Liquid biopsy-based analysis by ddPCR and CAPP-Seq in melanoma patients.

BACKGROUND: The development of BRAF/MEK inhibitors in patients with metastatic melanoma harboring BRAF mutations has garnered attention for liquid biopsy to detect BRAF mutations in cell-free DNA (cfDNA) using droplet digital PCR (ddPCR) or next-generation sequencing methods. OBJECTIVE: To investigate gene mutations in tumor DNA and cfDNA collected from 43 melanoma patients and evaluate their potential as biomarkers. METHODS: ddPCR and CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) techniques were performed to detect gene mutations in plasma cfDNA obtained from patients with metastatic melanoma. RESULTS: Gene variants, including BRAF, NRAS, TP53, GNAS, and MET, were detectable in the plasma cfDNA, and the results were partially consistent with the results of those identified in the tissues. Among the variants examined, copy numbers of MET mutations were consistent with the disease status in two melanoma patients. CONCLUSION: Liquid biopsy using CAPP-Seq and ddPCR has the potential to detect tumor presence and mutations, especially when tissue biopsies are unavailable. MET mutations in cfDNA may be a potential biomarker in patients with metastatic melanoma.

Existence of Staphylococcus aureus correlates with the progression of extramammary Paget's disease: potential involvement of interleukin-17 and M2-like macrophage polarization.

BACKGROUND: The microbiome plays an important role in the tumour microenvironment (TME). OBJECTIVES: In this study, we investigated the clinical significance of the microbiota in extramammary Paget's disease (EMPD). MATERIALS & METHODS: Patients with EMPD, treated between March 2007 and September 2019 at Kumamoto University Hospital, were investigated retrospectively. Inclusion criteria included: histological diagnosis of EMPD, inspection of the bacterial culture of the cancer lesion using swab sampling, and availability of sufficient tissue in paraffin blocks for immunohistochemistry. For the latter, primary antibodies against IL-17, CD163 and ionized calcium-binding adapter molecule 1 (Iba1) were used. RESULTS: Bacterial cultures of the cancer lesion revealed that Staphylococcus aureus (S. aureus) was highly prevalent in EMPD patients, with dermal invasion or lymph node metastasis, compared to patients without these findings. Furthermore, the number of IL-17-positive cells and CD163-positive M2-like macrophages (pro-tumour macrophages) were increased in EMPD tissues with S. aureus. Moreover, the number of IL-17-producing cells in EMPD tissues positively correlated with the accumulation of CD163-positive M2-like macrophages. In addition, the percentage of CD163-positive cells within Iba-1-positive macrophages (total macrophages) was also significantly elevated in EMPD tissues with S. aureus. CONCLUSION: Based on these findings, S. aureus may exacerbate the pathological condition of EMPD via the accumulation of IL-17 and M2-like macrophages.

GLP-1 increases Kiss-1 mRNA expression in kisspeptin-expressing neuronal cells.

Feeding-related metabolic factors exert regulatory influences on the hypothalamic-pituitary-gonadal axis. Glucagon-like peptide-1 (GLP-1) is an anorexigenic hormone synthesized from the ileum in response to food intake. The purpose of this study was to examine the direct effect of GLP-1 on hypothalamic kisspeptin and gonadotropin-releasing hormone (GnRH) expression using the rat clonal hypothalamic cell line rHypoE-8. GLP-1 significantly increased Kiss-1 mRNA expression in rHypoE-8 cells up to 1.94 ± 0.22-fold. This effect of GLP-1 on Kiss-1 gene expression was also observed in GT1-7 GnRH-producing neurons and in primary cultures of fetal rat brain. GLP-1 increased cAMP-mediated signaling, as determined by cAMP response element activity assays, but failed to activate extracellular signal-regulated kinase pathways. Another anorexigenic factor, leptin, similarly increased Kiss-1 mRNA levels up to 1.34 ± 0.08-fold in rHypoE-8 cells. However, combined treatment with GLP-1 and leptin failed to potentiate their individual effects on Kiss-1 mRNA expression. Gnrh mRNA expression was not significantly increased by GLP-1 stimulation in rHypoE-8, but kisspeptin significantly stimulated the expression of Gnrh mRNA in these cells. Our current observations suggest that the anorexigenic peptide GLP-1 directly regulates Kiss-1 mRNA expression in these hypothalamic cell lines and in neuronal cells of fetal rat brain and affects the expression of Gnrh mRNA.

