Lhb Gene Expression Research Articles

FOXO1 Transcription Factor Inhibits Luteinizing Hormone β Gene Expression in Pituitary Gonadotrope Cells

Synthesis of luteinizing hormone (LH) is tightly controlled by a complex network of hormonal signaling pathways that can be modulated by metabolic cues, such as insulin. One group of candidate genes that may be regulated by insulin signaling in pituitary gonadotrope cells is the FOXO subfamily of forkhead transcription factors. In this study we investigated whether FOXO1 is expressed in gonadotropes and if it can modulate LH β-subunit (Lhb) gene expression. We demonstrated that FOXO1 is expressed in murine gonadotrope cells and that insulin signaling increased FOXO1 phosphorylation and cytoplasmic localization in a PI3K-dependent manner. We also showed that FOXO1 repressed basal transcription and gonadotropin-releasing hormone (GnRH) induction of both the murine and human LHB genes in LβT2 cells, suggesting that FOXO1 regulation of LHB transcription may be conserved between rodents and humans. Although we did not detect FOXO1 binding to the proximal Lhb promoter, the FOXO1 DNA binding domain was necessary for the suppression, suggesting that FOXO1 exerts its effect through protein-protein interactions with transcription factors/cofactors required for Lhb gene expression. FOXO1 repression mapped to the proximal Lhb promoter containing steroidogenic factor 1 (SF1), pituitary homeobox 1 (PTX1), and early growth response protein 1 (EGR1) binding elements. Additionally, FOXO1 blocked induction of the Lhb promoter with overexpressed SF1, PTX1, and EGR1, indicating that FOXO1 repression occurs via these transcription factors but not through regulation of their promoters. In summary, we demonstrate that FOXO1 phosphorylation and cellular localization is regulated by insulin signaling in gonadotropes and that FOXO1 inhibits Lhb transcription. Our study also suggests that FOXO1 may play an important role in controlling LH levels in response to metabolic cues.

FOXO Regulation of Gonadotropin Hormone Synthesis in Pituitary Gonadotropes.

Synthesis of luteinizing hormone (LH) is tightly controlled by a complex network of hormonal signaling pathways that may be regulated by metabolic cues, such as insulin. One group of candidate genes that may act as metabolic sensors in pituitary gonadotrope cells is the FOXO subfamily of forkhead transcription factors. We investigated whether FOXO1 is expressed in gonadotropes and if it can modulate LH beta (Lhb) gene expression. We demonstrated that FOXO1 is expressed in murine gonadotrope cells and that insulin signaling increased FOXO1 phosphorylation and cytoplasmic localization. We also showed that FOXO1 repressed basal transcription and GnRH induction of both the murine and human LHB genes in LβT2 cells, indicating that FOXO1 regulation of LHB transcription may be conserved between rodents and humans. Although we did not detect FOXO1 binding to the proximal Lhb promoter, the FOXO1 DNA-binding domain was necessary for the suppression, suggesting that FOXO1 exerts its effect through protein-protein interactions with transcription factors/cofactors required for Lhb gene expression. FOXO1 repression mapped to the proximal Lhb promoter containing SF1, PTX1 and EGR1 binding elements. FOXO1 also blocked induction of the Lhb promoter with overexpressed SF1, PTX1 and EGR1, indicating that FOXO1 repression occurs via these transcription factors but not through regulation of their promoters. In summary, we demonstrate that FOXO1 phosphorylation and cellular localization is regulated by insulin signaling in gonadotropes and that FOXO1 inhibits Lhb transcription. Our studies suggest that FOXO1 may modulate LH hormone levels in response to metabolic cues.

CREB Binding Protein (CBP) Activation Is Required for Luteinizing Hormone Beta Expression and Normal Fertility in Mice

Normal function of the hypothalamic-pituitary-gonadal axis is dependent on gonadotropin-releasing hormone (GNRH)-stimulated synthesis and secretion of luteinizing hormone (LH) from the pituitary gonadotroph. While the transcriptional coactivator CREB binding protein (CBP) is known to interact with Egr-1, the major mediator of GNRH action on the Lhb gene, the role of CBP in Lhb gene expression has yet to be characterized. We show that in the LβT2 gonadotroph cell line, overexpression of CBP augmented the response to GNRH and that knockdown of CBP eliminated GNRH responsiveness. While GNRH-mediated phosphorylation of CBP at Ser436 increased the interaction with Egr-1 on the Lhb promoter, loss of this phosphorylation site eliminated GNRH-mediated Lhb expression in LβT2 cells. In vivo, loss of CBP phosphorylation at Ser436 rendered female mice subfertile. S436A knock-in mice had disrupted estrous cyclicity and reduced responsiveness to GNRH. Our results show that GNRH-mediated phosphorylation of CBP at Ser436 is required for Egr-1 to activate Lhb expression and is a requirement for normal fertility in female mice. As CBP can be phosphorylated by other factors, such as insulin, our studies suggest that CBP may act as a key regulator of Lhb expression in the gonadotroph by integrating homeostatic information with GNRH signaling.

