Different Mechanisms of c-Jun NH2-terminal Kinase-1 (JNK1) Activation by Ultraviolet-B Radiation and by Oxidative Stressors

Irradiation of mammalian cells with ultraviolet-B radiation (UV-B) triggers the activation of a group of stress-activated protein kinases known as c-Jun NH2-terminal kinases (JNKs). UV-B activates JNKs via UV-B-induced ribotoxic stress. Because oxidative stress also activates JNKs, we have addressed the question of whether the ribotoxic and the oxidative stress responses are mechanistically similar. The pro-oxidants sodium arsenite, cadmium chloride, and hydrogen peroxide activated JNK1 with slow kinetics, whereas UV-B potentiated the activity of JNK1 rapidly.N-acetyl cysteine (a scavenger of reactive oxygen intermediates) abolished the ability of all oxidative stressors tested to activate JNK1, but failed to affect the activation of JNK1 by UV-B or by another ribotoxic stressor, the antibiotic anisomycin. In contrast, emetine, an inhibitor of the ribotoxic stress response, was unable to inhibit the activation of JNK1 by oxidative stressors. Although UV-A and long wavelength UV-B are the spectral components of the ultraviolet solar radiation that cause significant oxidative damage to macromolecules, the use of a filter to eliminate the radiation output from wavelengths below 310 nm abolished the activation of JNK1 by UV. Our results are consistent with the notion that UV-B and oxidative stressors trigger the activation of JNK1 through different signal transduction pathways

Loss of Cellular K+ Mimics Ribotoxic Stress

The tumor promoter palytoxin has been found to activate the stress-activated protein kinase/c-Jun NH2-terminal kinase 1 (SAPK/JNK1), and it also potentiates, as demonstrated here, the p38/HOG1 mitogen-activated protein kinase and the upstream activator of SAPK/JNK1, SEK1/MKK4. In search of possible mechanisms for both the cytotoxicity and the activation of stress kinases by palytoxin, we found that palytoxin is a potent inhibitor of cellular protein synthesis. The inhibition of translation by palytoxin does not result from its direct binding to the translational apparatus. We have previously demonstrated that ribotoxic stressors (Iordanov, M. S., Pribnow, D., Magun, J. L., Dinh, T.-H., Pearson, J. A., Chen, S. L.-Y., and Magun, B. E. (1997) Mol. Cell. Biol. 17, 3373–3381) signal the activation of SAPK/JNK1 by binding to or covalently modifying 28 S rRNA in ribosomes that are active at the time of exposure to the stressor. Palytoxin acted as a ribotoxic stressor, inasmuch as it required actively translating ribosomes at the time of exposure to activate SAPK/JNK1. Palytoxin has been shown to augment ion fluxes by binding to the Na+/K+-ATPase in the plasma membrane of cells. To determine whether altered fluxes of either Na+ or K+ could be responsible for the effects of palytoxin on translation and on activation of SAPK/JNK1, cells were exposed to palytoxin in modified culture medium in which a major portion of the Na+ was replaced by either K+ or by choline+. The substitution of Na+ by K+ strongly inhibited the ability of palytoxin both to inhibit protein translation and to activate SAPK/JNK1, whereas the substitution of Na+ by choline+ did not. These results suggest that palytoxin-induced efflux of cellular K+ mimics ribotoxic stress by provoking both translational inhibition and activation of protein kinases associated with cellular defense against stress

D-MEKK1, the Drosophila orthologue of mammalian MEKK4/MTK1, and Hemipterous/D-MKK7 mediate the activation of D-JNK by cadmium and arsenite in Schneider cells

BACKGROUND: The family of c-Jun NH(2)-terminal kinases (JNK) plays important roles in embryonic development and in cellular responses to stress. Toxic metals and their compounds are potent activators of JNK in mammalian cells. The mechanism of mammalian JNK activation by cadmium and sodium arsenite involves toxicant-induced oxidative stress. The study of mammalian signaling pathways to JNK is complicated by the significant degree of redundancy among upstream JNK regulators, especially at the level of JNK kinase kinases (JNKKK). RESULTS: Using Drosophila melanogaster S2 cells, we demonstrate here that cadmium and arsenite activate Drosophila JNK (D-JNK) via oxidative stress as well, thus providing a simpler model system to study JNK signaling. To elucidate the signaling pathways that lead to activation of D-JNK in response to cadmium or arsenite, we employed RNA interference (RNAi) to knock down thirteen upstream regulators of D-JNK, either singly or in combinations of up to seven at a time. CONCLUSION: D-MEKK1, the fly orthologue of mammalian MEKK4/MTK1, and Hemipterous/D-MKK7 mediates the activation of D-JNK by cadmium and arsenite

ZAK is required for doxorubicin, a novel ribotoxic stressor, to induce SAPK activation and apoptosis in HaCaT cells

