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Program for Developmental and Reproductive Biology, Biomedicum Helsinki, and Departments of Bacteriology and Immunology, Haartman Institute (N.K.-O., J.B., M.K., J.K., K.V., J.P.K., O.R.), University of Helsinki, 00014 Helsinki, Finland; Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University (U.V., M.H., A.J.W.H.), Palo Alto, California 94305; School of Biological and Molecular Sciences, Oxford Brookes University (M.C., N.P.G.), Headington, Oxford, United Kingdom OX3 0BP; Department of Molecular Medicine, National Public Health Institute, Biomedicum Helsinki (V.M.O.), 00251 Helsinki, Finland; Ludwig Institute for Cancer Research, Uppsala Branch (A.M.), SE-752 37 Uppsala, Sweden; and Department of Cellular Biochemistry, Netherlands Cancer Institute (P.t.D.), 1066 CX Amsterdam, The Netherlands
Address all correspondence and requests for reprints to: Dr. Olli Ritvos, Biomedicum Helsinki, Room C502b, P.O. Box 63, Haartmaninkatu 8, University of Helsinki, 00014 Helsinki, Finland. E-mail: olli.ritvos{at}helsinki.fi.
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Abstract |
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PS2). However, unlike BMP-2, GDF-9 did not activate the phosphorylation of antiphospho-Smad1 antibody (PS1)-immunoreactive proteins in hGL cells. Infection of hGL cells with an adenovirus expressing Smad2 (Ad-Smad2) confirmed that GDF-9 activates specifically phosphorylation of the Smad2 protein. Infection of hGL cells with Ad-Smad7, which expresses the inhibitory Smad7 protein, suppressed the levels of both GDF-9-induced endogenous and adenoviral PS2-reactive proteins. Furthermore, GDF-9 increased the steady state levels of inhibin ßB-subunit mRNAs in hGL cells and strongly stimulated the secretion of dimeric inhibin B. Again, Ad-Smad7 blocked GDF-9-stimulated inhibin B production in a concentration-dependent manner. We identify here for the first time distinct molecular components of the GDF-9 signaling pathway in the human ovary. Our data suggest that GDF-9 mediates its effect through the pathway commonly activated by TGFß and activin, but not that activated by many BMPs. The results are also consistent with the suggestion that in addition to endocrine control of inhibin production by gonadotropins, a local paracrine control of inhibin production is likely to occur via oocyte-derived factors in the human ovary. |
Introduction |
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Although previous studies have provided important new information on GDF-9-regulated cellular events, the receptor systems and downstream signaling pathways used by GDF-9 have not been determined. Other TGFß family members are known to mediate their signals across the plasma membrane by binding to and activating distinct combinations of heteromeric complexes of specific type I and type II serine/threonine kinase receptors (reviewed in Ref. 15). Activated type I receptors initiate intracellular signaling through the phosphorylation of specific receptor-regulated Smad proteins (R-Smads). TGFß and activin can induce the phosphorylation of Smad2 and Smad3, whereas the phosphorylation of Smad1, Smad5, and Smad8 is induced by the BMPs. The activated Smads form heteromeric complexes with a common partner Smad4 and translocate into the nucleus where they, together with certain transcription factors and coregulators, modulate target gene expression. The inhibitory Smads, Smad6 and Smad7, antagonize the R-Smads either by competing with R-Smads for binding to type I receptors (Smad7) (16) or by preventing complex formation between activated R-Smads and Smad4 (Smad6) (17). Smad7 acts as a common inhibitor for R-Smads, whereas Smad6 appears to act more specifically as an inhibitor for BMP signaling (reviewed in Ref. 15). We recently reported that activin, TGFß, and BMP-2 regulate inhibin ßB-subunit mRNA expression and inhibin B production in human granulosa-luteal (hGL) cells (18, 19, 20) and that the mRNAs for Smad proteins are expressed in these cells (20). Here we report how GDF-9 affects Smad-mediated signaling pathways and inhibin B production in hGL cells.
