Tumor Necrosis Factor- Stimulates Lactate Dehydrogenase A ...

0013-7227/99/$03.00/0 Endocrinology Copyright © 1999 by The Endocrine Society

Vol. 140, No. 7 Printed in U.S.A.

Tumor Necrosis Factor-a Stimulates Lactate Dehydrogenase A Expression in Porcine Cultured Sertoli Cells: Mechanisms of Action ´ E GRATAROLI, JINGWEI JI, FAYC ¸ AL BOUSSOUAR, RENE MOHAMED BENAHMED

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INSERM, U-407, Faculte´ de Me´decine Lyon-Sud, F-69921 Oullins Cedex, France ABSTRACT In the present study, we investigated the regulatory action of tumor necrosis factor-a (TNFa) on lactate dehydrogenase A (LDH A), a key enzyme involved in lactate production. To this end, use was made of a primary culture system of porcine testicular Sertoli cells. TNFa stimulated LDH A messenger RNA (mRNA) expression in a dose (ED50 5 2.5 ng/ml; 0.1 nM TNFa)-dependent manner. This stimulatory effect was time dependent, with an effect detected after 6 h of TNFa treatment and maximal after 48 h of exposition (5-fold; P , 0.001). The direct effect of TNFa on LDH A mRNA could not be accounted for by an increase in mRNA stability (half-life 5 9 h), but was probably due to an increase in LDH A gene transcription. Inhibitors of protein synthesis (cycloheximide), gene transcription (actinomycin D and dichlorobenzimidazole riboside), tyrosine kinase (genistein), and protein kinase C (bisindolylmaleimide) abrogated

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N THE TESTICULAR seminiferous tubules, spermatogenesis is a complex process that is highly dependent upon the hormonally (FSH and testosterone) regulated Sertoli cells (1, 2). Sertoli cells provides regulatory factors, such as growth factors (3, 4) and nutrients (5), to the germ cells. Among nutrients are energy substrates such as lactate. Indeed, several observations have indicated that (postmeiotic) germ cells use Sertoli cell lactate rather than glucose as energy substrate (5). A similar metabolic cooperation involving lactate as an energy metabolite also occurs in other tissues such as the brain, particularly between astrocytes and neurons where astrocytes play a role comparable to that of testicular Sertoli cells (6, 7). Several biochemical steps are involved in lactate production, including glucose uptake, glycolysis, and the interconversion of lactate and pyruvate. Lactate dehydrogenase (LDH; EC.1.1.1.27) catalyzes this interconversion with nicotinamide adenine dinucleotide (NAD1) as coenzyme. Mammals have three different subunits of LDH that are encoded by three genes, ldh a, ldh b, and ldh c (8). 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

Received September 25, 1998. Address all correspondence and requests for reprints to: Dr. Mohamed Benahmed, INSERM U-407, Faculte´ de Me´decine Lyon-Sud, BP 12, F-69921 Oullins Cedex, France. E-mail: [email protected].

completely (actinomycin D, dichlorobenzimidazole riboside, cycloheximide, and genistein) or partially (bisindolylmaleimide) TNFa-induced LDH A mRNA expression. These observations suggest that the stimulatory effect of TNFa on LDH A mRNA expression requires protein synthesis and may involve a protein tyrosine kinase and protein kinase C. In addition, we report that LDH A mRNA levels were increased in Sertoli cells treated with FSH. However, although the cytokine enhances LDH A mRNA levels through increased gene transcription, the hormone exerts its stimulatory action through an increase in LDH A mRNA stability. The regulatory actions of the cytokine and the hormone on LDH A mRNA levels and therefore on lactate production may operate in the context of the metabolic cooperation between Sertoli and postmeiotic germ cells in the seminiferous tubules. (Endocrinology 140: 3054 –3062, 1999)

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 (8). LDH-B4 has a low Km for pyruvate and is allosterically inhibited by high levels of this metabolite, whereas the A4 isozyme has a higher Km for pyruvate and is not inhibited by it (9). 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 concentrations of pyruvate that are likely to be present during anaerobic glycolysis (9). In the context of the metabolic cooperation between Sertoli cell and postmeiotic germ cells, we have recently reported that 1) germ cells may control and direct lactate production in Sertoli cells via some signaling molecules, such as tumor necrosis factor-a (10); and 2) a redistribution of LDH isoforms occurs under tumor necrosis factor-a (TNFa) action in favor of LDH A, which preferentially catalyzes the conversion of pyruvate into lactate. In the present study, we extend this observation and further characterize the mechanisms involved in the stimulatory action of TNFa on LDH A expression in Sertoli cells.

