Nacα protects the larval fat body from cell death by maintaining cellular proteostasis in Drosophila.

Protein homeostasis (proteostasis) is crucial for the maintenance of cellular homeostasis. Impairment of proteostasis activates proteotoxic and unfolded protein response pathways to resolve cellular stress or induce apoptosis in damaged cells. However, the responses of individual tissues to proteotoxic stress and evoking cell death program have not been extensively explored in vivo. Here, we show that a reduction in Nascent polypeptide-associated complex protein alpha subunit (Nacα) specifically and progressively induces cell death in Drosophila fat body cells. Nacα mutants disrupt both ER integrity and the proteasomal degradation system, resulting in caspase activation through JNK and p53. Although forced activation of the JNK and p53 pathways was insufficient to induce cell death in the fat body, the reduction of Nacα sensitized fat body cells to intrinsic and environmental stresses. Reducing overall protein synthesis by mTor inhibition or Minute mutants alleviated the cell death phenotype in Nacα mutant fat body cells. Our work revealed that Nacα is crucial for protecting the fat body from cell death by maintaining cellular proteostasis, thus demonstrating the coexistence of a unique vulnerability and cell death resistance in the fat body.

Crucial role of iron in epigenetic rewriting during adipocyte differentiation mediated by JMJD1A and TET2 activity.

Iron metabolism is closely associated with the pathogenesis of obesity. However, the mechanism of the iron-dependent regulation of adipocyte differentiation remains unclear. Here, we show that iron is essential for rewriting of epigenetic marks during adipocyte differentiation. Iron supply through lysosome-mediated ferritinophagy was found to be crucial during the early stage of adipocyte differentiation, and iron deficiency during this period suppressed subsequent terminal differentiation. This was associated with demethylation of both repressive histone marks and DNA in the genomic regions of adipocyte differentiation-associated genes, 　including Pparg, which encodes PPARγ, the master regulator of adipocyte differentiation. In addition, we identified several epigenetic demethylases to be responsible for iron-dependent adipocyte differentiation, with the histone demethylase jumonji domain-containing 1A and the DNA demethylase ten-eleven translocation 2 as the major enzymes. The interrelationship between repressive histone marks and DNA methylation was indicated by an integrated genome-wide association analysis, and was also supported by the findings that both histone and DNA demethylation were suppressed by either the inhibition of lysosomal ferritin flux or the knockdown of iron chaperone poly(rC)-binding protein 2. In summary, epigenetic regulations through iron-dependent control of epigenetic enzyme activities play an important role in the organized gene expression mechanisms of adipogenesis.

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.

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 synergizes with PDGF to stimulate migration of mouse embryo fibroblasts through activation of p38MAPK with a PTX-sensitive manner.

The elucidation of the functional mechanisms of extracellular acidification stimulating intracellular signaling pathway is of great importance for developing new targets of treatment for solid tumors, and inflammatory disorders characterized by extracellular acidification. In the present study, we focus on the regulation of extracellular acidification on intracellular signaling pathways in mouse embryo fibroblasts (MEFs). We found extracellular acidification was at least partly involved in stimulating p38MAPK pathway through PTX-sensitive behavior to enhance cell migration in the presence or absence of platelet-derived growth factor (PDGF). Statistical analysis showed that the actions of extracellular acidic pH and PDGF on inducing enhancement of cell migration were not an additive effect. However, we also found extracellular acidic pH did inhibit the viability and proliferation of MEFs, suggesting that extracellular acidification stimulates cell migration probably through proton-sensing mechanisms within MEFs. Using OGR1-, GPR4-, and TDAG8-gene knock out technology, and real-time qPCR, we found known proton-sensing G protein-coupled receptors (GPCRs), transient receptor potential vanilloid subtype 1 (TRPV1), and acid-sensing ion channels (ASICs) were unlikely to be involved in the regulation of acidification on cell migration. In conclusion, our present study validates that extracellular acidification stimulates chemotactic migration of MEFs through activation of p38MAPK with a PTX-sensitive mechanism either by itself, or synergistically with PDGF, which was not regulated by the known proton-sensing GPCRs, TRPV1, or ASICs. Our results suggested that others proton-sensing GPCRs or ion channels might exist in MEFs, which mediates cell migration induced by extracellular acidification in the presence or absence of PDGF.

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.

