Activation of JNK and c-Jun is involved in glucose oxidase-mediated cell death of human lymphoma cells

Mitogen-activated protein kinases (MAPK) affect the activation of activator protein-1 (AP-1), which plays an important role in regulating a range of cellular processes. However, the roles of these signaling factors on hydrogen peroxide (H₂O₂)-induced cell death are unclear. This study examined the effects of H₂O₂ on the activation of MAPK and AP-1 by exposing the cells to H₂O₂ generated by either glucose oxidase or a bolus addition. Exposing BJAB or Jurkat cells to H₂O₂ affected the activities of MAPK differently according to the method of H₂O₂ exposure. H₂O₂ increased the AP-1-DNA binding activity in these cells, where continuously generated H₂O₂ led to an increase in mainly the c-Fos, FosB and c-Jun proteins. The c-Jun-NH₂-terminal kinase (JNK)-mediated activation of c-Jun was shown to be related to the H₂O₂-induced cell death. However, the suppression of H₂O₂-induced oxidative stress by either JNK inhibitor or c-Jun specific antisense transfection was temporary in the cells exposed to glucose oxidase but not to a bolus H₂O₂. This was associated with the disruption of death signaling according to the severe and prolonged depletion of reduced glutathione. Overall, these results suggest that H₂O₂ may decide differently the mode of cell death by affecting the intracellular redox state of thiol-containing antioxidants, and this depends more closely on the duration exposed to H₂O₂ than the concentration of this agent.     

Phosphorylation of microtubule-associated protein tau by isoforms of c-Jun N-terminal kinase (JNK)

Microtubule-associated protein tau in a hyperphosphorylated state is the major component of the filamentous lesions that define a number of neurodegenerative diseases commonly referred to as tauopathies. Hyperphosphorylation of tau at most sites appears to precede filament assembly. Many of the hyperphosphorylated sites are serine/threonine-proline sequences. Here we show that c-Jun N-terminal kinases JNK1, JNK2 and JNK3 phosphorylate tau at many serine/threonine-prolines, as assessed by the generation of the epitopes of phosphorylation-dependent anti-tau antibodies. Of the three protein kinases, JNK2 phosphorylated the most sites in tau, followed by JNK3 and JNK1. Phosphorylation by JNK isoforms resulted in a greatly reduced ability of tau to promote microtubule assembly. These findings extend the number of candidate protein kinases for the hyperphosphorylation of tau in Alzheimer's disease and other neurodegenerative disorders.     

The JNK/c-Jun signaling axis contributes to the TDP-43-induced cell death

Dysregulation of transactive response DNA-binding protein-43 (TDP-43) is closely linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). The contribution of the upregulation of TDP-43 expression to the pathogenesis has been strongly suggested by the observation that the level of TDP-43 expression is increased in both ALS and FTLD-U patients. We previously found that the low-grade (twice to five times more than the endogenous level) overexpression of TDP-43 induces neuronal cell death through the upregulation of Bim and CHOP expression and the downregulation of Bcl-xL expression. In this study, we further show that the low-grade overexpression of TDP-43 increases the level of phosphorylated c-Jun N-terminal kinase (JNK) and the co-incubation with a JNK inhibitor, the expression of a dominant-negative JNK, or the expression of a dominant-negative c-Jun inhibited the TDP-43-induced death in NSC34 motor neuronal cells. These data together suggest that the JNK/c-Jun signaling axis contributes to the TDP-43-induced cell death.     

