Role of Kisspeptin and Kiss1r in the Regulation of Prolactin Gene Expression in Rat Somatolactotroph GH3 Cells

Endocrine (2019) 63:101–111 https://doi.org/10.1007/s12020-018-1759-1

ORIGINAL ARTICLE

Role of kisspeptin and Kiss1R in the regulation of prolactin gene expression in rat somatolactotroph GH3 cells

1 1 1 1 1 1 Tomomi Hara ● Haruhiko Kanasaki ● Tuvshintugs Tumurbaatar ● Aki Oride ● Hiroe Okada ● Satoru Kyo

Received: 1 June 2018 / Accepted: 23 August 2018 / Published online: 25 September 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract Hypothalamic kisspeptin is a known principal activator of gonadotropin-releasing hormone neurons and governs the hypothalamic-pituitary-gonadal axis. Previous reports have shown that kisspeptin is also released into the hypophyseal portal circulation and directly affects the anterior pituitary. In this study, we examined the direct effect of kisspeptin on pituitary prolactin-producing cells. The rat pituitary somatolactotroph cell line GH3 expresses the kisspeptin receptor (Kiss1R); however, in these cells, kisspeptin failed to stimulate prolactin-promoter activity. When GH3 cells overexpressed Kiss1R, kisspeptin clearly increased prolactin-promoter activity, with a concomitant increase in extracellular signal-regulated kinase (ERK) and cAMP/protein kinase A (PKA) signaling pathways. In the experiments using GH3 cells overexpressing Kiss1R,

1234567890();,: 1234567890();,: kisspeptin did not potentiate thyrotropin-releasing hormone (TRH)-induced prolactin-promoter activity, but it potentiated the pituitary adenylate cyclase-activating polypeptide-induced prolactin-promoter activity, with a concomitant enhancement of ERK and PKA signaling pathways. Although the basal and TRH-induced prolactin-promoter activities were not modulated by increasing amounts of Kiss1R expression in GH3 cells, kisspeptin-stimulated prolactin-promoter activity was increased by the amount of Kiss1R overexpression. Endogenous Kiss1r mRNA expression in GH3 cells was signiﬁcantly increased by treatment with estradiol (E2) but not by TRH. In addition, kisspeptin’s ability to stimulate prolactin-promoter activity was restored after E2 treatment in non-transfected GH3 cells. Our current observations suggest that kisspeptin might have a direct effect on prolactin expression in the anterior pituitary prolactin-producing cells under the inﬂuence of E2, which may regulate Kiss1R expression and function.