Lipoprotein-associated lysolipids are differentially involved in high-density lipoprotein- and its oxidized form-induced neurite remodeling in PC12 cells.

Oxidatively damaged proteins and lipid peroxidation products have been shown to accumulate in the brain of neurodegenerative diseases, such as Alzheimer's disease and multiple sclerosis, and oxidized lipoprotein is considered to be toxic and neurodegenerative. However, the role of lipoprotein and its oxidized form in neurite remodeling has not been well understood. In the present study, we have aimed to clarify whether and, if so, how high-density lipoprotein (HDL) and oxidized HDL (oxHDL) affect neuritogenesis. In the presence of nerve growth factor, exposure of PC12 cells to either HDL or oxHDL induces a rapid neurite retraction, which is followed by re-outgrowth of neurites in either case; however, oxHDL-treated cells exhibit much longer outgrowths than do basal and HDL-treated cells. Thus, processes in the morphological changes of neuronal cells after lipoprotein treatment are composed of two phases: the reversible retraction phase and the extension phase. Characterization of the active fractions of lipids and experiments with desensitization and knockdown of receptors have indicated that the reversible retraction phase involves mainly sphingosine 1-phosphate for HDL and lysophosphatidic acid for oxHDL. The change in the components responsible for the retraction response is comparable with the change in sphingosine 1-phosphate and lysophosphatidic acid contents by the oxidation of HDL. In the extension phase, lysophosphatidylcholine, which is increased by the oxidation of HDL, may play a stimulatory role in neurite outgrowth. We conclude that lipoprotein and its oxidized form differentially regulate neuritogenesis through lipoprotein-associated lysolipid molecules.

Inhibition of interleukin-1ß production by extracellular acidification through the TDAG8/cAMP pathway in mouse microglia.

Interleukin-1ß (IL-1ß) is released from activated microglia and involved in the neurodegeneration of acute and chronic brain disorders, such as stroke and Alzheimer's disease, in which extracellular acidification has been shown to occur. Here, we examined the extracellular acidic pH regulation of IL-1ß production, especially focusing on TDAG8, a major proton-sensing G-protein-coupled receptor, in mouse microglia. Extracellular acidification inhibited lipopolysaccharide -induced IL-1ß production, which was associated with the inhibition of IL-1ß cytoplasmic precursor and mRNA expression. The IL-1ß mRNA and protein responses were significantly, though not completely, attenuated in microglia derived from TDAG8-deficient mice compared with those from wild-type mice. The acidic pH also stimulated cellular cAMP accumulation, which was completely inhibited by TDAG8 deficiency. Forskolin and a cAMP derivative, which specifically stimulates protein kinase A (PKA), mimicked the proton actions, and PKA inhibitors reversed the acidic pH-induced IL-1ß mRNA expression. The acidic pH-induced inhibitory IL-1ß responses were accompanied by the inhibition of extracellular signal-related kinase and c-Jun N-terminal kinase activities. The inhibitory enzyme activities in response to acidic pH were reversed by the PKA inhibitor and TDAG8 deficiency. We conclude that extracellular acidic pH inhibits lipopolysaccharide-induced IL-1ß production, at least partly, through the TDAG8/cAMP/PKA pathway, by inhibiting extracellular signal-related kinase and c-Jun N-terminal kinase activities, in mouse microglia.

Proton-sensing ovarian cancer G protein-coupled receptor 1 on dendritic cells is required for airway responses in a murine asthma model.

Ovarian cancer G protein-coupled receptor 1 (OGR1) stimulation by extracellular protons causes the activation of G proteins and subsequent cellular functions. However, the physiological and pathophysiological roles of OGR1 in airway responses remain largely unknown. In the present study, we show that OGR1-deficient mice are resistant to the cardinal features of asthma, including airway eosinophilia, airway hyperresponsiveness (AHR), and goblet cell metaplasia, in association with a remarkable inhibition of Th2 cytokine and IgE production, in an ovalbumin (OVA)-induced asthma model. Intratracheal transfer to wild-type mice of OVA-primed bone marrow-derived dendritic cells (DCs) from OGR1-deficient mice developed lower AHR and eosinophilia after OVA inhalation compared with the transfer of those from wild-type mice. Migration of OVA-pulsed DCs to peribronchial lymph nodes was also inhibited by OGR1 deficiency in the adoption experiments. The presence of functional OGR1 in DCs was confirmed by the expression of OGR1 mRNA and the OGR1-sensitive Ca(2+) response. OVA-induced expression of CCR7, a mature DC chemokine receptor, and migration response to CCR7 ligands in an in vitro Transwell assay were attenuated by OGR1 deficiency. We conclude that OGR1 on DCs is critical for migration to draining lymph nodes, which, in turn, stimulates Th2 phenotype change and subsequent induction of airway inflammation and AHR.