Role of the JNK/c-Jun/AP-1 signaling pathway in galectin-1-induced T-cell death

Galectin-1 (gal-1), an endogenous β-galactoside-binding protein, triggers T-cell death through several mechanisms including the death receptor and the mitochondrial apoptotic pathway. In this study we first show that gal-1 initiates the activation of c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 4 (MKK4), and MKK7 as upstream JNK activators in Jurkat T cells. Inhibition of JNK activation with sphingomyelinase inhibitors (20 μM desipramine, 20 μM imipramine), with the protein kinase C-δ (PKCδ) inhibitor rottlerin (10 μM), and with the specific PKCθ pseudosubstrate inhibitor (30 μM) indicates that ceramide and phosphorylation by PKCδ and PKCθ mediate gal-1-induced JNK activation. Downstream of JNK, we observed increased phosphorylation of c-Jun, enhanced activating protein-1 (AP-1) luciferase reporter, and AP-1/DNA-binding in response to gal-1. The pivotal role of the JNK/c-Jun/AP-1 pathway for gal-1-induced apoptosis was documented by reduction of DNA fragmentation after inhibition JNK by SP600125 (20 μM) or inhibition of AP-1 activation by curcumin (2 μM). Gal-1 failed to induce AP-1 activation and DNA fragmentation in CD3-deficient Jurkat 31-13 cells. In Jurkat E6.1 cells gal-1 induced a proapoptotic signal pattern as indicated by decreased antiapoptotic Bcl-2 expression, induction of proapoptotic Bad, and increased Bcl-2 phosphorylation. The results provide evidence that the JNK/c-Jun/AP-1 pathway plays a key role for T-cell death regulation in response to gal-1 stimulation.     

MMP-14 mediated MMP-9 expression is involved in TGF-beta1-induced keratinocyte migration

The importance of expression of matrix metalloproteinase (MMP) in keratinocyte migration is well established, but its role remains unclear. Here we investigated the function of MMP-14 in TGF-beta1-induced keratinocyte migration. TGF-beta1 stimulated cell migration and the expression of MMP-2, -9 in HaCaT human keratinocyte cells. When we lowered MMP-14 mRNA with siRNA, cell migration, and MMP-9 expression decreased. Furthermore, the MMP-14 siRNA also reduced activation of JNK in response to TGF-beta1, and a JNK-specific inhibitor decreased both cell migration and MMP-9 expression. Taken together, these results suggest that TGF-beta1-induced HaCaT cell migration is mediated by MMP-14, which regulates MMP-9 expression via JNK signaling.     

Cytosolic calcium is involved in the regulation of barley hemoglobin gene expression

Hemoglobin gene expression is upregulated during hypoxia. To determine whether the induction occurs via similar mechanisms that have been proposed for other hypoxically induced proteins, barley (Hordeum vulgare L.) aleurone layers were treated with various agents that interfere with known components of signal transduction. Ruthenium red, an organelle calcium channel blocker, inhibited anoxia-induced hemoglobin (Hb) and alcohol dehydrogenase (EC 1.1.1.1) (Adh) gene expression in a dose-dependent manner. The divalent ionophore, A23187, combined with EGTA also dramatically reduced anoxia-induced Hb and Adh expression. Normal induction of Hb by anoxia in EGTA-treated cells was restored by adding exogenous Ca²⁺ but not Mg²⁺, suggesting that cytosolic calcium is involved in Hb and Adh regulation. W-7, a calmodulin antagonist, did not affect anaerobically induced Hb and Adh expression even though it induced Hb under aerobiosis. A3, a protein kinase inhibitor, did not significantly affect anaerobically induced Hb, but did significantly upregulate the gene under aerobic conditions. The results indicate that calmodulin-independent anaerobic alteration in cytosolic Ca²⁺ and protein dephosphorylation are factors in Hb induction.     

The regulation of Bax by c-Jun N-terminal protein kinase (JNK) is a prerequisite to the mitochondrial-induced apoptotic pathway

The signaling mechanism by which JNK affects mitochondria is critical to initiate apoptosis. Here we show that the absence of JNK provides a partial resistance to the toxic effect of the heavy metal cadmium. Both wild type and jnk−/− fibroblasts undergoing death exhibit cytosolic cytochrome c but, unlike wild type cells, the JNK-deficient fibroblasts do not display increased caspase activity and DNA fragmentation. The absence of apoptotic death correlates with a specific defect in activation of Bax. We conclude that JNK-dependent regulation of Bax is essential to mediate the apoptotic release of cytochrome c regardless of Bid and Bim activation.     

