help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mukasa, C.
Right arrow Articles by Nawata, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mukasa, C.
Right arrow Articles by Nawata, H.
Endocrinology Vol. 144, No. 4 1603-1611
Copyright © 2003 by The Endocrine Society

ARTICLE

Activin Signaling through Type IB Activin Receptor Stimulates Aromatase Activity in the Ovarian Granulosa Cell-Like Human Granulosa (KGN) Cells

Chizu Mukasa, Masatoshi Nomura, Tomoko Tanaka, Kimitaka Tanaka, Yoshihiro Nishi, Taijiro Okabe, Kiminobu Goto, Toshihiko Yanase and Hajime Nawata

Department of Medicine and Bioregulatory Science (C.M., M.N., K.T., Y.N., T.O., K.G., T.Y., H.N.), Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582; Core Research for Evolutional Science and Technology (M.N., T.T., T.O., K.G., T.Y., H.N.), Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan

Address all correspondence and requests for reprints to: Masatoshi Nomura, Kyushu University, Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: nomura{at}med.kyushu-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In addition to a stimulatory effect on FSH production by the pituitary gland, activin is thought to have a paracrine or autocrine role in follicular development in the ovary, where it is produced. Recently, we established a human ovarian granulosa tumor cell line, KGN, which possesses in vivo characteristics of granulosa cells, namely the expression of functional FSH receptors and cytochrome P-450 aromatase. Here, we have demonstrated the activin signaling pathway and its role in KGN cells. A series of transient transfection experiments revealed that activin type IB receptor (ActRIB) is an essential component of the activin signaling pathway in KGN cells. Smad2 was found to act downstream of ActRIB as an intracellular signal transmitter. Smad7, but not Smad6, was an inhibitory Smad in the pathway. Finally, we show that FSH receptor expression and cytochrome P-450 (P-450) aromatase activity was up-regulated by activin stimulation through ActRIB in KGN cells. These results show that we have clarified the signaling mechanisms and the roles of activin in the human granulosa cell line, KGN. Activin signaling mediated by ActRIB-Smad2 system in the ovary may thus be essential for the regulation of follicular differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OVARIAN FOLLICULOGENESIS is controlled by a number of factors including the pituitary gonadotropin and the intraovarian TGF-ß family members such as activin, inhibin, bone morphogenetic protein (BMP), growth differentiation factor 9 (GDF9), and Müllerian-inhibiting substance (1). Therefore, it is necessary to elucidate the mechanism of how these local factors function and interact with the gonadotropin in the follicle to fully understand ovarian folliculogenesis.

Activin, a TGF-ß family member, was initially identified as a secreted protein from ovarian follicular fluid, which stimulates FSH production by the pituitary gland (2, 3). A growing number of studies have demonstrated its involvement in a wide variety of physiological processes, including embryogenesis and carcinogenesis, as well as reproduction (4). In the ovary, activin is expressed predominantly in the granulosa cell layer of follicles, suggesting important roles in processes such as folliculogenesis, steroid hormone production, and oocyte maturation as a paracrine or autocrine factor (5). The role of gonadotropin in ovarian follicle development is well established (6). It is apparent that both paracrine and autocrine growth factors have important roles in follicle development. Many in vitro studies using primary cultures of granulosa cells have shown that activin A has a direct effect on many ovarian granulosa cell functions. For example, it has a mitogenic effect on immature granulosa cells, as demonstrated by an increase in [3H]thymidine uptake in vitro (7). With respect to the relationship between activin and pituitary FSH, activin A has been demonstrated to induce FSH receptor mRNA (8, 9) and potentiate FSH-stimulated aromatase activity as well as progesterone and inhibin production in rat immature granulosa cells (10). These results strongly suggest the importance of activin in the regulation of both maturation and proliferation of granulosa cells through paracrine or autocrine mechanisms. Although many of these studies have provided valuable insights, the signaling mechanism of activin in ovarian granulosa cells is still not well understood, mainly because of the limitations in molecular biological analyses that use a primary culture system.

The basal components of the TGF-ß family signaling pathways are the type II and type I transmembrane serine and threonine kinase receptors and the cytoplasmic Smad proteins (11, 12, 13). Upon ligand binding, the type II receptor recruits and transphosphorylates the type I receptor. The type I receptor acts downstream of the type II receptor and has been shown to determine signaling specificity within the heteromeric receptor complex (14, 15). The type I receptor, activated by phosphorylation, subsequently propagates signals to the Smad pathway. Once phosphorylated, the receptor-regulated Smads (R-Smads) dissociate from the receptor complex, bind to the common Smad (Smad4) and enter the nucleus as a complex where they participate in the transcriptional regulation of the target genes. On the other hand, inhibitory Smads have been shown to block the phosphorylation of R-Smad by the type I receptor, or to block the hetero-oligomerization of R-Smad with Smad4 by binding to the type I receptor or R-Smad, thus interfering with the TGF-ß signaling (16, 17, 18). In addition to these Smad proteins, SARA (Smad anchor for receptor activation) has been identified as an essential intracellular component for activation of the Smad proteins by the type I receptor (19). Activin, like most of the TGF-ß family members, mediates its action by binding to a complex of type I and type II receptors. The type I receptors comprise type IA (ActRIA) and type IB (ActRIB), also known as the activin receptor-like kinases Alk2 and Alk4, respectively. The type II receptors comprise type IIA (ActRIIA) and type IIB (ActRIIB). Activated type I receptors propagate the signal to specific intracellular Smad proteins, namely Smad2 and/or Smad3. The precise roles for the different activin receptors and Smads are still not clear. The different usage of the type I and type II receptors and Smads may be responsible for the different actions of activin on different tissues and target genes, explaining their functional diversity. Thus, the delineation of the cellular components of the system may be necessary for understanding the regulation of numerous biological systems.

Recently, we established the human granulosa cell line, KGN. As reported previously (20), this cell line expresses the functional FSH receptors and has aromatase activity, reminiscent of ovarian granulosa cells in vivo. Thus, the KGN cell line could prove to be a very useful model for the study of the role of activin in the regulation of granulosa cell differentiation during folliculogenesis in the ovary. To clarify the signaling pathway and the role of activin in the ovarian granulosa cells, we have performed a detailed analysis of the KGN cells. We found that activin signaling was mainly transmitted though the ActRIB-Smad2 system, leading to the up-regulation of FSH receptor expression and aromatase activity in KGN cells. The activin signaling through the ActRIB-Smad2 may thus be essential for ovarian granulosa cell function and differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The human ovarian granulosa-like tumor cell line, KGN, was cultured in DMEM and Ham’s F-12 medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal bovine serum (Sera Laboratories Ltd., Sussex, UK), penicillin (100 U/ml), and streptomycin (100 µg/ml) in a 5% CO2 atmosphere at 37 C, as described previously (20).

