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BOR - Papers in Press, published online ahead of print December 27, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.012005
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BIOLOGY OF REPRODUCTION 68, 1877–1887 (2003)
DOI: 10.1095/biolreprod.102.012005
© 2003 by the Society for the Study of Reproduction, Inc.

Mechanisms of Hormone Action

Activin Signaling Pathways in Ovine Pituitary and LßT2 Gonadotrope Cells

Joëlle Dupont1,2, Judith McNeilly3, Anne Vaiman4, Sylvie Canepa2, Yves Combarnous2, and Catherine Taragnat2

Unité de Physiologie de la Reproduction et des Comportements,2 UMR INRA-CNRS, 37380 Nouzilly, France Medical Research Council Human Reproductive Sciences Unit,3 Center for Reproductive Biology, Edinburgh EH16 4SB, United Kingdom Centre de Recherches de Jouy,4 Département de Génétique Animale, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the pituitary, activin stimulates the synthesis and release of FSH. However, the activin receptor signaling pathways that mediate these effects are poorly known. We investigated these mechanisms in primary ovine pituitary cells (POP) and in the murine LßT2 gonadotrope cell line. POP cells and LßT2 cells express the different activin receptors (types IA, IB, IIA, and IIB) and the Smad proteins (Smad-2, -3, -4, and -7). In both POP and LßT2 cells, activin activated several signaling pathways: Smad-2, extracellular regulated kinase-1/2 (ERK1/2), p38, and phosphatidylinositol 3'-kinase (PI3K)/Akt. Phosphorylation of ERK1/2 and p38 were stimulated (3- to 6-fold) rapidly in 5 min, whereas activation of both Smad-2 and Akt (3- to 5-fold) occurred later, in 60 min. Activin also increased the association of activin receptor IIB with PI3K. Using specific inhibitors, we demonstrated that the activation of Smad-2 was partially blocked by the inhibition of PI3K but not by the inhibition of ERK1/2 or p38, suggesting a cross-talk between the Smad and PI3K/Akt pathways. In both POP and LßT2 cells, FSH expression and secretion in response to activin were not altered by the inhibition of PI3K/Akt, ERK1/2, or p38 pathways, whereas they were reduced by about 2-fold by expression of a dominant negative of Smad-2 or the natural inhibitory Smad-7 in LßT2 cells. These results indicate that activin activates several signaling pathways with different time courses in both POP and LßT2 cells, but only the Smad-2 pathway appears to be directly implicated in FSH expression and release in LßT2 cells.

activin, follicle-stimulating hormone, mechanisms of hormone action, pituitary, signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pituitary gonadotropins, FSH and LH, play a pivotal role in normal reproductive function. The gonadotrope cells in the anterior pituitary gland produce both of these hormones. FSH and LH are heterodimeric glycoproteins, each composed of a common {alpha} subunit and a unique ß subunit that confers biological specificity [1]. The synthesis and release of each gonadotropin are often discordant in various physiological situations such as the infantile period in the female rat [2], the estrous cycle in the ewe [3], and photoperiod in the hamster [4, 5]. However, the precise mechanisms of LH and FSH differential regulation are still unclear. Gonadotropin biosynthesis and secretion are regulated by hypothalamic factors, mainly GnRH, and by circulating gonadal steroids and polypeptides, including activin, inhibin, and follistatin [6].

Activin is a dimeric protein hormone synthesized as a homo- or heterodimer of the distinct ß subunits (ßA or ßB), which combine to form activin A (ßA-ßA), activin B (ßB-ßB), or activin AB (ßA-ßB). Inhibin is composed of an inhibin-specific {alpha} subunit combined with one of the activin ß subunits to form either inhibin A ({alpha}-ßA) or inhibin B ({alpha}-ßB) [7]. Activin is produced in various tissues and stimulates synthesis of FSH by direct action on gonadotrope cells, whereas inhibin is primarily produced by gonads and acts to counteract the effects of activin by reducing the secretion of FSH [8]. Inhibin blocks activin effects by competing with activin for receptor binding [9, 10]. Follistatin is a monomeric protein, structurally unrelated to the transforming growth factor (TGF) ß superfamily, that acts primarily by binding to activin and preventing its interaction with its receptor [11, 12]. Inhibin, activin, and follistatin are produced by the ovary and are released into the circulation. However, the activin and inhibin subunits and follistatin are also produced in the pituitary gland itself by the gonadotropes and folliculostellate cells in different species, including rat [13, 14], human [15], and sheep [16, 17]. Thus, these hormones may have autocrine/paracrine effects on synthesis and secretion of FSH.

