Effect of Hypoxia on the Release of Testosterone and Vascular Endothelial Growth Factor in Mouse TM3 Leydig cells

s Previous studies indicated that intermittent hypoxia resulted in an enhancement of plasma testosterone, increased response of Leydig cells to human chorionic gonadotropin (hCG), and angiogenesis in rat testes. Hypoxia has been shown to stimulate the expression of vascular endothelial growth factor (VEGF), which is a major mediator for angiogenesis and vasculogenesis. During hypoxia, VEGF promotes the angiogenesis in the testis. However, the effect of VEGF on the steroidogenesis of testosterone in Leydig cells is not clear. A mouse TM3 Leydig cell line has been used as a research model. Under hypoxic condition, the Leydig cells were incubated in an incubator chamber (95 % N2, 5 % CO2) for 1~24 hours. The cultured media were collected and assayed for testosterone by radioimmunoassay (RIA) and for VEGF by enzyme immunoassay (EIA). The cytosolic and nuclear proteins were extracted and intracellular protein expression was determined by Western blot. MTT test was used for detecting the proliferation of Leydig cells. The present data showed that the proliferation of Leydig cells was enhanced significantly under hypoxia condition. During hypoxia, administration of hCG or VEGF could stimulate proliferation of Leydig cells, but the stimulatory effect was abolished by the administration of anti-VEGF antibody. The basal release of VEGF was increased, and the response of VEGF production to hCG was also enhanced in hypoxic condition. Furthermore, induced expression of hypoxia inducible factor-1 α (HIF-1α) in Leydig cells resulted in an increase of VEGF release. Hypoxia failed to cause an increase of testosterone release in Leydig cells. Expression of phospho-extracellular signal-regulated kinase 1 and 2 (P-ERK1/2) was enhanced in response to hypoxia or hCG treatment. PD98059 (an inhibitor of MEK) inhibited the hCG or hypoxia-induced VEGF release and diminished the hCG-stimulated testosterone release. Meanwhile, the interaction between VEGF and testosterone were also investigated. Testosterone did not affect VEGF release in Leydig cells, but higher dose of VEGF could stimulate testosterone release in a dose dependent manner. Administration of anti-VEGF antibody abolished the stimulatory effect of VEGF on testosterone release. Expression of P-ERK1/2 was enhanced after treatment with VEGF. PD98059 inhibited VEGF-induced P-ERK1/2 expression. These data demonstrated that the enhancement of testosterone release during hypoxia was resulted from proliferation of Leydig cells by an increase of VEGF generation. Apparently, VEGF plays an important role in regulating steroidogenesis of testosterone in Leydig cells during hypoxia. Introduction Oxygen is essential for life in human and other mammals. Insufficient oxygen, hypoxia, in tissue or cell has profound physiological and pathological responses. Cellular hypoxia causes an induction of hypoxia-response genes, relate to the angiogenesis, oxygen transport and metabolism and so on (Adrian et al., 2001). Vascular endothelial growth factor (VEGF), one of the genes induced by hypoxia, is a key regulator for angiogenesis and vascular formation in vascular endothelial cells. VEGF was a homodimeric glycopepetide, had been characterized as a potential growth factor in angiogenesis of endothelial cells (Gospodarowicz et al., 1989). This growth factor was also called a vascular permeability factor (VPF), because of a 5,000 times permeability than histamine. It was been studied that, five human VEGF isoforms (VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206) have been characterized (Houck et al., 1991). VEGF121, VEGF145, and VEGF165 were considered as secreted VEGF isoforms, induced proliferation of endothelial cells mediated by VEGF receptor. Different type of VEGF receptor also been identified. VEGFR-1 (Flt-1) is thought to be a negative role in angiogenesis (Gille et al., 2000), but VEGFR-2 (Flk-1/KDR) is the main mediator of the mitogenic and angiogenenic effects of VEGF (Ferrara, 1999). It is well documented that, several transcription factors were involved in the response to hypoxia. For instance, Ap-1, NF-κB and HIF-1 have a potential role in induction of hypoxia-response genes (Faller et al., 1999). Hypoxia-inducible factor-1 (HIF-1) is a heterodimer that composed of the α and β subunit, also known as hypoxic response factor and aryl hydrocarbon receptor nuclear translocator (ARNT), respectively. In the hypoxic conditions, the transcriptional activity of HIF-1 is elevated and the expressions of hypoxia-response genes were also activated by HIF-1 through hypoxia-response elements (HREs) of genes, such as VEGF and erythropoietin (Epo). Previous studies have shown that, in hypoxic cells the stability of HIF-1α was regulated by Von Hippel-Lindau (VHL) protein mediated pathway (Adrian et al., 2001), and the activity might also be enhanced by p42/p44 MAPK mediated pathway (Edurne et al., 2000). Luteinzing hormone (LH) and follicle-stimulating hormone (FSH), released from anterior pituitary, were well known major regulators of testicular functions in male. Apart from these endocrine effectors, autocrine and paracrine control of Leydig cells has been studied in stimulatory or inhibitory role in steroidogenesis. Lejeune et al. showed that certain growth factors possessed positive (IGF-1, inhibin, activin) or negative (TGF-β, TGF-α/EGF, bFGF) regulation of LH/hCG receptor number and mRNA and steroidogenic enzyme mRNA and activites, to altered the responsiveness to LH in the immature porcine Leydig cells (Lejeune et al., 1996). Our studies have previously shown that intermittent hypoxia (12 % O2, 88 % N2) of male rats for 7~14 days caused an increase of plasma testosterone levels. Angiogenesis and vasodilation were observed in rat testicular tissues after intermittent hypoxia. It is plausible to postulate that the increase of plasma testosterone levels may result from certain angiogeneis-related growth factors, which were triggered by hypoxia. Therefore, in present study, using mouse Leydig cells, we investigated the effects of hypoxia on (a) cell proliferation, (b) release of testosterone and VEGF, and (c) basic cellular regulatory pathway involved. Materials and Methods Cell culture TM3 Leydig cells, a non-tumorogenic cell line derived from mouse testis, were obtained from the Culture Collection and Research Center (Food Industry Research and Development Institute, Taiwan, Republic of China). This cell line responds to luteinzing hormone (LH) by increasing testosterone production and secretion, through mechanisms similar to those encountered in freshly isolated cells. Cells were cultured in 1:1 mixture of Ham-12 and Dulbecco’s MEM (Sigma, St. Louis, MO, USA), containing 15 mM HEPES, 0.12 % NaHCO3 and supplemented with 0.45 % glucose, 5 % horse serum, 2.5 % fetal calf serum (Kibbutz Beit, Haemek, Israel) and 100 IU/ml potassium penicillin G + 100 μg/ml streptomycin sulfate (Sigma, St. Louis, MO, USA), in 75 cm flasks (Falcon, Franklin Lakes, NJ, USA). Cells were cultured at 37°C in humidified atmosphere of normoxic conditions (95 % Air and 5 % CO2) or placed in a modular incubator chamber (Billups-Rothenberg, NY, USA), flushed with hypoxic gas (95 % N2, and 5 % CO2). Effect of hypoxia on basal or hCG-stimulated cell proliferation in mouse TM3 Leydig cells Mouse TM3 Leydig cells (2,000 cells 200 μl) were preincubated for 48 hours in 96-well plates and then incubated for 1~16 hours with or without hCG at 1 IU/ml in normoxic or hypoxic condition. To test the role of hypoxia on VEGF-stimulated cell proliferation in TM3 Leydig cells, the cells were incubated with VEGF (5~20 ng/ml) in the hypoxic conditions. Moreover, to determine the proliferated effect in Leydig cells through VEGF receptor, the cells were incubated with hCG at 1 IU/ml or VEGF at 5 ng/ml in the presence or absence of anti-VEGF antibody at 0.1 μg/ml. At the end of incubation, the cells were performed to MTT assay for cell proliferation assessment. Effectr of hypoxia on basal and hCG-stimulated VEGF and testosterone secretion in mouse TM3 Leydig cells After preincubation for 48 hours in 12-well plates, the cells (10 cells ml) were incubated for 1~16 hours with or without hCG at 1 IU/ml in normoxic or hypoxic condition. At the end of incubation, the cultured medium was collected and stored at -20°C until analyzed for VEGF by ELISA and testodterone for ELISA. Furthermore, the cells (10 cells 10 ml) were seeded in 10 cm dishes (Falcon, Franklin Lakes, NJ, USA), and then incubated in normoxic or hypoxic condition in presence or absence of hCG at 1 IU/ml. After the treatments, the cells were collected, and the nuclear and cytoplasmic extracts were determined for protein expression by Western blot. Role of ERK1/2 on VEGF and testosterone release in mouse TM3 Leydig cells Mouse TM3 Leydig cells (10 cells 10 ml) were seeded in 10 cm dishes, and then incubated with or without hCG at 1 IU/ml for 1~16 hours in normoxic or hypoxic condition. At the end of incubation, the cells were extracted for cytoplasmic protein to determine ERK1/2 expression by Western blot. Furthermore, the cells (10 cells ml) were seeded in 12-well plates, and then incubated with or without hCG at 1 IU/ml plus 50 μM PD98059 (an inhibitor of MEK) or not in normoxic or hypoxic condition. The medium was collected and stored at -20°C until analyzed for VEGF by ELISA and for tstosterone by RIA. Effect of VEGF on testosterone release and ERK1/2 expression in mouse TM3 Leydig cells To ascertain the dose-dependent effect of VEGF, the cells (10 cells ml) were seeded in 12-well plates, and then incubated with VEGF (5~20 ng/ml) or not. The medium was collected and stored at -20°C until analyzed for tstosterone by RIA. Furthermore, the cells (10 cells 10 ml) were seeded in 10 cm dishes, and then incubated with or without hCG at 1 IU/ml, VEGF at 20 ng/ml in the presence or absence of PD98059 at 50 μM. At the end of incubation, the cells were extracted for cytoplasmic protein to determine ERK1/2 expression by Western blot. Antibodies and reagents Human chorionic gonadotropin (hCG) and mouse vascular endothelial growth factor (mVEGF) were purchased from Sigma Co. (Sigma, St. Louis, MO, USA). PD98059 was purchased from TOCRIS Cooksoon Co.. The first antibody, anti-HIF-1α (1:500 dilution, Cayman Chemicals) and anti-HIF-1β (1:2,000 dilution, Novus Biologicals) were used for Western blot. Other first antibody, anti-VEGF (1:200 dilution), anti-P-ERK1/2 (1:1,000 dilution), anti-β-actin (1:8,000 dilution) and anti-GAPDH (1:500 dilution) all were purchased from Santa Cruz Co.. The horseradish peroxidase-conjugated IgG, goat anti-rabbit IgG (1: 6000 dilution) and goat anti-mouse IgG (1: 8000 dilution) were purchased from ICN Pharmaceuticals, Inc. (Aurora, Ohio, U.S.A.). Cell proliferation assessment We used the modified colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay to quantified cell proliferation (Chung et al., 1999). Living cells reduced the yellow MTT to blue formazen, which was soluble in dimethyl sulfoxide (Wako, Osaka, Japan). In the culture medium, the intensity of blue staining was proportional to the number of cell alive at analysis. Described briefly, cells were incubated in 96-well microplates (Falcon, Franklin Lakes, NJ, USA) for 24 hours. Cells were plated at 2,000 cells per 200 μl per well with medium supplemented with 7.5 % serum. The culture medium was removed and replaced by serum free medium. Incubated for 24 hours, the culture medium was replaced again by serum free medium containing various drugs. After the treatments, the medium was removed and replaced by 50 μl 1 mg/ml MTT solution (Sigma, St. Louis, MO, USA). After a further 4-hour incubation period, the MTT solution was removed and replaced by 50 μl dimethyl sulfoxide and plates were shaken for 3 min. The optical density of each well was determined using microplate reader (Dynatech Laboratories, Chantilly, VG, USA) at a wavelength of 570 nm with a reference wavelength of 630 nm. VEGF ELISA Leydig cells were incubated under normoxic or hypoxic conditions for a period. At the end of incubation, the supernatant was collected, and VEGF levels were determined using Quantikine VEGF ELISA kit (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions. This kit specifically measures rodent VEGF164 and VEGF120 variants, and the limit of detection is 3 pg/ml. VEGF concentrations were normalized relative to cellular protein concentrations (Braford protein asssay). Testosterone RIA The concentrations of testosterone in medium were determined by RIA as described previously (Wang et al., 1994; Tsai et al., 1996). The anti-testosterone serum no. W8. was used, the sensitivity of testosterone RIA was 2 pg per assay tube. The intraand interassay coefficients of variance (CV) were 4.1 % (n = 6) and 4.7 % (n = 10), respectively. Nuclear and cytoplasmic protein extraction Nuclear and cytoplasmic protein extracts were prepared from Leydig cells using following method. Briefly, cells were scraped from cultured dishes, and harvested by centrifugation at 8,000 rpm at 4°C for 3 min. Cell pellets were resuspended in 100 μl hypotonic buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 0.1 % PMSF, 0.1 % aprotinin) in iced-bath for 5min. Followed by adding of 12.5 μl lysis buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 2.5 % NP-40), and the nuclei pelleted by centrifugation at 3,800 rpm at 4°C for 10 min. Collected the supernatants as the cytoplasmic fraction, and the nuclei pellets were resuspended in 50 μl of buffer C (20 mM HEPES, 0.45 M NaCl, 1 mM EDTA) while mixing for 20 min at 4°C followed by centrifugation at 14,000 rpm for 10 min at 4°C. The supernatants were stored at -70°C until analysis. Western blot Protein concentrations were determined by the Braford protein assay using BSA as the standared. Thirty microgram aliquots of nuclear and cytoplasmic proteins were separated by SDS-PAGE using 7.5~15 % polyacrylamide gels. Proteins were electroporetically transferred to polyvinylidene difluoride (PVDF) membranes (NEN Life Science Products, Inc., Boston, MA, USA) or nitrocellulose (NC) membrane (Schleoicher & Schuell, Inc., Keene, NH, USA) using a Trans-Blot SD semi-dry transfer cell (170-3940, Bio-Rad, Hercules, CA, USA). The membrane were washed in TBS-T buffer (0.242 % Tris-base, 0.8 % NaCl, 0.1 % Tween-20) and blocked in TBS-T containing 5 % nonfat dry milk for 2 hours at room temperature with gentle agitation. Then the membranes were incubated with first antibodies in TBS-T buffer containing 0.05 % BSA more then 16 hours at 4°C. Followed by incubated for 1 hour with horseradish peroxidase-conjugated secondary antibodies in 5 % nonfat dry milk of TBS-T buffer. The membranes were washed three times with TBS-T buffer, and then the blots were developed by enhanced chemiluminescence using ECL kit (ECL, Western blotting detection reagents, Amersham International, UK) and exposed to X-ray film. Using the computerized image analytic densitometry (Personal Densitometer, Molecular Dynamics, Sunyale, CA, USA) performed quantification of chemiluminescence signal data. Statistic analysis All data were expressed as mean ± SEM. Treatment means were tested for homogeneity using the analysis of variance (ANOVA), and the differences between the specific means were tested for the significance by Duncan's multiple range test (Steel & Torrie, 1960). The level of significance chosen was P < 0.05. Results Stimulatory effect of cell proliferation on basal or hCG-treatment under hypoxic conditions To study the effect of hypoxia on TM3 cell proliferation, cells were exposed to normoxic or hypoxic conditions for 1~16 hr. Proliferation status were assessed by MTT assay. As shown in Fig. 1, proliferation of TM3 cells was significantly increased by hCG-treatment for 16 hr in normoxic and hypoxic conditions. As expected, hypoxia caused a greater induction in TM3 cell proliferation than normoxia observed in 16 hr. Under hypoxic conditions, VEGF and hCG had more significant stimulatory effect on cell proliferation than normoxic conditions in a dose dependent manner (Fig. 2). To examine whether the VEGF receptor was involved in the induction of cell proliferation, we incubated the TM3 cell with anti-VEGF antibody. A similar inhibitory effect on cell proliferation was observed when the anti-VEGF antibody was added prior to stimulation of the cells with hCG, VEGF and hypoxic treatment (Fig. 3). Stimulation of VEGF release and expression by hCG in hypoxic conditions The effects of hypoxia on VEGF release by TM3 cells are shown in Fig. 4. After treating with hCG, the levels of medium VEGF were enhanced significantly at 4 hr in normoxia and at 2 hr in hypoxia. As stimulatory effect on TM3 cell proliferation, hypoxia also caused a significant elevation of VEGF release in basal status and hCG-treatment after 16 hr. Furthermore; to investigate whether the expression of VEGF was affected by hCG and hypoxia, the cells were incubated in normoxic or hypoxic conditions in the presence or absence of hCG. The data demonstrated that hypoxia didn’t cause an observably differential staining of VEGF in Western blots, but the hCG caused after treatment for 16 hr (Fig. 5). Previous studies had shown that HIF-1 was involved in several physiological responses to hypoxia. The transcriptional activity of HIF-1 mediated the specific genes transcription, such as the genes for glycolytic enzymes, erythropoietin, and VEGF (Adrian, 2001). As expected, HIF-1α expression was observed under hypoxic conditions, but the expression was barely detectable under normoxic conditions (Fig. 5). None of the treatments caused any change in the staining of HIF-1β, as previous knows that HIF-1β was consistent expression in the cells (Wang et al., 1995). Activation of ERK 1/2 by hCG and hypoxia in TM3 cells It had been shown that, in mouse Leydig cells, VEGF secretion could been mediated by an MEK 1/2-ERK 1/2 dependent pathway (Ravinder et al., 2003). Thus, we incubated the TM3 cells with the MEK inhibitor, PD98059, blocked the phosphorylation of ERK 1/2. Consequently, a significant altitude of enhancement in VEGF release in hypoxic conditions was abolished by PD98059 (Fig. 4). Takashi et al. had shown that hCG provokes a 2to 3-fold increase in the levels of phosphorylated ERK 1/2 in the MA-10 cells transiently transfected with an vector coding the wilid type hLHR (Takashi et al., 2002). Therefore, we tested the role of ERK 1/2 involved in testosterone production of TM3 cells. As expected, whatever in normoxic or in hypoxic conditions, PD98059 could inhibit the release of testosterone in hCG-stimulated cells (Fig. 6). Moreover, Minet et al. demonstrated that in human microvascular endothelial cells-1 (HMEC-1), ERK 1/2 were activated during hypoxia, and ERK 1 was needed for hypoxia-induced HIF-1 transcriptional activity (Minet et al., 2000). To verify the whether the expression of ERK 1/2 was affected by hypoxia in TM3 cells, we treated the cells with hCG in normoxic and hypoxic conditions. The results revealed that the expression of phospho-ERK 1/2 (P-ERK 1/2) were activated by hCG and hypoxia for 1 and 16 hr (Fig. 7). The levels of P-ERK 1/2 expression were judged by the staining of GAPDH, which consistent expression in the cells, as an internal control. VEGF increase testosterone release in TM3 cells It has been reported that some steroids, androgens, estrogens or gestagens are able to induce VEGF production in a variety of steroid-dependent cells. Johanna et al. had demonstrated that both estrogen and androgen stimulate the expression of VEGF by increasing gene expression and mRNA stability (Ruohola et al., 1999). Thus, the interaction between VEGF and testosterone were also investigated. Our results indicated that, the levels of VEGF were not altered in testosterone treatment at 10~10 M (data not shown). In a contrary, VEGF produced significant concentration dependent increase in testosterone production in TM3 cells (Fig. 8). Furthermore, administration of anti-VEGF antibody, to block the signaling through VEGF receptor, resulted in an inhibitory effect of testosterone release (Fig. 8). To further verify the MAPK signaling pathway associated with this stimulatory effect by VEGF on testosterone release, the expression of ERK 1/2 was also detected. Our results were shown in Fig. 9, the VEGF induced clear elevation of ERK 1/2 expression that was suppressed by MEK inhibitor, PD98059.