Induction of hepatic multidrug resistance-associated protein 3 by ethynylestradiol is independent of cholestasis and mediated by estrogen receptor.
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 multidrug resistance-associated protein 2 (Mrp2) is down-regulated. This study was undertaken to determine whether Mrp3 induction results from a direct effect of EE, independent of accumulation of any endogenous common Mrp2/Mrp3 substrates resulting from cholestasis and the potential mediation of estrogen receptor (ER). In in vivo studies, male rats were given a single, noncholestatic dose of EE (5 mg/kg s.c.), and basal bile flow and the biliary excretion rate of bile salts and glutathione were measured 5 hours later. This treatment increased Mrp3 mRNA by 4-fold, detected by real-time polymerase chain reaction, despite the absence of cholestasis. Primary culture of rat hepatocytes incubated with EE (1-10 µM) for 5 hours exhibited a 3-fold increase in Mrp3 mRNA (10 µM), consistent with in vivo findings. The increase in Mrp3 mRNA by EE was prevented by actinomycin D, indicating transcriptional regulation. When hepatocytes were incubated with an ER antagonist [7α,17β-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI182/780), 1 µM], in addition to EE, induction of Mrp3 mRNA was abolished, implicating ER as a key mediator. EE induced an increase in ER-α phosphorylation at 30 minutes and expression of c-Jun, a well-known ER target gene, at 60 minutes, as detected by Western blotting of nuclear extracts. These increases were prevented by ICI182/780. In summary, EE increased the expression of hepatic Mrp3 transcriptionally and independently of any cholestatic manifestation and required participation of an ER, most likely ER-α, through its phosphorylation.
Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, (2000) Rosario, Argentina.
María L
ML
Ruiz
Juan P
JP
Rigalli
Agostina
A
Arias
Silvina
S
Villanueva
Claudia
C
Banchio
Mary
M
Vore
Aldo D
AD
Mottino
Viviana A
VA
Catania
D000818:Animals; D001646:Bile / Q000378:metabolism; D001647:Bile Acids and Salts / Q000378:metabolism; D002478:Cells, Cultured; D002779:Cholestasis / Q000235:genetics / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000378:metabolism; D004967:Estrogens / Q000494:pharmacology*; D004997:Ethinyl Estradiol / Q000494:pharmacology*; D000077267:Fulvestrant; D005978:Glutathione / Q000378:metabolism; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D061251:Primary Cell Culture; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D060888:Real-Time Polymerase Chain Reaction; D013997:Time Factors; D015854:Up-Regulation
D001647:Bile Acids and Salts; D003609:Dactinomycin; D004958:Estradiol; D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D004967:Estrogens; D004997:Ethinyl Estradiol; D000077267:Fulvestrant; D005978:Glutathione; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; C114462:multidrug resistance-associated protein 3