Mitogen‑activated protein kinases are involved in hepatocanalicular dysfunction and cholestasis induced by oxidative stress

TOLEDO FD; BASIGLIO CL; BAROSSO IR; BOAGLIO AC; ZUCCHETTI AE; SANCHEZ POZZI EJ; ROMA MG

ARCHIVES OF TOXICOLOGY.

SPRINGER

Lugar: Berlin; Año: 2017 vol. 91 p. 2391 - 2403

0340-5761

In previous studies, we showed that the prooxidantmodel agent tert-butyl hydroperoxide (tBuOOH)induces alterations in hepatocanalicular secretory functionby activating Ca2+-dependent protein kinase C isoforms(cPKC), via F-actin disorganization followed by endocyticinternalization of canalicular transporters relevant to bileformation (Mrp2, Bsep). Since mitogen-activated proteinkinases (MAPKs) may be downstream effectors of cPKC,we investigated here the involvement of the MAPKs ofthe ERK1/2, JNK1/2, and p38MAPK types in these deleteriouseffects. tBuOOH (100 μM, 15 min) increased the proportionof the active, phosphorylated forms of ERK1/2,JNK1/2, and p38MAPK, and panspecific PKC inhibition withbisindolylmaleimide-1 (100 nM) or selective cPKC inhibitionwith Gö6976 (1 μM) prevented the latter two events.In isolated rat hepatocyte couplets, tBuOOH (100 μM, 15min) decreased the canalicular vacuolar accumulation of thefluorescent Bsep and Mrp2 substrates, cholylglycylamidofluorescein, and glutathione-methylfluorescein, respectively,and selective inhibitors of ERK1/2 (PD098059), JNK1/2(SP600125), and p38MAPK (SB203580) partially preventedthese alterations. In in situ perfused rat livers, these threeMAPK inhibitors prevented tBuOOH (75 μM)-inducedimpairment of bile flow and the decrease in the biliaryoutput of the Bsep and Mrp2 substrates, taurocholate, anddinitrophenyl-S-glutathione, respectively. The changes inBsep/Mrp2 and F-actin localization induced by tBuOOH,as assessed by (immuno)fluorescence staining followed byanalysis of confocal images, were prevented total or partiallyby the MAPK inhibitors. We concluded that MAPKsof the ERK1/2, JNK1/2, and p38MAPK types are all involvedin cholestasis induced by oxidative stress, by promotingF-actin rearrangement and further endocytic internalizationof canalicular transporters critical for bile formation.