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.
The authors declare that they have no competing interests.
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.
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.
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.
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.
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.
Authors’ original file for figure 112958_2012_1044_MOESM1_ESM.jpeg
Authors’ original file for figure 212958_2012_1044_MOESM2_ESM.jpeg
Authors’ original file for figure 312958_2012_1044_MOESM3_ESM.jpeg
Authors’ original file for figure 412958_2012_1044_MOESM4_ESM.jpeg
Authors’ original file for figure 512958_2012_1044_MOESM5_ESM.jpeg
Authors’ original file for figure 612958_2012_1044_MOESM6_ESM.jpeg
Authors’ original file for figure 712958_2012_1044_MOESM7_ESM.jpeg
Authors’ original file for figure 812958_2012_1044_MOESM8_ESM.jpeg
Livne PM, Sivan B, Karmazyn B, Ben-Meir D: Testicular torsion in the pediatric age group: diagnosis and treatment. Pediatr Endocrinol Rev. 2003, 1: 128-133. PubMed
Ringdahl E, Teague L: Testicular torsion. Am Fam Physician. 2006, 74: 1739-1743. PubMed
Powell JD, Elshtein R, Forest DJ, Palladino MA: Stimulation of hypoxia-inducible factor-1 alpha (HIF-1alpha) protein in the adult rat testis following ischemic injury occurs without an increase in HIF-1alpha messenger RNA expression. Biol Reprod. 2002, 67: 995-1002. 10.1095/biolreprod.101.002576. CrossRefPubMed
Salva A, Klinefelter GR, Hardy MP: Purification of rat leydig cells: increased yields after unit-gravity sedimentation of collagenase-dispersed interstitial cells. J Androl. 2001, 22: 665-671. PubMed
Brody JR, Kern SE: Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis. Biotechniques. 2004, 36: 214-216. PubMed
Wenger RH: Mammalian oxygen sensing, signalling and gene regulation. J Exp Biol. 2000, 203: 1253-1263. PubMed
Stroka DM, Burkhardt T, Desbaillets I, Wenger RH, Neil DA, Bauer C, Gassmann M, Candinas D: HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia. FASEB J. 2001, 15: 2445-2453. PubMed
Setchell BP, Waites GMH, Till AR: Variations in flow of blood within the epididymis and testis of the sheep and Rat. 1964, 203: 317-318. 10.1038/203317b0. Published online: 18 July 1964; | doi:
Iqbal S, Zhang S, Driss A, Liu Z-R, Kim H-RC, Wang Y, Ritenour C, Zhau HE, Kucuk O, Chung LWK, Wu D: PDGF upregulates Mcl-1 through activation of β-catenin and HIF-1α-dependent signaling in human prostate cancer cells. PLoS One. 2012, 7: e30764-10.1371/journal.pone.0030764. PubMedCentralCrossRefPubMed
- Myeloid cell leukemia-1 (Mc1-1) is a candidate target gene of hypoxia-inducible factor-1 (HIF-1) in the testis
Michael A Palladino
- BioMed Central
Neu im Fachgebiet Gynäkologie und Geburtshilfe
Meistgelesene Bücher aus dem Fachgebiet
Mail Icon II