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Endocrinology Vol. 147, No. 12 5932-5939
Copyright © 2006 by The Endocrine Society

Tumor Necrosis Factor-{alpha} Activates Transcription of Inducible Repressor Form of 3',5'-Cyclic Adenosine 5'-Monophosphate-Responsive Element Binding Modulator and Represses P450 Aromatase and Inhibin {alpha}-Subunit Expression in Rat Ovarian Granulosa Cells by a p44/42 Mitogen-Activated Protein Kinase-Dependent Mechanism

Victoria Morales1, I. Gonzalez-Robayna1, M. Pino Santana, Inmaculada Hernandez and Luisa F. Fanjul

Departamento de Bioquímica y Fisiología, Facultad de Medicina, Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria 35016, Spain

Address all correspondence and requests for reprints to: Luisa F. Fanjul, Department of Biochemistry and Physiology, Facultad de Medicina, Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, 35016, Spain. E-mail: lfanjul{at}dbbf.ulpgc.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The proinflammatory cytokine TNF{alpha} has important actions at the level of the ovary, including inhibition of P450 aromatase (P450AROM) activity and the secretion of inhibin, two proteins that are markers of the granulosa cell’s differentiated status. Because the transcription of both P450AROM and inhibin {alpha}-subunit can be suppressed in the ovary by the inducible repressor isoform of cAMP-responsive element binding modulator (ICER), we have investigated whether TNF{alpha} and its intracellular messenger ceramide can induce ICER expression and the mechanisms whereby the induction is accomplished. ICER mRNA levels were assessed by RT-PCR in granulosa cells treated with TNF{alpha}, the ceramide-mobilizing enzyme sphingomyelinase (SMase), or C6-cer, a cell-permeant ceramide analog. Rapid (3 h) yet transient increases in the four isoforms of ICER were observed in response to all treatments. Likewise, ICER protein measured by immunoprecipitation with a specific antibody increases after TNF{alpha}, SMase, or C6-cer treatment. The mandatory phosphorylation of cAMP-responsive element binding was also observed in response to TNF{alpha}, SMase, or C6-cer and shown to be prevented by the p44/42 MAPK-specific inhibitor PD098059 but no other kinase blockers. Activation of p44/42 MAPK by the cytokine and its messenger was subsequently demonstrated as well as the inhibition of ICER expression by PD098059. Finally, the blocking of p44/42 MAPK activation prevented TNF{alpha} inhibition of FSH-dependent increases in P450AROM and inhibin {alpha}-subunit mRNA levels, thus indicating that p44/42 MAPK-mediated ICER expression may be accountable for the effects of TNF{alpha} on the expression of both proteins.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE BEST CHARACTERIZED effect of TNF{alpha} is the ability to induce signals that trigger cell death (1, 2, 3), but this proinflammatory cytokine has pleiotropic effects in mammalian cells (4, 5, 6). TNF{alpha} is primarily secreted by monocytes and macrophages in response to bacterial lipopolysaccharide, and although resident or infiltrating macrophages secrete TNF{alpha} in the ovary (7), secretion by granulosa-luteal cells has also been reported (8, 9), and TNF{alpha} mRNA has been detected in ovarian tissue (10). Whatever is the source for the TNF{alpha} acting in the ovary, the cytokine has multiple and important effects in this organ that may be summarized as inhibition of FSH-dependent differentiation of granulosa cells (11, 12) and induction of apoptosis (13).

Two of the more prominent specific actions of TNF{alpha} in the ovary are the inhibition of FSH-stimulated P450 aromatase (P450AROM) catalytic activity and inhibin secretion by granulosa cells (14, 15). The acquisition of the ability to synthesize estrogens and inhibin is a hallmark of the granulosa cell preovulatory phenotype, because follicle development, ovulation, and cyclic secretion of pituitary hormones all rely to some extent on the production of adequate amounts of estrogens and inhibin at the different phases of the ovarian cycle (reviewed in Refs. 16, 17, 18, 19).

The transcription of P450AROM and inhibin {alpha}-subunit is tightly regulated by pituitary hormones. Upon binding to its G protein-coupled receptor in granulosa cells, FSH induces cAMP production, protein kinase A (PKA) catalytic subunit activation (20) and cAMP-responsive element binding protein (CREB) phosphorylation (21), resulting in the transcription of, among others, aromatase and inhibin {alpha}-subunit genes (22, 23, 24, 25). Likewise, LH receptor is coupled to cAMP generation and CREB phosphorylation (21), although during the preovulatory surge of this hormone, both aromatase and inhibin {alpha}-subunit expression are down-regulated (26, 27). In response to LH, the cAMP-inducible repressor isoform of cAMP-responsive element binding modulator (CREM) (ICER) (28) is expressed in the rat ovarian granulosa cells, where it possibly participates in the mechanism underlying the LH-triggered down-regulation of aromatase and {alpha}-inhibin genes (29, 30, 31).

TNF{alpha} effects initiate after the interaction with two 55- and 75-kDa receptors that are coexpressed in virtually all cell types (32). The sequence of events that immediately follow TNF{alpha} binding to its receptor is not completely understood but includes, in some instances, the activation of MAPKs (33, 34, 35).

Although CREB is the best characterized substrate of PKA (36, 37), CREB Ser133 phosphorylation may also be performed by Ca2+/calmodulin-dependent protein kinases and MAPKs (38, 39). That TNF{alpha} could induce CREB phosphorylation and ICER expression in granulosa cells seems reasonable in the light of the above mentioned observations and will possibly contribute to understanding the mechanism underlying TNF{alpha} effects on FSH-induced expression of P450AROM and inhibin {alpha}-subunit.

