Estrogen receptor-a mediates human multidrug resistance associated protein 3 induction by 17a-ethynylestradiol. Role of activator protein-1 Marı ´a Laura Ruiz a, *, Juan Pablo Rigalli a , Agostina Arias a , Silvina Stella Maris Villanueva a , Claudia Banchio b , Mary Vore c , Aldo Domingo Mottino a , Viviana Alicia Catania a a Instituto de Fisiologı´a Experimental (CONICET) – Facultad de Ciencias Bioquı´micas y Farmace ´uticas (UNR), Suipacha 570, (2000) Rosario, Argentina b Instituto de Biologı´a Molecular y Celular de Rosario (CONICET) – Facultad de Ciencias Bioquı´micas y Farmace ´uticas (UNR), Rosario, Argentina c Graduate Center for Toxicology, University of Kentucky, KY, USA 1. Introduction Multidrug resistance-associated protein 3 (MRP3/ABCC3) is a member of the superfamily of adenosine tri-phosphate (ATP)- binding cassette (ABC) transporters. It is expressed in several epithelial cells including hepatocytes [1]. Due to its basolateral localization it transports into blood a wide range of endogenous and exogenous compounds such as sulphated bile salts, bilirubin glucuronides, specific anti-cancer drugs such as methotrexate, etoposide, teniposide, etc. [2–6]. Under physiological conditions its expression is low, but it is highly inducible by drugs such as acetaminophen [7] and phenobarbital [8], or under several cholestatic situations such as later stages of primary biliary cirrhosis and extrahepatic cholestasis of different etiology [9]. Induction of MRP3 activity results in re-directioning the secretion of common MRP substrates from the apical to the basolateral pole. It is known that estrogens are involved in the pathogenesis of both oral contraceptive-induced cholestasis and cholestasis of pregnancy [10,11]. In previous work we demonstrated that 17a- ethynylestradiol (EE), a synthetic estrogen widely used in contraceptive formulations and in estrogen replacement therapies, up-regulates hepatic Mrp3 in rats [12,13]. We later demonstrated that induction of Mrp3 by EE in rat liver is independent of cholestasis and occurs via activation of ER [14], though the underlying mechanism down-stream ER remains unknown. The canonical model for ER-mediated regulation of gene expression involves the direct binding of dimeric ER to DNA sequences known as estrogen response elements (ERE), which are specific, inverted palindromic sequences. In addition, ER can indirectly associate with promoters through protein-protein interactions with other DNA-binding transcription factors such as specificity protein 1 Biochemical Pharmacology 86 (2013) 401–409 A R T I C L E I N F O Article history: Received 17 April 2013 Accepted 21 May 2013 Available online 6 June 2013 Keywords: Drug transporter AP-1 Estrogen Multidrug resistance Nuclear receptor c-JUN. A B S T R A C T Previously, we have demonstrated that 17a-ethynylestradiol (EE) induces rat multidrug-resistance associated protein 3 (Mrp3, Abcc3) expression transcriptionally through estrogen receptor-a (ER-a) activation. We explored the effect of EE on MRP3 expression of human origin. HepG2 cells were transfected with ER-a and incubated with EE (1–10–50 mM) for 48 h. MRP3 protein and mRNA levels were measured by Western blotting and Real time PCR, respectively. EE up-regulated MRP3 protein and mRNA at 50 mM only in ER-a(+)-HepG2 cells. The in silico analysis of mrp3 promoter region demonstrated absence of estrogen response elements, but showed several Ap-1 binding sites. We further evaluated the potential involvement of the transcription factors c-JUN and c-FOS (members of Ap-1) in MRP3 up-regulation. ER-a(+) HepG2 cells were incubated with EE and c-FOS and c-JUN levels measured by Western blotting in nuclear extracts. EE up-regulated only c-JUN. Experiments of overexpression and knock-down of c-JUN by siRNA further demonstrated that this transcription factor is indeed implicated in MRP3 upregulation by EE. Co-immunoprecipitation assay demonstrated that EE induces c-JUN/ER-a interaction, and chromatin immunoprecipitation assay showed that this complex is recruited to the AP-1 binding consensus element present at the position (À1300/À1078 bp) of human mrp3 promoter. We conclude that EE induces MRP3 expression through ER-a, with recruitment of ER-a in complex with c- JUN to the human mrp3 promoter. ß 2013 Elsevier Inc. All rights reserved. * Corresponding author at: Instituto de Fisiologı ´a Experimental (CONICET) – Facultad de Ciencias Bioquı ´micas y Farmace ´ uticas (UNR), Suipacha 570, (2000) Rosario, Argentina. Tel.: +54 341 4305799; fax: +54 341 4399473. E-mail addresses: ruiz@ifise-conicet.gov.ar (M.L. Ruiz), jprigalli@gmail.com (J.P. Rigalli), agoarias@yahoo.com.ar (A. Arias), villanueva@ifise-conicet.gov.ar (S.S.M. Villanueva), banchio@ibr-conicet.gov.ar (C. Banchio), maryv@emailuky.edu (M. Vore), aldomottino@yahoo.com.ar (A.D. Mottino), vcatania@fbioyf.unr.edu.ar (V.A. Catania). Contents lists available at SciVerse ScienceDirect Biochemical Pharmacology jo u rn al h om epag e: ww w.els evier.c o m/lo cat e/bio c hem p har m 0006-2952/$ – see front matter ß 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2013.05.025