Calmodulin-dependent Activation of ERK1

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 277, No. 5, Issue of February 1, pp. 3293–3302, 2002 Printed in U.S.A.

A Calcium/Calmodulin-dependent Activation of ERK1/2 Mediates JunD Phosphorylation and Induction of nur77 and 20␣-hsd Genes by Prostaglandin F2␣ in Ovarian Cells* Received for publication, November 15, 2001 Published, JBC Papers in Press, November 21, 2001, DOI 10.1074/jbc.M110936200

Carlos O. Stocco‡, Lester F. Lau§, and Geula Gibori‡¶ From the ‡Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, Illinois 60612 and the §Department of Molecular Genetics, University of Illinois College of Medicine, Chicago, Illinois 60607

In mammals the corpus luteum plays a central role in the regulation of cyclicity and maintenance of pregnancy. In the absence of fertilization and implantation, the corpus luteum loses the ability to secrete progesterone and undergoes luteolysis. Prostaglandin F2␣ (PGF2␣)1 is involved in the inhibition of * This work was supported by National Institutes of Health Grants HD11119 and HD12356 and by Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas, Argentina (to C. O. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¶ To whom correspondence and requests for reprints should be addressed: Dept. of Physiology and Biophysics (M/C 901), University of Illinois, 835 S. Wolcott Ave., Chicago, Il 60612-7342. Fax: 312-413-0159; E-mail: [email protected]. 1 The abbreviations used are: PGF2␣, prostaglandin F2␣; 20␣-HSD, 20␣-hydroxysteroid dehydrogenase; RT, reverse transcriptase; EMSA, electrophoretic mobility shift assay; DN, dominant-negative; PMA, phorbol 12-myristate 13-acetate; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; ERK, extracellular signalregulated kinase; JNK, c-Jun NH2-terminal kinase; CaM, calmodulin; CaM-K, Ca/CaM-dependent protein kinase; BAPTA/AM, 1,2-bis(2aminophenoxy)ethane-N,N,N⬘,N⬘-tetraacetic acid; PKC, protein kinase C; PKA, cAMP-dependent protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; PP2b, phosphatase 2B; luc, luciferase. This paper is available on line at http://www.jbc.org

progesterone production and luteal regression in many mammalian species. Thus, PGF2␣ causes luteal regression in domestic ruminants (1); accordingly, administration of PGF2␣ to cycling cows is a common practice used to synchronize the sexual cycle for the purpose of artificial insemination (2). In pregnant mice, the absence of PGF2␣ receptor causes a failure in parturition due to the lack of luteal regression and subsequent high levels of progesterone in circulation (3). Human luteal cells also produce and respond to PGF2␣ with a decrease in progesterone production (4 – 6). We have recently studied the mechanism by which PGF2␣ triggers the fall in progesterone production by the corpus luteum at the end of pregnancy in rodents. We have shown that PGF2␣ induces a rapid and massive expression of the luteal enzyme 20␣-hydroxysteroid dehydrogenase (20␣HSD) (7). This enzyme catabolizes progesterone into an inactive metabolite, 20␣-OH-progesterone, which cannot support pregnancy. Therefore, expression of 20␣-HSD results in decreased luteal progesterone secretion and parturition (8, 9). We have also established that PGF2␣ stimulation of the 20␣-hsd gene requires the transcription factor Nur77, which is induced by PGF2␣ in the corpus luteum of pregnant rats prior to the induction of 20␣-hsd (7). However, the signaling mechanism by which PGF2␣ stimulates nur77 and 20␣-hsd gene expressions remains largely unknown. PGF2␣-induced luteolysis is believed to be initiated through a receptor-mediated activation of phospholipase C, which generates inositol (1,4,5)P3 and diacylglycerol. These second messengers in turn increase free intracellular calcium ([Ca2⫹]i) and protein kinase C (PKC) activity, respectively (10, 11). The antisteroidogenic effects of PGF2␣ have been reported to be mediated by a PKC-dependent pathway, whereas loss of luteal cells appears to be due to [Ca2⫹]i (for review, see Ref. 1). It has also been demonstrated that PGF2␣ activates the mitogenactivated protein kinase (MAPK) signaling cascade in bovine (12) and human (4) luteal cells. However, little is known about the downstream signaling events that mediate the cellular responses or the signaling mechanisms by which PGF2␣ activates gene expression in the corpus luteum. Nur77 is an orphan nuclear steroid receptor and an immediate early gene whose synthesis is tightly regulated by extracellular signals. The regulation of Nur77 expression has been examined in other systems such as the adrenocortical derived cell line (Y1 cells), the pheochromocytoma cell line PC12, and immature thymocytes. In these systems, Nur77 expression is induced, respectively, by corticotropin via cAMP (13), by nerve growth factor and membrane depolarization via Ca2⫹ and AP1 proteins (14), and by T-cell receptors via Ca2⫹ (15). MAPK signaling has been shown to be involved in the induction of nur77 in excitable cells such as muscle and neuron cells (16, 17). These studies suggested to us that PKC, [Ca2⫹]i, or MAPK signaling may also be involved in PGF2␣ induction of Nur77 in luteal cells.

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We have previously demonstrated that prostaglandin F2␣ (PGF2␣) induces a rapid and transient expression of Nur77 in luteal cells. We have shown that Nur77 plays an important role in ovarian physiology by mediating the PGF2␣ induction of 20␣-HSD, a steroidogenic enzyme involved in the catabolism of progesterone. In this report we established, using luteinized granulosa cells, that PGF2␣ stimulates in vitro nur77 expression in a time- and dose-dependent manner. Serial 5ⴕ-deletion of the nur77 promoter revealed that the necessary and sufficient elements for PGF2␣ induction of Nur77 promoter activity are located between the nucleotides ⴚ86 and ⴚ33 upstream of the transcription start site, this region containing two AP1 elements. JunD binds to these AP1 sites, but its binding is not stimulated by PGF2␣. However, mutation of the AP1 sites as well as a dominant-negative JunD abolished nur77 induction by PGF2␣. PGF2␣ induces phosphorylation of JunD bound to the nur77 promoter. Stimulation of nur77 expression and JunD phosphorylation were prevented by inhibitors of calcium, calmodulin, or ERK1/2 kinase. PGF2␣induced ERK1/2 phosphorylation was prevented by calcium/calmodulin inhibitors. We conclude that activation of JunD through a calmodulim-dependent activation of ERK1/2 mediates nur77 induction by PGF2␣. Finally, we demonstrated that this molecular mechanism also mediates 20␣-hsd induction.