Comparison Between the Timing of JNK activation, c-Jun Phosphorylation, and Onset of Death Commitment in Sympathetic Neurones

Abstract: We have investigated the relationship between c-Jun N-terminal kinase (JNK) activity, apoptosis, and the potential of survival factors to rescue primary rat sympathetic neurones deprived of trophic support. Incubation of sympathetic neurones in the absence of nerve growth factor (NGF) caused a time-dependent increase in JNK activity, which became apparent by 3 h and attained maximal levels that were three- to fourfold higher than activity measured in neurones maintained for the same periods with NGF. Continuous culture in the presence of either NGF or the cyclic AMP analogue 4-(8-chlorophenylthio) cyclic AMP (CPTcAMP) not only prevented JNK activation from occurring, but also suppressed JNK activity that had been elevated by prior culture of the neurones in the absence of trophic support. When either NGF or CPTcAMP was added to cultures that had been initially deprived of neurotrophic support for up to 10 h, this resulted in complete suppression of total JNK activity, arrest of apoptosis, and rescue of >90% of the neurones that did not display apoptotic morphology by this time. However, when either agent was added after more protracted periods of initial neurotrophin deprivation (≥ 14 h), although this also resulted in near-complete suppression of total JNK activity and short-term arrest of apoptosis, not all of the neurones that appeared to be nonapoptotic at the time of agent addition were rescued. The lack of death commitment after 10 h of maintained JNK activity was not due to a late induction of c-Jun expression, because the majority of newly isolated sympathetic neurones had already been expressing high levels of c-Jun in their nuclei for several hours, yet were capable of being rescued by NGF. Elevation of JNK activity as a result of neurotrophic-factor deprivation was also associated with enhanced phosphorylation of c-Jun, assessed by immunoblot analysis and immunocytochemistry, and addition of NGF to cultures previously deprived of neurotrophic support resulted in a reversion of the state of phospho-c-Jun to that observed in cultures that had been maintained in the continuous presence of trophic support. We conclude that activation of JNK and c-Jun phosphorylation are not necessarily rate-limiting for apoptosis induction. In some neurones undergoing prolonged NGF deprivation, suppression of JNK activity and c-Jun dephosphorylation by NGF may be insufficient to effect their rescue. Thus, if c-Jun mediates death by increasing the expression of “death” genes, these must become effective very close to the death commitment point.     

Myeloperoxidase is involved in H2O2-induced apoptosis of HL-60 human leukemia cells

We examined the mechanism of H(2)O(2)-induced cytotoxicity and its relationship to oxidation in human leukemia cells. The HL-60 promyelocytic leukemia cell line was sensitive to H(2)O(2), and at concentrations up to about 20-25 micrometer, the killing was mediated by apoptosis. There was limited evidence of lipid peroxidation, suggesting that the effects of H(2)O(2) do not involve hydroxyl radical. When HL-60 cells were exposed to H(2)O(2) in the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), we detected a 12-line electron paramagnetic resonance spectrum assigned to the POBN/POBN(.) N-centered spin adduct previously described in peroxidase-containing cell-free systems. Generation of this radical by HL-60 cells had the same H(2)O(2) concentration dependence as initiation of apoptosis. In contrast, studies with the K562 human erythroleukemia cell line, which is often used for comparison with the HL-60, and with high passaged HL-60 cells (spent HL-60) studied under the same conditions failed to generate POBN(.). Cellular levels of antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase did not explain the differences between these cell lines. Interestingly, the K562 and spent HL-60 cells, which did not generate the radical, also failed to undergo H(2)O(2)-induced apoptosis. Based on this we reasoned that the difference in H(2)O(2)-induced apoptosis might be due to the enzyme myeloperoxidase. Only the apoptosis-manifesting HL-60 cells contained appreciable immunoreactive protein or enzymatic activity of this cellular enzyme. When HL-60 cells were incubated with methimazole or 4-aminobenzoic acid hydrazide, which are inhibitors of myeloperoxidase, they no longer underwent H(2)O(2)-induced apoptosis. Hypochlorous acid stimulated apoptosis in both HL-60 and spent HL-60 cells, indicating that another oxidant generated by myeloperoxidase induces apoptosis and that it may be the direct mediator of H(2)O(2)-induced apoptosis. Taken together these observations indicate that H(2)O(2)-induced apoptosis in the HL-60 human leukemia cell is mediated by myeloperoxidase and is linked to a non-Fenton oxidative event marked by POBN(.).     