Plasmids
The expression vectors for rat ActRIA, human ActRIB, and human ActRIBCA were kindly provided by Dr. Lawrence S. Mathews (University of Michigan, Ann Arbor, MI). A constitutively active form of ActRIB, designated ActRIBCA, was generated by the amino acid substitution of the threonine at position 206 with glutamine in human ActRIB cDNA (21). The expression vectors for Smad2, Smad3, and Smad4 were constructed by inserting their entire coding sequences into the pCDNA3 vector. The reporter plasmid 3TP-Lux, containing the three consecutive TPA responsive elements and TGF-ß responsive elements of the plasminogen activator inhibitor 1 (PAI-1) promoter, was kindly provided by Dr. J. Massague (Memorial Sloan-Kettering Cancer Center, New York, NY). The reporter plasmid AR3-Lux, containing the activin-responsive elements of the Mix 1 promoter and the expression vector for FAST1 were kindly provided by Dr. M. Whitman (Harvard Medical School, Boston, MA). The ActRIBCA expression vector was constructed by inserting a 1.6-kb cDNA fragment, which contains the entire human ActRIBCA coding sequence, between the HindIII and XbaI sites of the plasmid pEF-BOS (22). The ActRIBCA gene was tagged with a triple HA influenza virus hemagglutinin epitope at the carboxyl terminus and its expression was controlled by the human elongation factor-1{alpha} promoter. A 5.0-kb SalI-SalI fragment containing an internal ribosome-entry site (IRES)-ßgeo cassette (23) was inserted next to the HA epitope for G418 selection as described (24).

Luciferase assays
1 x 105 cells/well were transfected using 3 µl/well Superfect Transfection Reagents (QIAGEN, Hilden, Germany) with 1 µg/well 3TP-Lux reporter construct and 1 ng/well pRL-CMV (Promega Corp., Madison, WI) as an internal control. After incubation overnight in the optimum medium, the medium was changed to serum-containing culture medium. After another 24-h incubation of the cells in the presence or absence of activin A (100 ng/ml) or TGF-ß1 (50 ng/ml, Sigma, St. Louis, MO), luciferase activity was measured using the dual luciferase assay system (Promega Corp.) in a microLumat LB9507 luminometer. The luciferase activity was normalized for transfection efficiency using the Renilla luciferase activity from pRL-CMV. In other experiments, cells were cotransfected in the same way with the AR3-Lux reporter construct and the FAST1 expression vector.

mRNA analysis
KGN cells were cultured in a 10-cm dish containing 1.7 x 106 viable cells in 10 ml of medium. The cells were transfected with the appropriate expression vectors and cultured in the presence or absence of activin A (100 ng/ml) for 24 h followed by preparation of the total RNA using ISOGEN solution (Molecular Research Center, Inc., Tokyo, Japan). The final RNA pellet was dissolved in Tris-EDTA buffer. Total RNA was quantified by measuring the absorbance of the samples at 260 nm, and stored at -80 C until the assay. The first strand cDNA was synthesized using 1 µg total RNA using an RT-PCR kit (Stratagene, La Jolla, CA). To analyze the expression of the activin signaling components, including type I and type II activin receptors, Smads, and SARA, a sensitive RT-PCR was performed. PCR was carried out in a 50-µl reaction mixture containing MgCl2 (2.5 mM), deoxynucleotide triphosphate (0.3 mM) and 2.5 U of Taq DNA polymerase (Life Technologies, Inc.) under the following conditions: 30 or 35 cycles of denaturation at 93 C for 30 sec, annealing at 60 C for 30 sec and extension at 72 C for 1 min. Primer sets used in this study were as follows: ActRIA, 5'-AATGTTGCCGTGAAGATCTTC-3'/5'-CTGAGAACCATCTGTTGGGTA-3' (700 bp); ActRIB, 5'-CTGGCTGTCCGTCATGATGCA-3'/5'-CAATTCGCTCTCAGAGTCTCC-3' (684 bp); ActRIIA, 5'-ACCAGTGTTGATGTGGATCTT-3'/5'-TACAGGTCCATCTGCAGCAGT-3' (456 bp); ActRIIB, 5'-TTCTGCTGCTGTGAAGGCAAC-3'/5'-GAGGTCGCTCTTCAGCAAT ACA-3' (699 bp); Smad2, 5'-AGAAGTCAGCTGGTGGGTCTG-3'/5'-TCATGATGACTGTGAAGATCAGG-3' (370 bp); Smad3, 5'-GCTGGAAGAAGGGCGAGCAGA-3'/5'-CTTCATATTGAAGGCGAACTCAC-3' (299 bp); Smad4, 5'-TCTGGAGGTGGCCTGATCTTC-3'/5'-AAGTTGGCAGTGCTGGTAGCAT-3' (349 bp); Smad6, 5'-ACTGGATCTGTCCGATTCCAC-3'/5'-CGAAGTCGAACACCTTGATGG-3' (455 bp); Smad7, 5'-TGTGCAAAGTGTTCAGGTGGC-3'/5'-GTCCGAATTGAGCTGTCCGAG-3' (437 bp); SARA, 5'-AGAACATGCCTAATGGGTCTGG-3'/5'-CTGGGTCTTGCATTCCATA GG-3' (431 bp); FSH receptor, 5'-CTCAGGCTAGGGGTCAGAGA-3'/5'-CTGGTAGTTAGGATCACTAGC-3' (256 bp). The number in parentheses indicates the size of the products. Aliquots of the PCR products were electrophoresed in 2% agarose gels containing 0.5 mg/ml ethidium bromide and photographed under UV light using a positive/negative instant film (Polaroid 665, Nippon-Polaroid, Tokyo, Japan). The authenticity of the PCR products was confirmed by sequencing.

Quantitative analyses of the expressions of the activin receptors were performed using a LightCycler Roche (Mannheim, Germany) as described (25). The primer set for human ß-actin was supplied in the LightCycler-Primer set (Roche). The other primer sets used in this study were described above. RNA extraction, cDNA preparation and quantitative PCR were all performed in triplicate. For PCR, 2 µl each of the standard cDNA pool diluted 1:2, 1:20, 1:200, and 1:2000 (arbitrarily designated 1.0, 0.1, 0.01, and 0.001, respectively), the quality control cDNA pool diluted 1:25, and the sample cDNAs diluted 1:10 in sterile water or calibrator, were added to individual capillaries. Taq enzyme, deoxynucleotide triphosphate, reaction buffer, and SYBR GREEN I dye were supplied in the FastStart DNA Master SYBR Green I kit (Roche), of which 2 µl/capillary was added. Primer concentrations of 0.5 µM were added to each capillary. Magnesium concentrations (2–4 mM), annealing temperatures (58–62 C), and extension time (number of seconds = product size/25 plus 3) were determined for individual primer sets. The capillary volume was made up to 20 µl with sterile water. Forty cycles of PCR were programmed to ensure the threshold crossing point (cycle number) was attained. Fluorescence emission was monitored continuously during cycling. At the completion of cycling, melting curve analysis was carried out to establish the specificity of the amplicons produced. The level of expression of each mRNA and their estimated crossing points in each sample were determined relative to the standard preparation using the LightCycler computer software. The relative abundance of the mRNAs, expressed as fold changes, was extrapolated from crossing point data. A difference of 1 PCR cycle in crossing point number translates into a 2-fold change in mRNA expression.