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ERK1/2/JunD Mediates Luteal nur77 and 20␣-hsd Inductions by PGF2␣

In this investigation, we examined the mechanism by which PGF2␣ signals to the nur77 and 20␣-hsd genes in luteinized granulosa cells. We have shown that PGF2␣ induces nur77 expression through a Ca2⫹-CaM-dependent activation of the ERK1/2 MAP kinase pathway. ERK1/2 activation results in phosphorylation of the transcription factor JunD already bound to the Nur77 promoter. Nur77 activates the 20␣-hsd gene causing the expression of this progesterone-catabolizing enzyme. Finally, we provide evidence for a possible mechanism by which Ca2⫹-CaM may mediate ERK1/2 kinase activation by PGF2␣ in ovarian cells. EXPERIMENTAL PROCEDURES

FIG. 1. PGF2␣ induces Nur77 mRNA expression in a time- and dose-dependent manner in luteinized granulosa cells; effect of cycloheximide. A, luteinized granulosa cells were treated with 5 ␮M PGF2␣ for different times. B, cells were treated with different doses of PGF2␣ for 1 h. C, cells were treated with 5 ␮M PGF2␣ in absence (lanes 1, 2, 4, and 6) or presence (lanes 3, 5, 7, and 8) of cycloheximide (10 mg/ml) for different times. In A–C Nur77 mRNA levels were assessed by semiquantitative RT-PCR. Bars represent the mean ⫾ S.E. (n ⫽ 3). were incubated in reaction buffer (10 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 0.1 ␮g/ul of poly(dIdC), and 4% glycerol) for 20 min. Excess of unlabeled oligonucleotide or specific antibodies were added prior to addition of the labeled probe corresponding to the region ⫺45 to ⫺26 of the Nur77 promoter, and the incubation was continued for 20 min at 22 °C. The DNA-protein complexes were separated from the unbound DNA probe by nondenaturing PAGE (4% gel) at 4 °C, in 0.5⫻ Tris borate EDTA buffer. RNA Isolation and RT-PCR Analysis—Total RNA isolation and RTPCR reaction were performed as described previously (7). For co-amplification of Nur77 and L19 message, the primers used were 5⬘-TCT GCT CAG GCC TGG TGC TAC-3⬘ and 5⬘-GGC ACC AAG TCC TCC AGC TTG-3⬘ and 5⬘-GGA CAG AGT CCA AGG GTC CGC TGC AGTC-3⬘ and 5⬘-TCC AAG GGT CCG CTG CAG TC-3⬘, respectively. Statistical Analysis—One-way analysis of variance followed by the Tukey test was used for the statistical analysis of relative mRNA expression and luciferase activity data using the Prism software (GraphPad Software, Inc., San Diego, CA). Values were considered statistically significant at p ⬍ 0.05. RESULTS

PGF2␣ Induces Nur77 mRNA Expression in a Time- and Dose-dependent Manner in Luteinized Granulosa Cells—We first examined whether luteinized granulosa cells can be used in primary culture to explore PGF2␣ signaling to the nur77 gene. The results show that PGF2␣ induces nur77 expression in luteinized granulosa cells in a time- (Fig. 1A) and dose- (Fig. 1B) dependent manner. Effect of Cycloheximide on Nur77 Gene Regulation by PGF2␣—To examine whether PGF2␣ stimulation of nur77 de-


Chemicals—[␣-32P]Deoxycytidine triphosphate ([␣32P]dCTP) was purchased from Amershan Biosciences, Inc.; Advantage RT-for-PCR kit was purchased from CLONTECH (Palo Alto, CA); dNTP, ExTaq DNA polymerase, and ExTaq buffer were purchased from Takara Biomedicals (Shiga, Japan); the nucleotides used as primers in the RT-PCR analysis were obtained from Invitrogen; Western blotting Luminol Reagent was obtained from Santa Cruz Biotechnology (Santa Cruz, CA); Dulbecco’s modified Eagle’s medium:F-12 medium, nonessential amino acids, sodium pyruvate, trypsin-EDTA, antibiotics, and antimycotics were purchased from Mediatech (Herndon, VA). PGF2␣, D-glucose, TriReagent, aprotinin, leupeptin, phenylmethylsulfonyl fluoride, cycloheximide, and all other reagent-grade chemicals were purchased from Sigma. BAPTA/AM, KN93, KN62, staurosporine, calphostin C, PMA, phorbol 12,13-dibutyrate, W7, cyclosporin, FK506, PD98059, UO126, SB203580, SB202190, A23187, and ionomicyn compounds were obtained form Calbiochem. Cell Culture and Transient Transfection Assays—Luteinized granulosa cells were obtained and cultured as described previously (7). After 2 days of culture, medium was changed and the cells were transfected with different plasmids as indicated in the figure legend using LipofectAMINE (Invitrogen) according to the manufacturer’s protocol. The Nur77 promoter reported construct and the wild type and mutant JunD expression plasmids have been described previously (18). The ⫺33-bp Nur77-luc was generated by PCR following standard cloning techniques. The 2.5-kb 20␣-HSD-luc reporter construct has been described previously (19). 24 h after transfection, the cells were treated as indicated in the legends of Figs. 2, 4, 8, and 12. To harvest cells, each well was washed twice with ice-cold phosphate-buffered saline and immediately frozen at ⫺80 °C. For luciferase activity measurements, 80 ␮l of passive lysis buffer (Promega, Madison, WI) was added to each well. 20 ␮l of the cell lysate was used to measure the firefly luciferase activity driven by the Nur77 promoter using Promega’s luciferase Reporter system. Another 20 ␮l of the cell lysate was used to measure ␤-galactosidase activity using the CLONTECH ␤-galactosidase Assay System (CLONTECH). Relative light units were obtained by dividing the Nur77 promoter luciferase activity by the ␤-galactosidase activity. Western Blot Analysis—Nuclear extracts were extracted as described above, and the samples were processed by Western blot as described previously (7). The antibodies used were JunD (Santa Cruz Biotechnology), anti-phospho-JunD (Ser100) or c-Jun (Ser73), (Upstate Biotechnology, Lake Placid, NY), total ERK1/2 (Upstate Biotechnology), and phospho-ERK1/2 (New England Biolabs, Beverly, MA). The blots were exposed to primary and secondary antibodies according to manufacturer’s protocols. Protein-antibody complexes were visualized using Western blotting Luminol Reagent according to the manufacturer’s protocol (Santa Cruz Biotechnology). The band densities were determined by digital analysis using a Kodak Digital Science DC 120 Zoom Digital Camera and Kodak Digital Science 1D 2.0.2 software (Eastman Kodak Co.). Electrophoretic Mobility Shift Assay (EMSA)—To prepare nuclear cell extracts, 100-mm plates of luteinized granulosa cells at 70 – 80% of confluence were used. Cells were harvested in phosphate-buffered saline at 4 °C by scraping and centrifuging for 5 min at 12,000 ⫻ g. The cell pellet was resuspended in 400 ␮l of solution A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol) and placed in an orbital rocker for 20 min at 4 °C. The nuclear pellet was obtained by centrifugation for 30 s at 12,000 ⫻ g at 4 °C in an Eppendorf centrifuge and resuspended in solution B, which was similar to solution A except that it contained 420 mM NaCl and 5% (v/v) glycerol and no KCl. The solution was vigorously vortexed for 30 min at 4 °C and then centrifuged at 14,000 ⫻ g at 4 °C for 20 min. The supernatant containing nuclear extract was divided into aliquots and stored at ⫺80 °C. 2.5– 4.0 ␮g of nuclear extract


FIG. 2. PGF2␣ rapidly increases Nur77 promoter activity; effect of 5ⴕ deletions and point mutations. A, luteinized granulosa cells were transfected with one of several reporter constructs containing 5⬘-serial deletions in the Nur77 promoter region. 24 h later, cells were treated with PGF2␣ (5 ␮M) or vehicle for 6 h. B, cells were transfected with either the ⫺1200-, ⫺206-, ⫺86-, or ⫺33-bp Nur77-luc promoter and 24 h later treated with either vehicle or PGF2␣ (5 ␮M) for 1, 3, or 6 h. C: top, sequence of the region ⫺86 to ⫺33 of the Nur77 promoter showing the two AP1 sites found (underlined). Bottom, cells were transfected with either the wild type ⫺86 Nur77-luc promoter or the same promoter carrying mutations in one or both AP1 sites (mutations are in bold and underlined). 24 h later, cells were treated with PGF2␣ (5 ␮M) or vehicle (control) for 6 h. Transient expression of the reporter gene was quantified by a standard luciferase bioluminescence assay and normalized against ␤-galactosidase. Bars represent mean ⫾ S.E. of four experiments.

