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a Unité 407 de l'Institut National de la Santé et de la Recherche Médicale (INSERM U407), Faculté de Médecine Lyon-Sud, BP12, F-69921 Oullins Cedex, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) [8]. In a previous study, we have shown that lactate dehydrogenase (LDH) A4 activity may represent a key step in the stimulatory action of EGF on lactate production in cultured Sertoli cells [7]. LDH is formed by random association of four subunits to make homo- or heterotetramers. In mammals, these subunits are encoded by three loci: Ldh-a, Ldh-b, and Ldh-c [9, 10]. The C4 isozyme is unique among the LDH isozymes with respect to its restricted distribution within the germinal epithelium of the mammalian testes (reviewed in [11]), while LDH A and B subunits are ubiquitous in their occurrence and are found at characteristic levels in different tissues [9]. 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 [12].
In the study reported here, we extended and completed our previous observations demonstrating that the increase in LDH A4 activity was related to an increase in LDH A mRNA expression. We further characterized the mechanisms involved in EGF action on LDH A mRNA and compared these actions to those of other regulators of Sertoli cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dulbecco's modified Eagle's (DME)/Ham's F-12 medium (DME/F12) and Trizol reagent were obtained from Life Technologies (Cergy Pontoise, France). Collagenase/dispase was obtained from Boehringer (Mannheim, Germany). Actinomycin D (Act D), 5,6-dichloro-1-ß-ribofuranosyl benzimidazole (DRB), phorbol 12-myristate 13-acetate (PMA), and cycloheximide were purchased from Sigma Chemical Co. (St. Louis, MO) and used at the concentrations recommended to avoid cell toxicity. Calbiochem-Novabiochem Corporation (La Jolla, CA) was the source for bisindolylmaleimide (BIM). Porcine LDH A probe was kindly provided by Dr. S.S. Li (Laboratory of Genetics, Research Triangle Park, NC), rat glyceraldhyde 3-phosphate dehydrogenase (GAPDH) by Dr. J.M. Blanchard (Faculty of Sciences, Montpellier, France), and the 1.1-kilobase (kb) BamHI/EcoRI 18 S rRNA by Dr. Ann Ferguson (The University of Texas Medical Branch at Galveston).
Sertoli Cell Culture
Sertoli cells were isolated from immature porcine testes (2–3 wk old) using collagenase treatment as previously described [13]. Briefly, decapsulated testes were cut and then digested with 0.4 mg/ml collagenase (90–120 min at 32°C). Contaminating interstitial Leydig cells were released by a 20-min treatment with 1 M glycine, 2 mM EDTA, and 20 IU/ml deoxyribonuclease (DNase) in Ca2+-, Mg2+-free PBS (pH 7.2). Seminiferous tubules were then washed before incubation in DME/F12 containing collagenase (0.4 mg/ml) and DNase (0.05 mg/ml) for 30 min at 32°C to remove peritubular myoid cells. The supernatants were removed, and the tubular pellet was submitted to collagenase treatment as described above (0.4 mg/ml, 30 min, 32°C). Clumps were left to settle, the supernatants were discarded, and Sertoli cells were further washed several times. The resulting Sertoli cell populations were free of Leydig and germ cells [14] and contained between 2% and 5% peritubular myoid cells as evaluated by fibronectin, desmin, and alkaline phosphatase immunostaining (unpublished data).
Cells were counted in a Coulter counter (Coulter Electronics, Margency, France), plated in Falcon (Los Angeles, CA) 60-mm Petri dishes (5 x 106 cells per dish), and cultured at 32°C in a humidified atmosphere of 5% CO2, 95% air in DME/F12 (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). Cells were allowed to attach during an initial 24-h period in the presence of 0.5% of fetal calf serum.
