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Role of Sphingosine in the Tumor Necrosis Factor Stimulatory Effect on Lactate Dehydrogenase A Expression and Activity in Porcine Sertoli Cells

^a Institut National de la Sant;aae et de la Recherche M;aaedicale, INSERM U-407, Communications Cellulaires en Biologie de la Reproduction, Facult;aae de medecine Lyon-Sud, B.P. 12, F-69921 Oullins Cedex, France

ABSTRACT

In this study, the intracellular signaling mechanisms through which TNF increases LDH(A4) activity/expression in primary cultures of porcine testicular Sertoli cells were investigated. Studies were focused on sphingomyelin hydrolysis pathway. Treatment of [¹⁴C]serine-labeled cells with TNF (15 ng/ml, 0.8 nM) resulted in a transient decrease (20%) in cellular [¹⁴C]sphingomyelin and in an increase (27%) in [¹⁴C]sphingosine that remained elevated for at least 75 min. In the same experiments, no significant changes were detected in ceramide levels. Exogenous sphingosine stimulated LDH(A4) activity and LDHA expression in a dose-dependent manner (ED₅₀ = 8 µM of sphingosine). Such an increase in LDHA messenger RNA levels and LDH(A4) activity was detected at 24 h and was maximal after 48 h of treatment. Kinetically, the increase in LDH(A4) activity was similar whether Sertoli cells were treated with sphingosine (12 µM) or with TNF (20 ng/ml). Although sphingosine mimicked the action of TNF on Sertoli cells LDH(A4) activity and expression, the maximal stimulatory effect represented about 30% of TNF maximal activity. Sphingomyelinase, C2 ceramide, sphingosine 1-phosphate, N,N-dimethylsphingosine, and phosphorylcholine had no significant effect on LDHA expression/LDH(A4) activity. Exogenous C2 ceramide increased LDH(A4) activity only in cytokine-treated cells, suggesting its involvement as sphingosine precursor in TNF-stimulated LDH(A4) activity via the sphingomyelin hydrolysis pathway. The LDH(A4) activity stimulated by TNF was decreased by 36.2% by an inhibitor of sphingosine formation, NH4Cl (4 mM), supporting a role of sphingosine in the TNF effect. Moreover, bisindolylmaleimide (100 nM), a protein kinase C (PKC) inhibitor decreased significantly by 28.7% the TNF effect on LDH(A4) activity but had no effect on the stimulating action of sphingosine, suggesting that if PKC is involved in TNF action, the sphingosine effect on LDH(A4) is unrelated to the PKC activity or inhibition. Together, the present data suggest that in primary Sertoli cell cultures, TNF stimulating action on LDHA expression is partly exerted via sphingomyelin hydrolysis pathway, sphingosine being the active metabolite.

cytokines, Sertoli cells, signal transduction, testes

INTRODUCTION

Spermatogenesis is a complex molecular and cellular process that is sustained by a permanent dialogue between germ cells and somatic Sertoli cells under hormonal (luteinizing hormone/testosterone and follicle-stimulating hormone [FSH]) control [1, 2]. Such cell-to-cell communication appears to be mediated by several growth factor and cytokine families [3, 4]. One such cytokine, tumor necrosis factor alpha (TNF), has been reported to be produced in germ cells (round spermatids, [5]) and to affect Sertoli cell activity evaluated by several parameters, including aromatase activity, inhibin production [6], and insulin-like growth factor-binding protein 3 (IGF BP3) expression [7]. In a previous report [8], we demonstrated that TNF stimulates lactate production in primary cultures of purified porcine Sertoli cells through an increase in glucose uptake and a redistribution of lactate dehydrogenase (LDH) isoforms, which involves mainly an increase in the expression of LDHA messenger RNA (mRNA) levels and the activity of LDH(A4). Several observations have indicated that (postmeiotic) germ cells use Sertoli cell lactate rather than glucose as energy substrate [9]. Several biochemical steps are involved in lactate production, including glucose uptake, glycolysis, and the interconversion of lactate and pyruvate. Lactate dehydrogenase (EC.1.1.1.27) catalyzes this interconversion with nicotinamide adenine (NAD⁺) as coenzyme. Mammals have three different subunits of LDH that are encoded by three genes, ldh a, ldh b, and ldh c [10]. The A and B subunits form together five tetrameric isoenzymes: A4, A3B1, A2B2, A1B3, and B4. Although these hybrid forms occur in most tissues, the B-type subunit predominates in aerobic tissues such as heart and is superior for lactate oxidation, whereas the A-type subunit predominates in tissues that are subject to anaerobic conditions, such as skeletal muscle and liver, and is best suited for pyruvate reduction. The C4 isozyme is unique among the LDH isozymes with respect to its restricted distribution within the germinal epithelium of the mammalian testes [10]. Lactate dehydrogenase B4 has a low K_m for pyruvate and is allosterically inhibited by high levels of this metabolite, whereas the A4 isozyme has a higher K_m for pyruvate and is not inhibited by it [11]. The other isozymes have intermediate properties that vary with the ratio of their two types of subunits. The functional importance of LDH isozyme shifts is generally attributed to a need for increased A subunit-containing isozymes, which can derive more energy by reducing pyruvate to lactate. For this purpose, A-type LDH is more suitable than B-type LDH because, as mentioned, it is not inhibited by the high concentration of pyruvate that is likely to be present during anaerobic glycolysis [11]. An increase in the activity of such an isozyme after TNF treatment would therefore favor the conversion of pyruvate to lactate.