G Proteins and Autocrine Signaling Differentially Regulate Gonadotropin Subunit Expression in Pituitary Gonadotrope

Gonadotropin-releasing hormone (GnRH) acts at gonadotropes to direct the synthesis of the gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH). The frequency of GnRH pulses determines the pattern of gonadotropin synthesis. Several hypotheses for how the gonadotrope decodes GnRH frequency to regulate gonadotropin subunit genes differentially have been proposed. However, key regulators and underlying mechanisms remain uncertain. We investigated the role of individual G proteins by perturbations using siRNA or bacterial toxins. In LβT2 gonadotrope cells, FSHβ gene induction depended predominantly on Gα(q/11), whereas LHβ expression depended on Gα(s). Specifically reducing Gα(s) signaling also disinhibited FSHβ expression, suggesting the presence of a Gα(s)-dependent signal that suppressed FSH biosynthesis. The presence of secreted factors influencing FSHβ expression levels was tested by studying the effects of conditioned media from Gα(s) knockdown and cholera toxin-treated cells on FSHβ expression. These studies and related Transwell culture experiments implicate Gα(s)-dependent secreted factors in regulating both FSHβ and LHβ gene expression. siRNA studies identify inhibinα as a Gα(s)-dependent GnRH-induced autocrine regulatory factor that contributes to feedback suppression of FSHβ expression. These results uncover differential regulation of the gonadotropin genes by Gα(q/11) and by Gα(s) and implicate autocrine and gonadotrope-gonadotrope paracrine regulatory loops in the differential induction of gonadotropin genes.

The orphan nuclear receptors COUP-TFI and COUP-TFII regulate expression of the gonadotropin LHβ gene

Normal sexual development and reproductive function depend on precise temporal and quantitative expression of the pituitary gonadotropins, LH and FSH. LHβ-subunit gene expression is achieved by transcription factors acting at highly conserved and closely spaced cis-elements in the proximal 200 base pairs of the promoter. We now demonstrate that LHβ promoter activity is further regulated by the orphan nuclear receptors, chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and COUP-TFII). These data establish that COUP-TFs are expressed in primary pituitary gonadotropes and two gonadotrope-derived cell lines. COUP-TFs bind to two promoter regions in the LHβ gene which overlap but are distinct from two previously defined cis-elements for another orphan nuclear receptor, steroidogenic factor-1 (SF-1). Transient transfection studies demonstrated that COUP-TFs stimulate LHβ gene promoter activity in the absence of SF-1, but blunt SF-1-mediated stimulation of gene expression in a reporter construct containing both SF-1 cis-elements (GSEs). Evaluation of constructs containing mutations or truncations in the GSEs revealed a complex pattern of activation and inhibition by COUP-TF on this promoter, suggesting multiple mechanisms by which this factor modulates LHβ gene expression. To our knowledge, these data are the first to demonstrate COUP-TF expression and function in pituitary gonadotropes.

Regulation of Lhb and Egr1 gene expression by GNRH pulses in rat pituitaries is both c-Jun N-terminal kinase (JNK)- and extracellular signal-regulated kinase (ERK)-dependent.

