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Extracellular nucleotides, such as ATP, are released from cells in response to various stimuli and act as intercellular signaling molecules through activation of P2 receptors. Exposure to the ultraviolet radiation A (UVA) component of sunlight causes molecular and cellular damage, and in this study, we investigated the involvement of extracellular nucleotides and P2 receptors in the UVA-induced cellular response. Human keratinocyte-derived HaCaT cells were irradiated with a single dose of UVA (2.5 J/cm2), and ATP release and interleukin (IL)-6 production were measured. ATP was released from cells in response to UVA irradiation, and the release was blocked by pretreatment with inhibitors of gap junction hemichannels or P2X7 receptor antagonist. IL-6 production was increased after UVA irradiation, and this increase was inhibited by ecto-nucleotidase or by antagonists of P2Y11 or P2Y13 receptor. These results suggest that UVA-induced IL-6 production is mediated by release of ATP through hemichannels and P2X7 receptor, followed by activation of P2Y11 and P2Y13 receptors. Interestingly, P2Y11 and P2Y13 were associated with the same pattern of IL-6 production, though they trigger different intracellular signaling cascades: Ca2+-dependent and PI3K-dependent, respectively. Thus, IL-6 production in response to UVA-induced ATP release involves at least two distinct pathways, mediated by activation of P2Y11 and P2Y13 receptors.

Kupffer cells, which are resident macrophages in liver, can produce various cytokines and chemokines that induce hepatitis and liver fibrosis. It is suggested that extracellular ATP-induced activation of macrophage P2X7 receptor plays an important role in inflammation via release of pro-inflammatory mediators, but the role of P2X7 receptor in Kupffer cells remains unclear. Here, we show that activation of P2X7 receptor in Kupffer cells causes multiple inflammatory responses, using the clonal mouse Kupffer cell line (KUP5) that we previously established. Treatment of LPS-primed Kupffer cells with 3 mM ATP induced Ca(2+) influx, non-selective large pore formation, activation of MAPK, cell lysis, IL-1β release, prostaglandin E2 (PGE2) release, high mobility group box1 (HMGB1) release, and major histocompatibility complex (MHC) class I shedding. These events were significantly suppressed by pretreatment with P2X7 antagonist A438079, indicating involvement of P2X7 receptor activation in these inflammatory responses. Our results suggest that extracellular ATP-induced activation of P2X7 receptor of Kupffer cells plays multiple roles in the inflammatory response in liver. P2X7 receptor might be a new therapeutic target for treatment of liver diseases., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Ultraviolet A (UVA) radiation, the major UV component of solar radiation, can penetrate easily to the dermis, where it causes significant damage to cellular components by inducing formation of reactive oxygen species (ROS). On the other hand, extracellular ATP is released in response to various stimuli, and activates purinergic P2X7 receptor, triggering ROS production and cell death. Here, we examined the hypothesis that ATP release followed by activation of P2X7 receptor plays a role in UVA-induced oxidative cell damage, using human acute monocytic leukemia cell line THP-1. Indeed, UVA irradiation of THP-1 cells induced ATP release and activation of P2X7 receptor. Irradiated cells showed a rapid increase of both p67 phox in membrane fraction and intracellular ROS. Pretreatment with ecto-nucleotidase or P2X7 receptor antagonist blocked the UVA-initiated membrane translocation of p67 phox and ROS production. Furthermore, pretreatment with antioxidant or P2X7 receptor antagonist efficiently protected UVA-irradiated cells from caspase-dependent cell death. These findings show that autocrine signaling through release of ATP and activation of P2X7 receptor is required for UVA-induced stimulation of oxidative stress in monocytes.

Aliskiren is a substrate for P-glycoprotein (P-gp) and is metabolized via cytochrome P450 3A4 (CYP3A4). The aim of the present study was to assess whether P-gp influenced the pharmacokinetics of aliskiren and also if drug-drug interactions (DDIs) mediated through P-gp could be reproduced in cynomolgus monkeys. The study investigated the pharmacokinetics of aliskiren in mdr1a/1b gene-deficient (P-gp KO) and wild-type (WT) mice. The area under the plasma concentration-time curve (AUC) following the oral administration of aliskiren was 6.9-fold higher in P-gp KO mice than in WT mice, while no significant differences were observed in the AUC or total plasma clearance following the intravenous administration of aliskiren to P-gp KO mice. Then the pharmacokinetics of aliskiren were evaluated and DDIs between aliskiren and P-gp inhibitors, such as cyclosporin A (CsA) and zosuquidar, examined in cynomolgus monkeys. The AUC for aliskiren were 8.3- and 42.1-fold higher after the oral administration of aliskiren with the concomitant oral administration of zosuquidar and CsA at doses of 10 and 30 mg/kg, respectively. In contrast, the AUC after the intravenous and oral administration of aliskiren was not significantly affected by the oral administration of zosuquidar or intravenous administration of CsA, respectively. These results indicated that P-gp strictly limited the intestinal absorption of aliskiren in mice and monkeys, and also that the effects of intestinal P-gp inhibition by CsA or zosuquidar on the pharmacokinetics of aliskiren were sensitively reproduced in monkeys. In conclusion, aliskiren can be used as a sensitive substrate to evaluate intestinal P-gp inhibition in monkeys., (Copyright © 2014 John Wiley & Sons, Ltd.)

Recent studies suggest the effect of radiation is observed not only in irradiated cells but also in adjacent non-irradiated cells (bystander effect), although the mechanism has not yet been fully revealed. This bystander effect may be caused by intercellular communication via a gap junction or by messengers released from irradiated cells, such as reactive oxygen species, nitric oxide, or cytokines. However, an unknown mechanism is also possible in the bystander effect. On the other hand, it is known that extracellular ATP, ADP, uridine 5'-triphosphate (UTP), and uridine 5'-diphosphate (UDP), which are released from cells, act as intercellular signaling molecules by activating purinergic P2X and P2Y receptors (purinergic signaling). Recently, I have suggested these extracellular nucleotides may be novel mediators of a radiation-induced bystander effect, because our recent studies indicated that purinergic signaling is involved in important cellular responses to radiation. Our data indicate that ionizing irradiation causes activation of the transient receptor potential melastatin type 2 (TRPM2) channel, and then ATP is released from cells through the anion channel or connexin43 hemichannel mediated by the activation of a P2X7 receptor. The released nucleotides activate P2Y6 and P2Y12 receptors, which are involved in the DNA damage response after irradiation. Activation of the P2Y6 receptor is also involved in radiation-induced activation of the epithelial growth factor receptor-extracellular signal regulated protein kinase (EGFR-ERK)1/2 pathway and subsequent nuclear translocation of EGFR, which plays a role in DNA repair. Further, the induction of an antioxidant after irradiation is also mediated by the activation of the P2Y receptor. In conclusion, purinergic signaling could play an important role in the protective cellular response to ionizing irradiation.

Extracellular nucleotides, such as ATP, are released from cells and play roles in various physiological and pathological processes through activation of P2 receptors. Here, we show that autocrine signaling through release of ATP and activation of P2X7 receptor influences migration of human lung cancer cells. Release of ATP was induced by stimulation with TGF-β1, which is a potent inducer of cell migration, in human lung cancer H292 cells, but not in noncancerous BEAS-2B cells. Treatment of H292 cells with a specific antagonist of P2X7 receptor resulted in suppression of TGF-β1-induced migration. PC-9 human lung cancer cells released a large amount of ATP under standard cell culture conditions, and P2X7 receptor-dependent dye uptake was observed even in the absence of exogenous ligand, suggesting constitutive activation of P2X7 receptor in this cell line. PC-9 cells showed high motile activity, which was inhibited by treatment with ecto-nucleotidase and P2X7 receptor antagonists, whereas a P2X7 receptor agonist enhanced migration. PC-9 cells also harbor a constitutively active mutation in epidermal growth factor receptor (EGFR). Treatment with EGFR tyrosine kinase inhibitor AG1478 suppressed both cell migration and P2X7 receptor expression in PC-9 cells. Compared to control PC-9 cells, cells treated with P2X7 antagonist exhibited broadened lamellipodia around the cell periphery, while AG1478-treated cells lacked lamellipodia. These results indicate that P2X7-mediated signaling and EGFR signaling may regulate migration of PC-9 cells through distinct mechanisms. We propose that autocrine ATP-P2X7 signaling is involved in migration of human lung cancer cells through regulation of actin cytoskeleton rearrangement.