The RT-PCR fragment of human FSH receptor cDNA was subcloned into the pGEM-T Easy Vector (Promega Corp.). The plasmid was linearized and used as a template for [32P]-labeled riboprobe synthesis using an in vitro transcription system (Riboprobe System-SP6, Promega Corp.). The ribonuclease (RNase) protection assay was carried out according to the manufacturer’s instructions. Briefly, 20 µg of each total RNA and the riboprobe were hybridized followed by digestion with RNasese. The RNase-resistant hybrids were separated by electrophoresis in 5% polyacrylamide/8 M urea gels. The results were visualized by autoradiography and the signal intensities of the protected RNA bands were quantified by densitometric scanning (FUSIX BAS2000, Fuji Photo Film Co., Ltd., Tokyo, Japan).

Western blot analyses
Proteins were extracted by cell lysis in a buffer containing 50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 tablet/10 ml of protease inhibitor mix (Roche). Extracts were subjected to 10% SDS-PAGE, blotted, and probed with specific antibodies. The anti-HA antibody, 12CA5, was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Chemiluminescent signals were generated by incubation with the ECL reagent (Amersham Pharmacia Biotech, Buckinghamshire, UK). The antihuman P-450 aromatase antibody was kindly provided by Dr. N. Harada (Fujita Health School of Medicine, Toyoake, Japan).

Aromatase assay
The aromatase activity of KGN cells was determined as previously described (20). Briefly, KGN cells were transfected with the ActRIB expression plasmid and then plated on a 12-well multidish (Nalge Nunc International, Rochester, NY) in DMEM/Ham’s F-12 containing 10% fetal calf serum (FCS). At confluency, the culture medium was replaced with medium containing 10% dextran-coated charcoal-treated FCS (steroid-free FCS), and the cells were then incubated for another 24 h in the presence or absence of human FSH (hFSH; Sigma) or activin A at the concentration indicated in the figures. After treatment, the cells were further incubated with 11.4 nM [1ß-3H] androstenedione (NEN Life Science Products, Boston, MA; specific activity, 25.9 Ci/mmol) for 6 h. After incubation, the medium (1.0 ml) was transferred to tubes containing 0.5 ml ice-cold 30% (wt/vol) trichloroacetic acid, and then centrifuged to remove the precipitated protein. The cells were harvested using 0.25% trypsin-1 mM EDTA to determine the protein concentration. The amount of radioactivity in the [3H] H2O was corrected by subtracting the blank values from each sample. The cell protein content was determined using a micro bicinchoninic acid kit (Pierce Chemical Co., Rockford, IL) after the cells were dissolved in cell lysis buffer. The aromatase activity was expressed as picomoles per mg cell protein per hr incubation. Protein kinase inhibitor H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) was purchased from Seikagaku Corp. Co. Ltd. (Tokyo, Japan).

Statistics
All experiments were carried out at least three times. In the luciferase and the aromatase assays, each independent experiment was run in triplicate or duplicate plates and these were used to generate a single mean value, which was then used to generate the mean ± SD shown in the figures. All values represent the mean ± SD. Statistical significance was determined by one-factor ANOVA followed by a post hoc test (Fisher’s protected least significant difference test).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of activin signaling pathway components in KGN cells
To investigate the activin signaling pathway in KGN cells, we first investigated the expression of its components by a RT-PCR. As shown in Fig. 1Go, each product of the activin receptors and Smads amplified by RT-PCR was detected as a single band, and the size of each fragment was found to correspond to the predicted size. The authenticity of the PCR products was further confirmed by sequencing. All of the subtypes of activin type I and type II receptors, ActRIA, ActRIB, ActRIIA, and ActRIIB, were detected at 35 cycles of RT-PCR, consistent with previous reports that human granulosa cells express these four subtypes of receptors (26, 27). At 30 cycles, however, neither ActRIB nor ActRIIB were detected under the conditions employed in this study. To complete the signal transduction from the cell membrane to the nucleus, intracellular signaling molecules such as Smad proteins and SARA are required. In KGN cells, the mRNAs for these intracellular components, including Smad2, Smad3, Smad4, and SARA, were amplified as shown in Fig. 1Go. With regard to the inhibitory Smads, Smad7 was detected at 30 cycles, whereas Smad6 could not be amplified even at 40 cycles (data not shown). Taken together, we have detected mRNAs encoding all the known components required for activin signaling in KGN cells, suggesting the potential for activin signal transduction in KGN cells.



View larger version (34K):
[in this window]
[in a new window]
  		Figure 1. RT-PCR amplification of the known components of the activin signaling pathway. Total RNA from KGN cells was prepared and RT-PCR was performed as described in Materials and Methods. Ethidium bromide-stained PCR products were separated in a 2% agarose gel. Each of the products is listed above the panels. A, Activin receptors. PCR products at 30 or 35 cycles are shown. Note that all the subtypes of activin type I and type II receptors, ActRIA, ActRIB, ActRIIA, and ActRIIB, were detected at 35 cycles of RT-PCR. B, Smads and SARA. All the components except Smad6 are detected at 30 cycles.

 
Relative expression of the activin receptors in KGN cells
To quantitatively assess the relative expression levels of the activin receptors, crossing point comparisons of real-time PCR were performed and fold differences estimated. As summarized in Table 1Go, ActRIA and ActRIIA mRNAs were more highly expressed as the type I and type II activin receptors, respectively. The amounts of these receptors were 8-fold higher than those of ActRIB and ActRIIB, consistent with the results shown in Fig. 1Go. Next, the type I receptor employed for the functional receptor complexes and mediation of the action of activin in the granulosa cells was investigated.


View this table:
[in this window]
[in a new window]
  		Table 1. Quantitative analyses of the activin receptors by real-time PCR

 
Identification of ActRIB as the type I receptor for activin in KGN cells
To address the presence of the activin signaling pathway, KGN cells were transiently transfected with 3TP-Lux, a TGF-ß/activin-responsive reporter construct, and stimulated with either activin A or TGF-ß1 for 24 h before being measured for luciferase activity. Unexpectedly, activin had little effect on the luciferase activity, whereas TGF-ß increased it more than 3-fold as shown in Fig. 2Go, demonstrating a defective activin signaling pathway in KGN cells. Two isoforms of the type I receptors, ActRIA and ActRIB, have been identified by their ability to bind activin in the presence of the type II receptor (28). However, as these type I receptors appear to have different functions, it has been suggested that ActRIA transmits a BMP-like signal (29). Thus, we speculated that the low level of expression of ActRIB might be a reason for the defective activin signaling pathway in KGN cells. To address this possibility, KGN cells were transfected with each type I receptor along with the reporter construct as indicated in Fig. 2Go. As we expected, activin stimulation of KGN cells resulted in a 4-fold induction of the 3TP-Lux luciferase activity when the cells were cotransfected with ActRIB. The KGN cells became competent to activin stimulation by cotransfection of the ActRIB expression plasmid. These results suggest that activin signaling requires ActRIB, but not ActRIA, as a type I receptor in KGN cells.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 2. ActRIB is required for the activin signaling in KGN cells. 1 x 105 KGN cells were transiently transfected with 3TP-Lux (0.5 µg) with or without the indicated type I receptor (0.5 µg) and treated with either activin A (100 ng/ml) or TGF-ß1 (50 ng/ml) for 24 h before being measured for luciferase activity. In the presence of an increasing dosage of Smad7 (0.1–0.5 µg), the luciferase activity of the cells transfected with ActRIB followed by treatment with activin A (100 ng/ml) is decreased in a dose-dependent manner. The fold induction relative to the luciferase activity in the cells transfected with the reporter luciferase plasmid alone are shown. Each value indicates the mean ± SD of at least three separate experiments, with triplicate plates per point.

