Hikaru Sakamoto

Suppression of Leukotriene Formation in RBL-2H3 Cells That Overexpressed Phospholipid Hydroperoxide Glutathione Peroxidase

The overexpression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) by RBL-2H3 cells was used as the basis for an investigation of the effects of PHGPx on the formation of leukotrienes. The rates of production of leukotriene C4 (LTC4) and leukotriene B4(LTB4) in cells that overexpressed PHGPx were 8 times lower than those in a control line of cells. The reduction in rates of production of leukotrienes apparently resulted from the increase in the PHGPx activity since control rates of formation of leukotrienes could be achieved in PHGPx-overexpressing cells upon inhibition of PHGPx activity by diethyl malate. The conversion of radioactively labeled arachidonic acid to intermediates in the lipoxygenase pathway, such as 5- hydroxyeicosatetraenoic acid (5-HETE), LTC4, and LTB4, was strongly inhibited in PHGPx-overexpressing cells that had been prelabeled with [14C]arachidonic acid. PHGPx apparently inactivated the 5-lipoxygenase that catalyzed the conversion of arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) since 5-HPETE is a common precursor of 5-HETE, LTC4, and LTB4. The rates of formation of LTC4 and LTB4 in PHGPx-overexpressing cells returned to control rates upon the addition of a small amount of 12-HPETE. Flow cytometric analysis revealed that the rapid burst of formation of lipid hydroperoxides induced by A23187 was suppressed in PHGPx-overexpressing cells as compared with the control lines of cells. Subcellular fractionation analysis showed that the amount of PHGPx associated with nuclear fractions from PHGPx-overexpressing cells was 3.5 times higher than that from the control line of cells. These results indicate that PHGPx might be involved in inactivation of 5-lipoxygenase via reductions in levels of the fatty acid hydroperoxides that are required for the full activation of 5-lipoxygenase. Thus, in addition to its role as an antioxidant enzyme, PHGPx appears to have a novel function as a modulator of the production of leukotrienes.

Autocrine Regulation of UVA-Induced IL-6 Production via Release of ATP and Activation of P2Y Receptors

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.

Alteration of gene expressions by the overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (mtPHGPx).

To determine the effect on gene expression of trace levels of reactive oxygen species from mitochondria, we used the mRNA differential display technique to compare gene expression in two cell lines: M15, which overexpresses mitochondrial phospholipid hydroperoxide glutathione peroxidase (mtPHGPx), in rat basophilic leukemia RBL-2H3 cells, and a control cell line, S1. We isolated 27 differentially expressed genes, including 10 previously unreported sequences. These genes included cytoskeletal proteins (beta-tubulin, nonmuscle myosin alkali light chain, and vimentin), growth or proliferation regulators [growth differentiation factor 1 (Gdf-1), Rap1a, and inhibitor of growth 3 (Ing3)], and others. Although the expression of most of the isolated genes did not respond to ROS (hydrogen peroxide) or antioxidant (pyrolidine dithiocarbamate) treatment, the expression of Gdf-1 was downregulated by hydrogen peroxide treatment. Thus, low levels of ROS produced in mitochondria during normal cellular metabolism can modulate gene expression.

Calcium-dependent protein phosphorylation in morning glory hypocotyls

Abstract The membrane fraction isolated from hypocotyls of morning glory showed protein kinase activity, which catalysed phosphorylation of serine and threonine residues in the membrane proteins. The activity was stimulated by 0.1-1 μM Ca 2+ . Addition of calmodulin could not cause further stimulation, but reduced Ca 2+ -dependent protein kinase activity. The activity was restored by a calmodulin antagonist. The effect of calmodulin was not observed in phosphorylation of erogenous substrates by the protein kinase, suggesting that the function of an increased Ca 2+ -calmodulin complex may be to depress the phosphorylation of membrane proteins.

Net Increase of platelet membrane tyrosine specific-protein kinase activity by phorbol myristate acetate

Tyrosine protein kinase (TPK) activity in rabbit platelets after stimulation by phorbol myristate acetate (PMA) or thrombin was directly estimated by 32P incorporation from (gamma-32P)ATP (adenosine triphosphate) into synthetic peptide angiotensin II. By PMA-treatment a net increase of TPK activity was obtained, while thrombin acted on the TPK quickly but stimulation was limited within the range attained by the control after lengthy incubation. The responsive TPK to these stimulators was localized mainly in membrane but much less in cytosol. The specific activity of the particulate TPK was low in the sonicate of control ice cold platelets but increased about 6-fold when the platelets were incubated at 37 degrees C. On a brief contact of platelets with PMA at 37 degrees C the TPK was fully activated and reached a maximum value about 130% of the control. Determination of phosphotyrosine phosphatase in the stimulated platelet sonicate revealed that its participation in the above described increase of 32P-incorporation was meagre. The quick response suggested a possible role of TPK in the signal transduction through the platelet cell membrane.

Purinergic signaling mediates oxidative stress in UVA-exposed THP-1 cells

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 p67phox in membrane fraction and intracellular ROS. Pretreatment with ecto-nucleotidase or P2X7 receptor antagonist blocked the UVA-initiated membrane translocation of p67phox 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.