Activin exerts its biological effects by interacting with four types of transmembrane receptors (types IA, IB, IIA, and IIB) with protein serine/threonine kinase activity [18]. The type II receptors are involved in initial ligand binding leading to recruitment and phosphorylation of type I receptors by the kinase domain of type II receptors. Once phosphorylated, type I receptors exhibit kinase activity on Smad proteins. Smad-2 and Smad-3 are specific to the activin signaling and are phosphorylated by activated activin receptors on serine residues. Phosphorylation of Smad-2 and Smad-3 allows complex formation with Smad-4, a common effector shared by different TGFß family pathways [19]. Once formed the Smad-2/Smad-4 or Smad-3/Smad-4 complex translocates into the nucleus to activate transcription of specific target genes. However, the Smad signaling pathway might not be the sole pathway activated by the activin receptors. Other members of the activin family, such as TGFß1, activate other signaling pathways in addition to Smads, including the kinases extracellular regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3'-kinase (PI3K) [20]. The activin receptor subtypes and the Smad proteins have been characterized in the ovary [21, 22] and the pituitary, including the gonadotrope-derived LßT2 cell line [23–25]. However, no one has yet identified the activin receptor signaling pathways in the pituitary cells, particularly the pathway(s) involved in FSH expression and release. In the present study, we investigated the mechanism of action of activin in the primary ovine pituitary cells (POP) and in the mouse LßT2 cells as a model of mouse gonadotrope cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

The radionucleotides [{alpha}32P] dCTP (6000 Ci/mmol) and [{gamma}32P] ATP (6000 Ci/mmol) were obtained from Perkin Elmer Life Sciences (Boston, MA). Recombinant human activin and follistatin were purchased from R&D Systems (Minneapolis, MN). Dulbecco modified Eagle medium (DMEM), penicillin, streptomycin, and trypsin were obtained from Gibco-BRL Life Technologies (Gaithersburg, MD). Taq DNA polymerase was supplied by Promega (Madison, WI). Avian myeloblastosis virus reverse transcriptase and PI3K-specific inhibitor LY294002 were purchased from Sigma Chemical Co (St. Louis, MO). Both MEK1/2-specific inhibitor U0126 and p38 mitogen-activated protein kinase (MAPK)-specific inhibitor SB202190 were from Calbiochem (La Jolla, CA).

Rabbit polyclonal antibodies to phospho-Akt (Ser 473), Akt, phospho-ERK1/2 (Thr202/Tyr204), and phospho-p38 (Thr180/Tyr182) were purchased from New England Biolabs (Beverly, MA). Rabbit polyclonal antibodies to ERK2 (C14) and p38 (C20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to rabbit phospho-Smad-2 (Ser 465/467), rabbit Smad-2, and mouse p85 regulatory subunit of PI3K ({alpha}p85) were purchased from Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal antibodies to the different types of activin receptor ({alpha}ActR) were kindly provided by Dr. W. Vale (Salk Institute, La Jolla, CA). Monoclonal anti-actin (clone AC) was obtained from Sigma. All antibodies were used at 1:1000 dilution in Western blotting. For the immunoprecipitation study, {alpha}ActR-IIB was obtained from R&D Systems. The mouse Smad-7 cDNA and the dominant negative of Smad-2 (Smad2[3SA]) were kindly donated by Dr. P. ten Dijke (Ludwing Institute for Cancer Research, Uppsala, Sweden) and Dr. J. Wrana (Samuel Lunenfeld Research Institute, Toronto, Canada), respectively.

Cell Culture

LßT2 and MCF-7 cells were generously provided by Dr. P. Mellon (La Jolla, CA) and Dr. D. LeRoith (NIDDK, NIH, Bethesda, MD), respectively. Both cell lines were routinely cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, 100 µg/ml streptomycin, and 300 µg/ml L-glutamine in a humidified atmosphere of 95% air and 5% CO2. POP cells were prepared as previously described [26] by collagenase/DNase dispersion of the diced tissue from ewe (Ile de France) pituitary glands freshly obtained from a local abattoir. Cells were resuspended in DMEM without phenol red containing 5% FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin and then seeded in 48-well culture plates at a concentration of 4 x 105 cells/well or in 6-well tissue culture plates at a concentration of 3 x 106 cells/well. Cells were allowed to recover for 2 days. After two washes in medium without serum and an equilibration period of 2–3 h, test substances were added at the indicated concentrations and incubation times.

Gonadotropin Measurements

Concentrations of LH and FSH released into the culture medium of LßT2 cells were measured using rat reagents (rat LH: rLH-RP-3, rat FSH: rFSH-RP-2) supplied by NIDDK with samples for each hormone evaluated in duplicate in one assay [27]. The minimum detectable concentrations were 0.1 ng/ml for LH and 1 ng/ml for FSH, and the intra-assay coefficients of variation were <5%.

Concentrations of LH and FSH released into the culture medium of POP cells were measured using a specific two-site ELISA sandwich (enzyme-linked immunoassays [EIA]) that employed two anti-LH or anti-FSH monoclonal antibodies [28, 29]. Ovine LH (oLH, batch 1083; INRA, Nouzilly, France) and ovine FSH (oFSH, NIH RP2) were used as assay standards. The first antibody was directed against the ß subunit of the molecule, whereas the second biotinylated monoclonal antibody (Serotec MCA 1026 [30]) was directed against the {alpha} subunit. The detection limits of LH and FSH ELISAs were 0.1 ng/ml (2 pg/tube) and 0.4 ng/ml (20 pg/tube), respectively. The intra-assay coefficients of variation were 6.7% for oLH and 5.1% for oFSH. These oLH and oFSH EIA systems exhibited 0.01% and 0.07% cross-reactivity with highly purified oFSH and oLH, respectively.

