Follicle-Stimulating Hormone Activates p70 Ribosomal Protein S6 Kinase by Protein Kinase A-Mediated Dephosphorylation of Thr 421/Ser 424 in Primary Sertoli Cells
Charlotte Lécureuil,
Sophie Tesseraud,
Elodie Kara,
Nadine Martinat,
Amina Sow,
Isabelle Fontaine,
Christophe Gauthier,
Eric Reiter,
Florian Guillou and
Pascale Crépieux
Laboratoire de Physiologie de la Reproduction et des Comportements (C.L., E.K., N.M., A.S., I.F., C.G., E.R., F.G., P.C.), Institut National de la Recherche Agronomique/Centre National pour la Recherche Scientifique/Université de Tours/Haras Nationaux, Unité Mixte de Recherche 6175; and Recherches Avicoles (S.T.), Centre de Recherches de Tours, 37380 Nouzilly, France
Address all correspondence and requests for reprints to: P. Crépieux, Laboratoire de Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique/Centre National pour la Recherche Scientifique/Université de Tours/Haras Nationaux, Unité Mixte de Recherche 6175. E-mail: crepieux{at}tours.inra.fr.
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ABSTRACT
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FSH is a major hormonal input that drives Sertoli cells to their fully differentiated function in male reproduction. It is a physiologically important issue to define how FSH mediates its effects at the cellular level to regulate gene expression. FSH biological activities are transduced via a seven-spanned transmembrane receptor, the FSH-R, primarily leading to cAMP-dependent protein kinase A (PKA) activation and cAMP response element binding protein-mediated transcriptional responses. Nevertheless, the intracellular mechanisms interacting with PKA to control Sertoli cell differentiation by FSH are still incompletely defined. Here, we report that, in primary cultures of Sertoli cells isolated from prepubertal rats, FSH enhanced p70S6K enzymatic activity, in a PKA-dependent manner. p70S6K was constitutively phosphorylated on Thr 389, in a manner sensitive to inhibitors of phosphatidyl-inositide-3 kinase and mammalian target of rapamycin. But FSH could not enhance p70S6K phosphorylation on Thr 389. Rather, the hormone induced the dephosphorylation of Thr 421/Ser 424, located in the autoinhibitory domain of p70S6K, in a PKA-dependent manner. Consistently, FSH-induced phosphorylation of the S6 ribosomal protein, a cellular substrate of p70S6K, required PKA activity. In conclusion, these results show that FSH triggers unexpected regulations of p70S6K by dephosphorylation of Thr 421/Ser 424 mediated by PKA, and stimulates S6 phosphorylation, in Sertoli cells.
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INTRODUCTION
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IN VIVO, THE pituitary FSH promotes both proliferation and differentiation of its somatic target cells that reside in the gonads. In female, it drives progression of the ovarian follicle to the preovulatory phenotype (1). In the male rat, FSH stimulates Sertoli cell proliferation during fetal and perinatal life, until cells establish tight junctions to organize as a blood-testis barrier, which is completed by 19 d (2, 3). Starting at puberty, Sertoli cells coordinate the waves of spermatogenesis by providing germ cells with essential paracrine factors, while the hormone leads the testis to acquire its full-size and spermatogenic ability (4).
