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• W2092120029 abstract "We have previously demonstrated that prostaglandin F2α (PGF2α) 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 PGF2α induction of 20α-HSD, a steroidogenic enzyme involved in the catabolism of progesterone. In this report we established, using luteinized granulosa cells, that PGF2αstimulates in vitro nur77 expression in a time- and dose-dependent manner. Serial 5′-deletion of thenur77 promoter revealed that the necessary and sufficient elements for PGF2α induction of Nur77 promoter activity are located between the nucleotides −86 and −33 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 PGF2α. However, mutation of the AP1 sites as well as a dominant-negative JunD abolished nur77 induction by PGF2α. PGF2α induces phosphorylation of JunD bound to the nur77 promoter. Stimulation ofnur77 expression and JunD phosphorylation were prevented by inhibitors of calcium, calmodulin, or ERK1/2 kinase. PGF2α-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 PGF2α. Finally, we demonstrated that this molecular mechanism also mediates20α-hsd induction. We have previously demonstrated that prostaglandin F2α (PGF2α) 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 PGF2α induction of 20α-HSD, a steroidogenic enzyme involved in the catabolism of progesterone. In this report we established, using luteinized granulosa cells, that PGF2αstimulates in vitro nur77 expression in a time- and dose-dependent manner. Serial 5′-deletion of thenur77 promoter revealed that the necessary and sufficient elements for PGF2α induction of Nur77 promoter activity are located between the nucleotides −86 and −33 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 PGF2α. However, mutation of the AP1 sites as well as a dominant-negative JunD abolished nur77 induction by PGF2α. PGF2α induces phosphorylation of JunD bound to the nur77 promoter. Stimulation ofnur77 expression and JunD phosphorylation were prevented by inhibitors of calcium, calmodulin, or ERK1/2 kinase. PGF2α-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 PGF2α. Finally, we demonstrated that this molecular mechanism also mediates20α-hsd induction. prostaglandin F2α 20α-hydroxysteroid dehydrogenase reverse transcriptase electrophoretic mobility shift assay dominant-negative phorbol 12-myristate 13-acetate mitogen-activated protein mitogen-activated protein kinase extracellular signal-regulated kinase c-Jun NH2-terminal kinase calmodulin Ca/CaM-dependent protein kinase 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid protein kinase C cAMP-dependent protein kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase phosphatase 2B luciferase 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 F2α(PGF2α)1 is involved in the inhibition of progesterone production and luteal regression in many mammalian species. Thus, PGF2α causes luteal regression in domestic ruminants (1Niswender G.D. Juengel J.L. Silva P.J. Rollyson M.K. McIntush E.W. Physiol. Rev. 2000; 80: 1-29Crossref PubMed Scopus (774) Google Scholar); accordingly, administration of PGF2α to cycling cows is a common practice used to synchronize the sexual cycle for the purpose of artificial insemination (2Milvae R.A. Rev. Reprod. 2000; 5: 1-5Crossref PubMed Scopus (52) Google Scholar). In pregnant mice, the absence of PGF2α receptor causes a failure in parturition due to the lack of luteal regression and subsequent high levels of progesterone in circulation (3Sugimoto 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. Narumiya S. Science. 1997; 277: 681-683Crossref PubMed Scopus (531) Google Scholar). Human luteal cells also produce and respond to PGF2α with a decrease in progesterone production (4Tai C.J. Kang S.K. Choi K.C. Tzeng C.R. Leung P.C. J. Clin. Endocrinol. Metab. 2001; 86: 375-380PubMed Google Scholar, 5Miceli F. Minici F. Garcia Pardo M. Navarra P. Proto C. Mancuso S. Lanzone A. Apa R. J. Clin. Endocrinol. Metab. 2001; 86: 811-817Crossref PubMed Scopus (28) Google Scholar, 6Ottander U. Leung C.H. Olofsson J.I. Mol. Hum. Reprod. 1999; 5: 391-395Crossref PubMed Scopus (18) Google Scholar). We have recently studied the mechanism by which PGF2αtriggers the fall in progesterone production by the corpus luteum at the end of pregnancy in rodents. We have shown that PGF2αinduces a rapid and massive expression of the luteal enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD) (7Stocco C.O. Zhong L. Sugimoto Y. Ichikawa A. Lau L.F. Gibori G. J. Biol. Chem. 2000; 275: 37202-37211Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). This enzyme catabolizes progesterone into an inactive metabolite, 20α-OH-progesterone, which cannot support pregnancy. Therefore, expression of 20α-HSD results in decreased luteal progesterone secretion and parturition (8Gibori G. Adashi E.Y. The Ovary. Raven Press, Ltd., New York1993: 261-317Google Scholar, 9Albarracin C.T. Parmer T.G. Duan W.R. Nelson S.E. Gibori G. Endocrinology. 