was seen after the addition of a JunD antibody to the nuclear extracts from both control and PGF2␣-treated cells (Fig. 3A, lanes 5 and 6). The addition of c-Jun or c-Fos antibodies to the gel shift reaction did not cause a supershifted band (data not shown), confirming previous results showing that neither c-Jun nor c-Fos bind to the AP1 binding site located in the Nur77 promoter (14). These results indicate that JunD binds to the Nur77 promoter, but this binding is not stimulated by PGF2␣. When nuclear extracts were analyzed by SDS-PAGE and Western blot analysis using a pan JunD antibody, we confirmed (Fig. 3A, lower panel) that JunD is constitutively present in the nuclear fraction of granulosa cells and that its expression is not affected by PGF2␣. Since we could not demonstrate a causative relationship between nur77 expression and increased JunD DNA binding, we then hypothesized that PGF2␣ may increase JunD activity.


pends upon de novo protein synthesis, the effect of the protein synthesis inhibitor cycloheximide on the nur77 gene induction by PGF2␣ was examined. Luteinized granulosa cells were treated with 5 ␮M PGF2␣ in the presence (Fig. 1C, lanes 3, 5, 7, and 8) or absence (lanes 1, 2, 4, and 6) of cycloheximide (10 mg/ml) for different times. Treatment with PGF2␣ in the presence of cycloheximide induced significantly higher nur77 gene expression than PGF2␣ alone (Fig. 1C, lanes 3, 5, and 7 versus lanes 2, 4, and 6). Additionally, in the presence of cycloheximide, Nur77 mRNA levels remained elevated after 3 h of PGF2␣ treatment (Fig. 1C, lanes 5 and 7). Treatment with cycloheximide alone for 3 h increased nur77 expression (Fig. 1C, lane 8). PGF2␣ Rapidly Increases Nur77 Promoter Activity—To examine the mechanism of nur77 induction by PGF2␣, luteinized granulosa cells were transfected with a reporter construct containing the Nur77 promoter region spanning from ⫺1200 to ⫹120 bp (1.2-kb Nur7-luc), where ⫹1 represents the transcription start site. Cells were also transfected with a plasmid expressing ␤-galactosidase, allowing for normalization of transfection efficiencies. Using this promoter, we observed a 7-fold stimulation of luciferase activity following treatment with PGF2␣ (Fig. 2A). A marked decrease in PGF2␣ stimulation was observed upon deletion of the ⫺1200- to ⫺294-bp region. Further deletions of this promoter revealed that the minimal and necessary elements for PGF2␣ stimulation are located between the ⫺86 and ⫺33 region, since deletion of this region totally abolished the stimulatory effect of PGF2␣ (Fig. 2A). Time course experiments (Fig. 2B) revealed a rapid stimulation of the 1.2-kb Nur77-luc reporter construct by PGF2␣, with a 3-fold induction within 1 h of treatment. Maximal stimulation was observed 3 h after treatment with no further increase at 6 h. In contrast, when two smaller (⫺206 and ⫺86 bp) Nur77luc constructs were used, no stimulation was observed after 1 h of treatment. However, PGF2␣ stimulated the activity of these constructs 3 and 6 h after treatment in a time-dependent manner (Fig. 2B). As shown previously, no stimulation of the ⫺33-bp promoter was observed at any time studied (Fig. 2B). Analysis of the PGF2␣-sensitive region, nucleotides ⫺86 to ⫺33, revealed the presence of two putative AP1 binding sites (Fig. 2C). Mutations of either the distal (Fig. 2C, lane 3) or the proximal (Fig. 2C, lane 4) AP1 binding sites in the ⫺86-bp Nur77-luc construct profoundly decreased the induction of luciferase activity by PGF2␣, and mutation of both AP1 sites (Fig. 2C, lane 5) fully prevented PGF2␣ stimulation. JunD Constitutively Binds to the Nur77 Promoter but Is Active Only after PGF2␣ Treatment—To determine the transcription factors that bind to the AP1 sites, we performed gel shift assays using an oligonucleotide containing the proximal AP1 site as a probe. Nuclear extracts from luteinized granulosa cells treated with either PGF2␣ or vehicle both contained a specific protein able to bind to this probe (Fig. 3A, lanes 1 and 2). Addition of excess of a cold oligonucleotide containing the proximal AP1 site (lanes 3 and 4) or the distal AP1 site (data not shown) totally inhibited this binding, indicating that both AP1 sites may mediate the effect of PGF2␣ in agreement with the mutation studies showed in Fig. 2C. Studies by Sharma and Richards (20) using Western blot and gel shift analysis have revealed that JunD and FRA2 are major components of the AP1-DNA binding complex in luteinized granulosa cells, whereas c-jun and c-fos are expressed at low levels. Therefore, we examined first whether these AP1 proteins may bind to this AP1 site by adding specifics antibodies to the gel shift reaction. Whereas no supershift was observed with the FRA2 antibody (Fig. 3A, lanes 7 and 8), a strong supershift

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Transcriptional activity of JunD can be regulated by phosphorylation of serine residues located in the amino-terminal domain (21, 22). Most notable in this regard are regulatory phosphorylations occurring on Ser90 and Ser100 within the transactivation domain (23). Therefore, we next tested whether PGF2␣ induces the phosphorylation of the JunD protein bound to the Nur77 promoter, by adding a phospho-JunD (Ser100) antibody to the gel shift reaction. This antibody caused a prominent supershift when added to nuclear extract from PGF2␣treated cells (Fig. 3B, top panel: lane 4). However, this supershifted band was much less evident when nuclear extract from vehicle-treated cells were used. Accordingly, when nuclear extracts were analyzed by SDS-PAGE and Western blot analysis, the phospho-JunD protein was detectable only in cells treated with PGF2␣ (Fig. 3B, lower panel). The amount of total JunD remained the same regardless of PGF2␣ treatment, as shown by the use of a pan JunD antibody. JunD Mediates Nur77 Activation by PGF2␣—To confirm the participation of JunD in the induction of Nur77 by PGF2␣, we sought to determine whether the induction of nur77 expression could be altered by a dominant-negative mutant JunD (DNJunD). Luteinized granulosa cells were transfected with either a dominant-negative JunD expression vector, or with empty vector, and were treated with or without PGF2␣ (Fig. 4A). This DN-JunD protein is lacking its DNA binding domain but retains the capacity to form homodimers and heterodimers (14). As shown in Fig. 4A, left panel, the strong induction of the endogenous nur77 gene by PGF2␣ was inhibited by DN-JunD in a dose-dependent manner. Additional cells were co-transfected with the ⫺86-bp Nur77-luc reporter construct and with either

