Myeloid cell leukemia-1 (<it>Mc1</it>-<it>1</it>) is a candidate target gene of hypoxia-inducible factor-1 (<it>HIF</it>-<it>1</it>) in the testis

• Main Authors: Palladino Michael A, Shah Anoop, Tyson Rebecca, Horvath Jaclyn, Dugan Christine, Karpodinis Marie
• Format: Article
• Language: English
• Published: BMC 2012-12-01
• Series: Reproductive Biology and Endocrinology
• Subjects: Apoptosis; Torsion; Ischemia; Leydig cells
• Online Access: http://www.rbej.com/content/10/1/104

<p>Abstract</p> <p>Background</p> <p>Spermatic cord torsion can lead to testis ischemia (I) and subsequent ischemia-reperfusion (I/R) causing germ cell-specific apoptosis. Previously, we demonstrated that the hypoxia-inducible factor-1 (HIF-1) transcription factor, a key regulator of physiological responses to hypoxia, is abundant in Leydig cells in normoxic and ischemic testes. We hypothesize that testicular HIF-1 activates the expression of antiapoptotic target genes to protect Leydig cells from apoptosis. <it>In silico</it> analysis of testis genes containing a consensus hypoxia response element (HRE, 5’-RCGTG-3’) identified myeloid cell leukemia-1 (<it>Mcl</it>-<it>1</it>) as a potential HIF-1 target gene. The purpose of this study was to determine whether HIF-1 shows DNA-binding activity in normoxic and ischemic testes and whether <it>Mcl</it>-<it>1</it> is a target gene of testicular HIF-1.</p> <p>Methods</p> <p>The testicular HIF-1 DNA-binding capacity was analyzed <it>in vitro</it> using a quantitative enzyme-linked immunosorbent assay (ELISA) and electrophoretic mobility shift assays (EMSA). MCL-1 protein expression was evaluated by immunoblot analysis and immunohistochemistry. The binding of testicular HIF-1 to the <it>Mcl</it>-<it>1</it> gene was examined via chromatin immunoprecipitation (ChIP) analysis.</p> <p>Results</p> <p>The ELISA and EMSA assays demonstrated that testicular HIF-1 from normoxic and ischemic testes binds DNA equally strongly, suggesting physiological roles for HIF-1 in the normoxic testis, unlike most tissues in which HIF-1 is degraded under normoxic conditions and is only activated by hypoxia. MCL-1 protein was determined to be abundant in both normoxic and ischemic testes and expressed in Leydig cells. In a pattern identical to that of HIF-1 expression, the steady-state levels of MCL-1 were not significantly affected by I or I/R and MCL-1 co-localized with HIF-1α in Leydig cells. Chromatin immunoprecipitation (ChIP) analysis using a HIF-1 antibody revealed sequences enriched for the <it>Mcl</it>-<it>1</it> promoter.</p> <p>Conclusions</p> <p>The results demonstrated that, unlike what is observed in most tissues, HIF-1 displays DNA-binding activity in both normoxic and ischemic testes, and <it>Mcl</it>-<it>1</it> may be a key target gene of testicular HIF-1 with potential roles in the antiapoptotic protection of Leydig cells.</p>