To test this hypothesis, granulosa cells were stimulated with TNF{alpha} or with its intracellular messenger ceramide, and CREB phosphorylation and ICER expression were assessed and shown to be induced by the cytokine. Both ICER expression and the inhibitory effects of TNF{alpha} on FSH-induced increase in aromatase and inhibin {alpha}-subunit mRNA levels were shown to depend on p44/42 MAPK activation, because TNF{alpha} effects were counteracted by PD098059 (40), the specific inhibitor of the dual kinase.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and hormones
All culture media and Trizol were from Invitrogen (San Diego, CA). Recombinant human FSH was obtained from Serono Laboratories (Rockland, MA). TNF{alpha}, bacterial sphingomyelinase (SMase; from Staphylococcus aureus), C6-cer, H89, GF109203X, SB203580, PD098059, and annexin V fluorescein isothiocyanate/propidium iodine (PI) apoptosis detection system were purchased from Calbiochem (Barcelona, Spain). [35S]Methionine and [35S]cysteine (Translabel) were from ICN (Costa Mesa, CA). Antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA) (anti-CREM and anti-CREB) or Cell Signaling Technology-New England Biolabs (Beverly, MA) (anti-pCREB, anti-p42 MAPK, and anti-p-p44/42 MAPK). Avian myeloblastosis virus reverse transcriptase (M519), RNAsin ribonuclease inhibitor (N211), and Taq DNA polymerase were from Promega (Madison WI). Diethylstylbestrol, pregnant mare serum gonadotropin (PMSG), protease, and phosphatase inhibitors and all other reagents were from Sigma Chemical Co. (Madrid, Spain).

Animals and cell culture
Immature 19- to 21-d-old female Sprague Dawley rats were purchased from Charles River Laboratories (Barcelona, Spain) and kept on 12-h light, 12-h dark cycles with free access to food and water. Both housing conditions and experimental procedures were approved by the University of Las Palmas Committee on Animal Care that enforces European Union rule 86/609. Granulosa cells were obtained by follicle puncture from the ovaries of rats implanted with diethylstilbestrol for 3 d or with a single injection of PMSG (10 IU) and cultured for the time periods indicated for each experiment in McCoy’s 5a medium (modified, without serum).

Immunoprecipitation and Western blot analysis
To measure the cellular content of ICER protein, granulosa cells were incubated for 6 h in methionine- and cysteine-free McCoy’s 5a modified medium, with 250 µCi/ml [35S]methionine and [35S]cysteine. The cells were thereafter washed in PBS and incubated with TNF{alpha} (10 ng/ml), SMase (1 U/ml), or C6-cer (1 µM) for the times indicated in McCoy’s 5a medium supplemented with 4 mM methionine and 4 mM cysteine. To terminate the experiment, the cells were washed twice in ice-cold PBS and lysed for 30 min in 200 µl PBS containing 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 0.2% NaN3, and a mix of protease and phosphatase inhibitors (1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 15 mM iodoacetamide, and 5 µg/ml aprotinin). After centrifugation at 14,000 x g for 15 min, aliquots from the supernatant containing the same amount of [35S]methionine- and [35S]cysteine-labeled proteins (1–2 mg) were immunoprecipitated overnight at 4 C with anti-CREM antibody preabsorbed to protein A-Sepharose and used at twice the manufacturer’s recommendations to circumvent the low specificity for the ICER isoform. The labeled immunocomplexes were collected and subjected to SDS-PAGE. The dried gels were thereafter recorded as autoradiographs by photostimulatable-storage imaging followed by laser scanning in a Molecular Dynamics 400A PhosphoImager (Molecular Dynamics, Sunnyvale, CA).

Cell lysates were prepared with 1% Triton X-100, 10% glycerol in a buffer containing 150 mM NaCl, 1.5 mM MgCl2, 5 mM EDTA, 50 mM HEPES, and 1 mM phenylmethylsulfonyl fluoride. Proteins were resolved using 12% SDS-PAGE, transferred onto nitrocellulose membranes, and immunoblotted with the appropriate specific antibody. The membranes were thereafter developed with a peroxidase-conjugated goat antirabbit second antibody and the ELC Western blot analysis kit (Amersham Biosciences Europe GmbH, Barcelona, Spain). To serve as controls of protein loading, membranes were routinely stripped out of the antibodies using Restore Western Blot (Pierce Biotechnology, Rockford, IL) and reprobed with a new antibody against the nonphosphorylated forms of CREB and p42MAPK, at twice the concentration recommended by the manufacturer. Recording and visualizing were performed in an imaging system with cooled CCD capture system for chemiluminescence (Diana III from Raytest, Straubenhardt, Germany) using the software provided by the manufacturer (AIDA, 1D, 2D).

PCR
Total RNA was extracted from triplicate granulosa cell cultures for each treatment using a modified guanidinium isothiocyanate method, and samples were stored frozen (–70 C) in diethylpyrocarbamate-treated water until use. After drying and OD determination, RT was performed by standard protocols. Briefly, equal amounts of RNA (1 µg) were incubated for 75 min at 42 C in 20 µl (final volume) of 1x PCR buffer (10 mM Tris-HCl, 50 mM KCl, 5 mM MgCl2, and 0.1% Triton X-100, pH 9), 500 ng polydeoxythymidine primers, 1 mM dNTP, 5 U avian myeloblastosis virus reverse transcriptase, and 20 U RNAsin ribonuclease inhibitor. Each cDNA was amplified using specific oligonucleotides in 25 µl of 1x PCR buffer containing (final concentrations) 2.5 µM digoxigenin-dUTP, 0.625 U Taq DNA polymerase, and 125 ng (10–15 pmol each) of the appropriate specific primers for P450AROM, AROM-5'-TGCACAGGCTCGAGTATTTCC and AROM-3'-ATTTCCACAATGGG GCTGTCC; {alpha}-inhibin, INHIB-5'-GAGGATGTCTCCCAGGCCAT and INHIB-3'-CAGGTCT ATTCTGTGGA; or ICER, ICER-5'-ACTTAGGATCCACTGTGTACGGCCAAC and ICER-3'-GTTAAATAGAATTCACTAATCTGTTTTGGG. PCR products were detected by chemiluminescence, and because of the method’s high sensitivity, the thermal profile had to be adjusted as follows: 94 C for 1 min for denaturation and 1.5 min at 58 C and 72 C for annealing and elongation, respectively. Under these conditions, nonspecific background signals were reduced to undetectable levels and linearity ensured for up to 20 cycles for aromatase and {alpha}-inhibin, 29 cycles for ICER, and 16 cycles for the ribosomal protein L19 that was used as internal amplification control. The amplified products were resolved by 1.8% agarose gel electrophoresis and transferred to positively charged nylon membranes. Detection was performed with a commercially available digoxigenin luminescent detection kit (Roche Diagnostic SL, Barcelona, Spain) following the instructions of the manufacturer. The same system as in Western blot was used to visualize membranes.

