INVESTIGADORES
CROCENZI Fernando Ariel
congresos y reuniones científicas
Título:
Mitogen-activated protein kinases (MAPKS) of the p38 and ERK1/2 types are involved in estradiol 17ß-D-glucuronide (E17G)-induced cholestasis in rats
Autor/es:
BOAGLIO, ANDREA C; TOLEDO, FLAVIA D.; BAROSSO, ISMAEL R.; ZUCCHETTI, ANDRÉS ERNESTO; SÁNCHEZ POZZI, ENRIQUE J; CROCENZI, FERNANDO A.; ROMA, MARCELO G
Lugar:
Barcelona
Reunión:
Congreso; 47th Annual Meeting of the European Association for the Study of the Liver (EASL); 2012
Institución organizadora:
EASL
Resumen:
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 = 34), 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.