Radixin Is Required to Maintain Apical Canalicular Membrane Structure and Function in Rat Hepatocytes

Background & Aims: 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.