Over the past, large fields of investigations have been dedicated to define the cellular mechanisms governing FSH-dependent gene expression, during the development of Sertoli cells recapitulated in vitro (5, 6). The physiological functions of FSH are mediated by a seven-span transmembrane receptor primarily coupled to Gs upon hormone binding, and GTP-bound G
s stimulates its major effector, adenylate cyclase. Subsequently, a cAMP raise leads to activation of cAMP-dependent protein kinase A (PKA) (6). In differentiated Sertoli cells isolated from prepubertal rats, cAMP and PKA have been shown to be instrumental in stimulating FSH target gene expression. Recently, oligonucleotide microarray analysis has revealed numerous genes whose transcription could be regulated by FSH, in Sertoli cells isolated from prepubertal 20-d-old rats (7). Di-butyryl-cAMP mimicked FSH in enhancing the transcription of several of them, such as nerve growth factor-1B, prolactin-1, or pheochromocytoma cell-3, most likely by acting through cAMP response element binding protein (7). Furthermore, in the fetal and postnatal gonad, FSH stimulates Sertoli cell proliferation in a cAMP-dependent manner (3). Therefore, from the overwhelming literature on the subject, it can be assumed that most, if not all, biological effects of FSH are mediated by cAMP and PKA. The question that is addressed now is to understand at which level of the cellular machinery those cAMP/PKA-dependent mechanisms operate. Several data obtained in the last decade support the view that PKA could phosphorylate not only cAMP response element binding protein but also various signaling intermediates. For example, in Sertoli cells isolated from neonate rats, we previously reported that PKA regulated ERK 1, 2 MAPKs to enable a mitogenic response to FSH (8). These data are concordant with the notion that various signaling intermediates can be triggered by PKA in FSH-stimulated Sertoli cells. In addition, our current knowledge of FSH biological activities suggests that the hormone exerts not only transcriptional effects, but could also regulate genes posttranscriptionally. For example, FSH stabilizes the stem cell factor mRNA (9) and plays a general role in accelerating the nuclear export of mRNAs in Sertoli cells (10). A posttranscriptional regulation of the inhibin
-subunit gene expression by cAMP to mimic FSH action has also been reported (11). Furthermore, in granulosa cells, FSH appears to regulate the transcription factor hypoxia-inducible factor 1
at the posttranscriptional level (12).
Here, we sought to define new signaling intermediates that would require PKA to transduce FSH biological activities, in primary cultures of Sertoli cells isolated from premature 19-d-old rats. We focused on how FSH could regulate the p70 S6 kinase (p70S6K) pathway. This kinase catalyzes phosphorylation of the S6 protein, a component of the 40S subunit of the eukaryotic ribosome that plays a pivotal role in protein synthesis (13).
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RESULTS
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FSH Enhances p70S6K Activity in a PKA-Dependent Manner, But Does Not Increase p70S6K Phosphorylation on Thr389
After 60 min of stimulation, FSH significantly enhanced p70S6K enzymatic activity in Sertoli cells (Fig. 1A
). Likewise, cell stimulation with insulin for 60 min also led to increased p70S6K activity, as expected from previous results obtained in H4 hepatoma cells (14). Because cAMP/PKA-dependent signaling has been accounted for most FSH biological activities, we examined whether this pathway was also required for FSH-induced p70S6K activity. By preincubating Sertoli cells with the selective PKA inhibitor myristoylated PKI (Myr-PKI), we observed that FSH-stimulated p70S6K activity was significantly decreased, whereas it had no effect on activation by insulin, thus indicating that PKA was required for p70S6K activation by FSH, but not by insulin (Fig. 1A
). Activation of p70S6K is brought about by hierarchical phosphorylations of key residues that reside in distinct regulatory domains (15). One of them, Thr 389, is critical for kinase activation (16). Upon FSH stimulation, the level of p70S6K phosphorylation on Thr 389 remained constant, even when Sertoli cells were pretreated with the pharmacological PKA inhibitor H89 (Fig. 1B
). Stimulation by insulin induced an increase in p70S6K phosphorylation on Thr 389, which was insensitive to PKA inhibition. In conclusion, although required for FSH-stimulated p70S6K enzymatic activity, PKA does not contribute to p70S6K constitutive phosphorylation on Thr 389.

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Fig. 1. FSH Stimulates p70S6K Activity in Vitro in a PKA-Dependent Manner, But Does Not Enhance Phosphorylation of p70S6K on Thr 389
A, In vitro p70S6K activity was quantified in Sertoli cell lysates. Cells preincubated or not (DMEM) with 50 µM Myr-PKI for 30 min, prior stimulation by 100 ng/ml FSH or by 10 ng/ml insulin (Ins) for 60 min. Results are expressed as cpm, and the statistical analysis was carried out on raw data (n = 6). Shared superscripts indicate no significant difference, and different superscripts indicate significant differences, at the P > 0.05 level. B, Representative Western blots show the phosphorylation of p70S6K on Thr 389 in the corresponding whole cell extracts. Primary Sertoli cells isolated from 19-d-old rats were preincubated with 10 µM H89 for 30 min before stimulation by FSH or by insulin for the indicated period of time (n = 3). The values beneath the autoradiograms represent fold-stimulation of the signals in the different lanes vs. the basal level, after normalization with an anti-p70S6K antibody to correct for gel loading variations.
