Protein kinase C-dependent distribution of the multidrug resistance protein 2 from the canalicular to the basolateral membrane in human HepG2 cells.
The subcellular localization of hepatobiliary transport proteins directly affects the rate of bile formation, e.g., the conjugate export pump multidrug resistance protein 2 (MRP2) is regulated on a short-term scale by retrieval from and insertion into the canalicular membrane in the liver. This study reports on the effects of protein kinase C on MRP2 localization and activity in human hepatoblastoma HepG2 cells. MRP2 was detected in HepG2 cells by immunocytochemistry and Western blot analysis. Functional activity was assessed by confocal laser scanning microscopy using fluorescent MRP2 substrates. In untreated HepG2 cells MRP2 was almost exclusively localized at the apical membrane. Treatment of HepG2 cells with phorbol-12-myristate-13-acetate (PMA) resulted in a rapid decrease of apically localized MRP2 and a loss of more than 90% of pseudocanaliculi within 4 hours. This was accompanied by a reduced pseudocanalicular secretion of the MRP2 substrate glutathione-methylfluorescein. Interestingly, PMA treatment (1-100 nmol/L) led to the appearance of immunoreactive MRP2 at the basolateral membrane within 30 minutes. This was shown by its colocalization with MRP1, human dipeptidylpeptidase IV (DPPIV), and transfected rat Ntcp. The effects of PMA on MRP2 localization were sensitive to the protein kinase C (PKC) inhibitor Gö6850 but insensitive to inhibition of MEK by PD098059. Basolateral MRP2-appearance was not inhibited by cycloheximide or by disruption of microtubules or microfilaments. In rat livers cholestasis was induced by PMA (100 nmol) and MRP2 was detected at the basolateral membrane in some areas, colocalizing with Ntcp. The data suggest that retargeting of canalicular MRP2 to the basolateral membrane due to PKC activation may represent a novel mechanism that may contribute to cholestasis.
Kubitz
R
R
Department of Gastroenterology, Hepatology and Infectiology, Heinrich Heine University, Düsseldorf, Germany. RKubitz@t-online.de
Huth
C
C
Schmitt
M
M
Horbach
A
A
Kullak-Ublick
G
G
Häussinger
D
D
D018435:ATP Binding Cassette Transporter, Subfamily B; D001648:Bile Canaliculi / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011493:Protein Kinase C / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014018:Tissue Distribution / Q000187:drug effects; D014407:Tumor Cells, Cultured
D018435:ATP Binding Cassette Transporter, Subfamily B; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; C000715087:ABCC2 protein, human; C421109:multidrug resistance-associated protein 1