Analysis of apoptosis by flow cytometry
Granulosa cells (1 x 106 per tube) were obtained from the ovaries of immature rats that were injected 2 d before the experiment with 10 IU PMSG to induce aromatase expression and prevent apoptosis. The cells were incubated in McCoy’s 5a medium (modified, without serum), supplemented with FSH (0.002 IU/ml) and testosterone (10 ng/ml) to ensure the continuity of aromatase activation and provide aromatase substrate, respectively. The experiments were carried out for 6 h, and thereafter the cells were washed twice at 1000 x g with cold PBS and resuspended in 0.5 ml cold binding buffer. Annexin V-fluorescein isothiocyanate and PI were then added following the instructions provided by the manufacturer. The tubes were kept in the dark, and cytometric analysis (Epics XL; Coulter, Hialeah, FL) was conducted right after the end of incubations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TNF{alpha} inhibits P450AROM and inhibin {alpha}-subunit mRNA levels
We and others have previously reported that TNF{alpha} decreases the FSH-induced activity of P450AROM and the secretion of the inhibin {alpha}-ß dimer (15, 41). Although the changes in the catalytic activity of P450AROM and the rate of secretion of inhibin A should correlate with changes in the transcriptional rate of the enzyme and the inhibin {alpha}-subunit, we first wanted to test whether TNF{alpha} and its intracellular messenger ceramide did in fact inhibit FSH-induced increases in P450AROM and inhibin {alpha}-subunit mRNA. For that purpose, granulosa cells were cultured with FSH for 48 h, and TNF{alpha} (10 ng/ml), SMase (1 U/ml), or C6-cer (1 µM) was added 6 h before the completion of the experiment. As shown in Fig. 1Go, the cotreatment of granulosa cell cultures with TNF{alpha}, as well as with bacterial SMase that mobilizes ceramide or with the membrane-permeable analog of ceramide C6-cer, inhibits the FSH-induced increases in P450AROM and inhibin {alpha}-subunit mRNA levels. As can also be seen in Fig. 1Go, addition of cycloheximide counteracts TNF{alpha} and its intracellular messenger action, indicating that the effect is protein synthesis dependent.


Figure 1
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  		FIG. 1. TNF{alpha} inhibits P450AROM and {alpha}-inhibin subunit expression in granulosa cells. Granulosa cells (106 viable cells per well) were cultured with or without FSH (0.002 IU/ml) for 48 h in McCoy’s 5a medium. TNF{alpha} (10 ng/ml) (A), SMase (1 U/ml) (B), or C6-cer (1 µM) (C) were added 6 h before the completion of the experiment. Cycloheximide (CHX) (1 µM) was also added 3 h before the end of the experiment where indicated. P450AROM, {alpha}-inhibin subunit, and microsomal protein L-19 mRNA levels were assessed by RT-PCR as described in Materials and Methods. Triplicate dishes were used in each experiment, and the results of one of at least three different experiments are shown.

 
TNF{alpha}, SMase, and ceramide induce ICER expression
To test whether the protein synthesis-dependent effects of TNF{alpha} on P450AROM and inhibin {alpha}-subunit mRNA levels might be attributed to the repressor isoform of CREM (ICER), ICER mRNA and protein levels were assessed after 3, 6, and 12 h of treatment of granulosa cells with the cytokine or its intracellular messenger. Increases in ICER mRNA as determined by RT-PCR with the ICER gene-specific primers were found at 3 and 6 h after TNF{alpha} (Fig. 2AGo), SMase (Fig. 2BGo), or C6-cer (Fig. 2CGo) addition to cultured granulosa cells. After 12 h of treatment, ICER mRNA had already returned to basal, almost undetectable levels. ICER protein levels were also measured in extracts of granulosa cells labeled with [35S]methionine after immunoprecipitation with an anti-CREM antibody. A band with an apparent molecular mass of 14 Kda, presumably corresponding to ICER-II, was observed to increase at all the times tested, suggesting that in fact ICER is being synthesized in response to TNF{alpha}, ceramide mobilization, and the ceramide analog (bottom of Fig. 2Go, A–C).


Figure 2
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  		FIG. 2. TNF{alpha} increases ICER mRNA and protein levels in granulosa cells. Triplicate cultures of granulosa cells (106 viable cells per well) were treated with TNF{alpha} (10 ng/ml) (A), SMase (1 U/ml) (B), or C6-cer (1 µM) (C) for the times indicated in the figure. ICER mRNA levels were assessed by RT-PCR as described in Materials and Methods. ICER protein was determined after immunoprecipitation of protein extracts prepared from granulosa cells labeled with [35S]methionine. Similar results were obtained in three other experiments.