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TNFa REGULATES LDH A mRNA EXPRESSION Materials and Methods Materials DMEM/Ham’s F-12 medium (DMEM/F12) and TRIzol were obtained from Life Technologies (Eragny, France). Collagenase/dispase was obtained from Boehringer Mannheim (Mannheim, Germany). Human recombinant TNFa was purchased from Preprotech (Rocky Hill, NJ); its ED50, determined by the cytolysis of murine L929 cells in the presence of actinomycin D (Act D), was less than 0.05 ng/ml, corresponding to a specific activity of more than 2 3 107 U/mg. Act D, 5,6-dichlorobenzimidazole ribozide (DRB), phorbol 12-myristate 13-acetate (PMA), sphingosine, sphingosine-1-phosphate, C2-ceramide, and cycloheximide were purchased from Sigma Chemical Co. (St. Louis, MO) and used at the concentrations recommended to avoid cell toxicity. Calbiochem Novabiochem Corp. (La Jolla, CA) was the source for N,Ndimethylsphingosine (DMS) and bisindolylmaleimide (BIM). Okadaic acid (OA) and genistein were obtained from Euromedex (Souffelweyersheim, France). Porcine LDH A and LDH B probes were provided by Dr. S. S. Li (Laboratory of Genetics, Research Triangle Park, NC), and rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained from Dr. J. M. Blanchard (Faculty of Sciences, Monpellier University, Montpellier, France), respectively. Porcine FSH (USDA pFSHB-1) was provided by Dr. J. A. Proudman (USDA Agricultural Research Service), Animal Hormone Program (Beltsville, MD).

Isolation and culture of Sertoli cells Sertoli cells were isolated from immature porcine testes (2–3 weeks old) using collagenase treatment as described initially by Mather and Phillips (11). Testes were decapsulated, minced, washed in DMEM/F12, and submitted to collagenase dissociation (0.4 mg/ml, 90 –120 min at 32 C). Cells were recovered by mild centrifugation (200 3 g, 10 min), and after 5 min of sedimentation, the pellets of tubules obtained were washed several times by unit gravity in DMEM/F12 medium. Contaminating interstitial Leydig cells were released by a 20-min treatment (at room temperature) with 20 ml 1 m glycine, 2 mm EDTA, and 20 IU/ml deoxyribonuclease in Ca21- and Mg21-free PBS (pH 7.2). Tubules were then washed three times in DMEM/F12 by unit gravity before incubation in DMEM/F12 containing collagenase (0.4 mg/ml) and deoxyribonuclease (0.05 mg/ml) for 30 min at 32 C. The supernatants containing the peritubular myoid cells were removed, and the sedimented tubules were submitted to collagenase treatment as described above (0.4 mg/ml, 30 min, 32 C) until small clumps resulted. Clumps were left to settle, the supernatants were discarded, and Sertoli cells were further washed several times by unit gravity in DMEM/F12. The resulting Sertoli cell populations were free of Leydig and germ cells (12) and contained between 2–5% peritubular myoid cells, as evaluated by fibronectin, desmin, and alcalin phosphatase immunostainings (our unpublished data). Cells were counted in a Coulter counter (Coulter Electronics, Margency, France), plated in Falcon (Los Angeles, CA) 60-mm petri dishes (5 3 106 cells/dish), and cultured at 32 C in a humidified atmosphere of 5% CO2-95% air in DMEM/F12 (1:1) medium containing sodium bicarbonate (1.2 mg/ml), 15 mm HEPES, and gentamicin (20 mg/ml). This medium was supplemented with transferrin (5 mg/ml) and vitamin E (10 mg/ml).

Isolation of RNA and Northern blot hybridization Total RNA was isolated from Sertoli cells cultured in petri dishes using the Trizol reagent, a monophasic solution of phenol and guanidine isothiocyanate. This reagent is an improvement to the single step RNA isolation method developed by Chomczynski and Sacchi (13). Briefly, cells were lysed by adding 1 ml Trizol reagent and passing the cell lysate several times through a pipette. The homogenized samples were incubated for 5 min to permit the complete dissociation of nucleoprotein complexes. Chloroform (200 ml) was then added. After precipitation with isopropanol (500 ml), pellets were washed with 70% ethanol. After solubilization in sterile water, the amount of RNA was estimated by spectrophotometry at 260 nm. About 20 mg total RNA (denatured for 15 min at 65 C in the presence of 2.2 m formaldehyde, 12.5 m formamide, and 1 3 3(N-morpholino)-propanesulfonic acid were loaded on 1.2% agarose-2.2 m formaldehyde gel. After 5 h of migration in 0.02 m 3(N-