Interaction between kisspeptin and adenylate cyclase-activating polypeptide 1 on the expression of pituitary gonadotropin subunits: a study using mouse pituitary lbetaT2 cells.

We examined direct effect of kisspeptin on pituitary gonadotrophs. Kisspeptin-10 (KP10) significantly increased the promoter activities of the gonadotropin subunits, common alpha-glycoprotein (Cga), luteinizing hormone beta (Lhb), and follicle-stimulatinghormone beta (Fshb) in LbetaT2 cells overexpressing kisspeptin receptor (Kiss1r). KP10 and gonadotropin-releasing hormone (GnRH) increased gonadotropin subunit levels to similar degrees and combined treatment with GnRH and KP10 did not potentiate their individual effects. Adenylate cyclase-activating polypeptide 1 (ADCYAP1) also stimulates all three gonadotropin subunits. When cells were stimulated with both KP10 and ADCYAP1, expression of gonadotropin subunits was further increased compared to KP10 or ADCYAP1 alone. KP10 and GnRH dramatically increased serum response element (Sre) promoter levels but only slightly increased cAMP response element (Cre) promoter levels. Combined stimulation with KP10 and GnRH further increased Sre promoter levels. In contrast, ADCYAP1 slightly increased Sre promoter expression but did not modify the effect of KP10. However, ADCYAP1 increased Cre promoter to greater levels than KP10 alone, and combined treatment with KP10 and ADCYAP1 further increased Cre promoter expression. KP10 increased the expression of ADCYAP1 type I receptor (Adcyap1r) and the basal activity of the Cga promoter was increased at a higher Adcyap1r transfection level. The KP10-induced fold increase in all three gonadotropin subunit promoters was not altered by transfection with a higher amount of Adcyap1r vector. Our findings using model cells show that distinct signaling activation by ADCYAP1 potentiates the action of KP10. We also found that KP10 increases Adcyap1r expression.

How is GnRH regulated in GnRH-producing neurons? Studies using GT1-7 cells as a GnRH-producing cell model.

Hypothalamic secretion of gonadotropin-releasing hormone (GnRH) has been established as a principle pathway for initiating and integrating female reproductive function. GnRH stimulates the release of two gonadotropins-luteinizing hormone and follicle-stimulating hormone-from the anterior pituitary, which eventually stimulate the synthesis of sex steroids in association with follicular growth and ovulation. This reproductive control of the hypothalamic-pituitary-gonadal (HPG) axis also mediates gonadal feedback mechanisms. Although GnRH neurons certainly play a pivotal role in the HPG axis, the detailed mechanisms of their functional network, including regulatory systems, remain unknown. After the discovery of the indispensable role of kisspeptin in the development of human reproductive functions, our understanding of the neuroendocrine regulation of the HPG axis was revolutionized, and it is now recognized that kisspeptin acts upstream of GnRH and is responsible for sex steroid feedback mechanisms. Kisspeptin can stimulate gonadotropin release from the pituitary gland by stimulating GnRH release and GnRH antagonists prevent kisspeptin-induced gonadotropin release. Furthermore, it has been shown that GnRH neurons express kisspeptin receptors. Nevertheless, the detailed mechanisms underlying the regulation of homogeneous populations of GnRH neurons are still largely unknown because of the limitations of experimental models used for investigation. The hypothalamus consists of a complex network of distinct neuronal cells, and it is difficult to isolate single-cell populations of GnRH neurons. The establishment of GnRH-expressing cell lines has allowed us to examine the events happening at the single-cell level. In this review, we describe in vitro studies using a GnRH-producing cell model, GT1-7 cells, which have been used to examine how GnRH-producing cells respond to hypothalamic factors and how they are involved in GnRH synthesis.

Mutual regulation by GnRH and kisspeptin of their receptor expression and its impact on the gene expression of gonadotropin subunits.