Pulsatile GNRH regulates the gonadotropin subunit genes in a differential manner, with faster frequencies favoring Lhb gene expression and slower frequencies favoring Fshb. Early growth response 1 (EGR1) is critical for Lhb gene transcription. We examined GNRH regulation of EGR1 and its two corepressors, Ngfi-A-binding proteins 1 and 2 (NAB1 and NAB2), both in vivo and in cultured rat pituitary cells. In rats, fast GNRH pulses (every 30 min) stably induced Egr1 primary transcript (PT) and mRNA 2-fold (P < 0.05) for 1-24 h. In contrast, slow GNRH pulses (every 240 min) increased Egr1 PT at 24 h (6-fold; P < 0.05) but increased Egr1 mRNA 4- to 5-fold between 4 and 24 h. Both GNRH pulse frequencies increased EGR1 protein 3- to 4-fold. In cultured rat pituitary cells, GNRH pulses (every 60 min) increased Egr1 (PT, 2.5- to 3-fold; mRNA, 1.5- to 2-fold; P < 0.05). GNRH pulses had little effect on Nab1/2 PT/mRNAs either in vivo or in vitro. We also examined specific intracellular signaling cascades activated by GNRH. Inhibitors of mitogen-activated protein kinase 8/9 (MAPK8/9 [also known as JNK]; SP600125) and MAP Kinase Kinase 1 (MAP2K1 [also known as MEK1]; PD98059) either blunted or totally suppressed the GNRH induction of Lhb PT and Egr1 PT/mRNA, whereas the MAPK14 (also known as p38) inhibitor SB203580 did not. In summary, pulsatile GNRH stimulates Egr1 gene expression and protein in vivo but not in a frequency-dependent manner. Additionally, GNRH-induced Egr1 gene expression is mediated by MAPK8/9 and MAPK1/3, and both are critical for Lhb gene transcription.

Pulse Frequency-Dependent Gonadotropin Gene Expression by Adenylate Cyclase-Activating Polypeptide 1 in Perifused Mouse Pituitary Gonadotroph LbetaT2 Cells1

We examined how pulsatile stimulation with adenylate cyclase-activating polypeptide 1 (ADCYAP1) affected gonadotrophs. In static culture, gonadotropin-releasing hormone (GnRH) stimulated transcription of all the gonadotropin subunits. In contrast, ADCYAP1 increased common alpha-glycoprotein subunit gene (Cga) promoter activity but failed to increase luteinizing hormone beta (Lhb) and follicle-stimulating hormone beta (Fshb) promoters. Messenger RNAs for Lhb and Fshb were slightly but significantly increased by ADCYAP1 stimulation. The results of cotreatment of the cells with GnRH and ADCYAP1 was not different from the effects of GnRH alone on Lhb and Fshb transcriptional activities as well as on mRNA expressions. To determine the effect of pulsatile ADCYAP1 stimulation on gonadotropin subunit gene expression, perifused LbetaT2 cells were stimulated either at high frequency (5-min ADCYAP1 pulse every 30 min) or at low frequency (5-min ADCYAP1 pulse every 120 min). High-frequency ADCYAP1 pulses preferentially increased Lhb gene expression 2.29-fold +/- 0.15-fold, and low frequency pulses resulted in a 1.55-fold +/- 0.16-fold increase. Fshb gene expression was increased 1.87-fold +/- 0.3-fold by high-frequency ADCYAP1 pulses and 4.3-fold +/- 0.29-fold by low-frequency pulses. These results were similar to the frequency-specific effects of pulsatile GnRH. Follistatin (Fst) gene expression was specifically increased by high-frequency GnRH pulses. High-frequency ADCYAP1 pulses increased Fst to a larger extent (4.7-fold +/- 0.57-fold) than did low-frequency pulse (2.72-fold +/- 1.09-fold). ADCYAP1 receptor gene (Adcyap1r) expression was increased significantly following pulsatile GnRH regardless of pulse frequency. Low-frequency ADCYAP1 pulses, however, increased Adcyap1r expression (16.49-fold +/- 8.41-fold) to a larger extent than high frequency pulses did. In addition, high-frequency ADCYAP1 pulses specifically increased Gnrhr (GnRH receptor) expression by 4.38-fold +/- 0.81-fold; however, low-frequency pulses did not result in an increase. These results suggest that ADCYAP1, like GnRH, specifically regulates Lhb and Fshb subunit gene in a pulse frequency-specific manner. This regulation may involve alteration in numbers of GnRH and ADCYAP1 receptors as well as FST expression.

Rapid Effect of GNRH1 on Follicle-Stimulating Hormone Beta Gene Expression in LbetaT2 Mouse Pituitary Cells Requires the Progesterone Receptor1

