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Institute of Physiology, School of Medicine, National Yang-Ming University, 155 Linong Street, Section 2, Taipei 112, Taiwan, Republic of China
1 Agricultural Biotechnology Research Center,
2 Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, Republic of China
3 Division of Reproductive and Developmental Science, Queen’s Medical Research Institute, Edinburgh University, Edinburgh EH16 4TJ, UK
4 Institute of Molecular and Cellular Biology, School of Life Science, National Taiwan University, Taipei 106, Taiwan, ROC
(Requests for offprints should be addressed to F-C Ke; Email: fck{at}ccms.ntu.edu.tw; or to J-J Hwang; Email: jiuanh{at}ym.edu.tw)
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Abstract |
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In addition, TGFß1 had no effect on FSH-activated CREB and PI3K signaling mediators. We further found that rapamycin reduced the TGFß1-enhancing effect of FSH-stimulated steroidogenesis, yet it exhibited no effect on FSH action. Surprisingly, rapamycin displayed a suppressive effect at concentrations that had no effect on mTORC1 activity. Together, this study demonstrates a delicate interplay between cAMP/PKA and PI3K signaling in FSH and TGFß1 regulation of steroidogenesis in rat granulosa cells. Furthermore, we demonstrate for the first time that TGFß1 acts in a rapamycin-hypersensitive and mTORC1-independent manner in augmenting FSH-stimulated steroidogenesis in rat granulosa cells.
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Introduction |
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Recent studies suggest that at least two cellular signaling pathways are intertwined and obligatory in FSH action. The prototype of FSH signaling is that FSH first binds to specific, cell-surface G-protein-coupled receptors (GPCRs) and activates adenylyl cyclase leading to the production of cAMP, which teams up with cAMP-dependent protein kinase (PKA) and then triggers signaling cascades to regulate transcription of specific genes via the cAMP regulatory element-binding protein (CREB)–CREB-binding protein (CBP) complex (Mayr & Montminy 2001, Conkright & Montminy 2005). In addition, FSH can also activate the phosphatidylinositol-3-OH kinase (PI3K) pathway. FSH activates PI3K in rat granulosa cells leading to phosphorylation of Akt and serum and glucocorticoid-induced kinase (Sgk; Gonzalez-Robayna et al. 2000, Richards et al. 2002). This may be a crucial mechanism that enhances progesterone production (Zeleznik et al. 2003) and the expression of genes, such as aromatase, LH receptor, inhibin-
, and P450scc enzyme (Gonzalez-Robayna et al. 2000, Richards et al. 2002, Park et al. 2005). In addition, FSH enhances hypoxia-inducible factor-1 (HIF-1) activity through PI3K/Akt-dependent activation of mammalian target of rapamycin (mTOR), and HIF-1 activity is necessary for upregulation of FSH target genes, such as vascular endothelial growth factor (VEGF), inhibin-, and LH receptor (Alam et al. 2004). In addition to the typical Smad pathway (Derynck & Zhang 2003), TGFß can also activate PI3K/Akt pathway and this is implicated in the regulation of cell migration (Bakin et al. 2000), survival (Chen et al. 1998, Ju et al. 2005, Zocchi et al. 2005), and epithelial–mesenchymal transition process (Nawshad et al. 2005, Lien et al. 2006).
Akt is a central player in signal transduction activated in response to growth factors and is thought to contribute to many important cellular functions, including nutrient metabolism, cell growth, apoptosis, and modulating the activity of transcription factors (Brazil et al. 2004, Hanada et al. 2004, Woodgett 2005). Akt is subjected to phosphorylation regulation by phosphoinositide-dependent kinase 1 (PDK1) at the activation loop site, Thr308. Furthermore, full activation of Akt also requires phosphorylation of its Ser473 at carboxyl-terminal hydrophobic motif by kinase(s) such as integrin-linked kinase (ILK) and mTOR complex 2 (mTORC2; Brazil et al. 2004, Hanada et al. 2004, Sarbassov et al. 2005). mTOR is a conserved serine/threonine kinase, and there are two known mTOR complexes within cells, mTORC1 containing mTOR, GßL and raptor and mTORC2 containing mTOR, GßL, and rictor (Inoki et al. 2005, Martin & Hall 2005, Wullschleger et al. 2006). mTORC1 regulates cell growth through modulating transcription, and translation in part by regulating p70 ribosomal S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and mTORC2 is involved in actin polymerization and cell spreading. Additionally, mTORC1 is sensitive to rapamycin, and mTORC2 is not.
Several key observations suggest a potential link between Akt and transcriptional regulation. Akt directly phosphorylates FoxO transcription factors leading to nuclear export of FoxOs to cytoplasm and the release of their regulation of transcription (Burgering & Kops 2002, Tran et al. 2003). Ser256 of FoxO1 (forkhead homolog of rhabdomysarcoma, FKHR) and Ser253 of FoxO3a (forkhead-like protein-1, FKHRL1) are probably exclusively phosphorylated by Akt. FoxO family members participate in various cellular functions, including apoptosis, cell survival, stress detoxification, DNA repair, metabolism, and cell differentiation (Accili & Arden 2004). Three members of the forkhead family have been identified in the rodent ovary, FoxO1, FoxO3a, and FoxO4 (AFX; Kaestner et al. 2000, Brunet et al. 2001, Richards et al. 2002, Tran et al. 2003). Ablation of FoxO1 is embryonic lethal due to defective angiogenesis (Hosaka et al. 2004) and FoxO3a has a selective effect on ovarian function. Knockout of FoxO3a in mice causes a distinctive ovarian phenotype of global follicular activation leading to oocyte death, early depletion of functional ovarian follicles and secondary infertility, suggestive of premature ovarian failure (Castrillon et al. 2003, Hosaka et al. 2004). FSH through PI3K signaling induces rapid phosphorylation inactivation of FoxO1(Ser256), and this possibly leads to promotion of proliferation and differentiation of ovarian granulosa cells (Richards et al. 2002, Cunningham et al. 2003, Park et al. 2005). Together, these studies indicate that FoxOs are important regulators of follicular development, and that PI3K/Akt signaling is crucial for the upregulation of granulosa cell differentiation.