Hypothalamic kisspeptin plays a pivotal role in the regulation of the hypothalamic-pituitary-gonadal axis by stimulating gonadotropin-releasing hormone (GnRH) release into the portal circulation, with the subsequent release of gonadotropins. Kisspeptin and its receptor, the kisspeptin 1 receptor (Kiss1R), are also expressed in the pituitary gland. This study demonstrates the interaction between GnRH and kisspeptin within the pituitary gonadotrophs by altering their individual receptor expression. Our results show that kisspeptin and Kiss1R are expressed in the mouse pituitary gonadotroph cell line LßT2. Endogenous Kiss1R did not respond to kisspeptin and failed to stimulate gonadotropin LHß and FSHß expression in LßT2 cells; however, kisspeptin increased both LHß and FSHß promoter activity in Kiss1R-overexpressing LßT2 cells. Stimulating the cells with GnRH significantly increased Kiss1R expression, whereas kisspeptin increased the expression of the GnRH receptor (GnRHR) in these cells. Elevating the Kiss1R concentration led to an increase in the basal activities of gonadotropin LHß- and FSHß-subunit promoters. In addition, the level of kisspeptin-induced LHß promoter activity, but not that of FSHß, was significantly increased when a large number of Kiss1R expression vectors was introduced into the cells. The level of induction of GnRH-induced gonadotropin promoter activities was not significantly changed by increasing Kiss1R expression. Increasing the amount of GnRHR by overexpressing cellular GnRHR did not potentiate basal gonadotropin promoter activities; however, kisspeptin- and GnRH-stimulated increases in gonadotropin promoter activities were significantly potentiated (except GnRH-induced LHß promoters). The activities of serum response element-containing promoters were also modified in cells overexpressing Kiss1R or GnRHR. Our current observations demonstrate that GnRH and kisspeptin affect each other's function to stimulate gonadotropin subunit gene expression by reciprocally increasing the expression of their receptors.

Expression of GnRH and Kisspeptin in Primary Cultures of Fetal Rat Brain.

Genetic studies in humans or in vivo studies using animals have shown that kisspeptin released from the hypothalamus controls secretion of gonadotropin-releasing hormone (GnRH) from GnRH neurons, and subsequently GnRH induces gonadotropin secretion from the anterior pituitary. Kisspeptin did not stimulate GnRH expression in the GnRH-producing cell line GT1-7. Thus, we cultured GnRH and kisspeptin neurons from whole fetal rat brain and examined the regulation of GnRH and kisspeptin. Expression of GnRH messenger RNA (mRNA) was unchanged by estradiol (E2) treatment in these primary cultures. In contrast, kisspeptin mRNA expression was increased 2.00 ± 0.23-fold by E2 treatment. When these cultures were stimulated by kisspeptin-10, GnRH mRNA was significantly increased up to 1.51 ± 0.35-fold. Expression of GnRH mRNA was also stimulated 1.84 ± 0.33-fold by GnRH itself. Interestingly, kisspeptin mRNA was significantly increased up to 2.43 ± 0.40-fold by kisspeptin alone. In addition, kisspeptin mRNA expression was significantly increased by stimulation with GnRH (1.46 ± 0.21-fold). Our observations demonstrated that kisspeptin, but not GnRH, was upregulated by E2 and that kisspeptin stimulates GnRH mRNA expression in primary cultures of whole fetal rat brain. Furthermore, GnRH and kisspeptin stimulate their own neurons to produce GnRH or kisspeptin, respectively.

Retinoic acid and retinaldehyde dehydrogenase are not involved in the specific induction of the follicle-stimulating hormone ß subunit by trichostatin A, a selective inhibitor of histone deacetylase.

The selective histone deacetylase inhibitor, trichostatin A (TSA), increases follicle-stimulating hormone ß subunit (FSHß) mRNA expression but not α- and luteinizing hormone ß (LHß)-subunits in both the pituitary gonadotrophic cell line LßT2 and primary cultures of rat anterior pituitary cells. TSA increased histone acetylation in whole cell lysates in both cells. In addition, retinaldehyde dehydrogenases (RALDHs), which are retinoic acid (RA)-synthesizing enzymes, were induced by TSA in these cells. Anacardic acid, a histone acetyltransferase inhibitor that prevents histone acetylation, significantly inhibited TSA-induced FSHß mRNA expression as well as TSA-induced RALDH2 and RALDH3 mRNA expression. Similar to the effect of TSA, gonadotropin-releasing hormone stimulated RALDH expression in LßT2 cells. RA directly applied to the pituitary cells stimulated the transcriptional activity of the FSHß promoter. In addition, α- and LHß-subunit promoters were also activated by RA. Our results suggest that TSA specifically increases FSHß expression with a concomitant increase in RALDHs; however, RALDH and RA are not directly involved in the specific regulation of FSHß by TSA.