Deficiency of proton-sensing ovarian cancer G protein-coupled receptor 1 attenuates glucose-stimulated insulin secretion.

Ovarian cancer G protein-coupled receptor 1 (OGR1) has been shown as a receptor for protons. In the present study, we aimed to know whether OGR1 plays a role in insulin secretion and, if so, the manner in which it does. To this end, we created OGR1-deficient mice and examined insulin secretion activity in vivo and in vitro. OGR1 deficiency reduced insulin secretion induced by glucose administered ip, although it was not associated with glucose intolerance in vivo. Increased insulin sensitivity and reduced plasma glucagon level may explain, in part, the unusual normal glucose tolerance. In vitro islet experiments revealed that glucose-stimulated insulin secretion was dependent on extracellular pH and sensitive to OGR1; insulin secretion at pH 7.4 to 7.0, but not 8.0, was significantly suppressed by OGR1 deficiency and inhibition of G(q/11) proteins. Insulin secretion induced by KCl and tolbutamide was also significantly inhibited, whereas that induced by several insulin secretagogues, including vasopressin, a glucagon-like peptide 1 receptor agonist, and forskolin, was not suppressed by OGR1 deficiency. The inhibition of insulin secretion was associated with the reduction of glucose-induced increase in intracellular Ca(2+) concentration. In conclusion, the OGR1/G(q/11) protein pathway is activated by extracellular protons existing under the physiological extracellular pH of 7.4 and further stimulated by acidification, resulting in the enhancement of insulin secretion in response to high glucose concentrations and KCl.

Orally active lysophosphatidic acid receptor antagonist attenuates pancreatic cancer invasion and metastasis in vivo.

Pancreatic cancer is highly metastatic and has a poor prognosis. However, there is no established treatment for pancreatic cancer. Lysophosphatidic acid (LPA) has been shown to be present in effluents of cancers and involved in migration and proliferation in a variety of cancer cells, including pancreatic cancer cells, in vitro. In the current study, we examined whether an orally active LPA antagonist is effective for pancreatic cancer tumorigenesis and metastasis in vivo. Oral administration of Ki16198, which is effective for LPA(1) and LPA(3), into YAPC-PD pancreatic cancer cell-inoculated nude mice significantly inhibited tumor weight and remarkably attenuated invasion and metastasis to lung, liver, and brain, in association with inhibition of matrix metalloproteinase (MMP) accumulation in ascites in vivo. Ki16198 inhibited LPA-induced migration and invasion in several pancreatic cancer cells in vitro, which was associated with the inhibition of LPA-induced MMP production. In conclusion, Ki16198 is a promising orally active LPA antagonist for inhibiting the invasion and metastasis of pancreatic cancer cells. The inhibitory effects of the antagonist on invasion and metastasis in vivo may be partially explained by the inhibition of motility activity and MMP production in cancer cells.

Involvement of proton-sensing receptor TDAG8 in the anti-inflammatory actions of dexamethasone in peritoneal macrophages.

Dexamethasone (DEX), a potent glucocorticoid, increased the expression of T-cell death associated gene 8 (TDAG8), a proton-sensing G protein-coupled receptor, which is associated with the enhancement of acidic pH-induced cAMP accumulation, in peritoneal macrophages. We explored the role of increased TDAG8 expression in the anti-inflammatory actions of DEX. The treatment of macrophages with either DEX or acidic pH induced the cell death of macrophages; however, the cell death was not affected by TDAG8 deficiency. While DEX inhibited lipopolysaccharide-induced production of tumor necrosis factor-α, an inflammatory cytokine, which was independent of TDAG8, at neutral pH, the glucocorticoid enhanced the acidic pH-induced inhibition of tumor necrosis factor-α production in a manner dependent on TDAG8. In conclusion, the DEX-induced increase in TDAG8 expression is in part involved in the glucocorticoid-induced anti-inflammatory actions through the inhibition of inflammatory cytokine production under the acidic pH environment. On the other hand, the role of TDAG8 in the DEX-induced cell death is questionable.

