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

in Journal of Molecular Endocrinology
Correspondence should be addressed to L Zhu: zhuli85051796@163.com
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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 on Star promoter 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 by in vitro experiments with rat primary Leydig cells or TM3 cells exposed to hypoxia (1% O2), CoCl2 or 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 the Star promoter region and repress the expression of STAR. Mutation assays identified three HIF1-binding sites on mouse Star promoter. These findings indicate that HIF1 represses STAR transcription through directly binding to the Staar promoter at −2082/−2078, −2064/−2060 and −1910/−1906, leading to the negative regulation of testosterone synthesis.

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  • Supplemental Figure. Identification of real-time PCR products by electrophoresis and sequencing. 1, cDNA template. 2, RNA template. 3, no template. R, rat. M, mouse.
  • TABLE 1 Primers used in real time quantitative PCR experiments.
  • TABLE 2 Primers used in ChIP PCR experiments.

 

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    Testosterone level decline is accompanied by the accumulation of HIF1α protein. (A) After 4-, 8- or 12-h hypoxia (8% O2) treatment, mouse testis HIF1α protein levels were determined by Western blot, and serum testosterone concentrations were examined by ELISA (n = 10, ***P < 0.001 vs 0 h). (B) Histopathological changes were determined by H&E staining of testis from mice after hypoxia (8% O2) treatment for 0, 4, 8 or 12 h (n = 10). (C) Rat primary Leydig cells were exposed to 1% O2 for 0, 1, 2, 4, 8 and 16 h. HIF1α protein levels were determined by Western blot, and supernatant testosterone concentrations were examined by ELISA (n = 6, *P < 0.05 and ***P < 0.001 vs 0 h). (D, E or F) TM3 cells were exposed to 1% O2, 200 μM CoCl2 or 200 μM DFX for 0, 1, 2, 4, 8 and 16 h. HIF1α protein levels were determined by Western blot, and supernatant testosterone concentrations were examined by ELISA (n = 6, *P < 0.05, **P < 0.01, and ***P < 0.001 vs 0 h). DFX, deferoxamine. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0148.

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    Testosterone decline induced by HIF1 is recovered by pregenolone but not cAMP. (A) Testosterone levels after cAMP addition in the supernatant of TM3 cells with hypoxia, CoCl2 or DFX exposure (n = 6, ***P < 0.001). (B) Testosterone levels after pregenolone addition in the supernatant of TM3 cells with hypoxia, CoCl2 or DFX exposure (n = 6, *P < 0.05, ***P < 0.001). DFX, deferoxamine; Hyp, hypoxia; Nor, nomorxia.

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    Changes of testosterone synthesis-related protein after hypoxia, CoCl2 or DFX treatments. (A and B) Protein levels of HIF1α, VEGF-A, P450scc, 3b-HSD, Star and B-actin in mouse testis were determined by Western blot, and mRNA levels of Star, hsd3b, cyp11 and nr4a1 were detected by real-time PCR (n = 10, *P < 0.05, **P < 0.01, and ***P < 0.001 vs 0). (C and D) Protein levels of HIF1α, VEGF-A, P450scc, 3b-HSD, STAR and B-actin and mRNA levels of Star and Vegfa in primary Leydig cells (n = 6, **P < 0.01 and ***P < 0.001 vs 0). (E and F) Protein levels of HIF1α, VEGF-A, P450scc, 3b-HSD, STAR and B-actin and mRNA levels of Star and Vegfa in TM3 cells (n = 6, **P < 0.01 and ***P < 0.001 vs 0). DFX, deferoxamine; Hyp, hypoxia; Nor, nomorxia.

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    In situ detection of STAR and HIF1α levels in Leydig cells. (A) Histological sections of mouse testis were examined by immunofluorescence (n = 10). (B) In situ STAR and HIF1α in primary Leydig cells and TM3 cells were examined by immunofluorescence (n = 4). STAR was stained with Alexa 488 (green), HIF1α with Alexa 555 (red), and nucleus with DAPI (blue). DFX, deferoxamine; Hyp, hypoxia; Nor, nomorxia.

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    Repression of HIF1 upregulates STAR and testosterone. (A) Protein levels of HIF1α, STAR and B-actin in TM3 cells were determined by Western blot. (B) mRNA levels of Star were detected by real-time PCR and normalized to Actb (n = 6, *P < 0.05, ***P < 0.001). (C) In situ STAR and HIF1α in TM3 cells were examined by immunofluorescence (n = 4). STAR was stained with Alexa 488 (green), HIF1α with Alexa 555 (red) and nucleus with DAPI (blue). (D) Protein levels of HIF1α, STAR, and B-actin in TM3 cells were determined by Western blot (n = 4, *P < 0.05, ***P < 0.001). (E) mRNA levels of Star and Vegfa were detected by real-time PCR and normalized to Actb (n = 6, *P < 0.05, ***P < 0.001). (F) Testosterone levels in the supernatant of TM3 cells were determined by ELISA (n = 6, ***P < 0.001). (G) In situ STAR and HIF1α in TM3 cells were examined by immunofluorescence (n = 4). STAR was stained with Alexa 488 (green), HIF1α with Alexa 555 (red) and nucleus with DAPI (blue). A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0148.

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    HIF1α directly binds to the Star promoter region. (A) Probe sequences used. Numbers indicate the positions upstream of transcription start site. Capital letters show the putative HIF1-binding sites. (B) EMSA supershift experiments. Probes were labeled with biotin. Each probe was incubated with nuclear exact of TM3 cells. Shift arrows indicate the complex positions (lane 2, 7 and 12). Unlabeled probes were used as competitions and co-incubated with labeled probes and nuclear exact (lane 3, 8 and 13). HIF1 antibody was added to distinguish the combinations of HIF1 and probes. Supershift arrows indicate the complex positions (lane 4, 9 and 14). IgG was added into the mixture as negative control for HIF1 antibody (lane 5, 10 and 15). (C and D) ChIP experiments. IgG groups were operated as negative controls for HIF1 groups. PCR and real-time PCR were performed to amplify the ChIP products. Numbers in C indicate positions upstream of transcription start site. Vegfa was examined as positive control for ChIP. Enrichment effects of ChIP were performed in D by comparison to IgG (n = 3). (E) Luciferase reporter gene assay following transient transfection with Star (−1000/+38Luc or −2337/−1893Luc). The pRL-TK vector was co-transfected to normalize transfection efficiencies. Results were presented as a luciferase/Renilla ratio (n = 6, ***P < 0.001 vs Nor). DFX, deferoxamine; Hyp, hypoxia; Nor, nomorxia.

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    Binding sites of HIF1 on the Star promoter region are identified as −2082/−2078, −2064/−2060 and −1910/−1906. (A) Probe sequences. Numbers indicate positions upstream of transcription start site. Capital letters show the putative HIF1-binding sites. (B) EMSA experiments. Probes were labeled with biotin. Each probe was incubated with nuclear exact of TM3 cells. Shift arrows indicate the complex positions. Mutation probes were used to verify the specific combination. (C) Mutation sequences of putative HIF1-binding sites for Luciferase test. Numbers indicate positions upstream of transcription start site. (D and E) Luciferase reporter gene assay following transient transfection with Star (−2337/−1893Luc) and its mutations. The pRL-TK vector was co-transfected to normalize transfection efficiencies. Cells were treated with 1% O2 or 200 nM CoCl2. Results were presented as a luciferase/Renilla ratio (n = 6, **P < 0.01, ***P < 0.001).

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