Tumor necrosis factor alpha is a cytokine known to be a multifunctional mediator, with a wide range of biological responses in mammalian cells. This cytokine interacts with two cell surface receptors with apparent molecular weights of 55 kDa (TNF-R1) and 75 kDa (TNF-R2) [12]. Most of the TNF-induced responses are mediated by the activation of TNF-R1 [13], which has been identified in Sertoli cells [14]. Upon binding to its receptor subtypes, TNF evokes a complicated array of intracellular signals ([15] for review), including G protein-coupled activation of phospholipase A2, release of arachidonic acid, diacylglycerol production, and activation of protein kinase C (PKC), some of which may participate in the stimulation of LDH(A4) [16]. Among postreceptor events, the activation of sphingomyelinases, followed by the subsequent generation of a second-messenger ceramide moiety [12, 17, 18], seems to play an important role in the TNF-transducing system. Ceramide can be deacylated after ceramidase action to form sphingosine and subsequently, sphingosine 1-phosphate, via a specific sphingosine kinase [19]. In addition, evidence emerged during the past years that sphingolipid metabolites such as sphingosine [19, 20], ceramide [18], and sphingosine 1-phosphate [21] play an important role as intracellular signaling molecules for a variety of targets [22].

Although data concerning the various pathways of intracellular signal transduction mechanisms activated by TNF are now available in a set of different cell types [15, 23], there is no study devoted to the primary signaling pathway activated by TNF in Sertoli cells.

The aim of the present study was to examine the primary signaling pathway involved in TNF-induced LDH(A4) expression and activity in a model of primary cultures of porcine Sertoli cells. Specifically, here we focused our attention on the subsequent degradation of sphingomyelin into ceramide and sphingosine and on the role of these metabolites in mediating TNF action. Evidence is presented to indicate that in testicular Sertoli cells, 1) activation of sphingomyelin hydrolysis and generation of sphingosine occurs in response to the cytokine action and 2) sphingosine might be implicated in the stimulating effect of TNF on LDHA mRNA and LDH(A4) activity levels.

MATERIALS AND METHODS

Materials

Dulbecco modified Eagle medium-Ham F12 (DMEM-F12) medium was obtained from Gibco (Grand Island, NY). Human recombinant TNF (specific activity 2 x 10⁷ U/mg, determined by the cytolysis of murine L929 cells) was from Peprotech Inc (Rocky Hill, NJ). Phorbol 12-myristate 13-acetate (PMA), D-sphingosine, N-acetyl-D-sphingosine or C2-ceramide, sphingosine 1-phosphate, phosphorylcholine chloride, and bovine serum albumin (BSA) were from Sigma Chemical Co. (St Louis, MO). Calbiochem Novabiochem Corporation (La Jolla, CA) was the source for N,N-dimethylsphingosine and bisindolylmaleimide. The labeled compound L-[¹⁴C]serine (5.55 GBq or 150 mCi/mmol) was purchased from Du Pont-New England Nuclear (Les Ulis, France). Trizol was obtained from Life Technologies (Eragny, France).

Sertoli Cell Isolation and Culture

Pigs were routinely castrated within 3 wk of birth, and isolated Sertoli cells were prepared from porcine testes by collagenase treatment as described elsewhere [24].

Cells were plated in Falcon (Los Angeles, CA) 12-multiwell plates (10⁶ cells per dish) and cultured at 32°C in a humidified atmosphere of 5% CO₂, 95% air in DMEM-F12 medium (1:1) containing sodium bicarbonate (1.2 mg/ml), 15 mM HEPES, and gentamycin (20 µg/ml). This medium was supplemented with transferrin (5 µg/ml), and vitamin E (10 µg/ml). Exogenous lipid agents (ceramide, sphingosine, sphingosine-1-phosphate, and dimethylsulfide) were freshly diluted in sterile culture medium supplemented with BSA and were added in aliquots to a final concentration of 0.1% BSA, with control incubations receiving the same volume of medium and a similar final concentration of BSA.

