title: ERK1/2 and p38 MAPKs are complementarily involved in estradiol 17ß-D-glucuronide-induced cholestasis: crosstalk with cPKC and PI3K.
abstract: OBJECTIVE
  The endogenous, cholestatic metabolite estradiol 17ß-D-glucuronide (E(2)17G) induces endocytic internalization of the canalicular transporters relevant to bile formation, Bsep and Mrp2. We evaluated here whether MAPKs are involved in this effect.
  
  
  METHODS
  ERK1/2, JNK1/2, and p38 MAPK activation was assessed by the increase in their phosphorylation status. Hepatocanalicular function was evaluated in isolated rat hepatocyte couplets (IRHCs) by quantifying the apical secretion of fluorescent Bsep and Mrp2 substrates, and in isolated, perfused rat livers (IPRLs), using taurocholate and 2,4-dinitrophenyl-S-glutathione, respectively. Protein kinase participation in E(2)17G-induced secretory failure was assessed by co-administering selective inhibitors. Internalization of Bsep/Mrp2 was assessed by confocal microscopy and image analysis.
  
  
  RESULTS
  E(2)17G activated all kinds of MAPKs. The PI3K inhibitor wortmannin prevented ERK1/2 activation, whereas the cPKC inhibitor Gö6976 prevented p38 activation, suggesting that ERK1/2 and p38 are downstream of PI3K and cPKC, respectively. The p38 inhibitor SB203580 and the ERK1/2 inhibitor PD98059, but not the JNK1/2 inhibitor SP600125, partially prevented E(2)17G-induced changes in transporter activity and localization in IRHCs. p38 and ERK1/2 co-inhibition resulted in additive protection, suggesting complementary involvement of these MAPKs. In IPRLs, E(2)17G induced endocytosis of canalicular transporters and a rapid and sustained decrease in bile flow and biliary excretion of Bsep/Mrp2 substrates. p38 inhibition prevented this initial decay, and the internalization of Bsep/Mrp2. Contrarily, ERK1/2 inhibition accelerated the recovery of biliary secretion and the canalicular reinsertion of Bsep/Mrp2.
  
  
  CONCLUSIONS
  cPKC/p38 MAPK and PI3K/ERK1/2 signalling pathways participate complementarily in E(2)17G-induced cholestasis, through internalization and sustained intracellular retention of canalicular transporters, respectively.
authors.affiliation: Institute of Experimental Physiology, National Scientific and Technical Research Council/National University of Rosario, Rosario, Argentina.
authors.forename: Andrea C
authors.initials: AC
authors.lastname: Boaglio
authors.forename: Andrés E
authors.initials: AE
authors.lastname: Zucchetti
authors.forename: Flavia D
authors.initials: FD
authors.lastname: Toledo
authors.forename: Ismael R
authors.initials: IR
authors.lastname: Barosso
authors.forename: Enrique J
authors.initials: EJ
authors.lastname: Sánchez Pozzi
authors.forename: Fernando A
authors.initials: FA
authors.lastname: Crocenzi
authors.forename: Marcelo G
authors.initials: MG
authors.lastname: Roma
mesh_terms: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002779:Cholestasis / Q000139:chemically induced*; D004958:Estradiol / Q000031:analogs & derivatives* / Q000633:toxicity; D005260:Female; D022781:Hepatocytes / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D020935:MAP Kinase Signaling System / Q000502:physiology*; D018613:Microscopy, Confocal; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000502:physiology*; D018709:Statistics, Nonparametric; D013656:Taurocholic Acid; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
chemical_list: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters; C490548:Abcb11 protein, rat; C423013:Abcc2 protein, rat; C025483:estradiol-17 beta-glucuronide; D004958:Estradiol; D013656:Taurocholic Acid; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D048051:p38 Mitogen-Activated Protein Kinases