Mitogen-activated protein kinases (MAPKS) of the p38 and Erk1/2 types are involved in estradiol 17ß-D-glucuronide (E17G)-induced cholestasis in rats

BOAGLIO A.C.; TOLEDO F.D.; BAROSSO I.R.; ZUCCHETTI A.E.; SÁNCHEZ POZZI E.J.; CROCENZI F.A.; ROMA M.G.

Barcelona

Congreso; The International Liver Congress - 47th Annual Meeting of the European Association for the Study of the Liver (EASL); 2012

Interational Association for the Study of the Liver (IASL) - European Association for the Study of the Liver (EASL)

Background and Aims: E17G, an endogenous, cholestatic metabolite causally associated with pregnancy-induced cholestasis, induces endocytic internalization of canalicular transporters relevant to bile secretion (Bsep, Mrp2).We previously demonstrated that Ca2+-dependent PKC (cPKC)- and PI3K/Akt-dependent signaling pathways act complementarily by inducing internalization and intracellular retention of canalicular transporters, respectively (Hepatology 48:1885, 2008; Hepatology 52:1465, 2010). Since both signaling pathways can activate MAPKs, we ascertained here whether MAPKs are involved in E17G-induced cholestasis. Methods: Cholestasis was induced in the isolated, perfused rat liver (IPRL) by intraportal inyection of E17G (2 mmol/liver). The role of MAPKs was studied by perfusing the p38MAPK inhibitor SB203580 (SB, 250 nM) or the ERK1/2 inhibitor PD98059 (PD, 5 mM). Bsep/Mrp2 localization was studied by immunofluorescence, followed by confocal microscopy and image analysis. Functional status of Bsep/Mrp2 was assessed in isolated rat hepatocyte couplets (IRHCs) pretreated with SB (1 mM) or PD (5 mM), with or without E17G (200 mM), by measuring the canalicular vacuolar accumulation (cVA) of the fluorescent substrates cholyllysylfluorescein (CLF) and glutathione-methylfluorescein (GS-MF), respectively. Activation of MAPKs was evaluated by immunoblotting of their phosphorylated, active forms. Results: p38MAPK and ERK1/2 were both activated by E17G; p38MAPK activation was prevented by G¨o6976 (1 mM, cPKC inhibitor), whereas ERK1/2 activation was blocked by wortmannin (100 nM, PI3K inhibitor). In IPRLs, E17G diminished bile flow steadily (−69±14%, p < 0.001), and SB significantly prevented this drop (+126±18%, p < 0.001). Conversely, PD did not attenuate the initial bile-flow decay, but recovered bile flow progressively. Endocytic internalization of Bsep and Mrp2 was prevented by both PD and SB, when evaluated at the end of the perfusion period. In IRHCs (>200 per group, n = 3?4), E17G diminished cVA of CLF to 36±3% of control values (p < 0.01), and pretreatment with PD and SB partially attenuated this decrease (57±2% and 56±1%, respectively; p < 0.05). Protection was even higher when both inhibitors were co-administered (77±2%, p < 0.05), suggesting complementarity between p38MAPK and ERK1/2. cVA of GS-MF showed a similar pattern of change. Conclusions: E17G-induced cholestasis involves cooperative activation of cPKC/p38MAPK and PI3K/Akt/ERK1/2 signaling pathways. Whereas cPKC/p38MAPK mediates endocytic internalization of canalicular transporters, PI3K/Akt/ERK1/2 favors their intracellular retention.