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Centro de Investigaciones Endocrinológicas (CEDIE), Hospital de Niños ‘Ricardo Gutiérrez’, Gallo 1330, C1425EFD Buenos Aires, Argentina
Correspondence should be addressed to S B Cigorraga; Email: scigorraga{at}cedie.org.ar
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Interleukin-1ß (IL1ß) belongs to a set of local testicular regulators and is produced by interstitial macrophages and Leydig cells (Lin et al. 1993, Hayes et al. 1996). In addition, it has been shown that IL1 receptor type I and II mRNAs are constitutively expressed in Sertoli cell cultures (Gomez et al. 1997). Several roles of IL1ß in the regulation of Sertoli cell function have been previously studied (Khan & Nieschlag 1991, Okuda et al. 1995, Hoeben et al. 1996a, 1996b, Nehar et al. 1998, Meroni et al. 2000, Riera et al. 2001, Petersen et al. 2002). However, with few exceptions, these reports have not investigated the signal transduction pathways utilized by this cytokine to regulate Sertoli cell function. It is known that IL1ß activates a large number of signal transduction pathways in different cell types. One of the signals usually elicited by IL1ß, which is intimately associated to the inflammatory response, is the one that results in the translocation of the transcription factor NF
B to the nucleus (Sizemore et al. 1999). In this regard, we have previously shown that IL1ß promotes NF B activation and its nuclear translocation, as well as the expression of iNOS protein and subsequent NO production in the Sertoli cells (Meroni et al. 2000). In the latter report, we have also shown that this signal transduction pathway is partly responsible for the IL1ß-stimulated 
GTP activity observed in Sertoli cell cultures. Recently, a report appeared showing that in Sertoli cells, the expression of STAR-related (START) domain-containing proteins is regulated by IL1ß using mechanisms that involve the activation of c-Jun N-terminal kinase (JNK) and inducible COX-2 pathways (Ishikawa et al. 2005). So far, neither the participation of the above-mentioned nor of other signaling pathways in the regulation of additional Sertoli cell biological responses by IL1ß has been analyzed.
A few years ago, a link between IL1ß binding to its receptor and phosphatidyl inositol 3-kinase (PI3K) activation in several cell lines was observed (Reddy et al. 1997). PI3K is a key enzyme implicated in the regulation of a broad array of biological responses, including receptor-stimulated mitogenesis, oxidative burst, membrane ruffling, and glucose uptake (Rameh & Cantley 1999). Agonist-stimulated PI3K phosphorylates the D-3 position of the inositol ring of phospholipids, generating potential second messengers that participate in the activation of protein kinases such as protein kinase B (PKB; Vanhaesebroeck & Alessi 2000) and p70 S6 kinase (P70S6K; Proud 1996). PKB is activated by phospholipid binding and phosphorylation at Thr308 and Ser473 by specific kinases such as PDK1, a downstream effector of PI3K (Alessi et al. 1996). On the other hand, P70S6K, which was originally recognized as the kinase that regulates the multiple phosphorylation of the 40S ribosomal protein S6 in vivo, leads to up-regulation of ribosome biosynthesis and increases the translational capacity of the cell. Activation of P70S6K also involves the phosphorylation at multiple serine/threonine residues (Pullen & Thomas 1997). Recent studies have shown that at least two signaling pathways influence P70S6K activity. One of these pathways involves PI3K, PDK1, and perhaps also PKB (Martin et al. 2001). The other pathway that is essential for P70S6K activation involves the mammalian target of rapamycin (mTOR).
Another signaling pathway through which IL1ß apparently mediates its regulatory response in several cell types involves the ubiquitous MAPK pathway (Gould et al. 1995, Finch et al. 2001). MAPK cascades are typically organized in three-kinase architecture consisting of a MAPK, a MAPK activator (MEK, MKK, or MAPKK), and a MEK activator (MEK or MAPKK). Transmission of signals is achieved by sequential phosphorylation and activation of the components specific to a respective cascade. In mammalian systems, three principal MAPK modules have been identified. These include the ERK1/2 cascade, the JNK cascade, and the p38 MAPK cascade. The ERK1/2 cascade was first implicated in the regulation of cell growth and proliferation. However, it is presently known that this kinase also participates in the control of cellular metabolism and morphology, apoptosis, and carcinogenesis (Pearson et al. 2001).
