Protein Tyrosine Phosphatase Receptors N and N2 Control Pituitary Melanotroph Development and POMC Expression.

The neuroendocrine marker genes Ptprn and Ptprn2 encode protein tyrosine phosphatase receptors N and N2, 2 members of protein tyrosine phosphatase receptors void of enzymatic activity, and whose function and mechanism of action have not been elucidated. To explore the role(s) of Ptprn and Ptprn2 on the hypothalamic-pituitary-adrenal axis, we used mice in which both genes were knocked out (DKO). The focus in this study was on corticotrophs and melanotrophs from the anterior and intermediate lobes of the pituitary gland, respectively. In both sexes, DKO caused an increase in the expression of the corticotroph/melanotroph genes Pomc and Tbx19 and the melanotroph-specific gene Pax7. We also found in vivo and in vitro increased synthesis and release of beta-endorphin, alpha-melanocyte-stimulating hormone, and ACTH in DKO mice, which was associated with increased serum corticosterone levels and adrenal mass. DKO also increased the expression of other melanotroph-specific genes, but not corticotroph-specific genes. The dopaminergic pathway in the hypothalamus and dopaminergic receptors in melanotrophs were not affected in DKO mice. However, hyperplasia of the intermediate lobe was observed in DKO females and males, accompanied by increased proopiomelanocortin immunoreactivity per cell. These results indicate that protein tyrosine phosphatase receptor type N contributes to hypothalamic-pituitary-adrenal function by being involved in processes governing postnatal melanotroph development and Pomc expression.

Chromosomal-level assembly of Tokudaia osimensis, Tokudaia tokunoshimensis, and Tokudaia muenninki genomes.

Herein, we present the first high-quality long-read-based chromosome-level genome assemblies and gene annotations of the genomes of three endangered Tokudaia species: Tokudaia osimensis, Tokudaia tokunoshimensis, and Tokudaia muenninki. These species, which are endemic to different islands of the Ryukyu Islands, Japan, exhibited unique karyotypes and sex chromosomal characteristics. The genome assemblies generated using PacBio, Illumina, and Hi-C sequence data consisted of 13 (corresponded to 12 autosomes and one X chromosome), 23 (corresponded to 22 autosomes and one X chromosome), and 23 (corresponded to 21 autosomes and the neo- and ancestral X regions) chromosome-level scaffolds that contained 2,445, 2,477, and 2,661 Mbp of sequence data, respectively. Annotations of protein-coding genes were performed using RNA-Seq-based, homology-based, and Ab initio methods. BUSCO completeness values for every species exceeded 96% for genomes and 98% for genes. These data can be an important resource for contributing to our understanding of species genomes resulting from allopatric speciation and provide insights into mammalian sex-determination mechanisms and sex chromosome evolution.

Postnatal Development and Maintenance of Functional Pituitary Gonadotrophs Is Dependent on PI4-Kinase A.

Postnatal development of functional pituitary gonadotrophs is necessary for maturation of the hypothalamic-pituitary-gonadal axis, puberty, and reproduction. Here we examined the role of PI4-kinase A, which catalyzes the biosynthesis of PI4P in mouse reproduction by knocking out this enzyme in cells expressing the gonadotropin-releasing hormone (GnRH) receptor. Knockout (KO) mice were infertile, reflecting underdeveloped gonads and reproductive tracts and lack of puberty. The number and distribution of hypothalamic GnRH neurons and Gnrh1 expression in postnatal KOs were not affected, whereas Kiss1/kisspeptin expression was increased. KO of PI4-kinase A also did not alter embryonic establishment and neonatal development and function of the gonadotroph population. However, during the postnatal period, there was a progressive loss of expression of gonadotroph-specific genes, including Fshb, Lhb, and Gnrhr, accompanied by low gonadotropin synthesis. The postnatal gonadotroph population also progressively declined, reaching approximately one-third of that observed in controls at 3 months of age. In these residual gonadotrophs, GnRH-dependent calcium signaling and calcium-dependent membrane potential changes were lost, but intracellular administration of inositol-14,5-trisphosphate rescued this signaling. These results indicate a key role for PI4-kinase A in the postnatal development and maintenance of a functional gonadotroph population.

Functional Roles of DYRK2 as a Tumor Regulator.

The dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) regulates the induction of apoptosis and DNA repair, metastasis inhibition, cell cycle G1/S transition, protein scaffold stability for E3 ligase complexes, and embryogenesis. Owing to these functions, DYRK2 is thought to regulate tumorigenesis, and its function in cancer has been investigated. Notably, DYRK2 has been reported to function as a tumor suppressor; however, it has also been reported to act as an oncogene in some cancers. This discrepancy makes it difficult to elucidate the conserved functions of DYRK2 in cancer. Here, we reviewed the functions of DYRK2 in various cancers. Patient tissue samples were evaluated for each cancer type. Although some studies have used cell lines and/or xenografts to elucidate the mechanism of DYRK2 function, these studies are not sufficient to understand the role of DYRK2 in cancers. In particular, studies using genetically modified mice would help us to understand the reported functional duality of DYRK2 in cancer.