RNA Isolation and Northern Blot Analysis
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 on the single-step RNA isolation method developed by Chomczynski and Sacchi [15]. Briefly, cells were lysed by adding 1 ml of 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 was then added. After precipitation with isopropanol, pellets were washed with 70% ethanol. After solubilization in sterile water, the amount of RNA was estimated by spectrophotometry at 260 nm. For Northern blot analysis, about 20 µg total RNA from each dish was separated by electrophoresis on denaturing agarose gels and subsequently transferred to a nitrocellulose membrane (Hybond-C extra; Amersham, Little Chalfont, Bucks, UK). The blots were prehybridized for 4 h at 42°C and then hybridized overnight at 42°C with labeled probes (1–4 106 cpm/ml). Afterwards, membranes were washed four times in double-strength SSC (single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate), 0.1% SDS (15 min, room temperature), and then washed for 30 min at 55°C in 0.1-strength SSC, 0.1% SDS. Filters were exposed to Kodak X-OMAT films (Eastman Kodak, Rochester, NY) for 1–2 days at -70°C.
LDH A mRNA Stability Analysis
Cells were preincubated with or without EGF for 24 h before the addition of 25 µM DRB, to arrest new RNA synthesis. Cells were harvested at 0, 3, 6, 9, and 12 h after addition of DRB for RNA extraction and Northern analysis as described above.
The bands were quantified with a Bio Image densitometer (Millipore SA, Saint Quentin, France), normalized against a corresponding relative amount of 18 S mRNA in each sample, and expressed as relative densitometric units. The data are presented as mean ± SD of measurements from triplicate separate experiments. Differences between control and treated cells were assessed by Student's t-test for independent samples.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The stimulatory effect on LDH A mRNA was observed in a time- and dose-dependent manner. Indeed, EGF increased LDH A mRNA levels (Fig. 1A) after 12 h of exposure and was maximal at 36 h (2.9-fold induction, P < 0.001). Sertoli cells were exposed to various EGF concentrations (1–30 ng/ml) for 24 h, and total RNA was extracted. The data in Figure 1B show that the stimulatory effect of EGF on LDH A mRNA was dose-dependent as it was detectable at a concentration of 1 ng/ml EGF (P < 0.05) and was maximal at 10 ng/ml. The ED50 was 4 ng/ml (0.63 x 10-9 M EGF). The data in Figure 1 show that EGF treatment of Sertoli cells resulted in a (time- and dose-dependent) increase in mRNA coding for another glycolytic enzyme, GAPDH, usually used as a reporter gene. For this reason, 18 S (but not GAPDH) has been used in the present study as a reporter gene.
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Effect of EGF on LDH A Gene Expression and LDH A mRNA Half-Life
In this series of experiments, we tested the possibilities that EGF may control LDH A gene transcription and/or LDH A mRNA stability. As shown in Figure 2, the stimulatory effect of EGF (10 ng/ml, 12 h) on LDH A mRNA levels was completely abolished in the presence of two RNA synthesis inhibitors, Act D (5 µg/ml) and DRB (25 µM), suggesting that EGF probably exerts its stimulatory effect through a transcriptional mechanism.
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In order to assess the rates of degradation of LDH A mRNA transcripts, Sertoli cells were preincubated in the absence (control) or presence of EGF (10 ng/ml) for 24 h (Fig. 3). The transcriptional activity was then inhibited by treating Sertoli cells with DRB (25 µM). Cells were harvested at 0, 3, 6, 9, and 12 h after addition of DRB, and LDH A mRNA levels were quantified by Northern blot analysis. As shown in Figure 3, the decrease in LDH A mRNA levels was similar whether or not Sertoli cells were treated with EGF. In both conditions, LDH A mRNA decayed with an apparent half-life of 9 h. These observations suggest that EGF may control LDH A gene transcription but not LDH A mRNA stability.
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Requirement for a New Protein Synthesis for the Effect of EGF on LDH A mRNA Levels
To determine whether a new protein synthesis was required for the increase in LDH A mRNA levels induced by EGF, Sertoli cells were treated in the absence or presence of the protein synthesis inhibitor cycloheximide (CHX, 20 µg/ml), then treated with EGF (10 ng/ml) and harvested 12 h after treatment. Although CHX treatment alone did not affect LDH A mRNA level (Fig. 4), EGF caused a significant increase (P < 0.01), and this effect was abolished when CHX was present (P < 0.01). These results indicate that the EGF-stimulated LDH A mRNA level required a new protein synthesis.