Ovarian cancer G protein-coupled receptor 1-dependent and -independent vascular actions to acidic pH in human aortic smooth muscle cells.

Atherosclerosis is a chronic inflammation disease characterized by acidic micromilieu and the accumulation of numerous bioactive lipid mediators, such as lysophosphatidic acid (LPA) and prostaglandins, in the atherosclerotic lesion. Chronic acidification induced various effects on vascular smooth muscle cells, but the molecular mechanisms underlying these effects remain unknown. In this study, we examine the role of proton-sensing ovarian cancer G protein-coupled receptor 1 (OGR1) in extracellular acidification-induced regulation of cyclooxygenase (COX)-2 induction, PGI(2) production, MAPK phosphatase (MKP)-1 expression, and plasminogen activator inhibitor (PAI)-1 expression and proliferation in human aortic smooth muscle cells (AoSMCs). Experiments with knockdown with small interfering RNA specific to OGR1 and specific inhibitors for G proteins showed that acidification-induced COX-2 expression, PGI(2) production, and MKP-1 expression, but not PAI-1 expression and inhibition of proliferation, were dependent on OGR1 and mainly mediated by G(q/11) protein. LPA remarkably enhanced, through the LPA(1) receptor/G(i) protein, the OGR1-mediated vascular actions to acidic pH. In conclusion, acidic pH-induced vascular actions of AoSMCs can be dissected to OGR1-dependent and -independent pathways: COX-2 expression, PGI(2) production, and MKP-1 expression are mediated by OGR1, but PAI-1 expression and inhibition of proliferation are not. LPA, which is usually thought to be a proatherogenic lipid mediator, may exert antiatherogenic actions under acidic micromilieu through cross-talk between LPA(1)/G(i) protein and OGR1/G(q/11) protein.

Each one of certain histidine residues in G-protein-coupled receptor GPR4 is critical for extracellular proton-induced stimulation of multiple G-protein-signaling pathways.

GPR4, previously proposed as the receptor for sphingosylphosphorylcholine, has recently been identified as the proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways, including the G(s)-protein/cAMP, G(12/13)-protein/Rho, and G(q)-protein/phospholipase C pathways. In the present study, we examined whether extracellularly located histidine residues of GPR4 sense extracellular protons and, if so, whether a certain histidine residue is critical for coupling to the single or multiple signaling pathway(s). We found that the mutation of histidine residue at 79, 165, or 269 from the N-terminal of GPR4 to phenylalanine shifted the half-maximal effective concentration (EC(50)) of proton-induced signaling activities to the right, including cAMP accumulation, SRE promoter activity reflecting Rho activity, and NFAT promoter activity reflecting phospholipase C signaling activity, without an appreciable change in the maximal activities. These results suggest that the protonation of each one of histidine residues at 79, 165, and 269 in GPR4 may be critical for conformational change of the receptor for coupling to multiple intracellular signaling pathways through G-proteins.

Signaling pathways involved in DNA synthesis and migration in response to lysophosphatidic acid and low-density lipoprotein in coronary artery smooth muscle cells.

Low-density lipoprotein (LDL) and lysophosphatidic acid (LPA), one of the lipid components of lipoprotein, induced the DNA synthesis of coronary artery smooth muscle cells (CASMCs). The LDL- and LPA-induced DNA synthesis was markedly inhibited by the LPA receptor antagonist Ki16425, pertussis toxin, small interfering RNAs targeted for LPA1 receptors, and a potent calcineurin inhibitor cyclosporine A. It has been reported that LDL and LPA induced a migration response in a manner sensitive to Ki16425, pertussis toxin, and a LPA1 receptor-specific small interfering RNA. However, cyclosporine A was ineffective in inhibiting the migration response. Instead, an epidermal growth factor (EGF) receptor tyrosine kinase inhibitor markedly suppressed the migration response to LDL and LPA without having any significant effect on DNA synthesis. Thus, the LDL-induced stimulation of DNA synthesis and migration in CASMCs is mediated by its component LPA through LPA1 receptors and G(i/o)-proteins. Ca2+/calcineurin pathways and transactivation of EGF receptors mediate LPA1-receptor-induced DNA synthesis and migration, respectively.