 
TNF{alpha} and ceramide induce CREB phosphorylation by a P44/42 MAPK-dependent mechanism
Because of the presence of four CRE-like sequences in a second CREM gene intronic promoter that directs ICER transcription, ICER is a unique, cAMP-inducible member of the CREM family (28). Therefore, we next investigated whether the treatment of granulosa cells with TNF{alpha}, SMase, or C6-cer resulted in the phosphorylation of CREB. Figure 3AGo shows that, as expected, CREB phosphorylation occurs in granulosa cells 15–30 min after the treatment with TNF{alpha}, SMase, or C6-cer, thus providing the grounds to explain the increasing levels of ICER mRNA in response to the cytokine. Because no coupling to cAMP production has been attributed to any TNF receptor, we next used a set of different kinase inhibitors to explore the nature of the kinase involved in the observed phosphorylation of CREB. Figure 3BGo shows that the inhibitors of PKA (H89) and PKC (GF109203X) did not affect TNF{alpha}-induced CREB phosphorylation. SB203580 (43), an antagonist of p38 MAPK slightly decreased TNF{alpha} effects, whereas PD098059 (40), a specific inhibitor of p44/42 MAPKs, almost completely blocked the phosphorylation of CREB in response to TNF{alpha}, suggesting that the effect of TNF{alpha} on CREB phosphorylation is mediated by this kinase.


Figure 3
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  		FIG. 3. TNF{alpha} promotes CREB phosphorylation in granulosa cells. Granulosa cells were treated with TNF{alpha} (10 ng/ml), SMase (1 U/ml), or C6-cer (1 µM) for the times indicated (A) or for 30 min (B) with TNF{alpha} (10 ng/ml) alone or in combination with a pretreatment (60 min) with H89 (10 µM), GF109203X (1 µM), SB20203580 (20 µM), or PD098059 (10 µM). Phosphorylated levels of CREB, as well as unphosphorylated CREB used as total protein control, were assessed by immunoblot as described in Materials and Methods. Densitometric analysis of four experiments (mean ± SEM) was performed, and a representative immunoblot is shown.

 
To prove that in fact TNF{alpha} activates p44/42 MAPK in granulosa cells, triplicate cultures were stimulated with the cytokine, bacterial SMase, or C6-cer, and p44/42 MAPK activation was assessed using immunoblot with an antibody that specifically recognizes dually phosphorylated ERK1 and ERK2. Figure 4Go shows that TNF{alpha} (10 ng/ml) induces a rapid (5-min) phosphorylation of both kinases that reaches its maximum at 30 min after the treatment with the cytokine. Likewise, SMase (1 U/ml) and C6-cer (1 µM) activate both MAPKs during the first 30 min after they are added to the cells. Despite the discrete effect of SB203580 on TNF{alpha}-induced CREB phosphorylation, neither TNF{alpha} nor SMase or C6-cer were able to induce the activation of p38 MAPK (data not shown).


Figure 4
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  		FIG. 4. TNF{alpha}, SMase, and C6-cer activate p44/42 MAPK. Granulosa cells were treated with TNF{alpha} (10 ng/ml), SMase (1 U/ml), or C6-cer (1 µM) for the times indicated. Activation (phosphorylation) of p44/42 MAPK as well as total p42 MAPK were assessed by immunoblot with an antibody against p-Thr and p-Tyr sites and anti p42 MAPK, respectively. One of three other experiments is shown.

 
TNF{alpha}-induced ICER expression and P450AROM inhibition depends on p44/42 MAPK activation
To add support to the role of ERK1/ERK2 as mediators of TNF{alpha} effects, ICER, P450AROM, and inhibin {alpha}-subunit mRNA levels were assessed in granulosa cells pretreated for 48 h with FSH (0.002 IU/ml) and cotreated for the last 6 h of the experiment with TNF{alpha} (10 ng/ml) alone or with the different kinase inhibitors (added 1 h before TNF{alpha}). Neither PKA nor PKC or p38 MAPK inhibitors prevented TNF{alpha}-induced ICER expression (Fig. 5AGo). However, PD098059, the well known specific inhibitor of p44/42 MAPK, completely abolished the increases in ICER mRNA levels induced by TNF{alpha}. Likewise, PKA, PKC, or p38 MAPK inhibitors did not affect TNF{alpha} effects on P450AROM and inhibin {alpha}-subunit mRNA levels, whereas the expression of both genes was restored to the levels of control, TNF{alpha}-untreated cells, when the MAP dual kinase was inhibited, indicating the involvement of ERK1 and ERK2 in the TNF{alpha} effect.


Figure 5
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  		FIG. 5. TNF{alpha} induction of ICER expression and aromatase and inhibin {alpha}-subunit down-regulation depends on p44/42 MAPK activation. Granulosa cells were cultured 48 h with FSH (0.002 IU/ml) alone or with TNF{alpha} (10 ng/ml) that was added 6 h before the end of the experiment. H-89 (10 µM), GF109203X (1 µM), SB20203580 (20 µM), or PD098059 (10 µM) were added 60 min before TNF{alpha}. Levels of ICER (A), P450AROM (B, bottom) and inhibin {alpha}-subunit (B, top) mRNA were determined by RT-PCR. Densitometric analysis (mean ± SEM of OD arbitrary units is represented) was performed on the results of four different aromatase and inhibin {alpha}-subunit experiments. A representative RT-PCR is shown.

 
Effects of p44/42 MAPK inhibitors on TNF{alpha}-induced apoptosis
To prove a MAPK role on TNF{alpha}-induced apoptosis in granulose cells, flow cytometric analyses were performed with double-labeled (PI/annexin V) cells. Annexin V binds specifically to phosphatidylserine, which in live cells is located in the inner part of the cellular membrane. However, in the early phases of apoptosis, phosphatidylserine is exposed to the exterior, thus binding annexin V whose fluorescent signal (Fig. 6Go, lower right quadrants) provides a measure of the percentage of a cell population entering apoptosis. Live cells are also impermeable to PI, although in the late stages of apoptosis, cell membrane disruption allows for PI to enter the cell and bind DNA. The signal generated (Fig. 6Go, upper right quadrants) serves as an indicator of cells in the latter stages of apoptosis. As may be observed in Fig. 6Go, at time 0 of incubation, most (~99.7%) of the granulosa cells are live (lower right quadrant), whereas at the end of the experiment, a small number (~15%) of the cells undergoes spontaneous apoptosis, despite the presence of both FSH and testosterone.


