Heme oxygenase-1 arrests Leydig cells functions and impairs their regulation by histamine

in Journal of Molecular Endocrinology
Correspondence should be addressed to O P Pignataro: oppignataro@gmail.com
Restricted access

Testicular Leydig cells (LC) are modulated by several pathways, one of them being the histaminergic system. Heme oxygenase-1 (HO-1), whose upregulation comprises the primary response to oxidative noxae, has a central homeostatic role and might dysregulate LC functions when induced. In this report, we aimed to determine how hemin, an HO-1 inducer, affects LC proliferative capacity and whether HO-1 effects on LC functions are reversible. It was also evaluated if HO-1 interacts in any way with histamine, affecting its regulatory action over LC. MA-10 and R2C cell lines and immature rat LC were used as models. Firstly, we show that after a 24-h incubation with 25 µmol/L hemin, LC proliferation is reversibly impaired by cell cycle arrest in G2/M phase, with no evidence of apoptosis induction. Even though steroid production is abrogated after a 48-h exposure to 25 µmol/L hemin, steroidogenesis can be restored to control levels in a time-dependent manner if the inducer is removed from the medium. Regarding HO-1 and histamine interaction, it is shown that hemin abrogates histamine biphasic effect on steroidogenesis and proliferation. Working with histamine receptors agonists, we elucidated that HO-1 induction affects the regulation mediated by receptor types 1, 2 and 4. In summary, HO-1 induction arrests LC functions, inhibiting steroid production and cell cycle progression. Despite their reversibility, HO-1 actions might negatively influence critical phases of LC development and differentiation affecting their function as well as other androgen-dependent organs. What’s more, we have described a hitherto unknown interaction between HO-1 induction and histamine effects.

 

      Society for Endocrinology

Related Articles

Article Information

Metrics

All Time Past Year Past 30 Days
Abstract Views 113 113 86
Full Text Views 28 28 26
PDF Downloads 11 11 10

Altmetrics

Figures

  • View in gallery

    Effect of HEM on LC proliferation. MA-10 (A) and R2C (B) LC were incubated with increasing concentrations (1–25 µmol/L) of HEM or growth medium (GM) for 24 h. (C) Effect of HEM on normal ILCs proliferation. Rat ILCs were incubated with increasing concentrations of HEM (0.1–25 µmol/L) or 0.5 mg/L IGF-1 for 24 h. The cells were labeled with [3H]-thymidine during the last 16 h of the incubation. Scale bars are means ± s.e.m. for a representative (n = 3) octuplicate experiment. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001 vs control.

  • View in gallery

    Effect of HEM on MA-10 LC apoptosis. (A) MA-10 LC were incubated with 10 or 25 µmol/L HEM for 24 h, processed by TUNEL assay and analyzed by flow cytometry. Doxorubicin was used as a positive control for apoptosis. Scale bars are means ± s.e.m. for a representative (n = 3) triplicate experiment. ****P ≤ 0.0001 vs control. (B) Representative immunoblot for caspase-3 protein. The cells were incubated with or without 10 and 25 µmol/L HEM for 24 and 48 h. After treatments, the cells were lysed and subjected to Western blotting analysis.

  • View in gallery

    Effect of HEM on MA-10 cell cycle progression. Cells were incubated for 24 h in the absence or presence of 25 µmol/L HEM (A). Then, hemin was withdrawn, and the cells were maintained in fresh GM for another 48 h (B). After the incubation, cells were fixed, permeabilized and stained with PI. DNA content was analyzed by flow cytometry. Graphs show G1/G0, S, and G2/M cell cycle phases distribution. Scale bars are means ± s.e.m. for a representative (n = 3) triplicate experiment. ***P ≤ 0.001.

  • View in gallery

    Effect of long-term exposure to HEM on P4 production in MA-10 LC. The cells were incubated in the presence or absence of 10 µmol/L HEM for 24 or 48 h (A). Then, hemin was withdrawn, and the cells were maintained in fresh growth medium for another 36 or 48 h (B). Progesterone was measured by RIA. Scale bars are means ± s.e.m. for a representative (n = 3) triplicate experiment. **P ≤ 0.01 and ****P ≤ 0.0001 vs control.

