Molecular basis for hepatic multidrug resistance associated protein 3 induction by ethynylestradiol

MOTINO A, CATANIA V, RUIZ L Y BANCHIO C

Congreso; EASL,47th Annual Meeting of the European Association for the Study of the; 2012

  Multidrug resistance-associated protein 3 (Mrp3, Abcc3) expression and activity are up-regulated in rat liver after in vivo repeated administration of ethynylestradiol (EE), a cholestatic synthetic estrogen, whereas Mrp2 is down-regulated. Our aim was to determine if Mrp3 induction results from a direct effect of EE, independent of accumulation of any endogenous common Mrp2/Mrp3 substrates resulting from cholestasis. Male rats were given a single dose of EE (5 mg/kg, s.c.) and basal bile flow, biliary excretion rate of bile salts, and glutathione and serum alkaline phosphatase, were measured 5 h later. This treatment increased Mrp3 mRNA (Control: 100±13 vs EE: 431±147 % p< 0.05, n=5), despite the unchanged cholestatic parameters tested. Primary culture of rat hepatocytes incubated with 10 ìM EE for 5 h, exhibited an increase in Mrp3 mRNA (Control: 100±18 vs EE: 491±188% p< 0.05, n=5). The increase in Mrp3 mRNA by EE was prevented by actinomycin-D (5 ìg/ml), indicating transcriptional regulation. When hepatocytes were pre-incubated for 30 min with an estrogen receptor (ER) antagonist (ICI182/780, 1 ìM), induction of Mrp3 mRNA was totally abolished. Similarly, EE induced MRP3 in HepG2 cells only after transfection with ER-á. ER-á could modulate gene expression by binding to estrogen response elements (ERE), or indirectly through interaction with transcription factors such as Ap- An in silico analysis of MRP3 promoter region showed no presence of ERE binding sites but identified several Ap-1 binding sites. Co-transfection with plasmids encoding Ap-1 members (c- Jun plus c-Fos) and ER-á led to up-regulation of MRP3 expression (250% p< 0.05, n=3), with no response for the ER-á (-)/Ap-1 (+) group. These results suggest involvement of up-regulation of c-Fos or c-Jun by EE in MRP3 induction, and that their interaction with ER-á is necessary. Treatment of HepG2-ER-á(+) cells with EE increased the expression of c-Jun (500%, p< 0.05, n=3) but not c-Fos, as evaluated by western blotting of nuclear extracts. We also demonstrated by co-immunoprecipitation assays that c-Jun/ER-á interaction was increased by EE. In conclusion, EE induced Mrp3/MRP3 through a direct mechanism, requiring participation of ER-á in vivo repeated administration of ethynylestradiol (EE), a cholestatic synthetic estrogen, whereas Mrp2 is down-regulated. Our aim was to determine if Mrp3 induction results from a direct effect of EE, independent of accumulation of any endogenous common Mrp2/Mrp3 substrates resulting from cholestasis. Male rats were given a single dose of EE (5 mg/kg, s.c.) and basal bile flow, biliary excretion rate of bile salts, and glutathione and serum alkaline phosphatase, were measured 5 h later. This treatment increased Mrp3 mRNA (Control: 100±13 vs EE: 431±147 % p< 0.05, n=5), despite the unchanged cholestatic parameters tested. Primary culture of rat hepatocytes incubated with 10 ìM EE for 5 h, exhibited an increase in Mrp3 mRNA (Control: 100±18 vs EE: 491±188% p< 0.05, n=5). The increase in Mrp3 mRNA by EE was prevented by actinomycin-D (5 ìg/ml), indicating transcriptional regulation. When hepatocytes were pre-incubated for 30 min with an estrogen receptor (ER) antagonist (ICI182/780, 1 ìM), induction of Mrp3 mRNA was totally abolished. Similarly, EE induced MRP3 in HepG2 cells only after transfection with ER-á. ER-á could modulate gene expression by binding to estrogen response elements (ERE), or indirectly through interaction with transcription factors such as Ap- An in silico analysis of MRP3 promoter region showed no presence of ERE binding sites but identified several Ap-1 binding sites. Co-transfection with plasmids encoding Ap-1 members (c- Jun plus c-Fos) and ER-á led to up-regulation of MRP3 expression (250% p< 0.05, n=3), with no response for the ER-á (-)/Ap-1 (+) group. These results suggest involvement of up-regulation of c-Fos or c-Jun by EE in MRP3 induction, and that their interaction with ER-á is necessary. Treatment of HepG2-ER-á(+) cells with EE