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PKA Mediates FSH-Induced Dephosphorylation of p70S6K on Thr 421 and Ser424
Phosphorylation of Thr 421 and Ser 424, located in the autoinhibitory domain of p70S6K, is supposed to relieve conformational inhibition so that other phosphorylation sites, such as Thr 389, are subsequently exposed to signal transduction cascades, leading to full activity of the enzyme (16). Surprisingly, phosphorylation on Thr 421/Ser 424 was decreased upon FSH stimulation, whereas insulin stimulation enhanced it (Fig. 2A
). Therefore, in Sertoli cells treated by FSH, phosphorylation of Thr 421/Ser 424 may not be a prerequisite to p70S6K enzymatic activity. The same result was obtained when cells were stimulated by the cAMP-elevating agent forskolin (Fig. 2B
), suggesting that cAMP is sufficient to induce p70S6K dephosphorylation of Thr 421/Ser 424. Furthermore, preincubation of cells with Myr-PKI reverted FSH-induced dephosphorylation of p70S6K on Thr 421/Ser 424, but left insulin-stimulated phosphorylation on these sites unaltered (Fig. 2C
). These results show that p70S6K is constitutively phosphorylated on Thr 421/Ser 424 in Sertoli cells, and the FSH input stimulates p70S6K activity despite PKA-mediated dephosphorylation of p70S6K on Thr 421/Ser 424. Therefore, the regulatory mechanisms by which insulin and FSH activate p70S6K in Sertoli cells appear to differ.

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Fig. 2. FSH, But Not Insulin, Levels Off the Phosphorylation of p70S6K on Thr 421/Ser 424, in a PKA-Dependent Manner
Representative Western blots show the phosphorylation of p70S6K on Thr 421/Ser 424 in the corresponding whole cell extracts. The results are quantified as above. A, Sertoli cells were stimulated by FSH for the indicated periods of time, or by insulin for 60 min. B, Sertoli cells were stimulated for 30 min by FSH or by 10 µM forskolin (Fo) (n = 4). C, Sertoli cells were preincubated or not with 50 µM Myr-PKI for 30 min before stimulation for 30 min by FSH (n = 4), or by insulin for 60 min. *, The antibody also detects the p85 S6 kinase isoform.
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Inhibition of Phosphatidyl-Inositide-3 Kinase (PI-3 Kinase) and Mammalian Target of Rapamycin (mTOR) Pathways Alters Basal, and FSH-Induced p70S6K Activity
In other cell types stimulated by insulin, such as adipocytes, p70S6K has been reported to be phosphorylated after a PI-3 kinase-dependent signaling cascade (17, 18). Furthermore, in Sertoli cells, FSH is able to stimulate a PI-3 kinase-mediated response leading to protein kinase B phosphorylation (19). Therefore, we next asked whether PI-3 kinase was also involved in activation of p70S6K by FSH in Sertoli cells. First, the p70S6K enzymatic activity was dramatically impaired by pretreatment with the PI-3 kinase inhibitor LY294002 prior FSH stimulation, but the basal level was also decreased to a similar extent (Fig. 3A
). As expected (17), insulin-induced p70S6K activity was also decreased by pretreatment with LY294002 (Fig. 3A
).

