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

Abstract

In previous studies, we showed that the pro-oxidant model agent tert-butyl hydroperoxide (tBuOOH) induces alterations in hepatocanalicular secretory function by activating Ca²⁺-dependent protein kinase C isoforms (cPKC), via F-actin disorganization followed by endocytic internalization of canalicular transporters relevant to bile formation (Mrp2, Bsep). Since mitogen-activated protein kinases (MAPKs) may be downstream effectors of cPKC, we investigated here the involvement of the MAPKs of the ERK1/2, JNK1/2, and p38MAPK types in these deleterious effects. tBuOOH (100 µM, 15 min) increased the proportion of the active, phosphorylated forms of ERK1/2, JNK1/2, and p38MAPK, and panspecific PKC inhibition with bisindolylmaleimide-1 (100 nM) or selective cPKC inhibition with Gö6976 (1 μM) prevented the latter two events. In isolated rat hepatocyte couplets, tBuOOH (100 µM, 15 min) decreased the canalicular vacuolar accumulation of the fluorescent Bsep and Mrp2 substrates, cholylglycylamido fluorescein, and glutathione-methylfluorescein, respectively, and selective inhibitors of ERK1/2 (PD098059), JNK1/2 (SP600125), and p38MAPK (SB203580) partially prevented these alterations. In in situ perfused rat livers, these three MAPK inhibitors prevented tBuOOH (75 µM)-induced impairment of bile flow and the decrease in the biliary output of the Bsep and Mrp2 substrates, taurocholate, and dinitrophenyl-S-glutathione, respectively. The changes in Bsep/Mrp2 and F-actin localization induced by tBuOOH, as assessed by (immuno)fluorescence staining followed by analysis of confocal images, were prevented total or partially by the MAPK inhibitors. We concluded that MAPKs of the ERK1/2, JNK1/2, and p38MAPK types are all involved in cholestasis induced by oxidative stress, by promoting F-actin rearrangement and further endocytic internalization of canalicular transporters critical for bile formation.

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Arch Toxicol (2017) 91:2391–2403
DOI 10.1007/s00204-016-1898-1
ORGAN TOXICITY AND MECHANISMS
Mitogen‑activated protein kinases are involved
in hepatocanalicular dysfunction and cholestasis induced
by oxidative stress
Flavia D. Toledo1 · Cecilia L. Basiglio1 · Ismael R. Barosso1 · Andrea C. Boaglio1 ·
Andrés E. Zucchetti1 · Enrique J. Sánchez Pozzi1 · Marcelo G. Roma1
Received: 26 May 2016 / Accepted: 24 November 2016 / Published online: 2 December 2016
© Springer-Verlag Berlin Heidelberg 2016
output of the Bsep and Mrp2 substrates, taurocholate, and
dinitrophenyl-S-glutathione, respectively. The changes in
Bsep/Mrp2 and F-actin localization induced by tBuOOH,
as assessed by (immuno)fluorescence staining followed by
analysis of confocal images, were prevented total or par-
tially by the MAPK inhibitors. We concluded that MAPKs
of the ERK1/2, JNK1/2, and p38MAPK types are all involved
in cholestasis induced by oxidative stress, by promoting
F-actin rearrangement and further endocytic internalization
of canalicular transporters critical for bile formation.
Keywords Oxidative stress · Hepatocellular cholestasis ·
Canalicular transporters · Mitogen-activated protein
kinases · Actin cytoskeleton
Introduction
Oxidative stress (OS) is a common feature in most
hepatopathies, including drug and environmental toxin-
induced hepatotoxicity, hepatic ischemia–reperfusion
injury, viral and autoimmune hepatitis, alcoholic and non-
alcoholic steatohepatitis, and pathologies leading to hepatic
accumulation of either heavy metals, such as iron (hemo-
chromatosis) and copper (Wilson’s disease), or bile acids
(obstructive or functional cholestasis) (Copple et al. 2010;
Jaeschke et al. 2002).
In recent years, evidence has accumulated that, in chole-
static hepatopathies, a vicious circle occurs, since OS is
cholestatic in nature, thus aggravating the initial secretory
failure (Roma and Sanchez Pozzi 2008). Indeed, our group
demonstrated in isolated rat hepatocyte couplets (IRHCs)
that OS induces rapid endocytic internalization of the
bile salt export pump (Bsep, AKA: Abcc11) (Perez et al.
2006b), the main canalicular bile salt transporter. A similar
Abstract In previous studies, we showed that the pro-
oxidant model agent tert-butyl hydroperoxide (tBuOOH)
induces alterations in hepatocanalicular secretory function
by activating Ca2+-dependent protein kinase C isoforms
(cPKC), via F-actin disorganization followed by endocytic
internalization of canalicular transporters relevant to bile
formation (Mrp2, Bsep). Since mitogen-activated protein
kinases (MAPKs) may be downstream effectors of cPKC,
we investigated here the involvement of the MAPKs of
the ERK1/2, JNK1/2, and p38MAPK types in these deleteri-
ous effects. tBuOOH (100 µM, 15 min) increased the pro-
portion of the active, phosphorylated forms of ERK1/2,
JNK1/2, and p38MAPK, and panspecific PKC inhibition with
bisindolylmaleimide-1 (100 nM) or selective cPKC inhibi-
tion with Gö6976 (1 μM) prevented the latter two events.
In isolated rat hepatocyte couplets, tBuOOH (100 µM, 15
min) decreased the canalicular vacuolar accumulation of the
fluorescent Bsep and Mrp2 substrates, cholylglycylamido
fluorescein, and glutathione-methylfluorescein, respectively,
and selective inhibitors of ERK1/2 (PD098059), JNK1/2
(SP600125), and p38MAPK (SB203580) partially prevented
these alterations. In in situ perfused rat livers, these three
MAPK inhibitors prevented tBuOOH (75 µM)-induced
impairment of bile flow and the decrease in the biliary
Electronic supplementary material The online version of this
article (doi:10.1007/s00204-016-1898-1) contains supplementary
material, which is available to authorized users.
* Marcelo G. Roma
mroma@fbioyf.unr.edu.ar
1 Instituto de Fisiología Experimental (IFISE-CONICET),
Facultad de Ciencias Bioquímicas y Farmacéuticas,
Universidad Nacional de Rosario (UNR), Suipacha 570,
2000, Rosario, Argentina
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