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Death receptors 4 and 5 activate Nox1 NADPH oxidase through riboflavin kinase to induce reactive oxygen species-mediated apoptotic cell death.

ABSTRACT: Stimulation of the proapoptotic tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, death receptors 4 (DR4) and 5 (DR5), conventionally induces caspase-dependent apoptosis in tumor cells. Here we report that stimulation of DR4 and/or DR5 by the agonistic protein KD548-Fc, an Fc-fused DR4/DR5 dual-specific Kringle domain variant, activates plasma membrane-associated Nox1 NADPH oxidase to generate superoxide anion and subsequently accumulates intracellular reactive oxygen species (ROS), leading to sustained c-Jun N-terminal kinase activation and eventual apoptotic cell death in human HeLa and Jurkat tumor cells. KD548-Fc treatment induces the formation of a DR4/DR5 signaling complex containing riboflavin kinase (RFK), Nox1, the Nox1 subunits (Rac1, Noxo1, and Noxa1), TNF receptor-associated death domain (TRADD), and TNF receptor-associated factor 2 (TRAF2). Depletion of RFK, but not the Nox1 subunits, TRADD and TRAF2, failed to recruit Nox1 and Rac1 to DR4 and DR5, demonstrating that RFK plays an essential role in linking DR4/DR5 with Nox1. Knockdown studies also reveal that RFK, TRADD, and TRAF2 play critical, intermediate, and negligible roles, respectively, in the KD548-Fc-mediated ROS accumulation and downstream signaling. Binding assays using recombinantly expressed proteins suggest that DR4/DR5 directly interact with cytosolic RFK through RFK-binding regions within the intracellular death domains, and TRADD stabilizes the DR4/DR5-RFK complex. Our results suggest that DR4 and DR5 have a capability to activate Nox1 by recruiting RFK, resulting in ROS-mediated apoptotic cell death in tumor cells.

SUBMITTER: Park KJ

PROVIDER: S-EPMC3270986 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Bioaccumulative organohalogen chemicals, such as organochlorine (OC) insecticides, have been increasingly associated with disease etiology; however, the mechanistic link between chemical exposure and diseases, such as atherosclerosis, cancer, and diabetes, is complex and poorly defined. Systemic oxidative stress stemming from OC exposure might play a vital role in the development of these pathologies. Monocytes are important surveillance cells of the innate immune system that respond to extracellular signals possessing danger-associated molecular patterns by synthesizing oxyradicals, such as superoxide, for the purpose of combating infectious pathogens. We hypothesized that OC chemicals can be toxic to monocytes because of an inappropriate elevation in superoxide-derived reactive oxygen species (ROS) capable of causing cellular oxidative damage. Reactive oxyradicals are generated in monocytes in large part by NADPH oxidase (Nox). The present study was conducted to examine the ability of two chlorinated cyclodiene compounds, trans-nonachlor and dieldrin, as well as p,p'-DDE, a chlorinated alicyclic metabolite of DDT, to stimulate Nox activity in a human monocytic cell line and to elucidate the mechanisms for this activation. Human THP-1 monocytes treated with either trans-nonachlor or dieldrin (0.1-10 μM in the culture medium) exhibited elevated levels of intracellular ROS, as evidenced by complementary methods, including flow cytometry analysis using the probe DCFH-DA and hydroethidine-based fluorometric and UPLC-MS assays. In addition, the induced reactive oxygen flux caused by trans-nonachlor was also observed in two other cell lines, murine J774 macrophages and human HL-60 cells. The central role of Nox in OC-mediated oxidative stress was demonstrated by the attenuated superoxide production in OC-exposed monocytes treated with the Nox inhibitors diphenyleneiodonium and VAS-2870. Moreover, monocytes challenged with OCs exhibited increased phospho-p47(phox) levels and enhanced p47(phox) membrane localization compared to that in vehicle-treated cells. p47(phox) is a cytosolic regulatory subunit of Nox, and its phosphorylation and translocation to the NOX2 catalytic subunit in membranes is a requisite step for Nox assembly and activation. Dieldrin and trans-nonachlor treatments of monocytes also resulted in marked increases in arachidonic acid (AA) and eicosanoid production, which could be abrogated by the phospholipase A2 (PLA2) inhibitor arachidonoyltrifluoromethyl ketone (ATK) but not by calcium-independent PLA2 inhibitor bromoenol lactone. This suggested that cytosolic PLA2 plays a crucial role in the induction of Nox activity by increasing the intracellular pool of AA that activates protein kinase C, which phosphorylates p47(phox). In addition, ATK also blocked OC-induced p47(phox) serine phosphorylation and attenuated ROS levels, which further supports the notion that the AA pool liberated by cytosolic PLA2 is responsible for Nox activation. Together, the results suggest that trans-nonachlor and dieldrin are capable of increasing intracellular superoxide levels via a Nox-dependent mechanism that relies on elevated intracellular AA levels. These findings are significant because chronic activation of monocytes by environmental toxicants might contribute to pathogenic oxidative stress and inflammation.

Project description:Diesel exhaust particles (DEPs) are a main contributor to air pollution. Ultrafine DEPs can cause neurodegenerative diseases by increasing intracellular reactive oxygen species (ROS). Compared with other cells in the brain, oligodendrocytes responsible for myelination are more susceptible to oxidative stress. However, the mechanisms underlying ROS generation in oligodendrocytes and the susceptibility of oligodendrocytes to ROS by ultrafine DEPs remain unclear. Herein, we examined the effects of excessive ROS generated by NOX2, an isoform of the NADPH oxidase family, after exposure to ultrafine DEPs (200 μg/mL) on the survival of two types of oligodendrocytes-oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes (mOLs)--isolated from the brain of neonatal rats. In addition, mice were exposed to ultrafine DEP suspension (20 μL, 0.4 mg/mL) via the nasal route for 1 week, after which the expression of NOX2 and cleaved caspase-3 was examined in the white matter of the cerebellum. Exposure to DEPs significantly increased NOX2 expression and ROS generation in OPCs and mOLs. OPCs and mOLs clearly exhibited viability reduction, and a significant change in p53, Bax, Bcl-2, and cleaved caspase-3 expression, after DEP exposure. In contrast, treatment with berberine (BBR), an NOX2 inhibitor, significantly mitigated these effects. In mice exposed to DEP, the presence of NOX2-positive and cleaved caspase-3-positive oligodendrocytes was demonstrated in the cerebellar white matter; NOX2 and cleaved caspase-3 expression in the cerebellum lysates was significantly increased. BBR treatment returned expression of these proteins to control levels. These results demonstrate that the susceptibility of OPCs and mOLs to ultrafine DEPs is, at least in part, caused by excessive ROS produced by NOX2 and the sequential changes in the expression of p53, Bax, Bcl-2, and cleaved caspase-3. Overall, NOX2 inhibitor enhances the survival of two types of oligodendrocytes.

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Death receptors 4 and 5 activate Nox1 NADPH oxidase through riboflavin kinase to induce reactive oxygen species-mediated apoptotic cell death.

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