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Fig. 3. p70S6K Activity and Phosphorylation on Thr 389 Are Sensitive to PI-3 Kinase Inhibition, Whereas FSH-Induced Dephosphorylation of Thr 421/Ser424 Is Not
A, In vitro p70S6K activity was quantified in Sertoli cell lysates. Cells were preincubated or not (DMEM) with 50 µM LY294002 for 15 min before stimulation by FSH or by insulin for 60 min. Results are expressed as above (n = 3). Representative Western blots show the phosphorylation of p70S6K on Thr 389 (B) or on Thr 421/Ser424 (C and D) in the corresponding whole cell extracts. A control kinetics with FSH without pharmacological treatment has been done in each experimental condition but is not shown here for the sake of clarity. The results are quantified as above. Sertoli cells were preincubated or not with 50 µM LY294002 for 15 min (B and C), or with 10 nM rapamycin for 1 h (B and D) before stimulation by FSH or by insulin for the indicated period of time (n = 3).
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PI-3 kinase has been reported to impact primarily on Thr 389 to activate p70S6K (20). Because p70S6K was constitutively phosphorylated on Thr 389 and because pretreatment with LY294002 primarily impaired the basal enzymatic activity, we assumed that pretreatment with PI-3 kinase inhibitors could also affect the basal phosphorylation on Thr 389. Accordingly, we observed that constitutive phosphorylation of p70S6K on Thr 389 was abolished after pretreatment of cells with LY294002 (Fig. 3B
). In these conditions, insulin-induced p70S6K phosphorylation on Thr 389 was also abolished. Furthermore, the PI-3 kinase-related Ser/Thr kinase mTOR is also a potential Thr 389 kinase (16, 21). The immunosuppressant microlide rapamycin, which selectively inhibits mTOR, also impaired constitutive, as well as insulin-induced phosphorylation of p70S6K on Thr 389. In contrast to constitutive phosphorylation on Thr 389, FSH-induced dephosphorylation of p70S6K on Thr 421/Ser 424 was insensitive to PI-3 kinase inhibition, whereas insulin-stimulated phosphorylation of this site was decreased (Fig. 3C
). Similarly, rapamycin did not prevent FSH-induced dephosphorylation of p70S6K on Thr 421/Ser 424 either, but decreased insulin-stimulated phosphorylation on these sites (Fig. 3D
). Therefore, in Sertoli cells, p70S6K would be constitutively phosphorylated on Thr 389 in a PI-3 kinase-dependent manner, and inducibly dephosphorylated on Thr 421 and Ser 424 by FSH, in a PKA-dependent manner.
FSH Stimulates S6 Ribosomal Protein Phosphorylation in Primary Sertoli Cells
In the previous sections, p70S6K activity was assayed by the ability of the enzyme to phosphorylate in vitro a synthetic peptide from the 40S S6 ribosomal protein. S6 is a well-known target of p70S6K. It actively takes part in polysome assembly, when phosphorylated by kinase cascades in response to extracellular signals or amino acid uptake (22). To estimate whether the FSH-stimulated p70S6K activity was physiologically relevant in Sertoli cells, we next addressed whether the hormone could induce the phosphorylation of endogenously expressed S6 protein. As shown in Fig. 4A
, FSH induced phosphorylation of S6 on Ser 235/236, starting from 515 min to persist over 60 min of stimulation. In 3T3-L1 adipocytes, insulin is known to stimulate S6 phosphorylation as well (23). Likewise, in Sertoli cells, insulin also led to S6 phosphorylation with a maximum reached by 60 min (Fig. 4B
). Therefore, in Sertoli cells, both FSH and insulin enhanced S6 phosphorylation on Ser 235/236, with a slightly different kinetics.

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Fig. 4. FSH or Insulin Stimulates Phosphorylation of Ribosomal S6 Protein
The phosphorylation of ribosomal S6 protein on Ser 235/236 was analyzed in the corresponding whole cell extracts by Western blot. The results are quantified as above. A, Time-course stimulation of primary Sertoli cells by FSH (n = 9). B, The same cells were stimulated by insulin for the indicated periods of time (n = 3).