Immunoprecipitation and Immunoblotting

Cell lysates were solubilized and centrifuged as previously described [31]. Samples (2 mg of protein from the supernatants) were immunoprecipitated with either {alpha}p85 or {alpha}ActR-IIB at 1:1000 for 16 h at 4°C. The immune complexes were precipitated with protein G-agarose for 1 h at 4°C as previously described [31]. Immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Blots were blocked with 5% nonfat milk in Tris-buffered saline plus 0.1% Tween-20 for 1 h at room temperature and probed with either {alpha}p85 or {alpha}ActR-IIB. After extensive washing, immune complexes were detected with horseradish peroxidase conjugated with specific secondary antiserum (Amersham, Piscataway, NJ), followed by an enhanced chemiluminescence reaction. Densitometry was performed by scanning the radiographs and then analyzing the bands with the software MacBas 2.52 (Fuji PhotoFilm, Fuji Photofilm, Stamford, CT). When Western blotting was performed without immunoprecipitation, cell extracts (50 µg of protein) were directly subjected to SDS-PAGE.

PI3K Assay

PI3K activity was determined as previously described [31]. Cell lysates were immunoprecipitated overnight at 4°C with the ActR-IIB antibody (1:1000). PI3K activity was measured in the immunoprecipitates in the presence of phosphatidylinositol and labeled ATP. The radioactivity incorporated into phosphatidylinositol was quantified using a Storm PhosphoImager (Molecular Dynamics, Bondoufle, France).

Reverse Transcription Polymerase Chain Reaction

Total RNA from cells was extracted using TRIzol reagent according to the manufacturer's instructions (Gibco-BRL). Reverse transcription (RT) polymerase chain reaction (PCR) was performed to assay expression of ActR-IA, -IB, -IIA, and -IIB and Smad-2, -3, -4, and -7 genes in POP cells.

RT of total RNA (1 µg) was carried out in a 20-µl reaction mixture containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 200 µM of each deoxynucleotide triphosphate (Amersham), 50 pmol of oligo(dT)15, 5 U of RNase inhibitor, and 15 U of avian myeloblastosis virus reverse transcriptase. RT was carried out at 42°C for 1 h followed by incubation at 95°C for 5 min. Single-strand cDNAs were amplified with specific sets of primer pairs designed to amplify parts of the activin receptors and Smads as described in Table 1. PCRs were carried out using 2 µl of the RT reaction mixture in a volume of 50 µl containing 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 200 mM of each deoxynucleotide triphosphate, 10 pmol of each primer, and 1 U of Taq polymerase. The samples were processed for 35 PCR cycles (95°C for 1 min; 58°C for 1 min; 72°C for 1 min), with a final extension step at 72°C for 10 min. PCR products were visualized in an agarose gel (1.5%) stained with ethidium bromide. All the amplified ovine cDNA fragments were cloned using the PCR2.1 Topo cloning kit (Invitrogen, Carlsbad, CA) and automatically sequenced using an automated sequencer (ABI Perkinelmer, Cergypontoise, France). To evaluate the risk of amplification from genomic DNA potentially present in our total RNA preparations, all samples were amplified by PCR with each primer pair in the absence of reverse transcriptase. When no enzyme was present, no amplification product was detected after 35 cycles (data not shown).


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  		TABLE 1. Oligonucleotide primer sequences used for partial cloning and sequencing of ovine activin receptors and Smad mRNAs

Bacterial Artificial Chromosome Screening, Isolation, and Fluorescence In Situ Hybridization (FISH) Mapping on Ovine Chromosome

Metaphase chromosome spreads were obtained by standard procedures from primary sheep fetal lung fibroblasts (56,XY MFP2 [32]). The superpools of sheep bacterial artificial chromosome (BAC) library [33] were screened by PCR for three primer pairs corresponding to genes ActR-IA, ActR-IIA, ActR-IIB, and Smad-7. A total of four positive addresses were found in the library. The four clones were then mapped by FISH on metaphases (one for each of the four genes). BACs (400 ng/µl) were labeled by nick-translation (Kit Bionick, Roche, Penzberg, Germany), and the probes were mapped by FISH as previously described [34]. The R-banded chromosomes were identified according to the recommendation of the ISCNDB (2000).

