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Program for Developmental and Reproductive Biology, Biomedicum Helsinki and Departments of Bacteriology and Immunology, Haartman Institute, University of Helsinki (D.G.M., N.K.-O., S.My., S.D., O.R.), 00014 Helsinki, Finland; Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine (S.Ma., A.J.H.), Palo Alto, California 94305-5317; Research Center for Reproductive Health, Department of Obstetrics and Gynecology, University of Adelaide (R.B.G.), Woodville, South Australia 5011, Australia; and School of Biological and Molecular Sciences, Oxford Brookes University (N.P.G.), Headington, Oxford, United Kingdom OX3 0BP
Address all correspondence and requests for reprints to: Dr. Olli Ritvos, Biomedicum Helsinki, Room C502b, P.O. Box 63, University of Helsinki, Haartmaninkatu 8, 00014 Helsinki, Finland. E-mail: olli.ritvos{at}helsinki.fi.
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Introduction |
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Members of the TGF-ß superfamily signal by bringing together two different cell surface serine/threonine (Ser/Thr) kinase receptors, referred to as type I and type II receptors (reviewed in Refs. 17 and 18). The constitutively active type II receptor phosphorylates the type I receptor in the complex formed after ligand binding, which leads to activation of the type I receptor kinase activity. The best characterized substrates for the type I receptor are the intracellular Smad proteins, although other Smad-independent signaling pathways are also activated (19). The Smad proteins are subdivided into the receptor-regulated R-Smads (Smad1, -2, -3, -5, and -8), the inhibitory I-Smads (Smad-6 and -7), and the common partner Smad4 (20, 21). Upon phosphorylation of the R-Smads by the activated type I receptor, phosphorylated R-Smads form a heteromeric complex with Smad4, which modulates transcription after translocation to the nucleus. We have shown in our previous studies that all seven known type I Ser/Thr receptors, designated activin receptor-like kinases 1–7 (Alk1–7), activate endogenous and adenovirally overexpressed Smad1 and Smad2 proteins as well as inhibin B production in cultured human granulosa-luteal (hGL) cells when Alk1–7 are adenovirally overexpressed as constitutively active mutant Alk1–7 proteins (22). However, it has not yet been demonstrated which of the type I receptors are used for signaling the relevant TGF-ß family ligands in these primary cultures of human granulosa cells.
We have most recently investigated the process of GDF9 signaling in hGL cells (23) and rat diethylstilbestrol-treated granulosa cells (24). In both cell types, GDF9 treatment induced Smad2 phosphorylation and inhibin B production. In the TGF-ß superfamily, the type I receptors Alk4 (the activin type IB receptor) and Alk5 (the TGF-ß type I receptor) are principally associated with the activation of Smad2 and Smad3 (25). Recently, we have shown that signaling by GDF9 is mediated by the Alk5 receptor in COS-7 and cultured rat granulosa cells (26). We have also shown that GDF9 uses the type II BMP receptor (BMPRII) as its other signaling receptor in rat granulosa cells (27). However, although hGL cells endogenously express mRNAs of the type I receptors Alk4 (28) and Alk5 (29); the type II receptors TGF-ßRII (29), activin RII (28), and activin RIIB (28); and the BMPRII receptor (30), nothing is known about their functional ability to act as signaling mediators of any of the relevant TGF-ß family ligands in human ovarian cells.
In the current study we demonstrate the use of a Smad3 reporter (CAGA-luciferase) adenovirus in primary cultures of hGL cells to detect GDF9, TGF-ß, and activin responses. Also, from our adenoviral overexpression studies, we demonstrate the involvement of Alk5, but not Alk4, in Smad3 activation and inhibin B production in response to GDF9 treatment also in hGL cells. Finally, we show that adenoviral overexpression of wild-type receptors for TGF-ß or activin can be used to amplify the ligand response in hGL cells, which are refractory to nonviral methods of gene transfer.
