BIOLOGY OF REPRODUCTION (2015) 92(2):45, 1–8 Published online before print 17 December 2014. DOI 10.1095/biolreprod.114.123893
DS1, a Delta Subunit-Containing GABAA Receptor Agonist, Increases Gonadotropin Subunit Gene Expression in Mouse Pituitary Gonadotrophs1 Tselmeg Mijiddorj, Haruhiko Kanasaki,2 Unurjargal Sukhbaatar, Aki Oride, and Satoru Kyo Department of Obstetrics and Gynecology, Shimane University School of Medicine, Izumo, Japan
4-Chloro-N-[6,8-dibromo-2-(2-thienyl)imidazo[1,2-alpyridine-3-yl] (DS1) is a GABAA receptor agonist that selectively binds to delta subunit-containing GABAA 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. GnRHstimulated 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 GnRHincreased 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. follicle-stimulating hormone (FSH/FSH receptor), gonadotropinreleasing hormone (GnRH/GnRH receptor), luteinizing hormone (LH/LH receptor), neuroendocrinology
INTRODUCTION Pituitary gonadotropins, such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH), play a pivotal role in 1 This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, Culture, and Technology of Japan (to H.K. and A.O.). 2 Correspondence: Haruhiko Kanasaki, Department of Obstetrics and Gynecology, Shimane University School of Medicine, 89-1 Enya, Izumo City, Shimane Prefecture 693-8501, Japan. E-mail: [email protected]
Received: 30 July 2014. First decision: 25 August 2014. Accepted: 10 December 2014. Ó 2015 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363
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female reproduction by the stimulation of follicular growth, maturation of oocytes, and sex steroid synthesis in the gonads [1]. LH and FSH are principally regulated by gonadotropinreleasing hormone (GnRH), which is released from the hypothalamus in a pulsatile manner [2]. In addition, gonadotropins are also regulated by pituitary adenylate cyclaseactivating polypeptide (PACAP) [3, 4] as well as peptides produced in the gonads, such as inhibin, activin, and follistatin [5]. Although GnRH has been recognized as the most potent activator of the hypothalamic-pituitary-gonadal (HPG) axis, recent studies imply that hypothalamic kisspeptin neurons are the final component of the HPG axis and control GnRH release [6, 7]. Substantial evidence suggests that c-aminobutyric acid (GABA) neurons also exert a direct influence on GnRH neurons through its receptors [8, 9]. GnRH neurons receive abundant synaptic input from GABA neurons [10]. The inhibitory action of GABA on GnRH neurons was suggested by studies in which the administration of GABA antagonists increased gonadotropin release in rodents and sheep, probably by their action on GnRH neurons [11, 12]. Conversely, GABA also has a rapid excitatory effect on GnRH neurons [13, 14] and stimulates LH release [15]. The major neurotransmitter of the central nervous system, GABA acts through three structurally and pharmacologically distinct receptors: GABAA, GABAB, and GABAC. GABAA and GABAC are members of the ligand-gated Cl channel family, whereas GABAB receptors are coupled with G proteins [16]. In addition to their expression in the central nervous system, all three GABA receptor subtypes are also expressed by pituitary cells [17, 18]. Previous studies demonstrated that GABA inhibits prolactin release in vitro [19, 20]. In contrast, some reports showed the stimulatory effects of GABA on the secretion of adrenocorticotropic hormone, thyroid-stimulating hormone, and LH by pituitary cells [21, 22]. Several types of GABAA receptor subunits have been cloned and sequenced from mammals (a1–6, b1–3, c1–3, d, e, p, and h). Functional GABAA receptors are heteromeric pentamers, and muscimol is a specific agonist of GABAA receptors [23]. The majority of GABAA receptors consist of two a, two b, and one c subunit; however, less abundant populations of GABAA receptors contain the d subunit. Interestingly, for d subunit-containing GABAA receptors (dGABAA receptors), their expression is altered periodically during the estrous cycle in murine hippocampal neurons [24], and their expression is influenced by the ovarian steroid milieu [25]. d-GABAA receptors are located extra- or perisynaptically and are activated by ambient GABA [26]. In addition, dGABAA receptors are emerging as a valuable pharmacological target because they are not influenced by benzodiazepines [27]. Indeed, a previous study demonstrated that the d subunit of the GABAA receptor is expressed in the pituitary gland [28]. 4-Chloro-N-[6,8-dibromo-2-(2-thienyl)imidazo[1,2-alpyridine-3-yl] (DS1) is a compound that selectively and potently enhances GABA-evoked currents mediated by d subunit-
ABSTRACT
MIJIDDORJ ET AL.
containing GABAA (a4b3d) receptors. In this present study, we found that DS1 increases gonadotropin subunit gene expression in a pituitary gonadotroph cell line.
Reporter Plasmid Construction and Luciferase Assay The reporter constructs used in these experiments were generated by fusing 797/ þ5 of the rat LHb gene (LHb-Luc), 2000/ þ698 of the rat FSHb gene (FSHb-Luc), or 846/0 of the human aGSU gene (a-Luc) to the firefly luciferase (Luc) cDNA in pXP2 [29]. The plasmid containing 1164/ þ62 of the mouse GnRH receptor gene (GnRH-Luc) fused to firefly luciferase cDNA was generously provided by Dr. Ursula Kaiser (Brigham and Women’s Hospital and Harvard University, Boston, MA). The cells were transiently transfected by electroporation with 2.0 lg/well of gonadotropin subunit-Luc and 0.1 lg of pRL-TK (Promega) containing Renilla luciferase. When the activity of the promoter containing CRE or SRE was measured, the cells were transfected with either 2.0 lg of pCRE-Luc or pSRE-Luc, which have the CRE enhancer (34) or five tandem repeats of SRE, respectively, upstream of the firefly luciferase gene. After incubation with the test reagents, the cells were washed with ice-cold phosphate-buffered saline and lysed with passive lysis buffer (Promega). When an inhibitor was used, the cells were preincubated with the inhibitor alone for 60 min, then stimulated with the test reagents together with the inhibitor. After centrifugation at 15 000 3 g at 48C for 5 min, firefly and Renilla luciferase activity was measured in the supernatant with the DualLuciferase Reporter Assay System (Promega) using a luminometer (TD-20/20; Promega) according to the manufacturer’s protocol. Luciferase activity was normalized to that of Renilla luciferase activity to correct for transfection efficiency, with results expressed as the fold-increase compared to the unstimulated control.
