An Beum-Soo, Poon Song Ling, So Wai-Kin, Hammond Geoffrey L, Leung Peter C K
Department of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.
Biol Reprod. 2009 Aug;81(2):243-9. doi: 10.1095/biolreprod.109.076216. Epub 2009 Apr 8.
Gonadotropin-releasing hormone (GNRH) activates the progesterone receptor (PGR) in pituitary cells and accentuates gonadotropin expression. We show that GNRH1 increases Fshb mRNA levels in LbetaT2 mouse pituitary cells within 8 h and is three times more effective than GNRH2. By contrast, GNRH1 and GNRH2 do not affect Lhb gene expression in these cells. Within the same time frame, small interfering RNA (siRNA) knockdown of the PGR in LbetaT2 cells reduced GNRH1 activation of a PGR response element (PRE)-driven luciferase reporter gene and Fshb mRNA levels by >50%. Chromatin immunoprecipitation (ChIP) assays also demonstrated that PGR loading on the PRE within the Fshb gene promoter in LbetaT2 cells occurred within 8 h after GNRH1 treatment and was lost by 24 h. While the GNRH1-induced upregulation of the PRE reporter gene and Fshb mRNA levels was attenuated by cotreatment with protein kinase A (H-89) and protein kinase C (GF109203X) inhibitors, only GF109203X inhibited PGR phosphorylation at Ser249 in LbetaT2 cells. Immunoprecipitation assays also showed a progressive increase in the interaction between the PGR and its coactivator NCOA3 that peaked at 8 h coincident with the increase in Fshb mRNA after GNRH1 treatment. The siRNA-mediated knockdown of NCOA3 in LbetaT2 cells also reduced Fshb mRNA levels after GNRH1 treatment and loading of NCOA3 on the Fshb promoter PRE in a ChIP assay. We conclude that the rapid effect of GNRH1 on Fshb expression in LbetaT2 cells is mediated by PGR phosphorylation and loading at the PRE within the Fshb promoter together with NCOA3.
促性腺激素释放激素（GNRH）激活垂体细胞中的孕激素受体（PGR）并增强促性腺激素的表达。我们发现，GNRH1在8小时内可增加LbetaT2小鼠垂体细胞中Fshb mRNA水平，其效力是GNRH2的三倍。相比之下，GNRH1和GNRH2不影响这些细胞中Lhb基因的表达。在同一时间范围内，LbetaT2细胞中PGR的小干扰RNA（siRNA）敲低使PGR反应元件（PRE）驱动的荧光素酶报告基因的GNRH1激活以及Fshb mRNA水平降低了50%以上。染色质免疫沉淀（ChIP）分析还表明，GNRH1处理后8小时内，LbetaT2细胞中Fshb基因启动子内的PRE上出现了PGR结合，而在24小时时消失。虽然与蛋白激酶A（H-89）和蛋白激酶C（GF109203X）抑制剂共同处理可减弱GNRH1诱导的PRE报告基因和Fshb mRNA水平的上调，但只有GF109203X抑制了LbetaT2细胞中Ser249位点的PGR磷酸化。免疫沉淀分析还显示，PGR与其共激活因子NCOA3之间的相互作用逐渐增加，在8小时达到峰值，这与GNRH1处理后Fshb mRNA的增加相一致。LbetaT2细胞中NCOA3的siRNA介导的敲低也降低了GNRH1处理后的Fshb mRNA水平以及ChIP分析中NCOA3在Fshb启动子PRE上的结合。我们得出结论，GNRH1对LbetaT2细胞中Fshb表达的快速作用是由PGR在Fshb启动子内的PRE处的磷酸化和结合以及NCOA3介导的。
