Djurdjica Coss

Hormones in synergy: Regulation of the pituitary gonadotropin genes

The precise interplay of hormonal influences that governs gonadotropin hormone production by the pituitary includes endocrine, paracrine and autocrine actions of hypothalamic gonadotropin-releasing hormone (GnRH), activin and steroids. However, most studies of hormonal regulation of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the pituitary gonadotrope have been limited to analyses of the isolated actions of individual hormones. LHbeta and FSHbeta subunits have distinct patterns of expression during the menstrual/estrous cycle as a result of the integration of activin, GnRH, and steroid hormone action. In this review, we focus on studies that delineate the interplay among these hormones in the regulation of LHbeta and FSHbeta gene expression in gonadotrope cells and discuss how signaling cross-talk contributes to differential expression. We also discuss how recent technological advances will help identify additional factors involved in the differential hormonal regulation of LH and FSH.

A Novel AP-1 Site Is Critical for Maximal Induction of the Follicle-stimulating Hormone β Gene by Gonadotropin-releasing Hormone

Regulation of follicle-stimulating hormone (FSH) synthesis is a central point of convergence for signals controlling reproduction. The FSHβ subunit is primarily regulated by gonadotropin-releasing hormone (GnRH), gonadal steroids, and activin. Here, we identify elements in the mouse FSHβ promoter responsible for GnRH-mediated induction utilizing the LβT2 cell line that endogenously expresses FSH. The proximal 398 bp of the mouse FSHβ promoter is sufficient for response to GnRH. This response localizes primarily to an AP-1 half-site (–72/–69) juxtaposed to a CCAAT box, which binds nuclear factor-Y. Both elements are required for AP-1 binding, creating a novel AP-1 site. Multimers of this site confer GnRH induction, and mutation or internal deletion of this site reduces GnRH induction by 35%. The same reduction was achieved using a dominant negative Fos protein. This is the only functional AP-1 site identified in the proximal 398 bp, since its mutation eliminates FSHβ induction by c-Fos and c-Jun. GnRH regulation of the FSHβ gene occurs through induction of multiple Fos and Jun isoforms, forming at least four different AP-1 molecules, all of which bind to this site. Mitogen-activated protein kinase activity is required for induction of FSHβ and JunB protein. Finally, AP-1 interacts with nuclear factor-Y, which occupies its overlapping site in vivo.

FoxL2 Is Required for Activin Induction of the Mouse and Human Follicle-Stimulating Hormone β-Subunit Genes

Activin is a major physiological regulator of FSH. We identify FoxL2 as a critical component in activin induction of FSHbeta, both for the mouse gene, induction of which is Sma- and Mad-related protein (Smad) dependent, and for the human gene that is Smad independent. FoxL2 has been shown to regulate gonadotrope gene expression (GnRH receptor, alpha-glycoprotein subunit, porcine FSHbeta, and follistatin), but the mechanisms of action are not well understood. We identify novel sites required for activin action in both the mouse and human FSHbeta promoters, some of which bind FoxL2, and show that the FoxL2-binding element encompasses a larger region (12 bp) than the previously identified forkhead-binding consensus (7 bp). Remarkably, although required for activin induction, FoxL2 sites neither contribute to basal FSHbeta promoter activity nor confer activin response to a heterologous promoter; thus, they are neither classical activin-response elements nor is their role solely to recruit Smads to the promoter. FoxL2 overexpression can potentiate activin induction in gonadotropes and can confer activin responsiveness to FSHbeta in heterologous cells where this promoter is normally refractory to activin induction. Although Smad3 requires the presence of FoxL2 sites to induce mouse FSHbeta, even through its consensus Smad-binding element; the human promoter, which is induced by activin independently of Smad3, also requires FoxL2 sites for its induction by activin; thus the actions of FoxL2 are not exclusively through interactions with the Smad pathway. Thus, FoxL2 plays a key role in activin induction of the FSHbeta gene, by binding to sites conserved across multiple species.

A FoxL in the Smad house: activin regulation of FSH

Follicle-stimulating hormone (FSH), produced by pituitary gonadotrope cells, is required for maturation of ovarian follicles. The FSHbeta subunit is the limiting factor for production of mature hormone and provides biological specificity. Activin dramatically induces FSHbeta transcription and the secondary rise in FSH, important for follicular development, is dependent on this induction. Thus, regulation of FSHbeta levels by activin is crucial for female reproductive fitness. This review discusses activin signaling pathways, transcription factors and FSHbeta promoter elements required for activin responsiveness. Because FoxL2, a forkhead transcription factor, was recently shown to be instrumental in relaying activin signaling to the FSHbeta promoter, we focus in this paper on its role and the inter-relatedness of several key players in activin responsiveness on the FSHbeta promoter.