Keywords Kisspeptin ● Prolactin ● Kiss1R ● TRH ● PACAP

Introduction nucleus (AVPV) and the arcuate nucleus (ARC). Kisspeptin neurons in the AVPV region, in which the Kiss1 gene is Kisspeptin, which is encoded by the Kiss1 gene, is known upregulated by estradiol (E2), are known to be involved in for its principal role in reproductive function by regulating the E2-induced GnRH/luteinizing hormone (LH) surge, the hypothalamic-pituitary-gonadal axis, and it primarily whereas ARC kisspeptin neurons, which coexpress neuro- controls gonadotropin-releasing hormone (GnRH) neurons kinin B and dynorphin (Dyn) and in which E2 down- in the hypothalamus [1]. The Kiss1 gene and the kisspeptin regulates the Kiss1 gene, maintain pulsatile release of receptor (Kiss1R) are broadly distributed in the brain. In GnRH [2–5]. In addition to the two major populations, there rodents, kisspeptin neurons are located in the two different are other populations of kisspeptin neurons in the hypo- hypothalamic areas, the anteroventral periventricular thalamus, such as those in the ventromedial hypothalamus and paraventricular nucleus [6, 7]. Extrahypothalamic kisspeptin neurons have also been detected in bed nucleus of the stria terminals and median amygdala [6, 8]. Kisspeptin produced by the hypothalamus is known to be released into the peripheral circulation because kisspeptin * Haruhiko Kanasaki has been detected in the hypophyseal portal blood [9]. This [email protected] observation implies that the hypothalamic peptide kis- 1 Department of Obstetrics and Gynecology, Shimane University speptin directly modulates hormone secretion from the School of Medicine, Izumo 693-8501, Japan anterior pituitary as a hypothalamic factor. In addition, 102 Endocrine (2019) 63:101–111 kisspeptin and Kiss1R are expressed in peripheral organs Materials and methods outside the central nervous system [10, 11]. The pituitary gland also expresses the Kiss1 gene and Kiss1R [12], sug- Materials gesting that pituitary hormones might also be under the influence of kisspeptin in an autocrine and/or paracrine The following chemicals and reagents were sourced as fashion. follows: fetal bovine serum (FBS) and trypsin (GIBCO, The anterior pituitary gland is composed of five major Invitrogen, Carlsbad, CA); Dulbecco’s modified Eagle’s different hormone-secreting cells: corticotrophs, thyro- medium (DMEM), penicillin-streptomycin, TRH (Sigma- trophs, gonadotrophs, somatotrophs, and lactotrophs. These Aldrich, St. Louis, MO); pituitary adenylate cyclase- cells synthesize and secrete adrenocorticotropic hormone activating polypeptide 38 (PACAP38, Peptide Institute, (ACTH), thyroid-stimulating hormone (TSH), gonado- Osaka, Japan); kisspeptin-10 (KP10) (ANA SPEC, Fre- tropins (LH and follicle-stimulating hormone [FSH]), mont, CA); serum response element (SRE) and cAMP- growth hormone (GH), and prolactin under the influence of response element (CRE) firefly luciferase reporter genes relatively specific hypothalamic peptides such as (pSRE-Luc and pCRE-Luc) and pCI-neo (Promega, Madi- corticotropin-releasing hormone, thyrotropin-releasing hor- son, WI). mone (TRH), GnRH, and GH-releasing hormone. In addition to the primary secretagogues, several in vitro studies Cell culture support the hypothesis that hypothalamic kisspeptin can also act at the pituitary level and modulate pituitary func- GH3 cells were plated in 35-mm tissue culture dishes and tion. In rat, bovine, and porcine pituitary cultures, kis- incubated in high-glucose DMEM containing 10% heat- speptin stimulates the release of GH, prolactin, and LH [13– inactivated FBS and 1% penicillin-streptomycin at 37 °C in 15]. However, the first studies using cultured rat pituitary a humidified atmosphere of 5% CO2 in air. After 24 h, the cells and anterior pituitary fragments did not demonstrate culture medium was changed to high-glucose DMEM any direct effect on gonadotropin secretion [16, 17]. In containing 1% heat-inactivated FBS and 1% penicillin- addition, in vitro experiments using baboon pituitary cell streptomycin and incubated without (control) or with test cultures produced no evidence that ACTH and TSH release reagents for the indicated times. are modulated by kisspeptin, although LH and GH release was reported to be stimulated in this culture [18]. As for the Western blot analysis gonadotropin regulation by kisspeptin in pituitary gonadotrophs, we have previously reported that kisspeptin had a GH3 cell extracts were lysed on ice with RIPA buffer direct effect on the mouse pituitary gonadotroph cell line (phosphate-buffered saline [PBS], 1% NP-40, 0.5% sodium LβT2 and increased both LHβ and FSHβ-subunit-promoter deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) con- transcriptional activities [19]. taining 0.1 mg/mL phenylmethylsulfonyl fluoride, 30 mg/ Previous studies suggest that kisspeptin might have a mL aprotinin, and 1 mM sodium orthovanadate, scraped for direct effect on pituitary prolactin-producing cells. Kis- 20 s, and centrifuged at 14,000×g for 10 min at 4 °C. Protein speptin increases the prolactin release from cultured bovine concentration in the cell lysate supernatants was measured anterior pituitary cells, but its effect was less potent than using the Bradford method. Denatured protein (10 µg per that of TRH [20]. However, in another study using rat well) was resolved in 10% SDS polyacrylamide gel elec- anterior pituitary cell cultures, kisspeptin failed to modulate trophoresis gels according to standard protocols. Protein prolactin release [21]. In addition, kisspeptin has been was transferred onto polyvinylidene difluoride membranes shown to stimulate prolactin secretion and gene expression (Hybond-P PVDF, Amersham Biosciences, Little Chalfont, by directly acting at the pituitary level in goldfish;[22] UK), which were blocked for 2 h at room temperature in however, the capacity of this peptide to modulate Blotto (5% milk in Tris-buffered saline). Membranes were prolactin has not been confirmed in in vivo studies using incubated with anti-Kiss1R antibody (1:200 dilution; Santa goats [23]. Cruz Biotechnology, Inc., Dallas, TX) in Blotto overnight at In this study, we focused on the direct effect of kis- 4 °C and washed 3 times for 10 min per wash with Tris- speptin on prolactin-producing pituitary cells. We utilized buffered saline/1% Tween. Subsequent incubation with GH3 cells, which are a clonal strain of rat pituitary tumor horseradish peroxidase-conjugated monoclonal antibody and can synthesize and secrete both prolactin and GH [24]. was performed for 1 h at room temperature in Blotto, and We confirmed the presence of Kiss1R in these cells, and additional washes were performed appropriately. Following examined the direct effect of kisspeptin in these cells. enhanced chemiluminescence detection (Amersham Bios- ciences), membranes were exposed to X-ray film (Fujifilm, Tokyo, Japan). Extracts from rat anterior pituitary tissue Endocrine (2019) 63:101–111 103 were used as positive control, whereas extracts from COS7 BRL Life Technologies) according to the manufacturer’s cells, which are devoid of Kiss1R, were used as negative instructions. Total RNA of female rat anterior pituitary control [25]. tissue, which was excised under deep sodium pentobarbital anesthesia, was used as positive control. This protocol was Receptor overexpression approved by the committee of the Experimental Animal Center for Integrated Research in Shimane University The human GPR54 (Kiss1R) vector was generously pro- (IZ27-82). To obtain cDNA, 1.0 µg of total RNA was vided by Dr. Ursula Kaiser (Brigham and Women’s Hos- reverse transcribed using an oligo-dT primer (Promega), pital and Harvard Medical School, Boston, MA) and the and was prepared using a First-Strand cDNA Synthesis Kit PACAP type I receptor (PAC1R)-expressing vector (HA- (GIBCO, Invitrogen) in reverse transcription (RT) buffer. tagged PAC1R/pEF-BOS in pCAM17) was kindly provided The preparation was supplemented with 10 mM dithio- by Prof. A. Baba (Osaka University) [26]. Cells were threitol, 1 mM each dNTP, and 200 units of RNAse inhi- transiently transfected via electroporation with either bitor/human placenta ribonuclease inhibitor (Ribonuclease Kiss1R or PAC1R expression vectors. An empty vector Inhibitor, Code No. 2310, Takara, Tokyo, Japan) in a final (pCI neo) served as the mock control. volume of 10 μl. The reaction was incubated at 37 °C for 60 min. For the detection of Kiss1r mRNA, after PCR Transfections and luciferase assays amplification using primers for Kiss1r (sense: 5′- CTGCCACAGACGTCACTTTC-3′, antisense: 5′-ACA- The prolactin promoter reporter construct used was gener- TACCAGCGGTCCACACT-3′)[29], amplicons were ated by fusing −609/+12 of the prolactin gene to the firefly electrophoresed in a 2.0% agarose gel and visualized with luciferase cDNA in pGL3 (PRL-Luc), as previously ethidium bromide staining. cDNAs from rat anterior pitui- described [27]. To determine the extracellular signal-related tary tissues and COS7 cells were used as positive and kinase (ERK) and cAMP/protein kinase A (PKA)-mediated negative controls, respectively. Quantification of Kiss1r and signaling activity, pSRE-Luc (2.0 μg/well; contains tandem Prl mRNA was obtained through real-time quantitative repeats of the Sre enhancer [×4] upstream of the firefly PCR (ABI Prism 7000, Perkin Elmer Applied Biosystems, luciferase gene) and pCRE-Luc (2.0 μg/well; contains tan- Foster City, CA) following the manufacturer’s protocol dem repeats of the CRE enhancer [×4] upstream of the (User Bulletin No. 2), and utilizing Universal ProbeLibrary firefly luciferase gene) were applied. GH3 cells were tran- Probes and FastStart Master Mix (Roche Diagnostics, siently transfected by electroporation [28] with 2.0 μg/dish Mannheim, Germany). Using specific primers for Kiss1r of reporter construct and 0.1 μg of pRL-TK (Promega), [29] and Prl (sense: 5′-AATGACGGAAATA- which expresses Renilla luciferase, and plated in 35-mm GATGATTG-3′, antisense: 5′-CCAGTTATTAGTT- tissue culture dishes. When Kiss1R and PAC1R were GAAAVAGA-3′)[27], the simultaneous measurement of expressed in GH3 cells, Kiss1R- and PAC1R-expressing the mRNA of interest and GAPDH mRNA permitted nor- vectors were transiently transfected together with these malization of the amount of cDNA added per sample. For luciferase expression vectors. After stimulation, cells were each set of primers, a no-template control was included. The washed with ice-cold PBS and lysed with Passive Lysis thermal cycling conditions were 95 °C for 10 min for Buffer (Promega). Cell debris was pelleted by centrifuga- denaturation, followed by 40 cycles of 95 °C for 15 s and tion at 14,000×g for 10 min at 4 °C, and firefly luciferase 60 °C for 1 min. The cycle threshold (Ct) was determined and Renilla luciferase activities were measured in the using PRISM 7000 software and post-amplification data supernatants with the Dual-Luciferase Reporter Assay were analyzed using the delta-delta Ct method with System using a luminometer (TD-20/20) (Promega) Microsoft Excel. according to the manufacturer’s protocol. Firefly luciferase activity was normalized to Renilla luciferase activity to Statistical analysis correct for transfection efficiency, and the results are expressed as the fold increase compared to the unstimulated All experiments were independently repeated at least three control. All experiments were performed independently, times. Each experiment in each experimental group was three times, each with triplicate samples. performed using either triplicate samples (luciferase assay) or duplicate samples (real-time RT-PCR). Briefly, when we RNA preparation, reverse transcription, RT-PCR, and determined the mRNA expression, two samples were assayed real-time quantitative RT-PCR in duplicate. Six averages from three independent experiments were statistically analyzed. For the luciferase assay, three Total RNA from untreated or treated GH3 cells was samples were assayed in one experiment, and three averages extracted using commercially available TRIzol-S (GIBCO were statistically analyzed. Data are expressed as the mean ± 104 Endocrine (2019) 63:101–111