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Materials and Methods |
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Recombinant BMP-2 and TGFß1 were purchased from R\|[amp ]\|D Systems, Inc. (Minneapolis, MN). Fetal calf serum (FCS) was purchased from Euroclone Ltd. (Devon, UK). DMEM and Ham’s F-12 were purchased from Life Technologies, Inc. (Gaithersburg, MD). Anti-Flag M2 monoclonal antibody and puromycin were purchased from Sigma-Aldrich (St. Louis, MO). Smad1 and Smad2 activation was detected with rabbit antiphospho-Smad1 (PS1) and antiphospho-Smad2 (PS2) antibodies as previously described (21, 22). Heparin (Fragmin) was purchased from Pharmacia \|[amp ]\| Upjohn, Inc. (Stockholm, Sweden). BSA was purchased from Roche Molecular Biochemicals (Mannheim, Germany). Hybond C and Hybond N membranes were purchased from Amersham Pharmacia Biotech (Little Chalfont, UK). Fugene 6 was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Peroxidase-conjugated AffiniPure goat antirabbit IgG and rabbit antimouse IgG were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
Expression of mouse and rat recombinant GDF-9
At the initial stages of this study we used tagged and untagged recombinant rat GDF-9 that have been previously described (12). A cell line expressing fully processed mouse GDF-9 was also developed and used as an abundant source of bioactive recombinant GDF-9 protein. Mouse GDF-9 full-length cDNA (7) was subcloned into pEFIRES-P expression vector (23) and transfected into a human embryonic kidney cell line, HEK-293T, by Fugene 6 transfection reagent. Cells expressing high levels of the recombinant protein were selected with increasing concentrations of puromycin in DMEM supplemented with 10% FCS, 2 mmol/liter L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. The recombinant protein was produced into serum-free harvesting medium (DMEM/Ham’s F-12, 1:1) supplemented with L-glutamine and antibiotics, 0.01% (vol/vol) BSA, and 100 µg/ml heparin. Levels of recombinant mouse GDF-9 were estimated in immunoblots using the purified N-tagged rat GDF-9 as a standard (12).
Generation of monoclonal antibodies for GDF-9
A synthetic peptide to annotated amino acids 420–450 of the C-terminal part of human GDF-9 (GenBank accession no. NP 005251) of sequence VPAKYSPLSVLTIEPDGSIAYKEYEDMIATKC was made using F-moc chemistry, coupled to tuberculin through the cysteine thiol using a heterobifunctional agent, and used to immunize female BALB/c mice by standard methods. After an initial immunization and two boosts at monthly intervals, the sera of the mice collected by tail bleed were tested against ELISA wells coated directly with the immunizing peptide. High responding mice were boosted iv, and 4 d later the spleens were removed and used for fusion to SP2/0 splenocytes by standard methods. Positive hybridomas were tested in Western blot against recombinant GDF-9. Monoclonal antibody 37 was purified by protein A chromatography using a high salt protocol. The antibody has minimal cross-reaction with preparations of recombinant GDF-9B.
Cell culture and stimulations
hGL cells were obtained with permission from women undergoing in vitro fertilization treatments. Granulosa cells from one to six patients were pooled and extracted as described previously (24). For Smad activation experiments, 200,000–300,000 hGL cells/well were plated on 6-well plates, and for inhibin B assays, 30,000–40,000 cells/well were plated on 24-well plates. The HepG2 cells were plated on 6-well plates at a density of 500,000 cells/well and were cultured overnight for control Smad activation experiments. Both cells were cultured in DMEM supplemented with 10% FCS at 37 C in a humidified atmosphere of 5% CO2 in air. hGL cells were stimulated with 3–300 ng/ml GDF-9, 10–25 ng/ml TGFß, or 25 ng/ml BMP-2, depending on the experiment, 2–3 d after plating. When recombinant adenoviruses were used, the cells were stimulated with the ligands 24 h postinfection. For Smad activation experiments, the cells were incubated in low serum medium containing 0.5% FCS for 3 h before stimulations. Stimulations for Smad activation were made in fresh low serum medium. For inhibin B assays the cells were washed once with PBS (pH 7.4) and stimulated in DMEM containing 2% FCS (the inclusion of FCS is essential for maintaining good cell viability up to 96 h of culture).
