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.     

TDAG8 is a proton-sensing and psychosine-sensitive G-protein-coupled receptor

T cell death-associated gene 8 (TDAG8) has been reported to be a receptor for psychosine. Ovarian cancer G-protein-coupled receptor 1 (OGR1) and GPR4, G-protein-coupled receptors (GPCRs) closely related to TDAG8, however, have recently been identified as proton-sensing or extracellular pH-responsive GPCRs that stimulate inositol phosphate and cAMP production, respectively. In the present study, we examined whether TDAG8 senses extracellular pH change. In the several cell types that were transfected with TDAG8 cDNA, cAMP was markedly accumulated in response to neutral to acidic extracellular pH, with a peak response at approximately pH 7.0-6.5. The pH effect was inhibited by copper ions and was reduced or lost in cells expressing mutated TDAG8 in which histidine residues were changed to phenylalanine. In the membrane fractions prepared from TDAG8-transfected cells, guanosine 5'-O-(3-thiotriphosphate) binding activity and adenylyl cyclase activity were remarkably stimulated in response to neutral and acidic pH. The concentration-dependent effect of extracellular protons on cAMP accumulation was shifted to the right in the presence of psychosine. The inhibitory psychosine effect was also observed for pH-dependent actions in OGR1- and GPR4-expressing cells but not for prostaglandin E(2)- and sphingosine 1-phosphate-induced actions in any pH in native and sphingosine 1-phosphate receptor-expressing cells. Glucosylsphingosine and sphingosylphosphorylcholine similarly inhibited the pH-dependent action, although to a lesser extent. Psychosine-sensitive and pH-dependent cAMP accumulation was also observed in mouse thymocytes. We concluded that TDAG8 is one of the proton-sensing GPCRs coupling to adenylyl cyclase and psychosine, and its related lysosphingolipids behave as if they were antagonists against protein-sensing receptors, including TDAG8, GPR4, and OGR1.     

TDAG8介导的细胞内信号传送通路及其功能

T细胞死亡相关基因8编码的受体蛋白/G蛋白偶联受体65(TDAG8)是G蛋白偶联受体家族成员之一,起初它被鉴定为脂质分子(鞘氨醇半乳糖苷)的受体;后来的研究证明TDAG8也具有感知细胞外的质子或pH的功能。因此,TDAG8是一种特殊的双配体受体。TDAG8受体广泛表达在免疫组织等正常组织和肿瘤组织中,具有潜在的重要生理作用。本文对TDAG8介导的细胞内信号通路及TDAG8功能研究进展进行综述。     

TDAG51 mediates the effects of insulin-like growth factor I (IGF-I) on cell survival

Insulin-like growth factor-I (IGF-I) receptors and insulin receptors belong to the same subfamily of receptor tyrosine kinases and share a similar set of intracellular signaling pathways, despite their distinct biological actions. In the present study, we evaluated T cell death-associated gene 51 (TDAG51), which we previously identified by cDNA microarray analysis as a gene specifically induced by IGF-I. We characterized the signaling pathways by which IGF-I induces TDAG51 gene expression and the functional role of TDAG51 in IGF-I signaling in NIH-3T3 (NWTb3) cells, which overexpress the human IGF-I receptor. Treatment with IGF-I increased TDAG51 mRNA and protein levels in NWTb3 cells. This effect of IGF-I was specifically mediated by the IGF-IR, because IGF-I did not induce TDAG51 expression in NIH-3T3 cells overexpressing a dominant-negative IGF-I receptor. Through the use of specific inhibitors of various protein kinases, we found that IGF-I induced TDAG51 expression via the p38 MAPK pathway. The ERK, JNK, and phosphatidylinositol 3-kinase pathways were not involved in IGF-I-induced regulation of TDAG51. To assess the role of TDAG51 in IGF-I signaling, we used small interfering RNA (siRNA) expression vectors directed at two different target sites to reduce the level of TDAG51 protein. In cells expressing these siRNA vectors, TDAG51 protein levels were decreased by 75-80%. Furthermore, TDAG51 siRNA expression abolished the ability of IGF-I to rescue cells from serum starvation-induced apoptosis. These findings suggest that TDAG51 plays an important role in the anti-apoptotic effects of IGF-I.     

Identification of T cell death-associated gene 8 (TDAG8) as a novel acid sensing G-protein-coupled receptor

T cell death-associated gene 8 (TDAG8) is a G-protein-coupled receptor mainly expressed in lymphoid organs and cancer tissues. TDAG8 shares high amino acid sequence homologies with recently reported proton-sensing G-protein-coupled receptors, G2A, OGR1, and GPR4. Here we have identified TDAG8 as a novel proton-sensing receptor. Upon acid stimulation, stably expressed TDAG8 was internalized from the plasma membrane. As a signaling pathway downstream of TDAG8, accumulation of cyclic AMP was observed in response to solutions with a pH value lower than 7.2. Furthermore, RhoA activation and actin rearrangement were elicited by acid-stimulated TDAG8. These results suggest that TDAG8 may play biological roles in immune response and cellular transformation under conditions accompanying tissue acidosis.     