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PKA Mediates S6 Phosphorylation by FSH
Because p70S6K activation and dephosphorylation relied on PKA, we next addressed whether S6 could also be regulated by cAMP and PKA in our cell model. First, we found that S6 phosphorylation was enhanced by di-butyryl-cAMP (Fig. 5A
), or by forskolin (Fig. 5B
). Furthermore, H89 inhibited FSH-induced S6 phosphorylation (Fig. 5C
). H89 is not exclusively selective for PKA but can also inhibit p70S6K activity (24), hence providing misleading data when examining S6 phosphorylation. For this reason, we sought to confirm this result by preincubating cells with the more selective Myr-PKI inhibitor. We found that Myr-PKI also exhibited an inhibitory effect on S6 phosphorylation (Fig. 5D
), confirming the involvement of PKA in the FSH-induced S6 response.

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Fig. 5. Phosphorylation of Ribosomal S6 Protein Is Mediated by cAMP and PKA
The phosphorylation of ribosomal S6 protein on Ser 235/236 was analyzed in the corresponding whole cell extracts by Western blot, and the results are quantified as above. A, Representative kinetics of S6 phosphorylation in response to 100 µM di-butyryl (db)-c-AMP (n = 5). B, Representative kinetics of S6 phosphorylation in response to 10 µM forskolin (n = 3). C, FSH-stimulated phosphorylation of ribosomal S6 protein is sensitive to PKA pharmacological inhibition. Primary Sertoli cells were stimulated by FSH for the indicated period of time after preincubation with 10 µM H89 for 30 min. A representative experiment is shown (n = 3). D, A representative experiment with Sertoli cells preincubated with 50 µM Myr-PKI for 30 min before stimulation by FSH for 30 min (n = 3).
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FSH Stimulation of S6 Phosphorylation Is Sensitive to PI-3 Kinase Inhibition
When preincubating Sertoli cells with the PI-3 kinase inhibitor wortmannin (Fig. 6A
), the phosphorylation of S6 induced by FSH was decreased and was more transient than in the control experiment. As expected (25), wortmannin robustly inhibited insulin-stimulated S6 phosphorylation. Likewise, when cells were preincubated with the other PI-3 kinase inhibitor, LY294002, S6 phosphorylation after FSH stimulation was almost abolished (Fig. 6A
). These results suggest that FSH-induced S6 phosphorylation was sensitive to PI-3 kinase inhibition. Next, we investigated whether mTOR could also be involved in this response. When preincubating Sertoli cells with rapamycin, FSH-stimulated S6 phosphorylation was impaired (Fig. 6A
), suggesting that mTOR could mediate the FSH-induced S6 response in Sertoli cells. As expected (26, 27, 28), rapamycin, like the PI-3 kinase inhibitors, exerted drastic inhibitory effects on insulin-stimulated S6 phosphorylation. Furthermore, the farnesylation inhibitor FTI 277 blocked FSH-stimulated S6 phosphorylation, suggesting that Rheb could also be involved. Farnesylation is required for full activity of several GTP-binding proteins, among which is Rheb, recently shown to signal upstream of mTOR (28). As expected (26, 27, 28), in control cells, FTI 277 also inhibited insulin-stimulated S6 phosphorylation.

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Fig. 6. FSH-Stimulated Phosphorylation of Ribosomal S6 Protein Is Sensitive to Inhibitors of the PI-3 Kinase and mTOR Pathways
Representative Western blot experiments of the corresponding whole cell extracts show the phosphorylation of ribosomal S6 protein on Ser 235/236. The results are quantified as above. A, Sertoli cells were stimulated by FSH or by insulin for the indicated period of time after preincubation with 100 nM wortmannin for 15 min (n = 4), or with 50 µM LY294002 for 15 min (n = 3), or with 10 nM rapamycin for 1 h (n = 3), or with 10 µM of the farnesylation inhibitor FTI 277 for 30 min (n = 2). B, Sertoli cells were stimulated by forskolin (Fo) for 60 min after preincubation with 100 nM wortmannin for 15 min.
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Because we had observed that cAMP could mimic FSH-induced S6 phosphorylation, we next stimulated Sertoli cells with forskolin, in the presence of PI-3 kinase inhibitors. Similarly to FSH, forskolin could no longer stimulate S6 phosphorylation in cells preincubated with wortmannin (Fig. 6B
). Thus, in primary Sertoli cells, in addition to a PKA-dependent component, PI-3 kinase-, Rheb- and mTOR-dependent signaling cascades also contribute to the stimulatory effect of FSH on ribosomal S6 protein.