Figure 6
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  		FIG. 6. Effects of p44/42 MAPK inhibitors on TNF{alpha}-induced apoptosis in granulosa cells. Granulosa cells were obtained by follicular puncture from the ovaries of immature rats that had been injected 2 d before with 10 IU PMSG. Cells were incubated in 6-ml Falcon culture tubes, washed, labeled with PI and annexin V as described in Materials and Methods, and counted immediately after the incubations. A, Control at time 0 of incubation; B, control at the end of the experiment; C, TNF{alpha} (10 ng/ml); D, cells pretreated for 60 min with PD098059 (10 µM) plus 6 h with TNF. The experiment was replicated twice, each using triplicate tubes.

 
After 6 h of incubation with TNF{alpha}, more than 60% of the cells are entering apoptosis and an additional 23% are at the terminal phase of the apoptotic process. The addition of PD098059 reduced to 34 and 12% the number of cells experiencing early and late apoptosis, respectively, thus demonstrating that p44/42 MAPK is part of the mechanism implicated in TNF{alpha} induction of apoptosis in granulosa cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We herein present experimental data that shows for the first time the induction of ICER expression in granulosa cells by TNF{alpha} and its intracellular messenger ceramide. To our knowledge, this is also the first time ICER induction in response to this cytokine has been reported in any cell or tissue.

We have also observed that in granulosa cells, phosphorylation of CREB in response to the cytokine and its messenger is achieved by a MAPK-dependent mechanism. Although CREB phosphorylation at the Ser133 residue (44) is preferentially performed by PKA (36, 37), the phosphorylation of CREB by Ca2+/calmodulin-dependent protein kinases and MAPKs has been also extensively documented (38, 39). Thus, p44/42-MAPK- or p38-MAPK-dependent CREB phosphorylation has been shown in vitro and in vivo in several cell types (45, 46, 47) and is supposed to be achieved both through independent (46) or confluent pathways, these latter involving the phosphorylation of mitogen- and stress-activated kinases, which serve as substrates for p44/42 and p38 MAPK (47).

A p38-MAPK-dependent increase in the amount of phosphorylated CREB, after TNF{alpha}, SMase, or ceramide treatment has been reported in MC/9 and U937 myeloid cells (48). Our results in granulosa cells point to p44/42 as the kinase involved in TNF{alpha} effects because both CREB phosphorylation in response to TNF{alpha} and TNF{alpha}-induced transcription of ICER can be blocked by PD098059, a highly specific inhibitor of this kinase (40). Despite the fact that c-Jun N-terminal kinase, p38 MAPK, and other stress-activated kinases, were initially thought to be the only MAPK family members activated by cytokines (49), we have shown here, and others elsewhere, that TNF{alpha} can phosphorylate ERK1/ERK2 (33, 34, 50). Likewise, phosphorylation of the dual kinase has been reported in response to ceramide in cultured astrocytes (51).

To act as a key regulator of cAMP-dependent genes that are expressed in a rhythmic pattern was initially thought to be ICER’s role (52), as a result of two distinctive features: 1) ICER is a repressor of cAMP-driven transcription because it competes with members of the bZIP superfamily of proteins that bind the CRE 8-bp palindromic sequence, and 2) four CRE-like elements in the CREM intronic promoter that directs ICER transcription enable ICER to down-regulate its own expression (28).

However, many other functions have been latter attributed to ICER, including regulation of insulin secretion (53), hormone receptor desensitization (54, 55), bone remodeling (56), and influencing T-cell and macrophage function (57, 58) and virus replication (59). ICER has also been proposed to be involved in the control of cellular proliferation (60, 61) and apoptosis in a limited number of cells and/or tissues (62, 63, 64).

Granulosa cells apoptosis is known to be determinant in ovarian follicular atresia (65), a process that is likely to be controlled by as many regulatory molecules as follicular growth (66). Because of the fact that decreases in P450AROM levels have been shown to be tightly associated with apoptosis in granulosa cells of atretic follicles and the death of these cells is largely prevented by treatment with estrogens (67, 68), estrogens have been proposed to be important promoters of granulosa cell survival. Thus, it is more than possible that ICER would be mediating TNF{alpha} effects on granulosa cell apoptosis by inhibiting FSH-induced P450AROM expression.

Other and more direct contributions to the activation of the apoptotic machinery by ICER could be subserving the TNF{alpha} proapoptotic effects in granulosa cells. Both in neurons and cardiomyocytes, the ICER proapoptotic role seems to be performed by suppressing cAMP-dependent expression of bcl-2 (63, 64). In the ovary, the ablation of bcl-2 results in a diminished and abnormal number of follicles, whereas the overexpression of this antiapoptotic gene leads to decreased ovarian somatic cell apoptosis and enhanced folliculogenesis (69, 70). These findings would suggest that granulosa cell survival and the rescue of follicles from atresia will be favored by an augmented bcl-2 expression. However, no increases in bcl-2 expression have been encountered in granulosa cells in response to any of the antiapoptotic regulatory molecules, including the major survival factor for these cells, the gonadotropic hormone FSH, whose receptor is coupled to cAMP generation (66). Therefore, it seems unlikely that increases in bcl-2 expression would play a prominent role in salvaging granulosa cells from apoptosis.

Nevertheless, it has been reported that TNF{alpha} induction of granulosa cell apoptosis could by partly a result of the suppression of constitutive bcl-2 expression (42); thus, the possibility exists, and may be deserving of future studies, that in the ovary, as in neurons and cardiomyocytes, the ICER synthesized in response to TNF{alpha} could be repressing constitutive bcl-2 expression.

In conclusion, although it is more than likely that several other pathways would be contributing to the signaling of TNF{alpha} in granulosa cells, CREB phosphorylation by p44/42 MAPK followed by ICER induction and the repression of FSH-driven aromatase transcription plays a significant role in TNF{alpha} induction of apoptosis in these cells.


   Footnotes
 
This work was supported by the Direccion General de Enseñanza Superior e Investigación Científica Grant 98/0234.

