Limits...
Na+/H+ exchanger regulatory factor 1 overexpression-dependent increase of cytoskeleton organization is fundamental in the rescue of F508del cystic fibrosis transmembrane conductance regulator in human airway CFBE41o- cells.

Favia M, Guerra L, Fanelli T, Cardone RA, Monterisi S, Di Sole F, Castellani S, Chen M, Seidler U, Reshkin SJ, Conese M, Casavola V - Mol. Biol. Cell (2009)

Bottom Line: Furthermore, wt NHERF1 increased RhoA activity and transfection of constitutively active RhoA in CFBE41o- cells was sufficient to redistribute phospho-ezrin to the membrane fraction and rescue both the F-actin content and the CFTR-dependent chloride efflux.Rho kinase (ROCK) inhibition, in contrast, reversed the wt NHERF1 overexpression-induced increase of membrane phospho-ezrin, F-actin content, and CFTR-dependent secretion.We conclude that NHERF1 overexpression in CFBE41o- rescues CFTR-dependent chloride secretion by forming the multiprotein complex RhoA-ROCK-ezrin-actin that, via actin cytoskeleton reorganization, tethers F508del CFTR to the cytoskeleton stabilizing it on the apical membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of General and Environmental Physiology, University of Bari, Bari, Italy.

ABSTRACT
We have demonstrated that Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) overexpression in CFBE41o- cells induces a significant redistribution of F508del cystic fibrosis transmembrane conductance regulator (CFTR) from the cytoplasm to the apical membrane and rescues CFTR-dependent chloride secretion. Here, we observe that CFBE41o- monolayers displayed substantial disassembly of actin filaments and that overexpression of wild-type (wt) NHERF1 but not NHERF1-Delta Ezrin-Radixin-Moesin (ERM) increased F-actin assembly and organization. Furthermore, the dominant-negative band Four-point one, Ezrin, Radixin, Moesin homology (FERM) domain of ezrin reversed the wt NHERF1 overexpression-induced increase in both F-actin and CFTR-dependent chloride secretion. wt NHERF1 overexpression enhanced the interaction between NHERF1 and both CFTR and ezrin and between ezrin and actin and the overexpression of wt NHERF1, but not NHERF1-DeltaERM, also increased the phosphorylation of ezrin in the apical region of the cell monolayers. Furthermore, wt NHERF1 increased RhoA activity and transfection of constitutively active RhoA in CFBE41o- cells was sufficient to redistribute phospho-ezrin to the membrane fraction and rescue both the F-actin content and the CFTR-dependent chloride efflux. Rho kinase (ROCK) inhibition, in contrast, reversed the wt NHERF1 overexpression-induced increase of membrane phospho-ezrin, F-actin content, and CFTR-dependent secretion. We conclude that NHERF1 overexpression in CFBE41o- rescues CFTR-dependent chloride secretion by forming the multiprotein complex RhoA-ROCK-ezrin-actin that, via actin cytoskeleton reorganization, tethers F508del CFTR to the cytoskeleton stabilizing it on the apical membrane.

Show MeSH

Related in: MedlinePlus

Effect of NHERF1 overexpression and its interaction with ezrin on the kinetics of CFTR internalization. Polarized cell monolayers grown on permeable filters were biotinylated at 0°C and warmed to 37°C for the indicated times and the labeled CFTR remaining at the cell surface was stripped by MESNA. Internalized CFTR signals were normalized to the respective surface CFTR signals and plotted as a function of time. The data points represent mean ± SE of three experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2801722&req=5

Figure 3: Effect of NHERF1 overexpression and its interaction with ezrin on the kinetics of CFTR internalization. Polarized cell monolayers grown on permeable filters were biotinylated at 0°C and warmed to 37°C for the indicated times and the labeled CFTR remaining at the cell surface was stripped by MESNA. Internalized CFTR signals were normalized to the respective surface CFTR signals and plotted as a function of time. The data points represent mean ± SE of three experiments.

Mentions: We have observed previously that NHERF1 overexpression in 16HBE14o- and CFBE41o- cell monolayers increases the surface apical expression of both wt and F508del CFTR as measured by apical biotinylation and concluded that this increase could explain the NHERF1-induced increase of CFTR activity measured in both 16HBE14o- and CFBE41o- cell monolayers (Guerra et al., 2005). Therefore, we next determined whether that increase of surface expression could be explained by a NHERF1-dependent regulation of wt and F508del CFTR endocytosis. To this end, we analyzed the rate of endocytosis in polarized cell monolayers of 16HBE14o- or 16HBE14o-expressing NHERF1-ΔERM monolayers, as well as in control CFBE41o- and in CFBE41o-/sNHERF1 monolayers by using the biotin protection assay described in Materials and Methods (Figure 3). In the control CFBE41o- cells, which have a low but measurable cell surface expression of F508del CFTR protein in the apical membrane (Guerra et al., 2005), a larger fraction of surface-labeled F508del CFTR was internalized in the first 2.5 min. The overexpression of wt NHERF1 (CFBE41o-/sNHERF1, triangles) decreased F508del CFTR internalization to levels similar to that found for wt CFTR in 16HBE14o- cells (diamonds). Moreover, transfection of 16HBE14o- cells with NHERF1-ΔERM (circles) greatly increased wt CFTR internalization to levels similar to those observed in control CFBE41o- (squares) cell monolayers, suggesting that NHERF1 greatly influences both wt CFTR and F508del CFTR internalization through its interaction with the actin cytoskeleton via ezrin.