FIG. 4. JunD mediates Nur77 activation by PGF2␣. A: left panel, endogenous Nur77 mRNA levels in luteinized granulosas cells transfected with none (⫺), 0.1, or 0.5 ␮g/well of a mutant JunD coding sequence, which lack a DNA binding domain. 24 h after transfection, the cells were treated for 1 h with PGF2␣ (5 ␮M), and Nur77 mRNA was measured using semiquantitative RT-PCR. A: right panel, cells were co-transfected with the ⫺86-bp Nur77-luc promoter and 0.1, 0.5, or 1 ␮g/well of a dominant-negative JunD expression (DNJunD) vector or an empty vector (⫺). 24 h later, cells were treated with PGF2␣ (5 ␮M) for 6 h before luciferase activity determination. B, the 1.2-kb (left) or the ⫺86-bp Nur77 (right) promoter were transfected with an empty plasmid (columns 1 and 2) or a wild type JunD coding sequence (columns 3 and 4) or a JunD coding sequence carrying a serine 100-to-alanine mutation at serine 100 (JunDAla; column 5). 24 h later, cells were treated with either PGF2␣ (columns 2, 4, and 5) or vehicle (columns 1 and 3) for 6 h before luciferase activity determination. Experiments were repeated three times; results from a representative set are shown. Bars represent mean ⫾ S.E.

the DN-JunD expression vector or empty vector. They were then treated with either vehicle or PGF2␣ (Fig. 4A, right panel). DN-JunD protein inhibited the PGF2␣-induced ⫺86-bp Nur77luc reporter construct activity in a dose-dependent manner (Fig. 4A, right panel). At a 1-␮g concentration the DN-JunD expression vector completely abolished this induction. To further investigate the role of JunD protein in the PGF2␣mediated stimulation of nur77, we overexpressed a wild type JunD protein in luteinized granulosa cells and treated the cells with either vehicle of PGF2␣. As shown in Fig. 4B, overexpression of JunD had no effect on either the 1.2-kb (left panel) or the ⫺86-bp (right panel) Nur77-luc reporter construct activity. However, when cells transfected with JunD were treated with PGF2␣, a remarkable increase in promoter activity was observed. The induction of Nur77 promoter activity in JunDtransfected and PGF2␣-treated cells was severalfold higher than in nontransfected cells treated with PGF2␣. These results indicate that JunD is necessary, but not enough, to induce nur77 expression and that phosphorylation of the already present JunD protein by PGF2␣ is essential for the activation of the nur77 gene. To further study the functional role of JunD Ser100 phosphorylation in the induction of nur77 by PGF2␣, a plasmid encoding a modified JunD protein in which the serine 100 residue


FIG. 3. JunD constitutively binds to the Nur77 promoter but is active only after PGF2␣ treatment. A: top panel, nuclear extracts from luteinized granulosa cells treated with either PGF2␣ (lanes 2, 4, 6, and 8) or vehicle (lanes 1, 3, 5, and 7) for 20 min were analyzed by EMSA using a probe containing the proximal AP1 site. Addition of 50⫻ excess of cold oligonucleotide (lanes 3 and 4), pan JunD antibody (lanes 5 and 6), or pan FRA2 antidody (lanes 7 and 8) is shown. Bottom panel, JunD levels determined by Western blot analysis of 30 ␮g of nuclear or cytosolic proteins from granulosa cells treated with either PGF2␣ (⫹) or vehicle (⫺) for 20 min. B: top panel, supershift EMSA using a phosphoJunD antibody specific for Ser100 (P-JunD) (lane 3 and 4). Bottom panel, Western blot of nuclear extract (30 ␮g) from granulosa cells treated with PGF2␣ or vehicle (20 min) using a phospho-JunD antibody (PJunD) or a pan JunD antibody (Total JunD). Experiments were repeated four times, and results from one representative experiment are shown.


has been replaced by alanine (JunDAla) was introduced into luteinized granulosa cell. Mutation of serine to alanine in the AP1 proteins prevents phosphorylation of the mutated residue (24). No synergism between the overexpression of JunDAla and PGF2␣ treatment in the induction of the 1.2-kb and ⫺86-bp Nur77-luc construct activity was observed (Fig. 4B), further establishing the importance of JunD phosphorylation in the PGF2␣ stimulation of nur77 expression. PGF2␣ Stimulation of Nur77 Relies on a Calcium-dependent Mechanism without Participation of PKC Signaling (Fig. 5)—We next examined the intracellular mechanism by which PGF2␣ may induce phosphorylation of JunD and nur77 expression. Two known intracellular mediators of PGF2␣ action in the corpus luteum are PKC and Ca2⫹ (1, 11, 25). To examine whether the PKC pathway is involved, we either treated granulosa cells with the PKC inhibitors (staurosporine or calphostin C), or we depleted PKC via a long term treatment with PMA. Then PGF2␣ was added to the medium at a concentration or 5 ␮M, and the cells were cultured for 1 h before mRNA isolation. As expected, PGF2␣ induced Nur77; however, neither staurosporine, calphostin C, nor sustained PMA treatment affected PGF2␣ stimulation of nur77 (Fig. 5A). To test whether Ca2⫹ is involved, we used the membrane-permeable Ca2⫹ chelator BAPTA/AM (25 ␮M), to inhibit free intracellular Ca2⫹, and either the Ca2⫹ ionophores A23187 (1 mM) or ionomicyn (2 ␮M) to increase intracellular Ca2⫹. As shown in Fig. 5B, PGF2␣ induction of Nur77 mRNA expression was completely prevented by BAPTA/AM, whereas A23187 was able to induce endogenous Nur77 expression as effectively as PGF2␣. Similar results were also obtained with ionomicyn (data not shown).

FIG. 6. Calcium/Calmodulin mediates the PGF2␣ effect on nur77 expression. A, luteinized granulosa cells were pretreated with different doses of W7 for 1 h and then challenged with PGF2␣ (5 ␮M, 1 h). Nur77 mRNA levels were examined by semiquantitative RT-PCR. B, cells were transfected with either the 1.2-kb or the ⫺86-bp Nur77-luc promoter. 24 h later, they were treated for 1 h with BAPTA/AM (25 ␮M) or W7 (50 ␮M) prior to treatment with PGF2␣ (5 ␮M, 6 h). Experiments were repeated four times. Results from one representative experiment are shown. Bars represent mean ⫾ S.E.

To further investigate the lack of participation of PKC activation in the induction of Nur77 by PGF2␣, luteinized granulosa cells were treated with two different PKC activators, PMA or phorbol 12,13-dibutyrate. Surprisingly, and in contrast to the results obtained using PKC inhibitors, activation of PKC induced nur77 gene expression (Fig. 5C, left panel). Since recent evidence revealed that activation of PKC by PMA promoted calcium influx in Chinese hamster ovarian (26) and glomerular mesangial (27) cells, we tested whether the induction of nur77 by PKC activation is through an increase in [Ca2⫹]i. Cells were treated with either PMA alone or PMA plus BAPTA/AM. Blocking intracellular calcium with BAPTA/AM completely abolished the PMA-induced stimulation of Nur77 mRNA expression (Fig. 5C, middle panel). Similar results were obtained with Nur77 promoter activity (Fig. 5C, right panel) in response to PMA and BAPTA. Taken together, these results indicate that Ca2⫹ mediates the effect of PGF2␣ on Nur77 expression, without participation of PKC. Calcium/Calmodulin Mediates PGF2␣ Effect on nur77 Expression—Since one of the most common mechanisms by which elevated intracellular calcium regulates cellular events is through its association with CaM (28), we examined whether this Ca2⫹-binding protein is involved in PGF2␣ signaling. As shown in Fig. 6A, treatment with W7, a specific inhibitor of CaM, decreased PGF2␣-induced nur77 expression in a dose-dependent manner. To confirm the participation of Ca2⫹-CaM in the induction of nur77, either the 1.2-kb or the ⫺86-bp Nur77luc constructs were transfected into luteinized granulosa cells. 24 h later, cells where treated with either BAPTA/AM or W7 for 1 h. Then PGF2␣ was added to the medium at a 5-␮M concentration, and the cells were incubated for 6 h before luciferase activity determination. As shown in Fig. 6B, PGF2␣ stimulation of both Nur77-luc constructs was prevented by either blocking intracellular Ca2⫹ or inhibiting CaM. Taken together these results indicate that Ca2⫹ and CaM are required for the induction of nur77 in luteinized granulosa cells. Raf and MEK Kinase Activities Are Requisite for PGF2␣ Induction of Nur77—Once we established the role of Ca2⫹-CaM