A B PCR Western blotting

Kiss1r mRNA Kiss1R

Rat pituitary GH3 cells COS7 cells Rat pituitary GH3 cells COS7 cells tissue tissue

Fig. 1 Expression of Kiss1R in GH3 cells. a Total RNA from GH3 anterior pituitary tissues were analyzed by SDS-PAGE followed by cells and rat anterior pituitary tissues were prepared and RT-PCR was immunoblotting and incubation with antibody against Kiss1R. The carried out for 40 cycles using Kiss1r-specific primers. PCR products bands were visualized using horseradish peroxidase-conjugated sec- were resolved in a 2.0% agarose gel and visualized with ethidium ondary antibody. Tissues from rat anterior pituitary and extracts from bromide staining. b Cell lysates (10 μg) from GH3 cells and rat COS7 cells were used as positive and negative controls, respectively standard error of the mean. Statistical analysis was performed A ∗∗ using one-way ANOVA and Bonferroni’s post hoc test. P < 3.5 0.05 was considered statistically significant. 3 2.5 2 Results 1.5 1 Expression of Kiss1R and the effect of kisspeptin on (Fold induction) 0.5 prolactin-promoter activity in GH3 cells PRL-Luciferase promoter 0 control Kp10 Kp10 Kp10 Kp10 TRH 1nM 10nM 100nM 1μM 10μM First, we examined whether GH3 cells express Kiss1R. RT- fi B ∗∗ PCR analysis using speci c primers for Kiss1r revealed that 4 Kiss1r mRNA could be detected in the extracts from rat 3.5 ∗ ∗∗ anterior pituitary tissues as well as GH3 cells (Fig. 1a). 3 2.5 Kiss1r mRNA was not detected in COS7 cells, which are a ∗ fibroblast-like cell line derived from monkey kidney. 2 Western blotting analysis using anti-Kiss1R antibody 1.5 1

revealed that Kiss1R protein was also expressed in GH3 (Fold induction) 0.5 cells (Fig. 1b). Next, we examined the direct effect of kis- 0 PRL-Luciferase promoter speptin on prolactin expression using GH3 cells. Stimulat- control Kp10 Kp10 Kp10 Kp10 TRH ing the GH3 cells with increasing concentrations of 1nM 10nM 100nM 1μM 10μM kisspeptin failed to modulate the transcriptional activity of Fig. 2 Effect of kisspeptin on the activity of the prolactin (PRL) the prolactin promoter. TRH, a known prolactin secreta- promoter. GH3 cells were transfected without (mock) a or with 2.0 µg b gogue, stimulated the prolactin promoters 3.02 ± 0.18-fold of Kiss1R-expressing vector , together with pRL-TK (0.1 µg) plus 2.0 µg of PRL-Luc vector. At 48 h after transfection, cells were treated (P < 0.01) in these cells (Fig. 2a). Because endogenous with increasing doses of kisspeptin (Kp10) for 6 h. A firefly luciferase Kiss1R did not respond to exogenous kisspeptin, we over- assay was performed to examine prolactin-promoter activity, which expressed Kiss1R in the GH3 cells. When GH3 cells were was normalized to Renilla luciferase activity, and is expressed as the transfected with Kiss1R-expressing vectors, the cells clearly fold induction over the unstimulated controls. Data are expressed as the mean ± standard error of the mean (three independent experiments responded to kisspeptin and increased the activity of the were performed using triplicate samples). *P < 0.05; **P < 0.01 vs. prolactin promoter. In Kiss1R-overexpressing GH3 cells, control kisspeptin stimulation significantly increased prolactin- promoter activity compared to the untreated cells: 1.69 ± 0.09-fold (P < 0.05) at 10 nM and 2.49 ± 0.11-fold (P < and CRE-luciferase promoter assays. SRE is a DNA domain 0.01) at 1 μM kisspeptin (Fig. 2b). in the promoter region that binds to ERK-mediated transcription factors, and SRE-promoter activity reflects ERK- Effect of kisspeptin on SRE and CRE-promoter mediated signaling pathway activity. The CRE promoter is activity in GH3 cells overexpressing Kiss1R a known target of the CRE-binding protein, and the CRE- luciferase reporter system reflects the activity of the cAMP/ To examine the signaling pathways activated by kisspeptin PKA signaling pathway. In mock-transfected GH3 cells, in Kiss1R-overexpressing GH3 cells, we performed SRE- neither the SRE nor the CRE promoter was activated by Endocrine (2019) 63:101–111 105

A P < 0.01

100 ∗∗ n.s. ∗∗ 6 ∗∗ ∗∗ 80 ∗∗ 5 ∗∗ ∗ 60 4 ∗ 3 40 2

20 (Fold induction) 1 (Fold induction) 0

0 PRL-Luciferase promoter control Kp10 Kp10 Kp10 control Kp10 TRH Kp10 PACAP PACAP SRE-Luciferase promoter + + 10nM 100nM 1μM TRH Kp10 Fig. 4 Effect of kisspeptin, TRH, and PACAP on prolactin-promoter B activity. GH3 cells were transfected with 2.0 µg of Kiss1R-expressing 120 ∗∗ and 2.0 µg of PAC1R-expressing vectors, together with 2.0 µg of PRL- Luc and pRL-TK (0.1 µg) vectors. Forty-eight hours after transfection, 100 cells were treated with 100 nM kisspeptin (Kp10), 100 nM TRH, ∗∗ 80 100 nM PACAP, or both Kp10 and TRH or Kp10 and PACAP for 6 h. ∗∗ A ﬁreﬂy luciferase assay was performed to examine prolactin- 60 promoter activity, which was normalized to Renilla luciferase activity and is expressed as the fold induction over the unstimulated con- 40 trols. Data are expressed as the mean ± standard error of the mean 20 (three independent experiments performed using triplicate samples).