Na+/H+ exchanger regulatory factor 1 overexpression-dependent increase of cytoskeleton organization is fundamental in the rescue of F508del cystic fibrosis transmembrane conductance regulator in human airway CFBE41o- cells.

Favia M, Guerra L, Fanelli T, Cardone RA, Monterisi S, Di Sole F, Castellani S, Chen M, Seidler U, Reshkin SJ, Conese M, Casavola V - Mol. Biol. Cell (2009)

Effect of NHERF1 overexpression and its interaction with ezrin on the kinetics of CFTR internalization. Polarized cell monolayers grown on permeable filters were biotinylated at 0°C and warmed to 37°C for the indicated times and the labeled CFTR remaining at the cell surface was stripped by MESNA. Internalized CFTR signals were normalized to the respective surface CFTR signals and plotted as a function of time. The data points represent mean ± SE of three experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2801722&req=5

Figure 3: Effect of NHERF1 overexpression and its interaction with ezrin on the kinetics of CFTR internalization. Polarized cell monolayers grown on permeable filters were biotinylated at 0°C and warmed to 37°C for the indicated times and the labeled CFTR remaining at the cell surface was stripped by MESNA. Internalized CFTR signals were normalized to the respective surface CFTR signals and plotted as a function of time. The data points represent mean ± SE of three experiments.
Mentions: We have observed previously that NHERF1 overexpression in 16HBE14o- and CFBE41o- cell monolayers increases the surface apical expression of both wt and F508del CFTR as measured by apical biotinylation and concluded that this increase could explain the NHERF1-induced increase of CFTR activity measured in both 16HBE14o- and CFBE41o- cell monolayers (Guerra et al., 2005). Therefore, we next determined whether that increase of surface expression could be explained by a NHERF1-dependent regulation of wt and F508del CFTR endocytosis. To this end, we analyzed the rate of endocytosis in polarized cell monolayers of 16HBE14o- or 16HBE14o-expressing NHERF1-ΔERM monolayers, as well as in control CFBE41o- and in CFBE41o-/sNHERF1 monolayers by using the biotin protection assay described in Materials and Methods (Figure 3). In the control CFBE41o- cells, which have a low but measurable cell surface expression of F508del CFTR protein in the apical membrane (Guerra et al., 2005), a larger fraction of surface-labeled F508del CFTR was internalized in the first 2.5 min. The overexpression of wt NHERF1 (CFBE41o-/sNHERF1, triangles) decreased F508del CFTR internalization to levels similar to that found for wt CFTR in 16HBE14o- cells (diamonds). Moreover, transfection of 16HBE14o- cells with NHERF1-ΔERM (circles) greatly increased wt CFTR internalization to levels similar to those observed in control CFBE41o- (squares) cell monolayers, suggesting that NHERF1 greatly influences both wt CFTR and F508del CFTR internalization through its interaction with the actin cytoskeleton via ezrin.

Bottom Line: Furthermore, wt NHERF1 increased RhoA activity and transfection of constitutively active RhoA in CFBE41o- cells was sufficient to redistribute phospho-ezrin to the membrane fraction and rescue both the F-actin content and the CFTR-dependent chloride efflux.Rho kinase (ROCK) inhibition, in contrast, reversed the wt NHERF1 overexpression-induced increase of membrane phospho-ezrin, F-actin content, and CFTR-dependent secretion.We conclude that NHERF1 overexpression in CFBE41o- rescues CFTR-dependent chloride secretion by forming the multiprotein complex RhoA-ROCK-ezrin-actin that, via actin cytoskeleton reorganization, tethers F508del CFTR to the cytoskeleton stabilizing it on the apical membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of General and Environmental Physiology, University of Bari, Bari, Italy.

ABSTRACT
We have demonstrated that Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) overexpression in CFBE41o- cells induces a significant redistribution of F508del cystic fibrosis transmembrane conductance regulator (CFTR) from the cytoplasm to the apical membrane and rescues CFTR-dependent chloride secretion. Here, we observe that CFBE41o- monolayers displayed substantial disassembly of actin filaments and that overexpression of wild-type (wt) NHERF1 but not NHERF1-Delta Ezrin-Radixin-Moesin (ERM) increased F-actin assembly and organization. Furthermore, the dominant-negative band Four-point one, Ezrin, Radixin, Moesin homology (FERM) domain of ezrin reversed the wt NHERF1 overexpression-induced increase in both F-actin and CFTR-dependent chloride secretion. wt NHERF1 overexpression enhanced the interaction between NHERF1 and both CFTR and ezrin and between ezrin and actin and the overexpression of wt NHERF1, but not NHERF1-DeltaERM, also increased the phosphorylation of ezrin in the apical region of the cell monolayers. Furthermore, wt NHERF1 increased RhoA activity and transfection of constitutively active RhoA in CFBE41o- cells was sufficient to redistribute phospho-ezrin to the membrane fraction and rescue both the F-actin content and the CFTR-dependent chloride efflux. Rho kinase (ROCK) inhibition, in contrast, reversed the wt NHERF1 overexpression-induced increase of membrane phospho-ezrin, F-actin content, and CFTR-dependent secretion. We conclude that NHERF1 overexpression in CFBE41o- rescues CFTR-dependent chloride secretion by forming the multiprotein complex RhoA-ROCK-ezrin-actin that, via actin cytoskeleton reorganization, tethers F508del CFTR to the cytoskeleton stabilizing it on the apical membrane.

Show MeSH
Related in: MedlinePlus