The P2X7 receptor (P2X7R) is an ATP-gated cation channel that is abundantly expressed in monocytes/macrophages. P2X7R activation by ATP results in various cellular responses including Ca(2+) influx, membrane pore formation, and cytokine secretion. Since P2X7R has low affinity for ATP, high concentrations of ATP (in the mM range) are generally required to activate this receptor in vitro. Functional expression of P2X7R has been detected in monocytes/macrophages obtained from different animal species including humans, rodents, dogs, and bovines, but so far it has not been detected in swine (Sus scrofa). In this study, we investigated the expression and functions of P2X7R in swine macrophages, which were isolated from mixed primary cultures of swine kidney or liver tissue. The P2X7R mRNA and protein expression observed in the swine macrophages was comparable to that seen in a c-myc-immortalized mouse kidney-derived clonal macrophage cell line (KM-1). However, extracellular ATP did not induce P2X7R-dependent sustained Ca(2+) influx, membrane pore formation, or the secretion of the bioactive cytokine interleukin-1β in the swine macrophages, whereas these responses were clearly observed in the mouse KM-1 cells after stimulation with millimolar concentrations of ATP as a positive control. These findings suggest that the ATP/P2X7R pathway is impaired in swine macrophages at least in the culture conditions used in the present study.

We have previously reported that P2Y11 receptor mediates IFN-γ-induced IL-6 production in human keratinocytes, suggesting the importance of purinergic signaling in skin inflammatory diseases. In this study, the involvement of various P2 receptors in IL-6 production induced by silica nanoparticle 30 (SNP30) was examined in a human keratinocyte cell line, HaCaT. Exposure to SNP30 increased IL-6 production in the cells. Ecto-nucleotidase (apyrase), a non-selective antagonist of P2Y receptors (suramin), and a selective P2Y11 receptor antagonist (NF157) all inhibited IL-6 production. Nucleotides such as ATP and UTP themselves also significantly increased IL-6 production in the cells. It was further confirmed that ATP was released from HaCaT cells exposed to SNP30. These results support the possible role of ATP in SNP30-induced IL-6 production by HaCaT cells. In conclusion, these data demonstrate that P2Y11 receptor also mediates SNP30-induced IL-6 production in human keratinocytes, confirming that the ATP-P2Y11 purinergic signaling is a common pathway of IL-6 production leading to induction of skin inflammatory diseases., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

There is extensive evidence that nanoparticles (NPs) cause adverse effects in multiple organs, including liver, though the mechanisms involved remain to be fully established. Kupffer cells are macrophages resident in the liver, and play important roles in liver inflammation induced by various toxic agents, including lipopolysaccharide (LPS). Interleukin-1 (IL-1) family members IL-1α,β are released from LPS-primed macrophages exposed to NPs, including silica NPs (SNPs), via activation of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasomes. Here, we investigated the mechanism of production of IL-1β via activation of inflammasomes in mouse Kupffer cell line KUP5, focusing on the role of purinergic signaling via P2X7 receptor. IL-1β production by LPS-primed KUP5 cells exposed to SNPs was increased dose-dependently, and was greatest in response to SNPs with a diameter of 30 nm (SNP30), as compared with 70-nm and 300-nm SNPs (SNP70 and SNP300). ATP release was also highest in cells exposed to SNP30. Treatment of LPS-primed KUP5 cells with ATP also induced a high level of IL-1β production, similar to that induced by SNP30. IL-1β production was significantly inhibited by apyrase (an ecto-nucleotidase) and A438079 (a P2X7 antagonist/ATP-release inhibitor). Production of reactive oxygen species (ROS) was confirmed in cells exposed to SNP30. In conclusion, ATP released from P2X7 receptor in response to stimulation of KUP5 cells with SNP30 induces ROS production via cell-membrane NADPH oxidase. The ROS causes activation of inflammasomes, leading to caspase-1-dependent processing of IL-1β., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Resident macrophages in the liver (Kupffer cells) produce various cytokines and chemokines, and have important roles in hepatitis and liver fibrosis. The cells are activated by various factors, for example lipopolysaccharide (LPS), which is an endotoxin and is high in the blood of patients with liver cirrhosis. Involvement of P2 receptors in the release of pro-inflammatory cytokines from Kupffer cells is little. In this study, we investigated purinergic signaling in the release of pro-inflammatory cytokines, such as IL-6 and TNF-α, from liver Kupffer cells of C57BL/6 mice (KUP5 cells). KUP5cells were isolated from C57BL/6 mice and cultivated with Dulbecco's modified Eagle's medium. The cells were stimulated with LPS. LPS-induced IL-6 production by KUP5 cells was suppressed significantly by pretreatments with non-selective P2 antagonist suramin, P2Y13antagonist MRS2211, and ecto-nucleotidase, whereas P2Y receptor agonists, significantly increased the IL-6 production. P2Y13knockdown reduced LPS-induced IL-6 production, but by less than 50%. These results would suggest that P2Y receptors including P2Y13and others, may involves in LPS-induced IL-6 production in Kupffer cells, leading to the liver inflammation. Therefore, we first showed the importance of purinergic signaling via P2Y receptors in the activation of Kupffer cells and liver injury, which is worthwhile in drug development for liver diseases.

Regulatory T cells (Treg) play a role in suppression of immune response, including anti-tumor immunity. We have recently reported that treatment of naïve CD4 T cells with adenosine A(2B) receptor antagonist PSB603 under Treg-skewing conditions inhibits expression of Foxp3, a marker of differentiation to Treg, without blocking IL-2 production or CD25 expression, which are activation markers, in CD4 T cells. We hypothesized that PSB603 suppresses cancer growth and metastasis by inhibiting induction of Treg, thereby facilitating anti-tumor immunity. In this study, we first examined the effect of PSB603 on tumor growth in B16 melanoma-bearing C57BL/6 mice. Administration of PSB603 significantly suppressed the increase of tumor volume as well as the increase of Treg population in these mice. The populations of CD4 and CD8 T cells were higher and splenic lymphocyte-mediated cytotoxicity towards B16 melanoma was significantly increased in PSB603-treated mice. We confirmed that PSB603 did not reduce the viability of B16 melanoma cells in vitro. Moreover, we also examined the effect of PSB603 on tumor metastasis in pulmonary metastasis model mice intravenously injected with B16 melanoma cells. The metastasis was also suppressed in PSB603-treated mice, in which the population of Treg was significantly lower. Overall, our results suggest that A(2B) receptor antagonist PSB603 enhances anti-tumor immunity by inhibiting differentiation to Treg, resulting in a delay of tumor growth and a suppression of metastasis.

We previously showed that nucleotide P2 receptor agonists such as ATP and UTP amplify γ-ray-induced focus formation of phosphorylated histone H2A variant H2AX (γH2AX), which is considered to be an indicator of DNA damage so far, by activating purine P2Y6 and P2Y12 receptors. Therefore, we hypothesized that these P2 receptors play a role in inducing the repair response to γ-ray-induced DNA damage. In the present study, we tested this idea by using human lung cancer A549 cells. First, reverse-transcription polymerase chain reaction (RT-PCR) showed that P2Y6 receptor is highly expressed in A549 cells, but P2Y12 receptor is only weakly expressed. Next, colony formation assay revealed that P2Y6 receptor antagonist MRS2578 markedly reduced the survival rate of γ-ray-exposed A549 cells. The survival rate was also significantly reduced in P2Y6-knock-down cells, compared with scramble siRNA-transfected cells. Since it has reported that phosphorylation of ERK1/2 after activation of EGFR via P2Y6 and P2Y12 receptors is involved in the repair response to γ-ray-induced DNA damage, we next examined whether γ-ray-induced phosphorylation of ERK1/2 was also inhibited by MRS2578 in A549 cells. We found that it was. Taken together, these findings indicate that purinergic signaling through P2Y6 receptor, followed by ERK1/2 activation, promotes the cellular repair response to γ-ray-induced DNA damage.