For determination of cell number, Sertoli cells were detached from the culture dishes with trypsin-EDTA and counted in a Coulter counter (Coulter Electronics, Margency, France). Cellular viability was assessed by means of trypan blue stain and by using a hemacytometer. The results were expressed in terms of percentage of viable cells per dish.

Lactate Dehydrogenase Activity Determination

After incubation of Sertoli cells, the cell extracts were prepared and collected as described elsewhere [8]. Total LDH activity [25] was determined by a spectrophotometric method with the Enzyline LDH-Kit (BioMerieux, Lyon, France). The results were expressed as milli-International Units of enzyme activity per 10⁶ cells.

Measurement of the Activity of LDH Isozymes

As previously described [8], the activity of LDH isozymes was determined by using agarose gel electrophoresis under nondenaturing conditions [26] with theTitan Gel LD-Kit isozyme procedure (Helena Laboratories, Beaumont, TX). The different bands were quantified by densitometric scanning with the Bioimage scanner (Millipore SA, Saint Quentin, France). LDH isozyme activity was expressed as a percentage of the total LDH activity and presented as an activity percentage (activity %) or in milli-international units of enzyme activity per 10⁶ cells.

Metabolic Labeling, Lipid Extraction, and Quantification

Sertoli cells were cultured for 3 days in serine-poor (50 µM) MEM-F10 medium (1:1) and labeled with [¹⁴C]serine (1 µCi/ml) for the last 48 h. After equilibration for 30 min in fresh medium supplemented with 25 mM serine [27], triplicate cultures were treated with TNF or a vehicle, and experiments were terminated by placing the culture plates on ice. Lipids from individual cultures (10⁶ cells) were extracted with 1 ml ice-cold methanol and transferred to glass tubes containing 2 ml chloroform and 0.6 ml HCl 0.01 N [28]; the organic and aqueous phases were separated by centrifugation. The organic phases were dried under nitrogen and redissolved in 100 µl chloroform, and individual lipid fractions were separated by sequential thin-layer chromatography on silica gel 60 plates (Merck, Darmstadt, Germany) with chloroform/methanol/acetic acid/water [ct notation OK?[xc(50/30/8/5, by volume) and then butanol/water/acetic acid (3/1/1, by volume) [29] as developing solvent systems. Identification of each labeled lipid was achieved with iodine vapors and by comparison with standards corresponding to sphingomyelin (Rf = 0.28), sphingosine1-phosphate (Rf = 0.58), sphingosine (Rf = 0.68), and ceramides (Rf = 0.84). Radiolabeled lipids were detected and quantified with a Bioscan 2000 thin-layer chromatography imaging system. With this scanner, values for radiolabeled lipids are given in counts per minute (cpm) and also as a percentage of the total cpm of each total individual lipids extract.

Analysis of Messenger RNA Levels

Total RNA was extracted from Sertoli cells cultured in Petri dishes with TRIzol, a monophasic solution of phenol and guanidine isothiocyanate. This reagent is an improvement over the single-step RNA isolation developed by Chomczynski and Sacchi [30, 31]. RNA was electrophoresed on a 1.2% agarose-2.2 M formaldehyde gel and transferred to nitrocellulose membrane Hybond-C Extra (Amersham, Saclay, France) in 10x SSC (1.5 M NaCl and 0.15 M sodium citrate). Hybridization, washing, and stripping of Hybond membranes were carried out according to the instructions of the manufacturer.

Complementary DNA (cDNA) probes (LDH-A, 1.5-kilobase [kb] Xho-EcoRI; GAPDH, 1.3-kb PstI) were labeled with 40 µCi [-³²P]deoxy-CTP (SA, 10⁹ dpm/µg DNA) with a random primers DNA labeling kit (Promega, Madison, WI). Labeled probes were used as described elsewhere [8]. The intensities of the autoradiographic bands were estimated by densitometric scanning with the Bioimage scanner (Millipore). The data were expressed as relative densitometry units of LDHA transcript per glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript used as an internal standard.