Gonadotropin-releasing hormone (GNRH) activates the progesterone receptor (PGR) in pituitary cells and accentuates gonadotropin expression. We show that GNRH1 increases Fshb mRNA levels in LbetaT2 mouse pituitary cells within 8 h and is three times more effective than GNRH2. By contrast, GNRH1 and GNRH2 do not affect Lhb gene expression in these cells. Within the same time frame, small interfering RNA (siRNA) knockdown of the PGR in LbetaT2 cells reduced GNRH1 activation of a PGR response element (PRE)-driven luciferase reporter gene and Fshb mRNA levels by >50%. Chromatin immunoprecipitation (ChIP) assays also demonstrated that PGR loading on the PRE within the Fshb gene promoter in LbetaT2 cells occurred within 8 h after GNRH1 treatment and was lost by 24 h. While the GNRH1-induced upregulation of the PRE reporter gene and Fshb mRNA levels was attenuated by cotreatment with protein kinase A (H-89) and protein kinase C (GF109203X) inhibitors, only GF109203X inhibited PGR phosphorylation at Ser249 in LbetaT2 cells. Immunoprecipitation assays also showed a progressive increase in the interaction between the PGR and its coactivator NCOA3 that peaked at 8 h coincident with the increase in Fshb mRNA after GNRH1 treatment. The siRNA-mediated knockdown of NCOA3 in LbetaT2 cells also reduced Fshb mRNA levels after GNRH1 treatment and loading of NCOA3 on the Fshb promoter PRE in a ChIP assay. We conclude that the rapid effect of GNRH1 on Fshb expression in LbetaT2 cells is mediated by PGR phosphorylation and loading at the PRE within the Fshb promoter together with NCOA3.

Progesterone Inhibits Basal and Gonadotropin-Releasing Hormone Induction of Luteinizing Hormone β-Subunit Gene Expression

LH and FSH play critical roles in mammalian reproduction by mediating steroidogenesis and gametogenesis in the gonad. Gonadal steroid hormone feedback to the hypothalamus and pituitary influences production of the gonadotropins. We previously demonstrated that progesterone differentially regulates the expression of the LH and FSH beta-subunits at the level of the gonadotrope: FSHbeta transcription is induced, whereas LHbeta is repressed. In this study, we investigated the mechanism of progesterone repression of LHbeta gene expression using immortalized gonadotrope-derived LbetaT2 cells. The progesterone suppression of both basal and GnRH-induced LHbeta gene expression occurs in a hormone- and receptor-dependent manner. Chromatin immunoprecipitation demonstrates that the hormone-bound progesterone receptor (PR) is recruited to the endogenous mouse LHbeta promoter. In addition, suppression requires both the amino-terminal and DNA-binding regions of PR. Furthermore, progesterone suppression does not require direct PR binding to the promoter, and, thus, PR is likely recruited to the promoter via indirect binding through other transcription factors. These data demonstrate that the molecular mechanism for progesterone action on the LHbeta promoter is distinct from FSHbeta, which involves direct PR binding to the promoter to produce activation. It also differs from androgen repression of LHbeta gene expression in that, rather than Sp1 or steroidogenic factor-1 elements, it requires elements within -300/-250 and -200/-150 that also contribute to basal expression of the LHbeta promoter. Altogether, our data indicate that progesterone feedback at the level of the pituitary gonadotrope is likely to play a key role in differential production of the gonadotropin genes.

Transcriptional regulation of follistatin expression by GnRH in mouse gonadotroph cell lines: Evidence for a role for cAMP signaling

GnRH applied continuously or in pulses of high frequency increases follistatin, and thereby differentially regulates FSH and LH. This study was conducted in αT3-1 and LβT2 gonadotroph cells to begin to understand the signaling pathways through which GnRH stimulates follistatin synthesis. GnRH increased follistatin expression and stimulated a follistatin-LUC reporter in LβT2 cells, but was inactive in αT3-1 cells. GnRH also increased cAMP levels and stimulated a cAMP-responsive promoter only in LβT2 cells. Forskolin stimulated follistatin in both cell lines. GnRH activation of follistatin was blocked by the PKA inhibitor H89 and by over-expression of a dominant-negative inhibitor of CREB (A-CREB). Activation was also suppressed by PKC depletion, and was reduced by the PKC inhibitor bisindolylmaleimide. The MEK inhibitor PD98059 blocked activation by GnRH or forskolin implying that MAPK contributes to cAMP/PKA-mediated activation of follistatin. When LβT2 cells were transfected with follistatin-LUC together with A-CREB, and perifused with GnRH, activation was blocked during continuous GnRH, but stimulation by hourly GnRH pulses was unaffected. These experiments provide evidence that GnRH stimulates follistatin through multiple signaling pathways, and that cAMP-CREB activation is obligatory when GnRH is applied continuously. The finding that follistatin transcription was CREB-dependent with continuous but not pulsatile GnRH implies that the mode of ligand activation of GnRH receptors modifies the transcriptional response by changing the signaling network. These results provide a mechanism linking GnRH pulsatility to the differential control of FSH-β and LH-β gene expression through follistatin.