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DISCUSSION
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A major issue to understand FSH physiological function in male is to define the signaling pathways that mediate its proliferative activity in neonate and those that allow the same hormone to guide its target cells toward differentiation progressively, in premature animal. This developmental pathway requires the sequential synthesis of various markers of the sertolian function, and more than a hundred of them are controlled by FSH. However, the signaling pathways involved are more or less defined. Some of them lead to transcriptional regulations, whereas some others could act at the posttranscriptional level. Here, using differentiated primary cultures of rat Sertoli cells, we have reported an original mechanism of regulation of p70S6K activation, dependent on PKA. The role of p70S6K in cell cycle progression has been extensively studied (reviewed in Ref. 29), but conversely, the involvement of p70S6K in differentiation is far less documented. Only recently has a potential role of p70S6K in differentiation been put to the forth, in the acquisition of a contractile phenotype by airway myocytes (30), as well as in myelomonocytic leukemic cell lines or in peripheral mature neutrophils stimulated by granulocyte-macrophage stimulating factor (31). The present study has revealed unexpected features of p70S6K regulation by FSH, in primary cultures of differentiated rat Sertoli cells. By using various pharmacological agents, we highlighted a cross talk between PKA and p70S6K signaling pathways. Cross talks between PKA and p70S6K have been previously shed to light in proliferating cells. For example, PKA inhibits the activity of p70S6K in arterial smooth muscle cells, to antagonize cell proliferation and possibly migration (32). In our cell model, PKA stimulates p70S6K activity, suggesting that this cross talk could have different biological outcomes in proliferating vs. differentiating cells.
The regulation of p70S6K activity is complex. Thr421/Ser424 are located in the autoinhibitory domain of p70S6K. Originally, phosphorylation of Thr421/Ser424 has been considered to be required for opening the kinase into a fully active conformation, which facilitates phosphorylation of Thr 389 (33), thus enabling docking of phosphoinositide-dependent protein kinase 1 and subsequent phosphoinositide-dependent protein kinase 1-mediated phosphorylation of Thr 229 in the activation loop of the kinase (34). For example, in neutrophils, a MAPK-dependent phosphorylation on Thr 421/Ser 424 and an mTOR-dependent phosphorylation on Thr 389 both cooperate to fully activate p70S6K, in response to granulocyte-macrophage stimulating factor (35). Nevertheless, accumulating evidence indicate that phosphorylation of Thr421/Ser424 is not necessarily concordant with activation of p70S6K. For instance, in mitotic cell lines the microtubule-damaging agent paclitaxel induces phosphorylation of p70S6K on Thr 421/Ser 424 while decreasing the enzyme activity (36). In addition, mitotic arrest appears to induce p70S6K inactivation despite increased multiple phosphorylations of the autoinhibitory tail (37). Our model further completes these data, and proposes that phosphorylation of p70S6K on Thr 421/Ser 424 can even be instrumental in inactivating the enzyme in some conditions. Our current view is that at least two signaling pathways contribute to p70S6K regulation in differentiated Sertoli cells (Fig. 7
). The first one is a PI-3 kinase- and mTOR-dependent pathway, involved in basal and in FSH-induced p70S6K activity. In agreement, FSH-stimulated phosphorylation of S6 is sensitive to PI-3 kinase and mTOR inhibition. Rheb, a target of the tuberous sclerosis complex gene product, which has recently been placed in a PI-3 kinase and mTOR-dependent cascade (27, 28, 38), could also intervene in FSH-stimulated S6 phosphorylation. The second signaling route leading to p70S6K activation depends on FSH and would require PKA activity. Either directly or not, PKA inhibits phosphorylation of p70S6K on Thr 421/Ser 424, leading to full p70 activity. In strong support of this view is the fact that inhibiting PKA by the selective