Interactions between Two Different G Protein-Coupled Receptors in Reproductive Hormone-Producing Cells: The Role of PACAP and Its Receptor PAC1R.

Gonadotropin-releasing hormone (GnRH) and gonadotropins are indispensable hormones for maintaining female reproductive functions. In a similar manner to other endocrine hormones, GnRH and gonadotropins are controlled by their principle regulators. Although it has been previously established that GnRH regulates the synthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH)-both gonadotropins-from pituitary gonadotrophs, it has recently become clear that hypothalamic GnRH is under the control of hypothalamic kisspeptin. Prolactin, which is also known as luteotropic hormone and is released from pituitary lactotrophs, stimulates milk production in mammals. Prolactin is also regulated by hypothalamic factors, and it is thought that prolactin synthesis and release are principally under inhibitory control by dopamine through the dopamine D2 receptor. In addition, although it remains unknown whether it is a physiological regulator, thyrotropin-releasing hormone (TRH) is a strong secretagogue for prolactin. Thus, GnRH, LH and FSH, and prolactin are mainly regulated by hypothalamic kisspeptin, GnRH, and TRH, respectively. However, the synthesis and release of these hormones is also modulated by other neuropeptides in the hypothalamus. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a hypothalamic peptide that was first isolated from sheep hypothalamic extracts based on its ability to stimulate cAMP production in anterior pituitary cells. PACAP acts on GnRH neurons and pituitary gonadotrophs and lactotrophs, resulting in the modulation of their hormone producing/secreting functions. Furthermore, the presence of the PACAP type 1 receptor (PAC1R) has been demonstrated in these cells. We have examined how PACAP and PAC1R affect GnRH- and pituitary hormone-secreting cells and interact with their principle regulators. In this review, we describe our understanding of the role of PACAP and PAC1R in the regulation of GnRH neurons, gonadotrophs, and lactotrophs, which are regulated mainly by kisspeptin, GnRH, and TRH, respectively.

Expression and Regulation of Pituitary Adenylate Cyclase-Activating Polypeptide in Rat Placental Cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) was first identified as a hypophysiotropic factor that regulates pituitary cell functions and has been subsequently shown to be widely distributed and have multiple functions. The PACAP is known to be expressed in placental tissues and is suggested to have a critical role in physiological function of the placenta. In addition to PACAP, the hypothalamic peptides kisspeptin and gonadotropin-releasing hormone (GnRH) are also expressed in placental cells. In this study, we used primary cultures of placental tissues from rats of 16 to 18 days gestation and examined the regulation and function of PACAP. The PACAP messenger RNA (mRNA) expression and PACAP-immunoreactive cells were detected in primary cultures of rat placental cells. The PACAP mRNA expression in placental cells was upregulated in the presence of the sex steroids estradiol and progesterone; however, their combined treatment failed to enhance their individual effects. When the cells were stimulated with kisspeptin, PACAP mRNA expression was increased. Similarly, GnRH had a stimulatory effect on PACAP expression. Conversely, kisspeptin expression in placental cells was increased by PACAP stimulation, whereas PACAP failed to stimulate GnRH mRNA expression in these cells. Finally, we found that PACAP had a stimulatory effect on human chorionic gonadotropin expression in placental cells. Our current observations suggest that the hypothalamic peptides PACAP, kisspeptin, and GnRH are interrelated and maintain placental functions.