Extended-Synaptotagmin 1 Enhances Liver Cancer Progression Mediated by the Unconventional Secretion of Cytosolic Proteins.

Extended-synaptotagmin 1 (E-Syt1) is an endoplasmic reticulum membrane protein that is involved in cellular lipid transport. Our previous study identified E-Syt1 as a key factor for the unconventional protein secretion of cytoplasmic proteins in liver cancer, such as protein kinase C delta (PKCδ); however, it is unclear whether E-Syt1 is involved in tumorigenesis. Here, we showed that E-Syt1 contributes to the tumorigenic potential of liver cancer cells. E-Syt1 depletion significantly suppressed the proliferation of liver cancer cell lines. Database analysis revealed that E-Syt1 expression is a prognostic factor for hepatocellular carcinoma (HCC). Immunoblot analysis and cell-based extracellular HiBiT assays showed that E-Syt1 was required for the unconventional secretion of PKCδ in liver cancer cells. Furthermore, deficiency of E-Syt1 suppressed the activation of insulin-like growth factor 1 receptor (IGF1R) and extracellular-signal-related kinase 1/2 (Erk1/2), both of which are signaling pathways mediated by extracellular PKCδ. Three-dimensional sphere formation and xenograft model analysis revealed that E-Syt1 knockout significantly decreased tumorigenesis in liver cancer cells. These results provide evidence that E-Syt1 is critical for oncogenesis and is a therapeutic target for liver cancer.

Common and female-specific roles of protein tyrosine phosphatase receptors N and N2 in mice reproduction.

Simultaneous knockout of the neuroendocrine marker genes Ptprn and Ptprn2, which encode the protein tyrosine phosphatase receptors N and N2, causes infertility in female mice while males are fertile. To elucidate the mechanism of the sex-specific roles of Ptprn and Ptprn2 in mouse reproduction, we analyzed the effects of their double knockout (DKO) on the hypothalamic-pituitary-gonadal axis. In DKO females, delayed puberty and lack of ovulation were observed, complemented by changes in ovarian gene expression and steroidogenesis. In contrast, testicular gene expression, steroidogenesis, and reproductive organs development were not significantly affected in DKO males. However, in both sexes, pituitary luteinizing hormone (LH) beta gene expression and LH levels were reduced, as well as follicle-stimulating hormone beta gene and gonadotropin-releasing hormone (GnRH) gene, while the calcium-mobilizing and LH secretory actions of GnRH were preserved. Hypothalamic Gnrh1 and Kiss1 gene expression was also reduced in DKO females and males. In parallel, a significant decrease in the density of immunoreactive GnRH and kisspeptin fibers was detected in the hypothalamic arcuate nucleus of DKO females and males. The female-specific kisspeptin immunoreactivity in the rostral periventricular region of the third ventricle was also reduced in DKO females, but not in DKO males. These data indicate a critical role of Ptprn and Ptprn2 in kisspeptin-GnRH neuronal function and sexual dimorphism in the threshold levels of GnRH required to preserve reproductive functions.

Turnover of mammal sex chromosomes in the Sry-deficient Amami spiny rat is due to male-specific upregulation of Sox9.

Mammalian sex chromosomes are highly conserved, and sex is determined by SRY on the Y chromosome. Two exceptional rodent groups in which some species lack a Y chromosome and Sry offer insights into how novel sex genes can arise and replace Sry, leading to sex chromosome turnover. However, intensive study over three decades has failed to reveal the identity of novel sex genes in either of these lineages. We here report our discovery of a male-specific duplication of an enhancer of Sox9 in the Amami spiny rat Tokudaia osimensis, in which males and females have only a single X chromosome (XO/XO) and the Y chromosome and Sry are completely lost. We performed a comprehensive survey to detect sex-specific genomic regions in the spiny rat. Sex-related genomic differences were limited to a male-specific duplication of a 17-kb unit located 430 kb upstream of Sox9 on an autosome. Hi-C analysis using male spiny rat cells showed the duplicated region has potential chromatin interaction with Sox9. The duplicated unit harbored a 1,262-bp element homologous to mouse enhancer 14 (Enh14), a candidate Sox9 enhancer that is functionally redundant in mice. Transgenic reporter mice showed that the spiny rat Enh14 can function as an embryonic testis enhancer in mice. Embryonic gonads of XX mice in which Enh14 was replaced by the duplicated spiny rat Enh14 showed increased Sox9 expression and decreased Foxl2 expression. We propose that male-specific duplication of this Sox9 enhancer substituted for Sry function, defining a novel Y chromosome in the spiny rat.

Agonist dependency of the second phase access of ß-arrestin 2 to the heteromeric µ-V1b receptor.

During the development of analgesic tolerance to morphine, the V1b vasopressin receptor has been proposed to bind to ß-arrestin 2 and the µ-opioid receptor to enable their interaction. However, direct evidence of such a high-order complex is lacking. Using bioluminescent resonance energy transfer between a split Nanoluciferase and the Venus fluorescent protein, the NanoBit-NanoBRET system, we found that ß-arrestin 2 closely located near the heteromer µ-V1b receptor in the absence of an agonist and moved closer to the receptor carboxyl-termini upon agonist stimulation. An additive effect of the two agonists for opioid and vasopressin receptors was detected on the NanoBRET between the µ-V1b heteromer and ß-arrestin 2. To increase the agonist response of NanoBRET, the ratio of the donor luminophore to the acceptor fluorophore was decreased to the detection limit of luminescence. In the first phase of access, ß-arrestin 2 was likely to bind to the unstimulated V1b receptor in both its phosphorylated and unphosphorylated forms. In contrast, the second-phase access of ß-arrestin 2 was agonist dependent, indicating a possible pharmacological intervention strategy. Therefore, our efficient method should be useful for evaluating chemicals that directly target the vasopressin binding site in the µ-V1b heteromer to reduce the second-phase access of ß-arrestin 2 and thereby to alleviate tolerance to morphine analgesia.

Species-Dependent Enhancement of Ovarian Cancer G Protein-Coupled Receptor 1 Activation by Ogerin.

Ogerin is a positive allosteric modulator of human and mouse ovarian cancer G protein-coupled receptors (OGR1s). In the present study, we found that ogerin differentially enhances the activation of OGR1 in various animal species. Amino acid residues of OGR1 that are associated with ogerin are conserved among the species. This suggests that other amino acid residues may be involved in the action of ogerin. Chimeric receptors between human and zebrafish OGR1s showed that the amino acid residues that determine the species specificity of ogerin-induced enhancement reside in the transmembrane and/or intracellular regions of OGR1. This result highlights the importance of first verifying the effectiveness of ogerin to the OGR1 of the species of interest at the cellular level prior to analyzing the physiological and pathophysiological roles of OGR1 in the species.

Characterization of molecular mechanisms of extracellular acidification-induced intracellular Ca2+ increase in LßT2 cells.

Extracellular acidification regulates endocrine cell functions. Ovarian cancer G protein-coupled receptor 1 (OGR1), also known as GPR68, is a proton-sensing G protein-coupled receptor and is activated by extracellular acidification, resulting in the activation of multiple intracellular signaling pathways. In the present study, we found that OGR1 was expressed in some gonadotropic cells in rat anterior pituitary and in LßΤ2 cells, which are used as a model of gonadotropic cells. When we reduced extracellular pH, a transient intracellular Ca2+ increase was detected in LßT2 cells. The Ca2+ increase was inhibited by a Gq/11 inhibitor and Cu2+, which is known as an OGR1 antagonist. We also found that extracellular acidification enhanced GnRH-induced Gaussia luciferase secretion from LßT2 cells. These results suggest that OGR1 may play a role in the regulation of gonadotropic cell function such as its hormone secretion.

Metals Differentially Activate Ovarian Cancer G Protein-Coupled Receptor 1 in Various Species.

Human, mouse, and zebrafish ovarian cancer G protein-coupled receptors (OGR1s) are activated by both metals and extracellular protons. In the present study, we examined whether pig, rat, chicken, and Xenopus OGR1 homologs could sense and be activated by protons and metals. We found that all homologs stimulated serum response element (SRE)-driven promoter activities when they are stimulated by protons. On the other hand, metals differentially activated the homologs. The results using chimeric receptors of human and zebrafish OGR1s indicate that the specificity of the metal-induced activation lies in the extracellular region. These results suggest that protons are an evolutionally conserved agonist of OGR1. However, the types of metals that activated the receptor differed among the homologs.

Manganese and cobalt activate zebrafish ovarian cancer G-protein-coupled receptor 1 but not GPR4.

Mammalian ovarian G-protein-coupled receptor 1 (OGR1) is activated by some metals in addition to extracellular protons and coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebrafish OGR1, zebrafish GPR4, and human GPR4 (zOGR1, zGPR4, and hGPR4, respectively) could sense the metals and activate the intracellular signaling pathways. On one hand, we found that only manganese and cobalt of the tested metals stimulated SRE-promoter activities in zOGR1-overexpressed HEK293T cells. On the other hand, none of the metals tested stimulated the promoter activities in zGPR4- and hGPR4-overexpressed cells. The OGR1 mutant (H4F), which is lost to activation by extracellular protons, did not stimulate metal-induced SRE-promoter activities. These results suggest that zOGR1, but not GPR4, is also a metal-sensing G-protein-coupled receptor in addition to a proton-sensing G-protein-coupled receptor, although not all metals that activate hOGR1 activated zOGR1.

Easy detection of hormone secretion from LßT2 cells by using Gaussia luciferase.

Reproduction is regulated by gonadotropins secreted from gonadotrophs. The production and secretion of gonadotropins are mainly regulated by gonadotropin-releasing hormone (GnRH). Agonists or antagonists that influence GnRH action on gonadotrophs are important to regulate reproduction; however, these factors have not been fully characterized due to the lack of simple and easy-to-use techniques to detect gonadotropin secretion from gonadotropin-producing cells. In the present study, we found that Gaussia luciferase (Gluc), which was expressed in LßT2 cells, can be secreted like a luteinizing-hormone (LH) upon stimulation with GnRH. The Gluc secreted into the medium was easily monitored as luminescence signals. The detection range of the GnRH-induced Gluc activity was comparable to that of the enzyme-linked immunosorbent assay for LH. In addition, when the Gluc was expressed in AtT20 cells, which produce adrenocorticotropic hormone (ACTH), the Gluc activity in the medium increased in parallel with the ACTH secretion upon stimulation with corticotropin-releasing hormone. Thus, the Gluc assay in the present study can be easily used for high-throughput screening of factors that influence LH or ACTH secretion from LßT2 or AtT20 cells, respectively.

Two zebrafish G2A homologs activate multiple intracellular signaling pathways in acidic environment.

Human G2A is activated by various stimuli such as lysophosphatidylcholine (LPC), 9-hydroxyoctadecadienoic acid (9-HODE), and protons. The receptor is coupled to multiple intracellular signaling pathways, including the Gs-protein/cAMP/CRE, G12/13-protein/Rho/SRE, and Gq-protein/phospholipase C/NFAT pathways. In the present study, we examined whether zebrafish G2A homologs (zG2A-a and zG2A-b) could respond to these stimuli and activate multiple intracellular signaling pathways. We also examined whether histidine residue and basic amino acid residue in the N-terminus of the homologs also play roles similar to those played by human G2A residues if the homologs sense protons. We found that the zG2A-a showed the high CRE, SRE, and NFAT activities, however, zG2A-b showed only the high SRE activity under a pH of 8.0. Extracellular acidification from pH 7.4 to 6.3 ameliorated these activities in zG2A-a-expressing cells. On the other hand, acidification ameliorated the SRE activity but not the CRE and NFAT activities in zG2A-b-expressing cells. LPC or 9-HODE did not modify any activity of either homolog. The substitution of histidine residue at the 174(th) position from the N-terminus of zG2A-a to asparagine residue attenuated proton-induced CRE and NFAT activities but not SRE activity. The substitution of arginine residue at the 32nd position from the N-terminus of zG2A-a to the alanine residue also attenuated its high and the proton-induced CRE and NFAT activities. On the contrary, the substitution did not attenuate SRE activity. The substitution of the arginine residue at the 10th position from the N-terminus of zG2A-b to the alanine residue also did not attenuate its high or the proton-induced SRE activity. These results indicate that zebrafish G2A homologs were activated by protons but not by LPC and 9-HODE, and the activation mechanisms of the homologs were similar to those of human G2A.

Extracellular acidification activates ovarian cancer G-protein-coupled receptor 1 and GPR4 homologs of zebra fish.

Mammalian ovarian G-protein-coupled receptor 1 (OGR1) and GPR4 are identified as a proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebra fish OGR1 and GPR4 homologs (zOGR1 and zGPR4) could sense protons and activate the multiple intracellular signaling pathways and, if so, whether the similar positions of histidine residue, which is critical for sensing protons in mammalian OGR and GPR4, also play a role to sense protons and activate the multiple signaling pathways in the zebra fish receptors. We found that extracellular acidic pH stimulated CRE-, SRE-, and NFAT-promoter activities in zOGR1 overexpressed cells and stimulated CRE- and SRE- but not NFAT-promoter activities in zGPR4 overexpressed cells. The substitution of histidine residues at the 12th, 15th, 162th, and 264th positions from the N-terminal of zOGR1 with phenylalanine attenuated the proton-induced SRE-promoter activities. The mutation of the histidine residue at the 78th but not the 84th position from the N-terminal of zGPR4 to phenylalanine attenuated the proton-induced SRE-promoter activities. These results suggest that zOGR1 and zGPR4 are also proton-sensing G-protein-coupled receptors, and the receptor activation mechanisms may be similar to those of the mammalian receptors.