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Recombinant activin A, BMP2, and TGF-ß1 were purchased from R&D Systems (Minneapolis, MN). Fetal calf serum (FCS) was purchased from Euroclone Ltd. (Devon, UK). DMEM and Ham’s F-12 were purchased from Invitrogen Life Technologies, Inc. (Gaithersburg, MD). Heparin (Fragmin) was purchased from Pharmacia & Upjohn (Stockholm, Sweden). BSA was purchased from Roche (Mannheim, Germany). FuGene 6 was purchased from Roche (Indianapolis, IN). Anti-FLAG M2 monoclonal antibody was purchased from Sigma-Aldrich Corp. (St. Louis, MO). Phospho-Smad3 antibody was provided by Dr. E. Leof (Mayo Clinic, Rochester, MN) (31). A stable cell line expressing fully processed mouse GDF9 was created by transfection of human embryonic kidney (HEK) 293T cell cultures, as described previously (23), and was used as the source of bioactive recombinant GDF9 protein. Levels of recombinant mouse GDF9 were estimated in immunoblots using a purified N-tagged rat GDF9 as a standard (32).
Expression plasmids and reporter gene constructs
The pGL3CAGA12-luciferase reporter plasmid was provided by Dr. C. H. Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden) (33). The pGL3BRE-luciferase reporter plasmid (34) and the expression plasmid pcDNA3-Smad3 were provided by Dr. P. ten Dijke (The Netherlands Cancer Institute, Amsterdam, The Netherlands).
hGL cell cultures
hGL cells were obtained with informed consent from women undergoing in vitro fertilization (IVF) treatments. For each experiment, cells from one to six patients were pooled, enzymatically dispersed, and separated from red blood cells by centrifugation through Ficoll-Paque as previously described (35). Thereafter, hGL cells were counted and plated at a density of 3–4 x104 cells/well on 24-well plates (Cellstar, Greiner Bio-one, Frickenhausen, Germany; final concentration, 3–4 x104 cells/ml). hGL cells were cultured in DMEM supplemented with 10% FCS, 2 mM L-glutamine, and antibiotics. Cells were cultured 2 d before adenovirus infections and 2–3 d before ligand stimulation experiments.
Adenovirus construction and infections
The recombinant adenoviruses Ad-CAGA9-luciferase (36) and Ad-Alk5/HA (37) were provided by Dr. P. ten Dijke (The Netherlands Cancer Institute). The Alk4 adenovirus was produced using the Transpose-Ad system (Q•Biogene Illkirch, France) (based on Ref. 38). The Alk4-coding sequence was obtained from the plasmid pCI-Alk4(ActRIB), provided by Dr. A. Klibanski (Massachusetts General Hospital, Boston, MA). The use of recombinant adenoviruses in hGL cultures has been recently optimized (22). We found in these previous studies that cytopathic effects are seen when purified recombinant adenoviruses are added to hGL cells at multiplicity of infection values approaching 300. These effects are characterized by cellular detachment and death. Hence, in the current studies we used an amount of the respective adenoviruses such that no signs of cytotoxic effects could be detected. Also, the purified Ad-CAGA9-luciferase adenovirus was titrated on hGL cells against the luciferase response to the following ligands, TGF-ß, activin, and GDF9. The minimum amount of virus required to obtain a maximum response for these ligands was then used in all subsequent assays. All viruses were amplified in transcomplemental 293 cells and purified with cesium chloride gradient ultracentrifugation as described previously (39). The purified viruses were stored in PBS with 10% glycerol at –70 C. Before adenovirus infection, hGL cells were cultured for 2–3 d. The hGL cells were infected by incubating 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.
