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Endocrinology, doi:10.1210/en.2006-0194
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Endocrinology Vol. 147, No. 10 4931-4940
Copyright © 2006 by The Endocrine Society

Phosphatase Activation by Epidermal Growth Factor Family Ligands Regulates Extracellular Regulated Kinase Signaling in Undifferentiated Hen Granulosa Cells

Dori C. Woods and A. L. Johnson

Department of Biological Sciences, The University of Notre Dame, Notre Dame, Indiana 46556

Address all correspondence and requests for reprints to: A. L. Johnson, Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556. E-mail: johnson.128{at}nd.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous work has demonstrated that epidermal growth factor family ligands, signaling through the MAPK/ERK pathway, prevent hen granulosa cell differentiation, in vitro, even in the presence of factors that promote differentiation (e.g. TGFß and FSH). The working hypothesis is that a release from tonic inhibitory ERK signaling is prerequisite for the initiation of hen granulosa cell differentiation. Initial results demonstrate that the ERK signaling pathway is desensitized after treatment with TGF{alpha} or betacellulin. Thus, studies were conducted to evaluate a role for MAPK phosphatases in the termination of ERK signaling in undifferentiated granulosa cells. Subsequent to ligand-induced translocation of ERK to the nucleus, de novo transcription and translation of one or more protein tyrosine or dual-specificity phosphatases results in dephosphorylation and localization of inactivated ERK within the nucleus. RT-PCR amplification reveals expression of the MAPK-selective phosphatases (MKP), MKP-1, -3, and dual-specificity phosphatase 5, in granulosa cells. TGF{alpha} induces expression (within 3 h) of mRNA encoding the ERK-selective nuclear phosphatase, dual-specificity phosphatase 5, and subsequently (by 20 h) induces mRNA encoding the cytoplasmic phosphatase, MKP-3. Increased expression of phosphatases is associated with the intracellular localization and dephosphorylation of ERK and is inhibited by the selective ERK inhibitor, U0126. In turn, regulation of phosphatase activity occurs via the ubiquitin-proteasome degradation pathway because treatment of cells with the proteasome inhibitor, Z-LLF-CHO, markedly promotes ERK dephosphorylation. These data provide direct evidence for ERK-mediated negative feedback due to regulation of phosphatase activity in undifferentiated granulosa cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A FUNDAMENTAL CHARACTERISTIC of hen ovarian granulosa cells is their potential to undergo functional differentiation and initiate steroidogenesis immediately after follicle selection. This process of differentiation is tightly regulated by the interaction of critically timed cell-signaling events, which includes the activation and regulation of the MAPK signaling cascade, and specifically the ERK. Although it has been well established that the ERK cascade regulates proliferation, differentiation, and apoptosis in a variety of cell types across species (1), the downstream mechanisms and regulation of ERK signaling in undifferentiated granulosa cells remains poorly understood. For instance, ERK signaling has been reported to either induce or inhibit progesterone production in granulosa cells, with the effect likely dependent on stage of follicle development (2, 3, 4). The list of ligands proposed to directly or indirectly signal via the ERK pathway in granulosa cells continues to grow, both in number and diversity (e.g. gonadotropins, bone morphogenic protein-15, growth differentiation factor-9, epidermal growth factor-family ligands, glucocorticoids). Moreover, ligand availability (endocrine, paracrine, and/or autocrine), receptor subtype(s) (ErbB1, -2, -3, -4) expressed, upstream signaling molecules (e.g. SOS, Grb2, Ras), duration and intensity of signal, cross-talk with additional signaling pathways, and/or compartmentalization of signaling components can ultimately determine the cellular response (5, 6, 7, 8, 9, 10).

Of the factors known to initiate ERK signaling in granulosa cells, members of the epidermal growth factor (EGF) family of ligands (e.g. EGF, TGF{alpha}, betacellulin) have been linked to the inhibition of FSH-induced actions in granulosa cells from various species. For example, treatment with EGF in nonluteinized bovine granulosa cells leads to attenuation of FSH-induced estradiol production (11), whereas ovarian arterial infusion with TGF{alpha} blocks FSH-induced progesterone production in the ewe (12). Additionally, treatment with TGF{alpha} has been demonstrated to oppose the actions of TGFß1 on augmentation of FSH receptor (FSHR) expression in the hen and rat (13, 14). Inhibition of phosphorylated mothers against decapentaplegic (Smad) signaling is attributed to the kinase activity of phosphorylated ERK, which has previously been shown to promote the phosphorylation of human Smad2 and Smad3 within a protein domain distinct from the TGFß-regulated phosphorylation sites and prevent the translocation of these regulatory Smads to the nucleus (15, 16). Furthermore, in undifferentiated hen granulosa cells collected from a cohort of 6- to 8-mm-diameter (before selection or prehierarchal) follicles, TGF{alpha}-induced ERK signaling prevents FSH-mediated induction of FSHR and LH receptor (LHR) mRNA and steroid acute regulatory (StAR) mRNA and protein expression, plus inhibits P450 side-chain cleavage mRNA expression and progesterone production (2, 8).