Data Analysis

Unless mentioned in the legend, all experimental data are presented as the mean ± SD of three separate determinations of three replicate cultures within each treatment group. The experiments reported here were repeated at least three times with different primary cultures of purified Sertoli cells. Each cell's preparation was obtained from a different animal group (about 12 immature pigs per group). Although cells behaved similarly in response to treatments in all experiments, the data vary between the different cell's preparations because members of each animal group are routinely castrated at different periods of the year. Also, a representative triplicate experiment of each series of experiments is presented. Statistical analysis was performed by one-way ANOVA. Differences between groups were determined by a Student-Newman-Keuls multiple-comparison test. For the sphingomyelin hydrolysis (Fig. 2) and for the LDHA mRNA stimulation with C2-ceramide (Fig. 5D), ANOVA analysis was followed by Fisher's protected least-significant difference (PLSD) as a multiple-comparison test. A P < 0.05 was considered significant.

View larger version (32K): [in this window] [in a new window]   FIG. 2. Effect of TNF on sphingomyelin turnover in cultured Sertoli cells. Sertoli cells were cultured for 3 days in serine-poor (50 µM) MEM-F10 medium (1:1) and labeled with [14C]serine (1 µCi/ml) for the last 48 h, as described in Materials and Methods. Cells were allowed to equilibrate for 30 min in fresh MEM-F10 medium, and thereafter, triplicate cultures were treated, for the time periods indicated, in the absence (white bars) or presence of 15 ng/ml TNF (hatched bars). The lipids were extracted and [14C]sphingomyelin (A) or [14C]sphingosine (B) or [14C]ceramide (C) separated and quantitated as described in Materials and Methods. Results are expressed as cpm % by comparison with total cpm (histograms A, B, and C) and by comparison with control (inserts A, B, and C). Histograms show representive results of three independent experiments. Values are mean ± SD of triplicate incubations. * Treated cells versus control cells, P < 0.05

View larger version (32K): [in this window] [in a new window]   FIG. 5. Dose response of the effect of C2-ceramide on LDH(A4) activity and LDHA mRNA expression in cultured Sertoli cells. Sertoli cells were incubated for 48 h in the absence (control) or presence of TNF (20 ng/ml, as positive control; A) and in the absence (control) or presence of increasing concentrations (0.1–10 µM) of C2-ceramide alone (A and B) and in combination (C and D) with TNF (15 ng/ml). In A and C, LDH(A4) activity was evaluated after separation by gel electrophoresis, whereas in B and D, LDHA mRNA levels were evaluated by Northern blot analysis as described in Materials and Methods. The figure shows a representative gel electrophoresis in A and C for LDH(A4) activity and a representative autoradiogram in B and D for LDHA mRNA. The histograms show representative results of three independent experiments. Values are mean ± SD of triplicate incubations

RESULTS

TNF Stimulation of Sphingomyelin Hydrolysis in Cultured Porcine Sertoli Cells

The effect of TNF on sphingomyelin turnover were examined in Sertoli cells labeled to isotopic steady state with [¹⁴C]serine-labeled sphingomyelin. In preliminary experiments, we determined that 15 ng/ml (0.8 nM) of TNF was an efficient dose (F = 8.01, P = 0.0049) for induction of sphingomyelin hydrolysis after an incubation of 15 min (Fig. 1).

View larger version (14K): [in this window] [in a new window]   FIG. 1. Effect of TNF on sphingomyelin hydrolysis. Sertoli cells were cultured for 3 days in serine-poor (50 µM) MEM-F10 medium (1:1) and labeled with [14C]serine (1 µCi/ml) for the last 48 h, as described in Materials and Methods. Cells were allowed to equilibrate for 30 min in fresh MEM-F10 medium, and thereafter, triplicate cultures were treated for 15 min in the presence of increasing concentrations (5–25 ng/ml) of TNF. The lipids were extracted and [14C]sphingomyelin separated and quantitated as described in Materials and Methods. The graph shows representative results of three independent experiments. Values are mean ± SD of triplicate incubations

In Figure 2, values are compared in the absence and in the presence of the cytokine at each time of treatment, and the data indicate that the hydrolysis of sphingomyelin (Fig. 2A) is induced (globally, F = 6.15, P = 0.0003) after approximately 15 min of exposure to TNF (15 ng/ml) and that more than 20% (P < 0.001; insert A) of the prelabeled sphingomyelin pool was degraded in comparison with control (untreated cells.) At the time of maximal sphingomyelin hydrolysis, the cellularly labeled sphingosine pool (Fig. 2B and corresponding insert; globally (F = 3.40, P = 0.0046) increased by 27%, was significant (P = 0.029) at 30 min (29%) in TNF-treated cells, and remained elevated for 60 min. Variations in the basal control values were observed at the different times of incubation. Such observations have been reported elsewhere in other models of primary cultured cells subjected to the same experimental conditions [27].