Inhibition of the renal sodium glucose cotransporter (SGLT) increases urinary glucose excretion (UGE) and thus reduces blood glucose levels during hyperglycemia. To explore the potential of new antihyperglycemic agents, we synthesized and determined the human SGLT2 (hSGLT2) inhibitory potential of novel substituted 3-benzylindole-N-glucosides 6. Optimization of 6 resulted in the discovery of 3-(4-cyclopropylbenzyl)-4-fluoroindole-N-glucoside 6a-4 (TA-1887), a highly potent and selective hSGLT2 inhibitor, with pronounced antihyperglycemic effects in high-fat diet-fed KK (HF-KK) mice. Our results suggest the potential of indole-N-glucosides as novel antihyperglycemic agents through inhibition of renal SGLT2.

Inhibition of renal sodium-dependent glucose cotransporter 2 (SGLT2) increases urinary glucose excretion (UGE), and thus reduces blood glucose levels in hyperglycemia. A series of N-glucosides was synthesized for biological evaluation as human SGLT2 (hSGLT2) inhibitors. Among these compounds, N-glucoside 9d possessing an indole core structure showed good in vitro activity (IC50=7.1 nM against hSGLT2). Furthermore, 9d exhibited favorable in vivo potency with regard to UGE in rats based on good pharmacokinetic profiles., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Transforming growth factor-ß1 (TGF-β1) is a multifunctional cytokine that is involved in various pathophysiological processes, including cancer progression and fibrotic disorders. Here, we show that treatment with TGF-β1 (5 ng/mL) induced downregulation of cyclooxygenase-2 (COX-2), leading to reduced synthesis of prostaglandin E2 (PGE2), in human lung cancer A549 cells. Treatment of cells with specific inhibitors of COX-2 or PGE2 receptor resulted in growth inhibition, indicating that the COX-2/PGE2 pathway contributes to proliferation in an autocrine manner. TGF-β1 treatment induced growth inhibition, which was attenuated by exogenous PGE2. TGF-β1 is also a potent inducer of epithelial mesenchymal transition (EMT), a phenotype change in which epithelial cells differentiate into fibroblastoid cells. Supplementation with PGE2 or PGE2 receptor EP4 agonist PGE1-alcohol, as compared with EP1/3 agonist sulprostone, inhibited TGF-β1-induced expression of fibronectin and collagen I (extracellular matrix components). Exogenous PGE2 or PGE2 receptor agonists also suppressed actin remodeling induced by TGF-β1. These results suggest that PGE2 has an anti-fibrotic effect. We conclude that TGF-β1-induced downregulation of COX-2/PGE2 signaling is involved in facilitation of fibrotic EMT response in A549 cells.

Canagliflozin (1), a novel inhibitor for sodium-dependent glucose cotransporter 2 (SGLT2), has been developed for the treatment of type 2 diabetes. To investigate the effect of replacement of the phenyl ring in 1 with heteroaromatics, C-glucosides 2 were designed, synthesized, and evaluated for their inhibitory activities against SGLT2. Of these, 3-pyridyl, 2-pyrimidyl or 5-membered heteroaryl substituted derivatives showed highly potent inhibitory activity against SGLT2, while 5-pyrimidyl substitution was associated with slightly reduced activity. In particular, 2g (TA-3404) had remarkable anti-hyperglycemic effects in high-fat diet fed KK (HF-KK) mice., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The Fc region of human IgG1 mediates effector function via binding to Fcγ receptors and complement activation. The H and L chains of IgG1 antibodies are joined by four interchain disulfide bonds. In this study, these bonds within the therapeutic IgG1 rituximab (RTX) were cleaved either by mild reduction followed by alkylation or by mild S-sulfonation; consequently, two modified RTXs - A-RTX (alkylated) and S-RTX (S-sulfonated) - were formed, and both were almost as potent as unmodified RTX when binding CD20 antigen. Unexpectedly, each modified RTX had a higher binding affinity for FcγRIIIA (CD16A) than did unmodified RTX. However, S-RTX and A-RTX were each less potent than RTX in an assay of antibody-dependent cellular cytotoxicity (ADCC). In this ADCC assay, each modified RTX showed decreased secretion of granzyme B, but no change in perforin secretion, from effector cells. These results provide significant information on the structures within IgG1 that are involved in binding FcγRIIIA, and they may be useful in the development of therapeutic antagonists for FcγRIIIA., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Radiation exposure causes DNA damage, and DNA repair systems are essential to rescue damaged cells. Although DNA damage or oxidative stress activates transient receptor potential melastatin 2 (TRPM2) and vanilloid 1 (TRPV1) cation channels, it has not been established whether these TRP channels are involved in cellular responses to radiation-induced DNA damage. Here, we investigated the contribution of TRPM2 and TRPV1 channels to γ-irradiation- and UVB-induced DNA damage responses in human lung cancer A549 cells., Methods: A549 cells were irradiated with γ-rays (2.0Gy) or UVB (5-10mJ/cm(2)). γH2AX foci, ATM activation, 53BP1 accumulation and EGFR expression were evaluated by immunofluorescence staining. Extracellular ATP concentration was measured by luciferin-luciferase assay. Knockdown of TRPM2 and TRPV1 expression was done by siRNA transfection., Results: γ-Irradiation-induced γH2AX focus formation, ATM activation, 53BP1 accumulation and EGFR nuclear translocation, which are all associated with DNA repair, were suppressed by knockdown of TRPM2 and TRPV1 channels in A549 cells. Release of ATP, which mediates DNA damage response-associated activation of P2Y receptors, was suppressed by pre-treatment with catalase or knockdown of TRPM2 channel, but not TRPV1 channel. Similarly, UVB-induced γH2AX focus formation was suppressed in TRPM2- and TRPV1-knockdown cells, while UVB-induced ATP release was blocked in TRPM2- but not TRPV1-knockdown cells., Conclusion: Our results suggest that the activation of TRPM2 channel, which mediates ATP release, and TRPV1 channel plays significant roles in the cellular responses to DNA damage induced by γ-irradiation and UVB irradiation., General Significance: Our results provide a new insight into the function of TRP channels from the viewpoint of radiation biology., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Activation of P2X7 receptor of dendritic cells plays a significant role in inflammation through production of cytokines such as IL-1β, and recent studies have suggested structural and functional interactions of P2X7 receptor with P2X4 receptor in macrophages. However, it is unknown whether P2X4 receptor modulates P2X7 functions in dendritic cells. Here, we present evidence that expression of P2X4 receptor is required for P2X7 receptor-dependent IL-1β and IL-18 release in mouse bone marrow-derived dendritic cells (BMDCs). We confirmed expression of both P2X7 receptor and P2X4 receptor in BMDCs. Treatment of BMDCs with 3 mM ATP caused a transient, P2X4-dependent elevation, or spike, of intracellular Ca(2+) level [Ca(2+)]i, followed by the sustained P2X7-dependent increase of [Ca(2+)]i. We performed knockdown of P2X4 receptor in BMDCs by transfection with short hairpin RNA targeting this receptor. The ATP-induced initial peak of [Ca(2+)]i was decreased in P2X4-knockdown cells (P2X4-KD). Further, we found that ATP-induced IL-1β and IL-18 release from LPS-primed BMDCs was suppressed by pretreatment with P2X7 antagonist A438079 or P2X4 antagonist TNP-ATP. The P2X7-dependent IL-1β and IL-18 release was significantly lower in P2X4-KD cells. Chelation of intracellular Ca(2+) also caused suppression of ATP-induced IL-1β and IL-18 release. These results suggest that P2X4 receptor-induced Ca(2+) influx is required for effective production of IL-1β and IL-18 via activation of P2X7 receptor in BMDCs. We conclude that co-expression of P2X4 receptor with P2X7 receptor in dendritic cells leads to enhancement of inflammation through facilitation of P2X7-dependent release of pro-inflammatory cytokines., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Extracellular ATP and P2 receptors are reported to be involved in interleukin-6 (IL-6) production by human keratinocytes, but the role of extracellular ATP in cytokine-induced IL-6 production remains unclear. In this study, we investigated the involvement of various P2 receptors in IL-6 production induced by the Th1 cytokine interferon-gamma (IFN-γ) in a human keratinocyte cell line, HaCaT. IFN-γ increased IL-6 production in HaCaT cells. A non-selective antagonist of P2Y receptors (suramin), a selective P2Y11 receptor antagonist (NF157), ecto-nucleotidase (apyrase), and a soluble adenylate cyclase inhibitor (KH7) all inhibited IL-6 production. It was further confirmed that ATP was released from HaCaT cells stimulated with IFN-γ. These results suggest that extracellular ATP and P2Y11 receptor are involved in IFN-γ-induced IL-6 production. Knockdown of P2Y11 receptor suppressed IL-6 production, strongly supporting this idea. In conclusion, these data demonstrate that P2Y11 receptor mediates IFN-γ-induced IL-6 production in human keratinocytes, and suggest the importance of purinergic signaling in IFN-γ-induced skin inflammatory conditions, such as psoriasis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

