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01.12.2012 | Research | Ausgabe 1/2012 Open Access

Reproductive Biology and Endocrinology 1/2012

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

Zeitschrift:
Reproductive Biology and Endocrinology > Ausgabe 1/2012
Autoren:
Michael A Palladino, Anoop Shah, Rebecca Tyson, Jaclyn Horvath, Christine Dugan, Marie Karpodinis
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1477-7827-10-104) contains supplementary material, which is available to authorized users.
Anoop Shah, Rebecca Tyson, Jaclyn Horvath, Christine Dugan and Marie Karpodinis contributed equally to this work.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

MAP conceived of the study, participated in its design and coordination and drafted the manuscript. AS carried out ELISA DNA-binding experiments and drafted appropriate portions of the manuscript. RT and JH carried out Mcl-1 western blot experiments, Mcl-1 immunocytochemistry and ChIP analysis and drafted appropriate portions of the manuscript. CD and MC performed EMSA experiments and drafted appropriate portions of the manuscript. All authors read the draft manuscript and approved the final manuscript.

Abstract

Background

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. In silico analysis of testis genes containing a consensus hypoxia response element (HRE, 5’-RCGTG-3’) identified myeloid cell leukemia-1 (Mcl-1) 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 Mcl-1 is a target gene of testicular HIF-1.

Methods

The testicular HIF-1 DNA-binding capacity was analyzed in vitro 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 Mcl-1 gene was examined via chromatin immunoprecipitation (ChIP) analysis.

Results

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 Mcl-1 promoter.

Conclusions

The results demonstrated that, unlike what is observed in most tissues, HIF-1 displays DNA-binding activity in both normoxic and ischemic testes, and Mcl-1 may be a key target gene of testicular HIF-1 with potential roles in the antiapoptotic protection of Leydig cells.
Zusatzmaterial
Authors’ original file for figure 1
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Authors’ original file for figure 8
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Literatur
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