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Stimulates Lactate Dehydrogenase A Expression in Porcine Cultured Sertoli Cells: Mechanisms of Action
INSERM, U-407, Faculté de Médecine Lyon-Sud, F-69921 Oullins Cedex, France
Address all correspondence and requests for reprints to: Dr. Mohamed Benahmed, INSERM U-407, Faculté de Médecine Lyon-Sud, BP 12, F-69921 Oullins Cedex, France. E-mail: benahmed{at}lsgrisn1.univlyon1.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF) 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. TNF
stimulated LDH A messenger RNA (mRNA) expression in a dose
(ED50 = 2.5 ng/ml; 0.1 nM TNF)-dependent
manner. This stimulatory effect was time dependent, with an effect
detected after 6 h of TNF treatment and maximal after 48 h
of exposition (5-fold; P < 0.001). The direct
effect of TNF on LDH A mRNA could not be accounted for by an
increase in mRNA stability (half-life = 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
completely (actinomycin D, dichlorobenzimidazole riboside,
cycloheximide, and genistein) or partially (bisindolylmaleimide)
TNF-induced LDH A mRNA expression. These observations suggest that
the stimulatory effect of TNF 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. |
Introduction |
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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 (NAD+) 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 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- (10); and
2) a redistribution of LDH isoforms occurs under tumor necrosis
factor- (TNF) 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
TNF on LDH A expression in Sertoli cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x
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,N-dimethylsphingosine (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 pFSH-B-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 x
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 Ca2+- and
Mg2+-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 x 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 µg/ml). This medium was supplemented with transferrin (5 µg/ml) and vitamin E (10 µg/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 µl) was then added. After
precipitation with isopropanol (500 µl), pellets were washed with
70% ethanol. After solubilization in sterile water, the amount of RNA
was estimated by spectrophotometry at 260 nm. About 20 µg total RNA
(denatured for 15 min at 65 C in the presence of 2.2 M
formaldehyde, 12.5 M formamide, and 1 x
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-morpholino)-propanesulfonic acid
running buffer, RNAs were transferred to nitrocellulose membrane
(Hybond-C extra, Amersham, Aylesbury, UK) by capillary
transfer with 10 x 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
µCi [-32P]deoxy-CTP using a random primed DNA
labeling kit (SA, 109 dpm/µg DNA; Promega Corp., Charbonnieres, France). The labeled probes were separated
from free nucleotides by filtration through a
diethylaminoethyl-cellulose column. After 4 h of prehybridization
at 42 C, filters were hybridized with labeled probe (1–4 x
106 cpm/ml) overnight at 42 C in 50% formamide, 5 x
SSPE (0.9 M NaCl, 0.05 M sodium phosphate, and
5 mM EDTA, pH 7.4), 5 x Denhardt’s solution (1 g
Ficoll, 1 g polyvinylpyrrolidone, and 1 g BSA/liter), 1%
SDS, and 100 µg/ml yeast RNA. Afterwards, membranes were washed four
times in 2 x 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 -70
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 ± SD.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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enhances LDH A mRNA levels 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 TNF 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 TNF
(0.1 nM). The stimulatory effect on LDH A mRNA was observed
in a time-dependent manner. TNF 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, TNF (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.
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stimulates LDH A gene transcription may
control LDH A gene transcription and/or LDH A mRNA stability. As shown
in Fig. 2, the stimulatory effect of
TNF (10 ng/ml, 18 h) on LDH A mRNA levels was completely
abolished in the presence of two inhibitors of transcription, Act D (5
µg/ml) and DRB (25 µM), suggesting that TNF exerts
its stimulatory effect through a transcriptional mechanism.
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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
Sertoli cells with DRB (25 µM). 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 TNF. In both
conditions, LDH A mRNA decayed with an apparent half-life 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
TNF 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
in Fig. 4, as the effects of OA and
TNF 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 TNF (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 TNF act at different levels (gene transcription
vs. mRNA stability) to increase LDH A mRNA amounts in
cultured Sertoli cells.
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on LDH A mRNA by
cycloheximide and genistein on LDH A mRNA expression,
TNF-stimulated Sertoli cells (10 ng/ml, 18 h) were incubated in
the presence of cycloheximide (20 µg/ml, 18 h) or genistein (10
µg/ml; 18 h). As shown in Fig. 5A, the accumulation of LDH A mRNA in response to TNF was completely
abolished in the presence of cyloheximide, suggesting that de
novo protein synthesis was required to mediate the TNF effect
on LDH A mRNA. The data presented in Fig. 5B indicate that the tyrosine
kinase inhibitor, genistein, abrogated the stimulatory action of TNF
on LDH A mRNA levels, suggesting an involvement of a tyrosine kinase(s)
in the action of the cytokine.