FIG. 5. PGF2␣ stimulation of Nur77 relies on a calcium-dependent mechanism. A, luteinized granulosa cells were pretreated with Me2SO as vehicle (⫺), staurosporine (Stp, 100 nM), or calphostin C (Cph, 1 ␮M) for 1 h or with PMA (1 ␮M) for 24 h prior to treatment with PGF2␣ (5 ␮M, 1 h). B, cells were cultured in the presence of either vehicle, PGF2␣, PGF2␣ plus BAPTA/AM (25 ␮M), or A23187 (1 mM) for 1 h. Nur77 mRNA levels were examined by semiquantitative RT-PCR. C: left panel, luteinized granulosa cells were treated for 1 h with PGF2␣ (5 ␮M), PMA (0.5 ␮M), or phorbol 12,13-dibutyrate (PDBu, 1 ␮M). C: middle panel, luteinized granulosa cells were incubated with either vehicle or BAPTA/AM (25 ␮M) for 1 h before continued incubation for 1 h with PMA (0.5 ␮M). C: left panel, cells were transfected with the 1.2-kb Nur77 promoter. 24 h later, cells were treated with either vehicle or BAPTA/AM (25 ␮M) before treatment with PMA (0.5 ␮M, 1 h). mRNA levels were examined by semiquantitative RT-PCR and reporter activity measured by bioluminescence. Results from one representative experiment are shown (n ⫽ 3). Bars represent means ⫾ S.E.

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in the PGF2␣ stimulation of Nur77, we next examined the possible downstream targets of Ca2⫹-CaM. Ca2⫹-CaM is known to activate several kinases and phosphatases that determine the final cellular response after an increase in [Ca2⫹]i (29). Of particular interest are phosphatase 2B (PP2b) and calcium/calmodulin-dependent kinase (CaM-K), which have been shown to be involved in Nur77 induction in others systems (30, 31). We used specific inhibitors of these proteins to test their participation in nur77 induction by PGF2␣. As shown in Fig. 7A, PGF2␣ stimulation of nur77 was not prevented by either of the two PP2b inhibitors tested, cyclosporin A (100 ng/ml) and FK506 (1 ␮M). Interestingly, neither of the CaM-K inhibitors, KN93 (Fig. 7B) or KN63 (data not shown), was able to prevent PGF2␣ stimulation of nur77. These results indicate that PP2b and CaM-K do not play a role in the PGF2␣ stimulation of nur77 in luteal cells. Since PGF2␣ was shown previously to activate the MAPK pathway in the corpus luteum (4, 30), and since this system can also be activated by Ca2⫹-CaM (32), we explored the possibility that MAPK may mediate the PGF2␣ stimulation of Nur77. To date there are three distinct groups of MAPKs: the extracellular signal-regulated kinases (ERK1/2), c-Jun NH2-terminal kinase (JNK), and P38 MAPK (p38MAPK) (reviewed in Ref. 33). We used several specific inhibitors to differentially block the different members of the MAPK pathway. To prevent ERK1/2 activation, we used PD98059 that blocks MEK1/2 activation by Raf kinase (34) and UO126, which inhibits directly the MEK1/2 kinase (35). p38MAPK was inhibited by treatment with low concentration of SB203580 (2–10 ␮M) or SB202190 (20 ␮M), whereas a higher concentration of SB203580 (30 ␮M) was used to prevent JNK activity (36). As shown in Fig. 8A, PGF2␣ stimulation of nur77 expression was prevented only when the cells were pretreated with the MEK1/2 inhibitors (PD98059 and UO126). No such inhibition was observed with either the p38MAPK or JNK inhibitors. Pretreatment with UO126 also inhibited the stimulation of both 1.2-kb and ⫺86-bp Nur77-luc construct activity by PGF2␣ (Fig. 8B). Inhibition of p38MAPK or JNK kinases had no effect on the induction of Nur77 promoter activity (data not shown). These results indicate that activation of ERK1/2 is required for nur77 induction by PGF2␣. PGF2␣ Induces Phosphorylation of ERK1/2 through a Ca2⫹dependent Mechanism—We next examined whether PGF2␣

FIG. 8. Extracellular regulated kinase 1 and 2 mediates the PGF2␣ effect. A, luteinized granulosa cells were treated for 1 h with vehicle (⫺) or PD98059, UO126, SB203580, or SB202190 at the indicated micromolar concentration, prior to treatment with PGF2␣ (5 ␮M, 1 h). Nur77 mRNA levels were assessed by semiquantitative RT-PCR. B, luteinized granulosa cells transfected with either the 1.2-kb (left) or the ⫺86-bp Nur77-luc promoter (right) were treated with UO126 at two concentrations for 30 min prior to PGF2␣ treatment (5 ␮M, 6 h). Experiments were repeated five times, and results from a representative set are shown. Bars represent mean ⫾ S.E.

could induce ERK1/2 kinase phosphorylation and whether increased [Ca2⫹]i or Ca2⫹-CaM may mediate this effect. Luteinized granulosa cells were treated with BAPTA/AM or W7 for 45 min, followed by the addition of PGF2␣ (5 ␮M) for 20 min. Activation of ERK1/2 is known to require phosphorylation of tyrosine 202 and threonine 204 (33). We therefore assessed ERK1/2 phosphorylation by Western blot analysis with an antibody that specifically recognizes the phosphorylated threonine 202 and tyrosine 204 of ERK1/2. The results in Fig. 9 show a strong activation of ERK1/2 in cells treated with PGF2␣, both in whole cells extracts (upper panel) as well as in nuclear and cytosolic fractions (lower panel). Pretreatment with BAPTA/AM and W7 inhibited the activation of ERK1 and ERK2 by PGF2␣, as evidenced by a very low phospho-ERK1/2 signal (Fig. 9). The amount of total ERK1/2 remained the same regardless of treatment, as shown by the use of a pan ERK1/2 antibody, which recognizes total ERK1 and ERK2. These findings indicate that PGF2␣ activates ERK1/2 and that this effect is Ca2⫹-CaM-dependent. CaM and ERK1/2 Mediate PGF2␣-induced Phosphorylation of JunD—Since we have shown that inhibitors of CaM or ERK1/2 prevent the induction of nur77 by PGF2␣, we next examined whether these agents can also mediate the PGF2␣induced phosphorylation of JunD. Cells were treated with BAPTA/AM, W7, or PD98059 for 45 min, followed by the addition of PGF2␣ (5 ␮M) for 20 min. Nuclear proteins were analyzed by SDS-PAGE and Western blot. As shown in Fig. 10, PGF2␣ induced JunD phosphorylation. Pretreatment with BAPTA/


FIG. 7. PP2b or calcium/calmodulin-dependent kinase (CaM-K) are not involved in the PGF2␣ stimulation of nur77 in luteal cells. Luteinized granulosa cells were pretreated for 1 h with cyclosporin A (cyclosporin A (100 ng/ml) or FK506 (1 ␮M) (A) or with KN93 (20 or 40 ␮M) (B). Control received vehicle Me2SO (⫺). Next the cells were challenged with PGF2␣ (5 ␮M, 1 h). Nur77 expression was assessed by semiquantitative RT-PCR. Experiments were repeated three times, and results from one representative experiment are shown. Bars represent mean ⫾ S.E.