MATERIALS AND METHODS Materials The following chemicals and reagents were obtained from the indicated sources: fetal bovine serum (FBS) and trypsin from GIBCO, Invitrogen; Dulbecco modified Eagle medium (DMEM), DS1, GnRH, GABA, and penicillin-streptomycin from Sigma Chemical Co.; muscimol, SKF97541, and H89 from Abcam; U0126, anti-phosphorylated ERK (anti-P-ERK) antibody, anti-ERK antibody, and the appropriate horseradish peroxidase-conjugated secondary antibodies from Santa Cruz Biotechnology; serum response element (SRE) and cAMP response element (CRE) firefly luciferase reporter plasmids (pSRE-Luc and pCRE-Luc) from Stratagene; and pCI-neo from Promega.
Cell Culture LbT2 cells (kindly provided by Dr. P. Mellon of the University of California, San Diego) were maintained in a static monolayer culture in highglucose DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin at 378C in a humidified atmosphere of 5% CO2 in 95% air. After 24 h, the culture medium was changed to DMEM containing 1% heat-inactivated FBS and 1% penicillin-streptomycin and then incubated without (for control) or with the test reagents for the indicated times.
Statistical Analysis All experiments were repeated independently at least three times. Each experiment was performed with triplicate samples in each experimental group. The values are expressed as the mean 6 SEM. The median effective
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FIG. 1. Effect of DS1 on gonadotropin subunit promoter activity. LbT2 cells were transfected with 2.0 lg of LHb-Luc (A), FSHb-Luc (B), a-Luc (C), or GnRHR-Luc (D) vectors together with 0.1 lg of pRL-TK plasmid. At 48 h after transfection, the cells were treated with increasing concentrations of DS1 for 6 h. A luciferase assay was performed to examine LHb (A), FSHb (B), and a (C) subunit and GnRH receptor (D) promoter activity, which was normalized to pRL-TK activity. The results are expressed as fold-stimulation over unstimulated cells and presented as the mean 6 SEM of three independent experiments, each performed with triplicate samples. *P , 0.05, **P , 0.01 vs. control.
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concentration was calculated following nonlinear regression analysis. Statistical analysis was performed using Prism 5.0 (GraphPad Software) based on the appropriate analysis of variance (two-way or one-way, repeated measures) followed by the Dunnett or Bonferroni test for multiple comparisons. P , 0.05 was considered to be statistically significant.
Effect of DS1 on GnRH-Increased Gonadotropin Subunit Promoter Activity DS1 increased the activity all of three gonadotropin subunit promoters, but its effects were less than those induced by GnRH stimulation. GnRH increased LHb promoter activity by 8.76- 6 0.77-fold. In the presence of DS1, LHb promoter activity increased to 12.62- 6 1.21-fold, a significant difference (Fig. 2A). Similarly, GnRH-increased LHb and a subunit promoter activity was significantly enhanced in the presence of DS1 (from 8.07- 6 1.60-fold to 13.5- 6 1.79-fold for FSHb and from 81.67- 6 3.24-fold to 120.97- 6 1.49-fold for the a subunit) (Fig. 2, B and C).
RESULTS Effect of DS1 on Gonadotropin Subunit Promoter Activity Before the current experiments were started, we confirmed that d subunit-containing GABAA receptors were expressed in the mouse gonadotroph cell line LbT2 and primary cultures of anterior pituitary (data not shown). First, we examined the effect of DS1 on LbT2 cells. LbT2 cells were treated with increasing concentrations of DS1, and the promoter activity of the gonadotropin LHb, FSHb, and a subunits was determined using a dual luciferase assay. DS1 increased the promoter activity of all three gonadotropin subunits. A significant increase in activity was obtained at 1 lM DS1, and a maximal peak of 2.77- 6 0.02-fold was observed at 10 lM DS1 for the LHb subunit (Fig. 1A). DS1 also increased FSHb and a subunit promoter activity, with a peak at 50 lM (of 2.07- 6 0.42-fold) for FSHb and at 100 lM (of 4.66- 6 0.57-fold) for the a subunit (Fig. 1, B and C).
Effect of other GABA Receptor Agonists on Gonadotropin Subunit Promoter Activity DS1 is a specific agonist of d subunit-containing GABAA receptors. A prototypic specific agonist for GABAA receptors, muscimol, did not increase either LHb or FSHb promoter activity and did not modulate the effect of GnRH (Fig. 3, A and B). Similarly, a specific agonist of GABAB receptors, SKF97541, did not increase LHb or FSHb promoter activity and did not modulate the effect of GnRH (Fig. 3, C and D). Conversely, although a natural GABA compound did not 3
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FIG. 2. Effect of DS1 on GnRH-induced gonadotropin promoter activity. LbT2 cells were transfected with 2.0 lg of LHb-Luc (A), FSHb-Luc (B), or a-Luc (C) vectors together with 0.1 lg of pRL-TK plasmid. At 48 h after transfection, the cells were treated with 10 nM GnRH, 50 lM DS1, or both for 6 h. A luciferase assay was performed to examine LHb (A), FSHb (B), and a (C) subunit promoter activity, which was normalized to pRL-TK activity. The results are expressed as fold-stimulation over unstimulated cells and presented as the mean 6 SEM of three independent experiments, each performed with triplicate samples. The differences between GnRH and GnRH plus DS1 were statistically significant for the LHb, FSHb, and a subunit promoters. *P , 0.05, **P , 0.01 vs. control.
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increase the basal activity of the LHb or FSHb promoters, GABA significantly enhanced GnRH-increased FSHb promoter activity (Fig. 4B). The induction of LHb promoter activity by GnRH was slightly enhanced in the presence of GABA, but this effect was not statistically significant (Fig. 4A).
Similar to the gonadotropin promoters, both SRE and CRE promoter activity induced by GnRH was enhanced in the presence of DS1 (Fig. 5, A and B). Effect of ERK and PKA Inhibitors on DS1-Increased Gonadotropin Subunit Promoter Activity
Effect of DS1 on SRE and CRE Promoter Activity
U0126, a specific inhibitor of the ERK signaling pathway, almost completely inhibited DS1-induced LHb and FSHb promoter activity (Fig. 6, A and B). Conversely, H89, a PKA inhibitor, did not have a significant effect on GnRH-increased gonadotropin promoter activity.