Recombinant adenoviruses
Generation of the recombinant Smad adenoviruses used in this study has been previously described (25), and their use in hGL cultures has been recently optimized (26). These serotype 5 adenoviruses express Smad proteins that contain a Flag tag in their N terminus. All viruses were amplified and titrated in transcomplemental 293T cells and purified with cesium chloride gradient ultracentrifugation as described previously (27). The purified viruses were stored at -70 C with 10% glycerol in PBS. Before adenovirus infections, hGL cells were cultured for 2–3 d. The hGL cells were infected by incubating them with the virus(es) at 37 C in serum-free DMEM supplemented with L-glutamine and antibiotics for 1 h, and DMEM containing 2% FCS was added on top to stop the infection. The cells were then incubated for 24 h before continuing the experiments. The multiplicity of infection (MOI) used in all experiments varied from 0.1–30. The expression of adenovirally produced Smads was detected with monoclonal anti-Flag M2 antibody.
Smad activation experiments and Western blot analysis
The cells were cultured for 48–72 h before endogenous Smad activation experiments or adenoviral infections. Infected cells were stimulated 24 h after infections with the stimulant concentrations mentioned above. Control cells were treated with conditioned medium. The cells were incubated in low serum medium (0.5% FCS) for 3 h before stimulations. Stimulations were performed in fresh low serum medium with stimulation times varying from 15 min to 3 h, and cells were thereafter recovered in ice-cold PBS and subsequently lysed in reducing SDS-PAGE sample buffer. The samples were sonicated for 10 sec (amplitude, 5 µm) as described previously (28), run in 10% SDS-PAGE gels, and blotted onto a Hybond C nitrocellulose membrane. The membranes were blocked for 1 h in 2.5% (wt/vol) nonfat dried milk in Tris-buffered saline/Nonidet P-40 [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Nonidet P-40] and incubated with the primary antibody in 2.5% milk in Tris-buffered saline/Nonidet P-40 overnight at 4 C (dilution, 1:15 000 for PS1 and PS2). After washing, the membranes were incubated with the secondary antibody, a peroxidase-conjugated anti-IgG (Jackson ImmunoResearch Laboratories, Inc.; 1:15,000), for 1 h. Immunoreactive proteins were detected using enhanced chemiluminescence reagents (Amersham Pharmacia Biotech). The adenoviral Smad proteins were detected with an -Flag M2 monoclonal antibody according to the manufacturer’s protocol.
RNA isolation and Northern blotting
Cytoplasmic RNAs from cultured hGL cells were extracted with the modified Nonidet P-40 lysis procedure (24, 29). RNA was quantitated by absorbance measurement at 260 nm. For Northern blots, 10–20 µg of the RNAs were size-fractionated in 1.5% agarose gels. Before transfers, even loading of the gels was checked based on RNA fluorescence at 254 nm, after which the RNAs were transferred to Hybond N nylon membranes.
Preparation and labeling of cDNA probes and filter hybridizations
For the detection of inhibin - and ßB-subunit mRNAs by Northern blot hybridization, double- and single-stranded cDNA probes were prepared as previously described (24, 29). A human cyclophilin cDNA was used as a loading control for the experiment (29). Northern blot hybridizations were performed for 16 h at 42 C, and the filters were washed three times for 20 min each time with 0.1–1x standard saline citrate/1% sodium dodecyl sulfate at 50 C. Thereafter, filters were exposed to a Fujifilm Ip-Reader Bio-Imaging Analyzer Bas 1500 (Fuji Photo Film Co., Ltd., Tokyo, Japan).
Inhibin B ELISA
hGL cells were cultured on 24-well plates in DMEM supplemented with 10% FCS for 48 h at 37 C in 5% CO2 in air before infections and stimulations. The infections were made as described above, with MOI values ranging from 0.1–30. The cells were carefully washed with PBS before adding the stimulants. The stimulations were made in DMEM supplemented with 2% FCS 24 h postinfection with the stimulant concentration mentioned above. The spent media were harvested after 24–96 h of incubation, and dimeric inhibin B concentrations were quantified from the medium using an inhibin B ELISA from Serotec (Oxford, UK) together with a signal amplification kit (Life Technologies, Inc.).