We have previously shown that IL1ß stimulates lactate production, glucose uptake, and lactate dehydrogenase (LDH) activity (Riera et al. 2001). However, the precise molecular and biochemical mechanisms involved in IL1ß regulation of the above-mentioned Sertoli cell functions remain largely unknown. Therefore, the aim of the present study was to determine: a) whether IL1ß is able to stimulate PKB-, P70S6K-, and ERK1/2-signaling pathways in Sertoli cells and b) to what extent these signaling pathways participate in the IL1ß regulation of lactate production in Sertoli cell.
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Wortmannin, LY294002, U0126, and PD98059 were purchased from Biomol (Plymouth Meeting, PA, USA) and rapamycin was purchased from New England Biolabs Inc. (Beverley, MA, USA). [2,6-3H]2-deoxy-D-glucose ([2,6-3H]2-DOG) was purchased from NEN (Boston, MA, USA). Kodak X-Omat S films were purchased from Eastman Kodak. All other drugs and reagents were obtained from Sigma-Aldrich.
Sertoli cell isolation and culture
Sertoli cells from 20-day-old Sprague–Dawley rats were isolated as previously described (Meroni et al. 1999). Briefly, decapsulated testes were digested with 0.1% collagenase and 0.006% soybean trypsin inhibitor in Hanks’ balanced salt solution for 5 min at room temperature. Seminiferous tubules were preserved, cut, and subjected to 1 M glycine–2 mM EDTA (pH 7.4) treatment to remove peritubular cells. The washed tubular pellet was then digested again with collagenase for 10 min at room temperature to remove germinal cells. The Sertoli cell suspension, collected by sedimentation, was resuspended in a culture medium consisting of a 1:1 mixture of Ham’s F12 and Dulbecco’s modified Eagle medium, supplemented with 20 mM HEPES, 100 IU/ml penicillin, 2.5 µg/ml amphotericin B, 1.2 mg/ml sodium bicarbonate, 10 µg/ml transferrin, 5 µg/ml insulin, 5 µg/ml vitamin E, and 4 ng/ml hydrocortisone. An adequate aliquot of this cell suspension was preserved and DNA content was determined immediately. DNA levels were used to standardize the cell density in the cultures (Schteingart et al. 1995). Sertoli cells were cultured in 6-, 24-, or 96-multiwell plates or in 25 cm2 tissue culture flasks at a constant density of 5 µg DNA/cm2, at 34 °C in a mixture of 5% CO2–95% air.
No myoid cell contamination was revealed in the cultures when an immunoperoxidase technique was applied to Sertoli cell cultures using a specific antiserum to 
-smooth muscle actin. Remaining cell contaminants were of germ cell origin and this contamination was below 5% after 48 h in culture, as examined by phase contrast microscopy.
Culture conditions
Sertoli cells were allowed to attach for 48 h in the presence of insulin, and the medium was replaced at this time with fresh medium without insulin.
Stimulation with IL1ß was performed on day 3 in the absence or presence of PI3K, mTOR, and MEK inhibitors (wortmannin, rapamycin, and PD98059) as indicated in figure legends. In all cases, the inhibitors were added 15 min prior to the addition of IL1ß. The 72-h conditioned media obtained on day 6 was used to evaluate the lactate and transferrin levels. Cells harvested on day 6 were used to determine LDH and GTP activities. To determine mRNA levels for LDH A, cells stimulated for 48 h and collected on day 5 were utilized.
For 2-deoxyglucose uptake studies, cells harvested on day 5 and pretreated for 1 h with IL1ß in the absence or presence of the above-mentioned inhibitors were used.
Cells harvested on day 6 and cultured on six-well plates, pretreated as indicated in the figure legends, were used for western blot analysis of phosphorylated PKB, P70S6K, and ERK1/2 (P-PKB, P-P70S6K, and P-ERK1/2) levels.