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Involvement of Protein Kinase C (PKC) in the Effect of EGF on LDH A mRNA Levels
As the activation of PKC, which might be involved in EGF action [16], was reported to increase the LDH A mRNA level [17], we investigated the possible involvement of PKC in mediating the EGF-induced LDH A mRNA level. BIM, an inhibitor of PKC, was employed in order to test whether it affected EGF (10 ng/ml)-induced LDH A mRNA, in comparison to PMA (50 nM)-stimulated LDH A mRNA (a positive control experiment). As shown in Figure 5, BIM (100 nM), as expected, impaired the PMA effect (P < 0.01) and reduced the EGF effect on LDH A mRNA expression by about 50% (P < 0.001).
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Interactions Between EGF, Hormones (FSH), and Cytokines (TNF-, Interleukin [IL] 1) in Regulating LDH A mRNA Expression
To examine the potential additive effects of TNF-, IL-1, FSH, and EGF on LDH A mRNA levels, Sertoli cells were cultured with 10 ng/ml EGF, 10 ng/ml TNF-, 2 ng/ml IL-1, and 200 ng/ml FSH. In combination with the other factors, EGF treatment resulted in a stimulation of LDH A mRNA to a level that was significantly (P < 0.05) greater than that observed with each factor alone (Fig. 6, A and B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene. The magnitude as well as the steady increase of the EGF-induced enhancement of LDH A mRNA over 36 h suggests that this effect is probably indirect and that de novo synthesis of protein might be required as CHX abrogated EGF-dependent LDH A mRNA accumulation in Sertoli cells. We report in the present study 1) that RNA synthesis inhibitors Act D and DRB blocked the induction of LDH A mRNA by EGF and 2) that the decay curves for the 1.5-kb LDH A mRNA transcript in Sertoli cells were not different in the absence or presence of EGF. These findings clearly indicate that EGF stimulated LDH A mRNA expression at a transcription level. That BIM, an inhibitor of PKC, partly reduced the level of LDH A mRNA stimulated by EGF suggests that this action is mediated (at least partly) by the protein kinase.
This study shows that EGF enhances LDH A mRNA levels in the nanomolar concentration range. This observation suggests that the stimulatory action of EGF potentially occurs within a concentration range that might be expected under physiological conditions. By using an immunohistochemistry approach, we have previously localized EGF and TGF predominantly in the porcine testicular somatic cells, while EGF receptor (EGFR) was present in somatic and germ cells during postnatal development [18]. In the rat testis, immunoreactive EGF precursor (Mr 140 000) and EGF (Mr 6000) have been identified in germ cells, and in Sertoli cells and germ cells, respectively [19]. EGF has been reported to influence Sertoli cells, Leydig cells, and peritubular myoid cells by affecting several specific parameters (reviewed in [20, 21]). Specifically, nanomolar concentrations of EGF induced a 2-fold increase of lactate production in rat [22] and porcine [5] Sertoli cells. The original in vivo observation by Tsutsumi et al. [23] suggesting that submandibular gland EGF may control testicular function in an endocrine fashion has suggested the possible role of circulating and (intratesticular) EGF in testicular physiology. These authors reported that removal of the salivary glands of the mouse results in a decrease of circulating EGF to an undetectable level, with a concomitant impairment of spermatogenesis not accompanied by a modification of the circulating levels of testosterone or FSH. These alterations of spermatogenesis can be reversed by administration of EGF.