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action on LDH A mRNA)- and time (Fig. 6B)-dependent manner.
To test whether PKC activation is involved in TNF stimulatory action
on LDH A mRNA expression, Sertoli cells were incubated with TNF (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) TNF-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 TNF on LDH A mRNA.
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action, we asked whether some of them may reproduce the
cytokine action on LDH A mRNA expression. Sphingosine appears partly
involved in TNF action, as shown in Fig. 7
. Indeed, sphingosine (10
µM) 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
TNF-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 (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
µg/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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-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 TNF. 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 TNF treatment, as we report here, corresponds to a functional
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 TNF-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 TNF-dependent 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 TNF; and 2) the decay of LDH A mRNA levels was similar
regardless of whether Sertoli cells were treated with TNF. 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
TNF on the ldh a gene.
TNF enhanced LDH A mRNA levels in a nanomolar concentration range.
Such a concentration is consistent with the amounts of TNF reported
to be secreted in the Sertoli cell environment (15) and with the
Kd range previously observed for TNF receptors in this
cell type (16). These observations suggest that the effect of the
cytokine on LDH A mRNA is probably exerted via a TNF 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 TNF 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 TNF-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 TNF 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 TNF activates PKC
activity in Sertoli cells remains to be demonstrated, such a
possibility is plausible in view of 1) the fact that TNF 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, 18 and our present data). Recent studies have
identified another pathway for TNF signaling that involves the
production of ceramide and other metabolites resulting from
sphingomyelin hydrolysis (19, 20, 21, 22). That TNF may use the
sphingomyelinase pathway (at least partly) to stimulate LDH A
expression in Sertoli cells is based on two observations: 1) TNF
action was partly mimicked by sphingosine and sphingosine-1-phosphate
(but not by ceramide and DMS); and 2) TNF 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, TNF may well use other signaling
pathways in Sertoli cells. Indeed, it has recently been reported that
in mouse Sertoli cells, TNF induced interleukin-6 (IL-6) production
and integrin ligand expression through p38 and JNK/SAPK (activated
c-Jun N-terminal protein kinase/stress-activated protein
kinase) pathways (23, 24).
Although there is increasing evidence that induction of gene expression
by TNF is mediated through activation of trans-acting
DNA-binding nuclear proteins, the mechanisms through which TNF
activates nuclear transcription factors potentially involved in LDH A
mRNA expression are unknown. TNF induced the expression of several
target genes through activation of not only a number of transcription
factors, including nuclear factor-
B 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, 27, 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 3'-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 important in
determining the levels of induced gene expression under the control of
TNF. The effects of TNF 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 ß2-adrenoreceptor (35) mRNAs. However, based on
our present findings, it is of interest that TNF may increase some
mRNAs levels without affecting their stability. Indeed, in the present
study, TNF 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 TNF in our
experimental model. The involvement of cis-acting elements
in the promoter and 3',5'-untranslated regions of the LDH A gene in
TNF 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 TNF action on LDH A mRNA involves some
intermediates, such as growth factors or cytokines coupled to their
intracellular transducing pathways. Indeed, that TNF 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- (TGF) receptors whose
activation triggered a cascade of kinase activation. In this context,
it has been found that treatment of cultured pancreatic carcinoma cells
with TNF induced expression of EGF receptor and its ligand TGF
(37). Induction of EGF and TGF mRNA by TNF has been also
previously described in human malignant epithelial cells (38). More
recently, it has been shown that TNF stimulated EGF receptor in
testicular (untransformed) peritubular myoid cells (39). In testicular
Sertoli cells, it is possible that the system EGF/TGF/EGF receptor
may mediate TNF action on LDH A expression, as 1) EGF, TGF, 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). TNF may also act through other cytokines, such as IL-6.
Indeed, it has been demonstrated that in cultured mouse Sertoli cells,
TNF treatment increased IL-6 production (23, 24). We have
investigated the hypothesis that the observed TNF 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 TNF
(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 TNF
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 TNF, 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 TNF ligand and receptors in
the same tumors.
In summary, using as a model cultured testicular Sertoli cells, we
reported here that TNF up-regulates LDH A mRNA levels through a
transcriptional activity, but not through mRNA stabilization.
Additionally, our data indicated that TNF may involve both PKC and
the sphingomyelin hydrolysis signaling pathways in stimulating LDH A
expression.