Doxorubicin is an anthracycline drug that is one of the most effective and widely used anticancer agents for the treatment of both hematologic and solid tumors. The stress-activated protein kinases (SAPKs) are frequently activated by a number of cancer chemotherapeutics. When phosphorylated, the SAPKs initiate a cascade that leads to the production of proinflammatory cytokines. Some inhibitors of protein synthesis, known as ribotoxic stressors, coordinately activate SAPKs and lead to apoptotic cell death. We demonstrate that doxorubicin effectively inhibits protein synthesis, activates SAPKs, and causes apoptosis. Ribotoxic stressors share a common mechanism in that they require ZAK, an upstream MAP3K, to activate the pro-apoptotic and proinflammatory signaling pathways that lie downstream of SAPKs. By employing siRNA mediated knockdown of ZAK or administration of sorafenib and nilotinib, kinase inhibitors that have a high affinity for ZAK, we provide evidence that ZAK is required for doxorubicin-induced proinflammatory and apoptotic responses in HaCaT cells, a pseudo-normal keratinocyte cell line, but not in HeLa cells, a cancerous cell line. ZAK has two different isoforms, ZAK-α (91 kDa) and ZAK-β (51 kDa). HaCaT or HeLa cells treated with doxorubicin and immunoblotted for ZAK displayed a progressive decrease in the ZAK-α band and the appearance of ZAK-β bands of larger size. Abrogation of these changes after exposure of cells to sorafenib and nilotinib suggests that these alterations occur following stimulation of ZAK. We suggest that ZAK inhibitors such as sorafenib or nilotinib may be effective when combined with doxorubicin to treat cancer patients

Different Mechanisms of c-Jun NH2-terminal Kinase-1 (JNK1) Activation by Ultraviolet-B Radiation and by Oxidative Stressors

Irradiation of mammalian cells with ultraviolet-B radiation (UV-B) triggers the activation of a group of stress-activated protein kinases known as c-Jun NH2-terminal kinases (JNKs). UV-B activates JNKs via UV-B-induced ribotoxic stress. Because oxidative stress also activates JNKs, we have addressed the question of whether the ribotoxic and the oxidative stress responses are mechanistically similar. The pro-oxidants sodium arsenite, cadmium chloride, and hydrogen peroxide activated JNK1 with slow kinetics, whereas UV-B potentiated the activity of JNK1 rapidly.N-acetyl cysteine (a scavenger of reactive oxygen intermediates) abolished the ability of all oxidative stressors tested to activate JNK1, but failed to affect the activation of JNK1 by UV-B or by another ribotoxic stressor, the antibiotic anisomycin. In contrast, emetine, an inhibitor of the ribotoxic stress response, was unable to inhibit the activation of JNK1 by oxidative stressors. Although UV-A and long wavelength UV-B are the spectral components of the ultraviolet solar radiation that cause significant oxidative damage to macromolecules, the use of a filter to eliminate the radiation output from wavelengths below 310 nm abolished the activation of JNK1 by UV. Our results are consistent with the notion that UV-B and oxidative stressors trigger the activation of JNK1 through different signal transduction pathways

An Epidermotypic Model of Interface Dermatitis Reveals Individual Functions of Fas Ligand and Gamma Interferon in Hypergranulosis, Cytoid Body Formation, and Gene Expression

Analysis of complex cutaneous reactions using animal models allows for the identification of essential or modulatory participants, i.e. cyto- and chemokines, or adhesion molecules. However, complex whole animal modeling is bound to obscure some specific contributions of individual players. Mouse models suggest that expression of Fas ligand (FasL) by donor T cells is essential for the cutaneous acute graft-versus-host reaction (aGvHR), a major complication following allogeneic hematopoietic stem cell transplantation. The role of FasL/Fas in human cutaneous GvHR is not known. To understand the mechanisms of cytotoxicity and inflammation in human cutaneous GvHR, we developed an organotypic model using reconstructed human epidermis (RHE) that was exposed to FasL, gamma-interferon (IFNγ), or both. The model recapitulated key histological hallmarks of cutaneous aGvHR, including interface dermatitis, appearance of cytoid bodies, hypergranulosis, and expression of ICAM-1. Cytoid body formation and expression of ICAM-1 was attributable entirely to IFNγ, whereas hypergranulosis was triggered by FasL. Both FasL and IFNγ triggered vacuolar degeneration of keratinocytes. The validity of the RHE model of GvHR was demonstrated by histological correlation with biopsied skin from aGvHD patients. FasL and IFNγ each elicited potent and specific pro-inflammatory genomic responses in RHE. Inhibition of caspase activity dramatically augmented the FasL-induced pro-inflammatory responses, suggesting an “apoptosis-versus-inflammation” antagonism in cutaneous aGvHR and other lichenoid dermatoses

Role of Apoptotic Signaling Pathways in Regulation of Inflammatory Responses to Ricin in Primary Murine Macrophages

