Sustained internalization of the hepatocanalicular transporter multidrug resistance-associated protein 2 (Mrp2) in cholestasis leads to its exacerbated proteasomal degradation.

MEDEOT A.C.; ANDERMATTEN R.B.; SALAS G.; SCHUCK V.S.; BAROSSO I.R.; CROCENZI F.A.; ROMA M.G.

Mar del Plata

Congreso; Reunión de Sociedades de Biociencias (SAIC, SAI, SAFIS) 2022; 2022

SAIC, SAI, SAFIS

Exacerbated endocytosis of canalicular carriers, including Mrp2, is a main mechanism involved in cholestasis, as has been shown with the model cholestatic agent, taurolithocholate (TLC) (Crocenzi et al. Gut 52: 1170, 2003). We hypothesized that this exacerbated internalization is followed by accelerated degradation, thus explaining the unchanged carrier protein expression despite increased synthesis observed in chronic cholestatic diseases. We therefore tested here whether sustained Mrp2 internalization leads to accelerated degradation, and if so, which degradation mechanism is involved. Mrp2 protein expression was quantified by Western blot in sandwich-cultured rat hepatocytes (SCRH) incubated with TLC (2.5 μM), or vehicle (DMSO) in controls (C), in the presence of cycloheximide to block “de novo” Mrp2 synthesis. TLC exposure for 12 hs induced no change in Mrp2 expression as compared to C, whereas a significant decrease was observed at 24 h (69±4%; n=7) and 48 h (66±8%; n=3); p<0.05 vs. C. Pretreatment with the proteosomal inhibitor MG132 (10 μM) for 30 min, followed by TLC exposure for 24h, fully prevented this decrease (TLC: 68±4%, p<0.05 vs. C; MG132: 92±7 %; MG132+TLC: 106±6%, p<0.05 vs TLC; n=3). A similar pattern was observed by Mrp2 immunostaining followed by confocal microscopy. This prevention was not observed when SCRH were pretreated with the lysosomal inhibitor pepstain A (50 μM). Mrp2 transport function assays were carried out by preloading SCRHs with chloromethylfluorescein diacetate (CMFDA), and measuring the initial transport rate (ITR) of its fluorescent metabolite, GSH-S-methylfluorescein (GS-MF), a Mrp2 substrate. ITR, expressed as percent of C, was reduced by TLC (-25%), and this effect was prevented by MG132 (+6%). We conclude that sustained endocytosis of Mrp2 in cholestasis leads to its exacerbated proteosomal degradation. This finding could be relevant to understand the impairment of hepatocanalicular transporters in chronic cholestasis.