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treatment for 12 h. As shown in Fig. 11, blocking calcium or inhibiting either CaM or ERK1/2 prevented PGF2␣ induction of 20␣-HSD reporter construct activity (Fig. 11, A and B), whereas the PKA inhibitor used as control had no affect. Last (Fig. 11C), we examined whether JunD mediates the PGF2␣ induction of 20␣-HSD in ovarian cells by overexpressing a DN-JunD protein. Cells were transfected with the 2.5-kb 20␣HSD-luc promoter with either a DN-JunD expression vector or empty vector. 24 h later, cells were challanged with PGF2␣ for 12 h prior to the determination of luciferase activity. As shown in Fig. 11C, overexpression of the dominant-negative JunD completely abolished the induction of 20␣-HSD promoter activity by PGF2␣. DISCUSSION

FIG. 10. Calcium and ERK1/2 mediates the PGF2␣-induced phosphorylation of JunD. Luteinized granulosa cells were treated with BAPTA/AM, W7, or PD98059 for 45 min, followed by the addition of PGF2␣ at a concentration of 5 ␮M. The cells were incubated for 20 min before harvesting and lysing. Nuclear proteins (30 ␮g) were analyzed by SDS-PAGE and Western blot using an anti-phospho-JunD (Ser100) antibody (P-JunD) or a pan JunD antibody (T-JunD). Densitometric analysis of the bands was used to calculate the ratio between phospho- and total JunD protein. The experiment was repeated twice, and a representative blot is shown.

AM, W7, or PD98059 significantly reduced this effect. In accordance with our previous results indicating that neither p38MAPK nor JNK are involved in Nur77 stimulation, pretreatment of luteinized granulosa cells with 30 ␮M SB203580 did not reduce JunD phosphorylation. The use of a pan JunD antibody demonstrated that no change in total amount of JunD occurred after any of these treatments. These results indicate that Ca2⫹CaM and ERK1/2 mediate PGF2␣-induced phosphorylation of JunD. PGF2␣ Induction of 20␣-hsd Gene Expression—As mentioned in the introduction, the major function of Nur77 is to induce 20␣-HSD expression in ovarian cells (7). Therefore we examined whether the mechanism of nur77 induction described in the present report is also involved in the stimulation of 20␣HSD by PGF2␣. Luteinized granulosa cells transfected with the 2.5-kb 20␣-HSD-luc reporter construct (19) were treated with either Ca2⫹ (BAPTA/AM), CaM (W7), ERK1/2 (PD98059, UO126), or PKA (H9) inhibitors for 30 min followed by PGF2␣


FIG. 9. PGF2␣ induces phosphorylation of ERK1/2 through a Ca2ⴙ-dependent mechanism. Luteinized granulosa cells were treated with BAPTA/AM or W7 for 1 h. PGF2␣ was then added to the medium at a final concentration of 5 ␮M 20 min before harvesting and lysing the cells. Total, nuclear, or cytosolic protein fractions (30 ␮g) were analyzed by SDS-PAGE and Western blot using a pan ERK1/2 antibody (T-ERK1/2) or an anti-phospho-ERK1/2 antibody (P-ERK1/ 2). The experiment was repeated three times, and a representative blot is shown.

All our results taken together and summarized in Fig. 12 reveal that PGF2␣ induces the expression of nur77 through a Ca2⫹-CaM-dependent mechanism. The Ca2⫹-CaM complex formed upon PGF2␣ treatment causes activation of the ERK1/2 MAP kinase pathway. ERK1/2 phosphorylates the transcription factor JunD constitutively bound to its cognate binding site in the nur77 gene; increasing its transactivational activity. The Nur77 generated then acts to activate the 20␣-HSD gene expression, leading to the conversion of progesterone to 20␣OH-progesterone, a steroid unable to sustain pregnancy. Phosphorylation of JunD protein bound to the Nur77 promoter by the PGF2␣-activated ERK1/2 kinases is a remarkable finding that reveals alternative mechanisms by which these kinases can activate transcription through the AP1 family of proteins. ERK1/2-mediated AP-1 activation predominantly occurs at a transcriptional level (reviewed in Ref. 33). In contrast, our data suggest that ERK1/2 can also act through a posttranslational mechanism, stimulating the transactivation potential of pre-existing JunD proteins via alterations in their phosphorylation pattern. JunD is a classically constitutively expressed member of the AP1 family of the transcription factor in contrast to what is generally observed for c-jun and c-fos genes, which are considered immediate early genes. Also, in contrast to c-Jun, whose regulation by phosphorylation is well documented (21, 22), little is known about phosphorylation changes modulating the activity of JunD. JNK kinase, known to phosphorylate c-Jun, also phosphorylates JunD, although with less efficiency (37). Using 308 mouse keratinocytes, Rosenberger et al. (38) have recently observed that okakaic acid increases phosphorylation of JunD, which could be reduced by an inhibitor of ERK1/2 MAP kinase. In the present report, we have demonstrated that PGF2␣, through an ERK1/2-dependent mechanism, induces phosphorylation of the serine 100 residue in the JunD protein, resulting in an increase of its transcriptional activity in luteal cells. Sharma and Richards (20) have previously demonstrated that JunD is the major functional component of the AP1-DNA binding complex in luteinized granulosa cells. However, no functional target genes have been found or proposed for this constitutively expressed member of the AP1 family in luteal cells. Here, we show that PGF2␣ phosphorylation of the constitutively expressed JunD is crucial for nur77 induction. JunD overexpression had no effect on Nur77 promoter activity in the absence of PGF2␣ treatment, whereas a 16-fold increase in promoter activity was observed in these cells following treatment with PGF2␣. This increase in promoter activity was three times greater than that observed in cells transfected with an empty vector and treated with PGF2␣. Furthermore when a JunD construct containing a serine 100-to-alanine mutation protein was transfected, this synergism was not observed. Analysis of the Nur77 promoter revealed that its activity is rapidly induced by PGF2␣, within an hour, but only when the

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FIG. 12. Mechanism of Nur77 and 20␣-HSD inductions by PGF2␣ in luteal cells. PGF2␣ induces the expression of nur77 through a Ca2⫹-CaM-dependent mechanism. The Ca2⫹-CaM complex formed upon PGF2␣ treatment causes activation of the ERK1/2 MAP kinase pathway. ERK1/2 phosphorylates JunD, increasing its transcriptional activity. Expression of Nur77 activates the 20␣-HSD promoter leading to the expression of this progesterone-catabolizing enzyme.

