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

in Journal of Molecular Endocrinology
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  • 1 Laboratorio de Endocrinología Molecular y Transducción de Señales, Instituto de Biología y Medicina Experimental (IBYME) – Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
  • 2 Unidad de Biología Celular, Institut Pasteur, Montevideo, Uruguay
  • 3 Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina

Correspondence should be addressed to O P Pignataro: oppignataro@gmail.com

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.

Abstract

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.

Introduction

Leydig cells (LC) comprise the androgen-secreting population of the testis, which develops during fetal and post-natal life in the interstitium of the gonad. Testosterone produced both pre- and postnatally is essential for the correct development and function of the male reproductive system (Ye et al. 2017). Several paracrine/autocrine factors, apart from the canonical control by the hypothalamic–pituitary–gonadal axis, have been credited with steroidogenesis and proliferation-regulating functions in LC and other steroidogenic populations (Del Punta et al. 1996, Mondillo et al. 2005, Li et al. 2017, Zhao et al. 2017 and others). Concertedly, these factors fine-tune LC proficiency in testosterone production in accordance with the demands met in different developmental stages. In the present report, the antioxidant enzyme heme oxygenase-1 is studied as an inducible autocrine/paracrine factor that can affect LC functions, even impairing their regulation by histamine, a biogenic amine thoroughly studied by our group and others in the context of LC physiopathology.

Oxidative stress is generated by diverse factors such as ionizing and UV radiation, alcohol, metals, aging and others (Pisoschi & Pop 2015). Resulting free radicals have unstable electron configuration; hence, they can damage essential macromolecules (Yoshikawa & Naito 2002). This detrimental effect can be countered by the activation of signal transduction pathways that protect from oxidative injury. One of the main antioxidant effectors is heme oxygenase-1 (HO-1) (Loboda et al. 2016). HO-1 induction exerts a pivotal role in the maintenance and re-establishment of cellular homeostasis (Ryter et al. 2006) triggering a cytoprotective response that includes not only the degradation of potentially toxic heme, but also the generation of three different biologically active by-products that participate in cellular adaptation to stress: biliverdin, that is swiftly converted into bilirubin (a prominent antioxidant), Fe+2 and carbon monoxide (CO), a powerful gasotransmitter (Wegiel et al. 2014). HO-1 deficiency in mouse models and in the scarce cases reported in humans confirm the importance of this enzyme in the preservation of health (Soares & Bach 2009, Dunn et al. 2014). Steroidogenesis impairment by hemin, a well-known HO-1 inducer, has been already reported by our group. In MA-10 tumoral LC line and normal adult LC, this effect is mediated by the inhibition of cytochrome P450scc (CYP11A) activity as well as a reduction in StAR protein levels (Piotrkowski et al. 2009).

Histamine (HA), on the other hand, has long been studied by our group in the context of LC regulation (being the first work published: Mondillo et al. 2005). In LC, this biogenic amine’s actions are exerted by three different receptors subtypes (HRH1, HRH2 and HRH4) (Jones & Kearns 2011). HA dual concentration-dependent effect on steroidogenesis has been described in MA-10 LCs and in purified rat LC. HA positively regulates steroidogenesis at low concentrations via HRH2 and negatively at high concentrations via HRH1 and HRH4 (Mondillo et al. 2005, Abiuso et al. 2014). Similar results were observed in R2C LCs (Abiuso et al. 2018). In addition, it has been shown that NOS activation is the main intracellular mechanism by which HA downregulates steroidogenesis through HRH1 in MA-10 LCs (Mondillo et al. 2009). Likewise, HA positively modulates LC proliferation through HRH2 and negatively through HRH4 in MA-10 LCs (Pagotto et al. 2012, Abiuso et al. 2014). Inhibition of proliferation in R2C LCs, as well as in isolated progenitor and immature LCs is mediated by HRH4 (Abiuso et al. 2014, 2018). It has also been reported that HA and histidine decarboxylase (HDC; enzyme that catalyzes the conversion of histidine into histamine) are present in higher levels in neonatal testis than in adults (Zieher et al. 1971, Pagotto et al. 2012). In Hdc-knockout mice, the absence of HA impairs testosterone production, which might lead to deficiencies in testicular embryonic ontogenesis (Mondillo et al. 2007). This exhaustive characterization of HA effects on LC strongly supports the idea of histamine having a central role in the regulation of LC development and male reproductive functions.