Transient transfections and luciferase assays
HEK-293T cells were cultured in DMEM supplemented with 10% FCS, 2 mmol/liter L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37 C in 5% CO2. The 293T cells were plated at low confluence on 24-well plates and grown overnight before transfections. Transfections were performed in 0.5 ml medium with 100 ng/well CAGA12-luciferase reporter construct (33) or the BMP response element (BRE)-luciferase reporter construct (34), and 10 ng/well ß-galactosidase reporter plasmid using the FuGene 6 transfection reagent (Roche, Basel Switzerland). Twenty-four hours later the cells were treated with TGF-ß, activin A, BMP2, or GDF9 in 1% FCS/DMEM for 24 h. The cells were then lysed into 1x passive lysis buffer, and luciferase activity was measured with luciferase assay reagent (Promega Corp., Madison, WI) and normalized to ß-galactosidase activity. Data are the mean ± SEM of triplicate determinations from representative experiments, relative to an adjusted value of 1.0 for the mean of the control wells without Alk4 or Alk5 adenoviral infection.
Smad3 activation experiments and Western blot analysis
293T cells (106 cells) were cultured overnight in six-well plates in DMEM containing 10% FBS and transiently transfected in OptiMEM (Invitrogen Life Technologies, Inc., Carlsbad, CA) for 4 h with 500 ng or 2 µg/well of either pcDNA3 or pcDNA3-FlagSmad3 using Lipofectamine 2000 (Invitrogen Life Technologies, Inc.). After 20 h in DMEM containing 10% FBS, transfected cells were starved for at least 3 h in serum-free medium to minimize basal Smad activity. The cells were then treated with 300 ng/ml GDF9 for 60 min and washed once on ice with chilled PBS before cell lysis in Laemmli buffer containing ß-mercaptoethanol. Cells were gently sonicated on ice for 15 sec with an MSE sonicator (Sanyo Corp., Osaka, Japan) and boiled for 3 min. Cellular proteins were separated on 7.5% SDS-PAGE gels (Bio-Rad Laboratories, Inc., Hercules, CA) and electroblotted onto Hybond-P membranes (Amersham Biosciences, Piscataway, NJ). For detection of phosphorylated Smad3 and FlagSmad3, membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween and 5% fat-free dry milk. After blocking nonspecific binding, membranes were incubated with antiphospho-Smad3 antibody (31) (dilution, 1:3000) or M2 antibody (dilution, 1:1000) at 4 C overnight. The secondary antirabbit and antimouse antibodies were used following the manufacturer’s instructions (Amersham Biosciences). Immunoreactive proteins were detected using enhanced chemiluminescence (ECL kit, Amersham Biosciences).
Inhibin B ELISA
Measurement of dimeric inhibin B by ELISA was carried out as previously described (22). Briefly, hGL cells were cultured in 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 performed as described above. Ligand treatments (10 ng/ml TGF-ß and 250 ng/ml GDF9) were carried out in DMEM supplemented with 2% FCS 24 h postinfection; the spent medium was harvested after 96 h of incubation, and dimeric inhibin B concentrations were quantified from the medium using an inhibin B ELISA (Serotec, Oxford, UK) together with a signal amplification kit (Invitrogen Life Technologies). Data are the mean ± SEM of triplicate determinations from representative experiments, relative to an adjusted value of 1.0 for the mean of control wells without adenoviral infection.
Statistical analysis of bioassay data
For the data shown in Figs. 1–4
, the statistical significance of each reported effect was calculated based on the log values of triplicate experiments using a t test. Significance was accepted if P 
0.05. For Figs. 1 and 2, the t value 
(T, C) was calculated for each treatment sample group T compared with the control sample group C. For Figs. 3 and 4, the t value (Tn-cn, T0-c0) was calculated for each treatment sample group T at adenoviral concentration n compared with the treatment sample group without adenovirus application. These values were normalized by subtracting the mean value c of the control sample group C corresponding to the same adenoviral concentrations. The effect of the normalization was to subtract the treatment-independent effects of the adenovirus application.
