Involvement of p38 mitogen-activated protein kinase and apoptosis signal-regulating kinase-1 in nitric oxide-induced cell death in PC12 cells

Although nitric oxide (NO) plays key signaling roles in the nervous systems, excess NO leads to cell death. In this study, the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and apoptosis signal-regulating kinase-1 (ASK1) in NO-induced cell death was investigated in PC12 cells. NO donor transiently activated p38 MAPK in the wild type parental PC12 cells, whereas the p38 MAPK activation was abolished in NO-resistant PC12 cells (PC12-NO-R). p38 MAPK inhibitors protected the cells against NO-induced death, whereas the inhibitors were not significantly protective against the cytotoxicity of reactive oxygen species. Stable transfection with dominant negative p38 MAPK mutant reduced NO-induced cell death. Stable transfection with dominant negative mutant of ASK1 attenuated NO-stimulated activation of p38 MAPK and decreased NO-induced cell death. These results suggest that p38 MAPK and its upstream regulator ASK1 are involved in NO-induced PC12 cell death.     

ASK3, a novel member of the apoptosis signal-regulating kinase family, is essential for stress-induced cell death in HeLa cells

Apoptosis signal-regulating kinase 1 (ASK1) and ASK2 are both members of mitogen-activated protein kinase kinase kinase (MAP3K) family that are implicated in apoptotic cell death, stress responses, and various diseases. We have determined that NT2RI3007443, TESTI4031745, SGK341, and human MAP3K15 are all transcribed from the same genomic locus, which we designate “ASK3 gene” based on sequence homology to ASK1 and ASK2. NT2RI3007443, TESTI4031745, and SGK341 displayed distinct expression profiles among human tissues. TESTI4031745 was expressed in relatively high levels. The expression of TESTI4031745 was increased in rectum tumor and Alzheimer’s disease hippocampus and decreased in kidney tumor and Alzheimer’s disease frontal lobe. NT2RI3007443 showed moderate levels of ubiquitous expression in normal adult tissues. They did not drastically change in diseases except for increase in cirrhosis liver. Expression of SGK341 was restricted. It was highly expressed in fetal brain, and moderately expressed in normal hippocampus, pancreas, spleen, lung, and kidney. Further, its expression was dramatically increased in hepatic cirrhosis and decreased in lung tumor. Target proteins encoded by NT2RI3007443 and TESTI4031745 were translated in cell-free protein synthesis system. They exhibited protein kinase activity indicated by ATP consumption and phosphorylation of Syntide 2 as a substrate. We demonstrated that knockdown of ASK3 protected HeLa cells against cytotoxicity induced by anti-Fas monoclonal antibody, TNF-alpha, or oxidative stress. These findings suggest that “ASK3 gene” is a novel member of apoptosis signal-regulating kinases and that it plays a pivotal role in the signal transduction pathway implicated in apoptotic cell death triggered by cellular stresses. It can be a putative therapeutic drug target for multiple human diseases.     

Ghrelin inhibits apoptosis signal-regulating kinase 1 activity via upregulating heat-shock protein 70

Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), which has been originally isolated from rat stomach. It has been reported that ghrelin inhibited apoptosis in several cells, such as cardiomyocytes, endothelial cells, adipocyte, adrenal zona glomerulosa cells, pancreatic beta-cells, osteoblastic MC3T3-E1 cells, intestinal epithelial cells and hypothalamic neurons. However, it is unknown whether heat-shock protein 70 (HSP70) or apoptosis signal-regulating kinase 1 (ASK1) is the important target molecule which mediates the anti-apoptotic effects of ghrelin. We show that ghrelin inhibited ASK1 activity induced by sodium nitroprusside (SNP), inhibited ASK1-mediated caspase 3 activation and apoptosis in PC12 cells. Ghrelin promoted expression of HSP70. Quercetin, an inhibitor of HSP70, blocked the effects of ghrelin on ASK1 activity. Thus, ghrelin inhibits ASK1-mediated apoptosis and ASK1 activation by a mechanism involving induction of HSP70 expression. The results of the present study suggest the therapeutic potential of ghrelin for some pathological processes or disorders.     

