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.
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.
Favia
Maria
M
Department of General and Environmental Physiology, University of Bari, Bari, Italy.
Guerra
Lorenzo
L
Fanelli
Teresa
T
Cardone
Rosa Angela
RA
Monterisi
Stefania
S
Di Sole
Francesca
F
Castellani
Stefano
S
Chen
Mingmin
M
Seidler
Ursula
U
Reshkin
Stephan Joel
SJ
Conese
Massimo
M
Casavola
Valeria
V
D000199:Actins / Q000378:metabolism; D000818:Animals; D001980:Bronchi / Q000166:cytology*; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D004705:Endocytosis; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D015398:Signal Transduction; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D000099149:Ezrin
D000199:Actins; D002712:Chlorides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein; D000099149:Ezrin; C093349:cystic fibrosis transmembrane conductance regulator delta F508; C093376:sodium-hydrogen exchanger regulatory factor; C071058:moesin; C071776:radixin