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Previously we have shown that GDF9 activates Smad2 in hGL cells (23) and in primary cultures of rat granulosa cells (24). Smad2 activation is initiated by induction of the Ser/Thr kinase activity of the TGF-ß type I receptor Alk5 or the activin type IB receptor Alk4 (25). The activation of these two receptors is also associated with the phosphorylation (and, hence, activation) of Smad3 (25). Indeed, we have just reported that GDF9 activates Smad3 via Alk5 in P19, COS-7, and cultured rat granulosa cells (26). The activation level of Smad3 was simply monitored by the CAGA-luciferase reporter plasmid, which contains repeats of the Smad3 response element (CAGA) in front of the luciferase cDNA (33). In the current study we initially wanted to investigate whether GDF9 could activate the CAGA-luciferase reporter in some readily transfectable human cell line. We found that the human embryonic kidney cell line HEK-293T was very responsive to GDF9, as measured by induction of the CAGA-luciferase reporter activity (P 0.01 for GDF9 vs. control; Fig. 1A
). In the same cell line, the BMP-responsive reporter, BRE-luciferase, was only activated by BMP2 (P 0.01 for BMP vs. control) and not by GDF9 (Fig. 1B). Activation of the CAGA promoter in 293T cells by GDF9 was concentration dependent, with a 50% effective dose of 250 ng/ml (Fig. 1C). GDF9 bioactivity could be neutralized by preincubation with either the BMPRII ectodomain (27) or a monoclonal antibody specific for GDF9 (8) (data not shown). Because GDF9 activates the Smad3-dependent CAGA promoter, we examined the phosphorylation state of Smad3 in GDF9-treated 293T cells. As expected, GDF9 increased the phosphorylation level of recombinant Smad3 (Fig. 1D) detected after transfection of 293T cells with a FLAG-Smad3 expression plasmid and immunoblotting with an antiphospho-Smad3 antibody (31).
Activation of a Smad3-specific reporter in hGL cells by GDF9, TGF-ß, and activin
Because we observed activation of Smad3 by GDF9 in human 293T cells, we wanted to determine whether GDF9 activates Smad3 in primary cultures of hGL cells, in which we have previously seen activation of Smad2 by GDF9 (23). hGL cells are refractory to transfection by liposome-based reagents; hence, we made use of an adenovirus incorporating the reporter CAGA9-luciferase (36). Using adenoviral gene transduction, we introduced the CAGA-reporter into hGL cells and stimulated the cells with various ligands. GDF9, TGF-ß, and activin all activated the CAGA-luciferase reporter in hGL cells, and this response was dose dependent (Fig. 2). The lack of activation of the CAGA-luciferase reporter in hGL cells by BMP2 (Fig. 2C) demonstrates the specificity of the assay for Smad3-activating ligands. The results show that a specific and sensitive assay for Smad3 activation is thus able to be performed in hGL cells. Furthermore, when Smad3 is overexpressed in hGL cells via adenoviral gene transduction, the cellular response to GDF9 or TGF-ß is greatly potentiated (data not shown).
Adenoviral overexpression of wild-type Alk5 and Alk4 amplifies the CAGA-luciferase response of hGL cells to GDF9, TGF-ß, and activin, respectively
GDF9, being a member of the TGF-ß superfamily, is most likely to signal via the binding and activation of cell surface type I and II Ser/Thr kinase receptors, a major substrate of which are the Smad proteins, which act as transcription factors. Recently, we have shown that GDF9 activates Smad2 in hGL cells (23); in this study we also demonstrate Smad3 activation (Fig. 2). Smad2/3 activation is associated with induction of the Ser/Thr kinase activity of the TGF-ß type I receptor Alk5 or the activin type IB receptor Alk4. Previously, we demonstrated the expression of both Alk4 (28) and Alk5 (29) receptors in hGL cells. In the current study we investigated the effect of adenoviral-mediated overexpression of either Alk5 or Alk4 in hGL cells on the GDF9, TGF-ß, or activin response, as measured by Smad3 activation, via the CAGA-luciferase reporter. Overexpression of Alk5 potentiated the response of hGL cells to GDF9 stimulation as well as to stimulation by the Alk5 ligand TGF-ß, but not to activin (Fig. 3A). This potentiation was dependent on the dose of Alk5 adenovirus used to transduce the hGL cells. In contrast, the overexpression of Alk4 did not affect the GDF9 response of the transduced hGL cells, but strongly potentiated the activin response of these cells (Fig. 3B). Again, potentiation of the activin response was dependent on the dose of Alk4 adenovirus used to transduce hGL cells.
