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

Reproductive Biology and Endocrinology

Marie Karpodinis¹  Christine Dugan¹  Jaclyn Horvath¹  Rebecca Tyson¹  Anoop Shah¹  Michael A Palladino¹ 

[1] Department of Biology, Monmouth University, 400 Cedar Avenue, West Long Branch, NJ, 07764, USA

关键词: Leydig cells;    Ischemia;    Torsion;    Apoptosis;   

Others  :  1149710
DOI  :  10.1186/1477-7827-10-104

 received in 2012-08-08, accepted in 2012-11-27,  发布年份 2012

【 摘 要 】

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.

【 授权许可】

   
2012 Palladino et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Weidemann A, Johnson RS: Biology of HIF-1α. Cell Death Differ 2008, 15:621-627.
• [2]Yee Koh M, Spivak-Kroizman TR, Powis G: HIF-1 regulation: not so easy come, easy go. Trends Biochem Sci 2008, 33:526-534.
• [3]Ke Q, Costa M: Hypoxia-inducible factor-1 (HIF-1). Mol Pharmacol 2006, 70:1469-1480.
• [4]Semenza GL: Hypoxia-inducible factors in physiology and medicine. Cell 2012, 148:399-408.
• [5]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.
• [6]Ringdahl E, Teague L: Testicular torsion. Am Fam Physician 2006, 74:1739-1743.
• [7]Farias JG, Puebla M, Acevedo A, Tapia PJ, Gutierrez E, Zepeda A, Calaf G, Juantok C, Reyes JG: Oxidative stress in rat testis and epididymis under intermittent hypobaric hypoxia: protective role of ascorbate supplementation. J Androl 2010, 31:314-321.
• [8]Turner TT, Tung KS, Tomomasa H, Wilson LW: Acute testicular ischemia results in germ cell-specific apoptosis in the rat. Biol Reprod 1997, 57:1267-1274.
• [9]Lysiak JJ, Nguyen QAT, Kirby JL, Turner TT: Ischemia-reperfusion of the murine testis stimulates the expression of proinflammatory cytokines and activation of c-jun N-terminal kinase in a pathway to E-selectin expression. Biol Reprod 2003, 69:202-210.
• [10]Turner TT, Lysiak JJ, Shannon JD, Nguyen QAT, Bazemore-Walker CR: Testicular torsion alters the presence of specific proteins in the mouse testis as well as the phosphorylation status of specific proteins. J Androl 2006, 27:285-293.
• [11]Piret J-P, Mottet D, Raes M, Michiels C: Is HIF-1alpha a pro- or an anti-apoptotic protein? Biochem Pharmacol 2002, 64:889-892.
• [12]Palladino MA, Pirlamarla PR, McNamara J, Sottas CM, Korah N, Hardy MP, Hales DB, Hermo L: Normoxic expression of hypoxia-inducible factor 1 in Rat leydig cells in vivo and in vitro. J Androl 2011, 32:307-323.
• [13]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.
• [14]Lysiak JJ, Kirby JL, Tremblay JJ, Woodson RI, Reardon MA, Palmer LA, Turner TT: Hypoxia-inducible factor-1{α} is constitutively expressed in murine leydig cells and regulates 3{β}-hydroxysteroid dehydrogenase type 1 promoter activity. J Androl 2009, 30:146-156.
• [15]Kuschel A, Simon P, Tug S: Functional regulation of HIF-1α under normoxia–is there more than post-translational regulation? J Cell Physiol 2012, 227:514-524.
• [16]Free MJ, Schluntz GA, Jaffe RA: Respiratory Gas tensions in tissues and fluids of the male Rat reproductive tract. Biol Reprod 1976, 14:481-488.
• [17]Lysiak JJ, Nguyen QA, Turner TT: Fluctuations in rat testicular interstitial oxygen tensions are linked to testicular vasomotion: persistence after repair of torsion. Biol Reprod 2000, 63:1383-1389.
• [18]Aitken RJ, Roman SD: Antioxidant systems and oxidative stress in the testes. Oxid Med Cell Longev 2008, 1:15-24.
• [19]Kozopas KM, Yang T, Buchan HL, Zhou P, Craig RW: MCL1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to BCL2. Proc Natl Acad Sci U S A 1993, 90:3516-3520.
• [20]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.
• [21]Brody JR, Kern SE: Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis. Biotechniques 2004, 36:214-216.
• [22]Wenger RH: Mammalian oxygen sensing, signalling and gene regulation. J Exp Biol 2000, 203:1253-1263.
• [23]Lai VK, Afzal MR, Ashraf M, Jiang S, Haider HK: Non-hypoxic stabilization of HIF-Iα during coordinated interaction between Akt and angiopoietin-1 enhances endothelial commitment of bone marrow stem cells. J Mol Med 2012, 90:719-730.
• [24]Schipani E, Ryan HE, Didrickson S, Kobayashi T, Knight M, Johnson RS: Hypoxia in cartilage: HIF-1alpha is essential for chondrocyte growth arrest and survival. Genes Dev 2001, 15:2865-2876.
• [25]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.
• [26]Bianciardi P, Fantacci M, Caretti A, Ronchi R, Milano G, Morel S, von Segesser L, Corno A, Samaja M: Chronic in vivo hypoxia in various organs: hypoxia-inducible factor-1alpha and apoptosis. Biochem Biophys Res Commun 2006, 342:875-880.
• [27]BelAiba RS, Djordjevic T, Bonello S, Flügel D, Hess J, Kietzmann T, Görlach A: Redox-sensitive regulation of the HIF pathway under non-hypoxic conditions in pulmonary artery smooth muscle cells. Biol Chem 2004, 385:249-257.
• [28]Pagé EL, Chan DA, Giaccia AJ, Levine M, Richard DE: Hypoxia-inducible factor-1alpha stabilization in nonhypoxic conditions: role of oxidation and intracellular ascorbate depletion. Mol Biol Cell 2008, 19:86-94.
• [29]Paick JS, Park K, Kim SW, Park JW, Kim JJ, Kim MS, Park JY: Increased expression of hypoxia-inducible factor-1α and connective tissue growth factor accompanied by fibrosis in the rat testis of varicocele. Actas Urol Esp 2012, 36:282-288.
• [30]Dehne N, Brüne B: HIF-1 in the inflammatory microenvironment. Exp Cell Res 2009, 315:1791-1797.
• [31]Imtiyaz HZ, Simon MC: Hypoxia-inducible factors as essential regulators of inflammation. Curr Top Microbiol Immunol 2010, 345:105-120.
• [32]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. Published online: 18 July 1964; doi:
• [33]Thomas LW, Lam C, Edwards SW: Mcl-1; the molecular regulation of protein function. FEBS Lett 2010, 584:2981-2989.
• [34]Kim S-H, Ricci MS, El-Deiry WS: Mcl-1: a gateway to TRAIL sensitization. Cancer Res 2008, 68:2062-2064.
• [35]Yu EZ, Li Y-Y, Liu X-H, Kagan E, McCarron RM: Antiapoptotic action of hypoxia-inducible factor-1 alpha in human endothelial cells. Lab Invest 2004, 84:553-561.
• [36]Piret J-P, Minet E, Cosse J-P, Ninane N, Debacq C, Raes M, Michiels C: Hypoxia-inducible factor-1-dependent overexpression of myeloid cell factor-1 protects hypoxic cells against ter-butyl hydroperoxide-induced apoptosis. J Biol Chem 2005, 280:9336-9344.
• [37]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.