Cell extracts and western blot analysis
Cells were washed once with PBS at room temperature. Then, 200 µl PBS containing 20 µl protease inhibitor cocktail from Sigma-Aldrich (P-8340) and 2 mM phenylmethylsulfonylfluoride were added to the cells. Cells were then placed on ice and disrupted by ultrasonic irradiation. Protein content in the samples was determined by Lowry’s assay (Lowry et al. 1951). Two hundred microliters of 2xLaemmli buffer (4% w/v SDS, 20% v/v glycerol, 10% v/v 2-mercaptoethanol, 0.004% w/v bromophenol blue, and 0.125 M Tris–HCl, pH 6.8) were added and thoroughly mixed (Laemmli 1970). Samples were immersed in a boiling water bath for 5 min and then immediately settled on ice. Forty micrograms of protein aliquots were seeded in each lane. Proteins were resolved in 10% SDS-PAGE (10% acrylamide/bisacrylamide for the resolving gel and 4.3% acrylamide/bisacrylamide for the stacking gel) in a mini protean 3 cell (Bio-Rad). After SDS-PAGE, gels were equilibrated in transfer buffer for 10 min and electrotransferred at 100 V for 60 min onto PVDF membranes (Hybond-P, Amersham Pharmacia Biotech) using a mini trans-blot cell (Bio-Rad). Membranes were probed with commercial kits (phosphoplus Akt (Ser473) antibody kit, phosphoplus P70 S6 kinase (Thr421/Ser424) antibody kit, and phosphoplus p44/42 MAPK (Thr202/Tyr204) antibody kit; New England Biolabs Inc.) that allow specific recognition of both total (T-PKB, T-P70S6K, and T-ERK1/2) and phosphorylated (P-PKB, P-P70S6K, and P-ERK1/2) PKB, P70S6K, and ERK1/2. A 1:1000 dilution of primary antibodies, as indicated by the kit manufacturer, was used. The intensities of the autoradiographic bands were estimated by densitometric scanning using NIH Image software (Scion Corporation, Frederick, MD, USA). Levels of the corresponding total PKB, P70S6K, and ERK 1/2 served as loading controls.
Measurement of 2-DOG uptake
Glucose transport was studied using the uptake of the labeled non-metabolizable glucose analog 2-DOG. Cells were washed thrice with glucose-free PBS at room temperature. Then, Sertoli cells were incubated at 34 °C in 0.5 ml glucose-free PBS containing [2,6-3H]2-DOG (0.5 µCi/ml) for 30 min. Unspecific uptake was determined in incubations performed in the presence of a 10 000-fold higher concentration of unlabeled 2-DOG. At the end of the incubation period, dishes were placed on ice and extensively washed with ice-cold PBS until no radioactivity was present in the washings. Cells were then dissolved in 0.5 M sodium hydroxide and 0.4% sodium deoxycholate, and counted in a liquid scintillation spectrophotometer. Parallel cultures receiving identical treatments to those performed before the glucose uptake assay were destined to DNA determinations. 2-DOG incorporation into the cells was normalized, considering the amount of DNA in the cultures.
Lactate determination
Lactate was measured by a standard method involving conversion of NAD+ to NADH, determined as the rate of increase of absorbance at 340 nm. A commercial kit from Sigma-Aldrich was used.