Dissociation of Janus Kinase 2 and Signal Transducer and Activator of Transcription 5 Activation after Treatment of Nb2 Cells with a Molecular Mimic of Phosphorylated Prolactin1

We have previously demonstrated that phosphorylated PRL acts as an antagonist to the Nb2 proliferative activities of unmodified PRL. A molecular mimic of phosphorylated PRL, which substitutes an aspartate residue for the normally phosphorylated serine (serine 179), has the same properties. Because it takes less than one fourth the amount of phosphorylated hormone, or the aspartate mutant, to block the proliferative activity of unmodified hormone, we have investigated whether the high potency of the aspartate mutant is achieved by the production of an alternate and interfering intracellular signal cascade. Nb2 cells were exposed to 5 or 500 ng/ml human NIDDK PRL, wild-type recombinant PRL (unmodified PRL), or aspartate mutant PRL (pseudophosphorylated PRL) for 1, 5, or 10 min at 37 C. At 5 ng/ml and 10 min, wild-type recombinant PRL showed greater activation of Janus kinase 2 (JAK 2) than the NIDDK preparation. This is consistent with a previous report of higher proliferative activity for the wild-type hor...

GnRH Induces the c-Fos Gene via Phosphorylation of SRF by the Calcium/Calmodulin Kinase II Pathway

Despite extensive studies on GnRH regulation of the gonadotropin subunit genes, very little is known about mechanism of induction of intermediary immediate early genes, such as c-Fos, that are direct targets of GnRH signaling and that upon induction, activate transcription of gonadotropin genes. Although c-Fos is induced by a variety of stimuli in other cell types, in the gonadotropes, only GnRH induces c-Fos and through it FSHβ. Thus, understanding the specificity of c-Fos induction by GnRH will provide insight into GnRH regulation of FSHβ gene expression. GnRH induction of c-Fos in LβT2 cells requires the serum response factor (SRF)-binding site, but not the Ets/ELK1 site. This is in contrast to c-Fos induction by growth factors in other cells, which activate c-Fos transcription via phosphorylation of ELK1 and require the ELK1-binding site. The SRF site alone is sufficient for induction by GnRH, whereas induction by 12-tetradecanoylphorbol-13-acetate (TPA) requires both the ELK1 and SRF sites. Although ELK1 site is not required, upon GnRH stimulation, ELK1 interacts with SRF and is recruited to the SRF site. GnRH phosphorylates ELK1 through ERK1/2 and p38 MAPK, which correlates with the signaling pathways necessary for c-Fos and FSHβ induction. GnRH also causes phosphorylation of SRF through calmodulin-dependent kinase II (CamKII), which leads to increased binding to its site. CamKII activation is sufficient for phosphorylation of SRF and for induction of the c-Fos gene through the SRF site. Thus, GnRH uses a combination of growth factor signaling and the CamKII pathway to induce c-Fos to regulate FSHβ gene expression in gonadotrope cells.

Kisspeptin regulates gonadotropin genes via immediate early gene induction in pituitary gonadotropes.

Kisspeptin signaling through its receptor, Kiss1R, is crucial for many reproductive functions including puberty, sex steroid feedback, and overall fertility. Although the importance of Kiss1R in the brain is firmly established, its role in regulating reproduction at the level of the pituitary is not well understood. This study presents molecular analysis of the role of kisspeptin and Kiss1R signaling in the transcriptional regulation of the gonadotropin gene β-subunits, LHβ and FSHβ, using LβT2 gonadotrope cells and murine primary pituitary cells. We show that kisspeptin induces LHβ and FSHβ gene expression, and this induction is protein kinase C dependent and mediated by the immediate early genes, early growth response factor 1 and cFos, respectively. Additionally, kisspeptin induces transcription of the early growth response factor 1 and cFos promoters in LβT2 cells. Kisspeptin also increases gonadotropin gene expression in mouse primary pituitary cells in culture. Furthermore, we find that Kiss1r expression is enhanced in the pituitary of female mice during the estradiol-induced LH surge, a critical component of the reproductive cycle. Overall, our findings indicate that kisspeptin regulates gonadotropin gene expression through the activation of Kiss1R signaling through protein kinase C, inducing immediate early genes in vitro, and responds to physiologically relevant cues in vivo, suggesting that kisspeptin affects pituitary gene expression to regulate reproductive function.

Prolactin receptor antagonists.