Regulation of kisspeptin and gonadotropin-releasing hormone expression in rat placenta: study using primary cultures of rat placental cells.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) and kisspeptin in the hypothalamus are thought to be crucial components of the hypothalamic-pituitary-gonadal (HPG) axis and maintain reproductive function. These neuropeptides are also expressed in the placenta, where they may contribute to placental physiology. In this study, we examined how these peptides are regulated within the placenta. METHODS: We used primary cultures of placental tissue from rats of 16-18 days gestation. After stimulation with estradiol, GnRH, kisspeptin, and neurokinin B (NKB), changes in placental GnRH, kisspeptin, and human chorionic gonadotropin (hCG) mRNA expression were evaluated by real-time quantitative RT-PCR analysis. RESULTS: Immunocytochemical analysis showed that rat placental cells contained cells expressing kisspeptin or GnRH. GnRH and kisspeptin mRNA expression was significantly increased in placental cells in the presence of estradiol; NKB mRNA expression was also stimulated by estradiol. Stimulation of the cells with kisspeptin failed to stimulate GnRH mRNA expression. Conversely, both GnRH itself and NKB increased GnRH mRNA expression. Kisspeptin mRNA expression was not increased by kisspeptin itself; however, GnRH and NKB significantly increased kisspeptin mRNA expression. hCG expression was increased in the presence of estradiol. In addition, kisspeptin, GnRH, and NKB could stimulate the expression of hCG mRNA in placental cells. CONCLUSIONS: Our experiments using primary cultures of rat placental cells showed that GnRH, kisspeptin, and NKB expression was enhanced by estradiol, and unlike in the hypothalamus, kisspeptin did not control the expression of GnRH in placental cells. NKB might be located upstream of kisspeptin and GnRH, and these neuropeptides might be involved in the induction of hCG expression in placental cells.

Role of thyrotropin-releasing hormone in prolactin-producing cell models.

Thyrotropin-releasing hormone (TRH) is a hypothalamic hypophysiotropic neuropeptide that was named for its ability to stimulate the release of thyroid-stimulating hormone in mammals. It later became apparent that it exerts a number of species-dependent hypophysiotropic activities that regulate other pituitary hormones. TRH also regulates the synthesis and release of prolactin, although whether it is a physiological regulator of prolactin that remains unclear. Occupation of the Gq protein-coupled TRH receptor in the prolactin-producing lactotroph increases the turnover of inositol, which in turn activates the protein kinase C pathway and the release of Ca(2+) from storage sites. TRH-induced signaling events also include the activation of extracellular signal-regulated kinase (ERK) and induction of MAP kinase phosphatase, an inactivator of activated ERK. TRH stimulates prolactin synthesis through the activation of ERK, whereas prolactin release occurs via elevation of intracellular Ca(2+). We have been investigating the role of TRH in a pituitary prolactin-producing cell model. Rat pituitary somatolactotroph GH3 cells, which produce and release both prolactin and growth hormone (GH), are widely used as a model for the study of prolactin- and GH-secreting cells. In this review, we describe the general action of TRH as a hypophysiotropic factor in vertebrates and focus on the role of TRH in prolactin synthesis using GH3 cells.

Trichostatin A reduces GnRH mRNA expression with a concomitant increase in retinaldehyde dehydrogenase in GnRH-producing neurons.

Trichostatin A (TSA) is a selective inhibitor of mammalian histone deacetylase and is widely used to modify the ability of DNA transcription factors to bind DNA within chromatin by interfering with histone deacetylation. In the GnRH-producing neuronal cell line GT1-7, TSA significantly reduced expression of GnRH mRNA. Kisspeptin, a known regulator of GnRH release, failed to increase GnRH mRNA expression and did not modify TSA-induced reduction of GnRH expression. TSA, but not kisspeptin, increased histone acetylation in whole-cell lysates and significantly stimulated the expression of retinaldehyde dehydrogenase (RALDH), a retinoic acid (RA)-synthesizing enzyme that is known to be involved in cell differentiation. In addition, treatment of the GT1-7 cells with RA dose-dependently inhibited the expression of GnRH mRNA. Whereas, TSA-induced reduction of GnRH mRNA was not modulated by treatment with the pan-RA receptor inverse agonist BMS493 or the RA metabolism inhibitor liarozole. Our current results suggest that the RALDH and RA might not be directly involved in the reduction of GnRH expression induced by TSA, however these substances could be a novel regulator of GnRH.

