Mitsutoshi Tsukimoto

The Activation of P2X7 Receptor Impairs Lysosomal Functions and Stimulates the Release of Autophagolysosomes in Microglial Cells

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-1β 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 Ca2+-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.

γ-Irradiation induces P2X7 receptor-dependent ATP release from B16 melanoma cells

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.

Involvement of SLC17A9-dependent vesicular exocytosis in the mechanism of ATP release during T cell activation.

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 Ca2+ elevation and IL-2 production. Furthermore, bafilomycin A, ectonucleotidase, and P2Y6 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 Ca2+ 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.

Autocrine Regulation of Macrophage Activation via Exocytosis of ATP and Activation of P2Y11 Receptor

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.

Regulation of P2X7-dependent inflammatory functions by P2X4 receptor in mouse macrophages.

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.

Autocrine regulation of TGF-β1-induced cell migration by exocytosis of ATP and activation of P2 receptors in human lung cancer cells.

Summary TGF-&bgr;1 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-&bgr;1-induced actin remodeling associated with cell migration. Treatment with TGF-&bgr;1 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-&bgr;1-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-&bgr;1-induced migration and actin remodeling. These results indicate the involvement of TGF-&bgr;1-induced ATP release in cell migration, at least in part, through activation of P2X7 receptors. TGF-&bgr;1 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-&bgr;1 failed to induce release of ATP from SLC17A9-knockdown cells. TGF-&bgr;1-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-&bgr;1-induced migration of lung cancer cells.

Involvement of P2X4 receptor in P2X7 receptor-dependent cell death of mouse macrophages.

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.

Involvement of Purinergic Signaling in Cellular Response to γ Radiation

Abstract 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 γ radiation using human HaCaT keratinocytes. The concentration of ATP in culture medium was increased after γ irradiation (0.1–1.0 Gy), suggesting that radiation induces ATP release from cells. Intracellular Ca2+ 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 γ radiation and nucleotides (ATP and UTP). Both γ 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 (P2Y6 antagonist) and apyrase. These results suggest that both released nucleotides and activation of P2Y receptors are involved in γ-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.

Repeated 0.5-Gy gamma irradiation attenuates autoimmune disease in MRL-lpr/lpr mice with suppression of CD3+CD4-CD8 -B220+ T-cell proliferation and with up-regulation of CD4+CD25+Foxp3+ regulatory T cells

Abstract Tago, F., Tsukimoto, M., Nakatsukasa, H. and Kojima. S. Repeated 0.5 Gy Gamma Irradiation Attenuates Autoimmune Disease in MRL-lpr/lpr Mice with Suppression of CD3+CD4−CD8−B220+ T-Cell Proliferation and with Up-regulation of CD4+CD25+Foxp3+ Regulatory T Cells. Radiat. Res. 169, 59–66 (2008). MRL-lpr/lpr mice are used as a model of systemic lupus erythematosus. We previously reported attenuation of autoimmune disease in MRL-lpr/lpr mice by repeated γ irradiation (0.5 Gy each time). In this study, we investigated the mechanisms of this attenuation by measuring the weight of the spleen and the population of highly activated CD3+CD4−CD8−B220+ T cells, which are characteristically involved in autoimmune pathology in these mice. Splenomegaly and an increase in the percentage of CD3+CD4−CD8−B220+ T cells, which occur with aging in nonirradiated mice, were suppressed in irradiated mice. The high proliferation rate of CD3+CD4−CD8−B220+ T cells was suppressed in the irradiated animals. The production of autoantibodies and the level of IL6, which activates B cells, were also lowered by radiation exposure. These results indicate that progression of pathology is suppressed by repeated 0.5-Gy γ irradiation. To uncover the mechanism of the immune suppression, we measured the regulatory T cells, which suppress activated T cells and excessive autoimmune responses. We found that regulatory T cells were significantly increased in irradiated mice. We therefore conclude that repeated 0.5-Gy γ irradiation suppresses the proliferation rate of CD3+CD4−CD8−B220+ T cells and the production of IL6 and autoantibodies and up-regulates regulatory T cells.

P2X4 receptor regulates P2X7 receptor-dependent IL-1β and IL-18 release in mouse bone marrow-derived dendritic cells

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.