Estrogen receptor-α mediates human multidrug resistance associated protein 3 induction by 17α-ethynylestradiol. Role of activator protein-1

Previously, we have demonstrated that 17 alpha-ethynylestradiol (EE) induces rat multidrug-resistance associated protein 3 (Mrp3, Abcc3) expression transcriptionally through estrogen receptor-alpha (ER-alpha) activation. We explored the effect of EE on MRP3 expression of human origin. HepG2 cells were transfected with ER-alpha and incubated with EE (1-10-50 mu M) 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 p14 only in ER-alpha(+)-HepG2 cells. The in silico analysis of mip3 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-alpha(+) 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-alpha interaction, and chromatin immunoprecipitation assay showed that this complex is recruited to the AP-1 binding consensus element present at the position (-1300/-1078 bp) of human mrp3 promoter. We conclude that EE induces MRP3 expression through ER-alpha, with recruitment of ER-alpha in complex with c-JUN to the human mrp3 promoter. (C) 2013 Elsevier Inc. All rights reserved.