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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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NHERF1 overexpression relocalizes phospho-ERM proteins in CFBE41o- cells. (A) Phospho-ezrin distribution was analyzed with a polyclonal phospho-ERM [ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558)] antibody: 1) A representative Western blot analysis for P-ezrin at Thr567 (top band, arrowhead corresponding to the phosphorylated ezrin) and total ezrin in membrane, cytosolic, and cytoskeletal fractions of control CFBE41o- cells (ctrl), CFBE41o-/sNHERF1 (sNHERF1), and CFBE41o- transfected with NHERF1-ΔERM cDNA (ΔERM); and 2) graphic representation of phospho-ezrin subcellular distribution, expressed as the phospho-ezrin/total ezrin ratio normalized in each fraction to the level in nontransfected CFBE41o- cells, designated as 1. Data represent means ± SE, n = 7; *p < 0.01, versus nontransfected CFBE41o- cells. 3) Confocal immunofluorescence microscopy was performed on polarized monolayers of CFBE41o-, CFBE41o-/sNHERF1, and CFBE41o- transfected with NHERF1-ΔERM cDNA grown on permeable filters. Phospho-ERM was detected by polyclonal antibody and the images are in the vertical (xz) plane. ap, location of apical region; bl, location of basal region. Bars, 10 μm. (B) Phospho-ERM localization in 16HBE14o- cell monolayers. Confocal immunofluorescence microscopy was performed in polarized 16HBE14o- cells grown on permeable filters. Bars, 10 μm.
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Figure 6: NHERF1 overexpression relocalizes phospho-ERM proteins in CFBE41o- cells. (A) Phospho-ezrin distribution was analyzed with a polyclonal phospho-ERM [ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558)] antibody: 1) A representative Western blot analysis for P-ezrin at Thr567 (top band, arrowhead corresponding to the phosphorylated ezrin) and total ezrin in membrane, cytosolic, and cytoskeletal fractions of control CFBE41o- cells (ctrl), CFBE41o-/sNHERF1 (sNHERF1), and CFBE41o- transfected with NHERF1-ΔERM cDNA (ΔERM); and 2) graphic representation of phospho-ezrin subcellular distribution, expressed as the phospho-ezrin/total ezrin ratio normalized in each fraction to the level in nontransfected CFBE41o- cells, designated as 1. Data represent means ± SE, n = 7; *p < 0.01, versus nontransfected CFBE41o- cells. 3) Confocal immunofluorescence microscopy was performed on polarized monolayers of CFBE41o-, CFBE41o-/sNHERF1, and CFBE41o- transfected with NHERF1-ΔERM cDNA grown on permeable filters. Phospho-ERM was detected by polyclonal antibody and the images are in the vertical (xz) plane. ap, location of apical region; bl, location of basal region. Bars, 10 μm. (B) Phospho-ERM localization in 16HBE14o- cell monolayers. Confocal immunofluorescence microscopy was performed in polarized 16HBE14o- cells grown on permeable filters. Bars, 10 μm.

Mentions: Therefore, we next examined phospho-ezrin distribution in both control CFBE41o- and CFBE41o-/sNHERF1 cell monolayers with a phospho-ERM [ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558)] antibody. Results obtained in cell fractionation experiments measuring the expression of phosphorylated ezrin (top band, arrowhead corresponding to the phosphorylated ezrin) and total ezrin in the membrane, cytosolic, and cytoskeletal fractions by Western blotting (Figure 6A, top) showed that the overexpression of wt NHERF1 (CFBE41o-/sNHERF1 cells) increased the ratio of phosphorylated to total ezrin in the membrane fraction, and this did not occur in either control CFBE41o- cells or those transfected with NHERF1-ΔERM. These results are in line with the findings that phosphorylated, activated ERM proteins are preferentially targeted to the actin rich membrane structures (Matsui et al., 1998; Hayashi et al., 1999). Confocal analysis of polarized monolayers on permeable filters (Figure 6A, bottom), confirmed that phospho-ERM proteins were lowly and randomly expressed in control CFBE41o- cells, whereas stable overexpression of wt NHERF1 (CFBE41o-/sNHERF1 cells) led to a significant increase of phospho-ERM expression mainly at the apical region. Interestingly, this apical redistribution of phospho-ERM was dependent on the capability of NHERF1 to interact with the N terminus of ezrin because CFBE41o- cells transfected with the cDNA encoding the NHERF1-ΔERM construct displayed a phospho-ERM distribution similar to that observed in control CFBE41o- cells. Altogether, these results suggest that the overexpression of NHERF1 regulates the intramolecular association of ezrin and its subsequent activation and recruitment in the apical region. Importantly, the hypothesis that NHERF1 overexpression in CFBE41o- cells restores a “normal” phenotype is supported by the finding that confocal analysis of phospho-ERM distribution in 16HBE14o- cells (Figure 6B) was similar to that found in the CFBE41o-/sNHERF1 cells.