1994; 134: 2453-2460Crossref PubMed Scopus (80) Google Scholar). We have also established that PGF2α stimulation of the 20α-hsd gene requires the transcription factor Nur77, which is induced by PGF2α in the corpus luteum of pregnant rats prior to the induction of 20α-hsd (7Stocco C.O. Zhong L. Sugimoto Y. Ichikawa A. Lau L.F. Gibori G. J. Biol. Chem. 2000; 275: 37202-37211Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). However, the signaling mechanism by which PGF2α stimulates nur77 and20α-hsd gene expressions remains largely unknown. PGF2α-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 (10Leung P.C. Steele G.L. Endocr. Rev. 1992; 13: 476-498Crossref PubMed Scopus (216) Google Scholar, 11McCracken J.A. Custer E.E. Lamsa J.C. Physiol. Rev. 1999; 79: 263-323Crossref PubMed Scopus (595) Google Scholar). The antisteroidogenic effects of PGF2α 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. 1Niswender G.D. Juengel J.L. Silva P.J. Rollyson M.K. McIntush E.W. Physiol. Rev. 2000; 80: 1-29Crossref PubMed Scopus (774) Google Scholar). It has also been demonstrated that PGF2α activates the mitogen-activated protein kinase (MAPK) signaling cascade in bovine (12Chen D.B. Westfall S.D. Fong H.W. Roberson M.S. Davis J.S. Endocrinology. 1998; 139: 3876-3885Crossref PubMed Scopus (77) Google Scholar) and human (4Tai C.J. Kang S.K. Choi K.C. Tzeng C.R. Leung P.C. J. Clin. Endocrinol. Metab. 2001; 86: 375-380PubMed Google Scholar) luteal cells. However, little is known about the downstream signaling events that mediate the cellular responses or the signaling mechanisms by which PGF2α 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 (13Wilson T.E. Mouw A.R. Weaver C.A. Milbrandt J. Parker K.L. Mol. Cell. Biol. 1993; 13: 861-868Crossref PubMed Scopus (232) Google Scholar), by nerve growth factor and membrane depolarization via Ca2+ and AP1 proteins (14Yoon J.K. Lau L.F. Mol. Cell. Biol. 1994; 14: 7731-7743Crossref PubMed Scopus (53) Google Scholar), and by T-cell receptors via Ca2+ (15Woronicz J.D. Calnan B. Ngo V. Winoto A. Nature. 1994; 367: 277-281Crossref PubMed Scopus (508) Google Scholar). MAPK signaling has been shown to be involved in the induction of nur77 in excitable cells such as muscle and neuron cells (16van den Brink M.R. Kapeller R. Pratt J.C. Chang J.H. Burakoff S.J. J. Biol. Chem. 1999; 274: 11178-11185Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 17Sakaue M. Adachi H. Dawson M. Jetten A.M. Cell Death Differ. 2001; 8: 411-424Crossref PubMed Scopus (32) Google Scholar). These studies suggested to us that PKC, [Ca2+]i, or MAPK signaling may also be involved in PGF2α induction of Nur77 in luteal cells. In this investigation, we examined the mechanism by which PGF2α signals to the nur77 and20α-hsd genes in luteinized granulosa cells. We have shown that PGF2α 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 20α-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 PGF2α in ovarian cells. [α-32P]Deoxycytidine triphosphate ([α32P]dCTP) was purchased from Amershan Biosciences, Inc.; Advantage RT-for-PCR kit was purchased fromCLONTECH (Palo Alto, CA); dNTP, ExTaqDNA 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). PGF2α, d-glucose, Tri-Reagent, 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. Luteinized granulosa cells were obtained and cultured as described previously (7Stocco C.O. Zhong L. Sugimoto Y. Ichikawa A. Lau L.F. Gibori G. J. Biol. Chem. 2000; 275: 37202-37211Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). 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 (18Woronicz J.D. Lina A. Calnan B.J. Szychowski S. Cheng L. Winoto A. Mol. Cell. Biol. 1995; 15: 6364-6376Crossref PubMed Scopus (193) Google Scholar). The −33-bp Nur77-luc was generated by PCR following standard cloning techniques. The 2.5-kb 20α-HSD-luc reporter construct has been described previously (19Zhong L. Ou J. Barkai U. Mao J.F. Frasor J. Gibori G. Biochem. Biophys. Res. Commun. 1998; 249: 797-803Crossref PubMed Scopus (16) Google Scholar). 24 h after transfection, the cells were treated as indicated in the legends of Figs. 2, 4, 8, and12. 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 μl of passive lysis buffer (Promega, Madison, WI) was added to each well. 20 μl 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 μl of the cell lysate was used to measure β-galactosidase activity using the CLONTECH β-galactosidase Assay System (CLONTECH). Relative light units were obtained by dividing the Nur77 promoter luciferase activity by the β-galactosidase activity.Figure 4JunD mediates Nur77 activation by PGF2α. A:left panel, endogenous Nur77 mRNA levels in luteinized granulosas cells transfected with none (−), 0.1, or 0.5 μg/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 PGF2α (5 μm), 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 μg/well of a dominant-negative JunD expression (DNJunD) vector or an empty vector (−). 