On the other hand, evidence is accruing that subsequent inactivation of the ERK signaling pathway may be prerequisite to initiate final differentiation of granulosa cells. For instance, inhibition of ERK signaling with pharmacologic inhibitors leads to an increase in FSH- or forskolin-induced StAR and progesterone production in both an FSH-responsive immortalized rat granulosa cell line and a human granulosa tumor cell line (4, 17). In addition, inhibition of ERK signaling using the pharmacologic MAPK kinase (MEK) inhibitor, U0126, initiates differentiation in cultured granulosa cells from hen prehierarchal follicles and potentiates FSH-induced LHR and StAR expression plus gonadotropin-induced progesterone production (2, 13). To date, however, the physiological regulation (particularly termination) of ERK signaling has not been extensively studied in nontransformed ovarian granulosa cells from any species.

Although the ERK signaling pathway has been demonstrated to inhibit the differentiation-inducing effects of TGFß1 within hen granulosa cells, it was recently established that ERK signaling is also important for TGF{alpha}- and betacellulin (BTC)-mediated induction of TGFß1 mRNA expression (18). Whereas this finding appears paradoxical, it was hypothesized that a temporal regulation of ERK signaling allows for an acute stimulatory effect upon TGF{alpha}-mediated TGFß1 gene transcription, followed by the subsequent termination of ERK signaling that enables newly translated TGFß1 to enhance FSH induction of FSHR, and subsequently LHR.

Herein we provide evidence for the rapid dephosphorylation of ERK and termination of ERK signaling in undifferentiated avian granulosa cells due to tyrosine or dual-specificity phosphatases (DUSPs; can target either phosphorylated tyrosine or serine/threonine residues) induced by the ERK signaling cascade. These results implicate a feedback mechanism by which ERK is phosphorylated in response to EGF family ligands, is translocated to the nucleus and then is rapidly dephosphorylated to an inactivated state after ERK-mediated activation of one or more tyrosine or dual-specificity phosphatases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and reagents
Single-comb white Leghorn hens (Creighton Bros., Warsaw, IN) 25–35 wk of age, laying regular sequences of six or more eggs, were used in all studies described. Hens were individually housed in laying batteries with free access to feed (Purina Layena mash; Purina Mills, St. Louis, MO) and water, under a controlled photoperiod of 15 h light, 9 h dark (lights on at midnight). Approximate time of oviposition was monitored daily. Each hen was killed by cervical dislocation 16–18 h before midsequence ovulation at which time ovarian follicles were removed and placed immediately in sterile 1% saline solution. All procedures described herein were reviewed and approved by the University of Notre Dame Institutional Animal Care and Use Committee and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.

Recombinant human TGF{alpha} was from PeproTech (Rocky Hill, NJ), whereas BTC was purchased from R&D Systems (Minneapolis, MN). U0126 (a selective MEK inhibitor) (2) was purchased from BioMol (Plymouth Meeting, NJ), whereas the selective proteasome inhibitors, (benzyloxycarbonyl)-leu-leu-phenylalaninal (Z-LLF-CHO) and lactacystin, were from Calbiochem (La Jolla, CA). Okadaic acid and sodium orthovanadate were from Sigma Chemical Co. (St. Louis, MO).

Granulosa cell cultures
Prehierarchal follicles were removed from the ovary and grouped by diameter. Layers of undifferentiated granulosa layers from 6- to 8-mm follicles were combined and cells dispersed as previously described (8, 13). Where appropriate, an aliquot of dispersed cells was immediately frozen at –70 C (T0 control). Cells were incubated for up to 240 min in 12 x 75 mm polypropylene culture tubes (Fisher Scientific, Pittsburgh, PA) in a shaking water bath or cultured for 8 or 20 h in 12-well polystyrene culture plates (Becton Dickinson Labware, Franklin Lakes, NJ). Incubations and cultures were conducted at 40 C in an atmosphere of 5% CO2-95% air at a density of approximately 5.0 x 105/well in DMEM medium containing 2.5% fetal bovine serum, 0.1 mM nonessential amino acids, and 1% antibiotic-antimycotic reagent (Invitrogen, Carlsbad, CA). Where applicable, cells were pretreated with inhibitors (e.g. U0126, Z-LLF-CHO, lactacystin) for 60–180 min before the addition of TGF{alpha} or BTC. Concentrations of TGF{alpha}, BTC, cycloheximide, actinomycin D, U0126, and okadaic acid used have been previously established (8, 18, 19, 20, 21), whereas the doses of sodium orthovanadate, Z-LLF-CHO, and lactacystin were empirically determined.

PCR amplification of phosphatase cDNA from hen ovarian tissues
In an effort to establish a role for phosphatase activity within prehierarchal follicles, MAPK phosphatases (MKP-1, -2, -3, and DUSP5) were amplified from reversed-transcribed RNA (reverse transcription system, Promega, Madison, WI) collected from ovarian stromal, granulosa, and theca tissues. Primer pairs specific for the Gallus phosphatases are as follows: MKP-1 (also called DUSP1; GenBank accession no. AF026522) sense, 5'-GACTGCCGCTCCTTCTTCTCC-3', antisense, 5'-CGCTTCGTAACCTCCCTTGAG-3' (300 bp); MKP-2 (DUSP4; AF167296) sense, 5'-CTGCGGGAGATGGAGGGC-3', antisense, 5'-CGTCGTCAGGGTCTTGGTTTTAG-3' (432 bp); MKP-3 (DUSP6; AY278202) sense, 5'-TAGAAAGGGTCACACAGCACGG-3', antisense, 5'-CTCCTGGCAAACAAATACTCCAAG-3' (323 bp); DUSP5 (XM_421754) sense, 5'-ATTTCCCTGGAGAGGAGCGATG-3', antisense, 5'-AGAGGCGGCTTCTGTTTTGC-3' (564 bp). Amplification conditions were specific for each primer pair but included an initial denaturing for 3 min at 94 C followed by 45 sec denaturing at 94 C; 30 sec annealing at 51–62 C, depending on primer pair; and 90 sec extension at 72 C for 35 cycles using Taq DNA polymerase (Invitrogen). All PCR products were subsequently subcloned using the TOPO TA cloning kit for sequencing (Invitrogen) and sequenced for verification of nucleic acid identity.

