Mentions: We first investigated the impact of Cdc42 or N-WASP depletions upon CFTR channel function. We performed iodide efflux experiments 48 h after siRNA transfections: in N-WASP-depleted cells, CFTR activity decreased, whereas in Cdc42-depleted cells it increased (Fig. 7). In the first case, there are pronounced similarities to the consequences of wiskostatin treatment. As for Cdc42 depletion impact, it contrasts with the previously observed ML141inhibition of CFTR channel activation.

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