(Fold induction) *P < 0.05; **P < 0.01 vs. control. The difference between PACAP and 0 PACAP + Kp10 treatment was statistically significant (P < 0.01). n.s., control Kp10 Kp10 Kp10 difference was not statistically significant CRE-Luciferase promoter 10nM 100nM 1μM Fig. 3 Effect of kisspeptin on SRE- and CRE-promoter activities. GH3 cells were transfected with 2.0 µg of Kiss1R-expressing vector, toge- treatment with kisspeptin and TRH did not enhance their ther with pRL-TK (0.1 µg) and 2.0 µg of SRE-Luc a or CRE-Luc b individual effects (TRH alone, 3.18 ± 0.45-fold vs. kis- vector. Forty-eight hours after transfection, cells were treated with speptin + TRH, 2.87 ± 0.53-fold; not significant). However, increasing amounts of kisspeptin (Kp10) for 6 h. A firefly luciferase assay was performed to examine SRE and CRE-promoter activity, PACAP stimulated prolactin-promoter activity to a greater which was normalized to Renilla luciferase activity and is expressed as degree compared to that stimulated by kisspeptin (4.32 ± the fold induction over the unstimulated controls. Data are expressed 0.39-fold), and combined stimulation with kisspeptin and as the mean ± standard error of the mean (three independent experi- PACAP significantly further increased prolactin-promoter ments performed using triplicate samples). **P < 0.01 vs. control activity compared with that by kisspeptin or PACAP alone (PACAP alone, 4.32 ± 0.39-fold vs. kisspeptin + PACAP, kisspeptin stimulation (data not shown). When Kiss1R was 5.20 ± 0.11-fold; P < 0.01) (Fig. 4). Next, we determined overexpressed in these cells, both SRE- and CRE- pro- the SRE and CRE-promoter activities stimulated by kis- moters were dramatically activated by kisspeptin. At 1 μM speptin, TRH, and PACAP. Kisspeptin dramatically kisspeptin stimulation, SRE- and CRE- promoters were increased SRE-promoter activity (99.79 ± 10.27-fold) com- activated 66.46 ± 15.19-fold and 94.42 ± 9.62-fold, respec- pared with that by TRH stimulation (6.44 ± 0.70-fold) in tively, relative to the control (Fig. 3a, b). Kiss1R- and PAC1R-overexpressing GH3 cells. Combined stimulation with kisspeptin and TRH failed to potentiate the Effect of kisspeptin on TRH or PACAP-induced effect of kisspeptin alone (105.47 ± 19.62-fold). PACAP prolactin-promoter activity stimulated SRE-promoter activity to a lesser degree than that stimulated by kisspeptin (14.88 ± 1.30-fold), and com- TRH is a principal secretagogue for prolactin. In addition, bined stimulation with kisspeptin and PACAP significantly PACAP participates in prolactin regulation [30]. To clearly increased the SRE-promoter activity compared with that observe the effect of both kisspeptin and PACAP, both stimulated by kisspeptin alone (kisspeptin alone, 99.79 ± receptors were overexpressed for the experiments. In GH3 10.27-fold vs. kisspeptin + PACAP, 150.91 ± 23.71-fold; P cells overexpressing both Kiss1R and PAC1R, kisspeptin < 0.05) (Fig. 5a). The patterns of CRE-promoter activity and TRH similarly stimulated prolactin-promoter activity stimulation were distinct from those of the SRE promoter. by 2.60 ± 0.10-fold and 3.18 ± 0.45-fold, respectively, Both kisspeptin and TRH significantly increased CRE- compared with the unstimulated controls, but combined promoter activity 26.73 ± 0.91-fold and 71.69 ± 4.24-fold, 106 Endocrine (2019) 63:101–111

A Effect of increasing amounts of Kiss1R-expressing P < 0.05 vector transfection in GH3 cells on kisspeptin and n.s. 200 ∗∗ TRH-induced prolactin-promoter activity

150 ∗∗ ∗∗ Next, we examined how the cell responses changed 100 according to Kiss1R expression levels. GH3 cells were transfected with different amounts of Kiss1R expression 50 vector and stimulated with kisspeptin. The basal activity