cDNA probes and Northern blot analysis of MKP-3 and DUSP5 mRNA
The MKP-3- and DUSP5-specific cDNA probes were amplified, subcloned, and sequenced as described above. The 323-bp MKP-3 and 564-p DUSP5 probes were prepared from the subcloned coding region after an EcoRI digestion. Probes were prepared by random-prime labeling with [{alpha}32P]dCTP (3000 Ci/mmol; Amersham, Arlington Heights, IL) using the Megaprime DNA labeling system (Amersham). Northern blot images were visualized on phosphor imaging screens using the Storm 840 PhosphorImager and analyzed using the ImageQuant data reduction system (Molecular Dynamics, Inc., Sunnyvale, CA). All MKP-3 and DUSP5 mRNA data were standardized to 18S rRNA as previously described (19).

Western blot analysis of phosphorylated and total ERK2 protein
After granulosa cell incubation or culture, total cellular protein was prepared as previously described (2). A monoclonal antibody for the activated form of ERK, specifically recognizing the phosphorylated threonine and tyrosine residues (clone 12D4; Upstate Biotechnology, Inc., Lake Placid, NY), was used at a dilution of 1:3000, whereas a goat antimouse secondary antibody conjugated to horseradish peroxidase (Pierce, Rockford, IL) was diluted 1:10,000. Blots were subsequently incubated with ECL Western blotting detection reagent (Amersham) for 1 min, wrapped in film wrap, and exposed to x-ray film for 3–5 min. Blots were further analyzed using a polyclonal antibody for total ERK2 or ERK1/2 protein (Santa Cruz Biotechnology, Santa Cruz, CA) to standardize for protein loading and to monitor total ERK expression. The extent of antibody binding was quantitated by densitometry.

In vitro phosphatase assay
An in vitro phosphatase assay was modified from Kassel et al. (22) to monitor phosphatase activity in lysates from undifferentiated granulosa cells treated for 240 min in the absence or presence of TGF{alpha} (25 ng/ml). Briefly, nuclear-enriched cell lysates were prepared by harvesting cells into phosphatase assay buffer [10 mM EDTA, 10 mM EGTA, 50 mM HEPES (pH 7.6), 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 10 mg/ml aprotinin]. Lysates were subjected to four cycles of freezing in liquid N2 followed by thawing at 4 C and then further disrupted by repeated aspiration through a 25-gauge needle. Disrupted lysates were cleared of cellular debris by centrifugation at 13,000 x g for 15 min at 4 C. To assess the extent of phosphatase activity, 100 µg of total protein from cell lysates in which ERK phosphorylation was maximally induced (ERK activated; prepared from granulosa cells treated with TGF{alpha} for 20 min) was incubated for 15 min at 30 C with 100 µg total protein collected from lysates of either control granulosa cells incubated for 240 min or lysates from granulosa cells treated with TGF{alpha} for 240 min. In preliminary studies phosphatase activity was determined to be linear from 0 to 15 min of incubation (r = 0.90; P < 0.01). In a separate experiment, 100 µg of total protein from ERK activated cell lysates was incubated for 15 min at 30 C with 100 µg total protein from lysates collected from either untreated control granulosa cells or lysates from cells treated with Z-LLF-CHO for 240 min. Phosphatase reactions were terminated by placing the mixtures on ice and adding Laemmli sample buffer. The extent of phosphorylated ERK after incubations was assessed by Western blot analysis as described above.

Confocal microscopy
Dispersed granulosa cells from the second and third largest preovulatory follicles adhered within 2 h to coverslips coated with poly-L-lysine. Cells were subsequently treated with 25 ng/ml TGF{alpha} for 20 min or 4 h, with Draq5 (5 µM; Alexis Biochemicals, Carlsbad, CA) added during the last 5 min of incubation for visualization of the nucleus. Cells were rinsed with PBS and fixed with 2% paraformaldehyde for 45 min. Cells were rinsed again, incubated in blocking buffer (PBS containing 10% goat serum) and then incubated with the appropriate primary antibody for 2 h at room temperature. The omission of primary antibody at this stage served as a control for primary antibody specificity. Cells were washed before incubation with fluorescein isothiocyanate-conjugated secondary antibodies (Invitrogen). Coverslips were washed and mounted onto slides using antifade mounting medium (Invitrogen), and cells were visualized using a Bio-Rad MRC scanning confocal system.