Based on these data, we hypothesize that HO-1 induction might have an impact on LC functions, presumably affecting their regulation by histamine. We describe that HO-1 negatively modulates LC proliferation, inducing a reversible arrest in G2/M phase. Even though the steroidogenic function is swiftly recovered in the absence of hemin, we show that while HO-1 upregulation lasts, LC functions are completely abrogated. Additionally, they remain incapable of offering a normal proliferative or steroidogenic response to histamine and histamine receptor agonists.

Materials and methods

Materials

Cell culture supplies were obtained from Gibco (Thermo Fisher Scientific) and plasticware from Corning (Tewksbury, MA, USA) and BD Biosciences (Franklin Lakes, NJ, USA). Caspase-3 p11 monoclonal antibody (sc-271759) was from Santa Cruz Biotechnology and β-tubulin antibody from Sigma-Aldrich. Peroxidase-conjugated anti-mouse and anti-rabbit IgG antibodies were purchased from Vector. Progesterone antibody was a gift from Dr L Bussmann (IBYME-CONICET, BA, Argentina). [3H]-thymidine (20 Ci/mmol) and [1,2,6,7-3H]-progesterone were purchased from New England Nuclear Corporation (North Billerica, MA, USA). Collagenase type II (CLS-2; 125 IU/mg) was from Worthington Biochemical Corporation (Lakewood, CA, USA). The following chemicals were purchased from Sigma-Aldrich: hemin (HEM), histamine (HA), amthamine (AMTHA), 2-(3-trifluoromethylphenyl) histamine dimaleate (FMPH), VUF 8430 (VUF), dibutyryl adenosine 3′,5′-cyclic monophosphate (db-cAMP), doxorubicin (DOXO), propidium iodide (PI), phenylmethylsulfonyl fluoride (PMSF), leupeptin, aprotinin, pepstatin A, charcoal, bovine serum albumin (BSA), Triton X-100 and RNAse A. The following chemicals were from Bio-Rad Laboratories: Tris, glycine, Bradford reagent, acrylamide, bisacrylamide, Tween-20, tetramethylethylenediamine (TEMED), and SDS. Other reagents used were of the best grade available.

Methods

Culture of MA-10 and R2C Leydig tumor cells

MA-10 cell line (kindly provided by Mario Ascoli, University of Iowa, IA, USA) is a murine clonal strain of Leydig tumor cells. The origin and handling of MA-10 cells have been described (Ascoli 1981, Pignataro & Ascoli 1990a). Authentication was performed according to ATCC guidelines. MA-10 cells were grown in Dulbecco modified Eagle medium/Ham F-12 (DMEM/F12) containing 4.76 g/L of Hepes, 1.2 g/L of sodium bicarbonate, 1 mL/L of Gentamicin Reagent Solution and 15% v/v horse serum (growth medium) at 37°C and 5% CO2 incubator. A maximum of 15 passages were performed.

The R2C rat Leydig tumor cell line was purchased from ATCC (# 58649146). They were grown in F-12 Nutrient Mixture/HAM (Ham’s F12), containing 1.2 g/L of sodium bicarbonate, 2 mM of Glutamine, 100 U/mL of Penicillin and 100 µg/mL of Streptomycin, supplemented with 12.5% horse serum and 2.5% fetal bovine serum (growth medium), at 37°C in a 5% CO2 atmosphere. Experiments in both cell lines were performed in serum free medium.

Preparation of rat immature LCs (ILCs)

Purified ILCs were isolated from testis of 35-day-old male Sprague–Dawley rats (200–250 g; Charles River descendants, Animal Care Lab, IBYME, Buenos Aires, Argentina) as described (Abiuso et al. 2014). Animals were killed by CO2 asphyxia, according to protocols for animal use approved by the institutional animal care and use committee (IBYME-CONICET), which follows National Institutes of Health guidelines.