Most prolactin (PRL) in the circulation is produced by the pituitary. However, a wide variety of traditional target tissues of the hormone have also been shown to produce their own prolactin. The amount produced per cell is low, but may well be sufficient for autocrine/paracrine activity. Although dopamine agonists allow one to study the target tissue effects of pituitary PRL, other agents, such as PRL receptor (PRLR) antagonists, are needed to analyze autocrine/paracrine loops. With PRLR antagonists, it should be possible to dissect out the role of extrapituitary prolactin in both the normal physiology, and the pathophysiology of various tissues. In tissues where the locally produced PRL may promote disease, such antagonists have the potential to be important therapeutics.This article briefly, but critically, reviews current understanding of PRL-receptor interactions and initial signaling, and describes the development of both growth hormone (GH) and PRL antagonists within that context. In the final section, results with a very potent PRL antagonist further one theme of the article, which is whether the simple receptor dimerization model explains all signal transduction following PRLR binding.Most prolactin (PRL) in the circulation is produced by the pituitary. However, a wide variety of traditional target tissues of the hormone have also been shown to produce their own prolactin. The amount produced per cell is low, but may well be sufficient for autocrine/paracrine activity. Although dopamine agonists allow one to study the target tissue effects of pituitary PRL, other agents, such as PRL receptor (PRLR) antagonists, are needed to analyze autocrine/paracrine loops. With PRLR antagonists, it should be possible to dissect out the role of extrapituitary prolactin in both the normal physiology, and the pathophysiology of various tissues. In tissues where the locally produced PRL may promote disease, such antagonists have the potential to be important therapeutics. This article briefly, but critically, reviews current understanding of PRL-receptor interactions and initial signaling, and describes the development of both growth hormone (GH) and PRL antagonists within that context. In the final section, results with a very potent PRL antagonist further one theme of the article, which is whether the simple receptor dimerization model explains all signal transduction following PRLR binding.

Maternal prolactin composition can permanently affect epidermal γδT cell function in the offspring

There have been few studies aimed at determining the effects of maternal peptide hormones on the developing fetus and even fewer aimed at determining the long-term consequences of abnormalities in maternal hormone exposure. In this study, we have examined the effect of maternal prolactin (PRL) on the production, seeding and long-term function of a T lymphocyte subset for which the precursors are only present during fetal life. Using this system, we can determine long-term consequences of maternal hormone exposure without concern for the subsequent influence of the offsprings endocrine milieu. Recombinant versions of the two major forms of the pituitary hormone, PRL, were administered to rats throughout pregnancy. Administration of a molecular mimic of phosphorylated PRL (PP-PRL) resulted in a marked increase in the level of apoptosis in the thymus of newborn pups, an effect that was not duplicated by administration of unmodified PRL. The increased thymic apoptosis in the animals exposed to PP-PRL resulted in decreased epidermal seeding of γδT cells and a markedly decreased γδT cell-modulated epidermal response in the offspring. This decreased γδT cell modulated response persisted to adulthood. We conclude that maternal PRL composition during pregnancy can have a permanent effect on at least one component of the developing immune system.

GnRH Increases c-Fos Half-Life Contributing to Higher FSHβ Induction

GnRH is a potent hypothalamic regulator of gonadotropin hormones, LH and FSH, which are both expressed within the pituitary gonadotrope and are necessary for the stimulation of gametogenesis and steroidogenesis in the gonads. Differential regulation of LH and FSH, which is essential for reproductive fitness, is achieved, in part, through the varying of GnRH pulse frequency. However, the mechanism controlling the increase in FSH during the periods of low GnRH has not been elucidated. Here, we uncover another level of regulation by GnRH that contributes to differential expression of the gonadotropins and may play an important role for the generation of the secondary rise of FSH that stimulates folliculogenesis. GnRH stimulates LHβ and FSHβ subunit transcription via induction of the immediate early genes, Egr1 and c-Fos, respectively. Here, we determined that GnRH induces rapidly both Egr1 and c-Fos, but specifically decreases the rate of c-Fos degradation. In particular, GnRH modulates the rate of c-Fos protein turnover by inducing c-Fos phosphorylation through the ERK1/2 pathway. This extends the half-life of c-Fos, which is normally rapidly degraded. Confirming the role of phosphorylation in promoting increased protein activity, we show that a c-Fos mutant that cannot be phosphorylated by GnRH induces lower expression of the FHSβ promoter than wild-type c-Fos. Our studies expand upon the role of GnRH in the regulation of gonadotropin gene expression by highlighting the role of c-Fos posttranslational modification that may cause higher levels of FSH during the time of low GnRH pulse frequency to stimulate follicular growth.