Dietary daidzein and puerarin do not affect pituitary LH expression but exert uterotropic effects in ovariectomized rats

Objective: To investigate the potency of LH suppression, as an indirect measure of alleviation of postmenopausal vasomotor symptoms, as well as the uterotropic effects of two isoflavones: daidzein and puerarin in an ovariectomized (ovx) rat model and compare them with the effects of 17β-estradiol benzoate (E2B). Design: Eighty female Sprague–Dawley rats were ovx and divided into six different treatment groups and one control group (11–12 animals per group). Daidzein, puerarin and E2B were added to the soy free rodent chow in low and high doses (250 mg and 1000 mg per kg, 600 mg and 3000 mg per kg and 4.3 mg and 17.3 mg per kg, respectively). After 3 months of treatment, animals were sacrificed and using real time RT-PCR, pituitary LHβ and uterine IGF-1, PR and C3 mRNA levels were measured. Additionally serum LH levels were measured in a radioimmunoassay. Results: Both of our tested isoflavones at low and high doses had no effect on the expression of the pituitary LH at the mRNA and protein level. Only E2B at both doses significantly decreased pituitary LHβ gene expression and serum LH levels. Daidzein and puerarin at high dose increased significantly uterine weights. Uterine IGF-1 gene expression was only upregulated in puerarin high group. Uterine PR mRNA levels were higher in animals fed with low dose daidzein and high dose puerarin. Uterine C3 gene expression was upregulated in animals fed with daidzein and puerarin at high doses. Although statistically significant, all these effects were however very discrete compared to those of E2B at low and high doses. Conclusion: We speculate that due to the lack of LH suppressing effects in our model, it is very unlikely for daidzein and puerarin to alleviate vasomotor symptoms in postmenopausal women. In contrast, due to their uterotropic effects, high dose consumption of commercially available preparations containing daidzein or puerarin may expose women with an intact uterus to the risk of endometrial hyperplasia.

Reciprocal Cross Talk between Gonadotropin-Releasing Hormone (GnRH) and Prostaglandin Receptors Regulates GnRH Receptor Expression and Differential Gonadotropin Secretion

The asynchronous secretion of gonadotrope LH and FSH under the control of GnRH is crucial for ovarian cyclicity but the underlying mechanism is not fully resolved. Because prostaglandins (PG) are autocrine regulators in many tissues, we determined whether they have this role in gonadotropes. We first demonstrated that GnRH stimulates PG synthesis by induction of cyclooxygenase-2, via the protein kinase C/c-Src/phosphatidylinositol 3'-kinase/MAPK pathway in the LbetaT2 gonadotrope cell line. We then demonstrated that PGF(2alpha) and PGI2, but not PGE2 inhibited GnRH receptor expression by inhibition of phosphoinositide turnover. PGF(2alpha), but not PGI2 or PGE2, reduced GnRH-induction of LHbeta gene expression, but not the alpha-gonadotropin subunit or the FSHbeta subunit genes. The prostanoid receptors EP1, EP2, FP, and IP were expressed in rat gonadotropes. Incubations of rat pituitaries with PGF(2alpha), but not PGI2 or PGE2, inhibited GnRH-induced LH secretion, whereas the cyclooxygenase inhibitor, indomethacin, stimulated GnRH-induced LH secretion. None of these treatments had any effect on GnRH-induced FSH secretion. The findings have thus elaborated a novel GnRH signaling pathway mediated by PGF(2alpha)-FP and PGI2-IP, which acts through an autocrine/paracrine modality to limit autoregulation of the GnRH receptor and differentially inhibit LH and FSH release. These findings provide a mechanism for asynchronous LH and FSH secretions and suggest the use of combination therapies of GnRH and prostanoid analogs to treat infertility, diseases with unbalanced LH and FSH secretion and in hormone-dependent diseases such as prostatic cancer.

Insulin augments GnRH-stimulated LHβ gene expression by Egr-1

Previous studies have shown that insulin augments GnRH-stimulated LH synthesis and release from primary gonadotrophs. In this study, regulation of LHβ gene expression by GnRH and insulin was examined in LβT2 cells. Endogenous LHβ mRNA is stimulated 2.4-fold by insulin alone, 2.6-fold by GnRH alone, and 4.7-fold by insulin together with GnRH. This effect of insulin, like GnRH, mapped to sequences −140 to +1 in the mouse LHβ gene. Insulin together with GnRH stimulates activity of an LHβ-reporter gene 7.1-fold; whereas, GnRH alone or insulin alone stimulates the reporter activity 2.8- and 3.1-fold, respectively. Blocking the binding of Egr-1 to sequences −51 to −42 in the LHβ gene inhibits effects of insulin and GnRH. Insulin together with GnRH increases Egr-1 mRNA levels and total Egr-1 binding to LHβ DNA. These findings indicate that insulin may impact regulation of the reproductive axis at the level of the pituitary.