Northern Blot

Total RNAs from cells (20 µg) were separated by denaturing formaldehyde electrophoresis, transferred to a nylon membrane by capillar action overnight, and hybridized as previously described [35, 36]. The ß subunit probes for murine FSH and ovine FSH and LH were generated by RT-PCR using the following primers: murine FSHß sense, 5'-ATCTGGTGTATAAGGACCCA-3'; murine FSHß antisense, 5'-TCATTTCACTGAAGGAGCAG-3'; ovine FSHß sense, 5'-ATGAAGTCCGTCCAGTTCTG-3'; ovine FSHß antisense, 5'-TTATTCTCTGATGTCACTGA-3'; ovine LHß sense, 5'-ATGCTCCAGGGACTGCTGCT-3'; ovine LHß antisense, 5'-GCAGCTTGAGAAGTCTTTAT-3'. Membrane-incorporated radioactivity was quantified using a STORM PhosphoImager (Molecular Dynamics). The integrity and the quantification of different transcripts were assessed using the human RNA 18S probe as a control (Ambion, Austin, TX).

Transfection of LßT2 Cells

LßT2 cells were plated the day before transfection at a density of 4 x 105 cells/well in six-well culture plates. Cells were then transiently transfected using the lipofectamine transfection reagent (Gibco-BRL) according to the manufacturer's instructions with 1 µg of Smad-2 (3SA) or Smad-7 constructs or 1 µg of pcDNA3.1 empty vector as a control. After 24 h of transfection, cells were placed in serum-free DMEM for 16 h and then stimulated with human recombinant activin (10-8 M) for 12 h (for the study of FSHß mRNA expression) or 24 h (for the study of the gonadotropin secretion). Cells were then washed with ice-cold PBS, and total RNAs were extracted. To check the efficiency of the negative dominant of Smad-2 (3SA) and the inhibitory Smad-7, LßT2 cells transfected with Smad-2 (3SA), Smad-7, or the vector alone were stimulated with activin (10-8 M) for 60 min at 37°C, and the level of Smad-2 phosphorylation was determined by Western blot.

Statistics

All experimental data are presented as the mean ± SEM. The effects of activin on FSH and LH expression and secretion were analyzed using two-way ANOVA to determine the effects of activin and of the culture experiment. Post hoc comparisons were performed by Newmann-Keuls and Scheffé tests using the Statview 4.5 software (SAS Institute, Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Activin on Gonadotropin mRNA Expression and Protein Release in POP Cells

Activin induced a strong increase in FSHß mRNA expression that was visible by 6 h (3-fold, P < 0.05), reached its peak at 12 h (15-fold, P < 0.05), and then declined at 72 h (Fig. 1A). We observed no effect of activin treatment on LHß mRNA expression (Fig. 1A). Under the same experimental conditions, stimulation with activin led to a time-dependent increase in FSH release (Fig. 1B). Incubation for 24 h with 10-8 M activin reduced by about 50% the release of LH in POP cells (Fig. 1C). This reduction was observed up to 72 h of activin treatment.



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  		FIG. 1. A) Time course of activin stimulation (10-8 M) for the expression of ß subunits of FSH and LH mRNA in POP cells. POP cells were treated with 10-8 M activin for indicated times after pre-treatment with 10-8 M follistatin for 24 h. Total RNA was prepared, and a Northern blot was carried out using specific ovine probes for the ß subunits of FSH and LH. As a control, the membranes were stripped and reprobed with 18S RNA to confirm equal loading and to determine the stimulation by activin. The value of the control (no activin treatment) was defined as 1.0, and from this value FSHß and LHß values were calculated. Values represent means ± SEM (bar) from three independent experiments. B) Time course of activin-induced FSH and LH secretion in POP cells. After pretreatment with 10-8 M follistatin for 24 h, cells were incubated for indicated times in serum-free medium in the presence or absence of 10-8 M activin, and the amounts of FSH (B) and LH (C) secreted into the incubation medium were determined by ELISA. Values are means ± SEM (n = 3). *P < 0.05 vs. control

Ovine Pituitary Activin Receptors and Smad Expression

The presence of the activin receptors and their appropriate intracellular substrates including Smad-2, -3, -4, and -7 was investigated first in the POP cells at the level of messengers by RT-PCR. PCR products targeted for ActR-IA, -IIA, -IB, and -IIB and Smad-2, -3, -4, and -7 were obtained at expected sizes: 651, 701, 684, 786, 471, 234, 610, and 321 base pairs (bp), respectively (Fig. 2A). The specificity of the amplified products was assessed by direct sequencing. Each partial sequence has been submitted to GenBank (accession numbers in Table 1). Ovine activin receptors and Smad genes exhibit a high homology (85–95%) with human or mouse activin receptors and Smad genes, respectively. Using FISH technology, ActR-IA, -IIB, and -IIA and Smad-7 were localized at ovine chromosome regions 2q23, 19q13, 17q24–25, and 23q23–25, respectively (Table 1). The localization of these genes was similar to that of their human homologues.