 
Smad2 as a downstream signal transmitter of the activin/ActRIB signaling pathway
Currently, the intracellular signaling pathway of activin is indistinguishable from that of TGF-ß. Both Smad2 and Smad3 have been shown to mediate the activin signal as well as TGF-ß (30). It is widely accepted that R-Smads are activated through phosphorylation by their ligand-activated type I receptors. Thus, it is conceivable to speculate that the type I receptors specify the downstream R-Smad proteins. To assess the involvement of Smad proteins in activin signaling, KGN cells were transfected with AR3-Lux, an activin-responsive reporter construct, and the FAST1 expression plasmid together with different combinations of Smads and ActRIB as indicated in Fig. 3Go. When AR3-Lux was used as a reporter construct, activin stimulation without ActRIB transfection resulted in a slight induction of the luciferase activity (significantly different, P < 0.05). AR3-Lux seems to be more sensitive than 3TP-Lux to activin stimulation, consistent with a previous observation (31). To confirm the requirement for ActRIB in activin signaling, the luciferase activity from AR3Lux was measured with or without cotransfection of ActRIB. As shown in Fig. 3Go, the luciferase activity was increased more than 5-fold by cotransfection of ActRIB alone and was further increased up to 18-fold by additional activin stimulation. Overexpression of ActRIB may result in a slight activation of downstream signal leading to an increase of luciferase activity. Together, these results further confirmed that ActRIB is essential for activin signaling in KGN cells. To define the Smad proteins involved in the pathway downstream to ActRIB, the cells were transfected with each of the Smad expression vectors with or without ActRIB as indicated in Fig. 3Go. When the cells were transfected with Smad2, a 5-fold increase in basal luciferase activity and a 2-fold induction by activin stimulation were observed. This increase of the activity by the Smad2 expression vector was further enhanced by the presence of ActRIB as shown in Fig. 3Go, suggesting that ActRIB activates Smad2 as a downstream R-Smad. When the cells were transfected with Smad3, a further induction of luciferase activity by the treatment with activin A and cotransfection of ActRIB was not observed, although the basal luciferase activity was increased more than 10-fold. Thus, Smad3 is unlikely to be a substrate for ActRIB in KGN cells. Because the luciferase activity from AR3-Lux in these transfected cells was increased by TGF-ß stimulation (data not shown), Smad3 may be phosphorylated by the TGF-ß type I receptor in KGN cells. Taken together, activin signaling mediated by ActRIB is likely to employ Smad2, but not Smad3, as a downstream signal transmitter.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 3. Smad2 and ActRIB function cooperatively in the activin signaling pathway. Luciferase assays were performed on the lysates from cells transfected with AR3-Lux (0.5 µg) together with FAST1 (0.1 µg) and the indicated receptor and/or Smad in the presence or absence of activin A (100 ng/ml). In the presence of an increasing dosage of Smad7 (0.1–0.5 µg), the luciferase activity of the cells transfected with ActRIB followed by treatment with activin A (100 ng/ml) is decreased in a dose-dependent manner. The fold induction relative to the luciferase activity in the cells transfected with the reporter luciferase plasmid alone in the absence of activin A are shown. Each value indicates the mean ± SD of three experiments, with triplicate plates per point.

 
Smad7 inhibits the activin-induced transcriptional activity in the presence of ActRIB
So far, two inhibitory Smads (I-Smads), Smad6 and Smad7, have been identified by their ability to block either the TGF-ß/activin or BMP signaling pathways. While Smad6 seems to inhibit the BMP signal preferentially, Smad 7 acts as a general inhibitor of the TGF-ß family signaling pathways (16, 17, 18). One of the mechanisms by which I-Smad blocks the TGF-ß family signaling pathway is through their efficient interaction with the activated type I receptor, thereby preventing phosphorylation of R-Smads by the activated type I receptor. As shown in Fig. 2Go, in the presence of ActRIB, Smad7 inhibited the 3TP-Lux activity in a dose-dependent manner. On the other hand, Smad6 had no inhibitory effect on the activin signaling pathway in KGN cells. This inhibitory action of Smad7 was also observed on the transcription of AR3-Lux as shown in Fig. 3Go.

FSH receptor mRNA is induced by activin signaling
Activin has been shown to be involved in the regulation of FSH receptor expression in rat granulosa cells (8, 9). To determine whether the expression of the FSH receptor is regulated in a manner similar to that in primary cultures of rat granulosa cells, the FSH receptor expression in KGN cells was analyzed by both RT-PCR and a RNase protection assay. As shown in Fig. 4AGo, RT-PCR revealed that the amount of FSH receptor mRNA was increased by the activation of the activin signaling pathway. To confirm the induction of the FSH receptor mRNA, an RNase protection assay was performed as shown in Fig. 4BGo. The intensity of each protected band in Fig. 4BGo was quantified by densitometric scanning. As summarized in Fig. 4CGo, activation of the activin signaling pathway in KGN cells resulted in more than a 2-fold increase in FSH receptor mRNA.



View larger version (39K):
[in this window]
[in a new window]
  		Figure 4. FSH receptor mRNA is increased by activin signaling in KGN cells. Total RNA was prepared from cells transfected with or without ActRIB, in the presence or absence of activin A as indicated. A, RT-PCR was performed with specific primers for the FSH receptor. The same procedure without reverse transcriptase gives rise to no amplified signal. B, RNase protection assays (20 µg total RNA per lane) of RNA extracted from KGN cells cultured without treatment (control, lane 2), with transfection of ActRIB in the absence (lane 3) or presence (lane 4) of activin A. The riboprobe was hybridized with each total RNA followed by digestion with RNases. The RNase-resistant hybrids were separated by electrophoresis in a 5% polyacrylamide/8 M urea gel as indicated by the arrowhead. An undigested riboprobe is shown with an arrow (lane 1). The same experiments were carried out three times with the RNA prepared independently from triplicate plates. C, Intensities of protected RNA bands in (B) were quantified by densitometric scanning. The data are represented as mean ± SD. Histograms without common letters are statistically different, P < 0.01.

 
Activin signaling through ActRIB stimulates aromatase activity in KGN cells
To investigate whether activin signaling is involved in the change of cell fate, such as follicular maturation, the effect of activin stimulation on aromatase activity was analyzed in KGN cells. As shown in Fig. 5Go, transient transfection of the ActRIB expression vector followed by an addition of activin A in the medium resulted in a more than 2-fold increase of the aromatase activity in KGN cells. Next, we examined the possible interaction between FSH and the activin signaling pathways in the regulation of aromatase activity. FSH is well known to regulate aromatase expression through the A-kinase pathway, and indeed increased the aromatase activity by more than 4-fold in KGN cells as shown in Fig. 5Go. When the cells were treated with both FSH and activin A, a further increase in aromatase activity was observed. Although the mechanisms underlying the increase of aromatase activity by activin signaling remain to be clarified, the evidence that activin increases the FSH receptor level may provide one possible explanation. However, the fold induction of aromatase activity by the stimulation of the activin signaling pathway is still less extensive than the previous observations obtained using a primary culture of marmoset granulosa cells that showed induction by 10-fold (32). Because the expression of ActRIB is essential for activin responsiveness as described above, the transfection efficiency of the ActRIB expression plasmid, usually less than 20%, is likely to be the reason of the lower induction in this experiment. Therefore, we established stable cell lines expressing a constitutively active form of the ActRIB cDNA for further investigation of the role of activin signaling in KGN cells.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 5. Effect of activin A and FSH on aromatase activity in KGN cells. KGN cells were transfected with or without ActRIB and preincubated with or without activin A (100 ng/ml) or hFSH (50 ng/ml) for 12 h, and aromatase activity was assayed as described in Materials and Methods. Each value indicates the mean ± SD of four separate experiments, with duplicate plates per point. Histograms without common letters are statistically different, P < 0.05 (a vs. b), P < 0.01 (b vs. c), P < 0.01 (c vs. d).