Inhibition of interleukin‐1β production by extracellular acidification through the TDAG8/cAMP pathway in mouse microglia

Interleukin‐1β (IL‐1β) 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‐1β production, especially focusing on TDAG8, a major proton‐sensing G‐protein‐coupled receptor, in mouse microglia. Extracellular acidification inhibited lipopolysaccharide ‐induced IL‐1β production, which was associated with the inhibition of IL‐1β cytoplasmic precursor and mRNA expression. The IL‐1β 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‐1β mRNA expression. The acidic pH‐induced inhibitory IL‐1β 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‐1β 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.

     

An unusual dependence of human herpesvirus-8 glycoproteins-induced cell-to-cell fusion on heparan sulfate

Human herpesvirus-8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins-induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: (1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; (2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, (3) co-expression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis.     

Expression of EphA8‐Fc in transgenic mouse embryos induces apoptosis of neural epithelial cells during brain development

EphAs and ephrin‐As are expressed in multiple regions of the developing brain and have been implicated in regulating brain size. Here, we report the identification of a novel mechanism in which reverse signaling through ephrin‐As controls neural epithelial cell number in the developing brain. Ectopic expression of EphA8‐Fc in transgenic embryos induced apoptosis of neural epithelial cells, which was accompanied by a dramatic decrease in brain size. The number of ephrin‐A5‐expressing cells was significantly reduced in the brain region where EphA8‐Fc was ectopically expressed. Furthermore, in vitro culture of the dissociated neuroepithelial cells revealed that EphA8‐Fc enhanced apoptotic cell death of the ephrinA5‐expressing cells in a caspase‐dependent manner. Thus, our results suggest that reverse signaling through ephrin‐As is biochemically linked with caspase‐dependent proapoptotic signaling during early brain development. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 702–712, 2013     

Expression and function of proton-sensing G-protein-coupled receptors in inflammatory pain

Background

Chronic inflammatory pain, when not effectively treated, is a costly health problem and has a harmful effect on all aspects of health-related quality of life. Despite the availability of pharmacologic treatments, chronic inflammatory pain remains inadequately treated. Understanding the nociceptive signaling pathways of such pain is therefore important in developing long-acting treatments with limited side effects. High local proton concentrations (tissue acidosis) causing direct excitation or modulation of nociceptive sensory neurons by proton-sensing receptors are responsible for pain in some inflammatory pain conditions. We previously found that all four proton-sensing G-protein-coupled receptors (GPCRs) are expressed in pain-relevant loci (dorsal root ganglia, DRG), which suggests their possible involvement in nociception, but their functions in pain remain unclear.

Results

In this study, we first demonstrated differential change in expression of proton-sensing GPCRs in peripheral inflammation induced by the inflammatory agents capsaicin, carrageenan, and complete Freund's adjuvant (CFA). In particular, the expression of TDAG8, one proton-sensing GPCR, was increased 24 hours after CFA injection because of increased number of DRG neurons expressing TDAG8. The number of DRG neurons expressing both TDAG8 and transient receptor potential vanilloid 1 (TRPV1) was increased as well. Further studies revealed that TDAG8 activation sensitized the TRPV1 response to capsaicin, suggesting that TDAG8 could be involved in CFA-induced chronic inflammatory pain through regulation of TRPV1 function.

Conclusion

Each subtype of the OGR1 family was expressed differently, which may reflect differences between models in duration and magnitude of hyperalgesia. Given that TDAG8 and TRPV1 expression increased after CFA-induced inflammation and that TDAG8 activation can lead to TRPV1 sensitization, it suggests that high concentrations of protons after inflammation may not only directly activate proton-sensing ion channels (such as TRPV1) to cause pain but also act on proton-sensing GPCRs to regulate the development of hyperalgesia.     

Regulation of T-cell death-associated gene 51 (TDAG51) expression in human T-cells

T-cell death-associated gene 51 (TDAG51) has been described to regulate T-cell receptor/CD3-dependent induction of CD95/Fas and subsequent activation-induced cell death (AICD) in a murine T-cell hybridoma. Using well-defined pharmacological inhibitors, we investigated the regulation of TDAG51 expression in human T-cells and the correlation with cell death. TDAG51 was induced in resting T-cells, lymphoid cell lines and AICD-susceptible as well as AICD-resistant T-cell clones, and induction was inhibited by MAP-kinase inhibitors and PKC inhibitor G?6983. No correlation between the effects of inhibitors on TDAG51 expression and cell death was observed. The constitutive TDAG51 expression in five pancreatic carcinoma cell lines was reduced by MAP-kinase inhibitors but not by G?6983. Furthermore, the inducible overexpression of TDAG51 in TetOn Jurkat cells did not modulate cellular proliferation, phorbolester/ionomycin-induced growth arrest, or the expression of various cell surface molecules. Our results indicate that the expression of TDAG51 in human T-cells does not correlate with AICD.     