Inhibition of superoxide anion production by extracellular acidification in neutrophils.

Extracellular acidification inhibited formyl-Met-Leu-Phe- or C5a-induced superoxide anion (O(2)(-)) production in differentiated HL-60 neutrophil-like cells and human neutrophils. A cAMP-increasing agonist, prostaglandin E(1), also inhibited the formyl peptide-induced O(2)(-) production. The inhibitory action on the O(2)(-) production by extracellular acidic pH was associated with cAMP accumulation and partly attenuated by H89, a protein kinase A inhibitor. A significant amount of mRNAs for T-cell death-associated gene 8 (TDAG8) and other proton-sensing ovarian cancer G-protein-coupled receptor 1 (OGR1)-family receptors is expressed in these cells. These results suggest that cAMP/protein kinase A, possibly through proton-sensing G-protein-coupled receptors, may be involved in extracellular acidic pH-induced inhibition of O(2)(-) production.

Involvement of proton-sensing TDAG8 in extracellular acidification-induced inhibition of proinflammatory cytokine production in peritoneal macrophages.

Extracellular acidification inhibited LPS-induced TNF-alpha protein production, which was associated with an inhibition of TNF-alpha mRNA expression, in mouse peritoneal macrophages. The LPS-induced cytokine production was also inhibited by G(s) protein-coupled receptor agonists prostaglandin E(1) and isoproterenol. Among OGR1 family proton-sensing GTP-binding regulatory protein-coupled receptors, TDAG8, OGR1, and G2A are expressed in the cells. The inhibitory action by acidic pH on TNF-alpha production was significantly attenuated in macrophages from TDAG8(Tp/Tp) mice but not in those from OGR1(geo/geo) mice. Moreover, small interfering RNA specific to TDAG8, but not to G2A, clearly attenuated the acidification-induced inhibition of TNF-alpha production. On the other hand, the down-regulation or deficiency of TDAG8 hardly affected prostaglandin E(1)- or isoproterenol-induced actions. LPS-induced IL-6 production was also inhibited by extracellular acidification in a manner that was sensitive to TDAG8 expression. The acidic pH-induced inhibitory action on the cytokine production was significantly reversed either by a small interfering RNA specific to G(s) proteins or by a protein kinase A (PKA)-specific inhibitor H89. Indeed, a PKA-specific cAMP derivative inhibited LPS-induced cytokine production. Moreover, acidification induced cAMP accumulation in a TDAG8-specific way. We conclude that TDAG8, at least partly, mediates the extracellular acidification-induced inhibition of proinflammatory cytokine production through the G(s) protein/cAMP/PKA signaling pathway in mouse macrophages.

LPA1 receptors mediate stimulation, whereas LPA2 receptors mediate inhibition, of migration of pancreatic cancer cells in response to lysophosphatidic acid and malignant ascites.

Malignant ascites from pancreatic cancer patients has been reported to stimulate migration of pancreatic cancer cells through lysophosphatidic acid (LPA) and LPA(1) receptors. Indeed, ascites- and LPA-induced migration was inhibited by Ki16425, an LPA(1) and LPA(3) antagonist, in Panc-1 cells. Unexpectedly, however, in the presence of Ki16425, ascites and LPA inhibited cell migration in response to epidermal growth factor (EGF). The inhibitory migratory response to ascites and LPA was also observed in the cells treated with pertussis toxin (PTX), a G(i) protein inhibitor, and attenuated by a small interfering RNA (siRNA) specific to the LPA(2) receptor. The inhibitory LPA action was reversed by the regulators of G-protein signaling domain of p115RhoGEF, dominant-negative RhoA or C3 toxin. Indeed, LPA activated RhoA, which was attenuated by the siRNA against the LPA(2) receptor. Moreover, LP-105, an LPA(2) agonist, also inhibited EGF-induced migration in the PTX-treated cells. A similar inhibitory migration response through LPA(2) receptors was also observed in YAPC-PD, BxPC-3, CFPAC-1 and PK-1 pancreatic cancer cell lines. LPA also inhibited the invasion of Panc-1 cells in the PTX-treated cells in the in vitro Matrigel invasion assay. We conclude that LPA(2) receptors are coupled to the G(12/13) protein/Rho-signaling pathway, leading to the inhibition of EGF-induced migration and invasion of pancreatic cancer cells.