 
KGN-ActRIBCA cells show high aromatase activity
The expression vector for ActRIBCA was constructed as described in Fig. 6AGo. The expression of ActRIBCA is under the control of the EF1{alpha} promoter and a selectable marker is translated from a single fusion transcript. The IRES sequence provides cap-independent translation of a fusion protein of Neor and ß-galactosidase. The KGN cells were transfected with the construct and cultured for 2 wk in the presence of G418 at the concentration of 200 µg/ml. Several colonies were picked and cultured individually for another 2 wk under the same conditions to create cell lines. Finally, we established three independent clones, which fundamentally had the same characteristics (data not shown). For a detailed analysis, we used one of the stable transformants, designated KGN-ActRIBCA. Firstly, the expression of ActRIBCA was confirmed by Western blotting using the anti-HA antibody and by lacZ staining. As shown in Fig. 6BGo, KGN-ActRIBCA cells expressed triple HA-tagged ActRIBCA protein and ß-galactosidase, indicating a stable expression of the constitutively active form of ActRIB in KGN cells. As shown in Fig. 6DGo, the aromatase activity of KGN-ActRIBCA at a basal level was dramatically increased by about 20-fold and reached a level much higher than that of the KGN cells treated with FSH. High expression of aromatase in KGN-ActRIBCA cells was also confirmed by western blotting as shown in Fig. 6CGo. Interestingly, FSH treatment failed to increase the aromatase activity in KGN-ActRIBCA cells to the extent seen in KGN cells. These results suggested that activation of activin signaling stimulates aromatase activity not only by the increase of FSH receptor expression but also by a mechanism independent of FSH signaling. To ensure this explanation, aromatase activity was examined in the presence of an inhibitor of adenylate cyclase, H89. As shown in Fig. 6DGo, 5 x 10-6 M of H89 completely abolished the increase of aromatase activity induced by FSH stimulation in KGN cells. On the other hand, the aromatase activity in KGN-ActRIBCA cells was not completely suppressed by the same concentration of H89. The aromatase activity in KGN-ActRIBCA cells in the presence of 10-5 M H89 remained about 10-fold higher than that in KGN cells, suggesting that the stimulatory effect of activin on aromatase activity is likely to be independent of the A-kinase pathway.



View larger version (34K):
[in this window]
[in a new window]
  		Figure 6. Expression of the ActRIBCA transgene in KGN cells. A, Diagram of the ActRIBCA transgene expression vector. The human ActRIBCA cDNA tagged with a triple HA epitope at the 3' end of the coding sequence was inserted between the EF-1{alpha} promoter and IRESßgeo cassette. B, Expression of the HA-ActRIBCA protein in the transgenic KGN-ActRIBCA cells was analyzed by immunoblotting the cell extracts with 12CA5, a monoclonal antibody against the HA epitope. The arrowhead indicates the HA-ActRIBCA band (upper panel). ß-Galactosidase staining further confirmed the transgene expression in the KGN-ActRIBCA cells (lower panels). C, Expression of the P-450 aromatase protein was analyzed by immunoblotting the cell extracts with an antibody against human P-450 aromatase. The arrowhead indicates the P-450 aromatase band. D, Effect of H89 on aromatase activity in KGN cells and KGN-ActRIBCA cells. KGN cells and KGN-ActRIBCA cells were preincubated with or without hFSH (50 ng/ml) and H89 (10-6 to 10-5 M), as indicated in the figure, for 12 h, and aromatase activity was assayed as described in Materials and Methods. Each value indicates the mean ± SD of three experiments, with duplicate plates per point. *, P < 0.05; **, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To investigate the signaling pathway of activin, we first characterized the expression of the components of the activin signaling pathway, including the type I and type II activin receptors, Smads and SARA, in KGN cells. However, despite the presence of these molecules, the transient transfection assay using the 3TP-Lux plasmid revealed that the cells are almost refractory to activin stimulation. Very importantly, cotransfection of the ActRIB expression plasmid was found to be required for activation of the reporter gene, although ActRIB is expressed at a level that can be detected by an agarose electrophoresis after 35 cycles of amplification. On the other hand, ActRIA, another type I receptor for activin, is more abundantly expressed than ActRIB by 8-fold. These results clearly demonstrate that ActRIB, but not ActRIA, is the type I receptor that functionally mediates activin signaling in granulosa cells. Defective activin signaling in KGN cells may thus be explained by an insufficient expression of ActRIB. The targeted disruption of the genes encoding the activin signal components, such as type II activin receptors and Smad2, in mice have revealed their haploinsufficient phenotypes. This genetic evidence strongly suggests that the dosage of these signaling molecules is important for normal embryonic development (33, 34). Therefore, it is conceivable that the dosage of activin type IB receptor is also critical for the activin signaling pathway. In granulosa cells, there may be a threshold of activin signal required for the exertion of its function. Alternatively, the abundance of the signaling components may be an important determinant for the relative responsiveness of the granulosa cells to the growth factor. Indeed, this concept has been recently addressed by Drummond et al. (25). Namely, in the rat ovary, the abundance of activin receptors and Smad proteins in the granulosa cells has been shown to change during follicular development and also change dramatically during postnatal days. It is of interest whether the expression levels of the activin receptors and Smad2 are altered at different stages of follicular development in humans. In KGN-ActRIBCA cells, the amount of endogenous Smad2 is not changed, but it is activated and found to be preferentially located in the nucleus (data not shown).

Both activin and TGF-ß are known to activate both Smad2 and Smad3 (35). A recent study in KAR6 cells demonstrated that activin specifically induces the association of both Smad2 and Smad3 with the activin receptor complex consisting of ActRIB and ActRIIA (30). However, our study suggests a functional preference for Smad2 for activation by activin in KGN cells. Furthermore, our functional study suggested that activation of Smad2 by activin occurred in the presence of ActRIB. There may be a preference for usage of either Smad2 or Smad3 or both, depending on the cell type. It is clear from our study that Smad2 functions downstream of ActRIB in the activin signaling pathway in KGN cells.