It is important to understand the mechanisms that regulate macrophage activation to establish novel therapies for inflammatory diseases, such as sepsis; a systemic inflammatory response syndrome generally caused by bacterial lipopolysaccharide (LPS). In this study, we investigated the involvement of extracellular ATP-mediated autocrine signaling in LPS-induced macrophage activation. We show here that ATP release via exocytosis, followed by activation of P2Y11 receptor, is a major pathway of the macrophage activation, leading to release of cytokines. Treatment of human monocyte THP-1 cells with LPS induced rapid ATP release from cells, and this release was blocked by knockdown of SLC17A9 (vesicular nucleotide transporter, VNUT), which is responsible for exocytosis of ATP. ATP-enriched vesicles were found in cytosol of THP-1 cells. These data suggest the involvement of vesicular exocytosis in the release of ATP. Knockdown of SLC17A9, the P2Y11 antagonist NF157 or knockdown of P2Y11 receptor significantly suppressed both M1-type polarization and IL-6 production in THP-1 cells, indicating an important role of activation of P2Y11 receptor by released ATP in macrophage activation. Next, the effect of NF157 on LPS-induced immune activation was examined in vivo. Administration of LPS to mice caused increase of serum IL-1ß, IL-6, IL-12 and TNF-alpha levels at 3-24 h after the administration. Pre-treatment of LPS-treated mice with NF157 suppressed both elevation of proinflammatory cytokines in serum and M1 polarization of peritoneal/spleen macrophages. Moreover, post-treatment with NF157 at 30 min after administration of LPS also suppressed the elevation of serum cytokines levels. We conclude that vesicular exocytosis of ATP and autocrine, positive feedback through P2Y11 receptors is required for the effective activation of macrophages. Consequently, P2Y11 receptor antagonists may be drug candidates for treatment of inflammatory diseases such as sepsis.

The P2X7 receptor is not only involved in cell proliferation, but also acts as an adenosine 5'-triphosphate (ATP)-gated non-selective channel, and its expression is increased in human melanoma. An irreversible antagonist of P2X7, such as oxidized ATP (oxATP), might block P2X7 receptor-mediated ATP release and proliferative signaling. Therefore, we carried out basic studies to test this idea and to examine the feasibility of using oxATP to treat B16 melanoma. We first found that low-pH conditions (mimicking the hypoxia and acidosis commonly seen in solid tumors) induced P2X7 receptor-mediated ATP release from B16 melanoma cells. Then, we compared the proliferation rates of B16 melanoma wild-type cells and B16 P2X7 receptor-knockdown clone (P2X7-KDC) cells in the presence of P2X7 agonists. The proliferation rate, as well as the ATP release, of agonist-treated P2X7-KDC cells was lower than that of agonist-treated wild-type cells. Next, the effect of P2X7 antagonist oxATP on B16 melanoma cell growth was examined in vitro and in vivo. oxATP significantly decreased B16 melanoma cell proliferation in vitro, and also significantly inhibited tumor growth in B16 melanoma-bearing mice. These data indicate that extracellularly released ATP may serve as an intercellular signaling molecule. We propose that the P2X7 receptor is a promising target for treatment of solid tumors., (Copyright © 2012 Elsevier B.V. All rights reserved.)

TGF-β1 plays a key role in cancer progression through induction of various biological effects, including cell migration. Extracellular nucleotides, such as ATP, released from cells play a role in signaling through activation of P2 receptors. We show here that exocytosis of ATP followed by activation of P2 receptors play a key role in TGF-β1-induced actin remodeling associated with cell migration. Treatment with TGF-β1 facilitated migration of human lung cancer A549 cells, which was blocked by pretreatment with ecto-nucleotidase and P2 receptor antagonists. ATP and P2 agonists facilitated cell migration. TGF-β1-induced actin remodeling, which contributes to cell migration, was also suppressed by pretreatment with ecto-nucleotidase and P2 receptor antagonists. Knockdown of P2X7 receptor suppressed TGF-β1-induced migration and actin remodeling. These results indicate the involvement of TGF-β1-induced ATP release in cell migration, at least in part, through activation of P2X7 receptors. TGF-β1 caused release of ATP from A549 cells within 10 minutes. Both ATP-enriched vesicles and expression of a vesicular nucleotide transporter (VNUT) SLC17A9, which is responsible for exocytosis of ATP, were found in cytosol of A549 cells. TGF-β1 failed to induce release of ATP from SLC17A9-knockdown cells. TGF-β1-induced cell migration and actin remodeling were also decreased in SLC17A9-knockdown cells. These results suggest the importance of exocytosis of ATP in cell migration. We conclude that autocrine signaling through exocytosis of ATP and activation of P2 receptors is required for the amplification of TGF-β1-induced migration of lung cancer cells.