Analysis of LDH A mRNA levels
Total RNA was isolated from Sertoli cells cultured in 25 cm2 tissue culture flasks with TRI reagent, a monophasic solution of phenol and guanidine isothiocyanate. The amount of RNA was estimated by spectrophotometry at 260 nm. About 20 µg total RNA were electrophoresed on a 1% agarose–10% formaldehyde gel. After migration, RNAs were transferred to Hybond-N nylon membrane (Amersham Pharmacia Biotech) by capillary transfer with 20x SSC (20x stock solution: 3 M NaCl and 0.3 M sodium citrate, pH 7.4) and fixed with U.V. Stratalinker (Stratagene Cloning Systems, La Jolla, CA, USA). Complete rat LDH A 3 ' UTR cDNA cloned into pBluescript (Accession number NM 017025) was gently provided by R Jungmann, Northwestern University Medical School, Chicago, IL, USA. The probe obtained by digestion with Pst I–Bgl II (bases 1143–1534) was labeled with [-32P]deoxy-CTP (Amersham Pharmacia Biotech) using a random-primed labeling kit (Stratagene Cloning Systems). Blots were prehybridized for 5 h at 42 °C in 50% formamide, NaCl/Pi/EDTA (0.75 M NaCl, 20 mM sodium phosphate (pH 7.5), and 1 mM EDTA), 5x Denhardt solution, 10% dextran sulfate, 0.5% SDS, and 100 µg/ml herring sperm DNA. Hybridization was then performed overnight at 42 °C in the same hybridization buffer containing 1–4 x106 c.p.m./ml 32P-labeled probe. Membranes were washed twice in 2x SSC–0.5% SDS (20 min, room temperature) followed by two washes in 1x SSC–0.1% SDS (30 min, 65 °C). Membranes were exposed to Kodak X-Omat S films (Eastman Kodak) for 1–2 days at –70 °C. The intensities of the autoradiographic bands were estimated by densitometric scanning using NIH Image software (Scion Corporation). The 18S signal was used to standardize LDH A mRNA content.
LDH activity measurement
Cells were disrupted by ultrasonic irradiation in 0.9% NaCl and aliquots were preserved for DNA determinations. The remaining material was centrifuged at 15 800 g for 10 min. The supernatant was used to measure total LDH activity, which was determined by a routinely used spectrophotometric method (Wiener Laboratories, Rosario, Argentina). Results were expressed as mlU per µg DNA.
-Glutamyl transpeptidase assay
GTP activity was assayed by the method of Orlowsky & Meister (1963), using L--glutamyl-p-nitroanilide as substrate and glycylglycine as the acceptor molecule. Sertoli cell monolayers were disrupted by ultrasonic irradiation in 0.5 ml reaction buffer (0.1 M Tris buffer, 0.01 M MgCl2, and 0.02 M glycylglycine, pH 9). Adequate aliquots for DNA determinations were preserved and 5 mM substrate (L--glutamyl-p-nitroanilide) was added to the remaining material. The reaction was allowed to proceed for 120 min at 34 °C, and the enzymatic reaction was stopped by the addition of acetic acid up to a 1 M concentration. Samples were then centrifuged and absorbances determined in a spectrophotometer at 410 nm. Values were compared against a standard curve with increasing concentrations of p-nitroaniline. Results were expressed as pmol p-nitroaniline produced per min per µg DNA in the culture.
Cell viability test
A cell viability test was performed in cells cultured on 96-well plates and treated for 3 or 72 h with wortmannin (0.1 µM), LY294002 (25 µM), rapamycin (1 nM), U0126 (1 µM), or PD98059 (10 µM). A commercial kit (CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay, Promega Corporation) was used.
Transferrin determination
Rat transferrin (rTF) was measured by RIA as described by Handelsman et al.(1989). A polyclonal antibody raised against rTF in rabbits was used (Cappel Laboratories, Cochranville, PA, USA). The cross-reactivity of human transferrin in this assay is < 0.003%. Unconditioned medium containing 10 µg/ml human TF did not show any cross-reactivity in this assay. This RIA has a sensitivity of 3 ng/tube and intra- and interassay coefficients of variation are 7 and 16% respectively.
Other assays
DNA was determined by the method of Labarca & Paigen (1980).
Statistical analysis
All experiments were run in triplicates and repeated 3–4 times. One-way ANOVA and post hoc analysis using Tukey–Kramer’s multiple comparisons test were performed using GraphPad InStat version 3.00 for Windows 95 (GraphPad Software, San Diego, CA, USA). P values < 0.05 were considered statistically significant.
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). Pooled data obtained in three independent experiments performed with 50 ng/ml IL1ß revealed 4.4 ± 0.6-, 3.5 ± 0.3-, and 4.1 ± 0.9-fold stimulation (mean ± S.E.M.) in P-PKB, P-P70S6K, and P-ERK1/2 levels respectively.