Distribution and Subcellular Localization of High-Molecular-Weight Microtubule-Associated Protein-2 Expressing Exon 8 in Brain and Spinal Cord

Abstract: The expression of high-molecular-weight (HMW) microtubule-associated protein-2 (MAP-2) expressing exon 8 (MAP-2+8) was examined by immunoblotting during rat brain development and in sections of human CNS. In rat brain, HMW MAP-2+8 expression was detected at embryonic day 21 and increased during postnatal development. In adult rats, HMW MAP-2+8 comigrated with MAP-2a. In human adult brain, HMW MAP-2+8 was expressed in select neuronal populations, including pyramidal neurons of layers III and V of the neocortex and parahippocampal cortex, pyramidal neurons in the endplate, CA2 and subiculum of the hippocampus, and the medium-sized neurons of the basal ganglia. In the cerebellum, a subpopulation of Golgi neurons in the internal granular cell layer and most Purkinje cells were also stained. In the spinal cord staining was observed in large neurons of the anterior horn. Staining was present in cell bodies and dendrites but not in axons. At the ultra-structural level, HMW MAP-2+8 immunoreactivity was observed on mitochondrial membranes and in postsynaptic densities (PSDs) of some asymmetric synapses in the midfrontal cortex and spinal cord. Immunoblots of proteins isolated from enriched mitochondrial and PSD fractions from adult human frontal lobe and rat brains confirmed the presence of HMW MAP-2+8. The presence of HMW MAP-2+8 in dendrites and in close proximity to PSDs supports a role in structural and functional attributes of select excitatory CNS synapses.     

Small molecule antagonists of CCR8 inhibit eosinophil and T cell migration

In this study, we demonstrate that in addition to T lymphocytes, human naïve eosinophils and the differentiated eosinophil-like cell line, AML14.3D10 express CCR8 and respond to CCL1 through CCR8 engagement. The responsiveness of cells was dependent on maturation stage, since CCL1 induced pronounced chemotaxis only in differentiated CCR8 positive AML14.3D10 cells. Despite the low CCR8 surface expression, human naïve eosinophils respond with a chemotaxis to high concentration CCL1. We further describe that Th2 clones in a maturation dependent fashion produce autocrine CCL1, which renders them unresponsive to further stimulation. An innovative method to enrich primary CCR8 reactive T cells was developed which demonstrates that primary peripheral CCR8 expressing T cells respond significantly to CCL1.We have developed novel small molecule CCR8 antagonists that are effective in inhibiting calcium mobilization and chemotaxis in differentiated AML cells as well as in human primary CCR8 positive T cells. Importantly, we demonstrate that the compounds can be divided into two subgroups: (i) compounds that are functional agonists for calcium mobilization and chemotaxis (ii) compounds that are pure antagonists. We demonstrate that agonism of these compounds does not correlate with their antagonistic potency. Taken together, we have identified a novel set of CCR8 compounds with antagonistic properties that inhibit CCL1 driven chemotaxis in both CCR8 expressing eosinophils as well as primary human T cells.     

Splenic dendritic cell subsets prime and boost CD8 T cells and are involved in the generation of effector CD8 T cells

The ability of the dendritic cell (DC) subsets, CD8alpha+ and CD8alpha- DCs, to initiate a CD8 T cell response or to activate memory CD8 T cells and generate effector CD8 T cells has been controversial. In this study, we analyse the capacity of splenic DC subsets to induce CD8 T cell responses to a CD8 T cell epitope (pb9) of a malaria antigen. The administration of peptide-pulsed CD8alpha- or CD8alpha+ DCs primes and boosts a primed CD8 T cell response against the malaria epitope. In vitro, depletion of CD11c(+) DCs from mouse splenocytes, immunised with recombinant vaccinia virus Ankara (MVA) expressing pb9 epitope, significantly reduced the generation of pb9-specific IFNgamma producing effector CD8 T cells, indicating that splenic DCs are involved in the development of pb9-specific IFNgamma producing effector cells. Taken together, this result shows that both DC subsets have the ability to prime and boost CD8 T cell responses and are involved in the activation of memory CD8 T cells.     

