Fig. 4.

The −1800/−1766 region of GnRH-E1 is sufficient for androgen repression, and mutation of the TPA-responsive site abrogates repression of GnRH-E1. (A), Sequences of the −1800/−1766 region and mutations. Box: putative HRE; overline: TPA-responsive site; underline: Oct1-binding site; bold/underline: mutations. (B), GT1-7 cells were transiently transfected with the AR expression plasmid and a multimer of the −1800/−1766 sequence (4×1800/1766), or this multimer with 3-bp mutations in the putative HRE (mHRE, −1796/−1794) or the TPA-responsive site (mTPA, −1790/−1788). (C), GT1-7 cells were transiently transfected with the AR expression plasmid and 4×1800/1766, or this multimer with mutation in the Oct1-binding site (mOct1a). (D), GT1-7 cells were transiently transfected with the AR expression plasmid and GnRH-E1 on RSVp, or GnRH-E1 with 3-bp mutations in the putative HRE (mHRE) or the TPA-responsive site (mTPA). (E), GT1-7 cells were transiently transfected with the AR expression plasmid and GnRH-E1 on RSVp, or GnRH-E1 with a different mutation in the TPA-responsive site (m1792/1791). Cells were treated with 100 nM R1881 (closed bars) or ethanol vehicle (open bars) for 24 h and subjected to luciferase assay. Data are shown as relative luciferase units, relative to vehicle-treated 4×1800/1766 or GnRH-E1, and represent the mean, ± SEM, of at least three experiments done in quadruplicate. **P<0.01 and *** P<0.001 versus vehicle-treated.