A Calcium/Calmodulin-dependent Activation of ERK1/2 Mediates JunD Phosphorylation and Induction of nur77 and20 -hsd Genes by Prostaglandin F2 in Ovarian Cells

Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, Illinois 60612, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2002; 277(5):3293-302. DOI: 10.1074/jbc.M110936200
Source: PubMed


We have previously demonstrated that prostaglandin F(2alpha) (PGF(2alpha)) 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 PGF(2alpha) induction of 20alpha-HSD, a steroidogenic enzyme involved in the catabolism of progesterone. In this report we established, using luteinized granulosa cells, that PGF(2alpha) stimulates in vitro nur77 expression in a time- and dose-dependent manner. Serial 5'-deletion of the nur77 promoter revealed that the necessary and sufficient elements for PGF(2alpha) 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 PGF(2alpha). However, mutation of the AP1 sites as well as a dominant-negative JunD abolished nur77 induction by PGF(2alpha). PGF(2alpha) 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. PGF(2alpha)-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 PGF(2alpha). Finally, we demonstrated that this molecular mechanism also mediates 20alpha-hsd induction.

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Available from: Lester F Lau, Dec 03, 2015
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    • "ERK phosphorylation was present mainly in the nucleus where it could act to phosphorylate transcription factors. It was reported that ERK was activated in response to PGF 2␣ , and resulted in the induction of the transcription factor Nur77, which subsequently induced the expression of 20␣-HSD, an enzyme that is responsible for the conversion of P 4 to an inactive 20␣-metabolite in rat luteal cells and ovarian cells (Stocco et al., 2002). In PGF 2␣ -treated bovine luteal cells, the RAF/MAPK kinase (MEK) 1/ERK pathway was identified as a proximal signaling event required for the induction of early growth response 1 and ultimately the expression of transforming growth factor B1 (TGFB1) (Hou et al., 2008). "
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    ABSTRACT: The current study was conducted to evaluate the expression of ATF3, in association with the activation of mitogen-activated protein kinases (MAPK) during prostaglandin F2α analog (PGF)-induced luteal regression in rats. A sequential PMSG/hCG treatment paradigm was used to obtain a single, well-defined generation of corpora lutea (CL) in rats. Rats were treated with PGF for 0-4h on day 7 of pseudopregnancy. Results showed that serum progesterone (P4) concentrations declined in a time dependent manner. Western blot results revealed that ATF3 increased within 2h post-PGF injection. Phosphorylated ERK1/2 (p-ERK) and JNK (p-JNK) increased within 30min and then were gradually reduced in response to PGF. In contrast, the levels of phosphorylated p38 MAPK (p-p38) were not significantly altered. The immunostaining density for p-ERK decreased from the periphery to the center of the corpus luteum following treatment with PGF, while ATF3 was expressed uniformly in the nuclei of luteal steroidogenic cells. These results indicated that treatment with PGF in vivo could induce increases in MAPK phosphorylation, especially in p-ERK, which might be correlated with the increases in ATF3 expression and the decline in P4 concentrations. To our knowledge, this is the first study to provide evidence for temporal relationships between MAPK activation and ATF3 expression during PGF-induced luteal regression in the rat. Copyright © 2015 Elsevier GmbH. All rights reserved.
    Full-text · Article · Jan 2015 · Acta Histochemica
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    • "Nur77 binds as monomer to the NGF1-B response element (NBRE) and as a homodimer or heterodimer to the Nur response element (NurRE) [6], [7]. Previous studies demonstrated that LH, the regulator of testicular steroidogenesis, induces Nur77 gene expression in Leydig cells [8] and that Nur77 regulates the expression of steroidogenic genes, including steroid 21-hydroxylase, 20α-hydroxysteroid dehydrogenase, and P450c17 [2], [9], [10]. Furthermore, Nur77-binding regions have been defined within the promoters of rat P450c17 [2], mouse StAR [11], and human 3β-HSD type 2 (3β-HSD2) [12] genes. "