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Results |
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PS2-immunoreactive proteins in hGL cells
We studied first whether either the activin/TGFß or the BMP-activated Smad pathway would be activated by GDF-9 in hGL cells. We used both recombinant rat and mouse GDF-9 in these experiments. Figure 1A
shows that a high affinity monoclonal antibody raised against human GDF-9, mAb-37, readily detects both rat and mouse recombinant GDF-9 in Western blots analysis of the culture medium of GDF-9-expressing 293T cells. The mouse GDF-9 expressed in 293T cells appears to be fully processed, whereas the rat GDF-9 is a mixture of the processed mature region and the unprocessed protein precursor (12). However, untagged rat and mouse GDF-9 are equally potent stimulants when similar amounts of processed mature region proteins are present in the 293T-conditioned medium. The activation of different Smads was followed by Western blot analyses using PS1 and PS2 antibodies, which detect Smad1 and Smad2 proteins, respectively, that are C-terminally phosphorylated by distinct ligand-activated type I Ser/Thr kinase receptors. These antibodies are specific to the phosphorylated forms of the Smad proteins and do not recognize the unphosphorylated proteins (21, 22). With GDF-9 stimulation, PS2-reactive bands first appeared after 30 min of stimulation, and the strongest effect was detected at 75 min (Fig. 1B, top panel). In control samples harvested at the same time points, no PS2 immunoreactivity was detected (data not shown). GDF-9 induced the appearance of PS2-immunoreactive proteins in a concentration-dependent manner, with maximal effects seen with 150 ng/ml GDF-9 (Fig. 1B, lower panel). The upper panel of Fig. 1C shows that, like TGFß, GDF-9 induced the appearance of PS2-immunoreactive protein. The lower panel of Fig. 1C shows that neither GDF-9 nor TGFß induced the appearance of PS1-immunoreactive proteins, whereas the positive control BMP-2 did, as previously described (26). The PS2- and PS1-immunoreactive bands correspond to the expected molecular masses (
3 kDa) of the phosphorylated forms of Smad2 and Smad1, respectively. Immunofluorescence staining of PS2-reactive proteins showed that in control cells phosphorylation of the putative Smad2 is practically absent without ligand stimulation, whereas cells stimulated for 60 min with GDF-9 or TGFß show nuclear staining, but a part of the PS2 immunoreactivity remains in the cytoplasm (data not shown).
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To verify that GDF-9 is actually able to induce phosphorylation of the Smad2 protein, we introduced an exogenous Smad2 gene into hGL cells through adenovirus infection. Adenovirus-mediated transduction is a very effective method for introducing genes of interest into primary hGL cells, which are otherwise very difficult to transfect. Close to 100% transduction efficiencies in hGL cells can be reached, as detected by green fluorescent protein expressing adenoviruses (26). The adenovirally expressed Smad2 protein (Ad-Smad2) contains an N-terminal Flag tag that can be readily detected using an -Flag antibody in Western blots and can be distinguished from the endogenous protein because of its slightly slower mobility in electrophoresis. We infected hGL cells with Smad2 adenoviruses at various MOI values, stimulated the cells 24 h postinfection with recombinant GDF-9, and analyzed the samples by PS2 Western blotting. Recombinant GDF-9 activated the phosphorylation of both endogenous and exogenous Smad2 proteins (Fig. 2A). At MOI values of 5 (five viruses per cell) or lower, PS2-reactive bands were rarely seen in unstimulated cells, whereas with Smad2 viruses given at MOI values of 10–100, spontaneous activation of Smad2 phosphorylation was clearly detected. Receptor-regulated Smad proteins overexpressed excessively from expression plasmids have been previously shown to be spontaneously activated in other types of mammalian cells (30), and here Ad-Smad2 MOI values of 3–5 were chosen for most experiments to avoid excessive Smad2 protein expression. To study whether inhibitory Smads could block the GDF-9-induced Smad2 phosphorylation, we introduced increasing amounts of Ad-Smad6 or Ad-Smad7 (MOI ranging from 0.3–30) into hGL cells simultaneously with a constant amount of Ad-Smad2 (MOI 5). The GDF-9-induced phosphorylation of both the endogenous and the adenovirally expressed Smad2 proteins was gradually reduced close to the control level in cells infected with Ad-Smad7 (Fig. 2B), whereas Ad-Smad6 was very ineffective in suppressing Smad2 phosphorylation (Fig. 2C).
