Sex-dependent regulation of vertebrate somatic growth and aging by germ cells.

The function of germ cells in somatic growth and aging has been demonstrated in invertebrate models but remains unclear in vertebrates. We demonstrated sex-dependent somatic regulation by germ cells in the short-lived vertebrate model Nothobranchius furzeri. In females, germ cell removal shortened life span, decreased estrogen, and increased insulin-like growth factor 1 (IGF-1) signaling. In contrast, germ cell removal in males improved their health with increased vitamin D signaling. Body size increased in both sexes but was caused by different signaling pathways, i.e., IGF-1 and vitamin D in females and males, respectively. Thus, vertebrate germ cells regulate somatic growth and aging through different pathways of the endocrine system, depending on the sex, which may underlie the sexual difference in reproductive strategies.

Rapid reverse genetics systems for Nothobranchius furzeri, a suitable model organism to study vertebrate aging.

The African turquoise killifish Nothobranchius furzeri (N. furzeri) is a useful model organism for studying aging, age-related diseases, and embryonic diapause. CRISPR/Cas9-mediated gene knockout and Tol2 transposon-mediated transgenesis in N. furzeri have been reported previously. However, these methods take time to generate knockout and transgenic fish. In addition, knock-in technology that inserts large DNA fragments as fluorescent reporter constructs into the target gene in N. furzeri has not yet been established. Here, we show that triple-target CRISPR-mediated single gene disruption efficiently produces whole-body biallelic knockout and enables the examination of gene function in the F0 generation. In addition, we developed a method for creating the knock-in reporter N. furzeri without crossing by optimizing the CRISPR/Cas9 system. These methods drastically reduce the duration of experiments, and we think that these advances will accelerate aging and developmental studies using N. furzeri.

Zebrafish imaging reveals TP53 mutation switching oncogene-induced senescence from suppressor to driver in primary tumorigenesis.

Most tumours are thought to arise through oncogenic cell generation followed by additional mutations. How a new oncogenic cell primes tumorigenesis by acquiring additional mutations remains unclear. We show that an additional TP53 mutation stimulates primary tumorigenesis by switching oncogene-induced senescence from a tumour suppressor to a driver. Zebrafish imaging reveals that a newly emerged oncogenic cell with the RasG12V mutation becomes senescent and is eliminated from the epithelia, which is prevented by adding a TP53 gain-of-function mutation (TP53R175H) into RasG12V cells. Surviving RasG12V-TP53R175H double-mutant cells senesce and secrete senescence-associated secretory phenotype (SASP)-related inflammatory molecules that convert neighbouring normal cells into SASP factor-secreting senescent cells, generating a heterogeneous tumour-like cell mass. We identify oncogenic cell behaviours that may control the initial human tumorigenesis step. Ras and TP53 mutations and cellular senescence are frequently detected in human tumours; similar switching may occur during the initial step of human tumorigenesis.

The protective role of proton-sensing TDAG8 in the brain injury in a mouse ischemia reperfusion model.

Extracellular acidification in the brain has been observed in ischemia; however, the physiological and pathophysiological implications of the pH reduction remain largely unknown. Here, we analyzed the roles of proton-sensing G protein-coupled receptors, including T-cell death-associated gene 8 (TDAG8), ovarian cancer G protein-coupled receptor 1 (OGR1), and G protein-coupled receptor 4 (GPR4) in a mouse ischemia reperfusion model. Cerebral infarction and dysfunctional behavior with transient middle cerebral artery occlusion (tMCAO) and subsequent reperfusion were exacerbated by the deficiency of TDAG8, whereas no significant effect was observed with the deficiency of OGR1 or GPR4. We confirmed that the pH of the predicted infarction region was 6.5. TDAG8 mRNA was observed in Iba1-positive microglia in the mouse brain. The tMCAO increased the mRNA expression of tumor necrosis factor-α in the ipsilateral cerebral hemisphere and evoked morphological changes in microglia in an evolving cerebral injury. These tMCAO-induced actions were significantly enhanced by the TDAG8 deficiency. Administration of minocycline, which is known to inhibit microglial activation, improved the cerebral infarction and dysfunctional behavior induced by tMCAO in the TDAG8-deficient mouse. Thus, acidic pH/TDAG8 protects against cerebral infarction caused by tMCAO, at least due to the mechanism involving the inhibition of microglial functions.

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.

A missense mutation of Leu74Pro of OGR1 found in familial amelogenesis imperfecta actually causes the loss of the pH-sensing mechanism.