Progesterone radioimmunoassay

Progesterone (P4) was measured by RIA in suitable aliquots as described (Pignataro & Ascoli 1990b, Del Punta et al. 1996, Mondillo et al. 2005). All experiments were conducted with equal number of cells. The intra- and interassay variations were 8 and 14.2% respectively.

[3H]-thymidine proliferation assay

DNA synthesis was evaluated according to the amount of [3H]-thymidine incorporated by the cells as we previously described (Abiuso et al. 2014, Pagotto et al. 2014). Treatments with the corresponding additions (detailed in each figure) lasted for 24 h, with a pulse of 0.25 μCi/well [3H]-Thymidine during the last 16 h. Radioactivity was measured using Tri-carb 2800TR liquid scintillation analyzer (PerkinElmer).

Apoptosis assessment by TUNEL

Cells were plated at a density of 1.5 × 106 cells/well in six-well plates. They were treated on Day 2 with or without hemin for 24 and 48 h. At the end of the incubation period, nuclear DNA fragmentation was detected by the TUNEL method using a cell death detection kit (Sigma-Aldrich) according to the manufacturer’s instructions. Apoptosis was analyzed using a flow cytometer FACSCanto II (BD Biosciences) and data were processed with FloJo software (BD Biosciences).

Western blot analysis and immunodetection of proteins

Cells were cultivated and treated under the same conditions as those for radioimmunoassay. Western blot analysis and immunodetection of proteins were carried out as previously described (Piotrkowski et al. 2009). Caspase-3 p11 antibody dilution was 1:100. Detection was performed with ECL Plus Western Blotting Detection Reagents (Amersham, GE Healthcare Biosciences). The intensity of immunospecific bands was quantified using ImageJ Software (National Institutes of Health). To correct for equal loading and blotting, caspase 3 blots were reprobed with a β-tubulin antibody.

Cell cycle analysis by flow cytometry

For cell cycle analysis, cells were harvested from six-well plates 24 h after stimulation in the presence or absence of HEM (10 μmol/L or 25 μmol/L), washed and fixed with 75% v/v ethanol overnight at −20°C. They were washed and incubated with 40 μg/mL PI, 3.8 mmol/L sodium citrate and 100 μg/mL RNAse A for 30 min at RT. The intracellular PI fluorescence intensity was measured using a flow cytometer FACSCanto II (BD Biosciences) (10,000 events/sample) and analyzed with FloJo software (BD Biosciences). Viability was also approached by PI staining on the same day cells were harvested.

Statistical analysis

All experiments performed herein were repeated at least three times and the data were pooled. If heterogeneity of variance was detected by Bartlett test, logarithmic transformation was performed before the analysis. The data were then subjected to one-way ANOVA followed by Tukey test for multiple-range comparisons.

Results

Hemin inhibits LC proliferation

To evaluate the effect of hemin on cell proliferation, MA-10 and R2C LC were incubated with increasing concentrations of hemin (1 µmol/L to 25 µmol/L) for 24 h and [3H]-thymidine incorporation was determined as a measure of cell proliferation. Growth medium (GM) was used as positive control. Hemin inhibition of MA-10 and R2C cell proliferation in a concentration-dependent manner is depicted in Fig. 1A and B, with a maximum effect observed at 25 µmol/L hemin (29% on average in MA-10 and 51% on average in R2C cells) (P ≤ 0.0001). Consistent results were obtained when assessing cell proliferation indirectly with the MTT colorimetric assay in both cell lines (data not shown).

Figure 1
Figure 1

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.