A key component of the response to DNA damage caused by ionizing radiation is DNA repair. Release of extracellular nucleotides, such as ATP, from cells plays a role in signaling via P2 receptors. We show here that release of ATP, followed by activation of P2Y receptors, is involved in the response to γ-irradiation-induced DNA damage. Formation of phosphorylated histone variant H2AX (γH2AX) foci, which are induced in nuclei by DNA damage and contribute to accumulation of DNA-repair factors, was increased at 1-3h after γ-ray irradiation (2.0Gy) of human lung cancer A549 cells. Focus formation was suppressed by pre-treatment with the ecto-nucleotidase apyrase. Pre-treatment with ecto-nucleotidase inhibitor ARL67156 or post-treatment with ATP or UTP facilitated induction of γH2AX, indicating that extracellular nucleotides play a role in induction of γH2AX foci. Next, we examined the effect of P2 receptor inhibitors on activation of ataxia telangiectasia mutated (ATM; a protein kinase) and accumulation of 53BP1 (a DNA repair factor), both of which are important for DNA repair, at DNA damage sites. P2Y6 receptor antagonist MRS2578, P2Y12 receptor antagonist clopidogrel, and P2X7 receptor antagonists A438079 and oxATP significantly inhibited these processes. Release of ATP was detected within 2.5min after irradiation, but was blocked by A438079. Activation of ATM and accumulation of 53BP1 were decreased in P2Y6 or P2Y12 receptor-knockdown cells. We conclude that autocrine/paracrine signaling through P2X7-dependent ATP release and activation of P2Y6 and P2Y12 receptors serves to amplify the cellular response to DNA damage caused by γ-irradiation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Ionizing radiation induces biological effects not only in irradiated cells but also in non-irradiated cells, which is called the bystander effect. Recently, in vivo and in vitro experiments have suggested that both gap junction hemichannel connexin43 (Cx43) and extracellular adenosine triphosphate (ATP) released from cells play a role in the bystander effect. We have reported that γ-irradiation induces ATP release from B16 melanoma cells, which is dependent on the P2X(7) receptor. However, the mechanism of ATP release caused by irradiation remains unclear. We here show the involvement of Cx43 in P2X(7) receptor-dependent ATP release after 0.5 Gy γ-irradiation. Inhibitors of gap junction hemichannels and an inhibitory peptide for Cx43 (gap26), but not an inhibitory peptide for pannexin1 (Panx1), significantly blocked γ-irradiation-induced ATP release from B16 melanoma cells. We confirmed high expression of Cx43 mRNA in B16 melanoma cells. These results suggest involvement of Cx43 in radiation-induced ATP release. We found that after 0.5 Gy γ-irradiation tyrosine phosphorylation was significantly blocked by P2X(7) receptor antagonist, but not gap26, suggesting that tyrosine phosphorylation is a downstream event from the P2X(7) receptor. Since tyrosine kinase inhibitor significantly suppressed radiation-induced ATP release, tyrosine phosphorylation appears to play an important role in the Cx43-mediated ATP release downstream of the P2X(7) receptor. In conclusion, the Cx43 hemichannel, which lies downstream of the P2X(7) receptor, is involved in ATP release in response to radiation. Our results suggest a novel mechanism for radiation-induced biological effects mediated by both ATP and Cx43.

We examined the receptor-mediated effects of extracellular ATP on neuronal differentiation of PC12 cells, Neuro2a cells and MEB5 cells by using a series of receptor antagonists. The P2Y13 receptor antagonist MRS2211 significantly accelerated neurite outgrowth in all cases. Treatment with nerve growth factor (NGF) alone activated ERK1/2 in PC12 cells, and the activation was further increased by MRS2211. These results suggest involvement of P2Y13 receptor in suppression of neuronal differentiation. Thus, P2Y13 receptor antagonists might be candidates for treatment of neurodegenerative diseases., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Activation of the P2X7 receptor of macrophages plays an important role in inflammation. We recently reported that co-expression of P2X4 receptor with P2X7 receptor facilitates P2X7 receptor-mediated cell death via Ca(2+) influx. However, it remained unclear whether P2X4 receptor is involved in P2X7 receptor-mediated inflammatory responses, such as cytokine production. Here, we present evidence that P2X4 receptor modulates P2X7 receptor-dependent inflammatory functions. Treatment of mouse macrophage RAW264.7 cells with 1mM ATP induced high mobility group box 1 (HMGB1) release and IL-1β production via activation of P2X7 receptor. Knockdown of P2X4 receptor or removal of extracellular Ca(2+) suppressed ATP-induced release of both HMGB1 and IL-1β. On the other hand, knockdown of P2X4 receptor or removal of extracellular Ca(2+) enhanced P2X7-dependent LC3-II expression (an index of autophagy), suggesting that P2X4 receptor suppresses P2X7-mediated autophagy. Since LC3-II expression was inhibited by pretreatment with antioxidant and NADPH oxidase inhibitor, we examined P2X7-mediated production of reactive oxygen species (ROS). We found that activation of P2X7 receptor-mediated production of ROS was significantly facilitated in P2X4-knockdown cells, suggesting that co-expression of P2X4 receptor with P2X7 receptor may suppress anti-inflammatory function-related autophagy via suppression of ROS production. We conclude that co-expression of P2X4 receptor with P2X7 receptor enhances P2X7-mediated inflammation through both facilitation of release of cytokines and suppression of autophagy., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Interaction of P2X7 receptor with P2X4 receptor has recently been suggested, but it remains unclear whether P2X4 receptor is involved in P2X7 receptor-mediated events, such as cell death of macrophages induced by high concentrations of extracellular ATP. Here, we present evidence that P2X4 receptor does play a role in P2X7 receptor-dependent cell death. Treatment of mouse macrophage RAW264.7 cells with 1mM ATP induced Ca(2+) influx, non-selective large pore formation, activation of extracellular signal-regulated protein kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK), and cell death via activation of P2X7 receptor. P2X4-knockdown cells, established by transfecting RAW264.7 cells with two short hairpin RNAs (shRNAs) targeting P2X4 receptor, showed a decrease of the initial peak of intracellular Ca(2+) after treatment with ATP, though pore formation and the P2X7-mediated activation of ERK1/2 and p38 MAPK were not affected. Intriguingly, P2X4 knockdown resulted in significant suppression of cell death induced by ATP or P2X7 agonist BzATP. In conclusion, our results suggest that P2X4 receptor is involved in P2X7 receptor-mediated cell death, but not pore formation or MAPK signaling., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Natural products obtained from marine invertebrates such as sponges and tunicates are attractive sources of drugs. However, a critical obstacle in the development of these compounds is the problem of supply. In most cases, neither chemical synthesis nor mariculture of invertebrates is economically feasible. Due to structural similarities, many marine natural products are suspected to be produced by associated microorganisms. A favorable strategy for the production of such compounds is to use culturable microorganisms. Here we report that didemnin B, a tunicate-derived depsipeptide, has been isolated from a culturable bacterium, Tistrella mobilis YIT 12409.

We previously showed that low doses (0.25-0.5 Gy) of γ-rays elevated thioredoxin (Trx-1) in various organs of mice after whole-body irradiation. Also, it is reported that extracellular ATP, which is released in response to various stresses, regulates the expression of intracellular antioxidants through activation of P2 receptors. We have recently found that low-dose γ-rays induce ATP release from the exposed cells. However, it is not yet clear whether the radiation-induced extracellular ATP modulates the cellular redox balance. Here, we investigated whether γ-ray irradiation-induced release of extracellular ATP contributes to the induction of the cellular antioxidant Trx-1, using mouse macrophage-like RAW264.7 cells. Irradiation with γ-rays or exogenously added ATP increased the expression of Trx-1, and in both cases the increase was blocked by pretreatment with an ectonucleotidase, apyrase. Then, the involvement of ATP-dependent reactive oxygen species (ROS) generation in the increase in antioxidant capacity was examined. ATP stimulation promoted the generation of intracellular ROS and also increased Trx-1 expression. The increase in Trx-1 expression was significantly suppressed by pretreatment of the cells with antioxidants. In conclusion, the γ-ray irradiation-induced release of extracellular ATP may, at least in part, contribute to the production of ROS via purinergic signaling, leading to promotion of intracellular antioxidants as an adaptive response to an oxidative stress., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Raloxifene is extensively glucuronidated in humans, effectively reducing its oral bioavailability (2%). It was also reported to be glucuronidated in preclinical animals, but its effects on the oral bioavailability have not been fully elucidated. In the present study, raloxifene and its glucuronides in the portal and systemic blood were monitored in Gunn rats deficient in UDP-glucuronosyltransferase (UGT) 1A, Eisai hyperbilirubinemic rats (EHBRs), which hereditarily lack multidrug resistance-associated protein (MRP) 2, and wild-type rats after oral administration. The in vitro-in vivo correlation (IVIVC) of four UGT substrates (raloxifene, biochanin A, gemfibrozil, and mycophenolic acid) in rats was also evaluated. In Gunn rats, the product of fraction absorbed and intestinal availability and hepatic availability of raloxifene were 0.63 and 0.43, respectively; these values were twice those observed in wild-type Wistar rats, indicating that raloxifene was glucuronidated in both the liver and intestine. The ratio of glucuronides to unchanged drug in systemic blood was substantially higher in EHBRs (129-fold) than in the wild-type Sprague-Dawley rats (10-fold), suggesting the excretion of raloxifene glucuronides caused by MRP2. The IVIVC of the other UGT substrates in rats displayed a good relationship, but the oral clearance values of raloxifene and biochanin A, which were extensively glucuronidated by rat intestinal microsomes, were higher than the predicted clearances using rat liver microsomes, suggesting that intestinal metabolism may be a great contributor to the first-pass effect. Therefore, evaluation of intestinal and hepatic glucuronidation for new chemical entities is important to improve their pharmacokinetic profiles.