(Fold induction) ∗∗ ∗∗ and TRH or kisspeptin-induced fold induction of prolactin- 0 SRE-Luciferase promoter control Kp10 TRH Kp10 PACAP PACAP promoter activity were compared. Basal activity of the + + prolactin promoter was unchanged by transfection of TRH Kp10 increasing amounts of Kiss1R expression vector (Fig. 6a). TRH-induced fold induction of prolactin-promoter activity B was not modiﬁed by the dose of transfected Kiss1R P < 0.01 1400 ∗∗ expression vector (Fig. 6b). However, kisspeptin-stimulated 1200 prolactin-promoter activity was signiﬁcantly higher in the 1000 ∗∗ cells transfected with 2.0 and 4.0 μg of Kiss1R vector

800 n.s. (3.75 ± 1.27-fold and 2.44 ± 0.58-fold, respectively) com- 600 pared with that in cells transfected with 1.0 μg of Kiss1R 400 vector (1.66 ± 0.25-fold) (Fig. 6c). ∗∗ (Fold induction) ∗∗ 200 ∗∗ 0 Effect of E2 on Kiss1R expression and function

CRE-Luciferase promoter control Kp10 TRH Kp10 PACAP PACAP + + TRH Kp10 Next, we examined how endogenous Kiss1r mRNA is Fig. 5 Effect of kisspeptin, TRH, and PACAP on SRE- and CRE- regulated in GH3 cells. TRH (100 nM) did not stimulate promoter activities. GH3 cells were transfected with 2.0 µg of Kiss1R- Kiss1r gene expression in GH3 cells. However, treatment of expressing and 2.0 µg of PAC1R-expressing vectors, together with cells with 100 nM E2 significantly increased Kiss1r mRNA a b pRL-TK (0.1 µg) and 2.0 µg of SRE-Luc or CRE-Luc vectors. expression, which was increased 1.72 ± 0.2-fold compared Forty-eight hours after transfection, cells were treated with 100 nM kisspeptin (Kp10), 100 nM TRH, 100 nM PACAP, or both Kp10 and with untreated cells (Fig. 7a). Kiss1r mRNA was not TRH or Kp10 and PACAP for 6 h. A firefly luciferase assay was increased by concentrations of E2 lower than 100 nM (data performed to examine SRE and CRE-promoter activity, which was not shown). Furthermore, we found that GH3 cells acquired normalized to Renilla luciferase activity and is expressed as the fold responsiveness to kisspeptin after the 48-h treatment with induction over the unstimulated controls. Data are expressed as the mean ± standard error of the mean (three independent experiments 100 nM E2. GH3 cells that were not overexpressing Kiss1R performed using triplicate samples). **P < 0.01 vs. control. The dif- were treated with E2 for 48 h and then stimulated with ference in SRE-promoter activity between Kp10 and PACAP + Kp10 kisspeptin. In E2-treated GH3 cells, but not in untreated fi treatment was statistically signi cant (P < 0.05). The difference in GH3 cells, kisspeptin significantly increased Prl mRNA CRE-promoter activity between PACAP and PACAP + Kp10 treatment was statistically significant (P < 0.01). n.s., difference was not expression 1.90 ± 0.16-fold compared to unstimulated cells statistically significant (Fig. 7b).

Discussion respectively, and combined stimulation with kisspeptin and The importance of hypothalamic kisspeptin in the regulation TRH did not enhance the effect of TRH alone (TRH alone, of hypothalamic GnRH neurons has been well documented, 71.69 ± 4.24-fold vs. kisspeptin + TRH, 89.66 ± 2.60-fold; but accumulating evidence suggests that kisspeptin also not significant). PACAP more potently activated CRE- plays a role as a hypophysiotropic hormone and acts promoter activity, 798.28 ± 50.06-fold, compared with kis- directly within the pituitary gland. As for prolactin control speptin or TRH. Although TRH-induced CRE-promoter by kisspeptin at the pituitary level, previous in vivo studies activity was not modified in the presence of kisspeptin, showed divergent responses. Central intracerebroventricular PACAP-stimulated CRE-promoter activity was sig- injection of kisspeptin reduced the prolactin release in both nificantly potentiated in the presence of kisspeptin (PACAP male and female mice [31], while Szawka et al. observed an alone, 798.28 ± 50.06-fold vs. kisspeptin + PACAP, increase in prolactin by the same kisspeptin stimulation 1182.67 ± 58.97-fold; P < 0.01) (Fig. 5b). [21]. Stimulatory effects of kisspeptin on prolactin Endocrine (2019) 63:101–111 107

A 1.6 1.4 1.2 1 0.8 0.6

(basal activity) 0.4 0.2 PRL luciferase promoter PRL 0 mock kiss1R kiss1R kiss1R 1.0ug/well 2.0ug/well 4.0ug/well

B C P < 0.05 P < 0.05 4 6

3.5 5 3 2.5 4 2 3 1.5 2 1 0.5 1 (TRH-induced activity ) PRL luciferase promoter PRL (Kp10-induced activity ) 0 luciferase promoter PRL 0 mock kiss1R kiss1R kiss1R mock kiss1R kiss1R kiss1R 1.0ug/well 2.0ug/well 4.0ug/well 1.0ug/well 2.0ug/well 4.0ug/well