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shows that wortmannin and LY294002 decreased the ability of IL1ß to increase the levels of P-PKB and that rapamycin and PD98059 (C) did not modify it. Figure 2D shows that wortmannin and LY294002 did not modify basal P-PKB levels. On the other hand, Fig. 3 shows that rapamycin (A) and wortmannin (B) decreased IL1ß stimulation of P-P70S6K levels and that PD98059 (C) did not modify it. Figure 3D shows that rapamycin and wortmannin did not modify the low levels of basal P-P70S6K. Finally, Fig. 4 shows that PD98059 and U0126 decreased the ability of IL1ß to stimulate P-ERK1/2 levels (A and B), but neither wortmannin nor rapamycin (C) modified it. Figure 4D shows that PD98059 and U0126 did not modify the low levels of basal ERK1/2.
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GTP activity observed in Sertoli cell cultures. The participation of PI3K-, P70S6K-, and ERK1/2-signaling pathways in IL1ß regulation of GTP activity was evaluated in the present study. Table 1 shows that wortmannin, rapamycin, and PD98059 did not modify IL1ß stimulation of GTP activity.
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; IL1ß+ AG: 23.2 ± 3.8 µg/µg DNA, X ± S.D., *,indicate statistically significant differences, P < 0.05). The next set of experiments intended to determine whether IL1ß-stimulated PI3K/PKB, P70S6K, and ERK1/2 signaling pathways were relevant to the regulation of lactate production. The inhibitors wort-mannin, LY294002, rapamycin, PD98059, and U0126 were added to the cultures 15 min prior to the initiation of a 72-h incubation period with IL1ß. A cell viability test performed at the end of 3- and 72-h incubation periods showed that, except for LY294002 in 72-h incubations, the inhibitors used had no effect on cell viability (Table 2).
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shows that wortmannin and PD98059 decreased IL1ß-stimulated lactate production, whereas rapamycin did not modify it. In order to rule out that the lack of effect of rapamycin was due to a decrease in the biological activity of the inhibitor caused by incubations during prolonged periods of time, the effect of rapamycin on another Sertoli cell biological response was tested. Table 3 shows that rapamycin inhibited IL1ß stimulation of transferrin levels in a 72-h incubation period.
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shows that wortmannin decreased the ability of IL1ß to stimulate glucose uptake, while PD98059 and rapamycin did not modify it. On the other hand, Fig. 7 shows that while wortmannin and rapamycin were not able to modify IL1ß-stimulated LDH A mRNA levels, PD98059 exerted an inhibitory effect. U0126, another MEK inhibitor, also decreased IL1ß stimulation of LDH A mRNA levels (data not shown). In addition, PD98059 (10 µM) and U0126 (1 µM) inhibited IL1ß stimulation of LDH activity (basal: 25.1 ± 0.9*; IL1ß: 44.7 ± 3.1; IL1ß+ PD98059: 37.1 ± 1.1
; IL1ß+ U0126: 35.3 ± 2.1 mIU/µg DNA, X ± S.D., *,,indicate statistically significant differences, P < 0.05).
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In Sertoli cells, IL1ß regulates estradiol production (Khan & Nieschlag 1991), transferrin and gelatinase A secretion (Hoeben et al. 1996a, 1996b), lactate production (Nehar et al. 1998, Riera et al. 2001), IL6 expression (Okuda et al. 1995), GTP activity (Meroni et al. 2000) and proliferation (Petersen et al. 2002) among other functions. However, with few exceptions, the above-mentioned studies have not investigated the signal transduction pathways utilized by this cytokine to regulate Sertoli cell biological functions. As mentioned before, we have shown that IL1ß utilizes an NO-dependent pathway to regulate GTP activity (Meroni et al. 2000). In addition, Ishikawa et al.(2005) have shown that the expression of some proteins related to lipid transport is IL1ß regulated by activation of JNK and cyclooxygenase-2. They claimed in their report that IL1ß is not able to activate PKB and ERK1/2. In sharp contrast to Ishikawa et al. findings, we have observed that IL1ß induces time-dependent activation of PKB-, P70S6K-, and ERK1/2-signaling pathways in rat Sertoli cells. This discrepancy is difficult to explain considering that only subtle experimental differences, some related to the stimulation–time schedule, were detected between their work and ours. Activation of the above-mentioned signal transduction pathways reached maximal values in 15-min incubations for P-PKB and P-ERK1/2 and in 30-min incubations for P-P70S6K. As expected, inhibition of PI3K activity with wortmannin and LY294002 decreased P-PKB and P-P70S6K levels but did not modify P-ERK1/2 levels, inhibition of mTOR activity with rapamycin decreased P-P70S6K levels but did not modify P-PKB and P-ERK1/2 levels, and inhibition of MEK activity by PD98059 and U0126 decreased P-ERK1/2 levels but did not modify P-PKB and P-P70S6K levels. Thus, the use of the above-mentioned inhibitors in combination with IL1ß stimulation constituted a useful tool to analyze to what extent these pathways are involved in IL1ß regulation of Sertoli cell differentiated function.