Two I-Smads, Smad6 and Smad7, have been so far identified as antagonists of either the TGF-ß/activin or the BMP signaling pathway (16, 17, 18). Inhibitory Smads have been shown to elicit their antagonistic effects by interacting with activated type I receptors and thereby preventing phosphorylation of R-Smads, or by competing with activated R-Smads for complex formation with Smad4. Recently, another mechanism by which I-Smads block TGF-ß family signaling has been described. Smad7 was found to recruit Smurfs (Smad ubiquitination regulatory factors), members of the HECT family of E3 ubiquitin ligases, to the TGF-ß type I receptor, resulting in the degradation of the TGF-ß type I receptor protein (36, 37). We demonstrated, for the first time, the inhibitory effect of Smad7 on the activin signaling through ActRIB in KGN cells, namely that Smad7 was found to block the activin-induced 3TP-Lux response in a dose-dependent manner, in good agreement with a previous report that Smad7 can prevent the association of the pathway-specific Smads with ActRIB (30). Further study will be needed to elucidate which mechanism described above takes place in granulosa cells.

Ovarian granulosa cells undergo a complete differentiation process during the growth and maturation of ovarian follicles that depends on pituitary gonadotropin. In this regard, the maintenance of the FSH receptor expression in the granulosa cells is important for the follicular maturation, and thus fertility. However, very few factors have been shown to regulate the expression of the FSH receptor. Those increasing the expression are FSH and activin. FSH has been shown to increase the number of its own receptors on the cell surface, implying a positive regulatory loop in the FSH signaling pathway in the granulosa cells (38). This may explain, at least in part, the mechanism by which FSH receptor expression is maintained during follicular maturation. On the other hand, activin has also been shown to increase the number of FSH receptors (8, 9) and extend the half-life of the FSH receptor mRNA (39), thereby enhancing the FSH signal in granulosa cells from diethylstilbestrol-treated immature rats. It is noted that the fold induction of FSH receptor mRNA observed in KGN cells is comparable to these observations in rat granulosa cells. The response of granulosa cells to FSH changes dramatically during follicular growth in vivo. In small follicles, FSH regulates the proliferation of granulosa cells, whereas, as follicles mature to a preovulatory stage, FSH induces the expression of differentiation-specific genes such as CYP19 encoding P-450 aromatase. Although the precise molecular mechanism involved in this switch remains unknown, it has been suggested that there may be a signal(s) that interacts with the FSH signal and directs the cells to differentiation rather than proliferation. Activin A has been shown to be present exclusively in the granulosa cells of mature follicles and in the corpus luteum in the human ovary (40). On the basis of the expression profile of activin A in granulosa cells, this may be one of the signals that modulates the FSH responsiveness and promotes the maturation of granulosa cells (10). To address the effect of activin on the differentiation of granulosa cells, we investigated P-450 aromatase activity in KGN cells. Activin A treatment together with transient transfection of ActRIB resulted in a 2-fold increase of aromatase activity, which was further increased by additional FSH treatment. These results suggest that the activin signal through ActRIB has not only a pivotal role but also a synergism with the FSH signal on the expression of P-450 aromatase in granulosa cells. The molecular mechanism underlying the synergism of activin with the FSH signal on the expression of FSH receptor and P-450 aromatase is not well understood; however, studies of KGN cells will help clarify the molecular mechanism of a synergism between activin and FSH signals.

FSH receptor activation by binding its ligand results in an intracellular cAMP increase followed by an activation of the A-kinase (PKA) pathway, which is well known as a major signaling pathway for the stimulation of the P-450 aromatase gene expression. Indeed, the increase of aromatase activity induced by FSH in KGN cells was completely blocked by pretreatment with the PKA inhibitor H89 at 5 x 106 M. On the other hand, the aromatase activity of KGN-ActRIBCA cells was suppressed only 40% by pretreatment with H89 at even 105 M, becoming a level higher than that of KGN cells indicating that the activin signal, at least in part, stimulates aromatase activity through a PKA-independent pathway. One possible mechanism may be that the FSH and activin signaling pathways converge at the promoter of the aromatase gene to regulate its expression. To address this possibility, an analysis of the transcriptional regulation of the Ic promoter, used in the human granulosa cells as well as in KGN cells (41), will be required.

Several kinases other than PKA have recently been shown to be activated by FSH receptor stimulation in granulosa cells. Those intracellular kinases activated downstream of the FSH receptor are protein kinase B (PKB/Akt), serum and glucocorticoid-induced kinase, p42-p44 ERK, MAPK and p38 mitogen-activated protein kinase (42, 43, 44, 45). On the other hand, recent progress has revealed that Smad signaling is not merely determined by the activation of the class of TGF-ß receptors, but is also regulated through cross-talk with other kinase signaling cascades (for reviews, see Refs. 46, 47, 48). For example, TGF-ß has been demonstrated to phosphorylate p38MAPK in various types of cells (49, 50). Both TGF-ß and activin signaling share a subset of receptor-regulated Smads, Smad2 and Smad3, and thus it is conceivable that activin signaling also has cross-talk with the p38MAPK pathway. Recently, activin signaling was found to require the p38MAPK pathway to exert its inhibitory effect on cell growth in breast cancer cells (51). Interestingly, in the KGN-ActRIBCA cells, but not in the KGN cells, p38MAPK is strongly phosphorylated (data not shown), suggesting activin signal cross-talk with the p38MAPK pathway in granulosa cells. It is noted that both activin and FSH receptor signals converge on p38 MAPK. Future studies in the KGN cells will provide a better understanding of the cross-talk network between these kinase pathways as well as P-450 aromatase gene regulation.

In conclusion, we propose that activin produced by granulosa cells plays a pivotal role in the maturation of follicles by activating the ActRIB-Smad2 signaling pathway. The results of our current study clearly show that activin A can stimulate P-450 aromatase activity through pathways both dependent and independent of PKA phosphorylation.


   Acknowledgments
 
We would like to thank Dr. Y. Etoh (Ajinomoto Co. Inc., Central Research Laboratories, Kawasaki, Japan) for kindly providing the recombinant human activin A, and Drs. L. Mathews, J. Massague, and M. Whitman for providing the plasmids. This work was performed in part at the Kyushu University Station for Collaborative Research.


   Footnotes
 
Abbreviations: ActRIA, Activin type IA receptor; ActRIB, activin type IB receptor; BMP, bone morphogenetic protein; FCS, fetal calf serum; GDF9, growth differentiation factor 9; H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide; hFSH, human FSH; IRES, internal ribosome-entry site; I-Smad, inhibitory Smad; P-450, cytochrome P-450; RNase, ribonuclease; R-Smads, receptor-regulated Smads; SARA, Smad anchor for receptor activation.

Received September 17, 2002.