Previous studies demonstrate that TGFß1 augmented FSH-stimulated progesterone production (Dodson & Schomberg 1987, Ke et al. 2004, 2005), and increased key players in steroidogenesis, StAR protein, and P450scc enzyme (markers of differentiation) in rat ovarian granulosa cells (Ke et al. 2004, 2005). It is well established that FSH regulates granulosa cell functions mainly through cAMP–PKA pathway for induction of specific genes obligatory for differentiation events (Richards 2001) and, recent observations indicate that FSH can also activate PI3K pathway (Gonzalez-Robayna et al. 2000, Richards et al. 2002, Cunningham et al. 2003, Zeleznik et al. 2003, Alam et al. 2004, Park et al. 2005). The signaling crosstalk between FSH and TGFß receptors remains unclear. Therefore, this study was to explore the interrelationship of cAMP/PKA and PI3K/Akt signaling in TGFß1 and FSH-stimulated steroidogenesis in rat ovarian granulosa cells, and particularly the involvement of mTORC toward TGFß1 enhancement of FSH action was determined.
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Materials and Methods |
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Ovine FSH (oFSH-19-SIAFP) and equine chorionic gonadotropin (eCG) were purchased from the NHPP, NIDDK, and Dr A F Parlow (USA). Recombinant human TGFß1 was obtained from R&D System, Inc. (Minneapolis, MN, USA). Penicillin and streptomycin were from GIBCO Invitrogen Corporation. Antisera against progesterone (Lee & Sherwood 2005), StAR protein (Clark et al. 1994), and P450scc enzyme (Hu et al. 1991) were kindly provided by Dr O David Sherwood (University of Illinois, IL, USA), Dr Douglas M Stocco (Texas Tech University Health Sciences Center, Lubbock, TX, USA), and Dr Bon-Chu Chung (Academia Sinica, Taipei, Taiwan) respectively. Antibodies against phospho-Akt(Thr308), mTOR, phospho-mTOR (Ser2448), phospho-mTOR(Ser2481), S6K, phospho-S6K(Thr389), FoxO1, phospho-FoxO1(Ser256), and 4E-BP1 were from Cell Signaling Technology, Inc. (Beverly, MA, USA). Wortmannin, Akt1-GST-agarose, and antibodies against ILK, Sgk, phospho-Sgk(Ser255/Thr256), Akt, phospho-Akt(Ser473), FoxO3a, phospho-FoxO3a(Ser253), phospho-CREB(Ser133), and CREB were from Upstate Biotechnology Co. (Lake Placid, NY, USA). Mouse monoclonal antibody against ß-actin was from Sigma Chemical Co. PKAI (myristoylated protein kinase A inhibitor amide 14–22) and rapamycin were from Calbiochem (San Diego, CA, USA). 8-CPT-2'–O–Me-cAMP was from BioLog Life Science Institute (Bremen, Germany). Protein-A/G plus agarose beads were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals used were purchased from Sigma Chemical Co. unless otherwise stated.
Animals
Immature Sprague–Dawley rats (24–27 days) were obtained from the Animal Center at National Yang-Ming University (Taipei, Taiwan). Rats were maintained under controlled temperature (20–23 °C) and light conditions (14 h light:10 h darkness). Food (Lab Diet from PMI Feeds, Ins., St Louis, MO, USA) and water were available ad libitum. This study was conducted in accordance with the United States National Research Council’s Guide for the Care and Use of Laboratory Animals and institutional guidelines.
Cell culture and treatment
Isolation and culture of ovarian granulosa cells from eCG-treated immature rats was performed as previously described (Hwang et al. 1996, Ke et al. 2005). Granulosa cells were plated into 24-well plates coated with matrigel (derived from Engelbreth–Holm–Swarm sarcoma tumors; Sigma Chemical Co.) at approximately 5 x 105 viable cells per well in 500 µl growth medium (DMEM/F12 medium containing 2 µg/ml bovine insulin, 0.1% fatty acid-free BSA, 100 U/ml penicillin, and 100 µg/ml streptomycin) and allowed to attach for 24 h at 37 °C, 5% CO2–95% air. Cultured cells were then washed twice and incubated in 500 µl incubation medium (DMEM/F12 containing 0.1% lactalbumin hydrolysate) for 24 h before the beginning of treatment. Cells were pretreated with PKAI, wortmannin or rapamycin for 1 h, and then treated with FSH, 8-Br-cAMP, and/or TGFß1 for an additional 48 h. The doses of drugs used throughout the study had no obvious cytotoxic effect. At the end of incubation, conditioned media were collected, cleared by centrifugation, and stored at –70 °C until the performance of the progesterone enzyme-linked immunoassay. Cell number was determined using the crystal violet assay as previously described (Gillies et al. 1986).