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  		FIG. 2. Expression of different forms of activin receptor and Smads in POP cells. A) Total RNAs extracted from freshly isolated ovine pituitary cells were subjected to RT-PCR using primer pairs designed to amplify fragments from ActR-IA, -IIA, -IB, or -IIB and Smad-2, -3, -4, and -7 transcripts. The expected sizes of the different amplified products are indicated on the right. B) Lysates from POP and MCF-7 cells transfected or not with bovine ActR-IA, -IB, -IIA, and -IIB cDNAs as control were analyzed with polyclonal antibodies directed against each of the activin receptor forms. Samples contained equal levels of protein, as confirmed by reprobing each membrane with an anti-{alpha}-actin antibody

We also performed Western blot analyses to detect the different forms of ovine activin receptor. Extracts of POP cells contained immunoreactive materials of the appropriate molecular masses for ActR-IA (60 kDa), ActR-IB (55 kDa), ActR-IIA (80 kDa), and ActR-IIB (60 kDa) (Fig. 2B). The MCF-7 cell line (human mammary gland origin) was used as a nonpituitary control, and the cells were transfected or not with each of the activin receptor cDNAs (Fig. 2B). Transfected MCF-7 cells for each activin receptor form expressed more of the corresponding receptor than did the untransfected cells (Fig. 2B). Their sizes were similar to those found in POP cells.

Activin Activates Different Signaling Pathways in POP Cells and LßT2 Cells

In different cell lines, activin is thought to signal primarily via the Smad transduction pathway. However, TGFß, which is related to activin, activates several other downstream signaling pathways, including the MAPK ERK1/2 and p38, the PI3K/Akt kinases, and the Smad pathway [20]. First, we tested whether these kinases were activated by activin in POP cells. Activin induced phosphorylation of Smad-2 within 30 min, achieving a 3-fold stimulation at 60 min (Fig. 3A). Activin also stimulated Akt phosphorylation with a similar time course (Fig. 3B). In contrast, activin-induced ERK1/2 and p38 phosphorylations were much quicker, reaching 6- and 3-fold at 5 min, respectively, and were then abolished after 60 min (Fig. 3, C and D). Similar amounts of Smad-2, Akt, ERK2, and p38 were loaded in all lanes. A similar time course of Smad-2, Akt, ERK1/2, and p38 phosphorylation was observed in the mouse LßT2 gonadotrope cell line and in the human MCF-7 breast cancer cell line (Fig. 3, A–D), suggesting that the activin effect on the activation of the signaling pathways is ligand specific but not cell specific. Cotreatment with 10-8 M follistatin and 10-8 M activin resulted in complete abrogation or strong attenuation of the response of all signaling pathways to activin (data not shown).



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  		FIG. 3. Activin activates Smad-2, Akt, ERK1/2, and p38 signaling pathways in POP, LßT2, and MCF-7 cells. After pretreatment with 10-8 M follistatin for 24 h, cells in serum-free medium were treated with exogenous 10-8 M activin for indicated times. Total proteins (40 µg) from freshly isolated POP, LßT2, and MCF-7 cells were electrophoresed on 10% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes. Phosphorylation of Smad-2, Akt, ERK1/2, and p38 were analyzed with polyclonal antibodies raised against phospho-Smad-2, -Akt, -ERK1/2, and -p38, respectively. To determine the level of phosphorylation, all the blots were stripped and reprobed with antibodies against the Smad-2, Akt, ERK2, and p38 proteins, and the ratio of phosphorylation:total protein was determined and plotted as the percentage of stimulation as compared with the control (unstimulated cells). Values represent means ± SEM from three independent experiments

Coimmunoprecipitation of ActR-IIB with PI3K in POP and LßT2 Cells

The potential involvement of the PI3K/Akt pathway in activin receptor signaling was further investigated. We examined whether the p85 regulatory subunit of PI3K could directly interact with ActR-IIB in both POP and LßT2 cells. The interaction between the p85 regulatory subunit of PI3K and the other types of activin receptors has not been studied. We observed one band of 85 kDa (Fig. 4A, right) and one band of 60 kDa (Fig. 4A, left) corresponding to p85 and ActR-IIB, respectively, in both LßT2 and POP cells. The coimmunoprecipitation of ActR-IIB and the regulatory subunit p85 of PI3K indicates that these two proteins are associated in the basal and activated states of ActR-IIB in both LßT2 and POP cells. Furthermore, after measurements of the intensities of the bands using the MacBas software, it appeared that the association of p85 and ActR-IIB was increased by 2-fold (P < 0.05) in response to activin treatment. These results occurred in the absence of any change in the level of ActR-IIB (Fig. 4A, left) or p85 (Fig. 4A, right). In the following experiments, the possibility that PI3K activity is associated with ActR-IIB was investigated in both cell types. Cell lysates were immunoprecipitated with {alpha}ActR-IIB, and PI3K activity was measured in precipitated products (Fig. 4B). In both cell types, PI3K activity was present; after normalization (100% = activity in unstimulated cells), activin treatment significantly (P < 0.05) increased PI3K activity associated with ActR-IIB (Fig. 4B).