Pituitary homeobox 1 (Pitx1) stimulates rat LHβ gene expression via two functional DNA-regulatory regions

Luteinizing hormone (LH) plays a central role in the reproductive axis, stimulating both gonadal steroid biosynthesis and the development of mature gametes. Over the past decade, significant progress has been made in characterizing the transcription factors and associated DNA-regulatory sites which mediate expression of the LH beta-subunit gene (LHbeta). One of these factors, pituitary homeobox 1 (Pitx1), has been shown to stimulate LHbeta gene promoter activity, both alone and in synergy with the orphan nuclear receptor, steroidogenic factor-1 (SF-1), and the early growth response gene 1 (Egr-1). Prior reports have attributed the Pitx1 response to a cis-element located at position -101 in the rat LHbeta gene promoter. While investigating the role of Pitx1 in regulating rat LHbeta gene expression, we observed a small, but significant, residual Pitx1 response despite mutation or deletion of this site. In the studies presented here, we identify the presence of a second functional Pitx1 region spanning positions -73 to -52 in the rat LHbeta gene promoter. Based on electrophoretic mobility shift assay, Pitx1 binds to both the initially described 5'Pitx1 site as well as this putative 3'Pitx1 region. In transient transfection analysis, mutation of the LHbeta-3'Pitx1 site significantly blunted Pitx1 responsiveness, with elimination of the Pitx1 response in a construct containing mutations in both Pitx1 cis-elements. We also analyzed the importance of each of these Pitx1 sites for providing functional synergy with SF-1 and with Egr-1. We observed a markedly decreased synergistic response with mutation of the 5'Pitx1 site with further loss following mutation of the 3'Pitx1 site. In contrast, functional interaction between Pitx1 and Egr-1 persisted with mutation of both Pitx1 regions. We conclude that Pitx1 stimulates the rat LHbeta gene promoter via two Pitx1 DNA-regulatory regions. These results further our understanding of the molecular mechanisms that regulate expression of this critical reproductive gene promoter.

Small nuclear RING finger protein stimulates the rat luteinizing hormone-beta promoter by interacting with Sp1 and steroidogenic factor-1 and protects from androgen suppression.

GnRH controls expression of the LH subunit genes, alpha and LHbeta, with the LHbeta subunit regulated most dramatically. Two enhancer regions, distal and proximal, on the rat LHbeta gene promoter cooperate for full basal expression and GnRH stimulation. It has been hypothesized that the transcription factors binding to these regions, Sp1, Egr-1, and steroidogenic factor 1 (SF-1), may interact directly or indirectly via a coactivator. One such coactivator may be small nuclear RING finger protein (SNURF), which is expressed in pituitary tissue and the LbetaT2 gonadotrope cell line. In transfection experiments in LbetaT2 cells, SNURF stimulated basal expression of LHbeta and increased overall GnRH stimulation. SNURF specifically stimulated LHbeta, with no effect on the alpha-subunit promoter. SNURF interacts with Sp1 and SF-1, but not Egr-1, in pull-down experiments. Point mutations or deletions of SNURF functional domains demonstrated that Sp1 and SF-1 interactions with SNURF are required for SNURF stimulatory effects on the LHbeta promoter. Endogenous SNURF is associated with the LHbeta promoter on native chromatin, suggesting that it plays a physiological role in LHbeta gene expression. SNURF also binds the androgen receptor, and SNURF overexpression overcomes androgen suppression of GnRH-stimulated LHbeta but not alphasubunit promoter activity. SNURF mutations that disrupt Sp1 or SF-1 binding eliminate rescue by SNURF. We conclude that SNURF may mediate interactions between the distal and proximal GnRH response regions of the LHbeta promoter to stimulate transcription and can also protect the promoter from androgen suppression.