Effect of overexpression of type I receptors Alk5 and Alk4 in hGL cells on GDF9-induced inhibin B production
Previously we have shown that GDF9 stimulates the production of inhibin B by hGL cells (23) and rat granulosa cells (24). Hence, we examined the effect of overexpression of either Alk5 or Alk4 in hGL cells on the GDF9 response, as measured by the level of inhibin B in the culture medium. Overexpression of Alk5 strongly potentiated (
4.5-fold) the response of hGL cells to GDF9 (Fig. 4A), whereas the TGF-ß-stimulated inhibin B response was less affected by the Alk5 expression level. The effect of overexpression of Alk4 on the GDF9 response was minimal (less than doubled; Fig. 4B). Overexpression of Alk4 alone was enough to cause an increase in the level of inhibin B produced by hGL cells, and the effect of GDF9 treatment on these cultures was only additive in terms of inhibin B production.
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In the current study we have made use of plasmid reporter constructs specific for the TGF-ß/activin Smad3 pathway (CAGA-luciferase) (33) or the BMP Smad1/5/8 pathway (BRE-luciferase) (34) and have used the easily transfectable human cell line, HEK-293T, as a model system to investigate the activation of these reporter plasmids. We found that GDF9, TGF-ß, and activin all activate the CAGA-luciferase reporter in 293T cells. Furthermore, although BMP2 activates the BRE-luciferase reporter in these cells, GDF9 does not, and TGF-ß and activin actually inhibit the basal activation level of this reporter. Such an inhibitory effect on the basal activity of the BRE-luciferase reporter was also recently observed for TGF-ß- and myostatin-treated HepG2 cells (42). Also, by transfecting the 293T cells with a Smad3 expression plasmid, we have been able to demonstrate phosphorylation of Smad3 in response to GDF9 treatment. Given the results of Smad3 activation in 293T cells, we made use of an adenovirus to incorporate a CAGA-luciferase reporter cassette into hGL cells, which are refractory to common transfection methods, but susceptible to adenovirus transduction. Indeed, recombinant adenoviruses have proven to be very efficient in expressing exogenous gene products in human (22) and rat (43) primary granulosa cells. Use of the Ad-CAGA9-luciferase adenovirus (36) enabled monitoring of Smad3 activation in hGL cells, an assay in which GDF9, TGF-ß, and activin were all active, but BMP2 was not. This is the first time to our knowledge that a transcriptional reporter assay has been carried out in cultures of primary granulosa cells via adenoviral transduction. It is important to note that GDF9 is capable of activating both Smad2 (23) and Smad3 (the current study) in hGL cells, because these Smads have different downstream effects (44). Interestingly, Smad3-null mice are viable, but have reduced fertility compared with wild-type mice (45, 46). Given the results presented in the current study, this decreased fertility may be due to decreased GDF9 signaling. Consistent with the important role of the GDF9 pathway in initial follicle recruitment, decreases in Smad3 expression in Smad3-null mice did not affect the size of the primordial follicle pool at birth, but did alter the growth of primordial follicles to the antral stage.