Ovarian cancer G protein-coupled receptor 1 (OGR1), also known as GPR68, is a proton-sensing G protein-coupled receptor (GPCR) coupling to Gq/11/phospholipase C/Ca2+ signaling pathways. The specific histidine residues at the extracellular surface of OGR1 are suggested to be involved in the proton sensing. Later, some metal ions, including nickel ion (Ni2+), are also indicated to be OGR1 ligands. OGR1 polymorphic variants have recently been found in three families with amelogenesis imperfecta, which suggested that OGR1 is required for the process of dental enamel formation. One of these families possesses a missense mutation from leucine to proline at 74 (L74P) of OGR1. In the present study, we characterized HEK293 cells with L74P OGR1 (L74P-OGR1) and hemagglutinin (HA)-tag, as compared with cells with wild-type OGR1 (WT-OGR1) and HA-tag. We found that either acidic pH or NiCl2 induced intracellular Ca2+ mobilization and morphological change in WT-OGR1-transfected cells; however, the extracellular stimulus-induced actions were severely damaged in L74P-OGR1-transfected cells. We further confirmed that either WT-OGR1 or L74P-OGR1 is localized mainly in the surface of the cells, but only WT-OGR1 is internalized in response to acidification or NiCl2. Thus, the L74P-OGR1 protein may be distributed in the plasma membranes but severely damaged in the receptor functions. We speculate that L74P in the second transmembrane domain in OGR1 may result in conformational changes in the receptor, thereby disturbing the sensing extracellular signals, i.e., protons or metal ions, and/or transducing them to the intracellular signaling machinery through G proteins.

Involvement of GPR4 in increased growth hormone and prolactin expressions by extracellular acidification in MtT/S cells.

Hormone-secreting pituitary adenomas show unregulated hormonal hypersecretion and cause hyperpituitarism. However, the mechanism of the unregulated hormone production and secretion has not yet been fully elucidated. Solid tumors show reduced extracellular pH, partly due to lactate secretion from anaerobic glycolysis. It is known that extracellular acidification affects hormone secretion. However, whether and how the extracellular acidification influences the unregulated hormone production and secretion remain unknown. In the present study, we found that GPR4, a proton-sensing G protein-coupled receptor, was highly expressed in MtT/S cells, a growth hormone-producing and prolactin-producing pituitary tumor cell line. When we reduced the extracellular pH, growth hormone and prolactin mRNA expressions increased in the cells. Both increased expressions were partially suppressed by a GPR4 antagonist. We also found that extracellular acidification enhanced growth hormone-releasing factor-induced growth hormone secretion from MtT/S cells. These results suggest that GPR4 may play a role in hypersecretion of the hormone from hormone-producing pituitary tumors. A GPR4 antagonist will be a useful tool for preventing the hypersecretion.

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.

The proton-sensing G protein-coupled receptor T-cell death-associated gene 8 (TDAG8) shows cardioprotective effects against myocardial infarction.

Myocardial infarction (MI) is an ischaemic heart condition caused by the occlusion of coronary arteries. Following MI, lactic acid from anaerobic glycolysis increases and infiltrating immune cells produce severe inflammation, which leads to acidosis in the ischaemic heart. However, the physiological implication of this pH reduction remains largely unknown. T-cell death-associated gene 8 (TDAG8) is a proton-sensing G protein-coupled receptor found on cardiac macrophages that recognise increases in extracellular protons. We demonstrated that TDAG8 negatively regulates the transcription of the chemokine Ccl20. The infarcted hearts of TDAG8 KO mice showed an increase in CCL20 expression and the number of infiltrating IL-17A-producing γδT cells that express CCR6, a receptor for CCL20. Accordingly, excessive IL-17A production, which is linked to the functional deterioration after MI, was observed in MI-operated TDAG8 KO mice. The survival rate and cardiac function significantly decreased in TDAG8 KO mice compared with those in wild-type mice after MI. Thus, our results suggest that TDAG8 is a key regulator of MI and a potential therapeutic target.

Alamandine reduces leptin expression through the c-Src/p38 MAP kinase pathway in adipose tissue.

OBJECTIVE: Obesity is associated with an increased risk of diabetes mellitus, hypertension, and renal dysfunction. Angiotensin 1-7 and alamandine are heptameric renin angiotensin system peptide hormones. Further, alamandine levels increase with renal dysfunction. In the cardiovascular system, angiotensin 1-7 and alamandine produce similar improvements and counterbalance angiotensin II in regulating vascular function. We aimed to determine whether the effect of alamandine on leptin expression and secretion in adipocytes was similar to that of angiotensin 1-7. APPROACH AND RESULTS: We studied isolated peri-renal visceral adipose tissue and peri-renal isolated visceral adipocytes from male Wistar rats. Angiotensin II from 0.01 to 10nM had no effect on leptin expression. Angiotensin 1-7 (1 nM) increased leptin secretion and expression, whereas alamandine (1 nM) decreased leptin secretion and expression in adipose tissue and isolated adipocytes and reduced blood leptin levels in vivo. These effects were mediated by Gq, c-Src, p38 mitogen-activated protein, and IκB activation. Additionally, alamandine induced nitric oxide expression via inducible nitric oxidase synthase and plasminogen activator inhibitor 1 expression in adipose tissue and isolated adipocytes. CONCLUSIONS: Angiotensin 1-7 and alamandine produced opposing effects on leptin expression and secretion in adipose tissue. This result suggests that the action of Mas (angiotensin 1-7 receptor) and Mas-related G-protein coupled receptor D in adipocytes exhibited opposing actions similar to angiotensin II type 1 and type 2 receptors.