A DNA domain in the promoter region, SRE binds ERKmediated transcription factors, whereas the CRE promoter is a target of the cAMP/protein kinase A (PKA) pathway. GnRH drastically increased SRE and CRE promoter activity by 20.786 0.87-fold and 13.74- 6 2.46-fold, respectively. DS1 increased SRE promoter activity by 2.08- 6 0.19-fold. Increased ERK phosphorylation was confirmed by Western blot analysis using a phosphorylation-specific antibody, but the increase in phosphorylation induced by DS1 was limited compared to that induced by GnRH stimulation (DS1, 3.16- 6 0.72-fold; GnRH, 19.02- 6 1.36-fold; data not shown). CRE promoter activity was also stimulated by DS1 (2.25- 6 0.29fold). The effect of DS1 on the SRE and CRE promoters was significant, but limited compared to the effect of GnRH.
DISCUSSION Schally et al. [19] first isolated GABA from the hypothalamus in 1997, and subsequent authors proposed that GABA is the major inhibitory neurotransmitter of the central nervous system. It also acts as a signaling gate to the reproductive system via its direct influence on GnRH neurons. Generally, the secretion and synthesis of gonadotropins are understood to be mainly under the control of hypothalamic 4
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FIG. 3. Effect of other GABA receptor agonists, muscimol and SKF97541, on gonadotropin promoter activity. LbT2 cells were transfected with 2.0 lg LHb-Luc (A and C) or FSHb-Luc (B and D) vectors together with 0.1 lg of pRL-TK plasmid. At 48 h after transfection, the cells were treated with 10 nM GnRH and 50 lM muscimol, a prototypic specific agonist of GABAA receptors (A and B), or 50 lM SKF97541, a GABAB receptor agonist (C and D), or both for 6 h. A luciferase assay was performed to examine LHb (A and C) and FSHb (B and D) subunit promoter activity, which was normalized to pRL-TK activity. The results are expressed as fold-stimulation over unstimulated cells and presented as the mean 6 SEM of three independent experiments, each performed with triplicate samples. The difference between GnRH and GnRH plus muscimol and between GnRH and GnRH plus SKF97541 for both LHb and FSHb promoters was not statistically significant (n.s.). **P , 0.01 vs. control.
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GnRH. Ovarian factors, such as inhibin, activin, and follistatin, also act as regulators of gonadotropins [30]. In addition, previous observations support the role of hypothalamic PACAP in the control of gonadotropin subunit expression [31, 32]. In the present study, we applied DS1, a GABAA a4b3d receptor agonist, and found that DS1 stimulation increased the gene expression of the gonadotropin subunits a, LHb, and FSHb in a pituitary gonadotroph cell line. GnRH receptor transcriptional activity was also stimulated by this compound. In addition, we found that the GnRH-induced increase of gonadotropin subunit expression was enhanced in the presence of DS1, suggesting that DS1 participates in the regulation of gonadotropin subunit gene expression by itself and also positively modulates other receptor-mediated increases of gonadotropin expression. This stimulation of gonadotropin subunit expression was not observed with a prototypic
specific agonist of GABAA receptors, muscimol, or a natural GABA compound, suggesting the possibility that the gonadotropin subunit stimulating effect of DS1 is mediated only by d subunit-containing GABAA receptors. The possibility exists that the stimulation of gonadotropin subunit expression by DS1 might not be through GABAA receptors, and we should interpret the current results carefully. Indeed, muscimol is regarded as a compound that activates all GABAA receptor subtypes [33]. However, it was reported that the high-affinity binding sites of muscimol differ greatly according to the distribution of GABAA receptors in the brain and that the sensitivity of d subunit-containing GABAA receptors to muscimol differs by gender [34]. Thus, it is appropriate to think muscimol does not evoke the same effect on d subunitcontaining GABAA receptors as DS1. Because the potency of muscimol and universal GABA compounds to stimulate d
FIG. 5. Effect of DS1 on SRE and CRE promoter activity. LbT2 cells were transfected with 2.0 lg of SRE-Luc (A) or CRE-Luc (B) and 0.1 lg of pRL-TK plasmid. At 48 h after transfection, the cells were treated with 10 nM GnRH, 50 lM DS1, or both for 6 h. A luciferase assay was performed to examine SRE and CRE promoter activity, which was normalized to pRL-TK activity and is expressed as the fold-difference of activation over unstimulated controls. The difference between GnRH and GnRH plus DS1 for both the SRE and CRE promoters was statistically significant (P , 0.01 and P , 0.05). **P , 0.01 vs. control.
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FIG. 4. Effect of a natural GABA compound on gonadotropin subunit promoter activity. LbT2 cells were transfected with 2.0 lg of LHb-Luc (A) or FSHbLuc (B) vectors together with 0.1 lg of pRL-TK plasmid. At 48 h after transfection, the cells were treated with 10 nM GnRH, 50 lM of a natural GABA compound, or both for 6 h. A luciferase assay was performed to examine LHb (A) and FSHb (B) subunit promoter activity, which was normalized to pRLTK activity. The results are expressed as fold-stimulation over unstimulated cells and presented as the mean 6 SEM of three independent experiments, each performed with triplicate samples. The difference between GnRH and GnRH plus GABA for the LHb promoter was not statistically significant (n.s.). The difference between GnRH and GnRH plus GABA for the FSHb promoter was statistically significant (P , 0.01). **P , 0.01 vs. control.