Accepted for publication January 6, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Findlay JK, Drummond AE, Dyson ML, Baillie AJ, Robertson DM, Ethier JF 2002 Recruitment and development of the follicle; the roles of the transforming growth factor-ß superfamily. Mol Cell Endocrinol 191:35–43[CrossRef][Medline]
  2. Ling N, Ying SY, Ueno N, Shimasaki S, Esch F, Hotta M, Guillemin R 1986 Pituitary FSH is released by a heterodimer of the ß-subunits from the two forms of inhibin. Nature 321:779–782[CrossRef][Medline]
  3. Vale W, Rivier J, Vaughan J, McClintock R, Corrigan A, Woo W, Karr D, Spiess J 1986 Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature 321:776–779[CrossRef][Medline]
  4. Leburn JJ, Chen Y, Vale WW 1997 Receptor serine kinases and signaling by activins and inhibins In: Aono T, Sugino H, Vale W, eds. Inhibin, activin and follistatin, regulatory function in system and cell biology. New York: Serono Symposia Publications; vol 1:1–20
  5. Peng C, Mukai ST 2000 Activins and their receptors in female reproduction. Biochem Cell Biol 78:261–279[CrossRef][Medline]
  6. McGee EA, Hsueh AJ 2000 Initial and cyclic recruitment of ovarian follicles. Endocr Rev 21:200–214[Abstract/Free Full Text]
  7. Miro F, Hillier SG 1996 Modulation of granulosa cell deoxyribonucleic acid synthesis and differentiation by activin. Endocrinology 137:464–468[Abstract]
  8. Xiao S, Robertson DM, Findlay JK 1992 Effects of activin and follicle stimulating hormone (FSH)-suppressing protein/follistatin on FSH receptors and differentiation of cultured rat granulosa cells. Endocrinology 131:1009–1016[Abstract]
  9. Nakamura M, Nakamura K, Igarashi S, Tano M, Miyamoto K, Ibuki Y, Minegishi T 1995 Interaction between activin A and cAMP in the induction of FSH receptor in cultured rat granulosa cells. J Endocrinol 147:103–11010[Abstract]
  10. Findlay JK 1993 An update on the roles of inhibin, activin, and follistatin as local regulators of folliculogenesis. Biol Reprod 48:15–23[Abstract]
  11. Heldin C-H, Miyazono K, ten Dijke P 1997 TGF-ß signalling from cell membrane to nucleus through SMAD proteins. Nature 390:465–471[CrossRef][Medline]
  12. Derynck R, Feng X-H 1997 TGF-ß receptor signaling. Biochem Biophys Acta 1333:105–150
  13. Massague J, Chen YG 2000 Controlling TGF-ß signaling. Genes Dev 14:627–644[Free Full Text]
  14. Macias-Silva M, Abdollah S, Hoodless PA, Pirone R, Attisano L, Wrana JL 1996 MADR2 is a substrate of the TGF ß receptor and its phosphorylation is required for nuclear accumulation and signaling. Cell 87:1215–1224[CrossRef][Medline]
  15. Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin CH, Miyazono K, ten Dijke P 1997 TGF-ß receptor-mediated signalling through Smad2, Smad3 and Smad4. EMBO J 16:5353–5362[CrossRef][Medline]
  16. Hayashi H, Abdollah S, Qiu Y, Cai J, Xu YY, Grinnell BW, Richardson MA, Topper JN, Gimbrone Jr MA, Wrana JL, Falb D 1997 The MAD-related protein Smad7 associates with the TGF ß receptor and functions as an antagonist of TGF ß signaling. Cell 89:1165–1173[CrossRef][Medline]
  17. Imamura T, Takase M, Nishihara A, Oeda E, Hanai J, Kawabata M, Miyazono K 1997 Smad6 inhibits signaling by the TGF-ß superfamily. Nature 389:622–626[CrossRef][Medline]
  18. Nakao A, Afrakhte M, Moren A, Nakayama T, Christian JL, Heuchel R, Itoh S, Kawabata M, Heldin NE, Heldin CH, ten Dijke P 1997 Identification of Smad7, a TGFß-inducible antagonist of TGF-ß signalling. Nature 389:631–635[CrossRef][Medline]
  19. Tsukazaki T, Chiang TA, Davison AF, Attisano L, Wrana JL 1998 SARA, a FYVE domain protein that recruits Smad2 to the TGF ß receptor. Cell 95:779–791[CrossRef][Medline]
  20. Nishi Y, Yanase T, Mu Y-M, Oba K, Ichino I, Saito M, Nomura M, Mukasa C, Okabe T, Goto K, Takayanagi R, Kashimura Y, Haji M, Nawata H 2001 Establishment and characterization of a steroidogenic human granulosa-like tumor cell line, KGN, that expresses functional follicle-stimulating hormone receptor. Endocrinology 142:437–445[Abstract/Free Full Text]
  21. Willis SA, Zimmerman CM, Li LI, Mathews LS 1996 Formation and activation by phosphorylation of activin receptor complexes. Mol Endocrinol 10:367–379[Abstract]
  22. Mizushima S, Nagata S 1990 pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res 18:5322[Free Full Text]
  23. Mountford P, Zevnik B, Duwel A, Nichols J, Li M, Dani C, Robertson M, Chambers I, Smith A 1994 Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression. Proc Natl Acad Sci USA 91:4303–4307[Abstract/Free Full Text]
  24. Gu Z, Nomura M, Simpson BB, Lei H, Feijen A, van den Eijnden-van Raaij J, Donahoe PK, Li E 1998 The type I activin receptor ActRIB is required for egg cylinder organization and gastrulation in the mouse. Genes Dev 12:844–857[Abstract/Free Full Text]
  25. Drummond AE, Le MT, Ethier J-F, Dyson M, Findlay JK 2002 Expression and localization of activin receptors, Smads, and ß-glycan to the postnatal rat ovary. Endocrinology 143:1423–1433[Abstract/Free Full Text]
  26. Eramaa M, Hilden K, Tuuri T, Ritvos O 1995 Regulation of inhibin/activin subunit messenger ribonucleic acids (mRNAs) by activin A and expression of activin receptor mRNAs in cultured human granulosa-luteal cells. Endocrinology 136:4382–4389[Abstract]
  27. Peng C, Ohno T, Koh LY, Chen VTS, Leung PCK 1999 Human ovary and placenta express messenger RNA for multiple activin receptors. Life Sci 64:983–994[CrossRef][Medline]
  28. Mathews LS 1994 Activin receptors and cellular signaling by the receptor serine kinase family. Endocr Rev 15:310–325[CrossRef][Medline]
  29. Yamashita H, ten Dijke P, Huylebroeck D, Sampath TK, Andries M, Smith JC, Heldin CH, Miyazono K 1995 Osteogenic protein-1 binds to activin type II receptors and induces certain activin-like effects. J Cell Biol 130:217–226[Abstract/Free Full Text]
  30. Lebrun J-J, Takabe K, Chen Y, Vale W 1999 Roles of pathway-specific and inhibitory Smads in activin receptor signaling. Mol Endocrinol 13:15–23[Abstract/Free Full Text]
  31. Funaba M, Mathews LS 2000 Identification and characterization of constitutively active Smad2 mutants: evaluation of formation of Smad complex and subcellular distribution. Mol Endocrinol 14:1583–1591[Abstract/Free Full Text]
  32. Hillier SG, Miro F 1993 Local regulation of primate granulosa cell aromatase activity. J Steroid Biochem Mol Biol 44:435–439[CrossRef][Medline]
  33. Nomura M, Li E 1998 Smad2 role in mesoderm formation, left-right patterning and craniofacial development. Nature 393:786–790[CrossRef][Medline]
  34. Song J, Oh SP, Schrewe H, Nomura M, Lei H, Okano M, Gridley T, Li E 1999 The type II activin receptors are essential for egg cylinder growth, gastrulation, and rostral head development in mice. Dev Biol 213:157–16935[CrossRef][Medline]
  35. Piek E, Heldin C-H, ten Dijke P 1999 Specificity, diversity, and regulation in TGF ß superfamily signaling. FASEB J 13:2105–2124[Abstract/Free Full Text]
  36. Kavsa P, Rasmussen RK, Causing CG, Bonni S, Zhu H, Thomsen GH, Wrana JL 2000 Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGFß receptor for degradation. Mol Cell 6:1365–1375[CrossRef][Medline]
  37. Ebisawa T, Fukuchi M, Murakami G, Chiba T, Tanaka K, Inamura T, Miyazono K 2001 Smurf1 interacts with transforming growth factor-ß type I receptor through Smad7 and induces receptor degradation. J Biol Chem 276:12477–12480[Abstract/Free Full Text]
  38. Findlay JK, Drummond AE 1999 Regulation of the FSH receptor in the ovary. Trends Endocrinol Metab 10:183–188[CrossRef][Medline]
  39. Tano M, Minegishi T, Nakamura K, Karino S, Ibuki Y, Miyamoto K 1997 Transcriptional and post-transcriptional regulation of FSH receptor in rat granulose cells by cyclic AMP and activin. J Endocrinol 153:465–473[Abstract]
  40. Wada M, Shintani Y, Kosaka M, Sano T, Hizawa K, Saito S 1996 Immunohistochemical localization of activin A and follistatin in human tissues. Endocr J 43:375–385[Medline]
  41. Mu YM, Yanase T, Nishi Y, Takayanagi R, Goto K, Nawata H 2001 Combined treatment with specific ligands for PPAR{gamma}:RXR nuclear receptor system markedly inhibits the expression of cytochrome P450arom in human granulosa cancer cells. Mol Cell Endocrinol 181:239–248[CrossRef][Medline]
  42. Gonzalez-Robayna IJ, Falender AE, Ochsner S, Firestone GL, Richards JS 2000 Follicle-stimulating hormone (FSH) stimulates phosphorylation and activation of protein kinase B (PKB/Akt) and serum and glucocorticoid-induced kinase (Sgk): evidence for A kinase-independent signaling by FSH in granulosa cells. Mol Endocrinol 14:1283–1300[Abstract/Free Full Text]
  43. Das S, Maizels ET, DeManno D, St. Clair E, Adam SA, Hunzicker-Dunn M 1996 A stimulatory role of cyclic adenosine 3', 5'-monophosphate in follicle-stimulating hormone-activated mitogen-activated protein kinase signaling pathway in rat ovarian granulosa cells. Endocrinology 137:967–974[Abstract]
  44. Cameron MR, Foster JS, Bukovsky A, Wimalasena J 1996 Activation of mitogen-activated protein kinases by gonadotropins and cyclic adenosine 5'-monophosphates in porcine granulosa cells. Biol Reprod 55:111–119[Abstract]
  45. Maizels ET, Cottom J, Jones JCR, Hunzicker-Dunn M 1998 Follicle stimulating hormone (FSH) activates the p38 mitogen-activated protein pathway, inducing small heat shock protein phosphorylation and cell rounding in immature rat ovarian granulosa cells. Endocrinology 139:3353–3356[Abstract/Free Full Text]
  46. Zhang Y, Derynck R 1999 Regulation of Smad signalling by protein associations and signalling crosstalk. Trends Cell Biol 9:274–279[CrossRef][Medline]
  47. ten Dijke P, Miyazono K, Heldin C-H 2000 Signaling inputs converge on nuclear effectors in TGF-ß signaling. Trends Biochem Sci 25:64–70[CrossRef][Medline]
  48. Wakefield LM, Roberts AB 2002 TGF-ß signaling: positive and negative effects on tumorigenesis. Curr Opin Genet Dev 12:22–29[CrossRef][Medline]
  49. Hanafusa H, Ninomiya-Tsuji J, Masuyama N, Nishita M, Fujisawa J, Shibuya H, Matsumoto K, Nishida E 1999 Involvement of the p38 mitogen-activated protein kinase pathway in transforming growth factor-ß-induced gene expression. J Biol Chem 274:27161–27167[Abstract/Free Full Text]
  50. Watanabe H, de Caestecker MP, Yamada Y 2001 Transcriptional cross-talk between Smad, ERK1/2, and p38 mitogen-activated protein kinase pathways regulates transforming growth factor-ß-induced aggrecan gene expression in chondrogenic ATDC5 cells. J Biol Chem 276:14466–14473[Abstract/Free Full Text]
  51. Cocolakis E, Lemay S, Ali S, Lebrun J-J 2001 The p38 MAPK pathway is required for cell growth inhibition of human breast cancer cells in response to activin. J Biol Chem 276:18430–18436[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
S. Gondo, T. Okabe, T. Tanaka, H. Morinaga, M. Nomura, R. Takayanagi, H. Nawata, and T. Yanase
Adipose Tissue-Derived and Bone Marrow-Derived Mesenchymal Cells Develop into Different Lineage of Steroidogenic Cells by Forced Expression of Steroidogenic Factor 1
Endocrinology, September 1, 2008; 149(9): 4717 - 4725.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
T. Miyoshi, F. Otsuka, J. Suzuki, M. Takeda, K. Inagaki, Y. Kano, H. Otani, Y. Mimura, T. Ogura, and H. Makino
Mutual Regulation of Follicle-Stimulating Hormone Signaling and Bone Morphogenetic Protein System in Human Granulosa Cells
Biol Reprod, June 1, 2006; 74(6): 1073 - 1082.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G. Chen, M. Nomura, H. Morinaga, E. Matsubara, T. Okabe, K. Goto, T. Yanase, H. Zheng, J. Lu, and H. Nawata
Modulation of Androgen Receptor Transactivation by FoxH1: A NEWLY IDENTIFIED ANDROGEN RECEPTOR COREPRESSOR
J. Biol. Chem., October 28, 2005; 280(43): 36355 - 36363.
[Abstract] [Full Text] [PDF]

