Low concentrations mono-butyl phthalate stimulates steroidogenesis by facilitating steroidogenic acute regulatory protein expression in mouse Leydig tumor cells (MLTC-1)

Di-n-butyl phthalate (DBP) is one of the most dominant phthalate esters and is widely distributed environmental contaminant. Although previous studies have demonstrated that DBP led to a variety of male reproductive abnormalities similar to those caused by androgen receptor antagonists, DBP and its active metabolite, mono-butyl phthalate (MBP), have been demonstrated no affinity for the androgen receptor, but rather exert anti-androgenic effect by altering testosterone biosynthesis. Furthermore, all these results were obtained from very high administrations of DBP or MBP. The purpose of this study was to determine the onset and the site of action of relatively low concentration of MBP on steroidogenesis in vitro. The mouse Leydig tumor cells (MLTC-1) was employed as a cellular model to investigate the effect of MBP on steroidogenesis. Various concentrations of MBP (1, 10, 100 and 1000nmol/l) and its solvent dimethyl sulfoxide (DMSO) were added to the medium for 24h followed by stimulation of some compounds such as human chorionic gonadotrophin (hCG), cholera toxin (CT), forskolin, cAMP analog 8-Br-cAMP, 22(R)-hydroxycholesterol (22R-HC) and pregnenolone. Progesterone in the medium and amounts of intracellular cAMP were measured by RIA. Expression of steroidogenic acute regulatory protein (StAR) was monitored by real-time PCR and Western blotting. The results revealed that the increases of progesterone production in the presence of hCG, CT, forskolin and 8-Br-cAMP were augmented by MBP. In contrast, the levels of intracellular cAMP exhibited no statistical significance when MLTC-1 cells were treated as above. These results implied that the site in the steroid biosynthesis pathway affected by MBP occurs after PKA activation in MLTC-1 cells. Moreover, supplementing the medium with 22R-HC and pregnenolone as progesterone precursors for P450 side chain cleavage enzyme (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD), respectively, resulted in no rise in progesterone production, making clear that MBP did not influence the P450scc and 3beta-HSD but on the rate-limiting step, cholesterol transportation into mitochondria. In fact, the above results were confirmed by the upgraded StAR expression in MBP-treated cells. These data support that MBP promotes steroid hormone production by facilitating StAR expression in MLTC-1 cells.     

On the mechanisms involved in the regulation of the cell-surface receptors for human choriogonadotropin and mouse epidermal growth factor in cultured Leydig tumor cells

The MA-10 cells are a clonal strain of mouse Leydig tumor cells that have receptors for human choriogonadotropin (hCG) and mouse epidermal growth factor (mEGF). Exposure of the cells to hCG results in a reduction in the number of surface hCG receptors, and little or no change in the number of surface mEGF receptors. On the other hand, exposure of the cells to mEGF results in a reduction in the number of both surface mEGF receptors and surface hCG receptors. In order to study these phenomena, we assumed that the number of surface receptors is determined by the rate at which receptors appear at the surface and by the rate of receptor internalization. When these rates were measured, we found that hCG and mEGF reduce their respective surface receptors by increasing the rate of receptor internalization, and that mEGF reduces the surface hCG receptors by decreasing the rate of appearance of the receptor.     

Epidermal growth factor activates steroid biosynthesis in cultured Leydig tumor cells without affecting the levels of cAMP and potentiates the activation of steroid biosynthesis by choriogonadotropin and cAMP

The studies presented herein were designed to investigate the effects of mouse epidermal growth factor (mEGF) on steroid biosynthesis in a clonal strain of cultured murine Leydig tumor cells (designated MA-10). We show that in short-term incubations (up to 8 h), mEGF activates steroid biosynthesis without affecting cAMP levels. The maximal activation of steroid biosynthesis by mEGF (about 10-fold) is, however, much lower than the maximal activation detected with human choriogonadotropin (hCG) or cAMP analogues (about 1000-fold). We also show that mEGF has two (opposing) effects on the activation of steroidogenesis by hCG. Initially, it transiently attenuates the increase in intracellular cAMP and steroid biosynthesis provoked by submaximal concentrations of hCG. At later times, however, it potentiates the stimulatory effects of submaximal concentrations of hCG on steroid biosynthesis in a synergistic fashion. Last, we show that mEGF and submaximal concentrations of cAMP analogues also activate steroidogenesis in a synergistic fashion and that the degree of synergism attained with cAMP analogues plus mEGF is much higher than that attained with hCG plus mEGF. Taken together, our results show that mEGF (i) activates steroidogenesis without affecting cAMP levels and (ii) modulates the activation of steroidogenesis by the cAMP second messenger system.     

Analysis of the developmental expression of small VCP-interacting protein and its interaction with steroidogenic acute regulatory protein in Leydig cells

Small VCP-interacting protein (SVIP) is a 9-kDa protein that is composed of 76 amino acids, and it plays a role in the endoplasmic reticulum-associated protein degradation (ERAD) pathway. Recent studies have shown that SVIP is an androgen-responsive protein and its expression is regulated by androgens. Because no data are available regarding the cellular localization and expression of SVIP in the mouse testis, where androgens are highly expressed, immunohistochemistry and western blotting were performed. In the fetal testis, we found that moderate but consistent staining of SVIP is present in the cytoplasm of Leydig cells. In prepubertal and adult life, SVIP remains present in Leydig cells as well as in the cytoplasm of some peritubular and Sertoli cells. From postnatal day 15 onward, SVIP is strongly expressed in the cytoplasm of Leydig cells.Furthermore, TM3, MA-10 Leydig and Sertoli cell lines were also used to evaluate the expression of SVIP. To identify the interacting partners, such as steroidogenic acute regulatory (STAR) protein, colocalization studies were performed by fluorescence microscopy, showing that STAR colocalized with SVIP in the adult mouse testis. The expression changes of STAR were studied by using SVIP siRNAs in Leydig cell line cultures. Depletion of SVIP resulted in decreased expression of STAR. Additionally, the number and size of lipid droplets were significantly increased in SVIP-depleted Leydig cells. Taken together, our data identify SVIP as a marker of Leydig cell lineage and as a regulator of STAR protein expression and lipid droplet status in Leydig cells.     

Interaction of thyroid hormone and steroidogenic acute regulatory (StAR) protein in the regulation of murine Leydig cell steroidogenesis

The steroidogenic acute regulatory (StAR) protein, a novel phosphoprotein, is a crucial factor involved in intramitochondrial cholesterol transportation, the rate-limiting step in steroidogenesis. The present investigations were undertaken to elucidate involvement of thyroid hormone and StAR protein in the regulation of steroidogenesis in mouse Leydig cells. Treatment of cells with triiodothyronine (T₃) coordinately augmented the levels of StAR protein, StAR mRNA, and steroid production, and these responses were progressively dependent on expression of steroidogenic factor 1 (SF-1). With regard to steroidogenesis and StAR expression, the T₃ response requires both on-going mRNA and protein synthesis. In addition, the effects of T₃ were acutely modulated at the steroidogenic machinery and luteinizing hormone receptor (LHR) function, while these levels were suppressed following longer periods of exposure to T₃. Furthermore, the inhibition of SF-1 expression by DAX-1 markedly abolished T₃-mediated StAR expression in a time frame, which was consistent with decreased steroid biosynthesis. Specific involvement of SF-1 was further confirmed by assessing the 5′-flanking region of the mouse StAR gene, which identified a region between −254 and −110 bp that was essential for T₃ function. Importantly, it was found that the SF-1 binding site at position −135 bp of the 5′-flanking region was greatly involved in T₃-mediated reporter activity. Electrophoretic mobility shift assays (EMSA) also demonstrated involvement of SF-1 in T₃ function. The relevance of T₃-mediated LHR function was investigated in mice rendered hypo-and hyperthyroid, which accounted for up-regulation in the former and down-regulation in the latter group, respectively. These findings demonstrate a key role of thyroid hormone in maintaining mouse Leydig cell function, where thyroid hormone and StAR protein coordinately regulate steroid hormone biosynthesis.     

Effect of different steroidogenic stimuli on protein phosphorylation and steroidogenesis in MA-10 mouse Leydig tumor cells.

Numerous studies have indicated that treatment of Leydig cells with gonadotropin results in increased levels of intracellular cAMP, binding of cAMP to and activation of protein kinase A, phosphorylation of proteins, synthesis of new proteins and eventually, stimulation of steroidogenesis. In addition, recent studies have indicated that protein phosphorylation is an indispensable event in the production of steroids in response to hormone stimulation in adrenal cells. Because of the important role of phosphorylation in steroidogenic regulation, we investigated the effects of human chorionic gonadotropin (hCG), dibutyryl cyclic AMP (dbcAMP), forskolin and the phorbol ester, phorbol-12-myristate 13-acetate (PMA) on protein phosphorylation in MA-10 mouse Leydig tumor cells. Cells were stimulated with different steroidogenic compounds in the presence of [32P]orthophosphoric acid for 2 h and phosphoproteins analyzed by two-dimensional polyacrylamide gel-electrophoresis (PAGE). Results demonstrated an increase in the phosphorylation of four proteins (22 kDa, pI 5.9; 24 kDa, pI 6.7 and 30 kDa, pI 6.3 and 6.5) in response to 34 ng/ml hCG, 1 mM dbcAMP and 100 microM forskolin. Conversely, treatment of cells with PMA increased the phosphorylation of only one of these proteins (30 kDa, pI 6.3). At least two of these proteins (30 kDa, pI 6.5 and 6.3) appear to be identical to proteins which we and others have shown to be synthesized in response to trophic hormone stimulation in adrenal, luteal and Leydig cells. In addition, they also appear to be identical to adrenal cell mitochondrial proteins demonstrated to be phosphorylated in response to ACTH. These data indicate that proteins similar to those phosphorylated in adrenal cells in response to ACTH are phosphorylated in hormone stimulated testicular Leydig cells and that these proteins may be involved in steroidogenic regulation.     

Galectin-1: biphasic growth regulation of Leydig tumor cells

Galectin-1 (Gal-1) is a widely expressed beta-galactoside-binding protein that exerts pleiotropic biological functions. To gain insight into the potential role of Gal-1 as a novel modulator of Leydig cells, we investigated its effect on the growth and death of MA-10 tumor Leydig cells. In this study, we identified cytoplasmic Gal-1 expression in these tumor cells by cytofluorometry. DNA fragmentation, caspase-3, -8, and -9 activation, loss of mitochondrial membrane potential (DeltaPsim), cytochrome c (Cyt c) release, and FasL expression suggested that relatively high concentrations of exogenously added recombinant Gal-1 (rGal-1) induced apoptosis by the mitochondrial and death receptor pathways. These pathways were independently activated, as the presence of the inhibitor of caspase-8 or -9 only partially prevented Gal-1-effect. On the contrary, low concentrations of Gal-1 significantly promoted cell proliferation, without inducing cell death. Importantly, the presence of the disaccharide lactose prevented Gal-1 effects, suggesting the involvement of the carbohydrate recognition domain (CRD). This study provides strong evidence that Gal-1 is a novel biphasic regulator of Leydig tumor cell number, suggesting a novel role for Gal-1 in the reproductive physiopathology.     

Inhibition of gonadotropin-responsive adenylate cyclase in MA-10 Leydig tumor cells by epidermal growth factor

We have previously shown that mouse epidermal growth factor (mEGF) attenuates the increase in intracellular cAMP provoked by human choriogonadotropin (hCG) in MA-10 Leydig tumor cells (Ascoli, M., Euffa, J., and Segaloff, D. L. (1987) J. Biol. Chem. 262, 9196-9203). The studies presented herein were designed to investigate the mechanism(s) responsible for this phenomenon. We show that mEGF attenuates the increase in cAMP accumulation provoked by hCG primarily, if not entirely, by inhibiting adenylate cyclase activity. This phenomenon has some specificity for the agonist used, but it is not cell-specific. Thus, mEGF inhibited hCG-activated adenylate cyclase in MA-10 cells and in rat luteal cells but had no effect on the forskolin-activated enzyme in MA-10 cells or the isoproterenol-activated enzyme in rat luteal cells.     

Apical epidermal growth factor receptor signaling: regulation of stretch-dependent exocytosis in bladder umbrella cells

The apical surface of polarized epithelial cells receives input from mediators, growth factors, and mechanical stimuli. How these stimuli are coordinated to regulate complex cellular functions such as polarized membrane traffic is not understood. We analyzed the requirement for growth factor signaling and mechanical stimuli in umbrella cells, which line the mucosal surface of the bladder and dynamically insert and remove apical membrane in response to stretch. We observed that stretch-stimulated exocytosis required apical epidermal growth factor (EGF) receptor activation and that activation occurred in an autocrine manner downstream of heparin-binding EGF-like growth factor precursor cleavage. Long-term changes in apical exocytosis depended on protein synthesis, which occurred upon EGF receptor-dependent activation of mitogen-activated protein kinase signaling. Our results indicate a novel physiological role for the EGF receptor that couples upstream mechanical stimuli to downstream apical EGF receptor activation that may regulate apical surface area changes during bladder filling.     

Brain-derived neurotrophic factor: A steroidogenic regulator of Leydig cells

The brain-derived neurotrophic factor (BDNF) was first recognized for its roles in the peripheral and central nervous systems, and its complex functions on mammalian organs have been extended constantly. However, to date, little is known about its effects on the male reproductive system, including the steroidogenesis of mammals. The purpose of this study was to elucidate the effects of BDNF on testosterone generation of Leydig cells and the underlying mechanisms. We found that BDNF-induced proliferation of TM3 Leydig cells via upregulation of proliferating cell nuclear antigen ( Pcna) and promoted testosterone generation as a result of upregulation of steroidogenic acute regulatory protein ( Star), 3b-hydroxysteroid dehydrogenase ( Hsd3b1), and cytochrome P450 side-chain cleavage enzyme ( Cyp11a1) both in primary Leydig cells and TM3 Leydig cells, which were all attenuated in Bdnf knockdown TM3 Leydig cells. Furthermore, the possible mechanism of testosterone synthesis was explored in TM3 Leydig cells. The results showed that BDNF enhanced extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation, and the effect was disrupted by Bdnf deletion. Moreover, PD98059, a potent selective inhibitor of ERK1/2 activation, compromised BDNF-induced testosterone generation and upregulation of Star, Hsd3b1, and Cyp11a1. The Bdnf knockdown assay, on the other hand, indicated the autocrine effect of BDNF on steroidogenesis in TM3 Leydig cells. On the basis of these results, we concluded that BDNF, acting as an autocrine factor, induced testosterone generation as a result of the upregulation of Star, Hsd3b1, and Cyp11a1 via stimulation of the ERK1/2 pathway.     

Androgenic regulation of epidermal growth factor in the mouse ventral prostate

Castration of adult male mice caused a marked reduction in the amount of immunoreactive epidermal growth factor (EGF) in the ventral prostate, and the treatment of such castrated mice with testosterone increased the EGF level significantly. Gel filtration of prostate extract showed that the immunoreactive EGF in the prostate had the same molecular weight (6,045) as the submandibular gland EGF. Moreover, its isoelectric point (pH 4.5) was almost similar to that (pH 4.55) of the submandibular gland peptide. These results suggest that under the control of androgens, mouse ventral prostate synthesizes EGF structurally and functionally identical to the submandibular gland EGF.