Enzyme-linked immunoassay
Progesterone levels in conditioned media were measured using an enzyme-linked immunoassay. Progesterone standard and enzyme substrate 2,2'-azino-bis(3-ethylbenzthiazoline 6-sulfonic acid) diammonium were purchased from Sigma Chemical Co. The protocol followed was that furnished in a commercial progesterone assay kit (Diagnostic Systems Laboratory, Webster, TX, USA). Progesterone–horseradish peroxidase conjugate was from Fitzgerald Industries International, Inc. (Concord, MA, USA). The absorbance of reaction products was measured at 410 nm using an ELISA reader (Dynatech MR50000, Worthing, West Sussex, UK).
Immunoblotting
Granulosa cells (approximately 5–6 x 106) were cultured in matrigel-coated 60 mm culture dishes, pretreated with PKAI, wortmannin, or rapamycin for 1 h, and then treated with 10 ng/ml FSH and/or 5 ng/ml TGFß1 for 30 min or 1 h to determine their effects on the activation of the PI3K downstream signaling molecules, including Akt, Sgk, mTOR, S6K, FoxO1, FoxO3a, 4E-BP1, and PKA signaling including CREB, and for 48 h to determine their effects on protein levels of StAR protein (Clark et al. 1994), P450scc enzyme (Hu et al. 1991), and 3ß-HSD enzyme (Thomas et al. 2002). Cell extracts were prepared in lysis buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% Na-deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 1 mM NaF, and aprotinin, leupeptin, and pepstatin of 1 µg/ml each). Cell lysates (40–60 µg protein each) were analyzed by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% milk/0.05% TBST (Tris-buffered saline with 0.05% Tween 20) for 60 min, the membranes were incubated with primary antibody overnight at 4 °C. The primary antibody was visualized using horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies and enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech UK Limited). Relative quantification of ECL signals on X-ray film was analyzed using a two-dimensional laser scanning densitometer (Molecular Dynamics, Sunnyvale, CA, USA).
ILK kinase activity assay
Rat granulosa cells were cultured as previously described, and given control vehicle, FSH or FSH plus TGFß1 for 30 min. Cells lysates were prepared as previously described, and ILK kinase activity assay was performed. Cell lysates (300 µg each) were immunoprecipitated with 4 µg mouse monoclonal anti-ILK antibody, overnight at 4 °C. The immune complexes were isolated with protein A/G plus agarose beads overnight at 4 °C, and washed thrice with washing buffer (50 mM HEPES (pH 7), 2 mM MgCl2, 2 mM MnCl2, 200 mM Na3VO4, and aprotinin, leupeptin, and pepstatin of 1 µg/ml each). The kinase activity assay was performed using 2 µg Akt1-GST-agarose as the substrate, and 200 µM ATP in the reaction buffer (50 mM HEPES (pH 7), 2 mM MgCl2, 2 mM MnCl2, 200 mM Na3VO4, and 200 mM NaF), and allowed to react for 45 min at 37 °C. Phosphorylation of the substrate was detected by immunoblotting using anti-phospho-Akt(Ser473) antibody.
Statistical analysis
Quantitative data were analyzed by ANOVA and Duncan’s multiple range tests at a significance level of 0.05 using the general linear model of the SAS program (SAS Institute Inc., Cary, NC, USA). Also, Student’s t-test was used to identify significant differences between two treatment groups.
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Results |
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PKAI (a PKA inhibitor, 2.5–20 µM) and wortmannin (a PI3K inhibitor, 1–4 µM) each dose dependently suppressed the FSH plus TGFß1-stimulated progesterone production in rat granulosa cells (Fig. 1
). Also, both PKAI (20 µM) and wortmannin (4 µM) alone decreased the FSH- and FSH plus TGFß1-stimulated progesterone production (Fig. 2). Interestingly, the combined treatment of PKAI and wortmannin exhibited similar inhibitory effects to those of either treatment alone (Fig. 2). It is worth noting that though 8-Br-cAMP (1 mM) mimicked the FSH effect in stimulating progesterone production, TGFß1 did not augment 8-Br-cAMP effect as it did on the FSH effect (Fig. 2). PKAI and wortmannin decreased the 8-Br-cAMP-stimulated progesterone production (potency, PKAI 
PKAI + wortmannin < wortmannin; Fig. 2). Additionally, the involvement of cAMP–GEF signaling pathway in progesterone secretion was examined by employing a cAMP–GEF activator, 8CPT-2Me-cAMP, which effectively discriminates between the cAMP–GEF and the PKA signaling pathways (Enserink et al. 2002). Unlike 8-Br-cAMP, 8CPT-2Me-cAMP (10–1000 µM) had no effect on progesterone production either in the absence or presence of TGFß1 (data not shown).
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). Here, for the first time, we demonstrate that TGFß1 enhanced the FSH-increased 3ß-HSD enzyme level (Fig. 3) and PKAI (20 µM) and wortmannin (4 µM) alone suppressed the FSH plus TGFß1-stimulated increases in the protein level of all the three players (Fig. 3). We also noticed that PKAI and/or wortmannin suppressed the FSH-increased 3ß-HSD enzyme level, yet they had no effect on the FSH-increased StAR protein level (Fig. 3). TGFß1 alone did not affect the content of all the three players, and PKAI and wortmannin did not alter their basal levels (data not shown). Consistent with the inhibitory effect on progesterone production, the combined treatment of PKAI and wortmannin exhibited similar suppression on FSH-increased 3ß-HSD enzyme level and FSH plus TGFß1-increased StAR protein, P450scc, and 3ß-HSD enzyme levels as those of either treatment alone (Fig. 3).
