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.