Mitochondrial TSPO Deficiency Triggers Retrograde Signaling in MA-10 Mouse Tumor Leydig Cells

The mitochondrial translocator protein (TSPO) has been shown to bind cholesterol with high affinity and is involved in mediating its availability for steroidogenesis. We recently reported that targeted Tspo gene deletion in MA-10 mouse tumor Leydig cells resulted in reduced cAMP-stimulated steroid formation and significant reduction in the mitochondrial membrane potential (ΔΨm) compared to control cells. We hypothesized that ΔΨm reduction in the absence of TSPO probably reflects the dysregulation and/or maintenance failure of some basic mitochondrial function(s). To explore the consequences of TSPO depletion via CRISPR-Cas9-mediated deletion (indel) mutation in MA-10 cells, we assessed the transcriptome changes in TSPO-mutant versus wild-type (Wt) cells using RNA-seq. Gene expression profiles were validated using real-time PCR. We report herein that there are significant changes in nuclear gene expression in Tspo mutant versus Wt cells. The identified transcriptome changes were mapped to several signaling pathways including the regulation of membrane potential, calcium signaling, extracellular matrix, and phagocytosis. This is a retrograde signaling pathway from the mitochondria to the nucleus and is probably the result of changes in expression of several transcription factors, including key members of the NF-κB pathway. In conclusion, TSPO regulates nuclear gene expression through intracellular signaling. This is the first evidence of a compensatory response to the loss of TSPO with transcriptome changes at the cellular level. The cells used for total RNA extraction were cultured in 6-well plates to 80% confluence. Total RNA samples from Wt and Tspo mutant cells were extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA) and then quantified with an Agilent 2100 bioanalyzer. The quantified RNA samples were used for NEBNext® (New England Biolabs, Ipswich, MA, USA) rRNA-depleted (Human/Mouse/Rat) stranded library preparation. Total transcripts of the cells were sequenced at McGill University and the Genome Quebec Innovation Center