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  		FIG. 4. Coimmunoprecipitation of ActR-IIB with PI3K in POP and LßT2 cells. A) Association of the p85 regulatory subunit of PI3K with ActR-IIB was determined from freshly isolated POP cells and LßT2 cells that were pretreated with 10-8 M follistatin for 24 h and then incubated in the absence or presence of 10-8 M activin for 60 min. ActR-IIB (left) or the p85 regulatory subunit of PI3K (right) was immunoprecipitated from whole cell lysates, and the samples were subjected to Western blotting with antibodies against p85 ({alpha}p85) or ActR-IIB ({alpha}ActR-IIB), respectively. Membranes were then reprobed with {alpha}ActR-IIB or {alpha}p85 to evaluate ActR-IIB or p85 levels in each lane. B) Following incubation of POP and LßT2 cells with or without 10-8 M activin for 60 min, cells lysates were immunoprecipitated with {alpha}ActR-IIB, and PI3K activity was measured in the presence of phosphatidylinositol (PI) and labeled ATP. The PI corresponding to the phosphorylated PI was quantified using a PhosphoImager, and the values are plotted. Results are representative of three independent experiments with each cell type

Presence of Cross-Talk Between Smad and PI3K/Akt Signaling Pathways in POP and LßT2 Cells

Because Smad proteins play a central role in the activin receptor pathway, we tested whether the other activin-responsive signaling pathways (particularly those with a quicker time course) were involved in activin-induced Smad-2 phosphorylation in POP and LßT2 cells. Because similar results were observed in both POP and LßT2 cells, Figure 5, A and B, illustrates the results obtained only for POP cells. A panel of pharmacological inhibitors including ERK1/2-, p38-, and PI3K-specific inhibitors was used to identify the signaling pathways potentially involved in the activation of Smad-2 by activin. In the presence of the ERK1/2 pathway inhibitor (U0126) or the p38 inhibitor (SB202190), activin-induced Smad-2 phosphorylation was unaltered (Fig. 5A). To ensure that the inhibitors were active on POP cells, these cells were treated for 60 min with the inhibitors prior to the activin stimulation. At 10 µM, U0126 was able to inhibit ERK1/2 phosphorylation induced by activin stimulation (Fig. 5B); similarly, 50 µM LY294002 and 50 µM SB202190 inhibited Akt and p38 phosphorylation, respectively (Fig. 5B). Western blot analyses, performed to detect all forms of ERK1/2, Akt, and p38 kinases, revealed that protein levels were similar in all samples. Because inhibition of activin-induced Smad-2 phosphorylation could not be detected with ERK1/2 and p38 inhibitors, we employed a specific PI3K/Akt inhibitor, LY294002, which fully inhibits PI3K activity and Akt phosphorylation. The addition of LY294002 did inhibit, but only by 50% (P < 0.05), activin-induced Smad-2 phosphorylation. Similar results were obtained in the LßT2 cells (data not shown).



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  		FIG. 5. Cross-talk between the Smad-2 and PI3K/Akt signaling pathway in POP cells. A) Effect of inhibitors U0126, SB202190, and LY294002 on activin-induced Smad-2 phosphorylation in POP cells. Cells pretreated with 10-8 M follistatin for 24 h were incubated for 60 min with the inhibiting concentration of each inhibitor (10 µM U0126, 50 µM SB202190, or 50 µM LY294002) and then stimulated or not with 10-8 M activin for an additional 60 min. Smad-2 phosphorylation was then detected by Western blot using specific antibodies for phosphorylated Smad-2. The levels of Smad-2 were monitored with anti-Smad-2 antibodies. Representative Western blots are shown with quantification (control medium without inhibitors and activin set as 1.0). The experiments were performed in triplicate. B) Inhibition of activin-induced ERK1/2, p38, and Akt phosphorylation by the MEK1/2 inhibitor U0126, the p38 inhibitor SB202190, and the PI3K inhibitor LY294002. POP cells were plated in six-well plates (3 x 106 cells/well). Cells pretreated with 10-8 M follistatin for 24 h were incubated with U0126, SB202190, or LY294002 for 60 min at the indicated concentrations and then treated or not treated with 10-8 M activin for 5 min (for ERK1/2 and p38) or 60 min (for Akt). ERK1/2, p38, and Akt phosphorylation was immunodetected with phosphoantibodies. The membranes were then stripped and reprobed with antibodies that detected the total proteins (control). Results shown are representative of two experiments

Activin-Induced FSH Expression and Secretion Require Smad-2 Phosphorylation in LßT2 Cells

