Osmotic cell shrinkage activates ezrin/radixin/moesin (ERM) proteins: activation mechanisms and physiological implications.
Hyperosmotic shrinkage induces multiple cellular responses, including activation of volume-regulatory ion transport, cytoskeletal reorganization, and cell death. Here we investigated the possible roles of ezrin/radixin/moesin (ERM) proteins in these events. Osmotic shrinkage of Ehrlich Lettre ascites cells elicited the formation of long microvillus-like protrusions, rapid translocation of endogenous ERM proteins and green fluorescent protein-tagged ezrin to the cortical region including these protrusions, and Thr(567/564/558) (ezrin/radixin/moesin) phosphorylation of cortical ERM proteins. Reduced cell volume appeared to be the critical parameter in hypertonicity-induced ERM protein activation, whereas alterations in extracellular ionic strength or intracellular pH were not involved. A shrinkage-induced increase in the level of membrane-associated phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] appeared to play an important role in ERM protein activation, which was prevented after PtdIns(4,5)P(2) depletion by expression of the synaptojanin-2 phosphatase domain. While expression of constitutively active RhoA increased basal ERM phosphorylation, the Rho-Rho kinase pathway did not appear to be involved in shrinkage-induced ERM protein phosphorylation, which was also unaffected by the inhibition or absence of Na(+)/H(+) exchanger isoform (NHE1). Ezrin knockdown by small interfering RNA increased shrinkage-induced NHE1 activity, reduced basal and shrinkage-induced Rho activity, and attenuated the shrinkage-induced formation of microvillus-like protrusions. Hyperosmolarity-induced cell death was unaltered by ezrin knockdown or after phosphatidylinositol 3-kinase (PI3K) inhibition. In conclusion, ERM proteins are activated by osmotic shrinkage in a PtdIns(4,5)P(2)-dependent, NHE1-independent manner. This in turn mitigates the shrinkage-induced activation of NHE1, augments Rho activity, and may also contribute to F-actin rearrangement. In contrast, no evidence was found for the involvement of an NHE1-ezrin-PI3K-PKB pathway in counteracting shrinkage-induced cell death.
Rasmussen
Maria
M
Department of Molecular Biology, University of Copenhagen, Copenhagen, Denmark.
Alexander
R Todd
RT
Darborg
Barbara V
BV
Møbjerg
Nadja
N
Hoffmann
Else K
EK
Kapus
András
A
Pedersen
Stine F
SF
D000199:Actins / Q000378:metabolism; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D019556:COS Cells; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism; D016923:Cell Death; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism*; D048429:Cell Size; D002522:Chlorocebus aethiops; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D003599:Cytoskeleton / Q000201:enzymology / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism*; D041681:NIH 3T3 Cells; D009997:Osmotic Pressure; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010766:Phosphorylation; D021381:Protein Transport; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012462:Saline Solution, Hypertonic / Q000378:metabolism; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism; D013552:Swine; D013997:Time Factors; D014162:Transfection; D054460:rho-Associated Kinases / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism; D000099149:Ezrin
D000199:Actins; D001639:Bicarbonates; D027682:Cation Transport Proteins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D012462:Saline Solution, Hypertonic; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein; D000099149:Ezrin; C490998:SLC9A1 protein, human; C071058:moesin; C071776:radixin