Received September 25, 1998.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-stimulated lactate production is linked to
lactate dehydrogenase A expression and activity increase in porcine
cultured Sertoli cells. Endocrinology 138:1964–1971 in mouse
spermatogenic cells. Endocrinology 133:389–396[Abstract]
antagonizes follicle-stimulating hormone action in cultured Sertoli
cells. Endocrinology 133:69–76[Abstract]
activates the sphingomyelin signal transduction
pathway in a cell free system. Science 255:1715–1718B by
phosphatidylcholine specific phospholipase C-induced << acidic >>
sphingomyelin breakdown. Cell 71:765–776[CrossRef][Medline]
increases tyrosine phosphorylation of a 23-Kda nuclear
protein in U937 cells through ceramide signaling pathway. Biochem
Biophys Res Commun 215:489–496[CrossRef][Medline]
induces interleukin 6
production and integrin ligand expression by distinct transduction
pathways. J Biol Chem 273:7566–7571 regulation of glucose transporter (Glut1)
mRNA turnover. J Biol Chem 272:1331–1337-induced glucose transporter (Glut-1) mRNA
stabilization in 3T3–L1 preadipocytes. J Biol Chem 267:8336–8341 of ß1-, ß2-,
and ß3-adrenoreceptor gene expression in 3T3–F422A adipocytes.
J Biol Chem 272:24514–24521 induces the expression of
transforming growth factor and the epidermal growth factor receptor
in human pancreatic cancer cells. Proc Natl Acad Sci USA 90:863–867 and epidermal growth factor receptor mRNA. J Biol Chem 268:2762–2766 regulates
plasminogen activator inhibitor 1 in rat testicular peritubular cells.
Endocrinology 138:1097–1105 and their
receptor during postnatal development and spermatogenesis of the boar
testis. Mol Cell Endocrinol 123:61–69[CrossRef][Medline]
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  X. Gueripel, M. Benahmed, and A. Gougeon Sequential Gonadotropin Treatment of Immature Mice Leads to Amplification of Transforming Growth Factor {beta} Action, Via Upregulation of Receptor-Type 1, Smad 2 and 4, and Downregulation of Smad 6 Biol Reprod, March 1, 2004; 70(3): 640 - 648. [Abstract] [Full Text] [PDF]
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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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R. Grataroli, D. Vindrieux, A. Gougeon, and M. Benahmed Expression of Tumor Necrosis Factor-{alpha}-Related Apoptosis-Inducing Ligand and Its Receptors in Rat Testis During Development Biol Reprod, June 1, 2002; 66(6): 1707 - 1715. [Abstract] [Full Text] [PDF]
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L. Benbrahim-Tallaa, E. Tabone, G. Tosser-Klopp, F. Hatey, and M. Benahmed Glutathione S-Transferase Alpha Expressed in Porcine Sertoli Cells Is under the Control of Follicle-Stimulating Hormone and Testosterone Biol Reprod, June 1, 2002; 66(6): 1734 - 1742. [Abstract] [Full Text] [PDF]
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 V. Pentikainen, K. Erkkila, L. Suomalainen, M. Otala, M. O. Pentikainen, M. Parvinen, and L. Dunkel TNF{alpha} Down-Regulates the Fas Ligand and Inhibits Germ Cell Apoptosis in the Human Testis J. Clin. Endocrinol. Metab., September 1, 2001; 86(9): 4480 - 4488. [Abstract] [Full Text] [PDF]
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 P. Cooper, S. Potter, B. Mueck, S. Yousefi, and G. Jarai Identification of genes induced by inflammatory cytokines in airway epithelium Am J Physiol Lung Cell Mol Physiol, May 1, 2001; 280(5): L841 - L852. [Abstract] [Full Text] [PDF]
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R.;a. Grataroli, F. Boussouar, and M. Benahmed Role of Sphingosine in the Tumor Necrosis Factor {alpha} Stimulatory Effect on Lactate Dehydrogenase A Expression and Activity in Porcine Sertoli Cells Biol Reprod, November 1, 2000; 63(5): 1473 - 1481. [Abstract] [Full Text]
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F. Boussouar and M. Benahmed Epidermal Growth Factor Regulates Glucose Metabolism Through Lactate Dehydrogenase A Messenger Ribonucleic Acid Expression in Cultured Porcine Sertoli Cells Biol Reprod, October 1, 1999; 61(4): 1139 - 1145. [Abstract] [Full Text]
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) or presence of TNF
) or OA (20 nM; 
) for 24 h, after which
25 µM DRB was added to control and treated cells. For
half-life determination, total RNA was isolated from control,
TNF
