CRISPR/Cas9‒Mediated Tspo Gene Mutations Lead to Reduced Mitochondrial Membrane Potential and Steroid Formation in MA-10 Mouse Tumor Leydig Cells

Abstract

The outer mitochondrial membrane translocator protein (TSPO) binds cholesterol with high affinity and is involved in mediating its delivery into mitochondria, the rate-limiting step in hormone-induced steroidogenesis. Specific ligand binding to TSPO has been shown to initiate steroid formation. However, recent studies of the genetic deletion of Tspo have provided conflicting results. Here, we address and extend previous studies by examining the effects of Tspo-specific mutations on steroid formation in hormone- and cyclic adenosine monophosphate (cAMP)-responsive MA-10 cells, using the CRISPR/Cas9 system. Two mutant subcell lines, nG1 and G2G, each carrying a Tspo exon2-specific genome modification, and two control subcell lines, G1 and HH, each carrying a wild-type Tspo, were produced. In response to dibutyryl cAMP, the nG1 and G2G cells produced progesterone at levels significantly lower than those produced by the corresponding control cells G1 and HH. Neutral lipid homeostasis, which provides free cholesterol for steroid biosynthesis, was altered significantly in the Tspo mutant cells. Interestingly, the mitochondrial membrane potential (ΔΨm) of the Tspo mutant cells was significantly reduced compared with that of the control cells, likely because of TSPO interactions with the voltage-dependent anion channel and tubulin at the outer mitochondrial membrane. Steroidogenic acute regulatory protein (STAR) expression was induced in nG1 cells, suggesting that reduced TSPO affected STAR synthesis and/or processing. Taken together, these results provide further evidence for the critical role of TSPO in steroid biosynthesis and suggest that it may function at least in part via its regulation of ΔΨm and effects on STAR.

Poly (ADP‐ribose) polymerase inhibition protects against myocardial ischaemia/reperfusion injury via suppressing mitophagy

“…Down‐regulation of CypD prevents MPTP opening and protects against the loss of ΔΨm . Conversely, the loss function of TSPO causes MPTP opening and leads to a reduced ΔΨm . Our results showed that both CypD and TSPO could be poly‐ADP‐ribosylated, but the effects of this modification on the functions of CypD and TSPO were not studied.…”

“…35 Conversely, the loss function of TSPO causes MPTP opening and leads to a reduced ΔΨm. 21,36 Our results showed that both CypD and TSPO could be poly-ADP-ribosylated, but the effects of this modification on the functions of CypD and TSPO were not studied. As reported, poly-ADP-ribosylation can serve as a marker for ubiquitinproteasomal system (UPS)-dependent protein degradation.…”

Translocator protein (18 kDa): an update on its function in steroidogenesis

“…These factors were not considered in the study by Selvaraj’s group . Moreover, we recently generated Tspo KO MA‐10 cell lines via CRISPR/Cas9 technology and observed that these cells make little to no steroids and that PK 11195 stimulation did not affect steroid biosynthesis in the TSPO‐deficient cells …”

“…[18][19][20][21] Moreover, we recently generated Tspo KO MA-10 cell lines via CRISPR/Cas9 technology and observed that these cells make little to no steroids and that PK 11195 stimulation did not affect steroid biosynthesis in the TSPO-deficient cells. 62 One of the main arguments used by Selvaraj and colleagues is that TSPO plays no role in steroidogenesis and the adrenal tumour H295R cells do not contain TSPO but produce steroids. 63 Indeed, this observation is cited as the "gold standard" to debunk the role of TSPO in steroid biosynthesis.…”

Cholesterol accumulation, lipid droplet formation, and steroid production in Leydig cells: Role of translocator protein (18‐kDa)

“…22 Consistent with this, knockout of Tspo in MA-10 cells by CRISPR/Cas9 technology resulted in little to no steroid (progesterone) production. 23 In vivo, rodents in which Tspo was knocked out did not produce corticosterone in response to adrenocorticotropic hormone (ACTH), 24 and had reduced total steroidogenic output and age-dependent androgen deficiency. 25 Additionally, the stimulation of TSPO by several TSPO-specific drug ligands was shown to increase steroid formation in MA-10 cells and primary Leydig cells in vitro, and in Leydig cells in vivo.…”