Fig. 6 Effects of Kiss1R overexpression on the basal levels and the expressed as basal a and the fold induction over unstimulated controls kisspeptin- and TRH-induced fold induction of prolactin-promoter in the mock-transfected group. The fold induction of TRH-stimulated activity. GH3 cells were transfected with 1.0–4.0 µg of Kiss1R- b and Kp10-stimulated c cells over unstimulated cells was calculated. expressing vector together with 2.0 µg of PRL-Luc and pRL-TK Data are expressed as the mean ± standard error of the mean (three (0.1 µg) vectors. Forty-eight hours after transfection, cells were treated independent experiments performed using triplicate samples). **P < with 100 nM TRH b and 100 nM kisspeptin (Kp10) c for 6 h. A firefly 0.01 vs. mock control. The differences between the 1.0 µg and higher luciferase assay was performed to examine prolactin (PRL) promoter amounts of Kiss1R-expressing cells in Kp10-induced prolactin-pro- activity, which was normalized to Renilla luciferase activity and moter activity were statistically significant (P < 0.05)

A B 2.5 2.5 ∗ ∗∗ 2 2

1.5 1.5

1 1 mRNA expression mRNA mRNA expression mRNA 0.5 (Fold induction) 0.5 (fold induction) Prl 0 0 Kiss1r control TRH 100nM E2 100nM control Kp10 control Kp 10

E2-non treatment E2- treatment 48H

Fig. 7 Effects of estradiol on Kiss1r mRNA expression and receptor quantitative real-time PCR after mRNA extraction and reverse tran- function. a GH3 cells were treated with 100 nM TRH and 100 nM scription. Samples for each experimental group were run in duplicate estradiol (E2) for 48 h. Kiss1r mRNA levels were measured by and normalized to GAPDH mRNA levels. Results are expressed as the quantitative real-time PCR after mRNA extraction and reverse tran- fold induction over unstimulated cells and presented as the mean ± scription. b GH3 cells were pre-treated in the presence or absence of standard error of the mean of three independent experiments, each 100 nM E2 for 48 h, and then stimulated with kisspeptin (Kp10) for an performed with duplicate samples. *P < 0.05; ** P < 0.01 vs. control additional 48 h. Prolactin (Prl) mRNA levels were measured by expression and release are also observed in the goldﬁsh pituitary cells. Because of the difﬁculty of isolating single- pituitary [22], but not in goats [23]. It was also reported that cell populations of pituitary lactotrophs from anterior kisspeptin could inhibit dopamine neurons in the hypotha- pituitary cells, we used the rat somatolactotroph cell line, lamus and modulate prolactin output [21]. GH3. These cells are a clonal strain of rat pituitary tumor In this study, we sought to clarify the action of kisspeptin and can synthesize and secrete both prolactin and GH [24]. at the single population of prolactin-producing lactotrophs GH3 cells respond to TRH and increase their synthesis and to evaluate the direct effect of kisspeptin on the anterior secretion of prolactin, but TRH reduces the synthesis of GH 108 Endocrine (2019) 63:101–111