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morpholino)-propanesulfonic acid running buffer, RNAs were transferred to nitrocellulose membrane (Hybond-C extra, Amersham, Aylesbury, UK) by capillary transfer with 10 3 SSC (1.5 m NaCl and 0.15 m sodium citrate) and fixed at 80 C for 2 h. The probes used for hybridization were a 1.5-kb Xho-EcoRI porcine LDH A complementary DNA (cDNA), a 1.4-kb XhoI-EcoRI porcine LDH B cDNA, and a 1.3-kb PstI rat GAPDH cDNA. Probes were labeled with 50 mCi [a-32P]deoxyCTP using a random primed DNA labeling kit (SA, 109 dpm/mg DNA; Promega Corp., Charbonnieres, France). The labeled probes were separated from free nucleotides by filtration through a diethylaminoethylcellulose column. After 4 h of prehybridization at 42 C, filters were hybridized with labeled probe (1– 4 3 106 cpm/ml) overnight at 42 C in 50% formamide, 5 3 SSPE (0.9 m NaCl, 0.05 m sodium phosphate, and 5 mm EDTA, pH 7.4), 5 3 Denhardt’s solution (1 g Ficoll, 1 g polyvinylpyrrolidone, and 1 g BSA/liter), 1% SDS, and 100 mg/ml yeast RNA. Afterwards, membranes were washed four times in 2 3 SSC-0.1% SDS (15 min, room temperature), followed by 30 min at 55 C. Filters were exposed to Kodak X-Omat film (Eastman Kodak Co., Rochester, NY) for 1–2 days at 270 C.

Statistical analysis The band densities were determined by scanning densitometric analysis using the BioImage scanner (Millipore Corp., Saint Quentin, France). The amount of RNA in each lane of each blot was internally standardized within a blot by assessing the amount of GAPDH messenger RNA (mRNA) per lane. Experiments were repeated at least three times with independent cell preparations. The statistical significance of the results was determined by Student’s t test when comparing data from three experiments. Data are presented as the mean 6 sd.

Results TNFa enhances LDH A mRNA levels

Sertoli cells were exposed to various TNFa concentrations (0.05–50 ng/ml) for 48 h, and total RNA was extracted. The data in Fig. 1A show that the stimulatory effect of TNFa on LDH A mRNA was dose dependent. It was detectable at a concentration of 0.8 ng/ml (0.04 nm; P , 0.001) and was maximal at 10 –12.5 ng/ml (0.5– 0.6 nm); the half-maximal (ED50) effect was observed at 2.5 ng/ml TNFa (0.1 nm). The stimulatory effect on LDH A mRNA was observed in a timedependent manner. TNFa increased LDH A mRNA levels (Fig. 1B) after 6 h of exposure and was maximal at 48 h (5-fold increase; P , 0.001). The data in Fig. 1C indicate that in similar experimental conditions, TNFa (0.05–50 ng/ml; 48 h) had no effect on LDH B mRNA levels, an observation supporting the specificity of the cytokine action on LDH A mRNA expression. TNFa stimulates LDH A gene transcription

In these series of experiments, we tested whether TNFa may control LDH A gene transcription and/or LDH A mRNA stability. As shown in Fig. 2, the stimulatory effect of TNFa (10 ng/ml, 18 h) on LDH A mRNA levels was completely abolished in the presence of two inhibitors of transcription, Act D (5 mg/ml) and DRB (25 mm), suggesting that TNFa exerts its stimulatory effect through a transcriptional mechanism. To test whether TNFa may also affect LDH A mRNA stability, Sertoli cells were first treated with the cytokine (10 ng/ml) or with OA (20 nm) for 24 h (Fig. 3), used here as a positive control, as this drug has been reported to preferentially affect LDH A mRNA stability in rat C6 glioma cells (14). The transcriptional activity was then inhibited by treating

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TNFa REGULATES LDH A mRNA EXPRESSION

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FIG. 2. The transcription inhibitors Act D and DRB prevent TNFainduced expression of LDH A in Sertoli cells. Sertoli cells were preincubated for 1 h in the presence or absence of Act D (5 mg/ml) or DRB (25 mM). The cells were then incubated for 18 h with TNFa (10 ng/ml) in the continued presence of the respective pharmacological agents. After the indicated times, total cellular RNA was isolated and analyzed by Northern blot with labeled cDNA probes for LDH A or GAPDH. The upper panel shows a representative autoradiogram; the lower panel shows histograms representing the mean 6 SD of three separate experiments.