In our experimental conditions, no significant change was detected in ceramide level in comparison with the case of corresponding control groups (Fig. 2C and corresponding insert).

Effect of Sphingomyelin Metabolites on Sertoli Cell LDH(A4) Activity

The results in Table 1 show that in control cells (untreated cells), LDH(A4) activity represents 2.2 ± 1.2% (3.1 ± 1.7 mU/10⁶ cells) of the total LDH activity. With a maximal dose of TNF (20 ng/ml), Sertoli cell LDH(A4) activity increased to 50.3 ± 9.1% (128.6 ± 23.2 mU per 10⁶ cells). Although TNF effects on LDH(A4) activity were previously reported as having a plateau from 11 to 50 ng/ml [8], in our study, effects of TNF on LDH(A4) activity are observed from 15 ng/ml. Therefore, 15 and 20 ng/ml were used as submaximal and maximal doses, respectively.

View this table: [in this window] [in a new window]   TABLE 1. Effect of TNF{} and sphingomyelin metabolites on total LDH and on LDH(A4) activities in cultured Sertoli cells.\su\a\r\

To determine whether sphingomyelin metabolites might affect LDH(A4) activity, the enzyme activity was evaluated in Sertoli cells treated during 48 h with neutral sphingomyelinase (0.001 to 1 U/ml) and with different analogs of sphingomyelin metabolites (sphingosine 1 to 20 µM; phosphorylcholine 1 to 15 µM; C2 ceramide 0.1 to 20 µM, N,N-dimethylsphingosine 1 to 12 µM; and sphingosine-1-phosphate 1 to 16 µM). Only the results with a maximal dose compatible with cellular viability are shown. As indicated in Table 1 (globally, F = 22.48; P < 0.0001 for LDH activity and F = 104.10, P < 0.0001 for LDH(A4) activity), sphingosine (15 µM) significantly stimulated LDH(A4) activity (20.3 ± 4.0% increase, i.e., 39.5 ± 7.8 mU/10⁶ cells, P < 0.05). In contrast, sphingomyelinase (0.5 U/ml), phosphorylcholine (10 µM), C2-Cer (10 µM), N,N-dimethylsphingosine (10 µM), and sphingosine-1 phosphate (15 µM) failed to stimulate LDH(A4) activity.

Sphingosine Stimulates LDH(A4) Activity and LDHA mRNA Expression

In these series of experiments, we further characterized the stimulatory effect of sphingosine on LDH(A4) activity and LDHA mRNA levels. As shown in Figure 3, sphingosine (2–20 µM) stimulated LDH(A4) activity in a dose-dependent manner (F = 16.25, P < 0.0001), with maximal and half-maximal (ED₅₀) effects observed at 15 µM (20.3 ± 4.0% increase, P < 0.05) and 8 µM of sphingosine, respectively (Fig. 3A). Under the same conditions, LDHA mRNA (1.5 kb) levels were increased in a dose-dependent manner (F = 26.41, P < 0.0001), and the maximal effect of sphingosine was again observed with 15 µM (P < 0.05; Fig. 3B).

View larger version (36K): [in this window] [in a new window]   FIG. 3. Dose response of the effect of sphingosine on LDH(A4) activity and LDHA mRNA expression in cultured Sertoli cells. Sertoli cells were incubated for 48 h in the absence or presence of increasing concentrations (2–20 µM) of sphingosine. In A, LDH(A4) activity was measured as a percentage of total LDH activity per 106 cells, and in B, LDHA mRNA levels were evaluated by Northern blot analysis, as described in Materials and Methods. Both in A and B, a representative gel electrophoresis (for LDHA4 activity) and autoradiogram (for LDHA mRNA) are shown. The histograms show representative results of three independent experiments. Values are mean ± SD of triplicate incubations. * Treated cells versus control cells, P < 0.05

The stimulatory action of sphingosine (12 µM) exerted on both LDH(A4) activity (F = 94.72, P < 0.0001; Fig.4A) and LDHA mRNA (F = 6.62, P < 0.003; Fig. 4B) was time dependent. Indeed, it was apparent at 24 h for LDH(A4) activity but was significant at 48 h (P < 0.05; Fig. 4A) and significantly detected at 24 h for LDHA mRNA (P < 0.05) (Fig. 4B). The increase was maximal at 48 h for both LDH(A4) activity and LDHA mRNA (Fig. 4, A and B).

