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 a ffects the rate of bile formation, e.g., the conjugate export pump multidru g resistance protein 2 (MRP2) is regulated on a short-term scale by retriev al from and insertion into the canalicular membrane in the liver. This stud y reports on the effects of protein kinase C on MRP2 localization and activ ity in human hepatoblastoma HepG2 cells. MRP2 was detected in HepG2 cells b y immunocytochemistry and Western blot analysis. Functional activity was as sessed by confocal laser scanning microscopy using fluorescent MRP2 substra tes. In untreated HepG2 cells MRP2 was almost exclusively localized at the apical membrane. Treatment of HepG2 cells with phorbol-12-myristate-13-acet ate (PMA) resulted in a rapid decrease of apically localized MRP2 and a los s 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 min utes. This was shown by its colocalization with MRP1, human dipeptidylpepti dase IV (DPPIV), and transfected rat Ntcp. The effects of PMA on MRP2 local ization were sensitive to the protein kinase C (PKC) inhibitor Go6850 but i nsensitive to inhibition of MEK by PD098059. Basolateral MRP2-appearance wa s not inhibited by cycloheximide or by disruption of microtubules or microf ilaments. In rat livers cholestasis was induced by PMA (100 nmol) and Mrp2 was detected at the basolateral membrane in some areas, colocalizing with N tcp. The data suggest that retargeting of canalicular MRP2 to the basolater al membrane due to PKC activation may represent a novel mechanism that may contribute to cholestasis.