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receptors for GABA are present in anterior pituitary cells [28], and GABA is synthesized and released in the pituitary [36]. The hormonal milieu affects the GABA-ergic control of GnRH output and eventually controls gonadotropin release. The electrical response to GABA in rat GnRH neurons was reportedly modulated differently according to the stage of the estrous cycle, diurnal rhythm, and sex [37]. Also, the response to GABA on GnRH/LH secretion in ewes differed during breeding and nonbreeding seasons [38, 39]. In addition, interesting phenomena were reported for d-GABAA receptors in previous studies. The expression of d-GABAA receptors in hippocampal neurons is not static; rather, it changes dynamically during the estrous cycle [24, 25, 40]. Fluctuations in neurosteroid levels during the estrous cycle are coupled with alterations of d-GABAA receptors. Estrogen is generally believed to have a stimulatory effect on gonadotropin synthesis and secretion at the pituitary level in vivo [41]; the enforcement of d-GABAA receptor signaling might be one of the phenomena induced by gonadotrophs following estrogen elevation. In the present study, we observed that DS1 increased the activity of intracellular signaling pathways, such as ERK and cAMP/PKA, in LbT2 cells. In addition, signal transduction
subunit-containing GABAA receptors is weak, they might fail to stimulate gonadotropins by themselves. Indeed, both muscimol and universal GABA compounds failed to stimulate SRE and CRE promoters (data not shown). In addition, DS1increased gonadotropin subunit promoter expression was not inhibited in the presence of the universal GABAA receptor antagonist bicuculline (data not shown). Conversely, both muscimol and universal GABA compounds tended to enhance the action of GnRH on gonadotropin subunit expression slightly, but not significantly. Only the potentiation of the effects of GnRH on FSHb subunit expression by universal GABA compounds was statistically significant. If muscimol or GABA compounds could bind d subunit-containing GABAA receptors potently, it is natural to think these compounds may induce some effects on the action of GnRH. Thus, we think that it is reasonable to hypothesize that the effects of DS1 are associated with d subunit-containing GABAA receptors. A number of studies have demonstrated the GABA-ergic neurons in the hypothalamus are involved in controlling GnRH release, so gonadotropin release from anterior pituitary cells is also controlled indirectly by GABA. GABA is released from tuberoinfundibular and other hypothalamic regions as well as from neurointermediate pituitary lobe axons [35]. In addition, 6
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FIG. 6. Effect of U0126, an MEK inhibitor, and H89, a PKA inhibitor, on the DS1-induced increase of gonadotropin promoter activity. LbT2 cells were transfected with 2.0 lg of LHb-Luc (A and C) or FSHb-Luc (B and D) vectors together with 0.1 lg of pRL-TK plasmid. At 48 h after transfection, the cells were treated with 50 lM DS1 and 10 lM U0126 (A and B) or 10 lM H89 (C and D), for 6 h. A luciferase assay was performed to examine LHb (A and C) and FSHb (B and D) subunit promoter activity, which was normalized to pRL-TK activity. The results are expressed as fold-stimulation over unstimulated cells and presented as the mean 6 SEM of three independent experiments, each performed with triplicate samples. The difference between DS1 and DS1 plus H89 for both the LHb and FSHb promoters was not statistically significant (n.s.). The difference between DS1 and DS1 plus U0126 for both the LHb and FSHb promoters was statistically significant (P , 0.01). **P , 0.01 vs. control.
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systems that were evoked by GnRH were augmented in the presence of DS1, with a concomitant increase in GnRHinduced gonadotropin subunit expression. The excitatory effect of GABAA receptors on GnRH-producing neurons was thought to be due to an increase in Ca2þ via the activation of L-type calcium channels [42], which results in chloride ion efflux and membrane depolarization [43]. In addition, in a whole-cell voltage-clamp study using stable cell lines expressing GABAA a4b3d receptors, DS1 potently enhanced GABA-evoked currents produced by chloride ion flux through GABA-gated ion channels [44]. A number of phenomena were evoked in LbT2 cells by DS1 alone, so DS1 probably exerts its stimulatory effect through specific subunits of GABAA receptors. Conversely, GnRH receptors couple with Gq protein, stimulate phospholipase C, and generate diacylglycerols, resulting in protein kinase C (PKC) activation and inositol phospholipid (IP3) turnover. Ultimately, PKC stimulates the ERK signaling pathway, and IP3 increases intracellular calcium levels by promoting calcium mobilization. GnRH also stimulates the cAMP/PKA pathway through Gs-coupled receptors [45]. Considering the observations that DS1 alone increases the activation of the ERK and cAMP/PKA signaling pathways, which were determined by SRE- and CRE-luciferase assays, and that DS1 potentiates the effect of GnRH on the ERK and cAMP/PKA signaling pathways, we suggest that d subunit-containing GABAA receptors share part of the same intracellular signaling pathways as GnRH receptors. The effect of DS1 on gonadotropin LHb and FSHb subunit expression was almost completely inhibited in the presence of a specific inhibitor of the ERK pathway, but not by H89, an inhibitor of PKA. This observation suggests that the stimulating effect of DS1 on gonadotropin subunit expression depends on the ERK signaling pathway. The involvement of the ERK signaling pathway on gonadotropin LHb [46, 47] and FSHb subunit [47] gene expression has been described previously. The inhibition of ERK using a specific inhibitor prevented both LHb and FSHb subunit expression in LbT2 cells [48], and the induction of MEKK, an upstream activator of ERK, increased LHb and FSHb subunit promoter activity without GnRH stimulation [49]. These observations clearly suggest that DS1 increases gonadotropin subunit expression in an ERK-dependent manner. In the present study, we found that DS1, a compound that selectively enhances the expression of d subunit-containing GABAA receptors, can increase gonadotropin subunit gene expression. The precise role of d-GABAA receptors in the expression of pituitary gonadotrophs remains to be elucidated; however, these receptors might play an important role in their regulation. In addition, DS1 could be developed as a novel therapy for activation of the HPG axis.