  • View in gallery

    Interaction between HEM and HA or specific HA receptor agonists in LC steroidogenesis. MA-10 (A and C) and R2C (B and D) cells were preincubated in the presence or absence of 10 µmol/L HEM for 30 min. Then, 1 nmol/L or 10 µmol/L HA or 1 µmol/L FMPH, AMTHA or VUF were added and the incubation continued for 5 h. Progesterone was measured by RIA. Scale bars are means ± s.e.m. for a representative (n = 3) triplicate experiment. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001 vs control.

  • View in gallery

    Interaction between HEM and HA or specific HA receptor agonists in LC proliferation. MA-10 (A and C) and R2C (B and D) cells were preincubated in the presence or absence of 25 µmol/L HEM for 30 min. Then, 1 nmol/L or 10 µmol/L HA or 1 µmol/L FMPH, AMTHA or VUF were added and the incubation continued for 24 h. The cells were labeled with [3H]-thymidine during the last 16 h. Scale bars are means ± s.e.m. for a representative (n = 3) octuplicate experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001 vs control.

References

  • AbiusoAMBBerenszteinEPagottoRMPereyraENMedinaVMartinel LamasDJBesio MorenoMPignataroOPMondilloC 2014 H4 histamine receptors inhibit steroidogenesis and proliferation in Leydig cells. Journal of Endocrinology 241253. (https://doi.org/10.1530/JOE-14-0401)

    • Search Google Scholar
    • Export Citation
  • AbiusoAMBVarelaMLHaro DurandLBesio MorenoMMarcosAPonzioRMBelgoroskyAPignataroOBerenszteinEet al. 2018 Histamine H4 receptor as a novel therapeutic target for the treatment of Leydig cell tumors in prepubertal boys. European Journal of Cancer 125135. (https://doi.org/10.1016/j.ejca.2017.12.003)

    • Search Google Scholar
    • Export Citation
  • AlbrechtMFrungieriMBGonzalez-CalvarSMeinekeVKöhnFMMayerhoferA 2005 Evidence for a histaminergic system in the human testis. Fertility and Sterility 10601063. (https://doi.org/10.1016/j.fertnstert.2004.12.002)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • AscoliM 1981 Characterization of several clonal lines of cultured leydig tumor cells: gonadotropin receptors and steroidogenic responses. Endocrinology 8895. (https://doi.org/10.1210/endo-108-1-88)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • AscoliM 1985 Functions and regulation of cell surface receptors in cultured Leydig tumor cells. In The Receptors Vol. II pp 367400. Academic Press. (https://doi.org/10.1016/B978-0-12-185202-3.50017-2)

    • Search Google Scholar
    • Export Citation
  • Bukowska-StrakovaKCieslaMSzadeKNowakWNStrakaRSzadeATyszka-CzocharaMNajderKKonturekASiedlarMet al. 2017 Heme oxygenase 1 affects granulopoiesis in mice through control of myelocyte proliferation. Immunobiology 846857. (https://doi.org/10.1016/j.imbio.2017.05.006)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • ChangTWuLWangR 2008 Inhibition of vascular smooth muscle cell proliferation by chronic hemin treatment. American Journal of Physiology: Heart and Circulatory Physiology 9991007. (https://doi.org/10.1152/ajpheart.01289.2007)

    • Search Google Scholar
    • Export Citation
  • ChenHStanleyEJinSZirkinBR 2014 Stem Leydig cells: from fetal to aged animals. Birth Defects Research: C Embryo Today 272283. (https://doi.org/10.1002/bdrc.20192.Stem)