increased the expression of c-Jun (500%, p< 0.05, n=3) but not c-Fos, as evaluated by western blotting of nuclear extracts. We also demonstrated by co-immunoprecipitation assays that c-Jun/ER-á interaction was increased by EE. In conclusion, EE induced Mrp3/MRP3 through a direct mechanism, requiring participation of ER-á á. ER-á could modulate gene expression by binding to estrogen response elements (ERE), or indirectly through interaction with transcription factors such as Ap- An in silico analysis of MRP3 promoter region showed no presence of ERE binding sites but identified several Ap-1 binding sites. Co-transfection with plasmids encoding Ap-1 members (c- Jun plus c-Fos) and ER-á led to up-regulation of MRP3 expression (250% p< 0.05, n=3), with no response for the ER-á (-)/Ap-1 (+) group. These results suggest involvement of up-regulation of c-Fos or c-Jun by EE in MRP3 induction, and that their interaction with ER-á is necessary. Treatment of HepG2-ER-á(+) cells with EE increased the expression of c-Jun (500%, p< 0.05, n=3) but not c-Fos, as evaluated by western blotting of nuclear extracts. We also demonstrated by co-immunoprecipitation assays that c-Jun/ER-á interaction was increased by EE. In conclusion, EE induced Mrp3/MRP3 through a direct mechanism, requiring participation of ER-á in silico analysis of MRP3 promoter region showed no presence of ERE binding sites but identified several Ap-1 binding sites. Co-transfection with plasmids encoding Ap-1 members (c- Jun plus c-Fos) and ER-á led to up-regulation of MRP3 expression (250% p< 0.05, n=3), with no response for the ER-á (-)/Ap-1 (+) group. These results suggest involvement of up-regulation of c-Fos or c-Jun by EE in MRP3 induction, and that their interaction with ER-á is necessary. Treatment of HepG2-ER-á(+) cells with EE increased the expression of c-Jun (500%, p< 0.05, n=3) but not c-Fos, as evaluated by western blotting of nuclear extracts. We also demonstrated by co-immunoprecipitation assays that c-Jun/ER-á interaction was increased by EE. In conclusion, EE induced Mrp3/MRP3 through a direct mechanism, requiring participation of ER-á á led to up-regulation of MRP3 expression (250% p< 0.05, n=3), with no response for the ER-á (-)/Ap-1 (+) group. These results suggest involvement of up-regulation of c-Fos or c-Jun by EE in MRP3 induction, and that their interaction with ER-á is necessary. Treatment of HepG2-ER-á(+) cells with EE increased the expression of c-Jun (500%, p< 0.05, n=3) but not c-Fos, as evaluated by western blotting of nuclear extracts. We also demonstrated by co-immunoprecipitation assays that c-Jun/ER-á interaction was increased by EE. In conclusion, EE induced Mrp3/MRP3 through a direct mechanism, requiring participation of ER-á á (-)/Ap-1 (+) group. These results suggest involvement of up-regulation of c-Fos or c-Jun by EE in MRP3 induction, and that their interaction with ER-á is necessary. Treatment of HepG2-ER-á(+) cells with EE increased the expression of c-Jun (500%, p< 0.05, n=3) but not c-Fos, as evaluated by western blotting of nuclear extracts. We also demonstrated by co-immunoprecipitation assays that c-Jun/ER-á interaction was increased by EE. In conclusion, EE induced Mrp3/MRP3 through a direct mechanism, requiring participation of ER-á á is necessary. Treatment of HepG2-ER-á(+) cells with EE increased the expression of c-Jun (500%, p< 0.05, n=3) but not c-Fos, as evaluated by western blotting of nuclear extracts. We also demonstrated by co-immunoprecipitation assays that c-Jun/ER-á interaction was increased by EE. In conclusion, EE induced Mrp3/MRP3 through a direct mechanism, requiring participation of ER-á á(+) cells with EE increased the expression of c-Jun (500%, p< 0.05, n=3) but not c-Fos, as evaluated by western blotting of nuclear extracts. We also demonstrated by co-immunoprecipitation assays that c-Jun/ER-á interaction was increased by EE. In conclusion, EE induced Mrp3/MRP3 through a direct mechanism, requiring participation of ER-á á interaction was increased by EE. In conclusion, EE induced Mrp3/MRP3 through a direct mechanism, requiring participation of ER-áá The increased expression of c-Jun and its further interaction with ER-á are likely involved.á are likely involved.