Data analysis
All experiments were independently replicated a minimum of three times. Standardized values were expressed as a fold difference (mean ± SEM) vs. incubated/cultured controls or freshly collected (T0; for Northern blots) unless otherwise stated. Data were analyzed using the Student’s t test or a one-way ANOVA without including data from the incubated/cultured control group (arbitrarily set to 1.0) combined with the Fisher’s protected least significant difference test for significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ligand-induced ERK desensitization
After a single TGF{alpha} or BTC treatment, levels of phosphorylated ERK were maximal at 20 min (1.50 ± 0.15- and 1.66 ± 0.07-fold vs. control, respectively; P < 0.05 by paired t test) and then declined up to 240 min after treatment (Fig. 1Go, A and B; stippled bars). Granulosa cells were initially treated with either ligand for 20, 60, 180, or 240 min and then challenged for an additional 20 min with the respective ligand. After this 20-min challenge, there was no secondary increase in phosphorylated ERK observed in cells pretreated for 180 and 240 min (Fig. 1Go, A and B; cross-hatched bars). This lack of responsiveness was observed whether or not the culture medium was replaced before the second challenge. By comparison, there was an observable return to maximal levels of phosphorylated ERK in cells initially treated with TGF{alpha} for 60 min and then challenged with TGF{alpha} for an additional 20 min, compared with a single treatment with TGF{alpha} for 60 min (1.67 ± 0.05-fold vs. 1.17 ± 0.08-fold, respectively, P < 0.05). Levels of total ERK protein were unchanged in response to treatments at any of the time points examined. Collectively, these results confirm the phosphorylation of ERK after a single exposure to an EGF family ligand and provide evidence for ligand-induced desensitization of ERK signaling in granulosa cells within 180 min of an initial treatment. This apparent desensitization was maintained for up to 20 h because a 20-min challenge with either TGF{alpha} or BTC after an 8- or 20-h preculture with the respective ligand failed to increase the level of ERK phosphorylation, compared with cells precultured in the absence of ligand (Fig. 2Go, A and B). Again, levels of ERK protein were unchanged after treatment.


Figure 1
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  		FIG. 1. Western blot analysis of phosphorylated ERK (ERK-p) in undifferentiated granulosa cells collected from 6- to 8-mm follicles. Granulosa cells were treated once with TGF{alpha} (25 ng/ml; panel A) or BTC (2 ng/ml; panel B) for 20, 60, 180, or 240 min (stippled bars) or treated with TGF{alpha} or BTC for 20, 60, 180, or 240 min and then challenged with the same dose of TGF{alpha} or BTC for an additional 20 min (cross-hatched bars). ERK-p data are standardized to total ERK protein and expressed as fold difference vs. the control (Con; incubated for 240 min without growth factor) cells. Values represent means ± SEM of three replicate experiments. A, B, C, a, and b, P < 0.01.

 

Figure 2
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  		FIG. 2. Granulosa cells were precultured for 8 or 20 h without (–) or with (+) TGF{alpha} (25 ng/ml; panel A) or BTC (2 ng/ml; panel B) and then challenged without (–) or with (+) TGF{alpha} or BTC for an additional 20 min. Phosphorylated ERK (ERK-p) data are standardized to total ERK protein and expressed as fold difference vs. untreated cultured (Con) cells. Values represent means ± SEM of three replicate experiments. A and B, P < 0.03.

 
Evidence that ERK dephosphorylation is dependent on de novo synthesis of phosphatase(s)
After a 1-h pretreatment without (Fig. 3AGo, stippled bars) or with (Fig. 3Go, cross-hatched bars) the mRNA transcription inhibitor, actinomycin D (ActD; 1 µg/ml; Fig. 3AGo) or the protein translation inhibitor, cycloheximide (CHX; 30 µM; Fig. 3BGo), TGF{alpha} stimulated ERK phosphorylation within 20 min to levels comparable with cells pretreated in the absence of inhibitor. By contrast, pretreatment with either inhibitor prevented the transient decline in ERK phosphorylation for up to 240 min after TGF{alpha} treatment. Similarly, pretreatment with the tyrosine phosphatase inhibitor, sodium orthovanadate (1 mM NaV4; Fig. 4AGo, cross-hatched bars) or the serine/threonine phosphatase inhibitor, okadaic acid (50 nM OA; Fig. 4BGo, cross-hatched bars) failed to significantly alter TGF{alpha}-induced ERK phosphorylation after a 20-min challenge, compared with cells pretreated without inhibitor. However, NaV4, but not OA, prevented the transient dephosphorylation of ERK for up to 240 min after TGF{alpha} treatment. Taken together, the results suggest that the transient dephosphorylation of ERK and termination of the ERK signaling pathway within 180 min is mediated, at least in part, by de novo transcription and translation of a tyrosine or dual-specificity phosphatase.


Figure 3
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  		FIG. 3. ActD (panel A) and CHX (panel B) prevent the transient decrease of phosphorylated ERK (ERK-p) after treatment of undifferentiated granulosa cells with TGF{alpha}. Cells were incubated in the absence (–) or presence (+) of the mRNA transcription inhibitor, ActD (1 µg/ml), or the protein translation inhibitor, CHX (30 µM), and treated with TGF{alpha} (25 ng/ml) for 20, 60, 180, or 240 min. Control (Con) cells were treated without (stippled bar) or with (cross-hatched bar) inhibitor for 1 h and then incubated without TGF{alpha} for 240 min. Data are standardized to ERK2 protein and expressed as fold difference vs. the Con (– inhibitor). Values represent means ± SEM from four (ActD) or three (CHX) replicate experiments. ns, P > 0.05 for Con cells incubated without (–) vs. with (+) inhibitor (by Student’s t test); A–C, P < 0.05; a–c, P < 0.01.