View larger version (32K): [in this window] [in a new window]   FIG. 4. Time course of sphingosine effect on LDH(A4) activity and LDHA expression in cultured Sertoli cells. Sertoli cells were incubated for different times (3–72h) in the absence or presence of sphingosine (12 µM) and of TNF (20 ng/ml; used here as a positive control). In A, LDH(A4) activity was measured as a percentage of total LDH activity per 106 cells, and in B, LDHA mRNA levels were evaluated by Northern blot analysis as described in Materials and Methods. Both in A and B, a representative gel electrophoresis (for LDHA4 activity) and autoradiogram (for LDHA mRNA) are shown. The histograms show representative results of three independent experiments. Values are mean ± SD of triplicate incubations. * Treated cells versus control cells, P < 0.05

It is of interest to note that kinetically, sphingosine (12 µM) action was comparable to if not similar to that of TNF (20 ng/ml) on LDH(A4) activity (Fig. 4A).

C2-Ceramide Stimulates LDH(A4) Activity and LDHA mRNA Expression Only in the Presence of TNF

Under the same experimental conditions, ceramide C2 (0.1–1.0 µM) had no significant effect on LDH(A4) activity (Fig. 5A) and LDHA mRNA expression (Fig. 5B). However, when Sertoli cells were incubated simultaneously, in the presence of TNF (15 ng/ml as submaximal dose) and ceramide C2 (0.05–5 µM), LDH(A4) activity increased significantly (F = 9.87, P < 0.0006), with a plateau at 1 µM (1.8-fold, P < 0.05), by comparison with TNF alone (100%; Fig. 5C). In the same conditions, LDHA mRNA levels were increased in a dose-dependent manner (F = 15.11, P < 0.0001), with maximal effect at 2 µM ceramide C2 by comparison with TNF alone (P < 0.04 with Fisher's PLSD as a multiple-comparison test; Fig. 5D). As expected, sphingosine (2–8 µM) also induced an increase in LDH(A4) activity and in LDHA mRNA expression (at 6 µM) when added in the presence of TNF (15 ng/ml; data not shown).

These observations suggest that exogenous ceramide was unable to stimulate LDHA mRNA expression and LDH(A4) activity by itself. In the presence of TNF, however, when sphingomyelin hydrolysis pathway is activated, exogenous ceramide can be used as a sphingosine precursor via ceramidase activation.

Effect of NH4Cl, a Suppressor of Sphingosine Formation, on TNF-Stimulated LDH(A4) Activity

The data in Figure 6 (globally, F = 11.82, P = 0.0026) show that the incubation of Sertoli cells with a lyso-osmotrophic agent, NH4Cl (4 mM) [32], induced a 36.2% decrease in 20 ng/ml TNF-stimulated LDH(A4) activity (P < 0.05). This result is not due to a decrease in number of Sertoli cells (F = 0.616, P = 0.556) and viability (F = 0.625, P = 0.550; Table 2). As expected, NH4Cl had no significant effect on sphingosine-stimulated LDH(A4) activity (Fig. 6). These observations support again the mediating role of sphingosine in TNF action.

View larger version (35K): [in this window] [in a new window]   FIG. 6. Effect of NH4Cl on LDH(A4) activity in cultured Sertoli cells. Sertoli cells were preincubated for 2 h with or without NH4Cl (4 mM), then for 46 h in the absence or presence of TNF (20 ng/ml) or sphingosine (12 µM). LDH(A4) activity was measured as described in Materials and Methods. The histogram shows representative results of three independent experiments. Values are mean ± SD of triplicate incubations. * TNF-alpha-NH4Cl-treated cells versus TNF-alpha-treated cells, P < 0.05

Effect of Bisindolylmaleimide, a PKC Inhibitor, on Sphingosine-Stimulated LDH(A4) Activity

The data in Figure 7 (globally, F = 35.78, P < 0.0001) show that a PKC inhibitor, bisindolylmaleimide (BIM, 100 nM) [33] failed (P > 0.05) to alter sphingosine (12 µM)-stimulated LDH(A4) activity in cultured Sertoli cells, suggesting that sphingosine action was probably unrelated to the PKC pathway.

