Peripheral-Type Benzodiazepine Receptor-Mediated Action of Steroidogenic Acute Regulatory Protein on Cholesterol Entry into Leydig Cell Mitochondria

Abstract

Hormone-induced steroid biosynthesis begins with the transfer of cholesterol from intracellular stores into mitochondria. Steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine receptor (PBR) have been implicated in this rate-determining step of steroidogenesis. MA-10 mouse Leydig tumor cells were treated with and without oligodeoxynucleotides (ODNs) antisense to PBR and StAR followed by treatment with saturating concentrations of human choriogonadotropin. Treatment with ODNs antisense but not missense for both proteins inhibited the respective protein expression and the ability of the cells to synthesize steroids in response to human choriogonadotropin. Treatment of the cells with either ODNs antisense to PBR or a transducible peptide antagonist to PBR resulted in inhibition of the accumulation of the mature mitochondrial 30-kDa StAR protein, suggesting that the presence of PBR is required for StAR import into mitochondria. Addition of in vitro transcribed/translated 37-kDa StAR or a fusion protein of Tom20 (translocase of outer membrane) and StAR (Tom/StAR) to mitochondria isolated from control cells increased pregnenolone formation. Mitochondria isolated from cells treated with ODNs antisense, but not missense, to PBR failed to form pregnenolone and respond to either StAR or Tom/StAR proteins. Reincorporation of in vitro transcribed/translated PBR, but not PBR missing the cholesterol-binding domain, into MA-10 mitochondria rescued the ability of the mitochondria to form steroids and the ability of the mitochondria to respond to StAR and Tom/StAR proteins. These data suggest that both StAR and PBR proteins are indispensable elements of the steroidogenic machinery and function in a coordinated manner to transfer cholesterol into mitochondria.

Impact of Aging on Steroid Hormone Biosynthesis and Secretion

“…PBR ligands stimulate steroidogenesis and promote translocation of cholesterol from OMM to the IMM in testicular Leydig cells, ovarian granulose cells, and adrenocortical cells [81][82][83][84][85]. Targeted disruption of the PBR/TSPO gene in rat Leydig R2C cells (PBR-deficient cells) blocked the cholesterol import into the mitochondria and dramatically reduced steroid production, whereas reintroduction of PBR/TSPO in this cell line restored steroidogenesis [86,87]. Likewise, mutation of a single amino acid residue in the "cholesterol recognition amino acid consensus" domain in the carboxyl-terminal region disrupts cholesterol binding and transfer to IMM [76,87].…”

“…Targeted disruption of the PBR/TSPO gene in rat Leydig R2C cells (PBR-deficient cells) blocked the cholesterol import into the mitochondria and dramatically reduced steroid production, whereas reintroduction of PBR/TSPO in this cell line restored steroidogenesis [86,87]. Likewise, mutation of a single amino acid residue in the "cholesterol recognition amino acid consensus" domain in the carboxyl-terminal region disrupts cholesterol binding and transfer to IMM [76,87]. PBR/TSPO is a component of the multimeric 140-200-kDa complex located on the OMM especially at the OMM-IMM contact sites.…”

“…Although a physical interaction between StAR and PBR/TSPO has not been established, FRET measurements indicated that StAR/StarD1 and PBR/TSPO come within the 100 A o of each other [89]. Moreover, Hauet et al [87] reported that isolated mitochondria from mouse Leydig MA-10 cells that express the Tom20/StAR fusion construct produce steroids at a maximal level, but if the cells are treated with PBRantisense oligonucleotides, their ability to synthesize the steroid is lost; on the contrary, re-introduction of recombinant PBR into the mitochondria in vitro rescued the steroidogenesis [87,90]. Liu et al, [91] provided evidence that hormonal stimulation of a Leydig cell line leads to formation of a StAR/StarD1-PKARI-PAP7-PBR/TSPO macromolecular signaling complex on the outer mitochondrial membrane that mediates the effect of hormones on mitochondrial cholesterol transport and steroidogenesis.…”

Deleterious Cholesterol Hydroperoxide Trafficking in Steroidogenic Acute Regulatory (StAR) Protein-expressing MA-10 Leydig Cells

“…These proteins contain a C-terminal segment of ϳ200 amino acids, the StAR-related lipid transfer (START) domain, which binds a single Ch molecule in highly selective fashion (9,10). StarD1, the family prototype, localizes in the Mito outer membrane (OM), and in conjunction with peripheral benzodiazepine receptor and other proteins (3,7,11), facilitates the translocation of incoming Ch to the inner membrane (IM) for processing by the P450scc system (2,3). Structural homologues of StarD1 have been identified (StarD1-D6), which probably function in the cytosol because they lack organelle-targeting sequences (6,(12)(13)(14).…”

Steroidogenesis in MA-10 Mouse Leydig Cells Is Altered via Fatty Acid Import into the Mitochondria1

“…incubation. RIA analysis was performed as previously mentioned [18], with antiprogesterone serum purchased from MP Biomedicals.…”