Because of its lethal effects, ease of preparation, and ability to be delivered by aerosolization, ricin has been developed as a lethal weapon by various terrorist groups. When introduced into the pulmonary system of rodents, ricin causes pathological changes in the lung that are known to occur in acute respiratory distress syndrome (ARDS). Early response cytokines such as TNF-α and IL-1 are known to play a critical role in the pathogenesis of ARDS. Ricin induces the release of these proinflammatory cytokines and the transcriptional activation of the genes that encode them in vitro and in vivo. Macrophages, considered to act as upstream regulators of inflammatory cascades, may play a central role in the pathogenesis and the development of ricin-induced ARDS because of their ability to make and secrete proinflammatory cytokines. Exposure of primary macrophages to ricin in vitro led to activation of stress-activated protein kinases, increased expression of proinflammatory mRNA transcripts, subsequent increase in the synthesis and secretion of TNF-alpha, and apoptotic cell death. Interestingly, macrophages required the engagement of the apoptotic cascade for the maximal synthesis and release of some proinflammatory mediators. This work identifies a cross talk between the apoptotic and inflammatory signaling pathways induced by ricin in primary macrophages

Fas Ligand-Induced Proinflammatory Transcriptional Responses in Reconstructed Human Epidermis

Fas ligand (FasL) exerts potent proapoptotic and proinflammatory actions on epidermal keratinocytes and has been implicated in the pathogenesis of eczema, toxic epidermal necrolysis, and drug-induced skin eruptions. We used reconstructed human epidermis to investigate the mechanisms of FasL-induced inflammatory responses and their relationships with FasL-triggered caspase activity. Caspase activity was a potent antagonist of the pro-inflammatory gene expression triggered by FasL prior to the onset of cell death. Furthermore, we found that FasL-stimulated autocrine production of epidermal growth factor receptor (EGFR) ligands, and the subsequent activation of EGFR and ERK1 and ERK2 mitogen-activated protein kinases, were obligatory extracellular steps for the FasL-induced expression of a subset of inflammatory mediators, including CXCL8/interleukin (IL)-8, ICAM-1, IL-1α, IL-1β, CCL20/MIP-3α, and thymic stromal lymphopoietin. These results expand the known physiological role of EGFR and its ligands from promoting keratinocyte mitogenesis and survival to mediating FasL-induced epidermal inflammation

Ultraviolet Radiation Triggers the Ribotoxic Stress Response in Mammalian Cells

The ribotoxic stress response, which is conserved between prokaryotes and eukaryotes, is a cellular reaction to cytotoxic interference with the function of the 3′-end of the large (23 S/28 S) ribosomal RNA. The 3′-end of the large rRNA is directly involved in the three sequential steps of translational elongation: the aminoacyl-tRNA binding, the peptidyl transfer, and the ribosomal translocation. In mammalian cells, the ribotoxic stress response involves activation of the stress-activated protein kinase/c-Jun NH2-terminal kinase and the p38 mitogen-activated protein kinase and transcriptional induction of immediate early genes such as c-fos and c-jun. Active ribosomes are essential mediators of the ribotoxic stress response. We demonstrate here that the transcriptional response of mammalian cells to ultraviolet radiation (UV response) displays the characteristics of a ribotoxic stress response, inasmuch as (i) the activation of stress kinases and gene expression in response to UV requires the presence of active ribosomes at the moment of irradiation; (ii) UV irradiation inhibits protein synthesis; and (iii) irradiation of cells with UV causes specific damage to the 3′-end of the 28 S rRNA. In contrast, the activation of the stress kinases by hyperosmolarity, by the DNA-cross-linking agent diepoxybutane, or by growth factors and cytokines does not depend on the presence of active ribosomes. Our results identify UV as a potential ribotoxic stressor and support the notion that some of the cellular signaling cascades in response to UV might be generated in the ribosome, possibly triggered by damage to rRNA

Fas Ligand-Induced Proinflammatory Transcriptional Responses in Reconstructed Human Epidermis

Fas ligand (FasL) exerts potent proapoptotic and proinflammatory actions on epidermal keratinocytes and has been implicated in the pathogenesis of eczema, toxic epidermal necrolysis, and drug-induced skin eruptions. We used reconstructed human epidermis to investigate the mechanisms of FasL-induced inflammatory responses and their relationships with FasL-triggered caspase activity. Caspase activity was a potent antagonist of the pro-inflammatory gene expression triggered by FasL prior to the onset of cell death. Furthermore, we found that FasL-stimulated autocrine production of epidermal growth factor receptor (EGFR) ligands, and the subsequent activation of EGFR and ERK1 and ERK2 mitogen-activated protein kinases, were obligatory extracellular steps for the FasL-induced expression of a subset of inflammatory mediators, including CXCL8/interleukin (IL)-8, ICAM-1, IL-1α, IL-1β, CCL20/MIP-3α, and thymic stromal lymphopoietin. These results expand the known physiological role of EGFR and its ligands from promoting keratinocyte mitogenesis and survival to mediating FasL-induced epidermal inflammation