Sertoli cells with DRB (25 mm). The decay of LDH A mRNA was evaluated at different times (0 –12 h). As shown in Fig. 3, the decrease in LDH A mRNA levels was similar regardless of whether Sertoli cells were treated with TNFa. In both conditions, LDH A mRNA decayed with an apparent halflife of 9 h. By contrast, and as expected, in the presence of OA, LDH A mRNA levels remained stable. Together, these observations clearly suggest that TNFa may control LDH A gene transcription, but not LDH A mRNA stability, although the latter might be positively regulated by OA. These results were further confirmed and extended by the data presented FIG. 1. TNFa enhances LDH A mRNA levels. Cultured Sertoli cells were exposed for 48 h to the indicated concentrations of TNFa. Total cellular RNAs were then extracted, and Northern blotting analysis was performed using 20 mg total RNA. A, Representative autoradiograms of three separate experiments corresponding to specific hybridization with LDH A and GAPDH cDNA probes; the latter was used to standardize LDH A mRNA content. Results are represented as the percentage of LDH A mRNA detected in control (untreated) Sertoli cells. B, Sertoli cells maintained in serum-free medium were exposed, or not, to TNFa (10 ng/ml) for 0 – 48 h. Results for TNFatreated cells are expressed as the percentage of the LDH A mRNA level detected in control Sertoli cells and represent the mean 6 SD of three separate experiments. C, The blot in A was stripped and then rehybridized with LDH B cDNA probe.


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FIG. 3. Effect of TNFa on LDH A mRNA stability. Sertoli cells were incubated in the absence (‚) or presence of TNFa (10 ng/ml; Œ) or OA (20 nM; E) for 24 h, after which 25 mM DRB was added to control and treated cells. For half-life determination, total RNA was isolated from control, TNFa-treated, or OA-treated Sertoli cells at 0, 1.5, 3, 6, 9, and 12 h after the addition of DRB. Twenty micrograms of total RNA from each time point were analyzed by Northern blot with radiolabeled LDH A cDNA probe as described in Materials and Methods. The data are plotted as the percentage of LDH A mRNA remaining relative to that present at time zero. The figure shows a representative pattern of four separate experiments.

in Fig. 4, as the effects of OA and TNFa were additive on LDH A mRNA expression. Indeed, OA enhanced LDH A mRNA expression in a dose-dependent manner (Fig. 4A) with a maximal effect at 30 nm; the ED50 effect was observed at 15 nm OA. When Sertoli cells were treated with OA (20 nm) and TNFa (10 ng/ml, 24 h), the effects of the two factors on LDH A mRNA were additive (Fig. 4B). These observations confirm that OA and TNFa act at different levels (gene transcription vs. mRNA stability) to increase LDH A mRNA amounts in cultured Sertoli cells. Modulation of the stimulatory effect of TNFa on LDH A mRNA by cycloheximide and genistein

To further characterize the potential mechanisms involved in the stimulatory effect of TNFa on LDH A mRNA expression, TNFa-stimulated Sertoli cells (10 ng/ml, 18 h) were incubated in the presence of cycloheximide (20 mg/ml, 18 h) or genistein (10 mg/ml; 18 h). As shown in Fig. 5A, the accumulation of LDH A mRNA in response to TNFa was completely abolished in the presence of cyloheximide, suggesting that de novo protein synthesis was required to mediate the TNFa effect on LDH A mRNA. The data presented in Fig. 5B indicate that the tyrosine kinase inhibitor, genistein, abrogated the stimulatory action of TNFa on LDH A mRNA levels, suggesting an involvement of a tyrosine kinase(s) in the action of the cytokine. Potential involvement of PKC and sphingomyelin pathways in TNFa action on LDH A mRNA

LDH A mRNA was increased after PKC activation in PMA-treated Sertoli cells. Indeed, PMA increased LDH A

FIG. 4. Induction of LDH A mRNA expression by OA in Sertoli cells. A, Sertoli cells were incubated for 24 h with increasing concentrations of OA (0 – 40 nM). Total RNA was extracted and analyzed by Northern blot, as described in Fig 1. B, Sertoli cells were pretreated for 1 h with 20 nM OA followed by a 24-h incubation with 10 ng/ml TNFa. Total RNA was extracted, and Northern blot analysis was performed as described in Materials and Methods. Results are expressed as the mean 6 SD of three separate experiments.

