Reactive nitrogen species-induced cell death requires Fas-dependent activation of c-Jun N-terminal kinase.

Nitrogen dioxide is a highly toxic reactive nitrogen species (RNS) recently discovered as an inflammatory oxidant with great potential to damage tissues. We demonstrate here that cell death by RNS was caused by c-Jun N-terminal kinase (JNK). Activation of JNK by RNS was density dependent and caused mitochondrial depolarization and nuclear condensation. JNK activation by RNS was abolished in cells lacking functional Fas or following expression of a truncated version of Fas lacking the intracellular death domain. In contrast, RNS induced JNK potently in cells expressing a truncated version of tumor necrosis factor receptor 1 or cells lacking tumor necrosis factor receptor 1 (TNF-R1), illustrating a dependence of Fas but not TNF-R1 in RNS-induced signaling to JNK. Furthermore, Fas was oxidized, redistributed, and colocalized with Fas-associated death domain (FADD) in RNS-exposed cells, illustrating that RNS directly targeted Fas. JNK activation and cell death by RNS occurred in a Fas ligand- and caspase-independent manner. While the activation of JNK by RNS or FasL required FADD, the cysteine-rich domain 1 containing preligand assembly domain required for FasL signaling was not involved in JNK activation by RNS. These findings illustrate that RNS cause cell death in a Fas- and JNK-dependent manner and that this occurs through a pathway distinct from FasL. Thus, avenues aimed at preventing the interaction of RNS with Fas may attenuate tissue damage characteristic of chronic inflammatory diseases that are accompanied by high levels of RNS.

Baculovirus P35 inhibits NO-induced apoptosis in activated macrophages by inhibiting cytochrome c release.

The baculovirus protein P35 inhibits apoptosis in a diverse range of animals such as insects, nematodes and mammals. Evidence suggests that P35 can inhibit members of caspase family proteases that are key mediators of mammalian apoptosis. We demonstrate that p35 inhibits activation-induced nitric oxide (NO)-mediated apoptosis in the RAW 264.7 mouse macrophages. Parent or vector-transfected RAW 264.7 cells underwent apoptosis when treated with a combination of cisplatin and interferon-gamma (IFN-gamma) or LPS and IFN-gamma in a NO-dependent manner. By contrast, RAW 264.7 cells stably expressing P35 did not undergo apoptosis when treated with a combination of cisplatin and IFN-gamma or LPS and IFN-gamma. Activation of parent, vector- or p35-transfected cells with cisplatin and IFN-gamma or LPS and IFN-gamma caused equivalent levels of inducible nitric oxide synthase (iNOS) expression and produced equal amounts of nitrite, which ruled out attenuated iNOS activity during P35-mediated protection. Rather, expression of P35 inhibited translocation of mitochondrial cytochrome c into cytosol, mitochondrial depolarization, activation of caspase-9 and caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP). These findings indicate that P35 inhibits NO-induced apoptotic cell death of activated macrophages by inhibiting mitochondrial cytochrome c release, which suggests that P35 has targets upstream of the caspase cascade in apoptosis.

Dynamic regulation of Src-family kinases by CD45 in B cells.

CD45 is a key protein tyrosine phosphatase regulating Src-family protein tyrosine kinases (Src-PTKs) in lymphocytes; precisely how it exerts its effect remains controversial, however. We previously demonstrated that CD45 negatively regulates Lyn in the WEHI-231 B-cell line. Here we show that negative regulation by CD45 is physiologically significant in B cells and that some CD45 is constitutively associated with glycolipid-enriched microdomains (GEMs), where it inhibits Src-PTKs by dephosphorylating both the negative and the positive regulatory sites. Upon B-cell receptor (BCR) ligation, however, CD45 dissociates from GEMs within 30 seconds, inducing phosphorylation of 2 regulatory sites and activation of Src-PTKs, but subsequently reassociates with the GEMs within 15 minutes. Disruption of GEMs with methyl-beta-cyclodextrin results in abrogation of BCR-induced apoptosis in WEHI-231 cells, suggesting GEMs are critical to signals leading to the fate determination. We propose that the primary function of CD45 is inhibition of Src-PTKs and that the level of Src-PTK activation and the B-cell fate are determined in part by dynamic behavior of CD45 with respect to GEMs.

