Steroid Receptor Coactivator-3 Is Required for Progesterone Receptor Trans-activation of Target Genes in Response to Gonadotropin-releasing Hormone Treatment of Pituitary Cells

Beum-Soo An,David M Selva,Geoffrey L Hammond,Adolfo Rivero-Muller,Nafis Rahman,Peter C.K Leung

doi:10.1074/jbc.m600743200

Beum-Soo An, David M Selva + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m600743200

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Abstract

Regulation of gonadotropin production involves interplay between steroids and neuropeptides, and we have examined the effects of gonadotropin-releasing hormones (GnRH I and GnRH II) on progesterone receptor (PR) activation in alphaT3-1 pituitary cells. Treatment with GnRHs activated a progester-one response element (PRE)-luciferase reporter gene, and this was blocked by protein kinase C and protein kinase A inhibitors but not by RU486. Treatment with GnRHs phosphorylated the PR at Ser(294) and increased PR translocation to the nucleus within 1 h. Interactions between the PR and several coactivators were examined, and treatment with GnRHs specifically induced PR-steroid receptor coactivator-3 (SRC-3) interactions within 8 h. In chromatin immunoprecipitation assays, recruitment of PR and SRC-3 by the PREs of the luciferase reporter gene or the gonadotopin alpha-subunit gene promoter was also increased by GnRHs within 8 h, while progesterone-induced recruitment of PR to the PREs occurred in association with much less SRC-3. A small interfering RNA knockdown of type I GnRH receptor levels reduced PR activation by GnRHs, while progesterone-dependent PR activation was unaffected. Moreover, small interfering RNA knockdown of SRC-3 abolished PRE-luciferase trans-activation by the PR in response to GnRHs. Collectively, these data indicate that PR activation by GnRHs in alphaT3-1 cells is type I GnRH receptor-mediated and that trans-activation of PR-responsive genes requires SRC-3 in this context.

Highlights

Transactivation of progesterone receptor (PR) by GnRH I and GnRH II in ␣T3-1 Cells— The ability of GnRHs to activate PR-mediated transcription in ␣T3-1 cells was studied in the absence or presence of P4
When these effects of GnRH I and II were studied in the presence of 10Ϫ7 M P4, this resulted in a synergistic increase in PR trans-activation of the reporter plasmid after an 8 h treatment (Fig. 1B)
These initial experiments led us to suspect that the temporal difference in stimulation of PR by GnRHs and P4 could be attributed to PR acting through ligand-independent and liganddependent pathways, respectively

Summary

The abbreviations used are

E2, estradiol; P4, progesterone; PR, progesterone receptor; PRE, progesterone response element; PKA, protein kinase A; GnRH I, gonadotropin releasing hormone; GnRHR, GnRH receptor; PKC, protein kinase C; GnRH II, second form of GnRH; RSV, Rous sarcoma virus; SRC, steroid receptor coactivator; siRNA, small interfering RNA; ChIP, chromatin immunoprecipitation. Cross-talk between the PR and GnRH I has been implicated in a GnRH I self-priming mechanism in the pituitary [29, 30], which is defined as an enhanced LH secretion by pituitary gonadotropes in response to a second stimulation by GnRH I [31]. This response appears to depend upon the capacity of estrogens to induce PR expression in gonadotropes [31], but it is completely absent in PR knock-out mice [32]. We have explored the effects of GnRH I and GnRH II on ligand-dependent and ligand-independent activation of the PR in ␣T3-1 mouse pituitary cells, in which GnRH I and II act via a common type I GnRHR

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION