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BOR - Papers in Press, published online ahead of print November 27, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.009795
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BIOLOGY OF REPRODUCTION 68, 1663–1673 (2003)
DOI: 10.1095/biolreprod.102.009795
© 2003 by the Society for the Study of Reproduction, Inc.

Testis

Regulation of Vascular Endothelial Growth Factor Production by Leydig Cells In Vitro: The Role of Protein Kinase A and Mitogen-Activated Protein Kinase Cascade1

Ravinder Jit Kaur Anand, Hans-Joachim Paust, Klaus Altenpohl, and Amal K. Mukhopadhyay2

Institute for Hormone and Fertility Research, University of Hamburg, Hamburg, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously reported the presence of vascular endothelial growth factor (VEGF) in testicular cells, and high concentrations of VEGF have been measured in semen, although its role in male reproduction remains obscure. In the present study we focus on understanding the mechanism of VEGF production by mouse Leydig cells cultured in vitro. Production of VEGF protein in medium by testicular cells was markedly increased by the addition of hCG in a time- and dose-dependent manner. Gonadotropin-stimulated VEGF production was mediated by cAMP-dependent protein kinase A (PKA), as evidenced by the effect of hCG being mimicked by 8Br-cAMP and being abolished in the presence of a PKA-specific inhibitor, H-89. Protein kinase C was not involved, as evidenced by phorbol 12-myristate 13-acetate having no influence on VEGF production by Leydig cells. In addition to hCG, atrial natriuretic peptide was also able to stimulate VEGF production, suggesting that cGMP is able to cross-activate PKA. A specific Src kinase inhibitor, PP2, could completely block the stimulatory effects of both gonadotropin and 8Br-cAMP on VEGF production by Leydig cells, implying an involvement of the Src kinase pathway. Furthermore, addition of U0126, an inhibitor of MEK 1/2, abolished the increase in VEGF production stimulated by both hCG and 8Br-cAMP. A similar inhibitory effect was observed by the addition of SB203580, a p38 mitogen-activated protein kinase inhibitor. Thus, in conclusion, Leydig cells are able to produce VEGF by a process under gonadotropic control, and PKA plays a key role in this process. Downstream of PKA, it appears that both MEK 1/2 and Src kinase-dependent pathways are involved, although further research will be necessary to determine the precise link between PKA and other kinases involved.

angiogenesis, protein kinase, testes, testicular microcirculation, testosterone, vascular permeability


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Testicular functions are known to be primarily regulated by the hypophyseal gonadotropins, LH and FSH, although a number of testicular paracrine/autocrine factors appear to be relevant as well [1]. Apart from these endocrine, paracrine, or autocrine effectors, the testicular microcirculation can also be considered to be an important determinant for the regulation of both gametogenesis and steroidogenesis taking place in two discrete compartments of testis; namely, seminiferous tubules and Leydig cell clusters in the interstitial spaces. A free exchange of precursors, metabolites, intratesticular regulatory factors, and endocrine substances within and between these two testicular compartments is assured by a highly developed microvasculature. Maintenance of microcirculation is an important local regulatory determinant of testicular functions. In this context, it has been proposed that vascular endothelial growth factor (VEGF), which has been shown to be present in the testes of humans [2] and mice [3], may play an important role. VEGF, a homodimeric glycopeptide that is structurally related to the platelet-derived growth factor, is a potent mitogenic growth factor for endothelial cells [4–6]. The permeability potency of this growth factor is 50 000 times greater than that of histamine [7, 8], and it is, hence, also called a vascular permeability factor. That VEGF can be involved in regulating transport of important blood borne factors has been supported by a recent report showing that VEGF plays a major role in the cellular transport of blood glucose [9]. Therefore, it is conceivable that VEGF plays a pertinent role in the maintenance of testicular functions by virtue of its ability to facilitate transport of blood borne hormones and nutritional elements. Also, it appears that the expression of VEGF within the testes must be finely regulated in order to be optimally effective, because an overexpression of VEGF in the testes leads to a massive disruption of spermatogenesis, resulting in infertility [10]. It may be thus proposed that a fine regulation of VEGF production in the testes is essential in order to facilitate and maintain cross talk among the endocrine, endothelial, and gametogenic compartments of the testes. However, the underlying mechanism of the regulation of VEGF production by testicular cells remains obscure. Therefore, in this study, using Percoll-purified Leydig cells, we carried out a detailed investigation in order to understand the basic cellular regulatory pathways involved in the production of VEGF by these cells in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Human choriogonadotropin and 8Br-cAMP were obtained from Boehringer-Mannheim (Mannheim, Germany), and rat atrial natriuretic peptide (ANP) was from Saxon Biochemicals GmbH (Hannover, Germany). Phorbol 12-myristate 13-acetate (PMA), penicillin-streptomycin, collagenase, normal goat serum (NGS), and 4',6'-diamidino-2-phenylidole (DAPI) were from Sigma (Steinheim, Germany). H-89 (N-2-(p-bromocinnamyl amino) ethyl-5-isoquinolinesulfonamide·2HCl) and U0126 (bisamino (2-aminophenyl) thiomethylene butanedinitrile) were from Biomol Research Laboratories Inc. (Hamburg, Germany). PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo 3,4-d pyrimidine) was purchased from Calbiochem (San Diego, CA). Albumin fraction V (from bovine serum) was from Merck (Darmstadt, Germany). All other reagents were obtained from commercial sources and were of the highest purity grade.

Preparation and Incubation of Mouse Leydig Cells

Testes from healthy adult (3 mo of age) NMRI mice supplied by Charles River Laboratories (Sulzfeld, Germany) were decapsulated, and mouse Leydig cells were isolated and purified as described by Schumacher et al. [11]. Briefly, the decapsulated testicular mass was suspended in Dulbecco modified Eagle medium (DMEM; Sigma) without sodium bicarbonate, with L-glutamine and 15 mM HEPES containing 1.2 g/L sodium bicarbonate, 0.5 g/L BSA, and 10 ml/L penicillin-streptomycin. The testicular suspension was flushed gently through a rubber tube fitted with a syringe so as to untangle the tubules. The homogeneous solution was filtered through nylon mesh and the filtrate was centrifuged at 80 x gav for 10 min at room temperature. The decanted supernatant was layered on top of the Percoll gradient and centrifuged for 25 min at 800 x gav at room temperature. Highly purified Leydig cells were aspirated from the third band, corresponding to a Percoll concentration of 38%–52% (v/v). The sedimented cell pellet was washed twice with DMEM using a low speed centrifugation (80 x gav for 10 min at room temperature) and finally suspended in a known volume of the same medium. The cell number was determined using a Neubauer chamber. The cells were plated at a density of 50 000 and 100 000 cells per well for testosterone and VEGF quantification, respectively, in a 96-well culture plate, and cultured for various durations as indicated.

Preparation and Incubation of Rat Leydig Cells

Decapsulated testicular masses from healthy adult (3 mo) Wister rats were treated with collagenase (0.25 mg/ml DMEM) for 30 min at room temperature essentially as previously described [12]. The homogeneous suspension thus obtained was filtered through nylon mesh and the filtrate containing a crude Leydig cell suspension was obtained. The rest of the procedure to purify the Leydig cells was essentially the same as that mentioned above for isolation, purification, and culture of mouse Leydig cells.

Staining of Leydig cells for Immunofluorescence

Cells were seeded at 2 x 105 cells/well in 4-well chambered coverglass slides (Nalge Nunc International Corp., Naperville, IL). At indicated time points the culture medium was aspirated and the cells were washed twice with PBS pH 7.2. Fixation employed 4% (vol/vol) paraformaldehyde in PBS for 20 min at room temperature followed by two washings with PBS. Cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature, and a blocking solution (PBS containing 5% NGS) was added and incubated for 30 min at room temperature with gentle shaking. Following this, relaxin-like factor (RLF) antiserum raised in rabbit against the recombinant mouse RLF precursor protein [13], diluted 1:1000 in PBS containing 2% NGS, was added and incubated for 2 h at room temperature with gentle shaking. The preimmune serum from the same animal was used at the same dilution in control wells incubated similarly. The cells were then washed three times with PBS for 5 min each. Cy3-conjugated affinity-purified goat anti-rabbit immunoglobulin G (Jackson Immunoresearch Laboratories, West Grove, PA) was used at 1:100 dilution in PBS containing 2% NGS for 1 h at room temperature with gentle shaking. Slides were thereafter protected from light. Cells were then washed three times with PBS for 5 min each. Nuclear counterstaining was performed with 0.1 µg/ml DAPI in PBS for 5 min followed by washing with PBS. Cells were observed and documented using a fluorescence microscope.

Detection of VEGF Expression by Polymerase Chain Reaction Analysis

Total RNA from Percoll-purified mouse and rat Leydig cells was prepared using the RNAqueous kit (Ambion, Wiesbaden, Germany). RNA (1.5 µg) from each isolate was reverse transcribed in oligo(dT)-primed reverse transcription reactions with superscript reverse transcriptase provided by Invitrogen (Karlsruhe, Germany) according to the manufacturer's instructions. Aliquots (2 µl) of reverse-transcribed cDNAs were used to amplify mRNA sequences coding VEGF isoforms. Primers were designed according to the published sequence for VEGF alternatively spliced isoforms VEGF-188, VEGF-164, and VEGF-120 (GenBank accession numbers NM031836, AF260425, and AF215726, respectively): sense primer 5'-GCACCCACGACAGAAGGG GAG-3' and antisense primer 5'-TCACCGCCTTGGCTTGTCACA-3'. The expected sizes of amplified VEGF isoforms are 569 base pairs (bp), 497 bp, and 365 bp for the three respective VEGF isoforms. Polymerase chain reaction assays (50 µl) were performed with 0.2 units of HotStarTaq DNA polymerase (Qiagen, Hilden, Germany) with a compatible buffer recommended by the manufacturer in the presence of 100 pmol of each primer. The first denaturation was performed for 15 min at 95°C (step 1). The successive denaturation steps were performed for 30 sec at 95°C, followed by annealing at 50°C for 1 min and extension at 72°C for 1 min (step 2). The second step consisted of 30 cycles. A final elongation for 10 min at 72°C was used to amplify transcripts. The amplified cDNA fragments were size-fractionated in 2% (wt/v) agarose gels and visualized by staining with ethidium bromide (0.2 ng/ml).

Quantitation of VEGF

Percoll-purified Leydig cells were cultured for indicated duration in the absence or presence of an addition of test compounds. At the end of incubation, the medium was aspirated, and VEGF concentration was determined using a 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 of VEGF per milliliter. According to the information provided by the manufacturer, this immunoassay has been calibrated against a highly purified Sf-21-expressed recombinant mouse VEGF.

Quantitation of Testosterone

In order to determine testosterone produced in the medium, the incubation was stopped by the addition of absolute ethanol (final alcohol concentration approximately 80%) followed by vortex mixing. The ethanol extract was evaporated to dryness, the residue was dissolved in 1 ml of modified essential medium containing 0.1% sodium azide, and the content of testosterone was measured with a specific ELISA based essentially on a radioimmunoassay procedure described previously [12, 14].

In Vitro Kinase Assay for cAMP-Dependent Protein Kinase Activity

Purified Leydig cells were washed twice with PBS (pH 7.4) and the cell pellets were stored at -80°C until further use. Leydig cell lysates for PKA activity measurement were prepared by suspending the cells in cold extraction buffer (pH 7.4) containing 25 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM mercaptoethanol, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 0.05% BSA, and then homogenized using a Dounce homogenizer. Cell lysates were centrifuged, and the supernatant was used in a nonradioactive assay for PKA (PKA Assay System, Promega, Madison, WI). On average, 2 x 106 cells/ml were used for each data point. Phosphorylated and nonphosphorylated forms of the PKA substrate peptide were separated by agarose gel electrophoresis and imaged with UV light.

Statistical Analysis

Data were analyzed using ANOVA with a post hoc Neuman-Keuls test or the Student t-test, whichever was applicable. Significance was set at P < 0.05, P < 0.01, or P < 0.001. In the figures, values are presented as means ± SEM, and n refers to the number of observations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PCR analysis using specific primers listed in Materials and Methods, and cDNA obtained by reverse transcription of RNA isolated from Percoll-purified Leydig cells revealed (Fig. 1) amplification of three fragments of 569 bp, 497 bp, and 365 bp, corresponding to the three splice variants of VEGF transcripts for VEGF188, VEGF164, VEGF120, respectively. It is noteworthy that in both rats and mice, VEGF164 appears to be predominantly expressed, whereas the expression of VEGF188 can be considered as minor.



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  		FIG. 1. Detection of VEGF expression by RT-PCR analysis. Expression of VEGF was analyzed at the mRNA level by RT-PCR. PCR products were size-fractionated on agarose gel, stained with ethidium bromide, and visualized with UV light. In the negative control (C), in which cDNA was substituted with water, no amplified DNA was detected. Transcripts coding VEGF isoforms in isolated Leydig cells from mouse (mLC) and rat (rLC) are indicated. Lane M represents markers, and sizes are indicated. The size of amplified products corresponding to different VEGF isoforms are indicated on the right-hand side

In the next experiment, Leydig cells isolated from the testes of rats (Fig. 2A) and mice (Fig. 2B) were cultured in the absence of any addition or in the presence of either hCG or 8Br-cAMP, and the amounts VEGF protein secreted into the culture media were measured with the help of a specific VEGF ELISA. There was a basal production of VEGF from the unstimulated cells. However, when the cells were stimulated with hCG or 8Br-cAMP, there was a significant increase in the amount of VEGF measured.



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  		FIG. 2. VEGF production by rat and mouse Leydig cells in response to hCG and 8Br-cAMP. Percoll-purified Leydig cells were cultured in the absence of any addition or in the presence of 5 ng/ml hCG or 1 mM 8Br-cAMP. After 20 h if incubation the medium was aspirated and VEGF concentration was determined with a specific ELISA. Each data point represents the mean ± SEM (n = 3). One representative experiment out of three is presented. Values are significant, *P < 0.05 compared with basal values

Having demonstrated that Leydig cells from both rats and mice can produce VEGF when stimulated with gonadotropin and its second messenger analogue, 8Br-cAMP, further experiments to analyze the underlying mechanism were conducted only with mouse Leydig cells. The time course experiment using mouse Leydig cells depicted in Figure 3 shows that only a minor increase in the amounts of VEGF measured in the medium of unstimulated cells could be observed over various durations of culture. In contrast, stimulation with either hCG or 8Br-cAMP resulted in a robust time-dependent increase in the amounts of VEGF measured.



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  		FIG. 3. Time course of VEGF production by mouse Leydig cells in response to hCG and 8Br-cAMP. Percoll-purified Leydig cells were cultured for the indicated duration in the absence of any addition or in the presence of 5 ng/ml hCG or 1 mM 8Br-cAMP. At the end of incubation, the medium was aspirated and VEGF concentration was determined with a specific ELISA. Each data point represents the mean ± SEM (n = 3). One representative experiment out of three is presented

Because ANP, like gonadotropin, is known [12, 14] to stimulate steroidogenesis in mouse Leydig cells, it was of interest to determine whether ANP is also able to stimulate VEGF production. The data in Figure 4 clearly show that ANP at a concentration of 100 nM was able to stimulate VEGF production to an extent comparable to that of hCG. ANP was not effective when used at a concentration of 10 nM.



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  		FIG. 4. Effect of ANP on VEGF production by mouse Leydig cells. Percoll-purified Leydig cells were cultured for 20 h in the absence of any addition or in the presence of 10 and 100 nM ANP, 5 ng/ml hCG, or 1 mM 8Br-cAMP. At the end of incubation, the medium was aspirated and VEGF concentration was determined with a specific ELISA. The data show the mean ± SEM of three independent observations. Values are significant, *P < 0.05 compared with basal values

Because all three agonists (hCG, ANP, and 8Br-cAMP) can stimulate testosterone production in these cells as well, it was of interest to discern whether or not the stimulation of VEGF production in response to these agonists was dependent on steroidogenesis. Our earlier studies demonstrated a loss of steroidogenic capacity of Leydig cells when cultured over a period beyond 2 days. Therefore, the cells were cultured for several days and were stimulated with 1 mM 8Br-cAMP every day for a period of 20 h for VEGF measurement or for a period of 3 h when steroid production was measured. Because testosterone production is a relatively rapid event following hormonal stimulation and is linear up to 10–12 h (unpublished data), we chose an incubation period of 3 h for experiments in which this steroid was measured. For VEGF production, we chose 20 h to allow the accumulation of sufficient proteins in the medium for detection in the assay. It is obvious from Figure 5A that the VEGF response of the Leydig cells to 8Br-cAMP remained unaffected over the entire period of culture. In contrast, a robust stimulation (Fig. 5B) of testosterone production in response to 8Br-cAMP was observed only at Day 0 (the day of preparation and plating of the cells) and Day 1. The steroidogenic response of the cells diminished markedly on Day 2 and was completely abolished thereafter. Thus, it is clear that the process of induction of VEGF production by Leydig cells is independent of steroidogenesis, because stimulation of VEGF production was retained even under conditions in which a total loss of steroidogenic response occurred. It is possible to argue that during the period of culture the Leydig cells may have disappeared and that some other cells, which were present as an impurity in the preparation, proliferated and took over. Therefore, we immunostained the cells with a specific antibody against RLF, a specific marker for adult-type Leydig cells [15], on the day of plating and on the final day of culture. The data presented in Figure 5C clearly show that on the day of plating (Day 0), more than 95% of cells stained positive for RLF based on comparison with DAPI counterstained cells, indicating that the Leydig cell preparation we used was highly pure. This relative abundance of Leydig cells also remain unchanged on Day 4 of culture.



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  		FIG. 5. VEGF production by Leydig cells cultured for a longer duration. Mouse Leydig cells were cultured for a period of 4 days, Day 0 being the day when cells were isolated and plated. The cells were stimulated on indicated days of culture with 1 mM 8Br-cAMP and the amounts of VEGF after 20 h of stimulation (A) or testosterone after 3 h of stimulation (B) produced in the culture medium were measured using specific ELISAs. Culture medium was replaced each day in the unstimulated cells in culture. The data show mean ± SEM (n = 3). Values are significant, *P < 0.001 compared with basal values. The immunostaining for the Leydig cell-specific marker RLF is presented in (C). The cells stained on the day of preparation and plating are depicted in a0, b0, and c0, and those cultured until Day 4 and stained thereafter are depicted in a4, b4, and c4. Panels a0 and a4 show control immunostaining in which preimmune serum from the same rabbit was used; b0 and b4 show immunostaining using a specific antibody for RLF, and c0 and c4 show nuclear counterstaining of cells with DAPI. Original magnification x70

Because 8Br-cAMP could mimic the effect of hCG, it appears conceivable that the PKA pathway is involved. Therefore, we examined the effect of the addition of varying concentrations of a specific PKA inhibitor, H-89, on VEGF (Fig. 6A) production in cells cultured for 20 h in the absence or in the presence of hCG or 8Br-cAMP. Although basal VEGF production in the absence of an agonist was affected only to a minor extent, the effect of hCG/8Br-cAMP was dose-dependently inhibited by H-89. At 30 µM, H-89 was able to completely abolish the agonist-stimulated VEGF production. In a parallel experiment (Fig. 6B), agonist-stimulated testosterone production was also inhibited by H-89 in a dose-dependent manner, as anticipated. Thus, the gonadotropin-induced VEGF production in Leydig cells depends on the activation of PKA.



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  		FIG. 6. Effect of the cAMP-dependent protein kinase inhibitor H-89 on VEGF (A) and testosterone (B) production by mouse Leydig cells. Mouse Leydig cells were preincubated in the absence or presence of various concentrations of H-89 for 1 h. This was followed by culturing the cells with or without 5 ng/ml hCG or 1mM 8Br-cAMP for a period of 20 or 3 h, respectively. At the end of the incubation, the culture medium was aspirated out of the wells and the amounts of VEGF produced were measured using a specific ELISA. In the experiment in which testosterone was measured, incubation was terminated with alcohol (final alcohol concentration approximately 80%) following no addition or addition of hCG or 8Br-cAMP after 3 h, and the amount of steroid produced was measured with a specific ELISA. Each data point represents the mean ± SEM (n = 3).

After establishing the role of cAMP-dependent protein kinase in gonadotropin-stimulated VEGF production by Leydig cells, it was of interest to determine whether protein kinase C (PKC) has any role to play in this process as well. Treatment of the cells with PMA, a PKC activator, which is known to reduce steroidogenesis in mouse Leydig cells [16], did not affect the amount of VEGF produced in cells stimulated with either hCG or 8Br-cAMP (Fig. 7). Therefore, PKA but not PKC, appears to play a role in the regulation VEGF formation in Leydig cells.



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  		FIG. 7. Action of the PKC activator, PMA, on mouse Leydig cells. Leydig cells were preincubated for 1 h with or without 0.2 M PMA before incubation without or with the addition of 5 ng/ml hCG or 1 mM 8Br-cAMP. After 20 h the culture medium was aspirated and the VEGF content in the medium was measured using a specific ELISA. The data show the mean ± SEM of three independent observations

Recently, Fredriksson et al. [17] have proposed that beta-adrenergic regulation of VEGF expression in brown adipocytes of mice is mediated through a PKA pathway that also requires activation of the Src kinase pathway. Therefore, we wondered whether this kinase could also be involved in Leydig cells. We used a specific Src tyrosine kinase inhibitor, PP2, and report (Fig. 8) that this inhibitor is able to block VEGF formation induced by both gonadotropin and 8Br-cAMP, but that it has no effect on basal release of VEGF.



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  		FIG. 8. Effect of the Src tyrosine kinase inhibitor PP2 on VEGF production by mouse Leydig cells. Percoll-purified Leydig cells were preincubated for 1 h in the absence or presence of 50 µM PP2. This was followed by no addition or addition of 5 ng/ml hCG or 1 mM 8Br-cAMP. After 20 h the culture medium was aspirated out of the wells (with no cells) and the amounts of VEGF produced were measured using a specific ELISA. The data show mean ± SEM of three independent observations. Values are significant, *P < 0.01 compared with the sample not treated with PP2.

Having demonstrated a role for Src kinase in the regulation of VEGF production by Leydig cells, the question naturally arises as to a possible involvement in this process of the components of the mitogen-activated protein (MAP) kinase cascade, particularly that of Erk 1/2 and p38 MAP kinase. Accordingly, we first examined the effect of the inhibitor U0126, which is specific for Erk 1/2, on VEGF production by Leydig cells. Addition of 25 µM U0126 abolished (Fig. 9) both gonadotropin and 8Br-cAMP stimulated increases in VEGF production. Basal production of VEGF remained unaffected. Thus the important role played by MEK 1/2 was underscored in the gonadotropin and cAMP-stimulated VEGF production by Leydig cells. Furthermore, SB203580, a p38 MAP kinase inhibitor, at concentrations of 30 and 60 µM, could also significantly inhibit hCG and 8Br-cAMP stimulated VEGF production by Leydig cells (Fig. 10).



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  		FIG. 9. Effect of the MEK 1/2 inhibitor U0126 on VEGF production by mouse Leydig cells. The cells were preincubated with U0126 for 1 h before incubation without any addition (basal) or with 5 ng/ml hCG or 1 mM 8Br-cAMP. After 20 h of incubation the culture medium was aspirated and the amount of VEGF secreted was measured using a specific ELISA. One representative experiment out of three is presented. Values are significant, *P < 0.01 compared with the sample not treated with U0126



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  		FIG. 10. Effect of the specific p38 MAP kinase inhibitor SB203580 on VEGF production by mouse Leydig cells. Percoll-purified Leydig cells were preincubated for 1 h in the absence or presence of 30 and 60 µM SB203580. This was followed by incubation of the cells without or with 5 ng/ml hCG or 1 mM 8Br-cAMP. After 20 h the culture medium was aspirated out of the wells and the VEGF produced was measured using a specific ELISA. The data show the mean ± SEM of three independent observations. One representative experiment out of three is presented. Values are significant, *P < 0.001 compared with the sample not treated with SB203580

Finally, we carried out a protein phosphorylation assay specific for PKA in the presence of all the kinase inhibitors employed in this study in order to exclude the possibility that some of the inhibitors used other than H-89 might also act at the level of PKA. As shown in Figure 11, the control (lane 1) with no source of kinase added, was completely devoid of any phosphorylated substrate, but the addition of a catalytic subunit of PKA (lane 2) resulted in a robust phosphorylation of the substrate. The addition of Leydig cell lysate alone (lane 3) resulted in a weak phosphorylation of the substrate, but when the cell lysate was added together with the activator cAMP, a marked phosphorylation of the substrate was observed by the appearance of a strong band of phosphorylated substrate (lane 4). This phosphorylation was almost completely abolished by the addition of 30 µM H-89 (lane 6), but not by 25 µM U0126 (lane 7), 50 µM PP2 (lane 8), or 60 µM SB203580 (lane 9), clearly indicating that except for H-89, no other inhibitor we used had any effect on cAMP-stimulated PKA activity in the Leydig cell lysate.



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  		FIG. 11. Specificity of inhibitors used for cAMP-dependent protein kinase activity in the lysate prepared from Leydig cells. Cell lysate (2 x 106 Leydig cells/ml) or the catalytic subunit of PKA was incubated with the fluorescent-labeled substrate peptide provided in the kit for 30 min at 22°C in a water bath in the presence or absence of cAMP (activator). Inhibitors were added where indicated. The reaction was terminated by heating the samples to 95°C for 10 min, cooled on ice-water, and 1 µl of 80% glycerin in 25 mM Tris-HCl buffer (pH 8.0) was added to each sample. An aliquot of the reaction mixture was loaded onto an 0.8% agarose gel. After 15–25 min the phosphorylated substrate carrying a net negative charge migrated toward the anode, whereas the nonphosphorylated substrate carrying the net positive charge moved toward the cathode. Electrophoretically separated phosphorylated from nonphosphorylated substrates were visualized through a UV transilluminator. Lane 1 shows the control with no source of PKA; lane 2, active catalytic subunit of PKA; lane 3, Leydig cell lysate without cAMP (activator); lane 4, Leydig cell lysate; lane 5, PKA plus 30 µM H-89; lane 6, Leydig cell lysate plus 30 µM H-89; lane 7, Leydig cell lysate plus 25 µM U0126; lane 8, Leydig cell lysate plus 50 µM PP2; lane 9, Leydig cell lysate plus 60 µM SB203580. Note that except for the sample in lane 3, all samples were supplemented with the activator (cAMP)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The main findings of this study are 1) that VEGF is expressed in both mouse and rat Leydig cells as evidenced by reverse transcription-PCR analysis, 2) that its production by Leydig cells could be stimulated by gonadotropin, 3) that cAMP-dependent protein kinase plays a key role in the gonadotropin-dependent increase in VEGF production, and 4) that greater VEGF production in response to activated PKA pathway is mediated by Src kinase and MAP kinase pathways involving an activation of MEK 1/2, Erk 1/2, and p38 MAP kinase.

The primary function of Leydig cells is to produce testosterone, which is responsible for the maintenance of spermatogenesis and secondary sexual characteristics. Testosterone production is mainly under LH control [1]. LH acts by binding to the cell surface seven-transmembrane G protein-coupled receptors and activating adenylate cyclase, leading to the production of cAMP, which in turn activates PKA.

Ergün et al. [2] showed that the Leydig and Sertoli cells of human testis are a source of VEGF and that both receptors specific for this growth factor, flt-1 and KDR (flk-1 in mice), were localized within the testis. In this study we demonstrated that primary cultures of mouse Leydig cells secrete an appreciable amount of VEGF and provide evidence for the first time that the rodent Leydig cells produce greater quantities of VEGF in response to gonadotropic stimulation. The action of LH was mimicked by 8Br-cAMP, augmenting the response of VEGF production, suggesting that the gonadotropin-mediated stimulation of VEGF production is dependent on the formation of second-messenger cAMP. Previously, in several cell types, forskolin analogues, cAMP analogues, or both have been used to demonstrate the induction of VEGF expression [18–28]. Our results agree well with the mechanism proposed in these published reports [19–22].

Because both steroidogenesis and VEGF production appear to be dependent on gonadotropin-stimulated cAMP formation, it is plausible to postulate that the increased steroid formation by Leydig cells in response to gonadotropic stimulation may be the trigger for the induction of VEGF. In a variety of steroid-dependent cells (e.g., in prostatic cells [29, 30], uterine cells [31, 32], and breast cancer cells [33]), steroids such as androgens, estrogens, or gestagens are able to induce VEGF production. However, in Leydig cells, ongoing steroidogenesis is not a prerequisite for the stimulation of VEGF production in response to the gonadotropin. Culturing of mouse Leydig cells over a period of 3 days leads to a complete cessation of steroidogenic response to 8Br-cAMP. In contrast, these cells cultured for a period of several days retained a full response of VEGF production. Of course, presumably because of the loss of gonadotropin receptors during the long-term culture, the cells could not be stimulated by the gonadotropin from culture Day 3 onward, but VEGF response to stimulation with 8Br-cAMP was completely intact. Furthermore, we show here that RLF can be a useful marker for ascertaining the purity of adult-type Leydig cells in testicular interstitial cell preparation, because no other testicular cells express this molecule [13, 15]. As Leydig cells lost their steroidogenic capacity over the days of culture, it was important to show that Leydig cells are indeed present throughout the culture. Using an antiserum against RLF, we could show that this indeed is the case because the cells continued to express RLF throughout. Thus it is obvious that a concomitant steroidogenesis is not essential for gonadotropin/cAMP-stimulated VEGF production by Leydig cells. However, our data do not exclude the possibility that steroids may be in a position to modulate this process. This will require further investigations.

A role for PKA in this process was confirmed by the observation that H-89, a specific inhibitor of PKA, effectively inhibited the gonadotropin and 8Br-cAMP-induced VEGF production by Leydig cells. It is interesting that not only gonadotropin, but also ANP, which is known to increase the testosterone production [12, 14], also stimulates VEGF production from the cells. It has been shown that cGMP is formed in Leydig cells via the membrane-associated guanylate cyclase in response to ANP, leading to greater steroidogenesis in these cells [34–36]. Schumacher et al. [34] have demonstrated that cGMP binds to the cAMP binding site on PKA and brings about a promiscuous activation of this kinase, thereby stimulating the steroidogenic cascade. Accordingly, we propose that a similar mechanism is responsible for ANP-induced VEGF production, as H-89 was able to abolish the effect of ANP on VEGF production as well (data not shown). This is the first demonstration of ANP being able to stimulate VEGF production. Because physiological target cells of natriuretic peptides are widespread in a variety of tissues, including the cardiovascular system, it will be interesting to determine whether these peptides are also able to regulate local VEGF concentrations in these tissues.

A role for PKC was discounted because treatment with PMA did not affect VEGF production either in unstimulated or in hCG/8Br-cAMP stimulated cells. At this concentration, PMA was, however, able to shift the dose-response curve for hCG-induced testosterone production to the left (data not shown), in accordance with a previous report [16]. Thus the regulation of VEGF production in mouse Leydig cells is independent of PKC. A role for PKC has been proposed in the induction of VEGF gene expression in a variety of cell lines [37–39], and a PKC responsive locus has been demonstrated in VEGF gene promoter. However, in Leydig cells, we did not observe a PKC-mediated effect. It is not immediately clear whether the observed differences could be explained by the fact that the current study was performed with primary cells, whereas previous demonstration of a role for PKC has been carried out in experiments using cell lines.

The Src family of nonreceptor tyrosine kinases plays important roles in various signal transduction pathways in many different cell types [40–44]. There is a growing body of evidence that Src is involved in the regulation of VEGF gene expression [17, 42]. Taylor et al. [43] were able to clearly demonstrate that Src tyrosine kinase has an important role to play in regulating LH-induced steroidogenesis in a murine tumor Leydig cell line (MA-10 cells). We are now able to show that inhibition of Src tyrosine kinase with PP2 interferes with the gonadotropin-stimulated VEGF secretion by primary Leydig cells. This nonreceptor tyrosine kinase has been previously demonstrated to be involved in cross talk among various signaling pathways, including those involving G-proteins [40, 41, 45]. Accordingly, an Src-dependent pathway also appears to be involved in the gonadotropin-mediated VEGF response in mouse Leydig cells. It is interesting that unlike the effect in MA-10 cells, PP2 had no effect on hCG/LH or 8Br-cAMP stimulated testosterone production in primary cultures of mouse Leydig cells.

Moreover, it has been shown in a variety of cells, such as vascular smooth muscle cells [46], that Src kinase acts as an upstream regulator of the MAP kinase pathway, because an inhibition of Src kinase by PP2 resulted in a decreased phosphorylation of Erk 1/2. In mouse Leydig cells, it was therefore of interest to examine whether the Erk 1/2 and p38 MAP kinase pathways were involved in regulating VEGF production.