Extracellular adenosine activates P1 receptors (A(1), A(2A), A(2B), A(3)) on cellular membranes. Here, we investigated the involvement of P1 receptor-mediated signaling in differentiation to regulatory T cells (Treg). Treg were induced in vitro by incubating isolated CD4(+)CD62L(+) naïve murine T cells under Treg-skewing conditions. Antagonists of A(1) and A(2B) receptors suppressed the expression of Foxp3, a specific marker of Treg, and the production of IL-10, suggesting the involvement of A(1) and A(2B) receptors in differentiation to Treg. We also investigated the effect of these antagonists on T cell activation, which is essential for differentiation to Treg, and found that A(1) antagonist, but not A(2B) antagonist, suppressed T cell activation. We conclude that A(1) and A(2B) receptors are both involved in differentiation to Treg, but through different mechanisms. Since A(2B) antagonist blocked differentiation to Treg without suppressing T cell activation, it is possible that blockade of A(2B) receptor would facilitate tumor immunity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The potent pro-inflammatory cytokine, interleukin-1β (IL-1β), is synthesized as an inactive 33-kDa precursor (pro-IL-1β) and is processed by caspase 1 into the bioactive 17-kDa mature form. The P2X7 receptor, an ATP-gated cation channel, plays an essential role in caspase 1 activation, production and release of mature bioactive 17-kDa form. We recently reported ATP induces the release of an unconventional 20-kDa form of IL-1β (p20-IL-1β) from lipopolysaccharide-primed microglial cells. Emerging evidence suggests physiological relevance for p20-IL-1β; however, the underlying mechanisms for its production and release remain unknown. Here, we investigated the pathways involved in the ATP-induced production of p20-IL-1β using lipopolysaccharide-primed mouse microglial cells. The activation of P2X7 receptor by ATP triggered p20-IL-1β production under acidic extracellular conditions. ATP-induced p20-IL-1β production was blocked by pepstatin A, a potent inhibitor of the lysosomal protease, cathepsin D. The removal of extracellular Ca(2+) inhibited the p20-IL-1β production as well as ATP-induced cathepsin D release via lysosome exocytosis. The acidic extracellular pH also facilitated the dilatation of membrane pore after ATP stimulation. Since facilitation of pore dilatation results in cytolysis accompanied with cytoplasmic pro-IL-1β leakage, our data suggest the leaked pro-IL-1β is processed into p20-IL-1β by cathepsin D released after ATP stimulation under acidic extracellular conditions., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Ultraviolet B (UVB) radiation induces inflammation in human skin. Extracellular nucleotides are released from cells in response to various stimuli and act as intercellular signaling molecules through activation of P2 receptors. In this study, we investigated the involvement of extracellular nucleotides and P2 receptors in UVB-radiation-induced inflammation using human keratinocyte-derived HaCaT cells. UVB radiation induced rapid ATP release from HaCaT cells; this was inhibited by pretreatment with anion transporter blockers or maxi-anion channel blockers. In addition, the radiation-induced activation of p38 MAPK was significantly blocked by pretreatment with ecto-nucleotidase (apyrase) or P2Y6 receptor antagonist (MRS2578). Expression of COX-2, mediated by activation of p38 MAPK, was also induced by UVB radiation. Both pretreatment with MRS2578 and knockdown of the P2Y6 receptor by siRNA transfection attenuated the induction of COX-2 in HaCaT cells exposed to UVB radiation. Our results indicate that UVB radiation evokes ATP release from human keratinocytes and also that activation of P2Y6 receptor mediates the UVB-radiation-induced activation of p38 MAPK and expression of COX-2. Thus P2Y6 receptor is a mediator of UVB-radiation-induced inflammatory responses in keratinocytes.

The EGF receptor (EGFR) is frequently expressed in tumors of epithelial origin. Although it is well known that ionizing radiation induces activation of EGFR, the mechanism remains unknown. Recently, we reported that activation of P2Y receptors is involved in γ-radiation-induced activation of extracellular signal-regulated kinase1/2 (ERK1/2), which is dependent on activation of EGFR. Here we focused on the mechanism of activation of EGFR in response to low-dose γ radiation, mainly in terms of the activation-associated formation of EGFR foci in A549 cells. Irradiation of cells with 0.1 Gy γ rays induced biphasic phosphorylation of EGFR and ERK1/2 as well as biphasic formation of EGFR foci. The radiation-induced focus formation of EGFR was abolished by ecto-nucleotidase, P2Y receptor antagonists and knockdown of P2Y6 receptor, suggesting the involvement of extracellular nucleotides and activation of P2Y6 receptors in this process. Further, a disintegrin and metalloprotease 17 (ADAM17) is expressed in A549 cells and an ADAM17 inhibitor significantly blocked the radiation-induced focus formation of EGFR. We conclude that activation of both P2Y6 receptors and ADAM17 mediates the low-dose γ-radiation-induced activation of EGFR, as evaluated in terms of focus formation, in A549 cells.

Long-term treatment with minocycline is known to induce pigmentation or discoloration in tissues but how remains unclear. We investigated the mechanism of pigmentation using B16 melanoma cells. First, we confirmed that intracellular melanin levels increased on minocycline treatment. Then, using the reverse transcriptase-polymerase chain reaction (RT-PCR), we found the expression of mRNA of tyrosinase, tyrosinase-related protein (TRP)-1 and TRP-2, to also be significantly increased by treatment with minocycline at 5 µg/ml for 72 h. These results suggest that the minocycline-induced stimulation of melanogenesis occurs at the transcriptional level. Western-blotting revealed slight phosphorylation of extracellular signal-regulated kinase (ERK) 30-60 min after the minocycline treatment. The mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 and the p38 inhibitor SB203580 were used to examine the signaling pathway associated with the mRNA expression of tyrosinase, TRP-1, or TRP-2 when B16 melanoma cells were treated with minocycline. The SB203580 inhibited the mRNA expression of tyrosinase and TRP-1, suggesting the minocycline-induced melanogensis occurred via a p38 signaling pathway.

It has recently been reported that activation of P2Y(1) receptor, one of the purine receptors, by extracellular nucleotides induces cytoprotection against oxidative stress. In this study, we examined the protective effect of ATP on the cell damage in human epidermal keratinocyte HaCaT cells exposed to H(2)O(2) via the P2Y receptor-mediated induction of intracellular antioxidants. The cells were damaged by exposure to H(2)O(2) in a dose- and time-dependent manner. The damage induced by 7.5 mM H(2)O(2) was blocked by pretreatment of the cells with ATP (1-10 µM). The protective effect of ATP was significantly reduced by P2Y receptor antagonists. Exogenously added ATP induced various intracellular antioxidants, including thiol-containing proteins, Cu/Zn superoxide dismutase (SOD) and thioredoxin-1, in HaCaT cells. In conclusion, it was found that ATP protected the cells from the H(2)O(2)-induced cell damages via the P2Y receptor-mediated induction of intracellular antioxidants.

Background: Bacterial meningitis is characterized by a marked predominance of polymorphonuclear leukocytes (PMNs: segmented granulocytes or neutrophils) in the cerebrospinal fluid (CSF), whereas aseptic meningitis is characterized by a predominance of mononuclear leukocytes (MNs: lymphocytes or monocytes). However, the pathophysiology of this predominance of PMNs in the CSF of patients with bacterial meningitis has never, to our knowledge, been clearly described., Methods: To investigate the predominant cell components of CSF from pediatric patients with bacterial meningitis, we investigated cell death in association with levels of tumor necrosis factor-alpha (TNF-α) in the CSF, using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay and flow cytometry., Results: The MTT assay of the CSF revealed that the PMNs had survived, while the MNs rapidly had undergone cell death. Although PMNs survived in CSF with high levels of TNF-α, PMN apoptosis was demonstrated with flow cytometry., Conclusions: The present study suggests that the pathophysiology of PMN predominance in the CSF of patients in the acute phase of bacterial meningitis is related to the rapid cell death of MNs and the survival of PMNs brought about by high levels of TNF-α.

