Gastroenterology

Gastroenterology

Volume 131, Issue 3, September 2006, Pages 878-884
Gastroenterology

Basic–liver, pancreas, and biliary tract
Radixin Is Required to Maintain Apical Canalicular Membrane Structure and Function in Rat Hepatocytes

https://doi.org/10.1053/j.gastro.2006.06.013Get rights and content

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.

Section snippets

Reagents

BD Adeno-X Expression Systems 2 was purchased from BD Biosciences (Bedford, MA). Alexa-conjugated secondary antibodies, TO-PRO 3, 5-chloromethylfluorescein diacetate (CMFDA), and Alexa 594–conjugated phalloidin were purchased from Molecular Probes (Eugene, OR). Cholylglycylamido-fluorescein (CGamF) was a gift from Alan Hofmann (San Diego, CA). The following antibodies were used: mouse anti-Mrp2 (Alexis Biochemicals, San Diego, CA), rabbit anti-radixin (Cell Signaling Technology, Beverly, MA),

Adenovirus-mediated siRNA Effectively Inhibits Radixin Expression in Sandwich-Cultured Rat Hepatocytes

To address the requirement for radixin in normal hepatocyte function, we attempted to inhibit radixin expression in collagen sandwich–cultured rat hepatocytes using adenovirus-mediated siRNA. We first tested the 4 siRNA target sequences in a normal rat liver cell line, clone 9, to evaluate their ability to suppress radixin. Immunoblot analysis in Figure 1A indicates that all 4 siRNAs individually and in combination reduced the level of radixin expression to 19%–34% of Ad-siControl–treated

Discussion

The principal finding in this study is that radixin is required for the maintenance of the structure of the apical bile canalicular membrane and the localization and function of apical canalicular membrane transport proteins. When this ERM protein is suppressed in cell culture, the once-established bile canalicular domain is disrupted markedly. This impairment in the normal configuration of the apical canalicular membrane was confirmed by the altered distribution of actin filaments. Actin

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    Supported by National Institutes of Health grants DK 25636 and P30-34989 (J.L.B.).

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