We have previously demonstrated the expression of mRNAs of Alk4 (28) and Alk5 (29) receptors in hGL cells, but we did not perform functional studies to determine which ligand signals they transmit in these cells. In the current study we investigated the effect of adenoviral-mediated overexpression of either Alk5 or Alk4 in hGL cells on the GDF9 response, as measured by the CAGA-luciferase reporter or by inhibin B production. The overexpression of Alk5 potentiated the response of hGL cells to GDF9 stimulation as well as to stimulation by the Alk5 ligand TGF-ß, although the effect on the TGF-ß response in the case of inhibin B production is less clear. The inhibin B response may well be dependent on some signaling molecules other than just activated Smads, unlike the CAGA-luciferase response. In the case of TGF-ß, the Alk5 expression level may well not be limiting for the inhibin B response, such that an increase in the Alk5 expression level may have a minimal effect. For the GDF9 response, the Alk5 expression level may be limiting; hence, there was good potentiation of the GDF9 response in both the CAGA-luciferase and inhibin B assays upon overexpression of Alk5. Such a situation is consistent with our previous results using COS-7 cells, which were not responsive to GDF9 without transfection with an Alk5 expression plasmid (26). The activin response in hGL cells was greatly potentiated by the overexpression of Alk4, but had no significant effect on the GDF9 or TGF-ß response.
Until recently, the only ligand known to use the type I receptor Alk5 in signaling was TGF-ß (17, 18). However, recently, myostatin (also known as GDF8) has been shown to activate both Alk5 and Alk4 as a complex with the activin type IIB receptor (42), and we have also shown that GDF9 signals via Alk5 in COS-7, mouse P19 cells, and rat granulosa cells (26). We have also previously shown that the BMPRII ectodomain is capable of binding GDF9 and neutralizing GDF9 bioactivity (27). Hence, it appears that GDF9 signals via a complex consisting of Alk5 and BMPRII. Such a combination of receptors has not been reported previously for any other member of the TGF-ß superfamily and may be significant in the ovarian specificity of GDF9 action. Interestingly, recently it was shown that the BMPRII is capable of signaling directly, as opposed to signaling via a type I receptor (47). However, it is not known whether such direct type II receptor signaling is important for the biological effects of GDF9.
Studies of primary human cell cultures are often very demanding, due at least partly to the difficulty in obtaining the cells. As a result of the procedure of oocyte aspiration from women undergoing IVF treatment, there is the possibility to obtain hGL cells, which we have used extensively for investigating fundamental aspects of human ovarian cell biology (22, 23, 28, 29, 30, 35). One difficulty in using these cells for in vitro studies of hormone/growth factor responses is that they are refractile to nonviral transfection methods (22) and, hence, transcriptional reporter assays. We have now shown that a reporter assay can be carried out in these cells by using an adenovirus containing the Smad3-specific response element CAGA (33) linked to the luciferase reporter-coding sequence. This reporter assay responds to the Smad3-activating ligands, TGF-ß and activin, as well as GDF9, which we recently demonstrated to be a Smad3-activating ligand in other cell types (26). Furthermore, the effects of such growth factors can be amplified by the overexpression of their cell surface receptors, which is potentially a useful approach for studying interactions of the members of the TGF-ß superfamily with hGL cells.
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First Published Online October 13, 2004
1 N.K.-O. and D.G.M. contributed equally to the manuscript. 
Abbreviations: Ad, Adenovirus; Alk, activin receptor-like kinase; BMP, bone morphogenetic protein; BMPRII, bone morphogenetic protein receptor type II; BRE, BMP response element; FCS, fetal calf serum; GDF9, growth differentiation factor-9; HEK, human embryonic kidney; hGL, human granulosa-luteal; I-Smad, inhibitory Smad; IVF, in vitro fertilization; R-Smad, receptor-regulated Smad; Ser/Thr, serine/threonine.
Received July 2, 2004.
Accepted September 28, 2004.
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PS3), followed by the secondary antibody and detection via enhanced chemiluminescence. For detecting the Flag peptide sequence, the blot that had been probed with the 