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)

NHERF1 overexpression relocalizes phospho-ERM proteins in CFBE41o- cells. (A) Phospho-ezrin distribution was analyzed with a polyclonal phospho-ERM [ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558)] antibody: 1) A representative Western blot analysis for P-ezrin at Thr567 (top band, arrowhead corresponding to the phosphorylated ezrin) and total ezrin in membrane, cytosolic, and cytoskeletal fractions of control CFBE41o- cells (ctrl), CFBE41o-/sNHERF1 (sNHERF1), and CFBE41o- transfected with NHERF1-ΔERM cDNA (ΔERM); and 2) graphic representation of phospho-ezrin subcellular distribution, expressed as the phospho-ezrin/total ezrin ratio normalized in each fraction to the level in nontransfected CFBE41o- cells, designated as 1. Data represent means ± SE, n = 7; *p < 0.01, versus nontransfected CFBE41o- cells. 3) Confocal immunofluorescence microscopy was performed on polarized monolayers of CFBE41o-, CFBE41o-/sNHERF1, and CFBE41o- transfected with NHERF1-ΔERM cDNA grown on permeable filters. Phospho-ERM was detected by polyclonal antibody and the images are in the vertical (xz) plane. ap, location of apical region; bl, location of basal region. Bars, 10 μm. (B) Phospho-ERM localization in 16HBE14o- cell monolayers. Confocal immunofluorescence microscopy was performed in polarized 16HBE14o- cells grown on permeable filters. Bars, 10 μm.
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Figure 6: NHERF1 overexpression relocalizes phospho-ERM proteins in CFBE41o- cells. (A) Phospho-ezrin distribution was analyzed with a polyclonal phospho-ERM [ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558)] antibody: 1) A representative Western blot analysis for P-ezrin at Thr567 (top band, arrowhead corresponding to the phosphorylated ezrin) and total ezrin in membrane, cytosolic, and cytoskeletal fractions of control CFBE41o- cells (ctrl), CFBE41o-/sNHERF1 (sNHERF1), and CFBE41o- transfected with NHERF1-ΔERM cDNA (ΔERM); and 2) graphic representation of phospho-ezrin subcellular distribution, expressed as the phospho-ezrin/total ezrin ratio normalized in each fraction to the level in nontransfected CFBE41o- cells, designated as 1. Data represent means ± SE, n = 7; *p < 0.01, versus nontransfected CFBE41o- cells. 3) Confocal immunofluorescence microscopy was performed on polarized monolayers of CFBE41o-, CFBE41o-/sNHERF1, and CFBE41o- transfected with NHERF1-ΔERM cDNA grown on permeable filters. Phospho-ERM was detected by polyclonal antibody and the images are in the vertical (xz) plane. ap, location of apical region; bl, location of basal region. Bars, 10 μm. (B) Phospho-ERM localization in 16HBE14o- cell monolayers. Confocal immunofluorescence microscopy was performed in polarized 16HBE14o- cells grown on permeable filters. Bars, 10 μm.
Mentions: Therefore, we next examined phospho-ezrin distribution in both control CFBE41o- and CFBE41o-/sNHERF1 cell monolayers with a phospho-ERM [ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558)] antibody. Results obtained in cell fractionation experiments measuring the expression of phosphorylated ezrin (top band, arrowhead corresponding to the phosphorylated ezrin) and total ezrin in the membrane, cytosolic, and cytoskeletal fractions by Western blotting (Figure 6A, top) showed that the overexpression of wt NHERF1 (CFBE41o-/sNHERF1 cells) increased the ratio of phosphorylated to total ezrin in the membrane fraction, and this did not occur in either control CFBE41o- cells or those transfected with NHERF1-ΔERM. These results are in line with the findings that phosphorylated, activated ERM proteins are preferentially targeted to the actin rich membrane structures (Matsui et al., 1998; Hayashi et al., 1999). Confocal analysis of polarized monolayers on permeable filters (Figure 6A, bottom), confirmed that phospho-ERM proteins were lowly and randomly expressed in control CFBE41o- cells, whereas stable overexpression of wt NHERF1 (CFBE41o-/sNHERF1 cells) led to a significant increase of phospho-ERM expression mainly at the apical region. Interestingly, this apical redistribution of phospho-ERM was dependent on the capability of NHERF1 to interact with the N terminus of ezrin because CFBE41o- cells transfected with the cDNA encoding the NHERF1-ΔERM construct displayed a phospho-ERM distribution similar to that observed in control CFBE41o- cells. Altogether, these results suggest that the overexpression of NHERF1 regulates the intramolecular association of ezrin and its subsequent activation and recruitment in the apical region. Importantly, the hypothesis that NHERF1 overexpression in CFBE41o- cells restores a “normal” phenotype is supported by the finding that confocal analysis of phospho-ERM distribution in 16HBE14o- cells (Figure 6B) was similar to that found in the CFBE41o-/sNHERF1 cells.

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