To exert its different effects, EGF utilizes different intracellular transducing systems. Although we do not know at the present time which of these systems are triggered to enhance LDH A mRNA level, there are several possibilities. It is well known that EGF acts on the target cells after binding to a specific membrane receptor. After ligand binding, EGFR undergoes dimerization, and the intracellular protein tyrosine kinase domain of the receptor becomes activated and the receptor becomes autophosphorylated. Consequently, EGFR is internalized and undergoes a destruction in endosomes or may recycle to the cell surface. EGF accelerates glucose consumption, and this effect has been related to its ability to enhance glucose uptake into the cells [5, 24]. Baulida et al. [25] have suggested that calcium cations play an important role in the regulation of glycolysis by EGF in A431 cells. This change correlated with an increase in phosphofructokinase-2 activity, which was not due to a change in the transcription or in the translation of the enzyme. It is well known that EGF by itself does not alter the basal level of cAMP in A431 cells [26]. On the other hand, some authors have related the metabolic effects of EGF to a mechanism that involves PKC as a part of the transduction pathway. Farese et al. [16] have suggested that the increase in 2-deoxyglucose uptake in myocytes is mediated through the activation of PKC by diacylglycerol. The mediation of PKC in the stimulation of lactate production by EGF in isolated hepatocytes was reported by Conricode and Ochs [27]. These authors also suggested that an increase in fructose-2,6-biphosphate might explain this effect, at least in part. Our present results indicating that EGF may use PKC as part of the transduction pathway is consistent with these observations. Nevertheless, direct evidence that EGF stimulates PKC activity in Sertoli cells still remains to be demonstrated.
Our present data suggest that EGF enhances LDH A mRNA expression by acting at a transcriptional level. EGF increases the transcriptional activities in various genes; expression of the ovine P-450 side chain cleavage enzyme gene (CYP11A1) is stimulated by EGF through the Activating Protein-1 (AP1)-binding site [28]. An AP1-binding site in the c-fos gene can mediate the induction of the c-fos gene by EGF in HeLa cells [29]. It is possible that EGF stimulates LDH A mRNA expression through enhancement of PKC activity and the AP1-binding site. Previous studies have identified the presence of cAMP- as well as 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive elements in the LDH A promoter, which regulate promoter function [30, 31]. The cAMP-responsive element in the LDH A promoter is probably involved in the stimulatory effect of FSH on cultured Sertoli cell LDH A mRNA expression that we recently reported [13]. Tian and coworkers [32, 33] have identified a region (bases 1478–1499) within the 3' untranslated region (3'UTR) in rat C6 glioma cells. This sequence acts as a uridine-rich instability element, and it functions specifically as a dominant stabilizer of LDH A mRNA half-life in response to activation of the protein kinase A signal transduction pathway. It has been demonstrated that differences in stability of primate and mouse LDH C transcripts in vitro and in tissue culture cell exist, and that the adenine-uridine-rich 3'UTR of primate LDH C transcript was involved in the modulation of mRNA stability [34]. Such an observation could be applied to LDH A transcript because the LDH A mRNA half-life estimated in rat C6 glioma cells and in porcine Sertoli cells are quite different (55 min vs. 9 h, respectively). Estimates of mRNA half-lives and stability using transcription inhibitors such as Act D and DRB must always be viewed with caution. Nevertheless, numerous studies have used this approach to assess the effects of hormones, drugs, and signaling molecules on the stability of a variety of mRNAs [35]. In general, the data obtained with Act D studies have been concordant with the results obtained from pulse-chase studies [36]. The relative magnitudes of the changes in half-lives (induced by hormones or other intermediates in signaling molecules such as cAMP) detected by the two methods are usually quite similar.