Stimulation of δ subunit-containing GABAA receptor by DS1 increases GnRH receptor expression but reduces GnRH mRNA expression in GnRH-producing GT1-7 cells.

Acting via ionotropic GABAA receptors, the neurotransmitter γ-aminobutyric acid (GABA) is an important modulator of gonadotropin-releasing hormone (GnRH) neurons. In the present study, we examined the effect of DS1, a GABAA α4ß3δ receptor agonist, on a strain of mouse hypothalamic immortalized GnRH neuronal cells, the GT1-7 cell line. DS1 increased the activities of serum-response element (SRE) and cAMP-response element (CRE) promoters, which reflect the activities of extracellular signal-regulated kinase and cAMP/protein kinase A (PKA) pathways, respectively. In G protein-coupled receptor 54 (GPR54)-overexpressing GT1-7 cells, both DS1 and kisspeptin-10 stimulated SRE promoter activity, and combined treatment with DS1 and kisspeptin further increased SRE promoter activity compared with DS1 or kisspeptin alone. Pituitary adenylate cyclase-activating polypeptide (PACAP) increased CRE promoter activity in PACAP type I receptor-overexpressing GT1-7 cells, with an effect similar to that of DS1 alone, and combined stimulation with PACAP and DS1 potentiated their individual effects. DS1 stimulated the transcriptional activity of GnRH receptor, and DS1 induced GnRH receptor mRNA and protein expression. PACAP-increased GnRH receptor expression was enhanced in the presence of DS1. However, DS1 significantly inhibited the basal expression of GnRH mRNA in GT1-7 cells. Our current observations suggest that DS1 exerts its stimulatory effect on the intracellular signal transduction system via GABAA α4ß3δ receptors in GnRH-producing neurons. Stimulation with DS1 increased the expression of GnRH receptor but decreased the basal expression of GnRH mRNA.

DS1, a delta subunit-containing GABA(A) receptor agonist, increases gonadotropin subunit gene expression in mouse pituitary gonadotrophs.

4-Chloro-N-[6,8-dibromo-2-(2-thienyl)imidazo[1,2-alpyridine-3-yl] (DS1) is a GABA(A) receptor agonist that selectively binds to delta subunit-containing GABA(A) alpha4beta3delta receptors. In the present study, we examined the effect of DS1 on pituitary gonadotropin subunit gene expression using the mouse pituitary gonadotroph cell line LbetaT2. DS1 increased the promoter activity of the gonadotropin subunits luteinizing hormone beta (LHbeta), follicle-stimulating hormone beta (FSHbeta), and alpha. Gonadotropin-releasing hormone (GnRH) receptor promoters were also activated by DS1. The effects of DS1 on gonadotropin subunit promoters were obvious, but they were less than those induced by stimulation with GnRH. GnRH-stimulated gonadotropin subunit promoters were enhanced in the presence of DS1. A prototypic specific agonist for GABAA receptors, muscimol, failed to increase LHbeta and FSHbeta subunit promoter activity and had no effect on GnRH-increased LHbeta and FSHbeta promoter activity. In addition, SKF97541, a specific agonist for GABAB receptors, did not modulate basal or GnRH-induced LHbeta and FSHbeta promoter activity. A natural GABA compound failed to increase gonadotropin promoter activity and potentiated the effect of GnRH on the FSHbeta promoter. DS1 increased the activity of serum response element (SRE) and cAMP response element (CRE) promoters, which reflect the activity of the extracellular signal-regulated kinase and cAMP/protein kinase A (PKA) pathways, and GnRH-increased SRE and CRE promoter activity was enhanced in the presence of DS1. A specific inhibitor of the ERK signaling pathway, U0126, prevented DS1-induced LHbeta and FSHbeta promoter activity almost completely; however, H89, a PKA inhibitor, did not modulate the effect of DS1. Our current observations demonstrate that the GABAA alpha4beta3delta receptor agonist DS1 can stimulate gonadotropin subunit gene expression in association with the ERK signaling pathway.

Trichostatin A specifically stimulates gonadotropin FSHß gene expression in gonadotroph LßT2 cells.

