Expression and function of NR5A1, NR5A2 and NR0B1 in mediating gonadotrophin-releasing hormone (GNRH) function in gonadotroph cell lines
SM Mirczuk1, B Poulet1, AN Chand1, JM Burrin2, & RC Fowkes1,
The nuclear receptors, NR5A1 (SF-1), NR5A2 (LRH-1) and NR0B1 (DAX-1), are key regulators of gene expression and development in endocrine tissues. NR5A1 regulates endocrine development and function, and is an important regulator of gene expression in pituitary gonadotrophs. In contrast, little is known regarding the role of NR5A2 in pituitary function. Using a range of anterior pituitary-derived cell lines and primary rat pituitaries, we have investigated the expression and action of NR5A1, NR5A2 and NR0B1 using RT-PCR, Western blotting and reporter gene assays. NR5A1 and NR0B1 were expressed in αT3-1 and LβT2 gonadotrophs by real-time PCR and Western blotting. Treatment with 100 nM GnRH, PMA (a PKC activator) or BayK (Ca2+ ionophore) for up to 24 h failed to alter either mRNA or protein expression. Reporter gene assays in αT3-1 cells revealed that neither the NR5A1 or NR0B1 promoters were affected by GnRH treatment, but over-expression of an NR5A1 expression vector dose-dependently enhanced NR0B1 transcription, suggesting NR0B1 is an NR5A1 target gene in gonadotrophs. Gel-shift analyses using nuclear extracts from αT3-1 cells showed members of the Sox-family of transcription factors bound the NR5A1 promoter, but that GnRH-treatment failed to alter Sox complex formation. GnRH failed to alter NR5A1 DNA binding activity, but phosphorylation of NR5A1 at Ser203 was enhanced in αT3-1 and LβT2 cells following stimulation with GnRH for 5 min. GnRH-stimulated glycoprotein hormone α-subunit (αGSU) transcription in αT3-1 and LβT2 cells was inhibited dose-dependently by over-expression of NR0B1, suggesting αGSU is negatively regulated by NR0B1 in gonadotrophs. NR5A2 was expressed in normal rat pituitary and rat thyrotroph αTSH cells by RT-PCR, but transcriptional activity of NR5A2-Gal4 fusion constructs were not affected by activators of Ca2+ signalling. Collectively, these data suggest pituitary expression of NR5A1, NR5A2 and NR0B1 is not subject to hormonal regulation by GnRH, but that GnRH can modify phosphorylation of NR5A1, and NR0B1 can inhibit GnRH-stimulated gene transcription in gonadotrophs. The full role of NR5A2 in the pituitary remains to be established.