mRNA levels in a dose (Fig. 6A)- and time (Fig. 6B)-dependent manner. To test whether PKC activation is involved in TNFa stimulatory action on LDH A mRNA expression, Sertoli cells were incubated with TNFa (10 ng/ml, 24 h) and/or PMA (50 nm) in the absence or presence of BIM (200 nm), an inhibitor of PKC. As shown in Fig. 6C, and as expected, BIM inhibited the PMA stimulatory effect, whereas it reduced only partly (27.2% decrease; P , 0.01) TNFa-stimulated LDH A mRNA expression. Together, the findings in Fig. 7 indicate that although activation of PKC was capable of increasing LDH A mRNA levels, such an activation accounted only for less than 30% of the stimulatory action of TNFa on LDH A mRNA. As different metabolites resulting from sphingomyelin hydrolysis have been suggested to be involved as intracellular signaling intermediates in TNFa action, we asked whether

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FIG. 5. Effects of cycloheximide and genistein on TNFa-induced LDH A mRNA expression. A, Sertoli cells were stimulated with 10 ng/ml TNFa (lane 2) or pretreated with cycloheximide (20 mg/ml) for 1 h and then stimulated with TNFa (lane 4). The control experiments include cells without treatment (lane 1) or treated with cycloheximide (lane 3) alone for 18 h. B, Sertoli cells were stimulated with 10 ng/ml TNFa (lane 2) for 18 h or pretreated with genistein (10 mg/ml) for 1 h and then stimulated with TNFa (lane 4). The control experiments include cells without treatment (lane 1) or treated with genistein (lane 3) alone. Total cellular RNAs were then extracted, and Northern blot analysis was performed as indicated in Fig. 1. Results are expressed as the mean 6 SD of three separate experiments.

some of them may reproduce the cytokine action on LDH A mRNA expression. Sphingosine appears partly involved in TNFa action, as shown in Fig. 7. Indeed, sphingosine (10 mm) and sphingosine-1-phosphate increased LDH A mRNA levels (2.5-fold increase; P , 0.01). Finally, among the different metabolites resulting from sphingomyelin hydrolysis, including the biologically active ceramide analog (C2-Cer), DMS, and exogenous sphingomyelinase, only sphingosine and sphingosine-1-phosphate were active on LDH A mRNA expression (Fig. 7). In addition to the stimulatory action of the sphingomyelin metabolites on LDH A mRNA, we found an increase in sphingosine production in TNFa-treated Sertoli cells (Grataroli, R., F. Boussouar, and M. Benahmed, unpublished data). Hormonal regulation of LDH A mRNA expression in Sertoli cells

FSH-induced LDH A mRNA expression was observed in a time-dependent manner. FSH increased LDH A mRNA

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FIG. 6. Induction of LDH A mRNA by PMA. A, Sertoli cells were treated for 24 h with different concentrations of PMA (0 –100 nM). B, Sertoli cells were incubated with 50 nM PMA for (0 – 48 h). C, Sertoli cells were preincubated for 1 h in the presence or absence of BIM (200 nM). The cells were then incubated for 24 h with TNFa (10 ng/ml) or PMA (50 nM) in the continued presence of the respective pharmacological agents. After the indicated time, total cellular RNA was isolated and analyzed by Northern blot with labeled cDNA probes for LDH A or GAPDH. For both A and B, the upper panel represents a representative autoradiogram, and the lower panel is a histogram representing the mean 6 SD of three separate experiments.

(Fig. 8A) after 4 h of exposure and was maximal at 48 h (4.6-fold increase; P , 0.001). Sertoli cells were incubated with various concentrations of FSH (0 –1 mg/ml) for 24 h. The data in Fig. 8B show that the effect of FSH on LDH A mRNA was dose dependent; the maximal effect was observed at 250 ng/ml. As shown in Fig. 8C, the decay curves for the 1.5-kb LDH A mRNA transcript in Sertoli cells were different in the absence and presence of FSH. FSH enhanced LDH A mRNA expression by enhancing mRNA stability. Discussion