View larger version (33K): [in this window] [in a new window]   FIG. 7. Effect of bisindolylmaleimide on LDH(A4) activity in cultured Sertoli cells. Sertoli cells were preincubated for 2 h with or without BIM (100 nM), then for 46 h in the absence or presence of PMA (50 nM) or TNF (20 ng/ml) or sphingosine (12 µM). LDH(A4) activity was measured as described in Materials and Methods. The histogram shows representative results of three independent experiments. Values are mean ± SD of triplicate incubations. * TNF-alpha-BIM-treated cells versus TNF-alpha-treated cells, and ** PMA-BIM-treated cells versus PMA-treated cells, P < 0.05

One must note that this inhibitor decreased significantly (-28.75%, P < 0.05) the effect of TNF (20 ng/ml) on LDH(A4) activity, suggesting an involvement of the PKC pathway in TNF-stimulated LDH(A4) activity. As a positive-control experiment, phorbol myristate acetate (PMA, 50 nM) action on LDH(A4) activity was inhibited by BIM (more than 70% decrease, P < 0.05).

DISCUSSION

The aim of our present study was to investigate the intracellular transducing pathways involved in TNF action on the expression and activity of LDH(A4), an enzyme involved in the formation of lactate. Several observations have indicated that lactate may represent a preferential energetic substrate for germ cells [9]. Indeed, 1) the inability of germ cells (particularly postmeiotic germ cells) to use glucose to energize their metabolism, 2) their preference for lactate as an energy source, and 3) the capacity of Sertoli cells to produce high amounts of lactate have generated a concept related to Sertoli cell-germ cell metabolic cooperation, with lactate playing a pivotal role [34–36].

Specifically, studies were focused on the sphingomyelin hydrolysis pathway. For this purpose, the experimental model used involved purified Sertoli cells obtained from porcine testes and cultured in a defined medium. Tumor necrosis factor alpha induces a wide variety of effects on many different cell types, and although different signaling pathways were shown to be activated by TNF, in general, the biological responses involved in such activations still remain to be identified. With regard to the experimental cell models used; a large amount of data has been generated in cell lines. Primary culture cells are probably more appropriate models for investigating intracellular signaling pathways involved in biological activity in that those cells are more representative of physiological conditions. By using this model, in reports elsewhere, we have already shown that under similar experimental conditions, TNF regulates Sertoli cell-specific activities without exerting a mitogenic or a cytotoxic effect on these cells [6–8]. In the present study, it is demonstrated that in Sertoli cells, 1) under the action of TNF, sphingomyelin, hydrolysis and generation of metabolites occur and 2) sphingomyelin metabolites such as sphingosine are involved, although partly, in the action of the cytokine on the activity/expression of LDH(A4).

In primary cultures of purified Sertoli cells, we found that TNF induced a transient hydrolysis of a significant fraction of the labeled sphingomyelin pool and that this was accompanied by increased levels of intracellular sphingosine, which remained elevated after a 1-h exposure to TNF. Sphingosine, which is a structural backbone and a breakdown product of cellular sphingolipids, has recently emerged as a lipid mediator in signal transduction pathways [19, 20]. The structural properties of this molecule allow for its incorporation [37] and rapid mobility in membranes and makes it accessible to different effector systems. Sphingosine elicits different responses in a wide variety of cell types. For example, this molecule has been involved in retinoblastoma protein dephosphorylation [38]; in inhibition of bovine parathyroid hormone secretion [39] in intracellular calcium mobilization and cellular proliferation of Swiss 3T3 fibroblasts [29]; in apoptosis in human myeloid leukemia cells [40]; and in the negative inotropic effects in adult mammalian cardiac myocytes [41].

On the basis of our present data, sphingosine production after TNF stimulation is a postreceptor event that appears to be involved in the stimulatory action of the cytokine on Sertoli cell LDH(A4) activity and LDHA expression. First, sphingosine increased LDH(A4) activity and LDHA mRNA levels in a time-dependent manner similar to that of TNF action. Second, the presence of ammonium chloride, an inhibitor of sphingosine formation [32], induced a decrease in TNF-stimulated LDHA. With regard to the amplitude of sphingosine action, the stimulatory effect of sphingosine on LDHA expression and activity mimicked only partly (about 30%) the action of TNF. Similarly, the effect of ammonium chloride reduced the cytokine action on LDHA activity by about 30%. Together, these observations indicate that sphingosine probably only partially mediates TNF action on LDHA activity/expression, representing about one-third of the whole cytokine activity.

