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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.

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Regulation of phospho-ezrin distribution by RhoA in CFBE41o- cells. A representative Western blot analysis for P-ezrin at Thr567 and total ezrin in membrane, cytosolic, and cytoskeletal fractions of control CFBE41o- cells transfected with the constitutively active mutant of RhoA, RhoA-V14, and histogram representation of phospho-ezrin subcellular distribution in CFBE41o- and CFBE41o- cells transfected with RhoA-V14, expressed as the ratio of phospho-ezrin/total ezrin normalized in each fraction to that in nontransfected CFBE41o- cells, designated as 1. Data are means ± SE, n = 5; **p < 0.001 versus nontransfected cells.
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Figure 7: Regulation of phospho-ezrin distribution by RhoA in CFBE41o- cells. A representative Western blot analysis for P-ezrin at Thr567 and total ezrin in membrane, cytosolic, and cytoskeletal fractions of control CFBE41o- cells transfected with the constitutively active mutant of RhoA, RhoA-V14, and histogram representation of phospho-ezrin subcellular distribution in CFBE41o- and CFBE41o- cells transfected with RhoA-V14, expressed as the ratio of phospho-ezrin/total ezrin normalized in each fraction to that in nontransfected CFBE41o- cells, designated as 1. Data are means ± SE, n = 5; **p < 0.001 versus nontransfected cells.

Mentions: RhoA is an important regulator of actin-based cytoskeletal organization (Tapon and Hall, 1997) and, in most cell types, is an upstream activator of ezrin (Matsui et al., 1998). In cell fractionation experiments, we observed that transfection with the dominant active mutant of RhoA, RhoA-V14, increased the ratio of phosphorylated to total ezrin in the membrane fraction of CFBE41o- cells similarly to that observed during NHERF1 overexpression (Figure 7). We then analyzed the role of RhoA in regulating F-actin content and CFTR-dependent chloride efflux. Transfection of control CFBE41o- cells with the dominant active RhoA-V14 induced a significant increase of cortical actin filaments (Supplemental Figure 6S) and strongly increased both F-actin content (Figure 8A, white bars) and CFTR-dependent chloride efflux (Figure 8B, white bars), whereas transfection with the dominant negative mutant of RhoA (RhoA-N19) had no effect. To assess if RhoA-V14-dependent rescue of CFTR activity resulted from a redistribution of F508del CFTR from the cytoplasm to the apical membrane, we performed confocal immunofluorescence measurements in nonpermeabilized CFBE41o- polarized cell monolayers stained with an mAb that recognizes a sequence in the first extracellular loop of the human CFTR protein (Bossard et al., 2007). As can be seen in Figure 8C, F508del CFTR was highly expressed on the apical membrane of CFBE41o- cell monolayers that had been transfected with RhoA-V14 bound to GFP, whereas nontransfected CFBE41o- monolayers did not show any signal for CFTR membrane expression. Altogether, these data suggest that in CFBE41o- cells, RhoA activity is very low and its activation is able to rescue both F-actin polymerization and F508del CFTR functional expression in the apical membrane.


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)

Regulation of phospho-ezrin distribution by RhoA in CFBE41o- cells. A representative Western blot analysis for P-ezrin at Thr567 and total ezrin in membrane, cytosolic, and cytoskeletal fractions of control CFBE41o- cells transfected with the constitutively active mutant of RhoA, RhoA-V14, and histogram representation of phospho-ezrin subcellular distribution in CFBE41o- and CFBE41o- cells transfected with RhoA-V14, expressed as the ratio of phospho-ezrin/total ezrin normalized in each fraction to that in nontransfected CFBE41o- cells, designated as 1. Data are means ± SE, n = 5; **p < 0.001 versus nontransfected cells.
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Related In: Results  -  Collection

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

Figure 7: Regulation of phospho-ezrin distribution by RhoA in CFBE41o- cells. A representative Western blot analysis for P-ezrin at Thr567 and total ezrin in membrane, cytosolic, and cytoskeletal fractions of control CFBE41o- cells transfected with the constitutively active mutant of RhoA, RhoA-V14, and histogram representation of phospho-ezrin subcellular distribution in CFBE41o- and CFBE41o- cells transfected with RhoA-V14, expressed as the ratio of phospho-ezrin/total ezrin normalized in each fraction to that in nontransfected CFBE41o- cells, designated as 1. Data are means ± SE, n = 5; **p < 0.001 versus nontransfected cells.
Mentions: RhoA is an important regulator of actin-based cytoskeletal organization (Tapon and Hall, 1997) and, in most cell types, is an upstream activator of ezrin (Matsui et al., 1998). In cell fractionation experiments, we observed that transfection with the dominant active mutant of RhoA, RhoA-V14, increased the ratio of phosphorylated to total ezrin in the membrane fraction of CFBE41o- cells similarly to that observed during NHERF1 overexpression (Figure 7). We then analyzed the role of RhoA in regulating F-actin content and CFTR-dependent chloride efflux. Transfection of control CFBE41o- cells with the dominant active RhoA-V14 induced a significant increase of cortical actin filaments (Supplemental Figure 6S) and strongly increased both F-actin content (Figure 8A, white bars) and CFTR-dependent chloride efflux (Figure 8B, white bars), whereas transfection with the dominant negative mutant of RhoA (RhoA-N19) had no effect. To assess if RhoA-V14-dependent rescue of CFTR activity resulted from a redistribution of F508del CFTR from the cytoplasm to the apical membrane, we performed confocal immunofluorescence measurements in nonpermeabilized CFBE41o- polarized cell monolayers stained with an mAb that recognizes a sequence in the first extracellular loop of the human CFTR protein (Bossard et al., 2007). As can be seen in Figure 8C, F508del CFTR was highly expressed on the apical membrane of CFBE41o- cell monolayers that had been transfected with RhoA-V14 bound to GFP, whereas nontransfected CFBE41o- monolayers did not show any signal for CFTR membrane expression. Altogether, these data suggest that in CFBE41o- cells, RhoA activity is very low and its activation is able to rescue both F-actin polymerization and F508del CFTR functional expression in the apical membrane.

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