Hypophyseal gene expression profiles of FSH-β, LH-β, and glycoprotein hormone-α subunits in Ictalurus punctatus throughout a reproductive cycle

A determination of the seasonal changes in the expression of the genes encoding the subunits of gonadotropic hormones is an important first step in the understanding of the molecular control of the onset of puberty and the reproductive cycle in fish. In this study, the abundance of transcripts encoding the glycoprotein hormone α (GpH-α), follicle-stimulating hormone β (FSH-β), and luteinizing hormone β (LH-β) subunits in pituitaries of female channel catfish were systematically tracked throughout an annual reproductive cycle. All three genes showed a concurrent elevation coinciding with the onset of ovarian recrudescence but then each showed a second elevation at different times of the ovarian cycle. In addition to the initial peak at recrudescence, the expression of FSH-β and GpH-α gene peaked again during mid- and late-vitellogenic growth, respectively. The LH-β gene expression remained low during the phases of regression and vitellogenic growth but was moderately elevated (7-fold) at the onset of ovarian recrudescence and dramatically elevated (36-fold) just prior to spawning (June–July) when the FSH-β levels were at their lowest. The expression patterns of FSH-β and LH-β are remarkably similar to the ovarian expression of their respective receptors.

Transcriptional regulation of the Luteinizing Hormone Beta-Subunit (LHβ) gene by Ptx1

Objective: Pituitary homeobox 1 (Ptx1) is critical for normal pituitary development and has more recently been shown to be important for expression of pituitary-specific genes in the adult, including the LHβ gene. Separate studies have demonstrated that the orphan nuclear receptors, steroidogenic factor-1 (SF-1) and chicken ovalbumin upstream promoter-transcription factor (COUP-TF), also mediate LHβ gene expression. The specific objective of this study was to further characterize transcriptional regulation of the LHβ gene by Ptx1, alone and in conjunction with SF-1 and the COUP-TFs. Design: LHβ promoter activity was measured in the presence or absence of the transcription factors of interest using a transient transfection-reporter system. Electrophoretic mobility shift assay (EMSA) was used to evaluate Ptx1 binding to LHβ gene sequences. Materials and Methods: A) Monkey kidney fibroblast cells (CV-1) were transiently transfected with rat LHβ promoter sequences subcloned into the luciferase reporter vector, pXP2. Cells were cotransfected with CMV-driven expression vectors for Ptx1, SF-1 and/or COUP-TF. Results are expressed as fold-change relative to expression in cells receiving equivalent amounts of empty expression vector. Statistical significance was determined by ANOVA followed by Student’s t test with significance set at p < 0.05. B) EMSA was used to determine the ability of a GST-Ptx1 fusion protein or endogenous Ptx1 (from the mouse gonadotrope cell line αT3–1) to bind to various rat LHβ promoter sequences. Results: A) Consistent with previous reports by our group and others, addition of Ptx1 significantly increased expression of region −797/+5 of the rat LHβ promoter (6-fold). Introduction of SF-1 also stimulated LHβ promoter activity (37-fold), with marked synergy in the presence of both factors (over 200-fold) (p < 0.01). Mutation of a previously identified Ptx1 cis-element at position -101 decreased (2.6-fold), but did not eliminate, the Ptx1 effect in a −207/+5 construct (p < 0.005 versus control). The Ptx1-SF-1 synergy was lost in this mutated construct. 5’deletion constructs further localized the residual Ptx1 response to nucleotides −82/+5, a region with three potential Ptx-1 cis-elements based on sequence homology to the consensus site. Overexpression of either COUP-TFI or COUP-TFII blunted the Ptx1 response by 75% and 50%, the SF-1 response by 50% and 30%, and the SF-1/Ptx1 response by 85% and 70%, respectively (p < 0.0001 in the presence versus the absence of the COUP-TFs). B) By EMSA, both endogenous Ptx1 and GST-Ptx1 bind to an oligonucleotide spanning the -101 LHβ-Ptx1 cis-element as confirmed by supershift of the protein-DNA complex with a Ptx1-specific antibody. The Ptx1 fusion protein also bound to the 3’Ptx1 region, although with lower affinity than to the −101 element. Oligonucleotides containing mutations in the putative 3’Ptx1 sites demonstrated loss of binding with mutation at position −65, but not at the other two sites. Conclusions: Ptx1 increases transcription of the rat LHβ gene via two cis-elements. SF-1 synergizes with Ptx1 at the 5’element, but apparently not at the 3’ element. Overexpression of the COUP-TFs blunts both the SF-1 response and the Ptx1 response, although the mechanism by which this occurs remains to be elucidated. These data further our understanding of both stimulatory and inhibitory control of gonadotropin gene expression. Supported by: R01 HD38089 (LMH, CDH, ZH) and HD19938 (K-HJ, UBK).