Trichostatin A (TSA) is a selective inhibitor of mammalian histone deacetylase. In the present study, TSA was found to selectively increase gene expression of the pituitary gonadotropin ß-subunit of follicle-stimulating hormone (FSH). Stimulation of mouse pituitary gonadotroph cell lines, LßT2, with TSA for 24 h resulted in no change in mRNA expression of the α- and LHß-subunit. On the other hand, FSHß-subunit mRNA expression was significantly increased in a dose-dependent fashion. Similarly, specific induction of the FSHß-subunit gene with TSA stimulation was observed in primary cultures of rat pituitary cells. Histone acetylation in whole cell lysates of LßT2 cells was significantly increased after TSA treatment, but not gonadotropin-releasing hormone (GnRH) treatment. The effect of TSA on FSHß mRNA expression was prominent compared to that of GnRH; however, TSA-stimulated FSHß mRNA expression was significantly reduced with combined TSA and GnRH treatment. TSA caused a slight increase in extracellular signal-regulated kinase (ERK) phosphorylation, while GnRH-increased ERK phosphorylation was potentiated in the presence of TSA. In addition, TSA, but not GnRH, significantly stimulated gene expression of retinaldehyde dehydrogenase 1 (RALDH1), a retinoic acid (RA) synthesizing enzyme involved in cell differentiation. These findings demonstrate that TSA specifically increases FSHß subunit gene expression with a concomitant increase in whole cell histone acetylation. Moreover, although GnRH is a stimulator of FSHß gene expression, it interfered with the stimulatory effect of TSA on FSHß mRNA expression, without modification of TSA-increased whole cell histone acetylation. This suggests that the mechanisms of TSA and GnRH-induced gonadotropin subunit gene expression are entirely distinct.

Expression of gonadotropin-inhibitory hormone receptors in mouse pituitary gonadotroph LßT2 cells and hypothalamic gonadotropin-releasing hormone-producing GT1-7 cells.

Gonadotropin-inhibitory hormone (GnIH) was first identified in quail as a novel neurohormone that acts directly on the anterior pituitary to inhibit gonadotropin release. GnIH inhibits not only gonadotropin release from the pituitary gland but also inhibits the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus. In this study, we examined how GnIH receptors were regulated in pituitary gonadotroph cells and GnRH-producing neurons in the hypothalamus. In the mouse pituitary gonadotroph cell line LßT2, GnRH increased expression of the GnIH receptor, G-protein coupled receptor 74 (GPR74). GnRH also stimulated the expression of GPR74 and GPR147 in primary cultures of rat anterior pituitary cells. In addition, when GnRH was administered to LßT2 cells in a pulsatile manner, low frequency GnRH pulse stimulation stimulated GPR74 and GPR147 expression more than did high frequency GnRH pulses. In the mouse hypothalamic GnRH-producing cell line GT1-7, hypothalamic kisspeptin did not significantly increase the expression of GnIH receptors. However, the intermittent administration of kisspeptin to GT1-7 cells significantly increased GPR74 and GPR147 mRNA expression. The overexpression of either constitutively active MEK kinase (MEKK) or protein kinase A (PKA) in LßT2 cells increased the expression of GPR74 mRNA. Conversely, in GT1-7 cells, although the overexpression of either MEKK or PKA failed to stimulate GnIH receptor expression, the combined overexpression of both kinases together increased GPR74 and GPR147 mRNA levels. Our current observations suggest that two central controllers of reproductive function, GnRH and kisspeptin, stimulate the expression of GnIH receptors in pituitary gonadotroph cells and hypothalamic GnRH neurons.

Kisspeptin induces expression of gonadotropin-releasing hormone receptor in GnRH-producing GT1-7 cells overexpressing G protein-coupled receptor 54.