Several studies from our laboratory have indicated that lactate production in Sertoli cells is under the control of different signaling molecules, including hormones such as FSH and insulin-like growth factor I, growth factors, and cytokines [20]. The local control, which is probably specific to the steps of the spermatogenic cycle, is more appropriate than the endocrine system in that it better takes into account the specific germ cell metabolic requirements. Indeed, the hormones such as FSH may act during the onset of puberty while the growth factors may act later in the more mature testes. Thus, it is possible that the postmeiotic germ cells and the somatic Sertoli cells, via the production of cytokines and growth factors, may control and direct the glucose metabolism in Sertoli cells toward the production of lactate, a key energy metabolite for germ cells. That the effects of these different factors, including EGF, TNF-, IL-1, and FSH, were additive suggests that these factors may act at different levels in stimulating LDH A mRNA expression. Such an additivity might be explained, at least for FSH and EGF, by the fact that the growth factor appears to stimulate the gene transcription (the present study) while the hormone stabilizes the transcript [13]. Furthermore, it is of interest to note that a high glycolytic rate even under aerobic conditions involving an increase in expression of several glycolytic enzymes has been reported in proliferating cells and tumor cells. For example, Greiner et al. [37] provided evidence that mitogen-stimulated rat thymocytes in vitro induced their glycolytic enzyme activities (hexokinase, 6-phosphofructo-1-kinase, pyruvate kinase, and lactate dehydrogenase) 8- to 10-fold in the S-phase of the cell cycle, which drives lactate formation. The transition from oxidative to glycolytic energy metabolism occurs as thymocytes undergo proliferation. On the basis of our present observations, EGF-treated Sertoli cells appear to exhibit a metabolic pattern with a high aerobic glycolytic rate that resembles that of proliferating cells. Because Sertoli cells do not proliferate in the adult testis, we suggest that the metabolic pattern they express is to fulfill the great demand of energy from germ cells in the context of Sertoli cell-germ cell metabolic cooperation.
Lactate is also an important energy metabolite in other normal tissues such as the embryo, the ovary, the eye, and the brain. In the brain, a metabolic cooperation involving lactate between neurons and astrocytes (astrocytes play a comparable supporting role to that of Sertoli cells) has been reported by Pellerin and Magistretti [38]. In the eye, lactate produced in the Müller cells by glycolysis is transported out of the cells and used by the photoreceptor cells to fuel oxidative phosphorylation [39].
Finally, we wish to mention that, although in the present study lactate was viewed only as an energy substrate, it is possible that this metabolite plays another crucial role in the testis physiology. Indeed, lactate, by creating an acidic microenvironment, may influence the mode of expression of certain genes, particularly the alternative splicing of some pre-mRNAs. In a recent study, we have shown that in mouse Sertoli cells, stem cell factor (SCF) pre-mRNA splicing might be affected by high concentrations of lactate. Lactate was shown to favor the switch of SCF splicing to the membrane form, a form that can promote germ cell survival and proliferation [40].
In summary, we have examined the regulation by EGF of the expression of LDH A transcript in cultured Sertoli cells and presented evidence that EGF up-regulates LDH A mRNA level, probably through a transcriptional mechanism in which PKC seems to be at play, at least partly, in mediating EGF action. These effects are exerted in the context of Sertoli cell-germ cell metabolic cooperation.
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FOOTNOTES |
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2 Correspondence. FAX: 33 4 78 86 31 16; benahmed{at}lsgrisn1.univ-lyon1.fr
Accepted: May 19, 1999.
Received: February 16, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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on lactate production in porcine Sertoli cells: glucose transport and lactate dehydrogenase isozymes as potential sites of action. Mol Cell Endocrinol 1993; 92:45–53.[CrossRef][Medline]-stimulated lactate production is linked to lactate dehydrogenase A expression and activity increase in porcine cultured Sertoli cells. Endocrinology 1997; 138:1964–1971. stimulates lactate dehydrogenase A expression in porcine cultured Sertoli cells: mechanisms of action. Endocrinology 1999; 140:3054–3062. and their receptor during postnatal development and spermatogenesis of the boar testis. Mol Cell Endocrinol 1997; 123:61–69.This article has been cited by other articles:
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  F. Boussouar, C. Mauduit, E. Tabone, L. Pellerin, P. J. Magistretti, and M. Benahmed Developmental and Hormonal Regulation of the Monocarboxylate Transporter 2 (MCT2) Expression in the Mouse Germ Cells Biol Reprod, September 1, 2003; 69(3): 1069 - 1078. [Abstract] [Full Text] [PDF]
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 S. Short, D. Tian, M. L. Short, and R. A. Jungmann Structural Determinants for Post-transcriptional Stabilization of Lactate Dehydrogenase A mRNA by the Protein Kinase C Signal Pathway J. Biol. Chem., April 21, 2000; 275(17): 12963 - 12969. [Abstract] [Full Text] [PDF]
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