Activation of PI3K/PKB pathway by FSH and basic fibroblast growth factor (bFGF) has been previously shown to be associated with the support of differentiated functions of Sertoli cells (Meroni et al. 2002, Riera et al. 2003). On the other hand, the ERK1/2 pathway, first recognized by its association with cell division, has been related to a diverse array of cellular processes. At 20 days of age in the rat, Sertoli cells have lost their ability to undergo mitosis. Therefore, it may be assumed that activation of the ERK1/2 pathway by IL1ß might also be related to the regulation of Sertoli cell differentiated functions as it was demonstrated for bFGF (Riera et al. 2003). Activation of P70S6K by IL1ß may be related to the maintenance of the differentiated state of Sertoli cells as well. In agreement with this latter hypothesis, a recent report describes P70S6K activation by FSH, the Sertoli cell tropic hormone (Lecureuil et al. 2005).
The present study shows that IL1ß stimulation of lactate production is inhibited by wortmannin and PD98059, while it is not modified by rapamycin. These results suggest that a PI3K/PKB- and an ERK1/2-dependent pathway participate in the regulation of lactate production by IL1ß. Since rapamycin may be lost during 72-h incubations, we looked for a positive control in order to rule out this possibility. We have analyzed the effect of rapamycin on IL1ß-stimulated transferrin production in 72-h incubations. We observed that rapamycin inhibits IL1ß stimulation of transferrin secretion suggesting that, on the one hand, an mTOR pathway participates in IL1ß regulation of transferrin production and, on the other hand, that the inhibitor is active over this period of time.
Glucose transport into the cell and the LDH isoenzyme system, which reversibly catalyzes the interconversion of pyruvate and lactate, are biochemical steps that participate in the regulation of lactate production. Facilitated Sertoli cell glucose transport across plasma membrane is mediated by the carrier proteins termed glucose transporters 1, 3, and 8 (GLUT1, GLUT3, and GLUT8), the glucose transporters so far demonstrated in this cell (Ulisse et al. 1992, Kokk et al. 2004, Carosa et al. 2005). As for the LDH isoenzyme system, increments in lactate production in rat Sertoli cells have been correlated with an increase in the LDH5 isoenzyme – containing four subunits A – and in LDH A mRNA levels. Particularly, increments in glucose uptake, LDH activity, and LDH A mRNA levels participate in the regulation of lactate production by IL1ß in the rat Sertoli cells (Riera et al. 2001). Considering that our results showed a participation of PI3K/PKB- and ERK1/2-signaling pathways in the regulation of lactate production by IL1ß, we decided to analyze the possible involvement of these signaling pathways in the regulation of the above-mentioned metabolic steps. Even though P70S6K did not seem to be involved in the regulation of lactate production, the participation of this signaling pathway in glucose uptake and LDH A mRNA levels deserved further investigation.
As for the regulation of glucose transport by IL1ß, we observed that wortmannin is the only inhibitor able to decrease IL1ß-stimulated glucose transport, suggesting the participation of a PI3K-dependent pathway. The latter pathway, originally described in relation to the mechanism of action of insulin (Okada et al. 1994, Czech & Corvera 1999), has been demonstrated to participate in the regulation of FSH- and bFGF-stimulated glucose uptake in rat Sertoli cells (Meroni et al. 2002, Riera et al. 2003). Altogether, these results suggest that activation of the PI3K/PKB-signaling pathway may constitute a universal mechanism that participates in the regulation of glucose transport into different cell types and by different extracellular signals.