 

Figure 4
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  		FIG. 4. Sodium orthovanadate (NaV4; panel A), but not OA (panel B), prevents the transient decrease of phosphorylated ERK (ERK-p) after treatment of undifferentiated granulosa cells with TGF{alpha}. Granulosa cells were incubated in the absence (–) or presence (+) of the tyrosine phosphatase inhibitor, NaV4 (1 mM; A) or the serine/threonine phosphatase inhibitor, OA (50 nm; B), for 1 h and then treated with TGF{alpha} for 20, 60, 180, or 240 min. Control (Con) cells were pretreated without (stippled bar) or with (cross-hatched bar) inhibitor for 1 h and then incubated without TGF{alpha}for 240 min. Data are standardized to total ERK2, and expressed as fold difference vs. the Con (– inhibitor). Values represent means ± SEM from three replicate experiments. ns, P > 0.05 for Con cells incubated without (–) vs. with (+) inhibitor (by Student’s t test); A–C, P < 0.05.

 
TGF{alpha}-induced phosphatase activity in undifferentiated granulosa cells
To investigate whether phosphatase activity is induced by TGF{alpha} after a 240-min treatment, enzyme activity was analyzed, in vitro, using lysates prepared from granulosa cells treated for 20 min with TGF{alpha} (a source of phosphorylated ERK substrate), combined with lysates from untreated control cells or cells treated with TGF{alpha} for 240 min. After a 15-min incubation of combined lysates, Western blot analysis revealed that the levels of phosphorylated ERK were significantly reduced by lysates from TGF{alpha} treatment for 240 min, compared with those from untreated controls (by 57.0 ± 5.0%, P < 0.03 by paired t test; Fig. 5Go). These results provide evidence for induced phosphatase activity 240 min after TGF{alpha} treatment.


Figure 5
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  		FIG. 5. Treatment with TGF{alpha} for 240 min induces phosphatase activity in undifferentiated granulosa cells. Western blot analysis of phosphorylated ERK (ERK-p) after coincubation of lysates collected from cells pretreated for 20 min with TGF{alpha} (ERK activated; a source of phosphorylated ERK substrate) with lysates from incubated control cells (open bar) or cells treated with TGF{alpha} for 240 min (stippled bar). Total ERK2 is presented to indicate even loading of protein. Densitometry data are standardized to total ERK2 and represent means ± SEM of three replicate experiments. *, P < 0.03 by Student’s t test.

 
Cellular localization of ERK
Immunofluorescent staining of phosphorylated ERK (Fig. 6Go, A–C) and ERK2 (Fig. 6Go, D–F) in granulosa cells reveals that ERK is phosphorylated and translocated to the nucleus by 20 min after TGF{alpha} treatment (arrows) and additionally that ERK is subsequently dephosphorylated by 240 min but remains largely localized within the nucleus. This finding supports the possibility for the expression of rapidly up-regulated nuclear MAPK phosphatase(s) capable of both ERK dephosphorylation and sequestration of inactivated ERK.


Figure 6
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  		FIG. 6. Cellular localization of phosphorylated ERK (ERK-p) after treatment of granulosa cells without (0 min) or with 25 ng/ml TGF{alpha} for 20 or 240 min. A–F, Granulosa cells were processed for confocal microscopy and immunofluorescently labeled for endogenous ERK-p using fluorescein isothiocyanate-conjugated secondary antibodies (top row; A–C) and ERK2 (bottom row; D–F). Images were taken across a single confocal plane at the cell body (x/y-axis). A–C, After treatment with TGF{alpha}, endogenous ERK is phosphorylated and translocated to the nucleus of all cells by 20 min (B, arrows) and is subsequently dephosphorylated by 240 min (C). D–F, ERK2 is present throughout the cytoplasm before TGF{alpha} treatment (D), is translocated to the nucleus after a 20-min treatment with TGF{alpha} (E, arrows), and remains localized within the nucleus after 240 min (F). Nuclear localization was verified by counterstaining with Draq5 (not shown). Bar, 10 µm.

 
Expression of phosphatase mRNA in undifferentiated granulosa cells
PCR amplification of cDNA from reverse-transcribed RNA isolated from hen ovarian tissues demonstrates the presence of MKP-1 and DUSP5 in stromal tissue but more importantly in granulosa and theca tissues from 6- to 8-mm follicles, whereas MKP-2 is detectable only in the stroma (Fig. 7AGo). Northern blot analysis reveals that DUSP5 (Fig. 7BGo), but not MKP-1 (data not shown), mRNA expression is significantly up-regulated after a 180-min incubation with TGF{alpha} (2.3-fold control, P < 0.01). There was no increase in levels of DUSP5 by 1 h of treatment, and after 20 h of treatment, DUSP5 mRNA had decreased to levels undetectable by Northern blot analysis (data not shown). Pretreatment with the MEK inhibitor, U0126, completely blocked TGF{alpha}-induced up-regulation of DUSP5, indicating that the TGF{alpha}-mediated effect is ERK specific.


Figure 7
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  		FIG. 7. Expression of dual-specificity phosphatases in granulosa and theca layers from 6- to 8-mm follicles plus ovarian stromal tissue. Panel A, RT-PCR shows that MKP-2 transcript (432 bp product) is detected only in ovarian stromal (Str) tissue, whereas MKP-1 (300 bp product) and DUSP5 (564 bp product) transcripts are expressed in Str, theca (Th), and granulosa (Gr) tissues. –, Negative (no template) control. Data are representative of three replicate amplifications using different reverse-transcribed templates. Panel B, Northern blot analysis of DUSP5 mRNA in granulosa cells after a 3-h incubation in the absence (Con) or presence of 25 ng/ml TGF{alpha} without or with a 1-h pretreatment with the selective MAPK inhibitor, U0126 (10 µM). Data are expressed as fold difference vs. freshly dispersed and frozen (T0) cells (not shown) and standardized to 18s rRNA. Values represent means ± SEM from three replicate experiments. A and B, P < 0.01.