Citation: Journal of Molecular Endocrinology 63, 3; 10.1530/JME-19-0063

Even though MA-10 and R2C cells are widely accepted models in the study of LCs functions, it is also relevant to test the involvement of HO-1 in normal LCs proliferation. Taking into account that adult LCs present minimal proliferative capacity (Chen et al. 2014), we performed experiments using a proliferating population of immature rat LCs (ILC) in culture, isolated from 35-day-old rats. ILC were incubated with increasing concentrations of hemin (0.1–25 µmol/L) for 24 h and [3H]-thymidine incorporation was determined. IGF-1 (0.5 mg/L) was used as a positive control. As shown in Fig. 1C, ILCs proliferation was strongly inhibited by hemin in a concentration-dependent manner, with a maximum effect observed at 25 µmol/L (80% in average) (P ≤ 0.0001).

Hemin does not induce apoptosis

Next, we explored whether the inhibitory effect of hemin on cell proliferation was due to apoptotic events. Considering that proliferation results were similar in all cell types, we continued working with MA-10 LC. They were cultured with 10 or 25 µmol/L hemin for 24 and 48 h and apoptosis was evaluated by TUNEL assay using flow cytometry. As shown in Fig. 2A, apoptosis levels were not different between the treated and control cells. Doxorubicin, an apoptosis inducer, significantly increased the proportion of apoptotic cells. Similar results were obtained in the case of cells incubated for 48 h (data not shown). Likewise, we did not detect caspase-3 cleavage products, meaning that the apoptotic pathway was not being activated when the cells were incubated with 10 or 25 µmol/L hemin for 24 and 48 h (Fig. 2B).

Figure 2
Figure 2

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.

Citation: Journal of Molecular Endocrinology 63, 3; 10.1530/JME-19-0063

Hemin arrests cell cycle in G2/M

To determine the effect of hemin on cell cycle progression, MA-10 cells were treated with 25 µmol/L hemin for 24 h, and cell cycle distribution was analyzed using PI staining and flow cytometry. Figure 3A shows a significant increase in the percentage of cells in G2/M phase induced by 25 µmol/L hemin (average percentage of cells: control 12.2; 25 µmol/L hemin, 25.1), with a concomitant decrease in the proportion of cells in G1 phase (average percentage of cells: control 62.3; 25 µmol/L hemin 42.0). Interestingly, when the cells were preincubated for 24 h with hemin and then maintained in culture for another 48 h with fresh GM in the absence of hemin, cell cycle distribution was equal to that of control cells. The percentage of cells in G2/M phase was 10% in control cells and 11.3% in those that had been exposed to 25 µmol/L hemin (Fig. 3B).

Figure 3
Figure 3

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.

Citation: Journal of Molecular Endocrinology 63, 3; 10.1530/JME-19-0063

Steroidogenesis inhibition by long-term hemin exposure is reversible

To explore whether the effects observed on steroidogenesis after a long-term HO-1 induction are reversible, MA-10 cells were incubated with 10 or 25 µmol/L hemin for 24 and 48 h. As shown in Fig. 4A, the incubation with hemin beyond 24 h totally abrogates steroidogenesis. To test if this effect was reversible, hemin was removed from the medium and the cells were further incubated with fresh GM for 36 or 48 h. As shown in Fig. 4B, P4 production was fully recovered to normal levels after a 48-h incubation. Noticeably, this recovery is explained by the normalization of StAR protein levels and CYP11A activity (data not shown).

Figure 4
Figure 4

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.

Citation: Journal of Molecular Endocrinology 63, 3; 10.1530/JME-19-0063

HO-1 induction impairs steroidogenesis regulation by HA

In order to characterize the interaction between HA and HO-1 in steroidogenesis regulation, MA-10 and R2C cells were preincubated with 10 µmol/L hemin for 30 min followed by a 5-h incubation with 1 nmol/L or 10 µmol/L HA. When MA-10 and R2C LC were co-incubated with hemin and 1 nmol/L HA, the stimulatory effect given by HA at this concentration is completely reverted even resulting in the inhibition P4 production. Concomitantly, 10 µmol/L HA inhibitory effect is potentiated by hemin (Fig. 5A and B).

Figure 5
Figure 5

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.