24 h later, cells were treated with PGF2α (5 μm) 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 PGF2α (columns 2,4, and 5) or vehicle (columns 1 and3) for 6 h before luciferase activity determination. Experiments were repeated three times; results from a representative set are shown. Bars represent mean ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Extracellular regulated kinase 1 and 2 mediates the PGF2α 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 PGF2α(5 μm, 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 PGF2αtreatment (5 μm, 6 h). Experiments were repeated five times, and results from a representative set are shown.Bars represent mean ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 12Mechanism of Nur77 and 20α-HSD inductions by PGF2α in luteal cells.PGF2α induces the expression of nur77 through a Ca2+-CaM-dependent mechanism. The Ca2+-CaM complex formed upon PGF2α treatment causes activation of the ERK1/2 MAP kinase pathway. ERK1/2 phosphorylates JunD, increasing its transcriptional activity. Expression of Nur77 activates the 20α-HSD promoter leading to the expression of this progesterone-catabolizing enzyme.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Nuclear extracts were extracted as described above, and the samples were processed by Western blot as described previously (7Stocco C.O. Zhong L. Sugimoto Y. Ichikawa A. Lau L.F. Gibori G. J. Biol. Chem. 2000; 275: 37202-37211Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). 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.). 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 μl 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 μg of nuclear extract 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 μg/ul of poly(dI-dC), 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. Total RNA isolation and RT-PCR reaction were performed as described previously (7Stocco C.O. Zhong L. Sugimoto Y. Ichikawa A. Lau L.F. Gibori G. J. Biol. Chem. 2000; 275: 37202-37211Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). 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. 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. We first examined whether luteinized granulosa cells can be used in primary culture to explore PGF2α signaling to thenur77 gene. The results show that PGF2αinduces nur77 expression in luteinized granulosa cells in a time- (Fig. 1A) and dose- (Fig. 1B) dependent manner. To examine whether PGF2αstimulation of nur77 depends upon de novo protein synthesis, the effect of the protein synthesis inhibitor cycloheximide on the nur77 gene induction by PGF2α was examined. Luteinized granulosa cells were treated with 5 μm PGF2α in the presence (Fig. 1C,lanes 3, 5, 7, and 8) or absence (lanes 1, 2, 4, and6) of cycloheximide (10 mg/ml) for different times. Treatment with PGF2α in the presence of cycloheximide induced significantly higher nur77 gene expression than PGF2α 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 PGF2α treatment (Fig. 1C, lanes 5and 7). Treatment with cycloheximide alone for 3 h increased nur77 expression (Fig. 1C, lane 8). To examine the mechanism of nur77 induction by PGF2α, 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 β-galactosidase, allowing for normalization of transfection efficiencies. Using this promoter, we observed a 7-fold stimulation of luciferase activity following treatment with PGF2α (Fig. 2A). A marked decrease in PGF2α 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 PGF2α stimulation are located between the −86 and −33 region, since deletion of this region totally abolished the stimulatory effect of PGF2α (Fig. 2A). Time course experiments (Fig. 2B) revealed a rapid stimulation of the 1.2-kb Nur77-luc reporter construct by PGF2α, 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) Nur77-luc constructs were used, no stimulation was observed after 1 h of treatment. However, PGF2αstimulated 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 PGF2α-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 PGF2α, and mutation of both AP1 sites (Fig. 2C,lane 5) fully prevented PGF2α stimulation. 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 PGF2α 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 PGF2α in agreement with the mutation studies showed in Fig. 2C. Studies by Sharma and Richards (20Sharma S.C. Richards J.S. J. Biol. Chem. 2000; 275: 33718-33728Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) 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, whereasc-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 was seen after the addition of a JunD antibody to the nuclear extracts from both control and PGF2α-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 (14Yoon J.K. Lau L.F. Mol. Cell. Biol. 1994; 14: 7731-7743Crossref PubMed Scopus (53) Google Scholar). These results indicate that JunD binds to the Nur77 promoter, but this binding is not stimulated by PGF2α. 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 PGF2α. Since we could not demonstrate a causative relationship betweennur77 expression and increased JunD DNA binding, we then hypothesized that PGF2α may increase JunD activity. Transcriptional activity of JunD can be regulated by phosphorylation of serine residues located in the amino-terminal domain (21Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2258) Google Scholar, 22Pyrzynska B. Mosieniak G. Kaminska B. J. Neurochem. 