The Nuclear Receptor NR2F2 Activates Star Expression and Steroidogenesis in Mouse MA-10 and MLTC-1 Leydig Cells1

Abstract

Testosterone production is dependent on cholesterol transport within the mitochondrial matrix, an essential step mediated by a protein complex containing the steroidogenic acute regulatory (STAR) protein. In steroidogenic Leydig cells, Star expression is hormonally regulated and involves several transcription factors. NR2F2 (COUP-TFII) is an orphan nuclear receptor that plays critical roles in cell differentiation and lineage determination. Conditional NR2F2 knockout prior to puberty leads to male infertility due to insufficient testosterone production, suggesting that NR2F2 could positively regulate steroidogenesis and Star expression. In this study we found that NR2F2 is expressed in the nucleus of some peritubular myoid cells and in interstitial cells, mainly in steroidogenically active adult Leydig cells. In MA-10 and MLTC-1 Leydig cells, small interfering RNA (siRNA)-mediated NR2F2 knockdown reduces basal steroid production without affecting hormone responsiveness. Consistent with this, we found that STAR mRNA and protein levels were reduced in NR2F2-depleted MA-10 and MLTC-1 cells. Transient transfections of Leydig cells revealed that a -986 bp mouse Star promoter construct was activated 3-fold by NR2F2. Using 5' progressive deletion constructs, we mapped the NR2F2-responsive element between -131 and -95 bp. This proximal promoter region contains a previously uncharacterized direct repeat 1 (DR1)-like element to which NR2F2 is recruited and directly binds. Mutations in the DR1-like element that prevent NR2F2 binding severely blunted NR2F2-mediated Star promoter activation. These data identify an essential role for the nuclear receptor NR2F2 as a direct activator of Star gene expression in Leydig cells, and thus in the control of steroid hormone biosynthesis.

MEF2 Cooperates With Forskolin/cAMP and GATA4 to Regulate Star Gene Expression in Mouse MA-10 Leydig Cells

“…The Ϫ131/Ϫ54-bp region is rich in transcription factor binding sites, including elements for SF1, AP1, NUR77, GATA4, and COUP-TFII (4,31,34,46). Mutation of several of these binding sites (COUP-TF, NUR77/SF1, and GATA) in the context of the Ϫ131-bp Star reporter (that no longer harbor the MEF2 element) impaired the MEF2/cAMP cooperation.…”

Molecular regulation of steroidogenesis in endocrine Leydig cells

“…In the testis, we [8,9] and others [11,119] found that COUP-TFII is expressed in some peritubular myoid cells and in Leydig stem cells giving rise to ALC. Because Coup-tfii À/À mice die before E10.5, an inducible inactivation strategy was used and revealed that COUP-TFII is essential for progenitor Leydig cell formation and their further maturation into functional adult Leydig cells [119].…”

“…FLC stem cells start to differentiate and gradually acquire the ability to synthesize androgens required for masculinization of the foetus [6] before disappearing after birth. The ALC population is derived from undifferentiated spindle-shaped, fibroblast-like mesenchymal Leydig stem cells that are believed to be present during fetal life [8][9][10][11]. The source of these cells has been identified as peritubular, interstitial, and/or peri-vascular in mouse and human testes [10][11][12].…”

“…1, the transcription factors already present in the cell include SF1 (NR5A1, Ad4BP) [123,[125][126][127], LRH1 (NR5A2) [128], COUP-TFII (NR2F2) [9], GATA4 [129,130], C/EBPb [130][131][132], SP1 [133], CREB/CREM [134,135], members of the AP1 family (cFOS and cJUN) [136][137][138][139], and MEF2 [140][141][142]. All these factors were shown to stimulate Star gene expression and thus steroidogenesis in Leydig cells.…”

MEF2 and NR2F2 cooperate to regulate Akr1c14 gene expression in mouse MA‐10 Leydig cells

“…Recently, the transcription factors myocyte enhancer factor 2 (MEF2) and chicken ovalbumin upstream promoter‐transcription factor II (NR2F2, COUP‐TFII) were identified in the mouse testis, including Leydig cells (Qin et al ., ; Daems et al ., ; Mendoza‐Villarroel et al ., ,b). The MEF2 family of transcription factors contains four members: MEF2A, 2B, 2C and 2D.…”