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To determine the involvement of PI3K signaling pathway in FSH and TGFß1-stimulated steroidogenesis in rat ovarian granulosa cells, we examined the ILK kinase activity and phosphorylation activation of the PI3K downstream signaling mediators. FSH stimulated ILK kinase activity within 30-min treatment, and the stimulatory effect is similar to that of FSH plus TGFß1 treatment (Fig. 4). Administration of FSH for 30 min to 1 h increased the phosphorylation of Akt(S473), mTOR(S2481), and S6K(T389), but not that of Akt(T308), Sgk(S255/T256), mTOR(S2448), and 4E-BP1 (Figs 5 and 6). We then chose 1-h treatment for the following experiments. FSH treatment for 1 h also increased the transcription factor phosphorylation of FoxO1(S256), FoxO3a(S253), and CREB(S133) (Fig. 7). The extent of FSH-induced phosphorylation of Akt(S473), mTOR(S2481), and S6K(T389), and transcription factors including FoxO1(S256), FoxO3a(S253), and CREB(S133) was similar to that of FSH plus TGFß1 treatment (Figs 5–7). The acute induction of FSH (± TGFß1) on the phosphorylation of PI3K pathway mediators (Akt, mTOR, S6K, FoxO1, and FoxO3a) disappeared at 24-h post-treatment (data not shown).
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and 7A). On the other hand, PKAI (20 µM) increased the basal phosphorylation of Akt(S473), mTOR(S2481), FoxO1(S256), and FoxO3a(S253) (Figs 6 and 7A). We also noticed an interesting observation that PKAI only reduced the FSH (± TGFß1)-stimulated phosphorylation of FoxO3a, and it had no significant effect on that of Akt(S473), mTOR(S2481), S6K(T389), and FoxO1(S256) (Figs 6 and 7A). Consistent with earlier studies (Richards 2001, Enserink et al. 2002), administration of FSH activated the PKA pathway as indicated by the increase in the phosphorylation of CREB(S133) and its suppression by PKAI (Fig. 7B). Wortmannin alone had no effect on the phosphorylation of CREB(S133), and surprisingly, PKAI suppressive effect disappeared in the presence of wortmannin (Fig. 7B). These results indicate a delicate intertwined regulation of FSH-induced PI3K and PKA signaling in rat granulosa cells. Involvement of mTOR complex (mTORC) in TGFß1 enhancement of FSH action
To determine the critical role of mTORC in FSH and TGFß1-stimulated steroidogenesis in rat granulosa cells, rapamycin (an mTORC1 inhibitor) was used. We demonstrate for the first time that rapamycin (10–20 to 10–9 M) significantly suppressed the FSH plus TGFß1-stimulated progesterone production, and rapamycin had no significant effect on FSH action (Fig. 8). Rapamycin at 10–9 M, but not 10–15 or 10–12 M, reduced the FSH (± TGFß1)-stimulated phosphorylation of S6K(T389) (Fig. 9A). This indicates that only 10–9 M rapamycin suppressed the mTORC1 activity. Consistent with progesterone production, rapamycin at doses of 10–15 and 10–9 M exhibited similar suppressive effect on FSH plus TGFß1-stimulated increases in the level of StAR protein, P450scc, and 3ß-HSD enzymes (Fig. 9B). Conversely, rapamycin exhibited no significant effects on FSH-increased progesterone production and the levels of StAR and 3ß-HSD (Figs 8 and 9B).
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). Interestingly, rapamycin (10–15 to 10–9 M) moderately decreased the FSH (± TGFß1)-stimulated phosphorylation of CREB(S133) (Fig. 10B).
