PARENT SESSION

(16) PROGESTERONE RECEPTOR IS TRANSCRIPTIONALLY ACTIVATED BY GONADOTROPIN-RELEASING HORMONES, GnRH I AND GnRH II, VIA A LIGAND-INDEPENDENT PATHWAY IN PITUITARY GONADOTROPH CELLS.

An, Beum-Soo¹, Kim, Ki-Yon¹, Selva, David M. , Hammond, Geoffrey L. ¹, Choi, Kyung-Chul ¹, Leung, Peter C. K. ¹, ¹ University of British Columbia, Vancouver, BC, Canada

ABSTRACT- Regulation of the hypothalamic-pituitary-gonadal axis involves a complex interplay of peptides and steroid hormones. The mechanisms of feedback regulation by sex steroids are complex and include effects at both hypothalamic and pituitary levels. To test the effects of gonadotropin-releasing hormone (GnRH) I and II on the activation of progesterone receptor (PR) in the mouse pituitary alphaT3-1 cell line, we analyzed the activity of progesterone response element (PRE) using a luciferase reporter gene assay, protein-protein interaction by immunoprecipitation, and protein-DNA interaction by ChIP assay. In addition, we investigated the nuclear translocation of PR by GnRH I and GnRH II. After transfection of pituitary alphaT3-1 with PRE-Luciferase vector, we treated the cells with GnRH I (10^-7 M) and II (10^-7 M), and evaluated transcriptional activities of PR by a luciferase assay. Both GnRH I and II induced the transcriptional activities of PR through the PRE in a ligand-independent manner. This event was blocked by specific PKC and PKA inhibitors, and not blocked by RU486, whereas the P4 effect on transcriptional activity was blocked by PKC, PKA inhibitors and RU486. We cotransfected the cells with a PRE vector and a GnRHR siRNA, and this reduced the effects of GnRH I and II on PRE activation. To elucidate the mechanism following treatments with GnRH I and II, the interactions between PR and several coactivators (SRC-1, SRC-2, SRC-3, CBP/p300 and PCAF) of PR were examined by immunoprecipitation and Western blotting analysis. ChiP assay indicated that treatment with GnRH I (10^-7 M) and II (10^-7 M) induced the interaction of SRC-3 with PRE promoter and SRC interaction with PR was increased by GnRHs in a time-dependent manner. Further, we observed that GnRHs also increased the translocation of PR to the nucleus from cytoplasm by immunocytochemistry. Taken together, these results indicate that GnRH I and II exert their effect on PR-mediated transcription in a ligand-independent manner, and that the effects of GnRHs on the PR activation may involve an interaction between PR and coactivators including SRC-3. [This work was supported by the Canadian Institutes of Health Research]

KEY WORDS: pituitary cells, GnRH and GnRH receptor, progesterone receptor, co-activators