Tissue and Cell 52 (2018) 78–91

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Tissue and Cell

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Insights into the role of estrogen-related receptors α, β and γ in tumor T Leydig cells ⁎ Malgorzata Kotula-Balaka, , Agnieszka Milona, Piotr Pawlickia, Malgorzata Opydo-Chanekb, Anna Pacwaa, Klaudia Lesniaka, Malgorzata Sekulaa, Marta Zarzyckac, Monika Bubkad, Waclaw Tworzydloe, Barbara Bilinskaa, Anna Hejmeja a Department of Endocrinology, Institute of Zoology and Biomedical Research, Jagiellonian University in Krakow, Gronostajowa 9, 30-387, Krakow, Poland b Department of Experimental Hematology, Institute of Zoology and Biomedical Research, Jagiellonian University in Krakow, Gronostajowa 9, 30-387, Krakow, Poland c Chair of Medical Biochemistry, Jagiellonian University Medical College, Kopernika 7, 31-034, Poland d Department of Glycoconjugate Biochemistry, Institute of Zoology and Biomedical Research, Jagiellonian University in Krakow, Gronostajowa 9, 30-387, Krakow, Poland e Department of Developmental Biology and Invertebrate Morphology, Institute of Zoology and Biomedical Research, Jagiellonian University in Krakow, Gronostajowa 9, 30-387, Krakow, Poland

ARTICLE INFO ABSTRACT

Keywords: In this study, we demonstrate, for the first time, estrogen-related receptor (ERR) regulation of the physiological Estrogen receptors and biochemical status of testicular tumor Leydig cells. Estrogen-related receptors In a mouse tumor Leydig cells, ERRs (α, β, and γ) were silenced via siRNA. Cell morphology and cell phy- Leydig cells siology (proliferation and observation of monolayer formation) were performed by inverted phase-contrast Proliferation microscope. Leydig cell functional markers (steroid receptors and signaling molecules) were examined by im- Monolayer formation munofluorescence and Western blotting. Additionally, progesterone secretion was assessed. Mitochondrial mass Signaling molecules and membrane potential were analyzed by flow-cytometry while cGMP and Ca2+ concentrations were analyzed using immunoenzymatic and colorimetric assays, respectively. These results revealed, ERRs indirectly regulate Leydig cell proliferation while ERRα and β affect cell monolayer formation. ERRs interact with canonical and membrane estrogen receptors (ERα,ERβ, and GPER), androgen receptor, metalloproteinase (MMP 9), protein kinase A (PKA), extracellular-regulated kinase (ERK), and neurogenic locus notch homolog protein 2 (Notch2). Depending on the type of ERR knocked down, coupled with estradiol treatment, changes in progesterone concentration and cGMP and Ca2+ concentrations constitute a microenvironment that may effect tumor Leydig cell characteristics. ERRs should be considered important factors in developing of innovating approaches that target pathological processes of testicular Leydig cells.

1. Introduction utilizing advanced microscopic, biochemical, and molecular conditions revealed the presence of two populations of Leydig cells; fetal and adult In 1850, Franz Leydig first described the testicular steroidogenic in the testis of mammals. cells that now bear his name (Leydig, 1850). For the next five decades, Androgens secreted by Leydig cells play a key function in male re- Leydig cells were extensively studied by light microscopy, and diverse production. They induce and maintain spermatogenesis, mediating speculations about their function were debated. In 1903, Ancel and their biological effects through the androgen receptor (AR), present in Bouin (1903) provided the first substantial evidence that Leydig cells Leydig, peritubular, and Sertoli cells, by autocrine, paracrine, and en- constituted an endocrine gland controlling male secondary sexual docrine pathways. The contribution of membrane AR in androgen characteristics (Bouin and Ancel, 1904). In the 1930s, testosterone was regulation of testicular cells has been recently reported (Bulldan et al., confirmed as a male hormone, and its endocrine actions were widely 2016). Estrogens, produced through androgen aromatization, are fun- studied together with the pituitary in the regulation of gonad function damental for the regulation of reproductive functions, thus ensuring (Christensen and Mason, 1965). However, direct evidence that Leydig proper male fertility (for review see Schulster et al., 2016). Estrogen cells produce androgens was not initially provided until the mid-1950s receptors alpha and beta (ERα and ERβ), along with their splice var- (Christensen and Mason, 1965; Wattenberg, 1958). Multiple studies iants and membrane associated G-protein-coupled functional ER

⁎ Corresponding author. E-mail address: [email protected] (M. Kotula-Balak). https://doi.org/10.1016/j.tice.2018.04.003 Received 14 March 2018; Received in revised form 8 April 2018; Accepted 9 April 2018 Available online 13 April 2018 0040-8166/ © 2018 Elsevier Ltd. All rights reserved. M. Kotula-Balak et al. Tissue and Cell 52 (2018) 78–91

(GPER), are abundant within testicular cells. In mammalian Leydig 2011). Classically, mitogenicity associated with estrogen receptor- cells, species-, age-, cellular origin (primary, tumor)- and experimental mediated cellular events is believed to be the mechanism by which condition (in vivo, in vitro)- specific patterns of localization and ex- estrogens contribute to tumorigenesis. A series of studies in rodents pression of ERα,ERβ and GPER have been reported (Fietz et al., 2016; reported that estrogen excess promotes hyperplasia, being generally Kotula-Balak et al., 2013; Mahmoud et al., 2015; Pelletier et al., 2000; accepted as proceeding into leydigioma (Cook et al., 1999). Woodward et al., 2002; Walker, 2011; Zarzycka et al., 2016). The co- The physiological effects of sex steroid hormones, along with gene expression of ERs and GPER is some tissues suggests the pathways they activity modulation, are mediated through rapid intracellular signaling activate may have a synergic, antagonistic or independent nature pathways including the activation of neurogenic locus notch homolog (Albanito et al., 2007). protein (Notch), extracellular signal-regulated kinase (ERK), mitogen- Recent studies examining various tissues suggest estrogen-related activated protein kinase (MAPK), metalloproteinases, generation of receptors (ERRs) type (α, β, and γ), belonging to the steroid hormone cAMP, mobilization of intracellular calcium (Ca2+) stores, and various receptor family, should be considered elements of sex steroid or/and interleukins and interferons (Brunsing and Prossnitz, 2011; Cheng estrogen signaling machinery. In 1988, Giguère et al. (1988), first re- et al., 2007; Filardo et al., 2000; Garcia and Hofmann, 2013; Holm ported ERRα. ERRβ was identified shortly after using ERRα cDNA as a et al., 2013; Lyng et al., 2000; Nwaneri et al., 2016; Scaling et al., 2014; probe. But due to its low expression level in most tissues and the early Tang et al., 2008). lethality of ERRβ knockout mice (Luo et al., 2003), ERRβ was not as Studies confirm that ERRs are regulators of cell energy metabolism intensively investigated as ERRα. The existence of ERRγ was uncovered via the supervision of mitochondrial, peroxisomal and endoplasmic only a decade later (Deblois and Giguère, 2008). The DNA sequences of reticulum processes, and these receptors are responsible for the in- ERRs is highly homologous to ERs. The action of ERRs and ERs in the duction and progression of cellular pathological events such as tumor regulation of multiple shared transcriptional targets interfere and/or development (for review see Huss et al., 2004 ; Deblois and Giguere, overlap, as ERRs can bind to functional estrogen response element 2011; Eichner and Giguère, 2011; Misawa and Satoshi Inoue, 2015; (ERE) in ER target genes (Huppunen and Aarnisalo, 2004). However, Misra et al., 2017). Therefore, herein we further explored the role of not much is known about ERR role and action in tissue physiology. ERRs in tumor Leydig cell physiology and molecular characteristics. More data are available on ERR implication in tissue pathology, e.g. tumor induction and progression (Bianco et al., 2012). Preliminary 2. Materials and methods evidence suggests that, in rat osteosarcoma cells and cervical cancer cells (HeLa), ERRs are regulated by components of fetal calf serum 2.1. Cell culture (Vanacker et al., 1999). In breast and prostate cancer cells, ERRs are activated by hormonal pharmacological agents (Roshan-Moniri et al., The mouse Leydig cell line MA-10 was a generous gift from Dr. 2014). Lacking an endogenous ligand, ERRs are reliant upon protein- Mario Ascoli (University of Iowa, Iowa City, IA), and was maintained protein interaction and post-translational modifications (Riggins, under standard technique (Ascoli, 1981). Middle passages of MA-10 2014). According to our previous studies, the expression of ERRs was cells were used for the study. The cells were grown in Waymouth’s significantly different following exposure of the primary or tumor media (Gibco, Grand Island, NY) supplemented with 12% horse serum

Leydig cells to xenoestrogens (polychlorinated napthalanes and poly- and 50 mg/l of gentamicin at 37 °C in 5% CO2. chlorinated biphenyls) (Milon et al., 2017; Pardyak et al., 2016). In- terestingly, the expression of ERR isoforms is shown to display diurnal 2.2. Estrogen related receptors knockdown using small interfering RNA rhythmicity in several tissues, including liver, skeletal muscle, white fat, kidneys, bones, and uterus (Yang et al., 2006). For transfection, Leydig cells were used at a confluence of 50–60% An unbalanced intratesticular ratio of active androgens and estro- in 6-well plates. Small interfering RNAs (siRNAs) (Life Technologies, gens can lead to various structural and functional abnormalities Gaithersburg, MD, USA) were introduced into MA-10 cells using throughout the reproductive system, including tumors (Rivas et al., jetPrime Transfection Reagent (Polyplus-transfection S.A., Bioparc, 2003). In both somatic and germ cells, androgen signaling contributes France), according to the manufacturer’s instructions. Briefly, cells to numerous processes, e.g. cell development, fate decisions, and dif- were incubated overnight in the indicated culture medium. The ferentiation, as well as organization of the blood-testis barrier (Garcia medium was removed after 24 h, and cells were rinsed once with PBS. and Hofmann, 2013; Mruk and Cheng, 2015). Interestingly, physiolo- Then, cells were cultured in serum-free, Opti-MEM, (Life Technologies) gical estradiol concentrations within the testis and semen, can exceed containing jetPrime buffer, jetPrime reagent, ERRα or ERRβ or ERRγ those found in the female vasculature (Luboshitzky et al., 2002; siRNA (Table 1) or non-targeting (control) siRNA, at a concentration of Hoffmann et al., 2010; Naderi and Safarinejad, 2003) thus, creating a 50 nM for 24 h. Specificity of siRNAs was checked in validation ex- specific microenvironment for testicular cell functions. Scarce data are periments with two different siRNAs for each of ERR genes (Table1). available on the molecular and biochemical characteristics of Leydig The culture medium was replaced with fresh medium after 24 h, and cell tumors. Carpino et al., (2007) have demonstrated that, in human cells were incubated for an additional hour, which promoted recovery. Leydig cell tumors varying expression patterns of ERα and ERβ are Then, cells were treated with 17β-estradiol (10 μM/ml) (Sigma Aldrich) observed in comparison to the control. In human leydigioma and rat for 24 h to analyze cell proliferation, monolayer formation, receptor tumor Leydig cells, authors additionally confirmed involvement of and enzyme protein expression (that was simultaneously measurement GPER in cell proliferation, growth and apoptosis (Chimetno et al., of efficacy of gene knockdown), and concentration of signaling

Table 1 siRNAs (Life Technologies) used for transfection assay.

siRNA siRNAα siRNAβ siRNAγ Negative control siRNA Positive control siRNA

used in this study cat.no. 4390771; siRNA ID s77021 cat.no. 4390771; siRNA ID cat.no. 4390771; siRNA ID cat.no. 4390843 cat.no. 4390849 s203075 s77027 used for validation cat.no.4390771; siRNA ID s77022 cat.no. 4390771; siRNA ID cat.no. 4390771; siRNA ID ————————— cat.no. AM4650 s77023 s77028

Abbreviations: ERR, αestrogen-related receptor alpha; ERRβ, estrogen-related receptor beta; ERRγ, estrogen-related receptor gamma.

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