large promoter was used (1.2 kb upstream of ⫹1). Deletion of the ⫺1200 to ⫺294 upstream region resulted in a 2-fold decrease in Nur77 promoter activity induction, indicating the presence of enhancers in this region. Similar findings have been described in T-cells, where induction of Nur77 promoter activity by increased [Ca2⫹]i is significantly affected by deletion of the region upstream of ⫺287 bp (18). These authors also found that a region of the Nur77 promoter spanning from ⫺307 to ⫺242 confers Ca2⫹ inducibility and cycloporin A sensitivity in T-cells (18). Our results indicate that PGF2␣ does indeed affect the activity of this region; however, nur77 induction in luteal cells is not cyclosporin A-sensitive, suggesting an unknown alternative mechanism of activation. We found that the minimal region necessary and sufficient for PGF2␣ induction is located downstream of the ⫺86 position. These results suggest that the regulation of Nur77 in luteal cells differs from that observed in immune cells. The ⫺206- or the ⫺86-bp Nur77-luc reporter construct was not rapidly induced by PGF2␣. On the contrary, PGF2␣ induced a sustained activation as compared

with the full-length promoter. This observation confirms a previous report indicating that different regions of the Nur77 promoter are responsible for either the early or delayed induction. Williams and Lau (39) demonstrated that nur77 can be induced rapidly within minutes as well as several hours after treatment. These authors also identified the ⫺86 to ⫹1 region as responsible for this delayed induction. Since nur77 transcription is rapidly suppressed following activation (13), it is possible that sequences located upstream of ⫺86 bp are responsible for this rapid repression. PGF2␣ was shown previously to cause PKC activation and increased [Ca2⫹]i. However, we have found no direct participation of PKC in the induction of Nur77, whereas an increase in intracellular Ca2⫹ appears to be a crucial event in the induction of Nur77 by PGF2␣. This conclusion is supported by our results showing a complete inhibition of Nur77 induction by the Ca2⫹ chelator, BAPTA, as well as the ability of two calcium ionophores to induce nur77 expression as efficiently as PGF2␣. CaM is a Ca2⫹-binding protein present in all eucaryotic cells, which is known to mediate Ca2⫹ effects (40). The ability of the CaM inhibitor, W7, to prevent induction of the nur77 gene by PGF2␣ indicates the participation of this protein in the PGF2␣ signaling pathway in luteal cells. It is clear, however, that PGF2␣-activated Ca2⫹-CaM complex does not involve the PP2b or CaM-K enzymes in luteal cells, although these proteins have previously been shown to play a role in Nur77 induction (36, 41). Instead our findings indicate that the PGF2␣-induced CaM activation leads to phosphorylation and activation of ERK1/2 kinase. PGF2␣ was previously shown to increase MAPK activity in human (4) and bovine (12) luteal cells. We have herein established that the ERK pathway is required for the induction of Nur77 by PGF2␣ in rat luteinized granulosa cells. Although it is possible that other components of the MAPK families (JNK and p38MAPK) may be activated by PGF2␣, our finding that specific inhibition of either MEK kinase with PD98059 or ERK1/2 kinase with UO126 was sufficient to abrogate PGF2␣-induced JunD phosphorylation as well as Nur77 gene activation suggests that ERK1/2 are the sole mediators of PGF2␣ action. The mechanism by which Ca2⫹-CaM activates the MAPK pathway is still not fully understood. The majority of the


FIG. 11. PGF2␣ induction of 20␣-HSD gene expression. Luteinized granulosa cells were transfected with the 2.5-kb 20␣-HSD-luc promoter. 24 h later, cells were treated with BAPTA/AM or W7 (A) or PD98059, UO126, or H9 (B) for 30 min. PGF2␣ was then added at a final concentration of 5 ␮M. The cells were incubated for 12 h before harvesting and lysing. C, cells were transfected with the 2.5-kb 20␣-HSD-luc promoter plus either an empty plasmid (⫺) or a dominant-negative mutant JunD expression vector (DNJunD). 24 h later, PGF2␣ was added to the medium at a final concentration of 5 ␮M. The cells were incubated for 12 h before harvesting and lysing. In all experiments transient expression of the reporter gene was quantified by a standard luciferase bioluminescence assay and normalized against ␤-galactosidase. Bars represent mean ⫾ S.E. of four experiments.