Kisspeptin signaling through its receptor is crucial for many reproductive functions. However, the molecular mechanisms and biomedical significance of the regulation of GnRH neurons by kisspeptin have not been adequately elucidated. In the present study, we found that kisspeptin increases GnRH receptor (GnRHR) expression in a GnRH-producing cell line (GT1-7). Because cellular activity of G protein-coupled receptor 54 (GPR54) and GnRHR was limited in GT1-7 cells, we overexpressed these receptors to clarify receptor function. Using luciferase reporter constructs, the activity of both the serum response element (Sre) promoter, a target for extracellular signal-regulated kinase (ERK), and the cyclic AMP (cAMP) response element (Cre) promoter were increased by kisspeptin. Although GnRH increased Sre promoter activity, the Cre promoter was not significantly activated by GnRH. Kisspeptin, but not GnRH, increased cAMP accumulation in these cells. Kisspeptin also increased the transcriptional activity of GnRHR; however, the effect of GnRH on the GnRHR promoter was limited and not significant. Transfection of GT1-7 cells with constitutively active MEK kinase (MEKK) and protein kinase A (PKA) increased GnRHR expression. In addition, GnRHR expression was further increased by co-overexpression of MEKK and PKA. The Cre promoter, but not the Sre promoter, was also further activated by co-overexpression of MEKK and PKA. GnRH significantly increased the activity of the GnRHR promoter in the presence of cAMP. The present findings suggest that kisspeptin is a potent stimulator of GnRHR expression in GnRH-producing neurons in association with ERK and the cAMP/PKA pathways.

Pituitary adenylate cyclase-activating polypeptide (PACAP) increases expression of the gonadotropin-releasing hormone (GnRH) receptor in GnRH-producing GT1-7 cells overexpressing PACAP type I receptor.

The present study demonstrates the action of pituitary adenylate cyclase-activating polypeptide (PACAP) on gonadotropin-releasing hormone (GnRH)-producing neuronal cells, GT1-7. Because we found the expression levels of PACAP type 1 receptor (PAC1R) to be low in these cells, we transfected them with PAC1R expression vector and observed the outcome. PACAP increased the activity of the serum response element (Sre) promoter, a target of extracellular signal-regulated kinase (ERK), as well as the cAMP response element (Cre) promoter in GT1-7 cells overexpressing PAC1R. We also observed ERK phosphorylation and cAMP accumulation upon PACAP stimulation. PACAP stimulated the promoter activity of GnRH receptor (GnRHR) with increasing levels of GnRHR proteins. Notably, the increase in GnRHR promoter activity from kisspeptin was potentiated in the presence of PACAP. A similar increasing effect of PACAP on the action of kisspeptin was observed for Cre promoter activity. On the other hand, the Sre promoter activated by kisspeptin was inhibited by co-treatment with kisspeptin and PACAP. Likewise, kisspeptin-increased GnRHR promoter activity and Cre promoter activity were both potentiated in the presence of cAMP, whereas the Sre promoter activated by kisspeptin was inhibited in the presence of cAMP. Our observations show that PACAP increases GnRHR expression and stimulates kisspeptin's effect on GnRHR expression in association with the cAMP/PKA signaling pathway in GT1-7 cells overexpressing PAC1R. In addition, PACAP was shown to have an inhibitory effect on ERK-mediated kisspeptin action.

Circulating kisspeptin and pituitary adenylate cyclase-activating polypeptide (PACAP) do not correlate with gonadotropin serum levels.

Kisspeptins are known to be the principle regulators of the hypothalamic-pituitary gonadal (HPG) axis. In addition, the role of pituitary adenylate cyclase-activating polypeptide (PACAP) in the regulation of pituitary gonadotropins has been elucidated. We measured plasma concentrations of kisspeptin and PACAP and determined whether the levels of these peptides varied in proportion to circulating gonadotropin levels. Plasma luteinizing hormone (LH) levels were higher in postmenopausal women and in patients with premature ovarian failure (POF) and lower in patients with idiopathic hypogonadotropic hypogonadism (IHH) compared with the LH level in normally menstruating women. Similarly, serum follicle-stimulating hormone levels were higher in postmenopausal women and in patients with POF but lower in pregnant women and patients with IHH compared with normally menstruating women. Plasma levels of kisspeptins were significantly higher in pregnant women compared with normally menstruating women. However, no significant differences were observed in postmenopausal women, patients with POF, and patients with IHH. On the other hand, plasma levels of PACAP were significantly lower in pregnant women, patients with POF, and in IHH patients when compared with normally menstruating women. No significant differences were observed in PACAP concentration between postmenopausal women and in normally menstruating women. Our observations suggest that the serum levels of kisspeptins and PACAP did not correlate with variations in serum gonadotropin levels.