Regulation of the G2/M phase of the cell cycle by sperm associated antigen 8 (SPAG8) protein

Sperm associated antigen 8 (SPAG8), a testis‐specific protein produced during male germ cell differentiation, was isolated from a human testis expression library using antibodies found in the serum obtained from an infertile woman. It was found to have a close functional relationship with microtubules. In this study, we generated a stably expressing SPAG8 CHO‐K1 cell line. Immunofluorescence confocal microscopy showed that SPAG8 was concentrated at the microtubule‐organizing center (MTOC) during prophase. As the cells progressed into metaphase, it co‐localized with α‐tubulin on the spindle. In anaphase, it was detected on both astral microtubules and mid‐zone. Following cytokinesis, SPAG8 resumed its localization on the MTOC. Meanwhile, flow cytometry analysis found that SPAG8 prolonged the G2/M phase of CHO‐K1 cells stably expressing SPAG8. Furthermore, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay showed that SPAG8 inhibited the proliferation of the stable cells. SPAG8 might be involved in the regulation of cell cycle by changing the phosphorylation level of Tyr15 on cdc2. These results suggest that SPAG8 might play a role in cell division during spermatogenesis. Copyright © 2009 John Wiley & Sons, Ltd.     

Temporal and spatial profile of caspase 8 expression and proteolysis after experimental traumatic brain injury

Recent studies have demonstrated that the downstream caspases, such as caspase 3, act as executors of the apoptotic cascade after traumatic brain injury (TBI) in vivo. However, little is known about the involvement of caspases in the initiation phase of apoptosis, and the interaction between these initiator caspases (e.g. caspase 8) and executor caspases after experimental brain injuries in vitro and in vivo. This study investigated the temporal expression and cell subtype distribution of procaspase 8 and cleaved caspase 8 p20 from 1 h to 14 days after cortical impact-induced TBI in rats. Caspase 8 messenger RNA levels, estimated by semiquantitaive RT-PCR, were elevated from 1 h to 72 h in the traumatized cortex. Western blotting revealed increased immunoreactivity for procaspase 8 and the proteolytically active subunit of caspase 8, p20, in the ipsilateral cortex from 6 to 72 h after injury, with a peak at 24 h after TBI. Similar to our previous studies, immunoreactivity for the p18 fragment of activated caspase 3 also increased in the current study from 6 to 72 h after TBI, but peaked at a later timepoint (48 h) as compared with proteolyzed caspase 8 p20. Immunohistologic examinations revealed increased expression of caspase 8 in neurons, astrocytes and oligodendrocytes. Assessment of DNA damage using TUNEL identified caspase 8- and caspase 3-immunopositive cells with apoptotic-like morphology in the cortex ipsilateral to the injury site, and immunohistochemical investigations of caspase 8 and activated caspase 3 revealed expression of both proteases in cortical layers 2-5 after TBI. Quantitative analysis revealed that the number of caspase 8 positive cells exceeds the number of caspase 3 expressing cells up to 24 h after impact injury. In contrast, no evidence of caspase 8 and caspase 3 activation was seen in the ipsilateral hippocampus, contralateral cortex and hippocampus up to 14 days after the impact. Our results provide the first evidence of caspase 8 activation after experimental TBI and suggest that this may occur in neurons, astrocytes and oligodendrocytes. Our findings also suggest a contributory role of caspase 8 activation to caspase 3 mediated apoptotic cell death after experimental TBI in vivo.     

Bid is cleaved upstream of caspase-8 activation during TRAIL-mediated apoptosis in human osteosarcoma cells

TRAIL induces apoptosis in many malignant cell types. In this study, we used the human papilloma virus (HPV) 16 E6 protein as a molecular tool to probe the TRAIL pathway in HCT116 colon carcinoma cells and U2OS osteosarcoma cells. Intriguingly, we found that while E6 protected HCT116 cells from TRAIL, U2OS cells expressing E6 remained sensitive to TRAIL. Furthermore, silencing FADD and procaspase-8 expression with siRNA did not prevent TRAIL-induced apoptosis in U2OS cells. However, siBid provided significant protection from TRAIL, and the cleavage kinetics of Bid and caspase-8 revealed that Bid was cleaved prior to the activation of caspase-8. Cathepsin B activity in U2OS cells was significantly activated shortly after exposure to TRAIL, and the cathepsin B inhibitor, CA074Me, inhibited both TRAIL- and anti-DR5-mediated apoptosis and delayed the cleavage of Bid. These findings suggest that TRAIL activates a pathway dependent on Bid, but largely independent of FADD and caspase-8, in U2OS cells.     

稳定表达人Eps8基因的胶质瘤U251细胞系的建立

应用亚克隆方法构建pEGFP-C3／Eps8真核表达载体,经测序鉴定后,用脂质体进行胶质瘤U251细胞的转染,应用G418筛选出稳定表达pEGFP-C3／Eps8和pEGFP—C3的细胞系,最后通过Western blot和荧光定位证明印娼在U251细胞中过量表达。本实验成功建立了稳定转染Eps8的U251细胞系,为进一步研究Eps8基因在胶质瘤中的功能奠定了良好的实验基础。