We discovered that C-glucosides 4 bearing a heteroaromatic ring formed metabolically more stable inhibitors for sodium-dependent glucose cotransporter 2 (SGLT2) than the O-glucoside, 2 (T-1095). A novel thiophene derivative 4b-3 (canagliflozin) was a highly potent and selective SGLT2 inhibitor and showed pronounced anti-hyperglycemic effects in high-fat diet fed KK (HF-KK) mice.

We recently reported that repeated 0.5-Gy gamma irradiation attenuates the pathology of collagen-induced arthritis. In this study, to investigate the mechanism further, we focused on changes in Treg/Th17 cells and changes in the production of antibody against an external antigen in response to gamma irradiation as well as on the radiosensitivity of Treg cells. DBA/1J mice were immunized with type II collagen to induce arthritis and exposed to low-dose gamma rays (0.5 Gy/week for 5 weeks). Production of IL6 and IL17 as well as autoantibody was suppressed by irradiation in the early phase of collagen-induced arthritis. The percentage of Treg cells was significantly increased by irradiation at 4, 6 and 8 weeks after the immunization. We also investigated the effect of repeated gamma radiation on the production of antibodies against an external antigen in ovalbumin-immunized BALB/c mice. We found that repeated 0.5-Gy gamma irradiation enhanced antibody production, accompanied by an increase of the antibody-producing plasma cell population and increased Th2-type cytokine secretion. We also found that the radiosensitivity of Treg cells did not differ from that of other T cells. These results suggest that a major mechanism of attenuation of the pathology of collagen-induced arthritis by repeated 0.5-Gy gamma irradiation is up-regulation of Treg cells concomitantly with suppression of IL6 and IL17 production.

Recent reports have shown that T cell receptor (TCR)-dependent ATP release from T cells is involved in production of interleukin-2 (IL-2) through activation of P2 receptors. Stimulation of TCR induces ATP release from T cells through gap junction hemichannels and maxianion channels, at least in part. However, the mechanisms of ATP release from activated T cells are not fully understood. Here, we studied the mechanisms of ATP release during TCR-dependent T cell activation by investigating the effects of various inhibitors on TCR-dependent ATP release from murine T cells. We found that not only anion channel and gap junction hemichannel inhibitors, but also exocytosis inhibitors suppressed the ATP release. These results suggest that ATP release from murine T cells is regulated by various mechanisms, including exocytosis. An inhibitor of exocytosis, bafilomycin A, significantly blocked TCR signaling, such as Ca(2+) elevation and IL-2 production. Furthermore, bafilomycin A, ectonucleotidase, and P2Y(6) receptor antagonist significantly inhibited production of pro-inflammatory cytokines from external antigen-restimulated splenocytes, indicating that vesicular exocytosis-mediated purinergic signaling has a significant role in TCR-dependent cytokine production. We also detected vesicular ATP in murine T cells and human T lymphoma Jurkat cells, both of which also expressed mRNA of SLC17A9, a vesicular nucleotide transporter. Knockdown of SLC17A9 in Jurkat cells markedly reduced ATP release and cytosolic Ca(2+) elevation after TCR stimulation, suggesting involvement of SLC17A9-dependent vesicular exocytosis in ATP release and T cell activation. In conclusion, vesicular exocytosis of ATP appears to play a role in T cell activation and immune responses.

Recent studies have suggested a bystander effect in nonirradiated cells adjacent to irradiated cells; however, the mechanism is poorly understood. In this study, we investigated the involvement of both extracellular nucleotides and activation of P2 receptors in cellular responses to gamma radiation using human HaCaT keratinocytes. The concentration of ATP in culture medium was increased after gamma irradiation (0.1-1.0 Gy), suggesting that radiation induces ATP release from cells. Intracellular Ca(2+) concentration was elevated when conditioned medium from irradiated cells was transferred to nonirradiated cells, and this elevation was suppressed by apyrase (ecto-nucleotidase), indicating the involvement of extracellular nucleotides in this event. Further, we examined the activation of ERK1/2 by gamma radiation and nucleotides (ATP and UTP). Both gamma radiation and nucleotides induced activation of ERK1/2. Next, the effect of inhibitors of P2 receptors on radiation-induced activation of ERK1/2 was examined. The activation of ERK1/2 was blocked by suramin (P2Y inhibitor), MRS2578 (P2Y(6) antagonist) and apyrase. These results suggest that both released nucleotides and activation of P2Y receptors are involved in gamma-radiation-induced activation of ERK1/2. We conclude that ionizing radiation induces release of nucleotides from cells, leading to activation of P2Y receptors, which in turn would result in a variety of biological effects.

The transcription factor nuclear erythroid-derived 2-related factor 2 (Nrf2) regulates expression of genes encoding antioxidant proteins involved in cellular redox homeostasis, while gamma-ray irradiation is known to induce reactive oxygen species in vivo. Although activation of Nrf2 by various stresses has been studied, it has not yet been determined whether ionizing irradiation induces activation of Nrf2. Therefore, we investigated activation of Nrf2 in response to gamma-irradiation in mouse macrophage RAW264.7 cells. Irradiation of cells with gamma-rays induced an increase of Nrf2 expression. Even 0.1 Gy of gamma-irradiation induced a translocation of Nrf2 from cytoplasm to the nucleus, indicating the activation of Nrf2 by low-dose irradiation. Expression of heme oxygenase-1, which is regulated by Nrf2, was also increased at 24 h after irradiation with more than 0.1 Gy of gamma-rays. Furthermore, the activation of Nrf2 was suppressed by U0126, which is an inhibitor of the extracellular signal regulated protein kinase 1/2 (ERK1/2) pathway, suggesting involvement of ERK1/2-dependent pathway in the irradiation-induced activation of Nrf2. Our results indicate that low-dose gamma-irradiation induces activation of Nrf2 through ERK1/2-dependent pathways.

Background: Ionizing irradiation causes not only growth arrest and cell death, but also release of growth factors or signal transmitters, which promote cancer malignancy. Extracellular ATP controls cancer growth through activation of purinoceptors. However, there is no report of radiation-induced ATP release from cancer cells. Here, we examined gamma-irradiation-induced ATP release and its mechanism in B16 melanoma., Methods: Extracellular ATP was measured by luciferin-luciferase assay. To investigate mechanism of radiation-induced ATP release, we pharmacologically inhibited the ATP release and established stable P2X(7) receptor-knockdown B16 melanoma cells using two short hairpin RNAs targeting P2X(7) receptor., Results: Cells were exposed to 0.5-8 Gy of gamma-rays. Extracellular ATP was increased, peaking at 5 min after 0.5 Gy irradiation. A selective P2X(7) receptor channel antagonist, but not anion transporter inhibitors, blocked the release of ATP. Further, radiation-induced ATP release was significantly decreased in P2X(7) receptor-knockdown cells. Our results indicate that gamma-irradiation evokes ATP release from melanoma cells, and P2X(7) receptor channel plays a significant role in mediating the ATP release., General Significance: We suggest that extracellular ATP could be a novel intercellular signaling molecule released from cancer cells when cells are exposed to ionizing radiation.

We investigated the protective effect of several heterocyclic pyrimidine compounds against ultraviolet B (UVB)-induced damage in human keratinocyte HaCaT cells, as well as the inhibitory effect on melanogenesis in B16 melanoma cells. One of the compounds examined, 2-piperadino-6-methyl-5-oxo-5,6-dihydro(7H)pyrrolo[3,4d]pyrimidine maleate (MS-818), showed low cytotoxicity even at 500 microM. At 50-500 microM, MS-818 dose-dependently suppressed the UVB (100 mJ/cm(2))-induced elevation of tumor necrosis factor alpha (TNF-alpha), one of the trigger cytokines for cell death, in HaCaT cells. In addition, MS-818 (100 microM) markedly inhibited melanogenesis in B16 melanoma cells via downregulation of tyrosinase expression mediated by microphthalmia-associated transcription factor (MITF) and extracellular signal-regulated kinase (ERK). In conclusion, MS-818 protected epidermal cells from UVB-induced damage and also suppressed melanogenesis in melanoma cells. It appears to be a good candidate for a new UVB-protective and whitening agent for application in cosmetics.

Diesel exhaust particles (DEP), a well-known air pollutant, exacerbate type I hypersensitivity conditions, such as asthma and pollen allergy. In this study, we examined the effect of diesel exhaust (DE) exposure on delayed-type hypersensitivity (DTH), a type IV hypersensitivity, induced with methyl-bovine serum albumin (mBSA) in C57BL/6 mice. Mice were exposed to DE containing DEP at a dose of 1.78 mg/m(3) in an inhalation chamber for 14 days. On Day 7, DTH mice and DE-exposed DTH mice were injected s.c. with 200 microl of 1.25 mg/ml mBSA emulsified with CFA in the dorsal region as initial sensitization. On Day 14, mice were injected s.c. into one footpad with 20 microl of 10 mg/ml mBSA dissolved in PBS as challenge. On Day15, footpad thickness and spleen weight were measured. Significant footpad swelling (%) was observed in DTH mice compared with normal control mice, and this swelling was significantly augmented by DE exposure. The levels of pro-inflammatory cytokines, including IFN-gamma, TNF-alpha, and IL-6, in DTH mice were significantly higher than in normal mice, and were also further enhanced by DE exposure. DE exposure increased production of IL-17, which enhances local tissue inflammation through up-regulation of pro-inflammatory cytokines, while production of IL-10, which inhibits local tissue inflammation through suppression of immune cell proliferation, was unchanged. No change was observed in the percentage of CD4(+)CD25(+)Foxp3(+)T regulatory (Treg) cells in splenic lymphocytes following DE exposure. IL-6 production was increased by DE, and this would facilitate the differentiation of naïve T cells to IL-17-producing Th17 cells, while concomitantly suppressing the competing differentiation pathway to IL-10-producing Treg cells. Our results indicate that DE inhalation may, in part, exacerbate the pathological symptoms of DTH and induction of pro-inflammatory cytokines such as IFN-gamma, TNF-alpha, IL-6 and IL-17.

Extracellular nucleotides and their metabolites activate ionotropic P2X and metabotropic P2Y receptors on the surface of various types of cells. Here, we investigated the involvement of P2X and P2Y receptor-mediated signaling in TCR-dependent T cell activation. Murine T cells were activated by stimulation of TCR, and both CD25 expression and interleukin (IL)-2 production were observed in activated T cells. Ecto-nucleotidase apyrase and P2Y6 antagonist MRS2578 significantly blocked the increases of both CD25 expression and IL-2 production, and P2X7 antagonists A438079 and oxidized ATP inhibited IL-2 production rather than CD25 expression, suggesting the involvement of P2Y6 and P2X7 receptors in different processes of T cell activation. MRS2578 also blocked TCR-dependent elevation of cytosolic Ca2+ in T cells. The P2X7 and P2Y6 receptors were expressed in murine CD4 T cells. In conclusion, our results indicate that activation of P2Y6 and P2X7 receptors contributes to T cell activation via TCR.

Expansion and commercialization of nanotechnology mean that it is important to understand the potential health hazards of manufactured nanoparticles. Here, we focused on the effect of fullerene, a type of nanoparticle already in commercial use, on delayed-type hypersensitivity (DTH) induced by methyl-bovine serum albumin (mBSA). Delayed-type hypersensitivity was induced with methyl-bovine serum albumin in female C57BL/6 mice. A colloidal suspension of crystalline C(60) (nano-C(60); average particle size 165 nm; 200 microL; 5.5 microg/mL) was injected intravenously twice, just before immunization and challenge with mBSA. Nano-C(60) treatment significantly attenuated footpad swelling, compared with that in DTH-disease control mice. Cytokine analysis indicated that nano-C(60) treatment switched the cytokine balance towards Th1-dominance. Pro-inflammatory cytokines IL-6 and IL-17 were significantly increased in DTH mice, and these increases were significantly suppressed by nano-C(60) treatment. Suppression of IL-17 by nano-C(60) was confirmed in an in vitro splenocyte culture. However, production of TNF-alpha was increased in DTH mice, and the increase was significantly enhanced by nano-C(60) treatment. The ratio of regulatory T (Treg) cells to total T (CD4+) cells was also significantly increased by nano-C(60) treatment, compared with that in DTH-disease control mice. Nano-C(60) treatment showed significant immunomodulatory effects in a mouse DTH model: IL-6 and IL-17 production was down-regulated, and the Treg cell ratio was up-regulated, concomitantly with attenuation of the pathology of DTH.

Recently, autophagy has been associated with the TLR signaling pathway to eliminate intracellular pathogens in the innate immune system. However, it is unknown if other pathways regulate autophagy during the immunologic response. Given the critical role of the purinergic P2X7 receptor (P2X7R) pathway during various immunologic functions (i.e., caspase activation and IL-1beta secretion), the principal objective here was to determine whether the P2X7R pathway may regulate autophagy in immune cells. We observed in both MG6 mouse microglial cells and primary microglia that activation of P2X7R by ATP increases the expression of microtubule-associated protein 1 light chain 3 (LC3)-II, the autophagosomal membrane-associated form of LC3, in an extracellular Ca(2+)-dependent manner. Consistent with this, immunohistochemistry showed extensive formation of LC3-immunopositive dots, and electron microscopy demonstrated accumulation of autophagosomes and autophagolysosomes in ATP-treated cells. Importantly, the up-regulation of LC3-II by P2X7R activation was not affected by autophagy inhibitors, such as 3-methyladenine and PI3K inhibitors. Furthermore, while lysosomal functions were impaired by ATP treatment, autophagolysosomal components were released into the extracellular space. Similarly, a phagocytosis assay using Escherichia coli BioParticles showed that phagosome maturation was impaired in ATP-treated cells and a robust release of LC3-immunopositive phagolysosomes was induced along with a radial extension of microtubule bundles. Taken together, the data suggest a novel mechanism whereby the P2X7R signaling pathway may negatively regulate autophagic flux through the impairment of lysosomal functions, leading to stimulation of a release of autophagolysosomes/phagolysosomes into the extracellular space.

Low- or intermediate-dose gamma radiation appears to have the capacity to ameliorate certain types of diseases, including allergic conditions, when examined under specific exposure conditions and with specific animal models, though the molecular mechanisms involved remain to be fully clarified. We investigated the anti-inflammatory effects of intermediate-dose gamma radiation by examining its effects on the activation state of p38 MAPK and the production of cytokines in mouse macrophage RAW264.7 cells. Dephosphorylation of both ERK1/2 and p38 MAPK was observed at 15 min after irradiation (0.5-1 Gy from a (137)Cs source) concomitant with a significant increase in the expression of MKP-1, which dephosphorylates ERK1/2 and p38 MAPK. Since activated p38 MAPK mediates TNF-alpha production, we examined the effect of radiation on LPS-induced activation of p38 MAPK and TNF-alpha production. The activation of p38 MAPK and production of TNF-alpha induced by LPS treatment were both suppressed in preirradiated cells. LPS-induced production of TNF-alpha was enhanced by knockdown of MKP-1. These results indicate that 0.5 Gy gamma radiation would cause up-regulation of MKP-1, leading to inactivation of p38 MAPK and suppression of TNF-alpha production, in cells of mouse macrophages cell line.