Citation: Journal of Molecular Endocrinology 63, 3; 10.1530/JME-19-0063

In order to assess which of HA receptors signaling is being affected by hemin actions, MA-10 and R2C cells were preincubated with 10 µmol/L hemin for 30 min followed by a 5-h incubation with specific agonists for each receptor namely, FMPH (2-(3-trifluoromethylphenyl)histamine dimaleate), AMTHA (amthamine) and VUF (VUF 8430) for HRH1, HRH2 and HRH4, respectively. As shown in Fig. 5C, in MA-10, the stimulatory effect produced by 1 µmol/L AMTHA was reverted by hemin (average percentage of control: AMTHA 1 µmol/L: 141.7, AMTHA plus hemin: 62.5. P ≤ 0.001). The inhibition of MA-10 steroidogenesis exerted by HRH1 and HRH4 was enhanced when the cells were incubated with FMPH or VUF plus hemin (average percentage of control: FMPH: 70.8, FMPH plus hemin: 39.6, VUF: 66.7, VUF plus hemin: 47.9. P ≤ 0.001). In R2C cells, HA and HO-1 pathways interact in the same manner as in MA-10 (Fig. 5D).

HO-1 induction impairs proliferation regulation by HA

HA and HO-1 interaction in LCs proliferation regulation was also assessed. When MA-10 and R2C cells were preincubated for 30 min with 25 µmol/L hemin and 1 nmol/L or 10 µmol/L HA were added for another 24 h, it was observed that the stimulatory effect exerted by 1 nmol/L HA was completely reverted by hemin, even resulting in P4 production inhibition. 10 µmol/L HA inhibitory effect was not affected by the preincubation with hemin (Fig. 6A and B).

Figure 6
Figure 6

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.

Citation: Journal of Molecular Endocrinology 63, 3; 10.1530/JME-19-0063

When MA-10 LCs cells were incubated with 1 µmol/L AMTHA (HRH2 agonist) and 25 µmol/L hemin, the stimulation of proliferation exerted by AMTHA was abolished by hemin (average percentage of control: AMTHA:110.8, AMTHA plus hemin:52.7). Incubation of cells with 1 µmol/L VUF plus hemin enhanced the inhibitory effect exerted by VUF alone (in percentage of control: VUF: 83.1 vs VUF + hemin: 52.0, on average) (Fig. 6C). In R2C LCs, the effect of HA receptor subtypes on cell proliferation has not been completely characterized. When R2C cells were incubated with 10 μmol/L FMPH (HRH1 agonist), R2C proliferation was inhibited and FMPH plus hemin enhanced the effect exerted by FMPH alone (average percentage of control: FMPH 79.3 vs FMPH plus hemin: 67.8). In the presence of 10 µmol/L AMTHA (HRH2 agonist) and 25 µmol/L hemin, we only observed hemin effect, since AMTHA had no effect on R2C cell proliferation. Finally, the incubation of cells with 10 µmol/L VUF plus hemin enhanced the inhibitory effect exerted by VUF alone (average percentage of control: VUF: 67.8 vs VUF + hemin: 56.3) (Fig. 6D).

Discussion

The correct synchronization of hormonal and non-hormonal signals is essential for the proper development and function of the testis and particularly of LC. Growing evidence supports the notion that histamine is part of this vital group of signals and has a central regulatory role over LC steroidogenic function and proliferative capacity through the activation of its different receptor subtypes (Albrecht et al. 2005, Mondillo et al. 2005, Khan & Rai 2007, Pagotto et al. 2012, Abiuso et al. 2014). Despite adult LC being a non-proliferating population, their development consists of sequentially occurring proliferation waves that require intact proliferative and steroidogenic capacity chiefly during embryonic phases of development (Ye et al. 2017). In the present report, we sought to determine whether LC functions and their regulation by histamine were impaired in a pro-oxidant scenario, such as exposure to pollutants/toxins, aging or microbial infection (Pisoschi & Pop 2015), where HO-1 is upregulated.