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41. Albanese C, Colin IM, Crowley WF, Ito M, Pestell RG, Weiss J, Jameson JL. The gonadotropin genes: evolution of distinct mechanisms for hormonal control. Recent Prog Horm Res 1996; 51:23–58 (discussion 59–61). 42. Sun W, Jarry H, Wuttke W, Kim K. Gonadotropin releasing hormone modulates gamma-aminobutyric acid-evoked intracellular calcium increase in immortalized hypothalamic gonadotropin releasing hormone neurons. Brain Res 1997; 747:70–77. 43. Hales TG, Sanderson MJ, Charles AC. GABA has excitatory actions on GnRH-secreting immortalized hypothalamic (GT1-7) neurons. Neuroendocrinology 1994; 59:297–308. 44. Wafford KA, van Niel MB, Ma QP, Horridge E, Herd MB, Peden DR, Belelli D, Lambert JJ. Novel compounds selectively enhance delta subunit containing GABAA receptors and increase tonic currents in thalamus. Neuropharmacology 2009; 56:182–189. 45. Liu F, Usui I, Evans LG, Austin DA, Mellon PL, Olefsky JM, Webster NJ. Involvement of both Gq/11 and Gs proteins in gonadotropin-releasing hormone receptor-mediated signaling in LbetaT2 cells. J Biol Chem 2002; 277:32099–32108. 46. Liu F, Austin DA, Mellon PL, Olefsky JM, Webster NJ. GnRH activates ERK1/2 leading to the induction of c-fos and LHbeta protein expression in LbetaT2 cells. Mol Endocrinol 2002; 16:419–434. 47. Harris D, Bonfil D, Chuderland D, Kraus S, Seger R, Naor Z. Activation of MAPK cascades by GnRH: ERK and Jun N-terminal kinase are involved in basal and GnRH-stimulated activity of the glycoprotein hormone LHbeta-subunit promoter. Endocrinology 2002; 143:1018–1025. 48. Purwana IN, Kanasaki H, Oride A, Miyazaki K. Induction of dual specificity phosphatase 1 (DUSP1) by gonadotropin-releasing hormone (GnRH) and the role for gonadotropin subunit gene expression in mouse pituitary gonadotroph LbetaT2 cells. Biol Reprod 2010; 82:352–362. 49. Yamada Y, Yamamoto H, Yonehara T, Kanasaki H, Nakanishi H, Miyamoto E, Miyazaki K. Differential activation of the luteinizing hormone beta-subunit promoter by activin and gonadotropin-releasing hormone: a role for the mitogen-activated protein kinase signaling pathway in LbetaT2 gonadotrophs. Biol Reprod 2004; 70:236–243.
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DS1, a Delta Subunit-Containing GABAA Receptor Agonist, Increases Gonadotropin Subunit Gene Expression in Mouse Pituitary Gonadotrophs1 Tselmeg Mijiddorj, Haruhiko Kanasaki,2 Unurjargal Sukhbaatar, Aki Oride, and Satoru Kyo Department of Obstetrics and Gynecology, Shimane University School of Medicine, Izumo, Japan
4-Chloro-N-[6,8-dibromo-2-(2-thienyl)imidazo[1,2-alpyridine-3-yl] (DS1) is a GABAA receptor agonist that selectively binds to delta subunit-containing GABAA 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. GnRHstimulated 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 GnRHincreased 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. follicle-stimulating hormone (FSH/FSH receptor), gonadotropinreleasing hormone (GnRH/GnRH receptor), luteinizing hormone (LH/LH receptor), neuroendocrinology
INTRODUCTION Pituitary gonadotropins, such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH), play a pivotal role in 1 This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, Culture, and Technology of Japan (to H.K. and A.O.). 2 Correspondence: Haruhiko Kanasaki, Department of Obstetrics and Gynecology, Shimane University School of Medicine, 89-1 Enya, Izumo City, Shimane Prefecture 693-8501, Japan. E-mail: [email protected]
Received: 30 July 2014. First decision: 25 August 2014. Accepted: 10 December 2014. Ó 2015 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363
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female reproduction by the stimulation of follicular growth, maturation of oocytes, and sex steroid synthesis in the gonads [1]. LH and FSH are principally regulated by gonadotropinreleasing hormone (GnRH), which is released from the hypothalamus in a pulsatile manner [2]. In addition, gonadotropins are also regulated by pituitary adenylate cyclaseactivating polypeptide (PACAP) [3, 4] as well as peptides produced in the gonads, such as inhibin, activin, and follistatin [5]. Although GnRH has been recognized as the most potent activator of the hypothalamic-pituitary-gonadal (HPG) axis, recent studies imply that hypothalamic kisspeptin neurons are the final component of the HPG axis and control GnRH release [6, 7]. Substantial evidence suggests that c-aminobutyric acid (GABA) neurons also exert a direct influence on GnRH neurons through its receptors [8, 9]. GnRH neurons receive abundant synaptic input from GABA neurons [10]. The inhibitory action of GABA on GnRH neurons was suggested by studies in which the administration of GABA antagonists increased gonadotropin release in rodents and sheep, probably by their action on GnRH neurons [11, 12]. Conversely, GABA also has a rapid excitatory effect on GnRH neurons [13, 14] and stimulates LH release [15]. The major neurotransmitter of the central nervous system, GABA acts through three structurally and pharmacologically distinct receptors: GABAA, GABAB, and GABAC. GABAA and GABAC are members of the ligand-gated Cl channel family, whereas GABAB receptors are coupled with G proteins [16]. In addition to their expression in the central nervous system, all three GABA receptor subtypes are also expressed by pituitary cells [17, 18]. Previous studies demonstrated that GABA inhibits prolactin release in vitro [19, 20]. In contrast, some reports showed the stimulatory effects of GABA on the secretion of adrenocorticotropic hormone, thyroid-stimulating hormone, and LH by pituitary cells [21, 22]. Several types of GABAA receptor subunits have been cloned and sequenced from mammals (a1–6, b1–3, c1–3, d, e, p, and h). Functional GABAA receptors are heteromeric pentamers, and muscimol is a specific agonist of GABAA receptors [23]. The majority of GABAA receptors consist of two a, two b, and one c subunit; however, less abundant populations of GABAA receptors contain the d subunit. Interestingly, for d subunit-containing GABAA receptors (dGABAA receptors), their expression is altered periodically during the estrous cycle in murine hippocampal neurons [24], and their expression is influenced by the ovarian steroid milieu [25]. d-GABAA receptors are located extra- or perisynaptically and are activated by ambient GABA [26]. In addition, dGABAA receptors are emerging as a valuable pharmacological target because they are not influenced by benzodiazepines [27]. Indeed, a previous study demonstrated that the d subunit of the GABAA receptor is expressed in the pituitary gland [28]. 4-Chloro-N-[6,8-dibromo-2-(2-thienyl)imidazo[1,2-alpyridine-3-yl] (DS1) is a compound that selectively and potently enhances GABA-evoked currents mediated by d subunit-
ABSTRACT
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containing GABAA (a4b3d) receptors. In this present study, we found that DS1 increases gonadotropin subunit gene expression in a pituitary gonadotroph cell line.