None of the authors has any information to disclose.

First Published Online August 31, 2006

1 V.M. and I.G.-R. have contributed equally to this work. Back

Abbreviations: CREB, cAMP-responsive element binding protein; CREM, cAMP-responsive element binding modulator; ICER, inducible repressor isoform of CREM; P450AROM, P450 aromatase; PI, propidium iodine; PMSG, pregnant mare serum gonadotropin; SMase, sphingomyelinase.

Received May 11, 2006.

Accepted for publication August 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cleveland JL, Ihle JN 1995 Contenders in FasL/TNF death signaling. Cell 81:479–482[CrossRef][Medline]
  2. Wallach D 1997 Cell death induction by TNF: a matter of self control. Trends Biochem Sci 22:107–109[CrossRef][Medline]
  3. Yeh WC, Hakem R, Woo M, Mak TW 1999 Gene targeting in the analysis of mammalian apoptosis and TNF receptor superfamily signaling. Immunol Rev 169:283–302[CrossRef][Medline]
  4. Beutler B, Cerami A 1988 Tumor necrosis, cachexia, shock and inflammation: a common mediator. Annu Rev Biochem 57:500–518
  5. Vilceck J, Lee TH 1991 Tumor necrosis factor. New insights into the molecular mechanisms of its multiple actions. J Biol Chem 266:7313–7316[Free Full Text]
  6. Baker SJ, Reddy EP 1996 Transducers of life and death: TNF receptor superfamily and associated proteins. Oncogene 12:1–9[Medline]
  7. Adashi EY 1990 The potential relevance of cytokines to ovarian physiology: the emerging role of resident ovarian cells of the white blood cell series. Endocr Rev 11:454–464[Abstract/Free Full Text]
  8. Zolti M, Meirom R, Shemesh M, Wollach D, Mashiach S, Shore L, Rafael ZB 1990 Granulosa cells as a source and target organ for tumor necrosis factor-{alpha}. FEBS Lett 261:253–255[CrossRef][Medline]
  9. Roby KF, Weed J, Lyles R, Terranova PF 1990 Immunological evidence for a human ovarian tumor necrosis factor-{alpha}. J Clin Endocrinol Metab 71:1096–1102[Abstract/Free Full Text]
  10. Sancho-Tello M, Perez-Roger I, Imakawa K, Tilzer L, Terranova PF 1992 Expression of tumor necrosis factor-{alpha} in the rat ovary. Endocrinology 130:1359–1364[Abstract/Free Full Text]
  11. Darbon JM, Oury F, Laredo J, Bayard F 1989 Tumor necrosis factor-{alpha} inhibits follicle-stimulating hormone-induced differentiation in cultured rat granulosa cells. Biochem Biophys Res Commun 163:1038–1046[CrossRef][Medline]
  12. Veldhuis JD, Garmey JC, Urban RJ, Demers LM, Aggarwal BB 1991 Ovarian actions of tumor necrosis factor-{alpha} (TNF-{alpha}): pleiotropic effects of TNF-{alpha} on differentiated function of untransformed swine granulosa cells. Endocrinology 129:641–648[Abstract/Free Full Text]
  13. Kaipia A, Sang-Young C, Eisenhauer K, Hsueh AJW 1996 Tumor necrosis factor-{alpha} and its second messenger ceramide, stimulate apoptosis in cultured ovarian follicles. Endocrinology 137:4864–4870[Abstract]
  14. Emoto N, Baird A 1988 The effect of tumor necrosis factor/cachectin on follicle-stimulating hormone-induced aromatase activity in cultured rat granulosa cells. Biochem Biophys Res Comm 153:792–798[CrossRef][Medline]
  15. Imai M, Yamamoto M, Otani H, Nakano R 1996 Cytokine modulation of inhibin secretion in cultured rat granulosa cells. J Endocrinol 151:449–457[Abstract/Free Full Text]
  16. Hsueh AJ, Adashi EY, Jones PB, Welsh TH 1984 Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr Rev 5:76–127[Abstract/Free Full Text]
  17. Tonetta SA, diZerega GS 1989 Intragonadal regulation of follicular maturation. Endocr Rev 10:205–229[Abstract/Free Full Text]
  18. Ackland JF, Schwartz NB, Mayo KA, Dodson RE 1992 Nonsteroidal signals originating in the gonads. Physiol Rev 72:731–787[Abstract/Free Full Text]
  19. Woodruff TK, Mather JP 1995 Inhibin, activin and the female reproductive axis. Annu Rev Physiol 57:219–244[CrossRef][Medline]
  20. Richards JS 1994 Hormonal control of gene expression in the ovary. Endocr Rev 15:725–751[Abstract/Free Full Text]
  21. Mukherjee A, Park-Sarge OK, Mayo KE 1998 Gonadotropins induce rapid phosphorylation of the 3',5'-cyclic adenosine monophosphate response element binding protein in ovarian granulosa cells. Endocrinology 137:3234–3245
  22. Hickey GJ, Chen S, Besman MJ, Shively JE, Hall PF, Gaddy-Kurten D, Richards JS 1988 Hormonal regulation, tissue distribution, and content of aromatase cytochrome P450 messenger ribonucleic acid and enzyme in rat ovarian follicles and corpora lutea: relationship to estradiol biosynthesis. Endocrinology 122:1426–1436[Abstract/Free Full Text]
  23. Fitzpatrick SL, Richards JS 1991 Regulation of cytochrome P450 aromatase messenger ribonucleic acid and activity by steroids and gonadotropins in rat granulosa cells. Endocrinology 129:1452–1462[Abstract/Free Full Text]
  24. Turner IM, Saunders PT, Shimasaki S, Hillier SG 1989 Regulation of inhibin subunit gene expression by FSH and estradiol in cultured rat granulosa cells. Endocrinology 125:2790–2792[Abstract/Free Full Text]
  25. Pei L, Dodson R, Schoderbek WE, Maurer RA, Mayo KE 1991 Regulation of the {alpha}-inhibin gene by cyclic 3',5'-monophosphate after transfection into granulosa cells. Mol Endocrinol 5:521–534[Abstract/Free Full Text]