In a previous work, we have described that HO-1 inhibits LC steroidogenesis by downregulating StAR and inhibiting CYP11A activity while 3β-hydroxysteroid dehydrogenase remains unaffected (Piotrkowski et al. 2009). Herein, we present novel evidence of HO-1 negative influence on LC proliferative capacity, employing two well-known LC lines and normal immature LC. In agreement, the observed negative effect on proliferation has been reported in other cell types (Seidel et al. 2010, Bukowska-Strakova et al. 2017, Kozakowska et al. 2018). Wagner and colleagues have proposed that HO-1 induction in preadipocytes augments ROS production, activates Akt2 and, consequently, inhibits cell proliferation (Wagner et al. 2017). In smooth muscle cells, HO-1 inhibits proliferation via CO-mediated inhibition of T-type Ca2+ channels (Duckles et al. 2015).

In this report, we demonstrate that the observed cell proliferation inhibition in MA-10 LC occurs without inducing apoptosis. This result is in line with several reports that pose HO-1 system activation as a mechanism for ameliorating apoptosis (Loboda et al. 2016). In particular, in TM3 LC line it has been reported that cadmium-derived apoptosis induction can be reverted by sulforaphane-mediated Nrf2/ARE pathway activation (Yang et al. 2019). Additionally, in an in vivo study, HO-1 induction has been proposed as the mechanism by which simvastatin inhibits apoptosis after ischemia reperfusion injury in the testis (Tu et al. 2011). Our present findings show that hemin arrests MA-10 cells in G2/M phase. Interestingly, Lee and colleagues have described a line of human pulmonary epithelial cells whose response to HO-1 consists in G2/M arrest and a marked resistance to oxidative noxae (Lee et al. 1996). We have also observed that when hemin is withdrawn, cell cycle progression promptly resumes. This recovery is supported by the fact that the exit from G2/M arrest in murine cells occurs in a few hours (van Vugt & Yaffe 2010). Besides, a report by Chang and colleagues that also describes HO-1-dependent arrest of cell cycle progression in vascular smooth muscle cells shows that proliferation is resumed after 1 week of hemin withdrawal (Chang et al. 2008).

As clearly depicted by our results, complete abrogation of MA-10 steroidogenic activity after a 48-h incubation with hemin can be restored to control levels if the inducer is removed from the medium. There are scarce reports on this respect. However, HO-1 inhibitory effect has already been reported in our model using short-term incubation protocols (Piotrkowski et al. 2009) and in adrenal steroidogenesis, where HO-1 inhibition leads to increased steroid secretion (Pomeraniec et al. 2004). Besides, it has been reported that in TM3 LC, decreased steroidogenesis induced by glutathione deficiency can be partially attributed to HO-1 induction (Li et al. 2016). Reversibility of hemin’s long-term effects, together with the absence of apoptosis induction, confirm that hemin effects are not lethal and might serve as a shielding mechanism against pro-oxidant insults, pausing cell functions until homeostasis is recovered. In line with this hypothesis, it has been reported that HO-1 acts as a protective factor in a variety of physiopathological contexts (Kusmic et al. 2014, Loboda et al. 2016, Li et al. 2019).

As mentioned earlier, the histaminergic system has been extensively studied by our group and others in the context of LC physiopathology. Histamine has shown to act differentially through its receptors subtypes, exerting a positive effect on steroidogenesis and proliferation via HRH2 and a negative effect via HRH4 (and HRH1 for steroidogenesis) (Mondillo et al. 2005, Pagotto et al. 2012, Abiuso et al. 2014). Despite there being reports that describe HA system’s antioxidant effect in some experimental models (Yadav et al. 2015, Sanna et al. 2017), to the best of our knowledge, a specific interaction between HA and HO-1 effects had not yet been described. To assess this interaction, we used both MA-10 LCs, which synthesize and secrete steroids in response to LH/hCG stimulation (Ascoli 1985), and R2C LCs, whose steroidogenic response is constitutively active (Freeman 1987, Rao et al. 2002). In both cases, when the cells were exposed both to hemin and HA, the biphasic effect that the amine exerts on steroidogenesis and proliferation is completely abrogated. Surprisingly, experiments with hemin and HA receptors 1, 2 and 4 agonists showed that regulation by all three receptors was being affected by HO-1 induction. These results suggest that hemin is masking HA regulation and that, as long as HO-1 induction lasts, LC remains incapable of offering a normal response. The way in which hemin interacts with HA and the different receptor agonists suggests that HO-1 induction might halt the steroidogenic pathway. CO, one of the products of heme degradation by HO-1, might serve as a possible candidate for this effect. It is widely known that some of the main steroidogenic enzymes are cytochrome P450 heme-containing proteins (Payne & Hales 2004) and that CO reversibly binds their metal centers reducing their enzymatic activity (Desmard et al. 2007, Rochette et al. 2013). We have obtained some preliminary results that support this hypothesis (work in progress).