Concerning LDH A mRNA levels, stimulation by IL1ß was impaired in the presence of the MEK inhibitors, but not in the presence of the PI3K inhibitor or the mTOR inhibitor. Confirming the previous result, we have observed that the MEK inhibitors also inhibited IL1ß-stimulated LDH activity. These results seem to indicate that, different from what was observed for glucose transport, an ERK1/2-dependent pathway participates in the regulation of LDH A mRNA levels and LDH activity. The latter results are similar to those previously observed for the regulation of LDH activity by bFGF (Riera et al. 2003).
In a previous report (Riera et al. 2001), we have shown that IL1ß increases total LDH activity and modifies the distribution profile of LDH isoenzymes by increasing LDH4 and LDH5 and decreasing LDH1 and LDH2. The mechanisms involved in the increase of LDH4 and LDH5 may be related to the observed increase in LDH A mRNA levels. Other authors have also related LDH A expression to LDH5 activity (Nehar et al. 1993, 1998). Since the MEK inhibitors decreased IL1ß stimulation of LDH A mRNA levels, we suggest that the inhibition of LDH activity is mainly due to a decrease in those isoenzymes that contain higher proportions of A subunits.
A kinetic study of IL1ß action on LDH A mRNA levels shows that maximal levels of mRNA are observed after a 48-h incubation period. These results are identical to those found by Nehar et al.(1998) for IL1 action in immature pig Sertoli cells. Prolonged treatments always raise the question of whether the observed effects of IL1ß are direct or indirect through the stimulation of other cytokines or prostaglandins. In this context, Nehar et al.(1998) have proposed that IL6 might be a mediator of IL1 response. In addition, Ishikawa & Morris (2006) have proposed that an autocrine-amplifying loop that involves COX-2-induced PGE2 and PGF2 production participates in the IL1ß-regulated Sertoli cell function. Considering that IL6, PGE2, and PGF2 stimulate ERK1/2 in other cell types (Lin et al. 2001, Kanda et al. 2005, Sales et al. 2005), the participation of this pathway in relation to these other hormones, but not to IL1ß, cannot be ruled out. However, taking into account that a preincubation with PD98059 or U0126 is necessary to observe the effect of the inhibitors on IL1ß stimulation of LDH A mRNA levels, it is tempting to speculate that IL1ß itself stimulates an ERK1/2 pathway that is related to the regulation of this biological response.
In this investigation, regulation of lactate production by IL1ß can be accounted for by the regulation of glucose uptake and LDH activity. However, other mechanisms such as modification of lactate export or the activity of enzymes of the glycolytic pathway (not analyzed in this paper) might also participate in the regulation of lactate production.
As previously mentioned, the action of IL1ß on GTP activity is partially mediated via cellular production of NO (Meroni et al. 2000). In the present study, we have observed that, on the one hand, stimulation of GTP activity by IL1ß is not inhibited by wortmannin, rapamycin, or PD98059 and, on the other hand, that an inhibitor of iNOS does not modify IL1ß stimulation of lactate production. Taken together, these results suggest that IL1ß utilizes PI3K/PKB- and ERK1/2-dependent pathways to regulate the different biochemical steps involved in the lactate production and an NO-dependent pathway to regulate the GTP activity.
In summary, our results suggest that IL1ß activates PKB-, P70S6K-, and ERK1/2-dependent pathways in rat Sertoli cells. Moreover, these results show that while IL1ß utilizes the PI3K/PKB pathway to regulate glucose transport, it utilizes the ERK1/2 pathway to regulate LDH activity and LDH A mRNA levels. This study reveals the different utilization of the pathways elicited by IL1ß to regulate distinct biochemical steps that are important to regulate lactate production in rat Sertoli cells. The overall picture suggests that different signal transduction pathways activated by the same extracellular signal can regulate specific biological responses.
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Funding
The work was supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica (PICTs 8681 and 25365) and CONICET (PIP 5479). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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References |
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