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Discussion |
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–3). And, though FSH increased progesterone production and protein levels of 3ß-HSD enzyme and StAR, only the former two FSH effects were sensitive to PKAI and wortmannin (Figs 2 and 3). Furthermore, the combined treatment of PKAI and wortmannin exhibited a similar extent of suppression to that of either treatment alone in FSH- and TGFß1 plus FSH-stimulated steroidogenesis (Figs 2 and 3). In addition to PKAI, wortmannin also suppressed 8-Br-cAMP-induced pro-gesterone production (Fig. 2). These results suggest a delicate intertwined regulation between cAMP/PKA and PI3K signaling mediators in FSH and TGFß1 regulation of steroidogenesis in rat granulosa cells. In addition, FSH may act through PI3K- and PKA-independent signaling in regulation of StAR protein level, and this awaits further study. Secondly, we made an interesting observation that PKAI reduced the FSH (± TGFß1)-increased phosphorylation of FoxO3a(S253) but not FoxO1(S256) (Fig. 7A), and this appears to be independent of Akt activity as FSH (± TGFß1)-increased Akt(S473) phosphorylation was not blocked by PKAI (Fig. 6). In addition, wortmannin suppressed the FSH (± TGFß1)-increased phosphorylation of Akt(S473), FoxO3a(S253), and FoxO1(S256) (Figs 6 and 7A). FoxO3a was recently reported to play a critical role in murine ovarian follicle development (Castrillon et al. 2003). These results suggest that FSH regulation of granulosa cell function may in part act through PI3K/Akt pathway to modulate the transcription factor activity of FoxO3a and FoxO1. Moreover, FSH may also act through PKA-dependent and Akt-independent signaling to regulate the activity of FoxO3a but not FoxO1. Thirdly, PKAI treatment suppressed the FSH-increased CREB(S133) phosphorylation as expected, but most interesting is that although wortmannin did not alter FSH-increased phospho-CREB(S133) levels, the PKAI suppressive effect disappeared in the presence of wortmannin (Fig. 7B). Since wortmannin targets all classes of PI3K including class III PI3K which is involved in the membrane–vesicle-trafficking system (Wymann et al. 2003) and GPCR-coupled PI3K
which participates in the receptor endocytosis (Naga Prasad et al. 2001), wortmannin may work through multi-mechanisms to modulate cAMP/PKA signaling, and yet enhance the non-cAMP/PKA signal induction of CREB(S133) phosphorylation. The signal discrimination on CREB(S133) phosphorylation between cAMP/PKA and non-cAMP/PKA stimuli may affect the specification of CREB target genes (Mayr & Montminy 2001). Also consistent with the previous study (Hillier et al. 1994), we have shown that FSH increased estradiol secretion in cultured rat ovarian granulosa cells, and TGFß1 augmented such action of FSH while TGFß1 alone had no effect (unpublished data). Figure 11 is a diagram of a proposed model regarding the molecular signaling of FSH and TGFß1-stimulated steroidogenesis in ovarian granulosa cells.
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and 7A). This indicates that FSH may not affect PDK1 activity in our system as the levels of phospho-Akt(T308) and phospho-Sgk(S255/T256) remained unchanged after FSH treatment. FSH-induced increases of Akt(S473) phosphorylation and its activity as indicated by FoxO1(S256) and FoxO3a(S253) phosphorylation may be mediated by the integrin signal mediator ILK. This is supported by the following evidence. First, this study shows that FSH increased ILK activity in rat granulosa cells (Fig. 4). Secondly, in spite of the controversial role of ILK to Akt(S473) phosphorylation (Brazil et al. 2004, Hanada et al. 2004, Woodgett 2005), our data agree with ILK conditional knockout and siRNA knockdown studies showing only Akt(S473) phosphorylation but not Akt(T308) phosphorylation was affected (Troussard et al. 2003). Finally, a recent report showed that mTORC2 (Rictor–mTOR complex) mediates PI3K-activated Akt(S473) phosphorylation. However, in contrast to ILK, Rictor siRNA knockdown affected both Akt(S473) and Akt(T308) phosphorylation (Sarbassov et al. 2005). Another PDK1 target is S6K1. Following S6K1(T389) phosphorylation, S6K1 requires T207 phosphorylation by PDK1 to have full activity (Pullen et al. 1998). S6K1(T389) was documented to be phosphorylated by mTORC1 (Raptor–mTOR complex). Phosphorylation of mTOR(S2448) as an in vivo target of S6K1 (Chiang & Abraham 2005, Holz & Blenis 2005) remained unchanged by FSH treatment in rat granulosa cells. Our results demonstrate a lack of stimulated-PDK1 activity by FSH treatment in rat granulosa cells, and this may implicate a distinct signal repertoire of different classes of PI3K in activation of downstream target, such as Akt and S6K1 (Vanhaesebroeck et al. 2005, Wymann & Marone 2005).
FSH has been reported to stimulate the expression of differentiation markers of rat granulosa cells (LH receptor, inhibin-, microtubule-associated protein 2D, and PKA type IIß regulatory subunit) via Akt-mTORC1 signaling that regulates translation and activation of HIF1 (Alam et al. 2004). In order to specify the role of mTORC1 in FSH-stimulated progesterone production in rat granulosa cells, rapamycin (a well-characterized inhibitor of mTORC1) was used in our system. Surprisingly, we found that rapamycin over a broad range of concentrations (10–20 to 10–9 M) exhibited no effect on FSH-stimulated steroidogenesis, and that rapamycin only suppressed the TGFß1 enhancing effect of FSH-stimulated steroidogenesis as indicated by protein levels of StAR, P450scc, and 3ß-HSD enzymes, and progesterone production (Figs 8 and 9B). Rapamycin at the concentration of 10–9 M did effectively block S6K1(T389) phosphorylation (Fig. 9A), yet it was without effect on FSH-increased progesterone production and protein levels of StAR and 3ß-HSD enzyme. Conversely, the rapamycin suppressive effect on TGFß1 enhancement of FSH action displayed an unusual broad effective range even at the concentrations that have no effect on S6K1(T389) phosphorylation (Figs 8 and 9). Therefore, this study indicates that TGFß1 enhancement of FSH action is specific and hypersensitive to rapamycin blockade and is independent of mTORC1 signaling.