Reporter Plasmid Construction and Luciferase Assay The reporter constructs used in these experiments were generated by fusing 797/ þ5 of the rat LHb gene (LHb-Luc), 2000/ þ698 of the rat FSHb gene (FSHb-Luc), or 846/0 of the human aGSU gene (a-Luc) to the firefly luciferase (Luc) cDNA in pXP2 [29]. The plasmid containing 1164/ þ62 of the mouse GnRH receptor gene (GnRH-Luc) fused to firefly luciferase cDNA was generously provided by Dr. Ursula Kaiser (Brigham and Women’s Hospital and Harvard University, Boston, MA). The cells were transiently transfected by electroporation with 2.0 lg/well of gonadotropin subunit-Luc and 0.1 lg of pRL-TK (Promega) containing Renilla luciferase. When the activity of the promoter containing CRE or SRE was measured, the cells were transfected with either 2.0 lg of pCRE-Luc or pSRE-Luc, which have the CRE enhancer (34) or five tandem repeats of SRE, respectively, upstream of the firefly luciferase gene. After incubation with the test reagents, the cells were washed with ice-cold phosphate-buffered saline and lysed with passive lysis buffer (Promega). When an inhibitor was used, the cells were preincubated with the inhibitor alone for 60 min, then stimulated with the test reagents together with the inhibitor. After centrifugation at 15 000 3 g at 48C for 5 min, firefly and Renilla luciferase activity was measured in the supernatant with the DualLuciferase Reporter Assay System (Promega) using a luminometer (TD-20/20; Promega) according to the manufacturer’s protocol. Luciferase activity was normalized to that of Renilla luciferase activity to correct for transfection efficiency, with results expressed as the fold-increase compared to the unstimulated control.
MATERIALS AND METHODS Materials The following chemicals and reagents were obtained from the indicated sources: fetal bovine serum (FBS) and trypsin from GIBCO, Invitrogen; Dulbecco modified Eagle medium (DMEM), DS1, GnRH, GABA, and penicillin-streptomycin from Sigma Chemical Co.; muscimol, SKF97541, and H89 from Abcam; U0126, anti-phosphorylated ERK (anti-P-ERK) antibody, anti-ERK antibody, and the appropriate horseradish peroxidase-conjugated secondary antibodies from Santa Cruz Biotechnology; serum response element (SRE) and cAMP response element (CRE) firefly luciferase reporter plasmids (pSRE-Luc and pCRE-Luc) from Stratagene; and pCI-neo from Promega.
Cell Culture LbT2 cells (kindly provided by Dr. P. Mellon of the University of California, San Diego) were maintained in a static monolayer culture in highglucose DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin at 378C in a humidified atmosphere of 5% CO2 in 95% air. After 24 h, the culture medium was changed to DMEM containing 1% heat-inactivated FBS and 1% penicillin-streptomycin and then incubated without (for control) or with the test reagents for the indicated times.
Statistical Analysis All experiments were repeated independently at least three times. Each experiment was performed with triplicate samples in each experimental group. The values are expressed as the mean 6 SEM. The median effective
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FIG. 1. Effect of DS1 on gonadotropin subunit promoter activity. LbT2 cells were transfected with 2.0 lg of LHb-Luc (A), FSHb-Luc (B), a-Luc (C), or GnRHR-Luc (D) vectors together with 0.1 lg of pRL-TK plasmid. At 48 h after transfection, the cells were treated with increasing concentrations of DS1 for 6 h. A luciferase assay was performed to examine LHb (A), FSHb (B), and a (C) subunit and GnRH receptor (D) promoter activity, which was normalized to pRL-TK activity. The results are expressed as fold-stimulation over unstimulated cells and presented as the mean 6 SEM of three independent experiments, each performed with triplicate samples. *P , 0.05, **P , 0.01 vs. control.
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concentration was calculated following nonlinear regression analysis. Statistical analysis was performed using Prism 5.0 (GraphPad Software) based on the appropriate analysis of variance (two-way or one-way, repeated measures) followed by the Dunnett or Bonferroni test for multiple comparisons. P , 0.05 was considered to be statistically significant.
Effect of DS1 on GnRH-Increased Gonadotropin Subunit Promoter Activity DS1 increased the activity all of three gonadotropin subunit promoters, but its effects were less than those induced by GnRH stimulation. GnRH increased LHb promoter activity by 8.76- 6 0.77-fold. In the presence of DS1, LHb promoter activity increased to 12.62- 6 1.21-fold, a significant difference (Fig. 2A). Similarly, GnRH-increased LHb and a subunit promoter activity was significantly enhanced in the presence of DS1 (from 8.07- 6 1.60-fold to 13.5- 6 1.79-fold for FSHb and from 81.67- 6 3.24-fold to 120.97- 6 1.49-fold for the a subunit) (Fig. 2, B and C).
RESULTS Effect of DS1 on Gonadotropin Subunit Promoter Activity Before the current experiments were started, we confirmed that d subunit-containing GABAA receptors were expressed in the mouse gonadotroph cell line LbT2 and primary cultures of anterior pituitary (data not shown). First, we examined the effect of DS1 on LbT2 cells. LbT2 cells were treated with increasing concentrations of DS1, and the promoter activity of the gonadotropin LHb, FSHb, and a subunits was determined using a dual luciferase assay. DS1 increased the promoter activity of all three gonadotropin subunits. A significant increase in activity was obtained at 1 lM DS1, and a maximal peak of 2.77- 6 0.02-fold was observed at 10 lM DS1 for the LHb subunit (Fig. 1A). DS1 also increased FSHb and a subunit promoter activity, with a peak at 50 lM (of 2.07- 6 0.42-fold) for FSHb and at 100 lM (of 4.66- 6 0.57-fold) for the a subunit (Fig. 1, B and C).
Effect of other GABA Receptor Agonists on Gonadotropin Subunit Promoter Activity DS1 is a specific agonist of d subunit-containing GABAA receptors. A prototypic specific agonist for GABAA receptors, muscimol, did not increase either LHb or FSHb promoter activity and did not modulate the effect of GnRH (Fig. 3, A and B). Similarly, a specific agonist of GABAB receptors, SKF97541, did not increase LHb or FSHb promoter activity and did not modulate the effect of GnRH (Fig. 3, C and D). Conversely, although a natural GABA compound did not 3
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FIG. 2. Effect of DS1 on GnRH-induced gonadotropin promoter activity. LbT2 cells were transfected with 2.0 lg of LHb-Luc (A), FSHb-Luc (B), or a-Luc (C) vectors together with 0.1 lg of pRL-TK plasmid. At 48 h after transfection, the cells were treated with 10 nM GnRH, 50 lM DS1, or both for 6 h. A luciferase assay was performed to examine LHb (A), FSHb (B), and a (C) subunit promoter activity, which was normalized to pRL-TK activity. The results are expressed as fold-stimulation over unstimulated cells and presented as the mean 6 SEM of three independent experiments, each performed with triplicate samples. The differences between GnRH and GnRH plus DS1 were statistically significant for the LHb, FSHb, and a subunit promoters. *P , 0.05, **P , 0.01 vs. control.