  26. Hickey GJ, Krasnow JS, Beattie WG, Richards JAS 1990 Aromatase cytochrome P450 in rat ovarian granulosa cells before and after luteinization: adenosine 3',5'-monophosphate-dependent and independent regulation. Cloning and sequencing of rat aromatase cDNA and 5' genomic DNA. Mol Endocrinol 4:3–12[Abstract/Free Full Text]
  27. Fitzpatrick SL, Carlone DL, Robker RL, Richards JAS 1997 Expression of aromatase in the ovary: down-regulation of mRNA by the ovulatory luteinizing hormone surge. Steroids 62:197–206[CrossRef][Medline]
  28. Molina CA, Foulkes NS, Lalli E, Sassone-Corsi P 1993 Inductibility and negative autoregulation of CREM: an alternative promoter directs the expression of ICER, an early response repressor. Cell 75:875–886[CrossRef][Medline]
  29. Kameda T, Mizutani T, Minegishi T, Ibuki Y, Miyamoto K 1999 Regulation of cAMP responsive element binding modulator isoforms in cultured rat ovarian granulosa cells. Biochim Biophys Acta 1445:31–38[Medline]
  30. Mukherjee A, Urban J, Sassone-Corsi P, Mayo KE 1998 Gonadotropins regulate inducible cyclic adenosine 3',5'-monophosphate early repressor in the rat ovary: implications for inhibin {alpha} subunit gene expression. Mol Endocrinol 12:785–800[Abstract/Free Full Text]
  31. Morales V, Gonzalez-Robayna I, Hernandez I, Quintana J, Santana P, Ruiz de Galarreta CM, Fanjul LF 2003 The inducible isoform of CREM (ICER) is a repressor of CYP 19 rat ovarian promoter. J Endocrinol 179:417–425[Abstract]
  32. Locksley RM, Killeen N, Lenardo MJ 2001 The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104:487–501[CrossRef][Medline]
  33. Waterman WH, Sha’afi RI 1995 Effects of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-{alpha} on tyrosine phosphorylation and activation of mitogen-activated protein kinases in human neutrophils. Biochem J 307:39–45[Medline]
  34. Engelman JA, Berg AH, Lewis RY, Lisanti MP, Scherer PE 2000 Tumor necrosis factor {alpha}-mediated insulin resistance, but not dedifferentiation, is abrogated by MEK1/2 inhibitors in 3T3–L1 adipocytes. Mol Endocrinol 14:1557–1569[Abstract/Free Full Text]
  35. Cheng N, Chen J 2001 Tumor necrosis factor-{alpha} induction of endothelial ephrin A expression is mediated by a p38 MAPK- and SAPK/JNK-dependent but nuclear factor-{kappa}B-independent mechanism. J Biol Chem 276:13771–13777[Abstract/Free Full Text]
  36. Gonzalez GA, Montminy MR 1989 Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59:611–617
  37. Yamamoto KK, Gonzalez GA, Biggs WH, Montminy MR 1988 Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB. Nature 334:494–498[CrossRef][Medline]
  38. Finkbeiner S, Tavazoie SF, Maloratsky A, Jacobs KM, Harris KM, Greenberg ME 1997 CREB: a major mediator of neurotrophin responses. Neuron 19:1031–1047[CrossRef][Medline]
  39. Ginty DD, Bonni A, Greeberg ME 1994 Nerve growth factor activates a Ras-dependent protein kinase that stimulates c-fos transcription via phosphorylation of CREB. Cell 77:713–725[CrossRef][Medline]
  40. Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR 1995 PD-098059 is a specific inhibitor of the activation of mitogen-activated protein kinase in vitro and in vivo. J Biol Chem 270:27489–27494[Abstract/Free Full Text]
  41. Santana P, Llanes L, Hernandez I, Gallardo G, Quintana J, Gonzalez J, Estevez F, Ruiz de Galarreta CM, Fanjul LF 1995 Ceramide mediates tumor necrosis factor effects on P450-aromatase activity in cultured granulosa cells. Endocrinology 136:2345–2348[Abstract]
  42. Sasson R, Winder N, Kees S, Amsterdam A 2002 Induction of apoptosis in granulose cells by TNF {alpha} and its attenuation by glucocorticoids involve modulation of Bcl-2. Biochem Biophys Res Commun 31:51–59
  43. Cuenda A, Rouse J, Doza YN, Meier R, Cohen P, Gallagher TF, Young PR, Lee JC 1995 SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett 364:229–233[CrossRef][Medline]
  44. Gonzalez GA, Yamamoto KK, Fisher WH, Karr D, Menzel P, Biggs WH, Vale WW, Montminy MR 1989 A cluster of phosphorylation sites on the cyclic AMP-regulated nuclear factor CREB predicted by its sequence. Nature 23:749–752
  45. Cohen P 1997 The search for physiological substrates of MAP and SAP kinases in mammalian cells. Trends Cell Biol 7:253–261
  46. Pierrat B, daSilva J, Mary J-L, Tomás-Zuber M, Lesslaur W 1998 RSK-B, a novel ribosomal S6 kinase family member, is a CREB kinase under dominant control of p38{alpha} mitogen-activated protein kinase (p38{alpha}MAPK). J Biol Chem 273:29661–29671[Abstract/Free Full Text]
  47. Deak M, Clifton AD, Lucocq JM, Alessi DR 1998 Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38 and may mediate activation of CREB. EMBO J 17:4426–4441[CrossRef][Medline]
  48. Scheid MP, Foltz IN, Young PR, Schrader JW, Duronio V 1999 Ceramide and cyclic adenosine monophosphate (cAMP) induce cAMP response element binding protein phosphorylation via distinct signaling pathways while having opposite effects on myeloid cell survival. Blood 93:217–225[Abstract/Free Full Text]