2000; 74: 42-51Crossref PubMed Scopus (43) Google Scholar). Most notable in this regard are regulatory phosphorylations occurring on Ser90 and Ser100 within the transactivation domain (23Yazgan O. Pfarr C.M. Cancer Res. 2001; 61: 916-920PubMed Google Scholar). Therefore, we next tested whether PGF2αinduces 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 PGF2α-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 PGF2α(Fig. 3B, lower panel). The amount of total JunD remained the same regardless of PGF2α treatment, as shown by the use of a pan JunD antibody. To confirm the participation of JunD in the induction of Nur77 by PGF2α, we sought to determine whether the induction ofnur77 expression could be altered by a dominant-negative mutant JunD (DN-JunD). Luteinized granulosa cells were transfected with either a dominant-negative JunD expression vector, or with empty vector, and were treated with or without PGF2α (Fig.4A). This DN-JunD protein is lacking its DNA binding domain but retains the capacity to form homodimers and heterodimers (14Yoon J.K. Lau L.F. Mol. Cell. Biol. 1994; 14: 7731-7743Crossref PubMed Scopus (53) Google Scholar). As shown in Fig. 4A,left panel, the strong induction of the endogenousnur77 gene by PGF2α 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 the DN-JunD expression vector or empty vector. They were then treated with either vehicle or PGF2α (Fig.4A, right panel). DN-JunD protein inhibited the PGF2α-induced −86-bp Nur77-luc reporter construct activity in a dose-dependent manner (Fig.4A, right panel). At a 1-μg concentration the DN-JunD expression vector completely abolished this induction. To further investigate the role of JunD protein in the PGF2α-mediated stimulation of nur77, we overexpressed a wild type JunD protein in luteinized granulosa cells and treated the cells with either vehicle of PGF2α. 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 PGF2α, a remarkable increase in promoter activity was observed. The induction of Nur77 promoter activity in JunD-transfected and PGF2α-treated cells was severalfold higher than in nontransfected cells treated with PGF2α. These results indicate that JunD is necessary, but not enough, to inducenur77 expression and that phosphorylation of the already present JunD protein by PGF2α is essential for the activation of the nur77 gene. To further study the functional role of JunD Ser100phosphorylation in the induction of nur77 by PGF2α, a plasmid encoding a modified JunD protein in which the serine 100 residue 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 (24Leppa S. Saffrich R. Ansorge W. Bohmann D. EMBO J. 1998; 17: 4404-4413Crossref PubMed Scopus (291) Google Scholar). No synergism between the overexpression of JunDAla and PGF2α 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 PGF2αstimulation of nur77 expression. We next examined the intracellular mechanism by which PGF2α may induce phosphorylation of JunD and nur77 expression. Two known intracellular mediators of PGF2α action in the corpus luteum are PKC and Ca2+ (1Niswender G.D. Juengel J.L. Silva P.J. Rollyson M.K. McIntush E.W. Physiol. Rev. 2000; 80: 1-29Crossref PubMed Scopus (774) Google Scholar, 11McCracken J.A. Custer E.E. Lamsa J.C. Physiol. Rev. 1999; 79: 263-323Crossref PubMed Scopus (595) Google Scholar, 25Davis J.S. Weakland L.L. Weiland D.A. Farese R.V. West L.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3728-3732Crossref PubMed Scopus (168) Google Scholar). 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 PGF2α was added to the medium at a concentration or 5 μm, and the cells were cultured for 1 h before mRNA isolation. As expected, PGF2α induced Nur77; however, neither staurosporine, calphostin C, nor sustained PMA treatment affected PGF2α stimulation of nur77 (Fig.5A). To test whether Ca2+ is involved, we used the membrane-permeable Ca2+ chelator BAPTA/AM (25 μm), to inhibit free intracellular Ca2+, and either the Ca2+ ionophores A23187 (1 mm) or ionomicyn (2 μm) to increase intracellular Ca2+. As shown in Fig. 5B, PGF2α induction of Nur77 mRNA expression was completely prevented by BAPTA/AM, whereas A23187 was able to induce endogenous Nur77 expression as effectively as PGF2α. Similar results were also obtained with ionomicyn (data not shown). To further investigate the lack of participation of PKC activation in the induction of Nur77 by PGF2α, luteinized granulosa cells were treated with two different PKC activators, PMA or phorbol 12," @default.
• W2092120029 created "2016-06-24" @default.
• W2092120029 creator A5008672669 @default.
• W2092120029 creator A5014230378 @default.
• W2092120029 creator A5051408647 @default.
• W2092120029 date "2002-02-01" @default.
• W2092120029 modified "2023-09-26" @default.
• W2092120029 title "A Calcium/Calmodulin-dependent Activation of ERK1/2 Mediates JunD Phosphorylation and Induction of nur77 and20α-hsd Genes by Prostaglandin F2α in Ovarian Cells" @default.
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