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increase the basal activity of the LHb or FSHb promoters, GABA significantly enhanced GnRH-increased FSHb promoter activity (Fig. 4B). The induction of LHb promoter activity by GnRH was slightly enhanced in the presence of GABA, but this effect was not statistically significant (Fig. 4A).
Similar to the gonadotropin promoters, both SRE and CRE promoter activity induced by GnRH was enhanced in the presence of DS1 (Fig. 5, A and B). Effect of ERK and PKA Inhibitors on DS1-Increased Gonadotropin Subunit Promoter Activity
Effect of DS1 on SRE and CRE Promoter Activity
U0126, a specific inhibitor of the ERK signaling pathway, almost completely inhibited DS1-induced LHb and FSHb promoter activity (Fig. 6, A and B). Conversely, H89, a PKA inhibitor, did not have a significant effect on GnRH-increased gonadotropin promoter activity.
A DNA domain in the promoter region, SRE binds ERKmediated transcription factors, whereas the CRE promoter is a target of the cAMP/protein kinase A (PKA) pathway. GnRH drastically increased SRE and CRE promoter activity by 20.786 0.87-fold and 13.74- 6 2.46-fold, respectively. DS1 increased SRE promoter activity by 2.08- 6 0.19-fold. Increased ERK phosphorylation was confirmed by Western blot analysis using a phosphorylation-specific antibody, but the increase in phosphorylation induced by DS1 was limited compared to that induced by GnRH stimulation (DS1, 3.16- 6 0.72-fold; GnRH, 19.02- 6 1.36-fold; data not shown). CRE promoter activity was also stimulated by DS1 (2.25- 6 0.29fold). The effect of DS1 on the SRE and CRE promoters was significant, but limited compared to the effect of GnRH.
DISCUSSION Schally et al. [19] first isolated GABA from the hypothalamus in 1997, and subsequent authors proposed that GABA is the major inhibitory neurotransmitter of the central nervous system. It also acts as a signaling gate to the reproductive system via its direct influence on GnRH neurons. Generally, the secretion and synthesis of gonadotropins are understood to be mainly under the control of hypothalamic 4
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FIG. 3. Effect of other GABA receptor agonists, muscimol and SKF97541, on gonadotropin promoter activity. LbT2 cells were transfected with 2.0 lg LHb-Luc (A and C) or FSHb-Luc (B and D) vectors together with 0.1 lg of pRL-TK plasmid. At 48 h after transfection, the cells were treated with 10 nM GnRH and 50 lM muscimol, a prototypic specific agonist of GABAA receptors (A and B), or 50 lM SKF97541, a GABAB receptor agonist (C and D), or both for 6 h. A luciferase assay was performed to examine LHb (A and C) and FSHb (B and D) subunit promoter activity, which was normalized to pRL-TK activity. The results are expressed as fold-stimulation over unstimulated cells and presented as the mean 6 SEM of three independent experiments, each performed with triplicate samples. The difference between GnRH and GnRH plus muscimol and between GnRH and GnRH plus SKF97541 for both LHb and FSHb promoters was not statistically significant (n.s.). **P , 0.01 vs. control.
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GnRH. Ovarian factors, such as inhibin, activin, and follistatin, also act as regulators of gonadotropins [30]. In addition, previous observations support the role of hypothalamic PACAP in the control of gonadotropin subunit expression [31, 32]. In the present study, we applied DS1, a GABAA a4b3d receptor agonist, and found that DS1 stimulation increased the gene expression of the gonadotropin subunits a, LHb, and FSHb in a pituitary gonadotroph cell line. GnRH receptor transcriptional activity was also stimulated by this compound. In addition, we found that the GnRH-induced increase of gonadotropin subunit expression was enhanced in the presence of DS1, suggesting that DS1 participates in the regulation of gonadotropin subunit gene expression by itself and also positively modulates other receptor-mediated increases of gonadotropin expression. This stimulation of gonadotropin subunit expression was not observed with a prototypic
specific agonist of GABAA receptors, muscimol, or a natural GABA compound, suggesting the possibility that the gonadotropin subunit stimulating effect of DS1 is mediated only by d subunit-containing GABAA receptors. The possibility exists that the stimulation of gonadotropin subunit expression by DS1 might not be through GABAA receptors, and we should interpret the current results carefully. Indeed, muscimol is regarded as a compound that activates all GABAA receptor subtypes [33]. However, it was reported that the high-affinity binding sites of muscimol differ greatly according to the distribution of GABAA receptors in the brain and that the sensitivity of d subunit-containing GABAA receptors to muscimol differs by gender [34]. Thus, it is appropriate to think muscimol does not evoke the same effect on d subunitcontaining GABAA receptors as DS1. Because the potency of muscimol and universal GABA compounds to stimulate d
FIG. 5. Effect of DS1 on SRE and CRE promoter activity. LbT2 cells were transfected with 2.0 lg of SRE-Luc (A) or CRE-Luc (B) and 0.1 lg of pRL-TK plasmid. At 48 h after transfection, the cells were treated with 10 nM GnRH, 50 lM DS1, or both for 6 h. A luciferase assay was performed to examine SRE and CRE promoter activity, which was normalized to pRL-TK activity and is expressed as the fold-difference of activation over unstimulated controls. The difference between GnRH and GnRH plus DS1 for both the SRE and CRE promoters was statistically significant (P , 0.01 and P , 0.05). **P , 0.01 vs. control.
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FIG. 4. Effect of a natural GABA compound on gonadotropin subunit promoter activity. LbT2 cells were transfected with 2.0 lg of LHb-Luc (A) or FSHbLuc (B) vectors together with 0.1 lg of pRL-TK plasmid. At 48 h after transfection, the cells were treated with 10 nM GnRH, 50 lM of a natural GABA compound, or both for 6 h. A luciferase assay was performed to examine LHb (A) and FSHb (B) subunit promoter activity, which was normalized to pRLTK activity. The results are expressed as fold-stimulation over unstimulated cells and presented as the mean 6 SEM of three independent experiments, each performed with triplicate samples. The difference between GnRH and GnRH plus GABA for the LHb promoter was not statistically significant (n.s.). The difference between GnRH and GnRH plus GABA for the FSHb promoter was statistically significant (P , 0.01). **P , 0.01 vs. control.