  49. Kyriakis JM, Avruch J 1996 Sounding the alarm: protein kinase cascades activated by stress and inflammation. J Biol Chem 271:24313–24316[Free Full Text]
  50. Suzuki K, Hino M, Hato F, Tatsumi N, Kitagawa S 1999 Cytokine-specific activation of distinct mitogen-activated protein kinase subtype cascades in human neutrophils stimulated by granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-{alpha}. Blood 93:341–349[Abstract/Free Full Text]
  51. Oh HL, Seok JY, Kwon CH, Kang SK, Kim YK 2006 Role of MAPK in ceramide-induced cell death in primary astrocytes. Neurotoxicology 27:31–38[CrossRef][Medline]
  52. Tehle JH, Foulkes FS, Molina CA, Simonnaux V, Pevet P, Sassone-Corsi P 1993 Adrenergic signals direct rhythmic expression of transcriptional repressor CREM in the pineal gland. Nature 365:314–320[CrossRef][Medline]
  53. Abderrahmani A, Cheviet S, Ferdaoussi M, Coppola T, Waeber G, Regazzi R 2006 ICER induced by hyperglycemia represses the expression of genes essential for insulin exocytosis. EMBO J 25:977–986[CrossRef][Medline]
  54. Monaco L, Foulkes NS, Sassone-Corsi P 1995 Pituitary follicle-stimulating hormone (FSH) induces CREM gene expression in Sertoli cells: involvement in long term desensitization of the FSH receptor. Proc Natl Acad Sci USA 92:10673–10677[Abstract/Free Full Text]
  55. Lalli E, Sassone-Corsi P 1995 Thyroid-stimulating hormone (TSH)-directed induction of the CREM gene in the thyroid gland participates in the long term desensitization of the TSH receptor. Proc Natl Acad Sci USA 92:9633–9637[Abstract/Free Full Text]
  56. Huening M, Yehia G, Molina CA, Christakos S 2002 Evidence for a regulatory role of inducible cAMP early repressor in protein kinase A-mediated enhancement of vitamin D receptor expression and modulation of hormone action. Mol Endo 16:2052–2064[Abstract]
  57. Bodor J, Habener JH 1998 Role of transcriptional repressor ICER in cyclic AMP-mediated attenuation of cytokine expression in human thymocytes. J Biol Chem 273:9544–9551[Abstract/Free Full Text]
  58. Mead JR, Hughes TR, Irvine SA, Singh NN, Ramji DP 2003 Interferon-{gamma} stimulates the expression of the inducible cAMP early repressor in macrophages through the activation of casein kinase 2. J Biol Chem 278:17741–17751[Abstract/Free Full Text]
  59. Colgin MA, Smith RL, Wilcos CL 2001 Inducible cyclic AMP early repressor produces reactivation of latent herpes simplex virus type 1 in neurons in vitro. J Virol 75:2912–2920[Abstract/Free Full Text]
  60. Servillo G, Penna L, Foulkes NS, Magni MV, Della Fazia MA, Sassone-Corsi P 1997 Cyclic AMP signaling pathway and cellular proliferation: induction of CREM during liver regeneration. Oncogene 14:1601–1606[CrossRef][Medline]
  61. Razavi R, Ramos JC, Yehia G, Schlotter F, Molina CA 1998 ICER-II{gamma} is a tumor suppressor that mediates antiproliferative activity of cAMP. Oncogene 17:3015–3019[CrossRef][Medline]
  62. Ruchaud S, Seite P, Foulkes NS, Sassone-Corsi P, Lanotte M 1997 The transcriptional repressor ICER and cAMP-induced programmed cell death. Oncogene 15:827–836[CrossRef][Medline]
  63. Jaworski J, Mioduszewska B, Sanchez-Capelo A, Figiel I, Habas A, Gozdz A, Proszynski T, Hetman M, Mallet J, Kaczmarek L 2003 Inducible cAMP early repressor, an endogenous antagonist of cAMP responsive element-binding protein, evokes neuronal apoptosis in vitro. J Neurosci 23:4519–4526[Abstract/Free Full Text]
  64. Tomita H, Nazmy M, Kajimoto K, Yehia G, Molina CA, Sadoshima J 2003 Inducible cAMP early repressor (ICER) is a negative-feedback regulator of cardiac hypertrophy and an important mediator of cardiac myocyte apoptosis in response to ß-adrenergic receptor stimulation. Circ Res 93:12–22[Abstract/Free Full Text]
  65. Hughes Jr FM, Gorospe WC 1991 Biochemical identification of apoptosis (programmed cell death) in granulosa cells: evidence for a potential mechanism underlying follicular atresia. Endocrinology 129:2415–2422[Abstract/Free Full Text]
  66. Kaipia A, Hsueh AJW 1997 Regulation of ovarian follicle atresia. Annu Rev Physiol 59:349–363[CrossRef][Medline]
  67. Tilly JL, Kowalski KI, Scomberg DW, Hsueh AJW 1993 Apoptosis in ovarian atretic follicles is associated with selective decreases in messenger ribonucleic acid transcripts for gonadotropin receptors and cytochrome P450 aromatase. Endocrinology 131:1670–1676
  68. Billig H, Furuta I, Hsueh AJ 1993 Estrogens inhibit and androgens enhance ovarian granulosa cells apoptosis. Endocrinology 133:2204–2212[Abstract/Free Full Text]
  69. Ratts VS, Flaws GA, Kolp R, Sorenson CM, Tilly JL 1995 Ablation of bcl-2 gene expression decreases the number of oocytes and primordial follicles in the postnatal female mouse gonad. Endocrinology 136:3665–3668[Abstract]
  70. Hsu SY, Lai RJ, Finegold M, Hsueh AJW 1996 Targeted overexpression of Bcl-2 in ovaries of transgenic mice leads to decreased follicle apoptosis, enhanced folliculogenesis, and increased germ cell tumorigenesis. Endocrinology 137:4837–4843[Abstract]




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