PPAR-gamma coactivator-1alpha regulates progesterone production in ovarian granulosa cells with SF-1 and LRH-1

Abstract

Previously, we demonstrated that bone marrow-derived mesenchymal stem cells (MSCs) differentiate into steroidogenic cells such as Leydig and adrenocortical cells by the introduction of steroidogenic factor-1 (SF-1) and treatment with cAMP. In this study, we employed the same approach to differentiate umbilical cord blood (UCB)-derived MSCs. Despite UCB-MSCs differentiating into steroidogenic cells, they exhibited characteristics of granulosa-luteal-like cells. We found that peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) was expressed and further induced by cAMP stimulation in UCB-MSCs. Consistent with these results, tissue-specific expression of Pgc-1alpha was observed in rat ovarian granulosa cells. PGC-1alpha binds to the NR5A family [SF-1 and liver receptor homolog-1 (LRH-1)] of proteins and markedly enhances their transcriptional activities. Reporter assays revealed that PGC-1alpha activated the promoter activities of SF-1 and LRH-1 target genes. Infection of KGN cells (a human cell line derived from granulosa cells) with adenoviruses expressing PGC-1alpha resulted in the induction of steroidogenesis-related genes and stimulation of progesterone production. PGC-1alpha also induced SF-1 and LRH-1, with the latter induced to a greater extent. Knockdown of Pgc-1alpha in cultured rat granulosa cells resulted in attenuation of gene expression as well as progesterone production. Transactivation of the NR5A family by PGC-1alpha was repressed by Dax-1. PGC-1alpha binds to the activation function 2 domain of NR5A proteins via its consensus LXXLL motif. These results indicate that PGC-1alpha is involved in progesterone production in ovarian granulosa cells by potentiating transcriptional activities of the NR5A family proteins.

Fig. 1.

Differentiation of UCB-MSCs into steroidogenic cells by SF-1. UCB-MSCs were transduced with GFP or SF-1 by retrovirus-mediated transfection. A, RT-PCR analysis of each gene in each clone cultured with or without 8-bromo-cAMP for 2 d. Lanes G–L represent granulosa-luteal cells from women undergoing oocyte retrieval for in vitro fertilization. B, Production of steroid hormones by UCB-MSCs stably expressing GFP or SF-1 in the presence (+) or absence (−) of 8-bromo-cAMP. ACTHR, ACTH receptor; FSHR, FSH receptor; LHR, LH receptor.

Fig. 2.

A, PGC-1α was induced in UCB-MSCs. The mRNA levels of each gene in MSCs were analyzed by RT-PCR. B–E, The expression and localization of the NR5A protein family and PGC-1α in the murine ovary. B, The mRNA levels of each gene were analyzed by RT-PCR. Lanes O and L represent an ovary and a liver, respectively. C–E, Localization of NR5A and Pgc-1α proteins in the murine ovary. Positive staining for Pgc-1α (C) and Lrh-1 (D) in nuclei of granulosa cells, and Sf-1 (E) in nuclei of granulosa and theca/interstitial cells. Absence of any nuclear staining in a control section incubated with nonimmune IgG and counterstained with eosin (F).

Fig. 3.

PGC-1α is a preferential coactivator for SF-1 and LRH-1. A, Dose-dependent effect of PGC-1α on the transactivation of SF-1 and LRH-1 in COS7 cells. B, Comparison of the coactivation ability between each coactivator on transactivation of SF-1 and LRH-1. COS7 cells were transfected with the 5×GAL4-E1B/Luc expression vector for 48 h. Luciferase activities were measured and relative activities were shown. Data are the mean ± sem of at least four independent experiments. DBD, DNA-binding domain.

Fig. 4.

PGC-1α induces SF-1- and LRH-1-induced promoter activities of steroidogenic genes (A–C) and Inhibin-α (D), but not the CYP19A1 gene (E). HEK293 cells were transiently transfected with each reporter and expression vector for 48 h. Luciferase activities were measured, and relative activities are shown. Data are expressed as the mean ± sem of at least four independent experiments.

Fig. 5.

PGC-1α induces steroidogenic genes and progesterone production in granulosa-like tumor KGN cells. KGN cells were infected with adenoviruses expressing GFP or PGC-1α. Gene expression of StAR (A), CYP11A1 (B), HSD3B2 (C), CYP19A1 (D), LRH-1 (E), and SF-1 (F) was measured by real-time PCR and normalized to 36B4 expression. Data are the mean ± sem of at least three independent experiments. G, Progesterone levels of each group in the medium were measured using ELISA. H–K, ChIP assays demonstrating the occupancy of PGC-1α on the promoters of StAR, CYP11A1, HSD3B2, and CYP19 genes. Assays were performed on GFP- (white column) or PGC-1α- (black column) infected KGN cell extracts using control IgG or specific antibody. Recovered chromatin was subjected to quantitative PCR analysis using primers encompassing proximal SF-1 and LRH-1 binding sites, 5 kb from the transcription initiation site of each gene. Data are the mean ± sem values of at least three independent experiments.

Fig. 6.

Pgc-1α is involved in the progesterone production of granulosa cells isolated from immature diethylstilbestrol-primed rats. Granulosa cells were infected with adenoviruses expressing GFP or Pgc-1α (A and B). A, Gene expression of StAR, P450scc, 3β-HSD, Hmgcs2, SR-BI, Inhibin-α, and Cyp19a was measured by quantitative PCR and normalized to 36B4 expression. Data are the mean ± sem of at least three independent experiments. B, Progesterone levels of each group in the medium were measured using ELISA. Granulosa cells were infected with adenoviruses expressing control and Pgc-1α short hairpin RNA, and cultured with or without FSH (C–E). C, Nuclear extracts from cells of each treatments were subjected to SDS-PAGE, and Western blot analysis was performed using each antibody. D, Progesterone levels of each group in the medium were measured using ELISA. E, Gene expression of StAR, P450scc, 3β-HSD, Hmgcs2, and inhibin-α was measured by real-time PCR and normalized to 36B4 expression. Data are the mean ± sem of at least two independent experiments. *, P < 0.05; **, P < 0.01.

Fig. 7.

Inhibition of Pgc-1α activity by Dax-1. A, Granulosa cells were isolated from immature diethylstilbestrol-primed rats and treated with FSH for the indicated times. Gene expression of each gene was measured by quantitative PCR. B and C, HEK293 cells were transiently transfected with each reporter and expression vector for 48 h. Luciferase activities were measured, and relative activities are shown. Data are the mean ± sem of at least four independent experiments. C, Schematic structure of PGC-1α and LXXLL constructs for mammalian two-hybrid interaction assays. D, Interaction of LXXLL-related peptides of DAX-1, PGC-1α, and its amino acid-substituted forms with SF-1 and LRH-1. Data are the mean ± sem of at least four independent experiments. DBD, DNA-binding domain.