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Figure 3A shows Northern blot analyses indicating that GDF-9 increased inhibin ßB-subunit mRNA steady state levels after 8 h of stimulation, whereas inhibin -subunit or ßA-subunit (data not shown) transcript levels were hardly affected. Subsequently, the concentrations of secreted dimeric inhibin B proteins were measured from the spent media of untreated and GDF-9-stimulated cells using a specific inhibin B ELISA. GDF-9 stimulated the production of inhibin B after 24 h, and the maximal effects were seen after 72 h (Fig. 3B). GDF-9 induced the production of inhibin B in a concentration-dependent manner, and maximal stimulations were seen at 300 ng/ml GDF-9 (Fig. 3C). As Ad-Smad7 suppressed GDF-9-induced Smad2 phosphorylation, we determined whether overexpression of this inhibitory Smad would affect GDF-9-stimulated inhibin B production. Increasing amounts of Ad-Smad7 (MOI values of 0.3–30) suppressed in a concentration-dependent manner GDF-9induced dimeric inhibin B production in hGL cells (Fig. 4, A and C), whereas Ad-Smad7 alone did not affect basal inhibin B levels (Fig. 4, B and D). No cytopathic effects could be detected at these MOI values.
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Discussion |
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Our present study focused on the postreceptor effects of GDF-9 by determining whether the Smad protein system used by many members of the TGFß family would be activated by this factor. Recent data obtained with rat granulosa cells identified the BMP receptor type II (BMPRII) as a component of the GDF-9 receptor system (33), and consequently, a BMP-like effect of GDF-9 on the Smad system might have been expected. However, our present data clearly indicate that GDF-9 activates Smad2 phosphorylation in hGL cells and thus acts in a very similar manner as TGFß and activin. The phosphorylation of Smad2 occurs as early as 30 min after GDF-9 stimulation, and a strong effect persists for at least 3 h. A similar time course of the effect of TGFß on Smad2 phosphorylation has been observed in other types of cells in culture (34). The effect of GDF-9 was concentration dependent, and 100–300 ng/ml doses were needed for maximal effects, suggesting that GDF-9 acts over a similar concentration range as activin (18, 26). Whereas BMP-2-activated endogenous Smad1 phosphorylation, we did not observe any effect of GDF-9 or TGFß on Smad1 phosphorylation in hGL cells. Introduction of exogenous Flag-tagged Smad2 proteins to hGL cells via recombinant adenoviruses directly confirmed that Smad2 is activated in the hGL cell context after GDF-9 stimulation. The hGL cells cannot be transfected easily with the use of common transfection methods, such as lipofection (our unpublished data), but recombinant adenoviruses have proven to be very efficient in expressing exogenous gene products in human and rat primary granulosa cells (26, 35). Adenoviral infection allowed us to effectively overexpress Smad2 proteins, and coinfection studies with respective viruses permitted simultaneous expression of exogenous Smad2 and the inhibitory Smad6 and Smad7 proteins in various combinations. The results suggest that Smad7 could completely quench GDF-9-induced Smad2 phosphorylation, whereas the BMP-Smad pathway inhibitor Smad6 had only weak effects. Thus, it seems that GDF-9 is better functionally classified as a TGFß or activin-like factor rather than a BMP-like factor. However, GDF-9 seems to show a more restricted target cell specificity than TGFß and activin, as the HepG2 hepatoma cells used in our experiments as positive controls for Smad2 activation were responsive to activin and TGFß, but not to GDF-9 (data not shown). As a further example of differential target cell response to GDF-9 compared with activin, our previous experiments have shown that GDF-9 does not induce mesoderm in the early Xenopus laevis embryo model, which is very responsive to activin (10). Taken together, GDF-9 causes activation of the Smad2 pathway in a TGFß/activin-like manner, but in a more target cell-specific fashion, whereas it is not able to activate the BMP-Smad pathway in the ovary. Our recent parallel studies with rat granulosa cells have also confirmed that GDF-9 activates endogenous Smad2, but not Smad1, proteins in rodent granulosa cells (36). Other recent rodent studies have indicated that Smad2 and Smad3 are expressed in the ovary at follicular stages that are probably regulated by GDF-9 (37, 38, 39). We and others have also shown the presence of the activin/TGFß Smads in the human ovary (20, 40). All of these recent studies indicate that the GDF-9-regulated Smads identified in this study are found in the mammalian ovary in GDF-9 target cells.