To study the signaling pathways involved in activin stimulation of FSHß mRNA expression and FSH release, the same specific inhibitors of PI3K, ERK1/2, and p38 were used. In both LßT2 (Fig. 6A) and POP (Fig. 6B) cells, none of the inhibitors (U0126, LY294002, and SB202190) had any effect on the increase in FSHß expression (left panel) and FSH release (right panel) in response to activin. This finding suggests that the ERK1/2, p38, and PI3K/Akt pathways are not involved in activin-induced FSH mRNA expression and secretion in either LßT2 or POP cells. To test the involvement of the Smad pathway, transient transfections were attempted with either a dominant negative of Smad-2 or the natural inhibitory Smad-7. No specific chemical inhibitors of Smad are presently available. Transfections did not perform well in POP cells (data not shown) but were efficient in LßT2 cells. Upon activin stimulation, Smad-2 phosphorylation was reduced by 60% and 70% after transfection of the Smad-2 dominant negative and Smad-7 constructs, respectively (data not shown). Upon activin stimulation, FSHß mRNA expression and FSH release increased by 10-fold (P < 0.001) and 2.8-fold (P < 0.001), respectively, in the LßT2 transfected cells with empty vector, whereas they increased by only 4.5-fold (P < 0.05) and 1.2-fold (P < 0.02) in the cells transfected with a Smad-2 dominant negative and by 4-fold (P < 0.05) and 1.3-fold (P < 0.05) in the cells transfected with the inhibitory Smad-7 (Fig. 6A). Activin did not affect LH secretion in LßT2 cells, and the decrease in LH secretion induced by activin was not altered by any of the inhibitors (U0126, LY294002, and SB202190) in POP cells (data not shown).



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  		FIG. 6. Effect of specific inhibitors and an overexpression of Smad-2 dominant negative and Smad-7 on FSHß mRNA expression and FSH secretion in response to activin in LßT2 (A) and POP (B) cells. LßT2 and POP cells were preincubated for 24 h with 10-8 M follistatin and then for 60 min with the specific inhibitors U0126 (10 µM), SB202190 (50 µM), or LY294002 (50 µM) and stimulated or not with 10-8 M activin for 12 h (for the FSHß mRNA expression analysis) or 24 h (for the study of FSH secretion). To determine whether the Smad pathway was involved in the FSH expression and secretion, LßT2 cells were transiently transfected with a dominant negative Smad-2 or the inhibitory Smad-7 or with the vector alone (empty vector) as a control experiment. The mRNA expression of FSHß in different conditions was determined by Northern blot in LßT2 cells (A, left) and POP cells (B, left). The amounts of FSH secreted in the incubation medium from LßT2 cells (A, right) and POP cells (B, right) were determined by RIA and ELISA, respectively. Values represent means ± SEM. **P < 0.001; *P < 0.05


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Activin is considered a potent stimulator of FSH expression and secretion in different species, including sheep [37]. In the present study, the stimulatory effect of activin on the release of FSH by ovine pituitary cells was accompanied by a simultaneous increase in mRNA levels for the ß subunit of FSH. Thus, these data indicate that activin modulates the biosynthesis of FSH at the transcriptional level in ovine and rat pituitary cells [38]. Activin also significantly reduced the release of LH by POP cells without affecting the mRNA level of LHß. Consistent with these data, Muttrikrishna and Knight [37] showed that activin exhibits a dose-dependent suppression of GnRH-induced LH release in rat pituitary cells. Furthermore, activin did not affect total LH content, indicating a lack of effect of activin on LH biosynthesis [37]. Thus, activin could affect the stability of LHß mRNA, the posttranslational processing of LH, or the storage of LH in POP cells. These data for the sheep stand in marked contrast to comparable data for rat pituitary cells. Activin has been reported either to increase LH secretion [39] or to have no effect on either basal or GnRH-induced LH release [37] in rat pituitary cells in vitro. In an effort to understand the mechanisms of action of activin on FSH expression and secretion, we characterized the elements of the response to activin in POP cells, i.e., the expression pattern of the different types of activin receptors and Smad proteins and the different signaling pathways activated by activin.

Various endogenous activin receptors and Smad proteins (i.e., ActR-IA, -IIA, -IB, and -IIB and Smad-2, -3, -4, and -7) were expressed in POP cells. Their respective transcripts were detected both in cultured pituitary cells and in intact pituitary (data not shown). Consistent with these results, Cameron et al. [24] localized by in situ hybridization the expression of ActR-IIA and ActR-IIB in the rat pituitary. These receptor subtypes have also been identified in gonadotrope cell lines, including the gonadotrope-derived {alpha}T3-1 [40] and LßT2 [25] lines. POP cells represent a heterogeneous population of cells and are difficult to manipulate in vitro. The LßT2 cells are transformed cells and consequently may differ from the gonadotrope cells. However, studies with LßT2 cells have greatly furthered the understanding of GnRH signaling [41], and these cells are useful for analyzing the complex intracellular signaling events that follow activin stimulation. Consequently, we used them here in addition to the POP cells to identify the activin receptor signaling pathway(s) involved in FSH expression and release.

We demonstrated that activin activates the Smad pathway and other signaling pathways, including the MAPKs ERK1/2 and p38 and the kinases PI3K and Akt in both POP cells and LßT2 cells. The same kinetics of activin activation of the different kinases were observed in both POP and LßT2 cells but also in the MCF-7 human breast cancer cells. Thus, activin seems to activate the same signaling pathways in a large set of target cells. Consistent with our results in various cell lines, activin and other members of the TGFß family have been reported to utilize other signaling pathways in addition to the Smad pathway to transduce their signal. In human T47D breast cancer cells, activin activates the p38 kinase pathway leading to cell growth arrest [42]. In endothelial cells, TGFß1 is essential for cell survival and formation of capillarylike structures through the stimulation of the PI3K (PI3K/Akt) and the p42/p44 MAPK pathways [20].