    • Search Google Scholar
    • Export Citation
  • Del PuntaKCharreauEHPignataroOP 1996 Nitric oxide inhibits Leydig cell steroidogenesis. Endocrinology 53375343. (https://doi.org/10.1210/endo.137.12.8940355)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • DesmardMBoczkowskiJPoderosoJMotterliniR 2007 Mitochondrial and cellular heme-dependent proteins as targets for the bioactive function of the heme oxygenase/carbon monoxide system. Antioxidants and Redox Signaling 21392155. (https://doi.org/10.1089/ars.2007.1803)

    • Search Google Scholar
    • Export Citation
  • DucklesHBoycottHEAl-OwaisMMEliesJJohnsonEDallasMLPorterKEGiuntiniFBoyleJPScraggJLet al. 2015 Heme oxygenase-1 regulates cell proliferation via carbon monoxide-mediated inhibition of T-type Ca 2 + channels. Pflugers Archiv 415427. (https://doi.org/10.1007/s00424-014-1503-5)

    • Search Google Scholar
    • Export Citation
  • DunnLLMidwinterRGNiJHamidHAParishCRStockerR 2014 New insights into intracellular locations and functions of heme Oxygenase-1. Antioxidants and Redox Signaling 17231742. (https://doi.org/10.1089/ars.2013.5675)

    • Search Google Scholar
    • Export Citation
  • FreemanDA 1987 Constitutive steroidogenesis in the R2C Leydig tumor cell line is maintained by the adenosine 3′,5′-cyclic monophosphate-independent production of a cycloheximide-sensitive factor that enhances mitochondrial pregnenolone biosynthesis. Endocrinology 124132. (https://doi.org/10.1210/endo-120-1-124)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • JonesBLKearnsGL 2011 Histamine: new thoughts about a familiar mediator. Clinical Pharmacology and Therapeutics 189197. (https://doi.org/10.1038/clpt.2010.256)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • KhanUWRaiU 2007 Differential effects of histamine on Leydig cell and testicular macrophage activities in wall lizards: precise role of H1/H2 receptor subtypes. Journal of Endocrinology 441448. (https://doi.org/10.1677/JOE-06-0225)

    • Search Google Scholar
    • Export Citation
  • KozakowskaMPietraszek-GremplewiczKCieslaMSeczynskaMBronisz-BudzynskaIPodkalickaPBukowska-StrakovaKLobodaAJozkowiczADulakJ 2018 Lack of heme oxygenase-1 induces inflammatory reaction and proliferation of muscle satellite cells after cardiotoxin-induced skeletal muscle injury. American Journal of Pathology 491506. (https://doi.org/10.1016/j.ajpath.2017.10.017)

    • Search Google Scholar
    • Export Citation
  • KusmicCBarsantiCMatteucciMVesentiniNPelosiGAbrahamNGL’AbbateA 2014 Up-regulation of heme oxygenase-1 after infarct initiation reduces mortality, infarct size and left ventricular remodeling: experimental evidence and proof of concept. Journal of Translational Medicine 89. (https://doi.org/10.1186/1479-5876-12-89)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • LeePJAlamJWiegandGWChoiAM 1996 Overexpression of heme oxygenase-1 in human pulmonary epithelial cells results in cell growth arrest and increased resistance to hyperoxia. PNAS 1039310398. (https://doi.org/10.1073/pnas.93.19.10393)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • LiWWuZQZhangSCaoRZhaoJSunZJZouW 2016 Augmented expression of gamma-glutamyl transferase 5 (GGT5) impairs testicular steroidogenesis by deregulating local oxidative stress. Cell and Tissue Research 467481. (https://doi.org/10.1007/s00441-016-2458-y)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • LiJZhouQMaZWangMShenWJAzharSGuoZHuZ 2017 Feedback inhibition of CREB signaling by p38 MAPK contributes to the negative regulation of steroidogenesis. Reproductive Biology and Endocrinology 19. (https://doi.org/10.1186/s12958-017-0239-4)

    • Search Google Scholar
    • Export Citation
  • LiSFujinoMTakaharaTKangXK 2019 Protective role of heme oxygenase-1 in fatty liver ischemia – reperfusion injury. Medical Molecular Morphology 6172. (https://doi.org/10.1007/s00795-018-0205-z)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • LobodaADamulewiczMPyzaEJozkowiczADulakJ 2016 Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism. Cellular and Molecular Life Sciences 32213247. (https://doi.org/10.1007/s00018-016-2223-0)

    • Search Google Scholar
    • Export Citation
  • MondilloCPatrignaniZRecheCRiveraEPignataroO 2005 Dual role of histamine in modulation of Leydig cell steroidogenesis via HRH1 and HRH2 receptor subtypes. Biology of Reproduction 899907. (https://doi.org/10.1095/biolreprod.105.041285)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • MondilloCFalusAPignataroOPapE 2007 Prolonged histamine deficiency in histidine decarboxylase gene knockout mice affects Leydig cell function. Journal of Andrology 8691. (https://doi.org/10.2164/jandrol.106.000257)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • MondilloCPagottoRMPiotrkowskiBRecheCGPatrignaniZJCymeryngCBPignataroOP 2009 Involvement of nitric oxide synthase in the mechanism of histamine-induced inhibition of Leydig cell steroidogenesis via histamine receptor subtypes in Sprague-Dawley rats. Biology of Reproduction 144152. (https://doi.org/10.1095/biolreprod.108.069484)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • PagottoRMMonzónCMorenoMBPignataroOPMondilloC 2012 Proliferative effect of histamine on MA-10 Leydig tumor cells mediated through HRH2 activation, transient elevation in cAMP production, and increased extracellular signal-regulated kinase phosphorylation levels. Biology of Reproduction 150. (https://doi.org/10.1095/biolreprod.112.102905)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • PagottoRMPereyraENMonzónCMondilloCPignataroOP 2014 Histamine inhibits adrenocortical cell proliferation but does not affect steroidogenesis. Journal of Endocrinology 1528. (https://doi.org/10.1530/JOE-13-0433)

    • Search Google Scholar
    • Export Citation
  • PayneAHHalesDB 2004 Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocrine Reviews 947970. (https://doi.org/10.1210/er.2003-0030)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • PignataroOPAscoliM 1990a Epidermal growth factor increases the labeling of phosphatidylinositol 3,4-bisphosphate in MA-10 Leydig tumor cells. Journal of Biological Chemistry 17181723.

    • Search Google Scholar
    • Export Citation
  • PignataroOPAscoliM 1990b Studies with insulin and insulin-like growth factor-I show that the increased labeling of Phosphatidylinositol-3,4-bisphosphate is not sufficient to elicit the diverse actions of epidermal growth factor on MA-10 Leydig tumor cells. Molecular Endocrinology 758765. (https://doi.org/10.1210/mend-4-5-758)

    • Search Google Scholar
    • Export Citation
  • PiotrkowskiBMonzónCMPagottoRMRecheCGBesioMCymeryngCBPignataroOP 2009 Effects of heme oxygenase isozymes on Leydig cells steroidogenesis. Journal of Endocrinology 155165. (https://doi.org/10.1677/JOE-09-0061)

    • Search Google Scholar
    • Export Citation
  • PisoschiAMPopA 2015 The role of antioxidants in the chemistry of oxidative stress: a review. European Journal of Medicinal Chemistry 5574. (https://doi.org/10.1016/j.ejmech.2015.04.040)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • PomeraniecYGrionNGaddaLPannunzioVPodestaEJCymeryngCB 2004 Adrenocorticotropin induces heme oxygenase-1 expression in adrenal cells. Journal of Endocrinology 113124. (https://doi.org/10.1677/joe.0.1800113)

    • Search Google Scholar
    • Export Citation
  • RaoRMJoYBabb-TarboxMSyapinPJStoccoDM 2002 Regulation of steroid hormone biosynthesis in R2C and MA-10 Leydig tumor cells: role of the cholesterol transfer proteins StAR and PBR. Endocrine Research 387394. (https://doi.org/10.1081/ERC-120016813)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • RochetteLCottinYZellerMVergelyC 2013 Carbon monoxide: mechanisms of action and potential clinical implications. Pharmacology and Therapeutics 133152. (https://doi.org/10.1016/j.pharmthera.2012.09.007)

    • Search Google Scholar
    • Export Citation
  • RyterSWAlamJChoiAMK 2006 Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. Physiological Reviews 583650. (https://doi.org/10.1152/physrev.00011.2005)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • SannaMDLucariniLDuranteMGhelardiniCMasiniEGaleottiN 2017 Histamine H4 receptor agonist-induced relief from painful peripheral neuropathy is mediated by inhibition of spinal neuroinflammation and oxidative stress. British Journal of Pharmacology 2840. (https://doi.org/10.1111/bph.13644)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • SeidelPGouletSHostettlerKTammMRothM 2010 DMF inhibits PDGF-BB induced airway smooth muscle cell proliferation through induction of heme-oxygenase-1. Respiratory Research 145. (https://doi.org/10.1186/1465-9921-11-145)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • SoaresMPBachFH 2009 Heme oxygenase-1: from biology to therapeutic potential. Trends in Molecular Medicine 5058. (https://doi.org/10.1016/j.molmed.2008.12.004)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • TuYPChuangSJChenSCLiuYHChenCFHourTC 2011 Simvastatin induces the expression of hemeoxygenase-1 against ischemia – reperfusion injury on the testes in rats. Toxicology Letters 242250. (https://doi.org/10.1016/j.toxlet.2011.09.016)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • van VugtMATMYaffeMB 2010 Cell cycle re-entry mechanisms after DNA damage checkpoints: giving it some gas to shut off the breaks! Cell Cycle 20972101. (https://doi.org/10.4161/cc.9.11.11840)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • WagnerGLindroos-christensenJEinwallnerEHusaJZapfTCLippKRauscherSGrögerMSpittlerALoeweRet al. 2017 HO-1 inhibits preadipocyte proliferation and differentiation at the onset of obesity via ROS dependent activation of Akt2. Scientific Reports 40881. (https://doi.org/10.1038/srep40881)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • WegielBNemethZCorrea-CostaMBulmerACOtterbeinLE 2014 Heme oxygenase-1: a metabolic nike. Antioxidants and Redox Signaling 17091722. (https://doi.org/10.1089/ars.2013.5667)

    • Search Google Scholar
    • Export Citation
  • YadavCHNajmiAKAkhtarMKhanamR 2015 Cardioprotective role of H3R agonist imetit on isoproterenol-induced hemodynamic changes and oxidative stress in rats. Toxicology Mechanisms and Methods 235240. (https://doi.org/10.3109/15376516.2014.997946)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • YangSHLiPYuLHLiLLongMLiuMDHeJB 2019 Sulforaphane protect against cadmium-induced oxidative damage in mouse Leydigs cells by activating Nrf2/ARE signaling pathway. International Journal of Molecular Sciences 630. (https://doi.org/10.3390/ijms20030630)

    • Search Google Scholar
    • Export Citation
  • YeLLiXLiLChenHGeRS 2017 Insights into the development of the adult Leydig cell lineage from stem Leydig cells. Frontiers in Physiology 430. (https://doi.org/10.3389/fphys.2017.00430)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • YoshikawaTNaitoY 2002 What is oxidative stress? Japan Medical Association Journal 271276.

  • ZhaoYLiHXWangKYanBYLiW 2017 Regulation of testicular steroidogenesis by Foxa3 via transcriptional modulation of ER a signaling in type 2 diabetes mellitus (T2DM). Biochemical and Biophysical Research Communications 786793. (https://doi.org/10.1016/j.bbrc.2017.06.118)

    • Search Google Scholar
    • Export Citation
  • ZieherLMDebeljukLIturrizaFManciniRE 1971 Biogenic amine concentration in testes of rats at different ages. Endocrinology 351354. (https://doi.org/10.1210/endo-88-2-351)

    • PubMed
    • Search Google Scholar
    • Export Citation

PubMed

Google Scholar