The present study further suggests that TGFß1 enhancement of FSH-stimulated steroidogenesis extends beyond FSH-activated PKA and PI3K signaling as supported by the following evidence. First, TGFß1 did not augment 8-Br-cAMP stimulation of progesterone production as it did on FSH effect in rat granulosa cells (Fig. 2). Secondly, in contrast to the TGFß effect on IGF-I signaling (Danielpour & Song 2006), our study shows that TGFß1 did not alter FSH-induced phosphorylation activation of cAMP–PKA signaling mediator (CREB) and PI3K signaling mediators (ILK, Akt, mTOR, S6K, and FoxOs) in rat granulosa cells (Figs 5–7). In addition, TGFß1 was reported not to alter FSH-increased cAMP levels in rat granulosa cells (Inoue et al. 2002). TGFß1 augmentation of FSH-stimulated steroidogenesis may signal through site(s) close to the level of FSH receptor activation other than downstream signal crosstalking, such as an interaction of Smad3–Akt (Conery et al. 2004, Remy et al. 2004, Song et al. 2006) or Smad3–PKA regulatory subunit (Zhang et al. 2004). Early reports have shown that TGFß1 attenuated FSH-induced downregulation of FSH receptors, and increased the expression of FSH receptors in granulosa cells (Gitay-Goren et al. 1993, Dunkel et al. 1994). In addition, recent progress indicates that the molecular mechanism of receptor endocytosis is pivotal on signal transduction (Miaczynska et al. 2004, Le Roy & Wrana 2005). The ratio of two main endocytic routes, clathrin-mediated endocytosis and raft/caveolar endocytosis, has been proposed to organize and coordinate the duration, intensity, integration, and compartmentalization of the core variable in cell signaling to determine the net outcome of signaling events (Polo & Di Fiore 2006). TGFß signaling has been demonstrated to be regulated through clathrin-mediated endocytosis (signaling) and raft/caveolar endocytosis (degradation) in a ligand-independent manner (Di Guglielmo et al. 2003). Also, rapamycin is known to bind to FKBP12 leading to the release of its inhibition on TGFß receptor type I in a ligand-independent manner (Chen et al. 1997). Whether rapamycin-induced activation of TGFß receptor type I affects the ratio of clathrin-mediated endocytosis to raft/caveolar endocytosis is unknown. The hypersensitive and mTORC1-independent effect of rapamycin on the TGFß1 facilitation of FSH-stimulated steroidogenesis in rat granulosa cells is worthy of further investigation. Clathrin-mediated endocytosis is also critical for GPCR signaling (Marchese et al. 2003); therefore, the cross-modulation between distinct receptors in trafficking routes may provide a possible mechanism for TGFß1 facilitation of FSH-stimulated steroidogenesis in rat granulosa cells.
Altogether, this study demonstrates a delicate interplay between cAMP/PKA and PI3K signaling in FSH and TGFß1 regulation of steroidogenesis in rat ovarian granulosa cells. Furthermore, we demonstrate for the first time that TGFß1 acts in a rapamycin-hypersensitive and mTORC1-independent manner in augmenting FSH-stimulated steroidogenesis in rat granulosa cells.
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Acknowledgements |
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References |
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Alam H, Maizels ET, Park Y, Ghaey S, Feiger ZJ, Chandel NS & Hunzicker-Dunn M 2004 Follicle-stimulating hormone activation of hypoxia-inducible factor-1 by the phosphatidylinositol 3-kinase/AKT/Ras homolog enriched in brain (Rheb)/mammalian target of rapamycin (mTOR) pathway is necessary for induction of select protein markers of follicular differentiation. Journal of Biological Chemistry 279 19431–19449.
Bakin AV, Tomlinson AK, Bhowmick NA, Moses HL & Artega C 2000 Phosphatidylinositol 3-kinase function is required for transforming growth factor ß-mediated epithelial to mesenchymal transition and cell migration. Journal of Biological Chemistry 275 36803–36810.
Brazil DP, Yang ZZ & Hemmings BA 2004 Advances in protein kinase B signaling: AKTion on multiple fronts. Trends in Biochemical Sciences 29 233–242.[CrossRef][ISI][Medline]
Brunet A, Park J, Tran H, Hu LS, Hemming BA & Greenberg ME 2001 Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a). Molecular and Cellular Biology 21 952–965.
Burgering BMT & Kops GJPL 2002 Cell cycle and death control: long liver forkheads. Trends in Biochemical Sciences 27 352–360.[CrossRef][ISI][Medline]
Castrillon DH, Miao L, Kollipara R, Horner JW & DePinho RA 2003 Suppression of ovarian follicle activation in mice by the transcription factor FoxO3a. Science 301 15–21.
Chen YG, Liu F & Massague J 1997 Mechanism of TGFbeta receptor inhibition by FKBP12. EMBO Journal 16 3866–3876.[CrossRef][ISI][Medline]
Chen RH, Su YH, Chuang RL & Chang TY 1998 Suppression of transforming growth factor-ß-induced apoptosis through a phosphatidylinositol 3-kinase/Akt-dependent pathway. Oncogene 17 1959–1968.[CrossRef][ISI][Medline]
Chiang GG & Abraham RT 2005 Phosphorylation of mammalian target of rapamycin (mTOR) at Ser-2448 is mediated by p70S6 kinase. Journal of Biological Chemistry 280 25485–25490.
Clark BJ, Wells J, King SR & Stocco DM 1994 The purification, cloning, and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR). Journal of Biological Chemistry 269 28314–28322.