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receptors for GABA are present in anterior pituitary cells [28], and GABA is synthesized and released in the pituitary [36]. The hormonal milieu affects the GABA-ergic control of GnRH output and eventually controls gonadotropin release. The electrical response to GABA in rat GnRH neurons was reportedly modulated differently according to the stage of the estrous cycle, diurnal rhythm, and sex [37]. Also, the response to GABA on GnRH/LH secretion in ewes differed during breeding and nonbreeding seasons [38, 39]. In addition, interesting phenomena were reported for d-GABAA receptors in previous studies. The expression of d-GABAA receptors in hippocampal neurons is not static; rather, it changes dynamically during the estrous cycle [24, 25, 40]. Fluctuations in neurosteroid levels during the estrous cycle are coupled with alterations of d-GABAA receptors. Estrogen is generally believed to have a stimulatory effect on gonadotropin synthesis and secretion at the pituitary level in vivo [41]; the enforcement of d-GABAA receptor signaling might be one of the phenomena induced by gonadotrophs following estrogen elevation. In the present study, we observed that DS1 increased the activity of intracellular signaling pathways, such as ERK and cAMP/PKA, in LbT2 cells. In addition, signal transduction
subunit-containing GABAA receptors is weak, they might fail to stimulate gonadotropins by themselves. Indeed, both muscimol and universal GABA compounds failed to stimulate SRE and CRE promoters (data not shown). In addition, DS1increased gonadotropin subunit promoter expression was not inhibited in the presence of the universal GABAA receptor antagonist bicuculline (data not shown). Conversely, both muscimol and universal GABA compounds tended to enhance the action of GnRH on gonadotropin subunit expression slightly, but not significantly. Only the potentiation of the effects of GnRH on FSHb subunit expression by universal GABA compounds was statistically significant. If muscimol or GABA compounds could bind d subunit-containing GABAA receptors potently, it is natural to think these compounds may induce some effects on the action of GnRH. Thus, we think that it is reasonable to hypothesize that the effects of DS1 are associated with d subunit-containing GABAA receptors. A number of studies have demonstrated the GABA-ergic neurons in the hypothalamus are involved in controlling GnRH release, so gonadotropin release from anterior pituitary cells is also controlled indirectly by GABA. GABA is released from tuberoinfundibular and other hypothalamic regions as well as from neurointermediate pituitary lobe axons [35]. In addition, 6
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FIG. 6. Effect of U0126, an MEK inhibitor, and H89, a PKA inhibitor, on the DS1-induced increase of gonadotropin promoter activity. LbT2 cells were transfected with 2.0 lg of LHb-Luc (A and C) or FSHb-Luc (B and D) vectors together with 0.1 lg of pRL-TK plasmid. At 48 h after transfection, the cells were treated with 50 lM DS1 and 10 lM U0126 (A and B) or 10 lM H89 (C and D), for 6 h. A luciferase assay was performed to examine LHb (A and C) and FSHb (B and D) subunit promoter activity, which was normalized to pRL-TK activity. The results are expressed as fold-stimulation over unstimulated cells and presented as the mean 6 SEM of three independent experiments, each performed with triplicate samples. The difference between DS1 and DS1 plus H89 for both the LHb and FSHb promoters was not statistically significant (n.s.). The difference between DS1 and DS1 plus U0126 for both the LHb and FSHb promoters was statistically significant (P , 0.01). **P , 0.01 vs. control.
GONADOTROPIN EXPRESSION BY DS1
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systems that were evoked by GnRH were augmented in the presence of DS1, with a concomitant increase in GnRHinduced gonadotropin subunit expression. The excitatory effect of GABAA receptors on GnRH-producing neurons was thought to be due to an increase in Ca2þ via the activation of L-type calcium channels [42], which results in chloride ion efflux and membrane depolarization [43]. In addition, in a whole-cell voltage-clamp study using stable cell lines expressing GABAA a4b3d receptors, DS1 potently enhanced GABA-evoked currents produced by chloride ion flux through GABA-gated ion channels [44]. A number of phenomena were evoked in LbT2 cells by DS1 alone, so DS1 probably exerts its stimulatory effect through specific subunits of GABAA receptors. Conversely, GnRH receptors couple with Gq protein, stimulate phospholipase C, and generate diacylglycerols, resulting in protein kinase C (PKC) activation and inositol phospholipid (IP3) turnover. Ultimately, PKC stimulates the ERK signaling pathway, and IP3 increases intracellular calcium levels by promoting calcium mobilization. GnRH also stimulates the cAMP/PKA pathway through Gs-coupled receptors [45]. Considering the observations that DS1 alone increases the activation of the ERK and cAMP/PKA signaling pathways, which were determined by SRE- and CRE-luciferase assays, and that DS1 potentiates the effect of GnRH on the ERK and cAMP/PKA signaling pathways, we suggest that d subunit-containing GABAA receptors share part of the same intracellular signaling pathways as GnRH receptors. The effect of DS1 on gonadotropin LHb and FSHb subunit expression was almost completely inhibited in the presence of a specific inhibitor of the ERK pathway, but not by H89, an inhibitor of PKA. This observation suggests that the stimulating effect of DS1 on gonadotropin subunit expression depends on the ERK signaling pathway. The involvement of the ERK signaling pathway on gonadotropin LHb [46, 47] and FSHb subunit [47] gene expression has been described previously. The inhibition of ERK using a specific inhibitor prevented both LHb and FSHb subunit expression in LbT2 cells [48], and the induction of MEKK, an upstream activator of ERK, increased LHb and FSHb subunit promoter activity without GnRH stimulation [49]. These observations clearly suggest that DS1 increases gonadotropin subunit expression in an ERK-dependent manner. In the present study, we found that DS1, a compound that selectively enhances the expression of d subunit-containing GABAA receptors, can increase gonadotropin subunit gene expression. The precise role of d-GABAA receptors in the expression of pituitary gonadotrophs remains to be elucidated; however, these receptors might play an important role in their regulation. In addition, DS1 could be developed as a novel therapy for activation of the HPG axis.
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