[24, 27]. We found that GH3 cells express Kiss1R. Because cyclase, leading to the accumulation of cAMP and sub- GH3 cells are a pituitary prolactin-producing cell model, it sequent activation of PKA;[38] PACAP has also been is plausible that normal prolactin-producing cells in the shown to activate ERK signaling pathways in a PKA- pituitary gland express Kiss1R and are directly influenced dependent manner [28]. We presume that combined treat- by hypothalamic kisspeptin. Unexpectedly, GH3 cells did ment with kisspeptin and TRH did not enhance their not respond to kisspeptin and failed to modulate the tran- prolactin-producing ability because Kiss1R and the TRH scriptional activity of the prolactin promoter. We sometimes receptor share common signaling pathways that are mainly encounter similar problems when we use immortalized-cell initiated by Gq protein and PLC. Indeed, stimulation of models. The pituitary gonadotroph cell line LβT2 expresses SRE- and CRE-promoter activities was not potentiated by Kiss1R, but these cells do not respond to kisspeptin without the combined treatment with kisspeptin and TRH. In con- Kiss1R overexpression [19]. Similarly, the mouse GnRH- trast, prolactin-promoter activity could be enhanced by producing cell model GT1-7 expresses Kiss1R, but kis- combined stimulation with kisspeptin and PACAP, with speptin failed to induce responses in these cells without concomitant enhancement of SRE- and CRE-promoter Kiss1R overexpression [32]. We postulate that endogenous activities, hypothetically because the main signal transduc- Kiss1R is reduced or not functional, probably due to cell tion systems are distinct between Kiss1R and the PACAP immortalization or multiple passages in these immortalized- receptor. cell models. Thus, we used GH3 cells overexpressing GH3 cells express Kiss1R, but they did not respond to Kiss1R as a prolactin-producing cell model in our experi- kisspeptin. Interestingly, the function of endogenous ments to determine the effect of kisspeptin. Kiss1R in these cells was recovered in the presence of When GH3 cells overexpressed Kiss1R, they clearly 100 nM E2. In addition, we found that the same con- responded to kisspeptin and increased prolactin-promoter centration of E2 could increase the expression of Kiss1R in activity. These observations clearly demonstrated that the these cells. Although it is still unclear whether our experi- kisspeptin/Kiss1R system in prolactin-producing cells has ments actually reflect the physiological situation, our cur- the ability to stimulate prolactin expression. Both SRE- and rent observations imply that E2 has some roles in prolactin- CRE-reporter luciferase activities were increased by kis- producing cells by modulating the expression and function speptin stimulation in GH3 cells overexpressing Kiss1R, of Kiss1R. The importance of E2 in the functional effects of suggesting that the overexpressed Kiss1R coupled with Gq kisspeptin was previously described. In ewes, primary and Gs proteins and increased both ERK and cAMP/PKA pituitary cell cultures responded to kisspeptin and increased signaling pathways. Previous studies revealed that kis- LH secretion only when the cells were obtained during the speptin can activate a variety of signals via Kiss1R, which follicular phase of the estrous cycle, while no response was includes Gq protein-involved activation of phospholipase C seen in cells from the luteal phase or from ovariectomized (PLC) and subsequent accumulation of inositol triphosphate animals [9]. In an ovariectomized rat model, pre-exposure (IP3), intracellular Ca2+ mobilization, and activation of to E2 was effective in achieving maximal LH release in protein kinase C. Kisspeptin also activates ERK, P38 response to kisspeptin [39]. Similarly, the effect of kis- MAPK, and PI3K/Akt [33]. Although early studies showed speptin on gonadotropin release in women is greater in the that Kiss1R does not couple with Gs protein and does not preovulatory phase but lower in the follicular phase of the increase cAMP accumulation [11, 34], it was subsequently menstrual cycle [40]. In addition, an in vitro study using shown that kisspeptin can increase cAMP accumulation in GnRH-producing GT1-7 cells demonstrated that E2 goldfish pituitary cells [35] and in GnRH-producing GT1-7 induced Kiss1R expression [41]. cells [32]. The prolactin-inducing ability of kisspeptin was altered Being the principal prolactin secretagogue, TRH could of by the amount of Kiss1R expression vector transfected into course stimulate prolactin-promoter activity in our experi- the GH3 cells. In short, kisspeptin-stimulated prolactin- ments. The hypothalamic peptide PACAP also works as a promoter activity increased with increasing amounts of prolactin-stimulating factor [36]. Interestingly, combined Kiss1R vector. A similar phenomenon was also observed in stimulation with kisspeptin and TRH failed to potentiate gonadotropin-producing cells. The mouse gonadotroph cell their individual effects on the prolactin promoter; however, line LβT2 expresses Kiss1R and increasing Kiss1R the combination of kisspeptin and PACAP further stimu- expression potentiates the ability of kisspeptin to increase lated prolactin-promoter activity compared to that stimu- LHβ-subunit promoter transcriptional activity [19]. lated individually. The TRH receptor couples with Gq Although the amount of Kiss1R vector did not modify the protein and activates PLC-mediated signaling pathways, basal and TRH-induced transcriptional capacities of the which includes IP3 accumulation or Ca2+ mobilization [37]. prolactin promoter in this study using GH3 cells, the basal PACAP receptors, such as the PACAP type I receptor, activity of both LHβ- and FSHβ-subunit promoters in LβT2 mainly couple with Gs protein, which binds to adenylate cells was modified [19]. The increase in Kiss1R number Endocrine (2019) 63:101–111 109 under the influence of E2 might introduce some other kisspeptin when we stimulated them with kisspeptin. Our influences on prolactin-producing cells. current observations suggest an important role of kisspeptin/ Regarding the inconsistencies between previous reports Kiss1R in the regulation of pituitary lactotroph functions. on the action of kisspeptin and our current observations of the effect of E2 on kisspeptin/Kiss1R functions, it is plau- Funding This work was supported in part by a Grant-in-Aid for Sci- fi sible that conflicting results concerning the direct effect of enti c Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 17K11237). kisspeptin on prolactin secretion and gene expression depend on the hormonal milieu of the experimental models. Compliance with ethical standards Developmental stage, male versus female, or day of the estrous cycle of the experimental female animals might Conflict of interest The authors declare that they have no conflict of determine the sensitivity of pituitary lactotrophs to kis- interest. speptin in the in vivo studies. Furthermore, kisspeptin may not only directly affect lactotrophs, but may also influence Ethical approval All applicable international, national, and/or institu- other regulators of prolactin. Indeed, it was reported that tional guidelines for the care and use of animals were followed. dopamine neurons, known negative regulators of prolactin, receive synaptic input from kisspeptin neurons and mod- References ulate prolactin secretion [21, 42, 43]. Furthermore, a recent study using a female rat model demonstrated that kisspeptin α 1. L. Pinilla, E. Aguilar, C. Dieguez, R.P. Millar, M. 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