Heme oxygenase is involved in H2O2-induced lateral root formation in apocynin-treated rice

Key message

Apocynin is a natural organic compound structurally related to vanillin. We demonstrated that hydrogen peroxide and heme oxygenase participated in apocynin-induced lateral root formation in rice.

Abstract

Apocynin, also known as acetovanillone, is a natural organic compound structurally related to vanillin. Information concerning the effect of apocynin on plants is limited. In this study, we examined the effect of apocynin on lateral root (LR) formation in rice. Treatment with apocynin induced LR formation and increased H₂O₂ production, but had no effect on nitric oxide production. Diphenyleneiodonium chloride, an inhibitor of H₂O₂ generating NADPH oxidase, was effective in reducing apocynin-induced H₂O₂ production and LR formation. Apocynin treatment also increased superoxide dismutase activity and decreased catalase activity. H₂O₂ application was able to increase the number of LRs. Moreover, H₂O₂ production caused by H₂O₂ and apocynin was localized in the root area corresponding to the LR emergence. Treatment with H₂O₂ and apocynin also increased heme oxygenase (HO) activity and induced OsHO1 mRNA expression. Lateral root formation and HO activity induced by H₂O₂ and apocynin were reduced by Zn protoporphyrin IX (the specific inhibitor of HO). Our data suggest that both H₂O₂ and HO are required for apocynin-induced LR formation in rice.     

Trans membrane domain IV is involved in ion transport activity and pH regulation of the NhaA-Na(+)/H(+) antiporter of Escherichia coli.

We have previously shown that the activity of NhaA is regulated by pH and found mutations that affect dramatically the pH dependence of the rate but not the K(m) (for Na(+) and Li(+)) of NhaA. In the present work, we found that helix IV is involved both in ion translocation as well as in pH regulation of NhaA. Two novel types of NhaA mutants were found clustered in trans membrane segment (TMS) IV: One type (D133C, T132C, and P129L) affects the apparent K(m) of NhaA to the cations with no significant effect on the pH profile of the antiporter; no shift of the pH profile was found when the activity of these mutants was measured at saturating Na(+) concentration. In contrast, the other type of mutations (A127V and A127T) was found to affect both the K(m) and the pH dependence of the rate of NhaA whether tested at saturating Na(+) concentration or not. These results imply that residues involved in the ion translocation of NhaA may (A127) or may not (D133, T132, and P129) overlap with those affecting the pH response of the antiporter. All mutants cluster in the N-terminal half of the putative alpha-helix IV, one type on one face, the other on the opposite. Cys accessibility test demonstrated that although D133C is located in the middle of TMS IV, it is inhibited by N-ethylmaleimide and is exposed to the cytoplasm.     

MPP(+)-induced mitochondrial dysfunction is potentiated by dopamine

MPP(+), the major metabolite of the Parkinsonism-inducing compound MPTP, responsible for the destruction of the nigrostriatal pathway in primates and rodents, has been assayed in isolated rat liver mitochondria in the presence of physiological concentrations of dopamine or analogous concentrations of melanin-dopamine. 5 microM MPP(+) in the presence of 70 microM dopamine or melanin-dopamine, but not alone, decreased the heat production and oxygen consumption of a mitochondrial suspension activated with succinate and ADP. Both dopamine and oxidized dopamine plus MPP(+) also decreased the mitochondrial reductive power measured with MTT. Mitochondrial swelling was observed, associated with an increase in membrane mitochondrial potential, as a synergistic effect between low concentrations of MPP(+) and dopamine. It is suggested that cytosolic dopamine, by itself or via its autooxidation products, may play a relevant role in the mitochondrial toxicity of MPP(+). A failure in the regulation of the storage/release of dopamine could aggravate a mitochondrial damage and trigger the neurodegenerative process underlying MPTP toxicity and Parkinson's disease.