The MAP kinase Erk 1/2 could be expected to mediate Src-dependent stimulation of VEGF expression. It has been suggested that in other cell types, VEGF expression (induced by other factors) is induced via the MAP kinase/Erk 1/2 kinase signaling cascade [42, 47–50]. We used a cell-permeable inhibitor of MEK 1/2 kinases, U0126. Both gonadotropin and 8Br-cAMP induced VEGF expression was completely abolished by the addition of 25 µM U0126. Also with PD98059, another inhibitor of MEK 1/2 kinases, we observed similar inhibitory effects (data not shown). It is thus obvious that also in mouse Leydig cells, VEGF secretion is mediated by an MEK 1/2-Erk 1/2 dependent pathway. A cross talk between the signals of MAP kinase/Erk 1/2 kinases and Src-tyrosine kinase may be postulated. In glioblastoma U87 and fibrosarcoma HT1080 cells, Src-dependent VEGF expression has been shown to be further mediated via Erk 1/2 MAP kinases [43, 50], and the Src-Erk 1/2 signaling cascade appears to be important for enhanced VEGF expression in other tumor cell types [48]. Here we report a similar mechanism that occurs in the primary cells as well, for enhanced VEGF production in response to a hormonal stimulation.

It is interesting that the p38 kinase (p38) specific inhibitor SB203580 was also found to completely abolish the hCG/LH or 8Br-cAMP-mediated stimulation of VEGF production. Erk 1/2 and p38 signaling have been demonstrated to operate simultaneously under acute conditions such as early response to adenovirus vectors [51]. Our observation deviates somewhat from that reported by Fredriksson et al. [17], who observed an involvement of the ß-adrenoreceptor/cAMP/PKA/Src kinase pathway in the norepinephrine-mediated induction of VEGF gene expression in mouse adipocytes but found that MAP kinases were not a part of the signaling mechanism. Our data clearly demonstrate that the MAP kinase-dependent pathway is indeed involved in our cell system. It is possible that this discrepancy could be explained by different types of cells used in these two studies.

In contrast to scanty information available on the role and regulation of VEGF in testes, its expression and regulation has been well-investigated in female reproductive tract tissues (see [52] for a review). In rat corpus luteum, expression of VEGF was demonstrated with in situ hybridization by Phillips et al. [53], and its role in regulating luteal function in pregnancy has been proposed [54]. VEGF has been localized both in human ovary and fallopian tubes [55], and it was found to be overexpressed in hyperthecotic ovarian stroma of Stein-Leventhal syndrome, in which it may also have a pathophysiological role [56]. A number of reports [57, 58] have shown that ovarian cells can produce VEGF in response to gonadotropic stimulation. However, in ovarian cells, the signaling mechanisms involved in gonadotropin-dependent regulation of VEGF expression and production has not yet been worked out in detail. It will be interesting to discern the similarities or differences in signaling pathways employed to regulate VEGF expression in the testes and in the ovaries, for which further research will be necessary.

In conclusion, our results clearly demonstrate the expression and secretion of VEGF by Leydig cells isolated from adult rats and mice. We have further established the occurrence of a hormonal regulation of VEGF production by Leydig cells. This regulation of VEGF production by Leydig cells is exclusively mediated by a gonadotropin receptor-induced cAMP-PKA signaling pathway. This pathway appears to use an Src-dependent signaling cascade that converges at the MEK 1/2 level, thereby proceeding through the Erk 1/2 signaling cascade so as to stimulate the expression of the VEGF gene and hence the protein. That both LH and FSH can stimulate the MAP kinase pathway, which involves phosphorylation of Erk 1/2 downstream of PKA, has been well established [59–61] in gonadal steroidogenic cells. More research will be necessary to clarify the role of Src tyrosine kinase in gonadotropin-mediated signal transduction in steroidogenic cells of testes and ovary.


   ACKNOWLEDGMENTS
 
We are grateful to Prof. Richard Ivell (Institute for Hormone and Fertility Research, Hamburg, Germany) for his generous gift of RLF antiserum and preimmune serum for immunostaining of Leydig cells. We thank Ms. Monika Kistler for introducing the cell preparation and culture methods, Ms. Annemarie Samalecos for introducing immunostaining, and Dr. Suleyman Ergün (Institute of Anatomy, UKE, Hamburg, Germany) for useful suggestions in the initial phase of this work.


   FOOTNOTES
 
1 Financial support to R.J.K.A. in the form of an Indo-German Fellowship from the Leidenberger-Müller Foundation is gratefully acknowledged. Back

2 Correspondence: A.K. Mukhopadhyay, Institute for Hormone and Fertility Research, Grandweg 64, D-22529 Hamburg, Germany. FAX: 49 40 561908 64; amal{at}ihf.de Back

Received: 26 July 2002.

First decision: 4 September 2002.

Accepted: 25 November 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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