antagonist Myr-PKI restored a significant phosphorylation of p70S6K on Thr 421/Ser 424 upon FSH stimulation but severely dampened p70S6K activity. How can the hormonal signal achieve this mechanism? Thr 421/Ser 424 have been reported to be targeted by ERK1, 2 (39), JNK or p38 MAPKs (36). Interestingly, with a comparable kinetics, FSH represses ERK1, 2 constitutive phosphorylation in Sertoli cells isolated from 19-d-old rats (8), in a PKA-dependent manner, suggesting that they could be the kinases involved. However, unpublished data from our group suggest that pharmacologically inhibiting ERK1, 2 MAPKs does not alter the basal phosphorylation of p70S6K on Thr 421/Ser 424. Alternatively, another possibility is that FSH would recruit a phosphatase, as evidenced in granulosa cells. For example, FSH stimulates the activity of the phosphotyrosine phosphatase PTP-20 to dephosphorylate Rho-GAP, thus leading to cytoskeleton rearrangements (40). Interestingly, FSH can also enhance the PKA-dependent phosphorylation of a phosphotyrosine phosphatase related to PTP-SL (41) to release active ERK1, 2 MAPKs (42). Whether a PKA-dependent protein phosphatase is involved in FSH-mediated p70S6K dephosphorylation on Thr 421/Ser 424 in Sertoli cells requires further investigations.

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Fig. 7. Comparison of the Mechanisms Leading to p70S6K Activation by FSH and by Insulin in Differentiating Sertoli Cells
p70S6K is constitutively phosphorylated on Thr 389, in a PI-3 kinase-dependent manner. FSH stimulation (plain arrows) leads to p70S6K activity and dephosphorylation of p70S6K on Thr 421/Ser 424, in a PKA-dependent manner. This mechanism contrasts with the way insulin activates p70S6K (dashed arrows), by phosphorylating both Thr 421/Ser 424 and Thr 389 in a PI-3 kinase-dependent manner. The arrows indicate a functional link, not a direct interaction.
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One intriguing aspect of our data is that in the same cell type, FSH and insulin trigger, respectively, dephosphorylation and phosphorylation of p70S6K on Thr 421/Ser 424, albeit both increasing enzymatic activity. Although it was not the topic of the present study to depict them all, it should be noted that several other phosphorylation sites have been mapped on p70S6K, which can modulate enzymatic activity. Whether their combinatorial phosphorylation could be differentially achieved through distinct insulin- or FSH-activated signaling pathways is worth to be examined in the future.
We have shown here that FSH stimulation leads to the rapid phosphorylation of ribosomal S6 protein, a well-identified cellular target of p70S6K. S6 is associated to the ribosomal 40S subunit, where it enhances the mRNA/ribosome association, and selects the mRNA to be translated. Furthermore, S6 is the sole among all the proteins of the 40S ribosomal subunit to be phosphorylated in response to extracellular signals, and the 40S subunit phosphorylated via S6 is recruited to the polysomes (22). Therefore, the fact that FSH leads to S6 phosphorylation could be in favor of a role of FSH in controlling translation. The identification of signaling pathways currently converging downward to ribosomal proteins is relevant to the notion that FSH, in addition to controlling its target genes at the transcriptional level, could also regulate gene expression at the level of translation. This conclusion is in agreement with the observations that FSH could regulate genes not only at the transcriptional level, but also posttranscriptionnally (9, 10, 11, 12).
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MATERIALS AND METHODS
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Pharmacological Reagents
Porcine FSH was purified by Dr. Jean Closset (Université de Liège, Liège, Belgium). Forskolin, insulin, di-butyryl-cAMP, wortmannin were purchased from Sigma Chemical Co. (St. Louis, MO), LY294002, rapamycin, were purchased from Cell Signaling Technology (Beverly, MA). H89 (N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline) was purchased from MP Biomedicals (Illkirch, France). Myr-PKI 1422 amide and farnesyltransferase inhibitor FTI 277 were from Calbiochem (San Diego, CA).
