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, GS; BAROSSO, IR; BOAGLIO, AC; LAROCCA, MC; SANCHEZ POZZI, EJ; ROMA, MG; CROCENZI, FA

Mar del Plata

Congreso; Reunión Anual de SAIC-SAI 2016; 2016

SAIC-SAI

Endocytosis of canalicular transporters is a key pathomechanism of E17G-induced cholestasis. In preliminary experiments in rat hepatocyte couplets, we reported that this process is prevented by specific inhibitors of the clathrin-dependent endocytosis (Medicina 74 Supl. III, 106, 2014). In physiological conditions, canalicular transporters are mostly presented in raft (high-cholesterol, caveolin-enriched) microdomains. Clathrin-dependent endocytosis requires both the presence of the cargo in non-raft (low-cholesterol, clathrin-enriched) microdomains and the recruitment of the adaptor protein AP2 to these domains. First, we evaluated whether E17G induces a shift of Bsep and Mrp2 from raft to non-raft microdomains, in order to allow for clathrin-dependent endocytosis from the latter domain. E17G (11 μmol/g b.w.) was administered to whole rats, and liver samples were taken after 20 min, when the maximum decrease of bile flowis reached. Highly purified plasma membrane fractions composed of raft or non-raft microdomains were obtained by ultracentrifugation from liver homogenates; enrichment of these fractions was assessed by western blot of the specific markers caveolin and clathrin, respectively. Western blot of Bsep and Mrp2 showed that E17G induced a switch between microdomains, leading to a high enrichment of these transporters in the non-raft fraction (Bsep: ~80% of total vs ~40% in controls; 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-cultured rat hepatocytes. AP2 knockdown completely prevented E17G-induced endocytosis of Bsep and Mrp2, as shown by confocal microscopy analysis. Thus, we proposed that E17G-induced endocytosis of Bsep and Mrp2 in rat livers involves a switch of these transporters from raft to non-raft canalicular microdomains, from where they areendocytosed by a clathrin-dependent mechanism.