In POP and LßT2 cells, Smad-2, ERK1/2, p38, and PI3K/Akt signaling pathways respond to activin with different time courses: a rapid activation of the MAPKs (ERK1/2 and p38) and a much slower activation for the Smad-2 and PI3K/Akt kinases. Using specific inhibitors, we showed that the Smad-2 phosphorylation is partly due to PI3K/Akt activation. Consistent with our result, a number of functional interactions between Smad and other signaling pathways have been reported. For example, recent work has demonstrated that activation of the p42/p44 MAPK pathway is involved in TGFß1-induced furin gene expression and suggests functional interactions between the Smad and the p42/p44 MAPK cascade pathway in this regulation [43]. Some reports indicate that Smad signaling converges with tyrosine kinase receptor signaling in response to growth factors and consequent activation of MAPK pathways [44]. TGFß activates Smad and p38 pathways, resulting in the formation of an active transcription complex composed of Smad-3/Smad-4 and the p38 nuclear target ATF-2 in myoblast C2C12 and human embryonic kidney 293 cells [45, 46]. In the present study, we demonstrated for the first time an interaction between ActR-IIB and the p85 regulatory subunit of PI3K. This association is strongly enhanced by activin in POP and LßT2 cells. The coimmunoprecipitation technique cannot be used to ascertain whether there is a direct or indirect association between these two components. Nevertheless, because ActR-IIB possesses proline-rich sequences [47], it is reasonable to assume that it is the binding site to SH3-domain-containing proteins, such as the p85 regulatory subunit of PI3K.

By measuring nuclear FSHß primary transcripts, activin has been shown to increase FSHß gene transcription [48]. However, the molecular mechanisms by which activin regulates FSHß gene transcription are unknown. In LßT2 and POP cells, the ERK1/2, p38, and the PI3K/Akt kinases were not involved in FSHß expression and FSH secretion. However, the expression of the Smad-2 dominant negative or the inhibitory Smad-7 strongly reduced the stimulatory effect of activin on FSHß expression and FSH secretion in the gonadotrope LßT2 cell line. The absence of total abolition of activin response for FSHß mRNA and FSH release in the presence of the dominant negative Smad-2 or the inhibitory Smad-7 may be explained by the fact that only about 30% of the cells were transfected in this experiment (data not shown). Thus, these results suggest that activin increases FSH expression and secretion by mechanisms that are dependent on Smad-2 in the LßT2 cells. Consistent with these results, scanning of the oFSHß 5.5-kilobase regulatory region of the oFSHß gene revealed at least 23 Smad consensus DNA-binding sites (5'-GTCTAGAC-3') with with six to eight conserved bases, reinforcing a possible involvement of Smad proteins in the activin regulation of oFSHß gene expression [49]. FSHß mRNA is known to have a much shorter half-life than LHß mRNA. The increase in the level of FSHß mRNA under activin stimulation might be due either to enhanced transcription and/or to stabilization of mRNA. The presence of Smad binding sequences in the oFSHß promoter suggests that the effects of activin arise at least partly at the transcription level. Nevertheless, we cannot rule out the possibility that part of the effect of activin is due to mRNA stabilization. This second effect could be mediated by an alternative to the Smad signaling pathway.

Activin activates various signaling pathways, including the ERK1/2, p38, PI3K, and Akt kinases in POP and LßT2 cells. Nevertheless, only a Smad protein seems to be directly implicated in FSHß expression and FSH secretion in LßT2 cells. The other kinases probably play other roles in pituitary cells. In particular, activin regulates a wide variety of cellular events, including cell proliferation, differentiation, and apoptosis [50]. Further investigation will enable us to discover the roles of the ERK1/2, p38, PI3K, Akt kinases in activin receptor signaling in pituitary cells.


   ACKNOWLEDGMENTS
 
We thank C. Gauthier for measurements of oLH and FSH in the culture medium, J. Fontaine for technical assistance, Dr. P. ten Dijke for the Smad-7 cDNA, Dr. J. Wrana for the dominant negative of Smad-2, Dr. J.F. Ethier for the cDNAs of bovine activin receptors, Dr. W. Vale for the rabbit polyclonal antibodies directed against the different types of activin receptors, Dr. J.F. Roser for the b-oLH antibodies, Dr. K. Henderson for b-oFSH, and Dr. A.F. Parlow for oFSH RP2. We thank Drs. P. Mellon and D. LeRoith for their gifts of LßT2 and MCF-7 cells, respectively. We also thank Drs. P. Monget, J. Simon, and L. Nicol for critically reading this manucript and Dr. P. Froment for assistance with the statistics.


   FOOTNOTES
 
1 Correspondence: Joëlle Dupont, Unité de Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, 37380 Nouzilly, France. FAX: 33 2 47 42 77 43; jdupont{at}tours.inra.fr Back

Received: 9 October 2002.

First decision: 6 November 2002.

Accepted: 12 December 2002.


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