Eosinophil peroxidase catalyzes JNK-mediated membrane blebbing in a Rho kinase-dependent manner.

Eosinophilic influx is characteristic of numerous inflammatory conditions. Eosinophil peroxidase (EPO) is a major enzyme present in eosinophils and upon degranulation, becomes released into the airways of asthmatics. As a result of its cationic nature and its ability to catalyze the formation of highly toxic oxidants, EPO has significant potential to induce cellular injury. The focus of the present study was to determine the cell-signaling events important in EPO-induced death of lung epithelial cells. In the presence of hydrogen peroxide and nitrite (NO2-; hereafter called EPO with substrates), EPO catalyzes the formation of nitrogen dioxide. EPO with substrates induced rapid and sustained activation of c-Jun-NH2-terminal kinase (JNK) and led to cell death, as was evidenced by enhanced mitochondrial depolarization, cytochrome c release, cleavage of caspases 9 and 3, poly-adenosine 5'-diphosphate ribosylation of proteins, the formation of single-stranded DNA, and membrane permeability. Moreover, EPO with substrates caused Rho-associated coiled coil-containing kinase-1-dependent dynamic membrane blebbing. Inhibition of JNK activity in cells expressing a dominant-negative JNK-1 construct (JNK-APF) prevented mitochondrial membrane depolarization and substantially decreased the number of cells blebbing compared with vector controls. The cellular responses to EPO with substrates were independent of whether NO2-, bromide, or thiocyanide was used as substrates. Our findings demonstrate that catalytically active EPO is capable of causing significant damage to lung epithelial cells in vitro and that this involves the activation of JNK.

Hydrogen peroxide signaling through tumor necrosis factor receptor 1 leads to selective activation of c-Jun N-terminal kinase.

Binding of tumor necrosis factor-alpha (TNFalpha) to its receptor, TNF-R1, results in the activation of inhibitor of kappaB kinase (IKK) and c-Jun N-terminal kinase (JNK) pathways that are coordinately regulated and important in survival and death. We demonstrated previously that in response to hydrogen peroxide (H2O2), the ability of TNFalpha to activate IKK in mouse lung epithelial cells (C10) was inhibited and that H2O2 alone was sufficient to activate JNK and induce cell death. In the current study, we investigated the involvement of TNF-R1 in H2O2-induced JNK activation. In lung fibroblasts from TNF-R1-deficient mice the ability of H2O2 to activate JNK was inhibited compared with fibroblasts from control mice. Additionally, in C10 cells expressing a mutant form of TNF-R1, H2O2-induced JNK activation was also inhibited. Immunoprecipitation of TNF-R1 revealed that in response to H2O2, the adapter proteins, TRADD and TRAF2, and JNK were recruited to the receptor. However, expression of the adaptor protein RIP, which is essential for IKK activation by TNFalpha, was decreased in cells exposed to H2O2, and its chaperone Hsp90 was cleaved. Furthermore, data demonstrating that expression of TRAF2 was not affected by H2O2 and that overexpression of TRAF2 was sufficient to activate JNK provide an explanation for the inability of H2O2 to activate IKK and for the selective activation of JNK by H2O2. Our data demonstrate that oxidative stress interferes with IKK activation while promoting JNK signaling, creating a signaling imbalance that may favor apoptosis.

Reactive nitrogen species and cell signaling: implications for death or survival of lung epithelium.

Reactive nitrogen species such as nitric oxide, peroxynitrite, and nitrogen dioxide have been implicated in the pathophysiology of inflammatory lung diseases. Yet, the molecular mechanisms and cell signaling events responsible for cellular injury remain to be elucidated. Two major signaling pathways, co-ordinately regulated and responsible for cell survival and cell death, involve nuclear factor kappa B and c-Jun-N-terminal kinase, respectively. A review of these pathways, their modes of action, and their importance in executing oxidative stress responses in lung epithelial cells are discussed.