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    ABSTRACT: Transforming growth factor- β1 (TGF-β1) has been reported to inhibit luteinizing hormone (LH) mediated-steroidogenesis in testicular Leydig cells. However, the mechanism by which TGF-β1 controls the steroidogenesis in Leydig cells is not well understood. Here, we investigated the possibility that TGF-β1 represses steroidogenesis through cross-talk with the orphan nuclear receptor Nur77. Nur77, which is induced by LH/cAMP signaling, is one of major transcription factors that regulate the expression of steroidogenic genes in Leydig cells. TGF-β1 signaling inhibited cAMP-induced testosterone production and the expression of steroidogenic genes such as P450c17, StAR and 3β-HSD in mouse Leydig cells. Further, TGF-β1/ALK5 signaling repressed cAMP-induced and Nur77-activated promoter activity of steroidogenic genes. In addition, TGF-β1/ALK5-activated Smad3 repressed Nur77 transactivation of steroidogenic gene promoters by interfering with Nur77 binding to DNA. In primary Leydig cells isolated from Tgfbr2flox/flox Cyp17iCre mice, TGF-β1-mediated repression of cAMP-induced steroidogenic gene expression was significantly less than that in primary Leydig cells from Tgfbr2flox/flox mice. Taken together, these results suggest that TGF-β1/ALK5/Smad3 signaling represses the expression of steroidogenic genes via the suppression of Nur77 transactivation in testicular Leydig cells. These findings may provide a molecular mechanism involved in the TGF-β1-mediated repression of testicular steroidogenesis.
    Full-text · Article · Aug 2014 · PLoS ONE
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    • "In addition, it was shown that the deletion of this promoter at the -289 bp positions significantly decreased the basal promoter activity [17]. PGF2α induces the expression of the nuclear orphan receptor and transcription factor Nur77 in the CL, which in turn leads to the stimulation of 20α-HSD, triggering a decrease in serum progesterone, which is essential for parturition [18]. Thus, Nur77 plays an important role in ovarian physiology by mediating the PGF2α-mediated induction of 20α-HSD in rats. "
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    ABSTRACT: Background Monkey 20α-hydroxysteroid dehydrogenase (20α-HSD) is a catabolic enzyme responsible for converting progesterone into biologically inactive 20α-hydroxyprogesterone, thereby playing a key role in the estrous cycle or pregnancy and allowing ovulation and parturition to occur in most mammalian animals. Monkey 20α-HSD was highly abundant in ovarian and placental tissues during the pre-ovulation and pre-parturition phase and was primarily localized in the syncytiotrophoblast of the placenta. In this study, we focused on the molecular characterization of the monkey 20α-HSD promoter region by conducting reporter assays in Chinese hamster ovary (CHO) K1 cells. Results A reporter assay using constructs of various lengths of the 5′-flanking region (-890-Luc, -513-Luc, -306-Luc, -273-Luc, and -70-Luc) revealed that a region corresponding to the activator protein 1 (Ap-1) located between -281 and -274 bp was essential for the transcriptional activity. Absence of the Ap-1 site in -273-Luc dramatically decreased the transcription levels to the control levels. When the reporter constructs were co-transfected with Ap-1 (Jun) and specificity protein (Sp-1) genes, the transcription activities of the constructs increased with the exception of -273 and -70, while that of the double construct was reduced compared to that of Ap-1 alone. Furthermore, mutational analysis demonstrated that a putative Ap-1 site played an important role in the expression of the reporter gene. These findings were confirmed by EMSA examining the interactions of the protein Ap-1 in a nuclear extract from CHO-K1 cells and the expression levels of the Ap-1 transcription factor in pre-parturition placenta and CHO-K1 cells. Although mut-1 and mut-2 of Ap-1 bound with nuclear extracts from CHO-K1 cells, the transcriptional activity of mut-3 was almost completely suppressed. Conclusions Our results indicate that the Ap-1 site (-281 → -274) (5′-TGTCTCAT-3′) plays a crucial role in the activation of the monkey 20α-HSD gene. Thus, we demonstrated that monkey 20α-HSD promoter activity is regulated by the transcription factor Ap-1 in CHO-K1 cells.
    Full-text · Article · Jul 2014 · BMC Biotechnology
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