With regard to the other metabolites resulting from sphingomyelin hydrolysis, our present studies employing biologically active ceramide analog (C2 ceramide) and sphingosine-1-phosphate suggested that neither the immediate precursor of sphingosine nor the immediate metabolite of sphingosine, respectively, are responsible for the stimulatory effect of TNF on LDHA expression and activity. Indeed, exogenous C2-ceramides failed to stimulate LDHA activity and expression, although the coincubation of Sertoli cells with both C2-ceramide and TNF increased TNF-induced stimulation of LDH(A4) activity and LDHA mRNA expression, suggesting that its potential capacity is included and metabolized in the sphingomyelin hydrolysis pathway stimulated by the cytokine. With regard to sphingosine-1-phosphate, its effect on LDHA activity was limited (6.6% of TNF effect) when compared with that of sphingosine. The failure of ceramides or sphingomyelinase to mimic biological effects triggered by the cytokine-induced sphingomyelin hydrolysis has been observed elsewhere in other cell types, for example, during prostaglandin biosynthesis in fibroblasts [42] and in granulosa cells [43]. Although we do not know at the present time the reasons for such a failure in Sertoli cells LDH(A4) activity/expression, two possibilities may be evoked. First, sphingomyelin hydrolysis leading to the production of ceramide may occur at several subcellular sites [44], suggesting that the TNF-induced ceramide production involved in LDHA stimulation may depend on the topology of its production an effect that cannot be mimicked by sphingomyelinase or C2-ceramide incubation alone. Second, it is possible that in some cell types, ceramidase activation by cytokines may provide a "switch" that will determine which sphingolipids (ceramide or sphingosine) will be enhanced by this cytokine to produce specific intracellular response(s). For example, such a possibility has been reported in IL-1ß-treated hepatocytes [45], in which it has been suggested that the activation of ceramidase by the low concentrations of IL-1ß down-regulate cytochrome P450 2C11 expression, whereas higher IL-1ß concentrations that induce 1-acid glycoprotein do not activate ceramidase activity and then allow ceramide accumulation. However, such a possibility for the bimodal regulation of ceramidase by TNF in Sertoli cells is still a hypothesis that remains to be investigated.

Because it has been shown in different cell systems that the biological effects of sphingosine are a consequence of the inhibition of protein kinase C [46], we have tested the effect of BIM, a PKC inhibitor [33], on sphingosine-stimulated LDH(A4) activity. The absence of alteration in sphingosine action in the presence of BIM suggests that the sphingosine effect was unrelated to PKC activity or inhibition. Similar observations have been reported in other systems. For example, the effects of sphingosine on cellular proliferation in Swiss 3T3 cells is clearly independent of PKC; the synergism between optimal concentrations of sphingosine and TPA suggested that these compounds did not share a common pathway for induction of mitogenesis [19]. However, besides these observations related to the sphingosine action, our results indicating that BIM decreased TNF effect similarly by about one-third also suggested a partial role of the PKC pathway in TNF-stimulated LDH(A4) activity. Moreover, the PKC pathway has been reported to play an active role in regulating LDHA mRNA stability [16]. Together, our present data indicate that, in terms of signaling pathways, TNF may utilize both sphingomyelin hydrolysis and PKC pathways in stimulating Sertoli cell LDH(A4) activity/expression. Together, these two systems may account for about 60% of the activity of the cytokine, suggesting that other signaling pathways might be used by TNF and remain to be identified. In this context, our recently published results reported a potential role of protein tyrosine kinase in Sertoli cell LDH(A4) expression [47]. Moreover, it is of interest to mention that recently, the mitogen-activated protein kinase pathway has been implicated in the stimulating action of TNF on IL6 production and integrin ligand expression in cultured mouse Sertoli cells [48]. It is possible that the nature and/or the amplitude of the involvement of these pathways and, more specifically, the effect of sphingomyelin metabolites used, would be different if other Sertoli cell functional parameters (e.g., FSH receptor, aromatase, inhibin, IGF BP3 expression) [6, 7] were selected. Such possibilities are currently being investigated in our laboratory.

In conclusion, we report that TNF stimulates LDHA expression and activity partly through the sphingomyelin hydrolysis pathway and, more specifically, via sphingosine production. This pathway is not the only one involved in the cytokine action because others, such as the PKC pathway, are also implicated. It is of interest to mention that our present findings were 1) generated in a model of (untransformed) primary cultures of purified testicular Sertoli cells and 2) related to the identification of primary signaling pathways involved in a key biological activity of Sertoli cells, in other words, production of lactate, a crucial energetic metabolite for germ cells.

ACKNOWLEDGMENTS

We are grateful to Dr. S.S.L. Li (Laboratory of Genetics, Research Triangle Park, NC) and Dr. J.M. Blanchard (Faculté des Sciences, Montpellier, France) for providing us with porcine LDH-A and GAPDH cDNA, respectively.

FOOTNOTES

First decision: 31 August 1999.

¹ Correspondence. FAX: 33 4 78 86 31 16; gratarol{at}esgrisn1.univ-lyon1.fr

Accepted: June 15, 2000.

Received: August 3, 1999.

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