 
Similar to MKP-1 and DUSP5, MKP-3 is expressed in granulosa and theca tissues from 6- to 8-mm follicles as well as ovarian stromal tissue (Fig. 8AGo). Moreover, MKP-3 mRNA expression is regulated by TGF{alpha} through an ERK-dependent mechanism (2.7-fold increase vs. control cultured cells, P < 0.05; Fig. 8BGo). However, in contrast to DUSP5, increased expression of MKP-3 mRNA is detected after a 20-h treatment but not after 180 min treatment (data not shown) with TGF{alpha}.


Figure 8
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  		FIG. 8. Expression and regulation of MKP-3. Panel A, RT-PCR amplification confirms the expression of MKP-3 (323 bp product) in stromal tissue (Str) plus granulosa (Gr) and theca (Th) tissues from 6- to 8-mm follicles. –, Negative (no template) control. Data are representative of three replicate amplifications using different reverse-transcribed templates. Panel B, Northern blot analysis of MKP-3 mRNA in granulosa cells after a 20-h incubation in the absence (Con) or presence of 25 ng/ml TGF{alpha} without or with a 1-h pretreatment with U0126 (10 µM). Data are expressed as fold difference vs. freshly dispersed and frozen (T0) cells (not shown) and standardized to 18s rRNA. Values represent means ± SEM of three replicate experiments. A and B, P < 0.05. Panel C, Confocal images of ERK2 indicate that TGF{alpha} (25 ng/ml) promotes the exit of ERK2 protein from the nucleus after 20 h of culture. Bar, 10 µm.

 
Inhibition of proteasome activity enhances phosphatase activity
To investigate whether proteasome activity regulates ERK signaling in primary cultured granulosa cells, undifferentiated granulosa cells were incubated with Z-LLF-CHO, a selective and reversible proteasome inhibitor (2, 10, and 20 µM) for 180 min to evaluate effects on basal levels of phosphorylated ERK (Fig. 9AGo). Whereas the 10- and 20-µM doses of Z-LLF-CHO caused an apparent complete dephosphorylation of ERK, there was no change in the levels of total ERK2 protein. A similar experiment conducted with 2.5, 10, or 50 µM of the selective but irreversible proteasome inhibitor, lactacystin, demonstrated that 10- and 50-µM doses also reduced basal phosphorylated ERK without altering levels of ERK2 (data not shown). Furthermore, pretreatment with Z-LLF-CHO (10 µM) for 180 min prevented any accumulation of phosphorylated ERK after a 20-min challenge with TGF{alpha} (Fig. 9BGo).


Figure 9
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  		FIG. 9. Inhibition of proteasome activity promotes phosphatase activity in undifferentiated granulosa cells. A, Granulosa cells were incubated without (0) or with 2, 10, or 20 µM of the reversible proteasome inhibitor, Z-LLF-CHO, for 240 min. Data are representative of three replicate blots. B, Granulosa cells were incubated without or with Z-LLF-CHO (10 µM) for 180 min before a 20-min treatment without (Con) or with 25 ng/ml TGF{alpha}. Data are representative of three replicate blots. C, Western blot and densitometric analysis of phosphorylated ERK (ERK-p) after the assay of phosphatase activity, in vitro. The phosphatase assay was performed by coincubation of lysates from untreated control cells incubated for 240 min or cells treated for 240 min with 10 µM Z-LLF-CHO with lysates from cells treated with TGF{alpha} for 20 min (ERK activated; a source of phosphorylated ERK substrate). Densitometry data are standardized to total ERK2 and represent means ± SEM from three replicate experiments. *, P < 0.02 vs. untreated control.

 
Finally, to investigate whether proteasome inhibitor-mediated ERK dephosphorylation is due to an accumulation of MAPK phosphatase(s), phosphatase activity was monitored, in vitro. Lysates from untreated control cells or cells treated with Z-LLF-CHO for 240 min were coincubated with lysates prepared from granulosa cells in which ERK phosphorylation had been induced by TGF{alpha} (ERK activated; a source of phosphorylated ERK substrate). Levels of phosphorylated ERK were dramatically reduced after incubation with lysates from Z-LLF-CHO cells treated for 240 min (by 91.5 ± 6.0%), compared with control cell lysates (P < 0.02 by paired t test; Fig. 9CGo). Collectively, these results indicate that the ubiquitin-proteasome degradation pathway represents a mechanism capable of rapidly regulating phosphatase activity, and by implication, ERK signaling.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results from the present studies combined with previously published data from hen granulosa cells (8, 13, 18) provide supportive evidence that activation and subsequent termination of ERK signaling precedes the initiation of granulosa cell differentiation in hen prehierarchal follicles and demonstrate that the termination of MAPK/ERK signaling is mediated, at least in part, by rapidly regulated phosphatase activity. An updated working model for these proposed events that ultimately lead to granulosa cell differentiation is presented in Fig. 10Go.