Effects of gonadotropin-releasing hormone agonist and testosterone on gonadotropin subunit gene expression in female red seabream (Pagrus major)

We examined the effects of gonadotropin-releasing hormone agonist (GnRHa) and testosterone (T) on the level of gonadotropin subunit mRNAs in the pituitary of ovariectomized or intact female red seabream. Ovariectomy induced increase of seabream (sb) GnRH, glycoprotein (GP) α and luteinizing hormone (LH) β mRNA levels. GnRHa treatment also stimulated GPα and LHβ mRNA levels. T treatment reduced GPα and LHβ mRNA expression probably via negative feedback action on sbGnRH. Both GnRHa and T treatment had no effect on follicle-stimulating hormone (FSH) β mRNA levels. These results suggest that the regulatory mechanisms of GPα and LHβ gene expression differ from those of FSHβ gene.

Effects of 4-nonylphenol on gene expression of pituitary hormones in juvenile Atlantic salmon ( Salmo salar)

Alkylphenols such as 4-nonylphenol (NP) are one of the wide variety of environmental chemicals reported to have estrogenic effects in both in vitro and in vivo studies. Induction of eggshell zona radiata proteins (Zrp) and vitellogenin (Vtg) mRNA and protein synthesis in the liver are widely used biomarkers for xenoestrogen exposure in fish. However, little work has been done to characterize the molecular effects of xenoestrogens on other potential target organs such as the pituitary. To evaluate pituitary effects and develop new potential biomarkers for xenoestrogens, the influences of NP and 17β-estradiol (E2) on the mRNA levels of pituitary gonadotropic hormone (GTH) beta subunits [leutinizing hormone beta (LHβ or GTH IIβ) and follicle stimulating hormone beta (FSHβ or GTH Iβ)], prolactin (PRL), growth hormone (GH) and the pituitary specific transcription factor (Pit-1) were investigated in individual male and female juvenile Atlantic salmon ( Salmo salar), 3 days after a single intraperitoneal (i.p.) injection. In one experiment, fish were injected with NP (125 mg/kg body weight (BW)) or E2 (5 mg/kg BW) and a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method was used to analyze LHβ and FSHβ mRNA levels. In the second experiment, fish were injected with three doses of NP (10, 50, 125 mg/kg BW) or a single dose of E2 (5 mg/kg BW) and Northern blot analysis was used to quantify LHβ, FSHβ, PRL, GH and Pit-1 mRNAs. Both NP (50 and 125 mg/kg BW) and E2 significantly induced LHβ mRNA levels ( P<0.01), but only in females. The highest dose of NP (125 mg/kg BW) significantly induced Pit-1 mRNA in males ( P<0.01). NP did not have significant effects on any of the other pituitary transcripts. NP induced LHβ mRNA synthesis in females by up to 6-fold and the changes appeared to correlate with the increases in hepatic Vtg and Zrp mRNA levels. The results show that LHβ mRNA assay in female juvenile salmonids may be used as a marker for pituitary effects of xenoestrogens. The data also suggest that NP may have the potential to perturb the regulation of LHβ gene expression by mimicking E2.

GnRH receptor signaling in tilapia pituitary cells: role of mitogen-activated protein kinase (MAPK)

The role of mitogen-activated protein kinase (MAPK, also known as extracellular signal regulated kinase; ERK) stimulation in gonadotropin-releasing hormone (GnRH) signaling was investigated in cultured pituitary cells of tilapia hybrids (Oreochromis niloticus x O. aureus). Exposure of the cells to salmon GnRH (sGnRH) resulted in a dose- and time-dependent elevation in ERK levels. The PKC activator, 1-O-tetradecanoyl phorbol-13-acetate (TPA) increased kinase levels, while addition of GnRH had no further effect. However, chronic exposure to TPA resulted in reduction of basal and GnRH-induced ERK elevation. When PKC was inhibited by GF109203X, the GnRH-elevated ERK levels were totally abolished. The role of MAPK activation on GPα, FSHβ and LHβ gene expression was determined by administration of MAPK-kinase (MEK) inhibitor (PD98059; PD). This inhibitor completely blocked GnRH-induced increases in ERK activity. Furthermore, it suppressed GPα and LHβ mRNA responses to GnRH, but had no effect on FSHβ transcript levels. PD also decreased basal LHβ mRNA levels. These results indicate that in tilapia pituitary cells, GnRH activates MAPK cascade in a PKC-dependent manner. ERK is involved in GnRH elevation of GPα and LHβ, but not in FSHβ genes transcription.