Abstract Body: Introduction: Reduced testosterone levels (hypogonadism) decrease bone density and muscle mass, impair sexual function, increase risks of obesity and cardiovascular disease. Testosterone is produced in Leydig cells of testis. MTP is required for lipoprotein biosynthesis. Transcription factor (TF) Srebp2 controls cholesterol biosynthesis. The roles of MTP and Srebp2 in testicular steroidogenesis are unknown.
Hypothesis: MTP and SREBP2 regulate cholesterol and steroid hormone biosynthesis by modulating binding of several TFs to steroidogenic genes, such as Hsd3b1.
Methods: MTP and SREBP2 deficient mouse Leydig MA10 cell lines were generated using CRISPR-Cas9 and steroid production measured. Pathways identified by RNA-Seq were validated by qPCR and Western blotting. Chromatin accessibility and TF binding were examined with ATAC-seq and confirmed by ChIP-qPCR.
Results: MTP inhibition, knockdown (KD), and genetic ablation in mouse MA10 cells increased progesterone secretion. MTP knockout (KO) upregulated cholesterol and steroid biosynthesis. Transcriptomics confirmed enrichment of cholesterol and steroidogenic pathways and showed increased Srebp2 expression in MTP KO clones. Srebf2 KD in these cells reduced cholesterol synthesis and progesterone production, suggesting MTP-mediated regulation requires Srebp2. ATAC-seq showed enhanced SREBP2 binding to cholesterol and steroid biosynthesis gene promoters. Moreover, Srebf2 KO reduced cholesterol metabolism gene expression and progesterone production; these effects were partially rescued by Srebf2 expression. Transcriptomics in Srebf2 KO cells revealed attenuated cholesterol and steroid biosynthesis pathways. ATAC-seq identified a Srebf2 binding motif in the Hsd3b1 gene and demonstrated that Srebf2 deficiency reduced chromatin accessibility of multiple TF-binding sites including SF-1, to steroidogenic gene promoters. Reduced SF-1 binding was confirmed by ChIP-qPCR.
Conclusions: MTP acts as a critical regulator of cholesterol homeostasis and steroidogenesis in mouse Leydig cells by regulating SREBP2 activity. SREBP2 acts as a critical transcriptional driver of steroidogenic gene expression by three mechanisms: providing cholesterol substrate, activating steroidogenic genes, and enhancing chromatin accessibility for other TFs. These findings identify a novel MTP–SREBP2 regulatory axis in steroidogenesis and suggest that inhibition of testicular MTP may represent a novel therapeutic approach to treat hypogonadism.