Figure 10
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  		FIG. 10. Proposed model for the role of phosphatase(s) in the termination of ERK signaling, an event prerequisite for the initiation of differentiation in hen granulosa cells. Acute effects are indicated by solid lines, whereas latent (MKP-3) or consequent (TGFß signaling) effects are indicated by dotted lines and light shading. EGF family ligands, produced in a paracrine (e.g. TGF{alpha}) or autocrine (e.g. BTC) fashion, bind with ErbB1 and/or ErbB4 receptors (18 ) to activate ERK signaling. Active ERK signaling has previously been shown to block Smad signaling (–) and TGFß-induced differentiation (8 ). Phosphorylated ERK (ERK-p) is rapidly translocated (within 20 min) to the nucleus (Fig. 6BGo) in which it initiates (+) the transcription of mRNA encoding TGFß1 (18 ) as well as the MAPK-selective phosphatases, DUSP5 and MKP-3 (Figs. 7BGo and 8BGo). Rapidly synthesized DUSP5 (perhaps combined with constitutive low levels of MKP-1 and MKP-3) dephosphorylates nuclear-localized ERK-p within 180 min (Fig. 6CGo). Induced MKP-3 occurs somewhat later (within 20 h) and is proposed to mediate the exit of ERK from the nucleus (Fig. 8CGo) plus affect a more prolonged desensitization of ERK signaling (Fig. 2Go). In turn, phosphatase activity is regulated by proteasome degradation (not depicted). Newly transcribed and translated TGFß1 can promote activation of TGFß receptor complexes (dashed lines). After the reduction of inhibitory ERK signaling, active signaling via regulatory (r) and common (co-) Smads promotes enhanced expression of FSHR to initiate the process of granulosa cell differentiation (13 ).

 
The present results confirm that after ligand-induced ErbB activation, ERK is transiently phosphorylated (Fig. 1Go) and rapidly translocated from the cytoplasm to the nucleus (Fig. 6BGo). Maximal phosphorylation of ERK occurs by 20 min because a challenge with TGF{alpha} or BTC at 20 or 60 min fails to increase levels of phosphorylated ERK above the levels detected at 20 min after a single treatment. By comparison, 180 min after an initial treatment with either ligand, levels of phosphorylated ERK have significantly declined, yet further ligand-induced accumulation of phosphorylated ERK is not observed. The absence of induced ERK phosphorylation after TGF{alpha} or BTC challenge provides evidence for MAPK desensitization and termination of ERK signaling. This result is associated with the localization of ERK to the nucleus (Fig. 6FGo). The finding that a challenge with TGF{alpha} after 60 min effectively induces a secondary increase in phosphorylated ERK (Fig. 1AGo) argues against receptor internalization as a primary cause of desensitization. Nevertheless, at this time it is not possible to eliminate receptor internalization as a secondary cause of MAPK/ERK desensitization.

Whereas prospective tyrosine or dual-specificity phosphatases responsible for TGF{alpha}-induced ERK inactivation have not previously been described in nontransformed granulosa cells, studies in several mammalian cell lines have demonstrated that the nuclear phosphatases, MKP-1, MKP-2, and DUSP5, and the predominately cytoplasmic phosphatase, MKP-3, can inactivate ERK signaling by either dephosphorylation or a combination of dephosphorylation and nuclear sequestration (23, 24, 25). In the present studies, ERK dephosphorylation and localization to the nucleus (Fig. 6Go, C and F) occurs by the time that TGF{alpha}-induced DUSP5 mRNA is elevated (Fig. 7BGo). In general, dual-specificity phosphatases inactivate ERK by the removal of phosphate groups from tyrosine and/or threonine residues (26). Whereas some nuclear dual-specific phosphatases (e.g. MKP-1 and -2) can dephosphorylate multiple MAPK substrates, including p38 and c-Jun N-terminal kinase (24), DUSP5 has selective affinity for ERK (25). Of note is the finding that MKP-2 mRNA expression is not detected in hen granulosa cells (Fig. 7AGo), nor is the protein detected by Western blot analysis in a human granulosa tumor cell line (27). Whereas MKP-1 has been shown to effectively dephosphorylate ERK in the nucleus (28), recent data indicate that the interaction between DUSP5 and ERK is 5- to 6-fold greater than that of MKP-1 and that overexpression of DUSP5, but not MKP-1, leads to nuclear retention of ERK (25). Using a catalytically inactive form of DUSP5 that contains the nuclear localization signal (NLS), it was determined that DUSP5 specifically anchors ERK within the nucleus and that this anchoring is dependent on both a kinase interaction motif (KIM) and the NLS (25). Both the conserved KIM and NLS domains are conserved within the Gallus DUSP5 coding sequence. Accordingly, the data presented herein suggest a significant role for DUSP5 in the inactivation and localization of ERK within the nucleus of hen granulosa cells. Furthermore, whereas activated MEK may be capable of entering the nucleus to phosphorylate ERK (25), nuclear-localized phosphatases can prevent accumulation of phosphorylated ERK.

