Radixin is required to maintain apical canalicular membrane structure and function in rat hepatocytes.
OBJECTIVE
Ezrin-radixin-moesin proteins are cross-linkers between the plasma membrane and actin filaments. Radixin, the dominant ezrin-radixin-moesin protein in hepatocytes, has been reported to selectively tether multidrug-resistance-associated protein 2 to the apical canalicular membrane. However, it remains to be determined if this is its primary function.


METHODS
An adenovirus-mediated short interfering RNA (siRNA) was used to down-regulate radixin expression in collagen sandwich-cultured rat hepatocytes and morphologic and functional changes were characterized quantitatively.


RESULTS
In control cultures, an extensive bile canalicular network developed with properly localized apical and basolateral transporters that provided for functional excretion of fluorescent cholephiles into the bile canalicular lumina. siRNA-induced suppression of radixin was associated with a marked reduction in the canalicular membrane structure as observed by differential interference contrast microscopy and F-actin staining, in contrast to control cells exposed to adenovirus encoding scrambled siRNA. Indirect immunofluorescence showed that apical transporters (multidrug-resistance-associated protein 2, bile salt export pump, and multidrug-resistance protein 1) dissociated from their normal location at the apical membrane and were found largely associated with Rab11-containing endosomes. Localization of the basolateral membrane transporter, organic anion transporting polypeptide 2 (Oatp2), was not affected. Consistent with this dislocation of apical transporters, the biliary excretion of glutathione-methylfluorescein and cholylglycylamido-fluorescein was decreased significantly in the radixin-deficient cells, but not in the control siRNA cells.


CONCLUSIONS
Radixin is essential for maintaining the polarized targeting and/or retaining of canalicular membrane transporters and is a critical determinant of the overall structure and function of the apical membrane of hepatocytes.
Wang
Wei
W
Liver Center, Yale University School of Medicine, New Haven, Connecticut 06520-8019, USA.
Soroka
Carol J
CJ
Mennone
Albert
A
Rahner
Christoph
C
Harry
Kathy
K
Pypaert
Marc
M
Boyer
James L
JL
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000235:genetics*; D005452:Fluoresceins / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D015870:Gene Expression; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D015151:Immunoblotting; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000235:genetics*; D008854:Microscopy, Electron; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats
D003598:Cytoskeletal Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D034741:RNA, Small Interfering; C090312:cholylglycylamidofluorescein; C069306:5-chloromethylfluorescein; C071776:radixin