In this report, we have examined the TNFa-dependent activation of LDH A mRNA expression by using primary cultures of purified Sertoli cells isolated from porcine testes. We present evidence that this expression was up-regulated by TNFa. The action of the cytokine on the LDH isozymes appears specific, in that it affects only the expression of LDH A, not that of LDH B. Such an increase in LDH A mRNA after TNFa treatment, as we report here, corresponds to a func-


FIG. 7. Effect of sphingomyelin metabolites on LDH A mRNA expression in Sertoli cells. The cultured Sertoli cells were incubated in the presence or absence of 10 ng/ml TNFa, 10 mM sphingosine, 10 mM sphingosine-1-phosphate, 5 mM C2-Cer, 10 mM DMS, or 0.5 IU/ml exogenous sphingomyelinase for 48 h. After the indicated time, total cellular RNA was isolated and analyzed by Northern blot with labeled cDNA probes for LDH A and GAPDH.

tional increase in LDH A4 protein and activity (which results in lactate release), as we previously reported (10). The magnitude of as well as the steady increase in the TNFa-induced enhancement of LDH A mRNA over 48 h suggests that this effect is probably indirect and that de novo synthesis of protein might be required as cycloheximide abrogated TNFadependent LDH A mRNA accumulation in Sertoli cells. In the present study, we reported that 1) transcription inhibitors such as actinomycin D and DRB blocked the induction of LDH A mRNA by TNFa; and 2) the decay of LDH A mRNA levels was similar regardless of whether Sertoli cells were treated with TNFa. Together, these findings indicate that the cytokine induced expression of LDH A mRNA through a transcriptional mechanism. Further studies based, for example, on pulse-chase or the nuclear run-on assays, have to be performed to confirm the direct transcriptional regulatory action by TNFa on the ldh a gene. TNFa enhanced LDH A mRNA levels in a nanomolar concentration range. Such a concentration is consistent with the amounts of TNFa reported to be secreted in the Sertoli cell environment (15) and with the Kd range previously observed for TNFa receptors in this cell type (16). These observations suggest that the effect of the cytokine on LDH A mRNA is probably exerted via a TNFa receptor(s), particularly via the p55 form, which has been reported to be present in Sertoli cells (15, 16). There is now general agreement that TNFa on binding to its receptor(s) activates different intracellular signaling pathways, including protein kinase C (PKC), protein kinase A (PKA), and sphingomyelinase. The intracellular transducing pathways involved in the TNFa-

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enhanced LDH A mRNA expression remain to be identified. It is possible that the PKC pathway might be partly involved in the stimulatory action of TNFa on LDH A mRNA levels, as suggested by the partial reduction of the cytokine action on LDH A mRNA by the PKC inhibitor, BIM. Although the specificity of BIM’s action on PKC remains to be confirmed, and direct evidence that TNFa activates PKC activity in Sertoli cells remains to be demonstrated, such a possibility is plausible in view of 1) the fact that TNFa activates and translocates PKC from the cytosol to the cell membrane in other cell types (17); and 2) the stimulatory action of PMA on LDH A mRNA levels (Refs. 14 and 18 and our present data). Recent studies have identified another pathway for TNFa signaling that involves the production of ceramide and other metabolites resulting from sphingomyelin hydrolysis (19 – 22). That TNFa may use the sphingomyelinase pathway (at least partly) to stimulate LDH A expression in Sertoli cells is based on two observations: 1) TNFa action was partly mimicked by sphingosine and sphingosine-1-phosphate (but not by ceramide and DMS); and 2) TNFa stimulates sphingosine production in cultured porcine Sertoli cells (Grataroli, R., F. Boussouar, and M. Benahmed, unpublished data). In addition to the PKC and sphingomyelinase pathways, TNFa may well use other signaling pathways in Sertoli cells. Indeed, it has recently been reported that in mouse Sertoli cells, TNFa induced interleukin-6 (IL-6) production and integrin ligand expression through p38 and JNK/SAPK (activated c-Jun Nterminal protein kinase/stress-activated protein kinase) pathways (23, 24). Although there is increasing evidence that induction of gene expression by TNFa is mediated through activation of trans-acting DNA-binding nuclear proteins, the mechanisms through which TNFa activates nuclear transcription factors potentially involved in LDH A mRNA expression are unknown. TNFa induced the expression of several target genes through activation of not only a number of transcription factors, including nuclear factor-kB and nuclear factor-IL-6, but also multiple response factors, including interferon response factor-1, interferon response factor-2, c-Fos, c-Jun, and c-Myc (25). Previous studies have identified the presence of cAMP- as well as 12-O-tetraphorbol 12-myristate 13-acetate-responsive elements in the LDH A promoter that regulate promoter function (14, 18, 26 –28). The cAMP response element recognizes hormone-inducible trans factors in rat ovarian cells (29). cAMP also induces expression of LDH A in rat glioma cells (30). The cAMP response element in LDH A promoter is probably involved in the stimulatory effect on Sertoli cell LDH A mRNA expression of hormones that classically use the cAMP/PKA pathway, such as FSH. We have shown here that FSH increases LDH A mRNA levels in cultured Sertoli cells. In addition to a potential regulatory mechanism at the transcriptional level, which, however, remains to be demonstrated, we show that FSH increases LDH A mRNA stability. Such an observation is compatible with that of Tian and co-workers, who recently identified a region within the 39-untranslated region with a 2-fold function: 1) it acts as a U-rich instability element; and 2) it functions specifically as a dominant stabilizer of LDH A mRNA half-life in response to activation of the PKA signal transduction (31). Both transcriptional and translational events appear to be