Primary Sertoli Cell Culture
Sertoli cells were isolated from testes 19-d-old Wistar rats (Iffa Credo, Lyon, France). Animals were treated in accordance with the Guide for Care and use of Laboratory Animals (National Institutes of Health guide). Sertoli cells were seeded in DMEM (Invitrogen Life Technologies Inc., Carlsbad, CA) complemented with streptomycin, glutamine, retinol, fungizone, vitamin E and human transferrin as reported previously (43). In average, our Sertoli cell cultures were 90% pure at 19 d postpartum, as previously quantified (44). Respective assays were performed the day after their initial seeding. Extracellularly released cAMP was quantified in each experimental condition, to ensure that the cells are healthy, despite the various pharmacological treatments.
Immunoblot Analysis and Antibodies
Five million cells per condition were lysed in 20 mM Tris (pH 7.8), 50 mM NaCl, 5 mM EGTA, 1% vol/vol Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 4 mM Na3VO4, 5 µg/ml leupeptin, 5 µg/ml pepstatin, and 5 µg/ml aprotinin. Sixty micrograms of proteins in total cell lysates were resolved by SDS-PAGE, electrophoretically transferred to a polyvinylidenedifluoride membrane (NEN Life Science Products, Boston, MA) and probed with the following primary antibodies: rabbit antiphospho-ribosomal protein S6 [Ser 235/236], antiphospho-p70 S6-kinase [Thr 389] or [Thr 421/Ser 424], all from Cell Signaling Technology Inc. Horseradish peroxidase-coupled antirabbit antibodies (Sanofi/Pasteur, Lyon, France) were used to detect antigen-antibody interactions by enhanced chemiluminescence (NEN Life Science Products). All the blotting membranes were re-probed with a rabbit anti-p70S6K polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) to normalize gel loading. All the autoradiograms were scanned and the OD of the signals was measured with the ImageMaster 1D Elite version 4 Software (Amersham Biosciences, Arlington Heights, IL).
p70S6K in Vitro Assay
After stimulation, Sertoli cells were disrupted on ice for 30 min in a lysis buffer containing 137 mM NaCl, 20 mM Tris HCl (pH 7.5), 1 mM MgCl2, 1 mM CaCl2, 150 mM Na3VO4, 1% Nonidet P-40, 10% glycerol, 2 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinine. p70S6K activity was measured in vitro by immune kinase assay according to the procedure in the p70S6K assay kit (Cell Signaling, Lake Placid, NY). Briefly, Sertoli cell lysates were immunoprecipitated with anti-p70S6K antibody and the specific enzyme activity of the protein was measured by estimating the phosphorylation of an artificial substrate (AKRRRLSSLRA) corresponding to an 11-amino acid sequence of the ribosomal protein S6 in the presence of
-S-33P-ATP.
Statistics
Measurements of p70S6K activity were carried out in triplicates, and expressed as mean values ± SEM of at least three independent experiments. Data were analyzed by the Mann-Whitney U test, using the Statistica 6.0 software. Values with P < 0.05 were considered to be statistically significant.
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ACKNOWLEDGMENTS
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The authors thank the technical assistance of the rat breeders Jean-Claude Braguer, Michel Vigneau, Christelle Badaire, and Claude Cahier. We are also indebted to Drs. Marie-Christine Maurel and Sophie Fauchécourt and to Vincent Piketty for critical reading of the manuscript, to Dr. Sabine Suire (Babraham Institute) for her constant support, and to Dr. Joëlle Dupont for kindly providing us with diverse reagents.
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FOOTNOTES
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C.L. and E.K. are funded by a fellowship from the Région Centre. This work was funded by the Institut National de la Recherche Agronomique, by the Centre National de la Recherche Scientifique, and by the Ligue contre le Cancer.
First Published Online March 17, 2005
Abbreviations: mTOR, Mammalian target of rapamycin; Myr-PKI, selective PKA inhibitor myristoylated PKI; PI-3 kinase, phosphatidyl-inositide 3-kinase; PKA, cAMP-dependent protein kinase A.
Received for publication July 16, 2004.
Accepted for publication March 7, 2005.
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