Clarke TR, Bain PA, Greco TL & Payne AH 1993 A novel mouse kidney 3 beta-hydroxysteroid dehydrogenase complementary DNA encodes a 3-ketosteroid reductase instead of a 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase. Molecular Endocrinology 7 1569–1578.[Abstract]
Conery AR, Cao Y, Thompson EA, Townsed CM Jr, Ko TC & Luo K 2004 Akt interacts directly with Smad3 to regulate the sensitivity to TGF-beta induced apoptosis. Nature Cell Biology 6 366–372.[CrossRef][ISI][Medline]
Conkright MD & Montminy M 2005 TORCs: transducers of regulated CREB activity. Trends in Cell Biology 15 457–459.[CrossRef][ISI][Medline]
Cunningham MA, Zhu Q, Unterman TG & Hammond JM 2003 Follicle-stimulating hormone promotes nuclear exclusion of the forkhead transcription factor Foxo1a via phosphatidylinositol 3-kinase in porcine granulosa cells. Endocrinology 144 5585–5594.
Danielpour D & Song K 2006 Cross-talk between IGF-I and TGF-beta signaling pathways. Cytokine and Growth Factor Reviews 17 59–74.[CrossRef][ISI][Medline]
Derynck R & Zhang YE 2003 Smad-dependent and Smad-independent pathways in TGF-ß family signaling. Nature 425 577–584.[CrossRef][Medline]
Di Guglielmo GM, Le Roy C, Goodfellow AF & Wrana JL 2003 Distinct endocytic pathways regulate TGF-beta signaling and turnover. Nature Cell Biology 5 410–421.[CrossRef][ISI][Medline]
Dodson WD & Schomberg DW 1987 The effect transforming growth factor-ß on follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells. Endocrinology 120 512–516.[Abstract]
Dorrington JH, Bendell JJ & Khan SA 1993 Interaction between FSH, estradiol-17 beta and transforming growth factor-beta regulate growth and differentiation in the rat gonad. Journal of Steroid Biochemistry and Molecular Biology 44 441–447.[CrossRef][ISI][Medline]
Dunkel L, Tilly JL, Shikone T, Nishimori K & Hsueh AJ 1994 Follicle-stimulating hormone receptor expression in the rat ovary: increases during prepubertal development and regulation by the opposing actions of transforming growth factors beta and alpha. Biology of Reproduction 50 940–948.[Abstract]
Eimerl S & Orly J 2002 Regulation of steroidogenic genes by insulin-like growth factor-1 and follicle-stimulating hormone: differential responses of cytochrome P450scc side-chain cleavage, steroidogenic acute regulatory protein, and 3ß-hydroxysteroid dehydrogenase/isomerase in rat granulosa cells. Biology of Reproduction 67 900–910.
Enserink JM, Christensen AE, de Rooij J, van Triest M, Schwede F, Genieser HG, Doskeland SO, Blank JL & Bos JL 2002 A novel Epac-specific cAMP analogue demonstrates independent regulation of Rap1 and ERK. Nature Cell Biology 4 901–906.[CrossRef][ISI][Medline]
Gillies RJ, Didier N & Denton M 1986 Determination of the cell number in monolayer cultures. Analytical Biochemistry 159 109–113.[CrossRef][ISI][Medline]
Gitay-Goren H, Kim IC, Miggans ST & Schombergm DW 1993 Transforming growth factor beta modulates gonadotropin receptor expression in porcine and rat granulosa cells differently. Biology of Reproduction 48 1284–1289.[Abstract]
Gonzalez-Robayna IJ, Falender AE, Ochsner S, Firestone GL & Richards JS 2000 FSH stimulates phosphorylation and activation of protein kinase B (PKB/Akt) and serum and glucocorticoid-induced kinase (Sgk): evidence for A-kinase independent signaling in granulosa cells. Molecular Endocrinology 14 1283–1300.
Hanada M, Feng J & Hemmings BA 2004 Structure, regulation and function of PKB/Akt-a major therapeutic target. Biochimica et Biophysica Acta 1697 3–16.[Medline]
Hillier SG, Whitelaw PF & Smyth CD 1994 Follicular oestrogen synthesis: the ‘two-cell, two-gonadotropin’ model revisited. Molecular and Cellular Endocrinology 100 51–54.[CrossRef][ISI][Medline]
Hirshfield AN 1991 Development of follicles in the mammalian ovary. International Review of Cytology 124 43–101.[ISI][Medline]
Holz MK & Blenis J 2005 Identification of S6 kinase 1 as a novel mammalian target of rapamycin (mTOR)-phosphorylating kinase. Journal of Biological Chemistry 280 26089–26093.
Hosaka T, Biggs WH III, Tieu D, Boyer AD, Varki NM, Cavenee WK & Arden KC 2004 Disruption of forkhead transcription factor (FOXO) family members in mice reveals their functional diversification. PNAS 101 2975–2980.
Hu MC, Guo IC, Lin JH, Chung BC, Hu MC, Guo IC, Lin JH & Chung BC 1991 Regulated expression of cytochrome P-450scc (cholesterol-side-chain cleavage enzyme) in cultured cell lines detected by antibody against bacterially expressed human protein. Biochemical Journal 274 813–817.[ISI][Medline]
Hwang JJ, Lin SW, Teng CH, Ke FC & Lee MT 1996 Relaxin modulates the ovulatory process and increases secretion of different gelatinases from granulosa and theca-interstitial cells in rats. Biology of Reproduction 55 1276–1283.[Abstract]
Ingman WV & Robertson SA 2002 Defining the action of transforming growth factor beta in reproduction. BioEssays 24 904–914.[CrossRef][ISI][Medline]
Inoki K, Ouyang H, Li Y & Guan KL 2005 Signaling by target of rapamycin protein in cell growth control. Microbiology and Molecular Biology Reviews 69 79–100.