Home page
 Integr. Comp. Biol.Home page
E. Oberdorster, J. Romano, and P. McClellan-Green
The Neuropeptide APGWamide as a Penis Morphogenic Factor (PMF) in Gastropod Mollusks
Integr. Comp. Biol., January 1, 2005; 45(1): 28 - 32.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
Y. Wu, S. Ghosh, Y. Nishi, T. Yanase, H. Nawata, and Y. Hu
The Orphan Nuclear Receptors NURR1 and NGFI-B Modulate Aromatase Gene Expression in Ovarian Granulosa Cells: A Possible Mechanism for Repression of Aromatase Expression upon Luteinizing Hormone Surge
Endocrinology, January 1, 2005; 146(1): 237 - 246.
[Abstract] [Full Text] [PDF]

Home page
 GENES CELLSHome page
S. Gondo, T. Yanase, T. Okabe, T. Tanaka, H. Morinaga, M. Nomura, K. Goto, and H. Nawata
SF-1/Ad4BP transforms primary long-term cultured bone marrow cells into ACTH-responsive steroidogenic cells
Genes Cells, December 1, 2004; 9(12): 1239 - 1247.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
J R V Silva, R van den Hurk, H T A van Tol, B A J Roelen, and J R Figueiredo
Gene expression and protein localisation for activin-A, follistatin and activin receptors in goat ovaries
J. Endocrinol., November 1, 2004; 183(2): 405 - 415.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow