Conditional steroidogenic cell-targeted deletion of TSPO unveils a crucial role in viability and hormone-dependent steroid formation

Abstract

Translocator protein (TSPO) is a key member of the mitochondrial cholesterol transport complex in steroidogenic tissues. To assess the function of TSPO, we generated two lines of Cre-mediated Tspo conditional knockout (cKO) mice. First, gonadal somatic celltargeting Amhr2-Cre mice were crossed with Tspo-floxed mice to obtain F1 Tspo Amhr2 cKO mice (Tspo fl/fl ;Amhr2-Cre /+). The unexpected Mendelian ratio of 4.4% cKO mice was confirmed by genotyping of 12.5-day-postcoitum (dpc) embryos. As Amhr2-Cre is expressed in gonads at 12.5 dpc, these findings suggest preimplantation selection of embryos. Analysis of expression databases revealed elevated levels of Amhr2 in two-and eight-cell zygotes, suggesting ectopic Tspo silencing before the morula stage and demonstrating elevated embryonic lethality and involvement of TSPO in embryonic development. To circumvent this issue, steroidogenic cell-targeting Nr5a1-Cre mice were crossed with Tspofloxed mice. The resulting Tspo fl/fl ;Nr5a1-Cre /+ mice were born at a normal Mendelian ratio. Nr5a1-driven Tspo cKO mice exhibited highly reduced Tspo levels in adrenal cortex and gonads. Treatment of mice with human chorionic gonadotropin (hCG) resulted in increased circulating testosterone levels despite extensive lipid droplet depletion. In contrast, Nr5a1-driven Tspo cKO mice lost their ability to form corticosterone in response to adrenocorticotropic hormone (ACTH). Important for ACTH-dependent steroidogenesis, Mc2r, Stard1, and Cypa11a1 levels were unaffected, whereas Scarb1 levels were increased and accumulation of lipid droplets was observed, indicative of a blockade of cholesterol utilization for steroidogenesis. TSPO expression in the adrenal medulla and increased epinephrine production were also observed. In conclusion, TSPO was found necessary for preimplantation embryo development and ACTH-stimulated steroid biosynthesis.translocator protein | anti-Mullerian hormone receptor type II | nuclear receptor subfamily 5 group A member 1 | knockout mice | steroidogenesis T he 18-kDa translocator protein (TSPO) is an outer mitochondrial membrane (OMM) protein, originally named the peripheral benzodiazepine receptor (PBR) due to its discovery as a high-affinity binding site for the benzodiazepine diazepam outside of the central nervous system (1). Pharmacological, biochemical, and structural research has revealed TSPO's ability to bind numerous classes of drugs (1-4). Additional investigations also demonstrated that TSPO is a high-affinity cholesterol-binding protein. Cholesterol binding is localized to the C-terminal portion of its fifth transmembrane helix at a conserved cholesterol recognition/association amino acid consensus motif (1, 5). This finding was further supported by recent structural data (6). A search for additional physiological ligands of TSPO revealed that both the tetrapyrole protoporphyrin IX, a heme precursor, and the polypeptide endozepine, a GABA A modulator, bind TSPO, suggesting a role for TSPO in numerous physiological processes (1, 2, 5). Interestingly...

TSPO: kaleidoscopic 18-kDa amid biochemical pharmacology, control and targeting of mitochondria

“…These studies not only question the role TSPO holds in steroidogenesis but contrast early attempts to generate Tspo -/-knockout mice, which resulted in embryonic lethality (5). However, the viability of mice has been shown to vary depending on methodology: Nr5a1-driven conditional knock-outs were born at a normal Mendelian ratio while Amhr2-Cre driven conditional knock-outs were born at a ratio of 4.4% (111). Global Tspo knockout phenotypes can range from lethal, when whole gene deletion is performed, to no phenotype, as in the case of cre-loxP technique, thus presenting the possibility that methodological differences may be the cause of such discrepancy (112).…”

“…Recently, however, a number of studies from independent groups have reported that TSPO global knock out mice are viable and that loss of the protein has no effect on basal steroid hormone biosynthesis (32,35,110,111). These studies not only question the role TSPO holds in steroidogenesis but contrast early attempts to generate Tspo -/-knockout mice, which resulted in embryonic lethality (5).…”

“…However, it is important to consider that compensatory mechanisms may play an important part in ensuring ongoing steroidogenesis in a TSPO null setting, and these may be different in the various strains, which may be attributed to methodology as highlighted already. In both global and conditional Tspo knock-out studies, several genes involved in steroidogenic processes were found to be upregulated including Cyp21a2 (110), Abca2 (110), and Scarb1 (111) in the adrenal glands and Lhcgr (111) in the testes ( TSPO is also attributed to immune regulation, with elevated expression observed in microglia and macrophages. Recently it was highlighted that TSPO is involved in cholesterol trafficking in macrophages, since its overexpression leads to increased transcription of proteins involved in cholesterol efflux, ACBA1 (ATP-binding cassette A1), PPARα (peroxisome-proliferator-activated receptor α) and LXRα (liver X receptor α), and corresponds with increased efflux of cholesterol to acceptors (113).…”

Primary Aldosteronism: Changing Definitions and New Concepts of Physiology and Pathophysiology Both Inside and Outside the Kidney

“…The initial and ratelimiting step involves the transport of cholesterol from the outer to the inner mitochondrial membrane by steroidogenic acute regulatory protein (StAR, encoded by STAR) (23,306). Evidence from recent studies in mice has raised the possibility that StAR works in conjunction with other proteins, including translocator protein (TSPO), to facilitate this shuttling process (141). At the inner mitochondrial membrane, cholesterol is converted to pregnenolone by the cholesterol side chain cleavage enzyme (P450scc, encoded by CYP11A1) (468).…”

Current status and future perspectives: TSPO in steroid neuroendocrinology

“…Due to Tspo sequence conservation from bacteria to humans, there has been some interest in studying the functional evolution of this gene (Fan et al 2012). TSPO2 (PBRL/Peripheral benzodiazepam-like) was identified as a gene that emerged from a duplication event preceding speciation of reptiles, birds and mammals (Nakazawa et al 2009).…”