(227) Hypoxia and HIF1α Regulation Cooperate to Modulate Cholesterol Metabolism in Leydig Cells

Luiz A. Berto Gomes¹; Olivia E. Smith¹; Mariusz P. Kowalewski^1 
1. Institute of Veterinary Anatomy, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland

Abstract Text:

Leydig cells, the primary testosterone-producing cells in the testis, function under low oxygen content and constitutively express hypoxia-inducible factor 1 alpha (HIF1α). Steroidogenesis relies on cholesterol transport into the mitochondria through steroidogenic acute regulatory (STAR) protein, where cholesterol is converted into pregnenolone and becomes available for  further steroidogenic steps. While the suppression of HIF1α has been associated with reduced STAR protein expression and decreased steroid output, the mechanisms underlying the role of oxygen availability and HIF1α activity on Leydig cell function remain unclear. To explore the importance of HIF1α in Leydig cells under different oxygen concentrations, RNA Sequencing was performed on MA-10 and MLTC-1 Leydig cells. Cells were stimulated with cAMP under normoxia (20%O₂), moderate (10%O₂), and severe hypoxia (1%O₂). Additionally, HIF1α expression was suppressed using Echinomycin under 20% and 10%O2. Transcriptomic analysis of differentially expressed genes (DEGs;p< 0.05, FDR< 0.01), validated by RT-qPCR, revealed significant effects of reduced oxygen levels and HIF1α on factors involved in STAR expression, HIF1α regulation, and cholesterol and fatty acid metabolism. Indeed, the results showed upregulated Jun(p< 0.01) and downregulated Nr5a1(p< 0.05) and Akap1(p< 0.01) expression, transcription factors and anchor proteins related to STAR expression, in stimulated cells cultured under 1%O₂. Furthermore, the suppression of HIF1α under normoxia and moderate hypoxia increased both Fos and Junb expression (p< 0.01), but reduced expression of transcription factors and kinases such as Atf1(p< 0.01), Gata4(p< 0.01), Nr5a1(p< 0.01), Prkca(p< 0.01), Prkce(p< 0.01), and Akap1(p< 0.05), highlighting HIF1α’s role in regulating factors related to STAR-mediated steroidogenesis. Regulators of HIF1α degradation, such as Egln1 and Vhl, were upregulated under moderate and severe hypoxia (p< 0.01), suggesting negative feedback to prevent excessive HIF1α accumulation. Despite Nfkb1(p< 0.05) upregulation in severe hypoxia, which has been reported to enhance Hif1a expression by binding to its promoter, Hif1a mRNA levels decreased (p< 0.01), suggesting post-transcriptional regulatory mechanisms to prevent uncontrolled HIF1α activity. Notably, blocking HIF1α under normoxia and moderate hypoxia led to increased Vhl(p< 0.01) and Zfp36(p< 0.05) expression and reduced Rcor1 and Nfkb1(p< 0.01). Only under moderate hypoxia was Hif1an(p< 0.05) expression increased, while Sin3a(p< 0.05) expression was suppressed. These findings suggest a compensatory mechanism modulating HIF1α expression, availability, and activity. Zfp36, encoding TTP, destabilizes Hif1a transcripts, and both Rcor1 and Sin3a (REST complex members) participate in Hif1a transcriptional repression. Finally, our analysis indicates that oxygen availability and HIF1α regulation in Leydig cells affects factors involved in cholesterol transport. Scarb1, encoding scavenger receptor class B type I (SR-BI), was significantly upregulated (p< 0.05) in all oxygen conditions following cAMP stimulation. Another factor, Lipe (encoding hormone-sensitive lipase, HSL), supporting the conversion of cholesterol esters into free cholesterol, was upregulated (p< 0.05) in cAMP-stimulated cells under normoxia and moderate hypoxia, and downregulated (p< 0.01) in severe hypoxia. Notably, both moderate and severe hypoxia increased Stard5 expression (p< 0.05), another cholesterol transport factor. Interestingly, the suppression of HIF1α in both normoxia and moderate hypoxia upregulated Lipe(p< 0.05), while simultaneously downregulated Scarb1(p< 0.01), Ldlr(p< 0.05) and Acaca(p< 0.05), suggesting that HIF1α regulates cholesterol uptake and mobilization to adapt to hypoxic conditions. Altogether, these findings indicate that cholesterol transport and availability are tightly regulated by oxygen levels and HIF1α activity, playing a crucial role in steroidogenesis in Leydig cells, and provides insight into the adaptation mechanisms of Leydig cells in low oxygen environments. (Supported by SNSF_grant Nr:310030_207895)