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.

Mutations in the pH-Sensing G-protein-Coupled Receptor GPR68 Cause Amelogenesis Imperfecta.

Amelogenesis is the process of dental enamel formation, leading to the deposition of the hardest tissue in the human body. This process requires the intricate regulation of ion transport and controlled changes to the pH of the developing enamel matrix. The means by which the enamel organ regulates pH during amelogenesis is largely unknown. We identified rare homozygous variants in GPR68 in three families with amelogenesis imperfecta, a genetically and phenotypically heterogeneous group of inherited conditions associated with abnormal enamel formation. Each of these homozygous variants (a large in-frame deletion, a frameshift deletion, and a missense variant) were predicted to result in loss of function. GPR68 encodes a proton-sensing G-protein-coupled receptor with sensitivity in the pH range that occurs in the developing enamel matrix during amelogenesis. Immunohistochemistry of rat mandibles confirmed localization of GPR68 in the enamel organ at all stages of amelogenesis. Our data identify a role for GPR68 as a proton sensor that is required for proper enamel formation.

Identification of a Potent and Selective GPR4 Antagonist as a Drug Lead for the Treatment of Myocardial Infarction.

GPR4, a pH-sensing G protein-coupled receptor, is highly expressed in endothelial cells and may be activated in myocardial infarction due the decreased tissue pH. We are interested in GPR4 antagonists as potential effective pharmacologic tools and/or drug leads for the treatment of myocardial infarction. We investigated the structure-activity relationship of a known GPR4 antagonist 1 as a lead compound to identify 3b as the first potent and selective GPR4 antagonist, whose effectiveness was demonstrated in a mouse myocardial infarction model.

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.

Protective Role of Proton-Sensing TDAG8 in Lipopolysaccharide-Induced Acute Lung Injury.

Acute lung injury is characterized by the infiltration of neutrophils into lungs and the subsequent impairment of lung function. Here we explored the role of TDAG8 in lung injury induced by lipopolysaccharide (LPS) administrated intratracheally. In this model, cytokines and chemokines released from resident macrophages are shown to cause neutrophilic inflammation in the lungs. We found that LPS treatment increased TDAG8 expression in the lungs and confirmed its expression in resident macrophages in bronchoalveolar lavage (BAL) fluids. LPS administration remarkably increased neutrophil accumulation without appreciable change in the resident macrophages, which was associated with increased penetration of blood proteins into BAL fluids, interstitial accumulation of inflammatory cells, and damage of the alveolar architecture. The LPS-induced neutrophil accumulation and the associated lung damage were enhanced in TDAG8-deficient mice as compared with those in wild-type mice. LPS also increased several mRNA and protein expressions of inflammatory cytokines and chemokines in the lungs or BAL fluids. Among these inflammatory mediators, mRNA and protein expression of KC (also known as CXCL1), a chemokine of neutrophils, were significantly enhanced by TDAG8 deficiency. We conclude that TDAG8 is a negative regulator for lung neutrophilic inflammation and injury, in part, through the inhibition of chemokine production.

Acidic environment augments FcεRI-mediated production of IL-6 and IL-13 in mast cells.

Although blood pH is maintained in a narrow range of around pH 7.4 in living organisms, inflammatory loci are characterized by acidic conditions. Mast cells tend to reside close to the surface of the body in areas such as the mucosa and skin where they may be exposed to exogenous acids, and they play an important role in immune responses. However, little is known about the effects of extracellular acidification on the functions of mast cell. Here, we found that extracellular acidification increased the dinitrophenyl-conjugated human serum albumin (DNP-HSA)-induced production of interleukin (IL)-6 and IL-13 in MC/9 cells or bone marrow-derived mouse mast cells sensitized with anti-DNP IgE. Extracellular acidification also inhibited migration of MC/9 cells toward DNP-HSA. In addition, acidic pH stimulated antigen-induced activation of p38 mitogen-activated protein kinase (MAPK) and protein kinase B (Akt). These findings suggest that extracellular acidification augmented antigen/IgE-induced and FcεRI-mediated production of IL-6 and IL-13 in mast cells, and that this was associated with the enhancement of p38 MAPK and Akt activation.

Characterization of Imidazopyridine Compounds as Negative Allosteric Modulators of Proton-Sensing GPR4 in Extracellular Acidification-Induced Responses.

G protein-coupled receptor 4 (GPR4), previously proposed as the receptor for sphingosylphosphorylcholine, has recently been identified as the proton-sensing G protein-coupled receptor (GPCR) coupling to multiple intracellular signaling pathways, including the Gs protein/cAMP and G13 protein/Rho. In the present study, we characterized some imidazopyridine compounds as GPR4 modulators that modify GPR4 receptor function. In the cells that express proton-sensing GPCRs, including GPR4, OGR1, TDAG8, and G2A, extracellular acidification stimulates serum responsive element (SRE)-driven transcriptional activity, which has been shown to reflect Rho activity, with different proton sensitivities. Imidazopyridine compounds inhibited the moderately acidic pH-induced SRE activity only in GPR4-expressing cells. Acidic pH-stimulated cAMP accumulation, mRNA expression of inflammatory genes, and GPR4 internalization within GPR4-expressing cells were all inhibited by the GPR4 modulator. We further compared the inhibition property of the imidazopyridine compound with psychosine, which has been shown to selectively inhibit actions induced by proton-sensing GPCRs, including GPR4. In the GPR4 mutant, in which certain histidine residues were mutated to phenylalanine, proton sensitivity was significantly shifted to the right, and psychosine failed to further inhibit acidic pH-induced SRE activation. On the other hand, the imidazopyridine compound almost completely inhibited acidic pH-induced action in mutant GPR4. We conclude that some imidazopyridine compounds show specificity to GPR4 as negative allosteric modulators with a different action mode from psychosine, an antagonist susceptible to histidine residues, and are useful for characterizing GPR4-mediated acidic pH-induced biological actions.

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.

Ionotropic and metabotropic proton-sensing receptors involved in airway inflammation in allergic asthma.

An acidic microenvironment has been shown to evoke a variety of airway responses, including cough, bronchoconstriction, airway hyperresponsiveness (AHR), infiltration of inflammatory cells in the lung, and stimulation of mucus hyperproduction. Except for the participation of transient receptor potential vanilloid-1 (TRPV1) and acid-sensing ion channels (ASICs) in severe acidic pH (of less than 6.0)-induced cough and bronchoconstriction through sensory neurons, the molecular mechanisms underlying extracellular acidic pH-induced actions in the airways have not been fully understood. Recent studies have revealed that ovarian cancer G protein-coupled receptor 1 (OGR1)-family G protein-coupled receptors, which sense pH of more than 6.0, are expressed in structural cells, such as airway smooth muscle cells and epithelial cells, and in inflammatory and immune cells, such as eosinophils and dendritic cells. They function in a variety of airway responses related to the pathophysiology of inflammatory diseases, including allergic asthma. In the present review, we discuss the roles of ionotropic TRPV1 and ASICs and metabotropic OGR1-family G protein-coupled receptors in the airway inflammation and AHR in asthma and respiratory diseases.

Acidic pH increases cGMP accumulation through the OGR1/phospholipase C/Ca(2+)/neuronal NOS pathway in N1E-115 neuronal cells.

Neuronal NO synthase (nNOS)-mediated cGMP accumulation has been shown to affect a variety of neuronal cell activities, regardless of whether they are detrimental or beneficial, depending on the amount of their levels, under the physiological and pathological situations. In the present study, we examined the role of proton-sensing G protein-coupled receptors (GPCRs), which have been identified as new pH sensors, in the acidic pH-induced nNOS/cGMP activity in N1E-115 neuronal cells. In this cell line, ovarian cancer G protein-coupled receptor 1 (OGR1) and G protein-coupled receptor 4 (GPR4) mRNAs are expressed. An extracellular acidic pH increased cGMP accumulation, which was inhibited by nNOS-specific inhibitors. Acidic pH also activated phospholipase C/Ca(2+) pathways and Akt-induced phosphorylation of nNOS at S1412, both of which have been shown to be critical regulatory mechanisms for nNOS activation. The acidic pH-induced activation of the phospholipase C/Ca(2+) pathway, but not Akt/nNOS phosphorylation, was inhibited by small interfering RNA specific to OGR1 and YM-254890, an inhibitor of Gq/11 proteins, in association with the inhibition of cGMP accumulation. Moreover cGMP accumulation was inhibited by 2-aminoethoxydiphenyl borate, an inhibitor of inositol 1,4,5-trisphosphate channel; however, it was not by wortmannin, a phosphatidylinositol 3-kinase inhibitor, which inhibited Akt/nNOS phosphorylation. In conclusion, acidic pH stimulates cGMP accumulation preferentially through the OGR1/Gq/11 proteins/phospholipase C/Ca(2+)/nNOS in N1E-115 neuronal cells. Akt-mediated phosphorylation of nNOS, however, does not appreciably contribute to the acidification-induced accumulation of cGMP.