ESTRADIOL 17 BETA-D-GLUCURONIDE (E17G) INDUCES SWITCH OF THE CANALICULAR TRANSPORTERS BSEP AND MRP2 FROM RAFT TO NON-RAFT MICRODOMAINS, FOLLOWED BY CLATHRIN-DEPENDENT ENDOCYTOSIS.

MISZCZUK, GISEL SABRINA; BAROSSO, ISMAEL R.; BOAGLIO, ANDREA C; LAROCCA, M CECILIA; SáNCHEZ POZZI, ENRIQUE J; ROMA, MARCELO G; CROCENZI, FERNANDO A.

Mar del Plata

Congreso; LXI REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA; 2016

Endocytosis of canalicular transporters is a key pathomechanismof E17G-induced cholestasis. In preliminary experiments in rat hepatocytecouplets, we reported that this process is prevented by specificinhibitors of the clathrin-dependent endocytosis (Medicina 74 Supl.III, 106, 2014). In physiological conditions, canalicular transportersare mostly presented in raft (high-cholesterol, caveolin-enriched)microdomains. Clathrin-dependent endocytosis requires both thepresence of the cargo in non-raft (low-cholesterol, clathrin-enriched)microdomains and the recruitment of the adaptor protein AP2 tothese domains. First, we evaluated whether E17G induces a shift ofBsep and Mrp2 from raft to non-raft microdomains, in order to allowfor clathrin-dependent endocytosis from the latter domain. E17G(11 ìmol/g b.w.) was administered to whole rats, and liver sampleswere taken after 20 min, when the maximum decrease of bile flowis reached. Highly purified plasma membrane fractions composedof raft or non-raft microdomains were obtained by ultracentrifugationfrom liver homogenates; enrichment of these fractions was assessedby western blot of the specific markers caveolin and clathrin, respectively.Western blot of Bsep and Mrp2 showed that E17G induced aswitch between microdomains, leading to a high enrichment of thesetransporters in the non-raft fraction (Bsep: ~80% of total vs ~40% incontrols; Mrp2: ~80% of total vs ~25% in controls). To evaluate thedependency of AP2 for E17G-induced canalicular transporter endocytosis,we performed siRNA knockdown of AP2 in sandwich-culturedrat hepatocytes. AP2 knockdown completely prevented E17G-inducedendocytosis of Bsep and Mrp2, as shown by confocal microscopyanalysis. Thus, we proposed that E17G-induced endocytosis ofBsep and Mrp2 in rat livers involves a switch of these transportersfrom raft to non-raft canalicular microdomains, from where they areendocytosed by a clathrin-dependent mechanism.