HIF 1 inhibits STAR transcription and testosterone synthesis in murine Leydig cells

Abstract

Hypoxia-inducible factor-1 (HIF1) is a critical transcription factor involved in cell response to hypoxia. Under physiological conditions, its ‘a’ subunit is rapidly degraded in most tissues except testes. HIF1 is stably expressed in Leydig cells, which are the main source of testosterone for male, and might bind to the promoter region of steroidogenic acute regulatory protein (STAR), which is necessary for the testosterone synthesis, according to software analysis. This study aims to identify the binding sites of HIF1 onStarpromoter and its transcriptional regulation of STAR to affect testosterone synthesis. Testosterone level and steroid synthesis-related proteins were determined in male Balb/C mice exposed to hypoxia (8% O2). While HIF1 was upregulated, the testosterone level was significantly decreased. This was further confirmed byin vitroexperiments with rat primary Leydig cells or TM3 cells exposed to hypoxia (1% O2), CoCl2or DFX to raise HIF1. The decline of testosterone was reversed by pregnenolone but not cAMP, indicating the cholesterol transport disorder as the main cause. In agreement, STAR expression level was decreased in response to HIF1, while 3b-hydroxysteroid dehydrogenase, 17b-hydroxysteroid dehydrogenase and p450scc did not exhibit significant changes. By ChIP, EMSA supershift and dual-luciferase reporter assays, HIF1 was found to bind to theStarpromoter region and repress the expression of STAR. Mutation assays identified three HIF1-binding sites on mouseStarpromoter. These findings indicate that HIF1 represses STAR transcription through directly binding to theStaarpromoter at −2082/−2078, −2064/−2060 and −1910/−1906, leading to the negative regulation of testosterone synthesis.