In conclusion, we have elucidated that a long-term HO-1 induction reversibly arrests LC functions, inhibiting steroid production and cell cycle progression. What is more, HO-1 impairs histamine regulatory effects on LC functions. The results obtained will help deepen the understanding on how oxidative stress, through HO-1 actions, might negatively influence critical phases of LC development and differentiation, affecting their function as well as other androgen-dependent organs.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This investigation was supported by grants from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PIP 392) and Universidad de Buenos Aires (UBA, UBACYT 2013-20020120100205) to O P P.

Acknowledgements

The authors would like to thank René Barón Foundation and Williams Foundation for the equipment donation to IBYME.

References

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    • PubMed
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    • PubMed
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    • PubMed
    • Search Google Scholar
    • Export Citation
  • Desmard M, Boczkowski J, Poderoso J & Motterlini R 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
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    • Export Citation
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    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.

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    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.

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    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.

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    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.

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    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.

  • Abiuso AMB, Berensztein E, Pagotto RM, Pereyra EN, Medina V, Martinel Lamas DJ, Besio Moreno M, Pignataro OP & Mondillo C 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
  • Abiuso AMB, Varela ML, Haro Durand L, Besio Moreno M, Marcos A, Ponzio RM, Belgorosky A, Pignataro O, Berensztein E, et 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
  • Albrecht M, Frungieri MB, Gonzalez-Calvar S, Meineke V, Köhn FM & Mayerhofer A 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
  • Ascoli M 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
  • Ascoli M 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-Strakova K, Ciesla M, Szade K, Nowak WN, Straka R, Szade A, Tyszka-Czochara M, Najder K, Konturek A, Siedlar M, et 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
  • Chang T, Wu L & Wang R 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
  • Chen H, Stanley E, Jin S & Zirkin BR 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 Punta K, Charreau EH & Pignataro OP 1996 Nitric oxide inhibits Leydig cell steroidogenesis. Endocrinology 53375343. (https://doi.org/10.1210/endo.137.12.8940355)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Desmard M, Boczkowski J, Poderoso J & Motterlini R 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
  • Duckles H, Boycott HE, Al-Owais MM, Elies J, Johnson E, Dallas ML, Porter KE, Giuntini F, Boyle JP, Scragg JL, et 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
  • Dunn LL, Midwinter RG, Ni J, Hamid HA, Parish CR & Stocker R 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
  • Freeman DA 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
  • Jones BL & Kearns GL 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
  • Khan UW & Rai U 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
  • Kozakowska M, Pietraszek-Gremplewicz K, Ciesla M, Seczynska M, Bronisz-Budzynska I, Podkalicka P, Bukowska-Strakova K, Loboda A, Jozkowicz A & Dulak J 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
  • Kusmic C, Barsanti C, Matteucci M, Vesentini N, Pelosi G, Abraham NG & L’Abbate A 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
  • Lee PJ, Alam J, Wiegand GW & Choi AM 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
  • Li W, Wu ZQ, Zhang S, Cao R, Zhao J, Sun ZJ & Zou W 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
  • Li J, Zhou Q, Ma Z, Wang M, Shen WJ, Azhar S, Guo Z & Hu Z 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
  • Li S, Fujino M, Takahara T & Kang XK 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
  • Loboda A, Damulewicz M, Pyza E, Jozkowicz A & Dulak J 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
  • Mondillo C, Patrignani Z, Reche C, Rivera E & Pignataro O 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
  • Mondillo C, Falus A, Pignataro O & Pap E 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
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