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FIG. 8. Effect of FSH on LDH A mRNA expression in Sertoli cells. A, Sertoli cells were incubated with 200 ng/ml FSH for (0 – 48 h). B, Sertoli cells were treated for 24 h with different concentrations of FSH (0 –1 mg/ml). C, Sertoli cells were incubated in the absence (‚) or presence of FSH (200 ng/ml; F) for 24 h, after which 25 mM DRB was added to control and treated cells. For mRNA half-life determination, total RNA was isolated from control or FSH-treated Sertoli cells at 0, 3, 6, 9, and 12 h after the addition of DRB. Twenty micrograms of total RNA from each time point were analyzed by Northern blot with radiolabeled LDH A cDNA probe as described in Materials and Methods. The data are plotted as the percentage of LDH A mRNA remaining relative to that present at time zero. The figure shows a representative pattern of three separate experiments.

important in determining the levels of induced gene expression under the control of TNFa. The effects of TNFa on the stability of many unstable mRNAs have been studied in different experimental conditions. Such inducible expression was shown to occur for IL-I (32), glucose transporter-1 (33, 34), and b2-adrenoreceptor (35) mRNAs. However, based on our present findings, it is of interest that TNFa may increase some mRNAs levels without affecting their stability. Indeed, in the present study, TNFa appears to act on LDH A transcription, but not on LDH A mRNA stability, although such a mRNA was potentially stabilized by okadaic acid (Ref. 14 and our present data). As OA, a potent additional tumor promoter, acts through specific inhibition of protein phosphatase-1 and -2A (for review, see Ref. 36), we might speculate that these phosphatases are probably not targeted by TNFa in our experimental model. The involvement of cisacting elements in the promoter and 39,59-untranslated regions of the LDH A gene in TNFa and FSH regulation of LDH A mRNA will be investigated using reporter constructs to further characterize the modes of action of these factors. It remains possible that TNFa action on LDH A mRNA involves some intermediates, such as growth factors or cytokines coupled to their intracellular transducing pathways. Indeed, that TNFa action on LDH A expression was suppressed by genistein suggests that the cytokine action may involve tyrosine kinase(s) activity. Although such a kinase(s) remains to be identified, there are some candidates and among them are receptor tyrosine kinases such as epidermal growth factor (EGF)/transforming growth factor-a (TGFa) receptors whose activation triggered a cascade of kinase activation. In this context, it has been found that treatment of cultured pancreatic carcinoma cells with TNFa induced ex-

pression of EGF receptor and its ligand TGFa (37). Induction of EGF and TGFa mRNA by TNFa has been also previously described in human malignant epithelial cells (38). More recently, it has been shown that TNFa stimulated EGF receptor in testicular (untransformed) peritubular myoid cells (39). In testicular Sertoli cells, it is possible that the system EGF/TGFa/EGF receptor may mediate TNFa action on LDH A expression, as 1) EGF, TGFa, and EGF receptor are expressed in Sertoli cells (40); and 2) EGF is able to increase LDH A expression in cultured Sertoli cells (our unpublished data). TNFa may also act through other cytokines, such as IL-6. Indeed, it has been demonstrated that in cultured mouse Sertoli cells, TNFa treatment increased IL-6 production (23, 24). We have investigated the hypothesis that the observed TNFa effects on LDH A mRNA expression were exerted via IL-6 production. We found that IL-6 had no effect on LDH A mRNA expression or LDH A4 activity and lactate production (our unpublished data). In the physiological context, lactate may play a key role in at least two conditions. Firstly, lactate is used as an energy substrate, particularly in different tissues, including the early embryo, the gonads, and the brain (6, 7). In the testes, the concept that Sertoli cells metabolize glucose to lactate for the use of germ cells arose because of the capability of cultured Sertoli cells to produce high amounts of lactate and the efficient use of lactate, but not glucose, by germ cells. These observations have led to the concept that one of the nurse cell functions of the Sertoli cells is to provide lactate for energy production in spermatocytes and spermatids (for review, see Ref. 5). A similar metabolic cooperation involving lactate as an energetic metabolite occurs in the brain, particularly between astrocytes and neurons, where astrocytes play a role