Inoue K, Nakamura K, Abe K, Hirakawa T, Tsuchiya M, Matsuda H, Miyamoto K & Minegishi T 2002 Effect of transforming growth factor beta on the expression of luteinizing hormone receptor in cultured rat granulosa cells. Biology of Reproduction 67 610–615.
Ju EM, Choi KC, Hong SH, Lee CH, Kim BC, Kin SJ, Kim IH & Park SH 2005 Apoptosis of mink lung epithelial cells by co-treatment of low-dose staurosporine and transforming growth factor-beta1 depends on the enhanced TGF-beta signaling and requires the decreased phosphorylation of PKB/Akt. Biochemical and Biophysical Research Communications 328 1170–1181.[CrossRef][ISI][Medline]
Kaestner KH, Knochel W & Matinex DE 2000 Unified nomenclature for the winged helix/forkhead transcription factors. Genes and Development 14 142–146.
Ke FC, Chuang LC, Lee MT, Chen YJ, Lin SW, Wang PS, Stocco DM & Hwang JJ 2004 The modulatory role of TGFß1 and androstenedione on FSH-induced gelatinase secretion and steroidogenesis in rat granulosa cells. Biology of Reproduction 70 1292–1298.
Ke FC, Fang SH, Lee MT, Sheu SY, Lai SY, Chen YJ, Huang FL, Wang PS, Stocco DM & Hwang JJ 2005 Lindane, a gap junction blocker, suppresses FSH and transforming growth factor ß1-induced connexin43 gap junction formation and steroidogenesis in rat granulosa cells. Journal of Endocrinology 184 555–566.
Lee HY & Sherwood OD 2005 The effects of blocking the actions of estrogen and progesterone on the rates of proliferation and apoptosis of cervical epithelial and stromal cells during the second half of pregnancy in rats. Biology of Reproduction 73 790–797.
Le Roy C & Wrana JL 2005 Signaling and endocytosis: a team effort for cell migration. Nature Reviews: Molecular Cell Biology 6 112–126.[CrossRef][ISI][Medline]
Lien SC, Usami S, Chien S & Chiu JJ 2006 Phosphatidylinositol 3-kinase/Akt pathway is involved in transforming growth factor-beta-1-induced phenotypic modulation of 10T1/2 cells to smooth muscle cells. Cell Signalling 18 1270–1278.[CrossRef][ISI][Medline]
Marchese A, Chen C, Kim YM & Benovic JL 2003 The ins and outs of G protein-coupled receptor trafficking. Trends in Biochemical Sciences 28 369–376.[CrossRef][ISI][Medline]
Martin DM & Hall MN 2005 The expanding TOR signaling network. Current Opinion in Cell Biology 17 158–166.[CrossRef][ISI][Medline]
Mayr B & Montminy M 2001 Transcriptional regulation by the phosphorylation-dependent factor CREB. Nature Reviews: Molecular Cell Biology 2 599–609.[CrossRef][ISI][Medline]
Miaczynska M, Pelkmans L & Zerial M 2004 Not just a sink: endosomes in control of signal transduction. Current Opinion in Cell Biology 16 400–406.[CrossRef][ISI][Medline]
Naga Prasad SV, Barak LS, Rapacciuolo A, Caron MG & Rockman HA 2001 Agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane by beta-adrenergic receptor kinase 1. A role in receptor sequestration. Journal of Biological Chemistry 276 18953–18959.
Nawshad A, Lagamba D, Polad A & Hay ED 2005 Transforming growth factor-beta signaling during epithelial-mesenchymal transformation: implications for embryogenesis and tumor metastasis. Cells, Tissues, Organs 179 22–23.
Park Y, Maizels ET, Feiger ZJ, Alam H, Peters CA, Woodruff TK, Unterman TG, Lee EJ & Jameson JL 2005 Induction of cyclin D2 in rat granulosa cells requires FSH-dependent relief from FOXO1 repression coupled with positive signals from Smad. Journal of Biological Chemistry 280 9135–9148.
Polo S & Di Fiore PP 2006 Endocytosis conducts the cell signaling orchestra. Cell 124 897–900.[CrossRef][ISI][Medline]
Pullen N, Dennis PB, Andjelkovic M, Dufner A, Kozma SC, Hemmings BA & Thomas G 1998 Phosphorylation and activation of p70s6k by PDK1. Science 279 707–710.
Remy I, Montmarquette A & Michnick SW 2004 PKB/Akt modulates TGF-beta signaling through a direct interaction with Smad3. Nature Cell Biology 6 358–365.[CrossRef][ISI][Medline]
Richards JS 2001 New signaling pathways for hormones and cyclic adenosine 3',5'-monophosphate action in endocrine cells. Molecular Endocrinology 15 209–218.
Richards JS, Sharma SC, Falender AE & Lo YH 2002 Expression of FKHR, FKHRL1, and AFX genes in the rodent ovary: evidence for regulation by IGF-I, estrogen, and the gonadotropin. Molecular Endocrinology 16 580–599.
Sarbassov DD, Guerrtin DA, Ali SM & Sabatini DM 2005 Phosphorylation a
, Control; 
, FSH; •, FSH + TGFß1.