Whereas the acutely regulated activity of nuclear phosphatase(s) can account for transient dephosphorylation and accumulation of ERK, granulosa cells remain unresponsive to further treatment with TGF{alpha} or BTC for up to 20 h after treatment (Fig. 2Go). By this time ERK protein is no longer exclusively localized within nucleus and ERK is once again dispersed throughout the cytoplasm (Fig. 8CGo). Interestingly, the predominant cytosolic localization of ERK 20 h after TGF{alpha} treatment occurs at a time that levels of MKP-3 mRNA are increased (Fig. 8BGo). Similar to DUSP5, MKP-3 specifically interacts with ERK through a conserved KIM located within the noncatalytic N terminus. In contrast to DUSP5, however, docking of phosphorylated ERK to the KIM of MKP-3 is required for catalytic activation of MKP-3 and dephosphorylation of ERK (23, 26, 29, 30, 31). Recent findings suggest that MKP-3 is not localized exclusively to the cytoplasm. Instead, the presence of an N terminal nuclear exclusion signal enables MKP-3 to be shuttled from the nucleus to the cytoplasm and provides the potential for MKP-3 to transport inactivated nuclear ERK back to the cytoplasm (23). This nuclear exclusion signal domain is conserved within the Gallus MKP-3 coding sequence. The biological implications of this include the maintenance of cytoplasmic ERK in a bound, inactivated state, which can result in prolonged desensitization of the ERK signaling pathway.

In a variety of transformed cells, including a human granulosa tumor cell line, it has been reported that inhibition of proteasome function leads to accumulation of MAPK phosphatase proteins and a decrease in phosphorylated ERK (e.g. Refs. 27 , 32). Accordingly, in concert with the regulation of de novo phosphatase expression, it was predicted that the presiding level of phosphatase activity within granulosa cells is also regulated by the rate of phosphatase degradation. Results from the present studies are consistent with a role for the ubiquitin-proteasome degradation pathway as a regulator of phosphatase activity because inhibition of proteasome activity eliminates basal, and prevents the accumulation of TGF{alpha}-induced, ERK phosphorylation (Fig. 9Go, A and B). These results are attributed to accrual of phosphatase(s) (as opposed to a loss of MEK activity) because increased phosphatase activity is detected in lysates of cells treated for 240 min with the proteasome inhibitor, Z-LLF-CHO (Fig. 9CGo). Similar results have been reported using a human granulosa cell tumor line, in which inhibition of proteasome activity with Z-LLF-CHO led to accumulation of MKP-1 and a reduction in levels of phosphorylated ERK (17). Moreover, studies demonstrated that ERK activation can negatively feed back on its own signaling by directly enhancing phosphatase stability. For instance, in the hamster fibroblast cell line, CCL39, ERK-induced phosphorylation of MKP-1 at serine residues 359 and 364 results in a reduction of proteasome-mediated degradation without modification of its phosphatase activity (33).

Finally, whereas attenuation of ERK signaling leads to differentiation of granulosa cells from hen prehierarchal follicles, this signaling pathway may also play a role in regulating the differentiation of granulosa cells derived from the germinal disc region of preovulatory follicles. Granulosa cells within the germinal disc (animal pole) region are considered essentially undifferentiated in that they are mitotically active, express comparatively high levels of EGF/ErbB1 receptor, and produce little to no progesterone. By comparison, granulosa cells distal to the germinal disc (vegetal pole) constitute the majority the follicular granulosa cells. Such cells are less proliferative, express lower levels of EGF/ErbB1 receptor, and are competent to produce large amounts of progesterone (34, 35, 36). This functional and spatial distinction of granulosa cells within the germinal disc region of preovulatory follicles has previously been proposed to result from the inhibitory effects of one or more EGF-family ligands derived from cells within the germinal disc and/or the germ cell (35).

In summary, these data represent the first to suggest that de novo transcription and translation of protein tyrosine and/or dual-specificity phosphatases may mediate the transient phosphorylation of ERK in undifferentiated granulosa cells after their induction and activation by EGF family member ligands. This acute regulation of ERK signaling is proposed to occur in conjunction with modulation of phosphatase activity by the ubiquitin-proteasome pathway. Among the phosphatases implicated in the acute (within 180 min) actions include (but are not necessarily limited to) DUSP5 and perhaps to a lesser extent MKP-1 and -3 but not MKP-2. It is also proposed that cytoplasmic MKP-3 may be integral in preventing further ligand-induced ERK signaling for up to 20 h. Accordingly, both the activation and subsequent inactivation of ERK signaling are critical for initiating granulosa cell differentiation subsequent to follicle selection (Fig. 10Go). The exact mechanism(s) responsible for a single prehierarchal follicle being selected into the preovulatory hierarchy of the hen per day remain obscure. Nevertheless, the model presented predicts that differentiation of the selected follicle is associated with granulosa-specific changes in the availability of one or more EGF family ligands (produced in an autocrine and/or paracrine fashion), expression of ErbB receptors, and activity of ERK-selective phosphatases.


   Acknowledgments
 
We thank M. Haugen for technical support and Drs. Crislyn D’Souza-Schorey and Holly Hoover for assistance with confocal microscopy studies.


   Footnotes
 
This work was supported by National Science Foundation Grant 0445949.

The authors D.C.W. and A.L.J. have nothing to declare related to the material being published.

First Published Online July 13, 2006

Abbreviations: ActD, Actinomycin D; BTC, betacellulin; CHX, cycloheximide; DUSP, dual-specificity phosphatase; EGF, epidermal growth factor; FSHR, FSH receptor; KIM, kinase interaction motif; LHR, LH receptor; MEK, MAPK kinase; MKP, MAPK phosphatase; NLS, nuclear localization signal; OA, okadaic acid; Smad, phosphorylated mothers against decapentaplegic; StAR, steroid acute regulatory; Z-LLF-CHO, (benzyloxycarbonyl)-leu-leu-phenylalaninal.

Received February 15, 2006.

Accepted for publication July 3, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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