comparable to that of testicular Sertoli cells (6, 7). Our present findings, demonstrating that TNFa (produced in spermatids) (15) stimulates the expression of LDH A, make this enzyme expression a key target in the metabolic cooperation between germ cells and Sertoli cells in the male gonad. It will be of interest to determine whether the cytokine also plays this role in other tissues, such as the brain. Secondly, besides its role as an exchangeable metabolic fuel, lactate, by creating an acidic microenvironment, may influence the mode of expression of certain genes, particularly the alternative splicing of pre mRNAs. Indeed, we have recently shown that in Sertoli cells, stem cell factor pre-mRNA splicing might be affected by the acidic microenvironment that results from the high amounts of lactate. Indeed, in mouse Sertoli cells, lactate was shown to favor the switch of SCF splicing to the membrane form of SCF, a form that can promote germ cell survival and proliferation (spermatogonia type A) (41). Finally, in pathology, cancer cells are also able to overproduce lactate aerobically. Alterations of the glycolytic pathway, including elevation of LDH, are thought to be hallmarks of cancer cells, which are able to produce lactate aerobically, a phenomenon known as the Warburg effect (42). The evaluation of LDH levels appeared useful in distinguishing between benign and malignant tumors, in predicting the response to therapy, and in judging prognosis (43). Although the exact role of LDH A in tumorogenesis remains to be clarified, it is of interest to note that LDH A has been reported to be a direct c-Myc-responsive gene that is involved in c-Myc-mediated cell transformation (44). Based on our present data indicating that TNFa enhances LDH A expression, it is tempting to speculate that the increase in LDH A expression in tumors might be related to an increase in the expression of some cytokines, such as TNFa, and/or their receptors in these tumors. However, although the expression of the cytokines and their receptors has been studied in different types of tumors, there are, to our knowledge, no published data reporting the coexpression of both LDH isozymes and TNFa ligand and receptors in the same tumors. In summary, using as a model cultured testicular Sertoli cells, we reported here that TNFa up-regulates LDH A mRNA levels through a transcriptional activity, but not through mRNA stabilization. Additionally, our data indicated that TNFa may involve both PKC and the sphingomyelin hydrolysis signaling pathways in stimulating LDH A expression. References 1. Sharpe RM 1994 Regulation of spermatogenesis. In: Knobil E, Neil JD (eds) The Physiology of Reproduction. Raven Press, New York, vol 1:1363–1434 2. Griswold MD 1995 Interactions between germ cells and Sertoli cells in the testis. Biol Reprod 52:211–216 3. Benahmed M 1996 Growth factors and cytokines in the testis. In: Comhaire FH (ed) Male Infertility. Chapman and Hall, London, pp 55–97 4. Gnessi L, Fabbri A, Spera G 1997 Gonadal peptides as mediators of development and functional control of the testis: an integrated system with hormones and local environment. Endoc Rev 18:541– 609 5. Grootegoed JA, Den Boer P 1987 Energy metabolism of spermatids: a review. In: Hamilton DW, Waites GMH (eds) Cellular and Molecular Events in Spermiogenesis. Cambridge University Press, Cambridge, pp 193–216 6. Pellerin L, Magistretti PJ 1994 Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization. Proc Natl Acad Sci USA 91:10625–10629 7. Pellerin L, Stolz M, Sorg O, Martin JL, Deschepper CF, Magistretti PJ 1997

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The Second Annual International Motor City Diabetes Symposium “From Beta Cell Dysfunction to Curing Type 1 Diabetes” Detroit, Michigan, October 29 –30, 1999 Organized by Wayne State University Comprehensive Diabetes Center. For information about the meeting, registration, and abstract submission, contact George Grunberger, M.D., at: [email protected], or visit the website: http://www.med.wayne.edu/diabetes/symposium99.