After demonstrating a stimulatory effect of GDF-9 on Smad2 phosphorylation, we determined how GDF-9 affects the inhibin system in hGL cells and whether the Smad system would be involved. Interestingly, Lanuza et al. (41) reported recently that denuded oocytes produce substances that stimulate inhibin A and B production in rat granulosa cells, and TGFß and activin were reported to have similar effects. The oocyte factor GDF-9 was found in this study to stimulate inhibin B production in hGL cells in a similar way as activin and TGFß did in previous studies (18, 19, 26, 42). In the present study GDF-9 induced a specific increase in ßBsubunit transcript levels in hGL cells. This subunit is apparently induced in these cells by GDF-9, activin (18), and TGFß (19), as well as BMP-2 (20). As the inhibin -subunit is available in abundance, selective stimulation of ßB-subunit expression seems to be sufficient to cause a clear increase in the amount of dimeric inhibin B produced by hGL cells after treatment with these factors. We have recently found that strong overexpression of Smad1 or Smad2 by recombinant adenoviruses stimulates inhibin B production in hGL cells, and activation of either pathway thus seems to be sufficient for the induction of inhibin B production in hGL cells (26). GDF-9 is likely to stimulate inhibin B production via the Smad2 pathway, and our present results, showing that GDF-9-stimulated inhibin B production is suppressed by Smad7 overexpression, would favor this hypothesis. The results do not, however, rule out the possibility that Smad-independent pathways could also be involved (43) in the cellular effect of GDF-9, but it seems clear that at least part of the effects of GDF-9 would be mediated through specific Smads. Interestingly, Su et al. (44) recently showed that some of the effects of GDF-9 on cumulus expansion in mouse ovary could be mediated through mitogen-activated kinases, possibly in a Smad-independent manner. Thus, it is anticipated that the effects of GDF-9 in the target cells are mediated via several parallel pathways, and cross-talk with the gonadotropin-activated pathways at various stages of folliculogenesis is likely to result in more complexity in the cellular response. To be able to study in detail the downstream signaling of GDF-9, the next challenge will be identification of the various components of the GDF-9 receptor complex. One of the type II receptors involved seems to be BMPRII, which is essential in mediating the effects of GDF-9 in rodent granulosa cells (33). We have previously shown that of the known type II Ser/Thr kinase receptors, BMPRII, TGFß receptor II, activin receptor type II (ActR-II) and ActR-IIB are expressed in hGL cells as well as ActR-I [activin receptor-like kinase (ALK) 2], ActR-IB (ALK4), BMPRIA (ALK3), and TßR-I (ALK5) of the type I receptors (18, 19, 20). BMPRIB (ALK6) is known to be expressed in sheep granulosa cells (45), and therefore, it is likely to be expressed in human cells as well. Further studies are in progress to identify which of these ALKs are involved in GDF-9 signaling and to determine what other possible components are needed in the receptor complex mediating the cellular effect of this ligand that appears to play a pivotal role in the regulation of cell growth and differentiation in the mammalian ovary.
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Acknowledgments |
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Footnotes |
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N.K.-o. and J.B. are recipients of Ph.D. fellowships from the Helsinki Biomedical Graduate School.
Abbreviations: ActR-II, Activin receptor type II; ALK, activin receptor-like kinase; BMP-2, bone morphogenetic protein-2; BMPR, bone morphogenetic protein receptor; FCS, fetal calf serum; GDF-9, growth differentiation factor-9; hGL, human granulosa-luteal; MOI, multiplicity of infection; PCOS, polycystic ovary syndrome; PS1, antiphospho-Smad1 antibody; PS2, antiphospho-Smad2 antibody; R-Smad, receptor-regulated Smad protein.
Received August 16, 2002.
Accepted November 3, 2002.
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promoter for creation of stable mammalian cell lines that express very high levels of recombinant proteins. Biochem Biophys Res Commun 252:368–372[CrossRef][Medline]
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