the PGF2␣ stimulation of 20␣-hsd expression in luteal cells. We have previously demonstrated that overexpression of a mutant Nur77 protein prevents PGF2␣ induction of 20␣-hsd (7). Here we have confirmed and further expanded upon our previous finding, to show that blockade of signaling pathway involved in nur77 induction also prevents 20␣-HSD promoter activation by PGF2␣. In summary, results of this investigation have unraveled the intracellular mechanism by which PGF2␣ regulates both nur77 and 20␣-hsd genes in the rat corpus luteum. REFERENCES 1. Niswender, G. D., Juengel, J. L., Silva, P. J., Rollyson, M. K., and McIntush, E. W. (2000) Physiol. Rev. 80, 1–29 2. Milvae, R. A. (2000) Rev. Reprod. 5, 1–5 3. Sugimoto, Y., Yamasaki, A., Segi, E., Tsuboi, K., Aze, Y., Nishimura, T., Oida, H., Yoshida, N., Tanaka, T., Katsuyama, M., Hasumoto, K., Murata, T., Hirata, M., Ushikubi, F., Negishi, M., Ichikawa, A., and Narumiya, S. (1997) Science 277, 681– 683 4. Tai, C. J., Kang, S. K., Choi, K. C., Tzeng, C. R., and Leung, P. C. (2001) J. Clin. Endocrinol. Metab. 86, 375–380 5. Miceli, F., Minici, F., Garcia Pardo, M., Navarra, P., Proto, C., Mancuso, S., Lanzone, A., and Apa, R. (2001) J. Clin. Endocrinol. Metab. 86, 811– 817 6. Ottander, U., Leung, C. H., and Olofsson, J. I. (1999) Mol. Hum. Reprod. 5, 391–395 7. Stocco, C. O., Zhong, L., Sugimoto, Y., Ichikawa, A., Lau, L. F., and Gibori, G. (2000) J. Biol. Chem. 275, 37202–37211 8. Gibori, G. (1993) in The Ovary (Adashi, E. Y., ed) pp. 261–317, Raven Press, Ltd., New York 9. Albarracin, C. T., Parmer, T. G., Duan, W. R., Nelson, S. E., and Gibori, G. (1994) Endocrinology 134, 2453–2460 10. Leung, P. C., and Steele, G. L. (1992) Endocr. Rev. 13, 476 – 498 11. McCracken, J. A., Custer, E. E., and Lamsa, J. C. (1999) Physiol. Rev. 79, 263–323 12. Chen, D. B., Westfall, S. D., Fong, H. W., Roberson, M. S., and Davis, J. S. (1998) Endocrinology 139, 3876 –3885 13. Wilson, T. E., Mouw, A. R., Weaver, C. A., Milbrandt, J., and Parker, K. L. (1993) Mol. Cell. Biol. 13, 861– 868 14. Yoon, J. K., and Lau, L. F. (1994) Mol. Cell. Biol. 14, 7731–7743 15. Woronicz, J. D., Calnan, B., Ngo, V., and Winoto, A. (1994) Nature 367, 277–281 16. van den Brink, M. R., Kapeller, R., Pratt, J. C., Chang, J. H., and Burakoff, S. J. (1999) J. Biol. Chem. 274, 11178 –11185 17. Sakaue, M., Adachi, H., Dawson, M., and Jetten, A. M. (2001) Cell Death Differ. 8, 411– 424 18. Woronicz, J. D., Lina, A., Calnan, B. J., Szychowski, S., Cheng, L., and Winoto, A. (1995) Mol. Cell. Biol. 15, 6364 – 6376 19. Zhong, L., Ou, J., Barkai, U., Mao, J. F., Frasor, J., and Gibori, G. (1998) Biochem. Biophys. Res. Commun. 249, 797– 803 20. Sharma, S. C., and Richards, J. S. (2000) J. Biol. Chem. 275, 33718 –33728 21. Karin, M. (1995) J. Biol. Chem. 270, 16483–16486 22. Pyrzynska, B., Mosieniak, G., and Kaminska, B. (2000) J. Neurochem. 74, 42–51 23. Yazgan, O., and Pfarr, C. M. (2001) Cancer Res. 61, 916 –920 24. Leppa, S., Saffrich, R., Ansorge, W., and Bohmann, D. (1998) EMBO J. 17, 4404 – 4413 25. Davis, J. S., Weakland, L. L., Weiland, D. A., Farese, R. V., and West, L. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3728 –3732 26. Sorin, B., Vacher, A. M., Djiane, J., and Vacher, P. (2000) J. Neuroendocrinol. 12, 910 –918 27. Ma, R., Pluznick, J., Kudlacek, P., and Sansom, S. C. (2001) J. Biol. Chem. 276, 25759 –25765 28. Means, A. R. (2000) Mol. Endocrinol. 14, 4 –13 29. Stull, J. T. (2001) J. Biol. Chem. 276, 2311–2312 30. Blaeser, F., Ho, N., Prywes, R., and Chatila, T. A. (2000) J. Biol. Chem. 275, 197–209 31. Liu, W., Youn, H. D., and Liu, J. O. (2001) Eur. J. Immunol. 31, 1757–1764 32. Enslen, H., Tokumitsu, H., Stork, P. J., Davis, R. J., and Soderling, T. R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10803–10808 33. Chang, L., and Karin, M. (2001) Nature 410, 37– 40 34. Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R. (1995) J. Biol. Chem. 270, 27489 –27494 35. Favata, M. F., Horiuchi, K. Y., Manos, E. J., Daulerio, A. J., Stradley, D. A., Feeser, W. S., Van Dyk, D. E., Pitts, W. J., Earl, R. A., Hobbs, F., Copeland, R. A., Magolda, R. L., Scherle, P. A., and Trzaskos, J. M. (1998) J. Biol. Chem. 273, 18623–18632 36. Cheng, Y., Zhizhin, I., Perlman, R. L., and Mangoura, D. (2000) J. Biol. Chem. 275, 23326 –23332 37. Kallunki, T., Deng, T., Hibi, M., and Karin, M. (1996) Cell 87, 929 –939 38. Rosenberger, S. F., Finch, J. S., Gupta, A., and Bowden, G. T. (1999) J. Biol. Chem. 274, 1124 –1130 39. Williams, G. T., and Lau, L. F. (1993) Mol. Cell. Biol. 13, 6124 – 6136 40. Sola, C., Barron, S., Tusell, J. M., and Serratosa, J. (2001) Int. J. Biochem. Cell Biol. 33, 439 – 455 41. Youn, H. D., Chatila, T. A., and Liu, J. O. (2000) EMBO J. 19, 4323– 4331


studies demonstrating that increased [Ca2⫹]i is able to activate ERK1 have been performed in excitable cells. For example, smooth muscle cell proliferation may occur via a Ca2⫹dependent activation of the MAPK pathway (42), and nerve growth factor activation of ERK in PC12 cells has been shown to require Ca2⫹ (43, 44). However, no studies to our knowledge have been conducted in endocrine cells. MAPK activation by tyrosine kinase receptors on critical tyrosine residues has been clearly established. The activation of MAPK by tyrosine kinase receptors has been clearly determined; these types of receptors cause MAPK phosphorylation on critical tyrosine residues. These residues then serve as docking sites for adaptor proteins such as Grb2, which in turn promote binding of the nucleotide exchange protein Sos. Sos protein promotes GDP/GTP exchange on Ras and thus switches it to the active form. Activated Ras then recruits Raf, the first kinase of the MAPK cascade, to the membrane, leading to sequential phosphorylation and activation of MEK and ERK (reviewed in Ref. 45). Most of the signal pathway involved in ERK activation downstream of increased [Ca2⫹]i are thought to converge at the level of Ras. Rusanescu et al. (46) have demonstrated that an increase in [Ca2⫹]i directly or indirectly induces Shc tyrosine phosphorylation and association with Grb2 and Sos. In addition protein tyrosine kinase 2 can also be activated by increased [Ca2⫹]i and then subsequently activate Ras (47). However, it is unlikely that these mechanisms mediate ERK1/2 activation in our system, since PGF2␣induced phosphorylation of ERK1/2 not only depends on Ca2⫹ but also on CaM. In agreement with our results there are reports suggesting that Ca2⫹ stimulation of Ras can also be mediated by CaM through activation of Ras-GTP exchange factors (48). Moreover, some CaM-binding Ras-like GTPase has been described (49). In our study, it is clear that inhibitors of the Ca 2⫹-CaM system totally prevented PGF 2␣induced ERK1/2 activation. Based on our findings that PD98059, an ERK1/2 kinase kinase (also known as Raf) inhibitor, blocked both JunD phosphorylation and nur77 induction, we postulate that ERK1/2 activation by CaM in luteal cells occurs upstream or at the level of Raf kinase. Chen and colleagues (12) have detected three isoforms of Raf kinase (A-Raf, B-Raf, and Raf-1 or c-Raf) in bovine luteal cells and have shown that Raf-1 and B-Raf, but not A-Raf, are activated by PGF2␣, further supporting the possibility that these kinases may be the target of PGF2␣-activated CaM. The participation of ERK1/2 in nur77 induction by PGF2␣ may also explain the superinduction of this transcription factor in the presence of protein inhibitors. Superinduction of immediate early genes by protein synthesis inhibitors can be manifested in three ways: (i) mRNA stabilization, (ii) activation of intracellular signaling cascades, and (iii) interference with transcriptional down-regulation (Ref. 50 and references therein). The existence of a labile repressor molecule, which would maintain genes such as nur77 in an inactive state, has been hypothesized. In this model, protein synthesis inhibitors deplete the cell of the short-lived repressor, causing superinduction. However, no such repressor molecules for nur77 have been identified to date. Protein synthesis inhibitors (anisomycin and cycloheximide) can also activate intracellular kinases, such as MAPK, similar to those activated by signal transduction cascades (51). This could potentially explain superinduction via direct promoter activation in the absence of a labile repressor. Further experiments will be necessary to determine the mechanism by which cycloheximide induces Nur77 in luteinized granulosa cells. Finally, we have demonstrated that the molecular mechanism by which PGF2␣ induces nur77 expression is involved in

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MECHANISMS OF SIGNAL TRANSDUCTION: A Calcium/Calmodulin-dependent Activation of ERK1/2 Mediates JunD Phosphorylation and Induction of nur77 and 20α-hsd Genes by Prostaglandin F2α in Ovarian Cells Carlos O. Stocco, Lester F. Lau and Geula Gibori

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J. Biol. Chem. 2002, 277:3293-3302. doi: 10.1074/jbc.M110936200 originally published online November 21, 2001