Epigallocatechin-3-gallate induces cell apoptosis of human chondrosarcoma cells through apoptosis signal-regulating kinase 1 pathway

Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. (-)-Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, has been shown to inhibit tumorigenesis and cancer cell growth in animal models. The aim of this study was to elucidate the mechanism of EGCG-induced apoptosis of human chondrosarcoma cells. EGCG induced cell apoptosis in human chondrosarcoma cell lines but not primary chondrocytes. EGCG induced upregulation of Bax and Bak, downregulation of Bcl-2 and Bcl-XL, and dysfunction of mitochondria in chondrosarcoma. We also found that the accumulation of reactive oxygen species (ROS) is a critical mediator in EGCG-induced cell death. EGCG induced apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation and its dissociation from 14-3-3. Treatment of chondrosarcoma cells with EGCG induced p38 and c-jun-NH2-kinase (JNK) phosphorylation. Transfection with ASK1 siRNA or p38 and JNK mutant antagonized the EGCG-induced cell apoptosis. Therefore, EGCG triggered ROS and activated the ASK1-p38/JNK pathway, resulting chondrosarcoma cell death. Importantly, animal studies revealed a dramatic reduction in tumor volume after 24 days of treatment. Thus, EGCG may be a novel anti-cancer agent for the treatment of chondrosarcoma.     

Discovery of potent apoptosis signal-regulating kinase 1 inhibitors via integrated computational strategy and biological evaluation

Abstract

Apoptosis signal-regulating Kinase 1 (ASK1) has been confirmed as a potential therapeutic target for the treatment of non-alcoholic steatohepatitis (NASH) disorder and the discovery of ASK1 inhibitors has attracted increasing attention. In this work, a series of in silico methods including pharmacophore screening, docking binding site analysis, protein-ligand interaction fingerprint (PLIF) similarity investigation and molecular docking were applied to find the potential hits from commercial compound databases. Five compounds with potential inhibitory activity were purchased and submitted to biological activity validation. Thus, one hit compound was discovered with micromolar IC₅₀ value (10.59?μM) against ASK1. Results demonstrated that the integration of computation methods and biological test was quite reliable for the discovery of potent ASK1 inhibitors and the strategy could be extended to other similar targets of interest.     

Knockdown of apoptosis signal-regulating kinase 1 modulates basal glycogen synthase kinase-3β kinase activity and regulates cell migration

GSK-3β is a basally active kinase. Axin forms a complex with GSK-3β and β-catenin; this complex promotes the GSK-3β-dependent phosphorylation of β-catenin, thereby inducing its degradation. However, the inhibition of GSK-3β provokes cell migration via the dysregulation of β-catenin. In this study, we determined that the level of apoptosis signal-regulating kinase 1 (ASK1) was lower in a metastatic breast cancer cell line, compared to that of non-metastatic cancer cell lines and the knockdown of ASK1 not only induces β-catenin activation via the inhibition of GSK-3β and collapsing the subsequent protein complex by regulating Axin dynamics, but also stimulates cell migration. Together, the blockage of the GSK-3β-β-catenin pathway resulting from the knockdown of ASK1 modulates the migration of breast cancer cells.     

Apoptosis signal-regulating kinase 1 is an intracellular inducer of p38 MAPK-mediated myogenic signalling in cardiac myoblasts

Myogenic differentiation is an essential process for the myogenesis in response to various extracellular stimuli. p38 MAPK is a core signalling molecule in myogenic differentiation. The activation of p38 MAPK is required for myogenic differentiation; however, the mechanism for this activation remains undefined. ASK1 is a member of the MAP3K family that activates both JNK and p38 MAPK pathways in response to an array of stresses such as oxidative stress, endoplasmic reticulum stress and calcium influx. Here, we reported that TNFα was significantly released from H9c2 cardiac myoblast in differentiation medium. Furthermore, the oxidant H₂O₂ acted as a messenger in the TNFα signalling pathway to disrupt the complex of ASK1-Trx, which was followed by the activation of ASK1 in cardiac myogenic differentiation. Subsequently, the activated ASK1 stimulated MKK3/6-p38MAPK signalling cascade to induce specific myogenic differentiation. In addition, exogenous TNFα added to the medium at physiological levels enhanced the ASK1-p38 MAPK signalling pathway through the increased generation of H₂O₂. Interestingly, inhibition of p38 MAPK abrogated the production of H₂O₂, suggesting that there might be a positive feedback loop in the myogenic-redox signalling pathway. These results indicate that ASK1 is a new intracellular regulator of activation of the p38 MAPK in cardiac myogenic differentiation.     

Interaction of apoptosis signal-regulating kinase 1 with isoforms of 14-3-3 proteins

Apoptosis signal-regulating kinase 1 (ASK1) is a critical mediator of apoptotic signaling pathways initiated by a variety of death stimuli. Its activity is tightly controlled by various mechanisms such as covalent modification and protein-protein interaction. One of the proteins that control ASK1 function is 14-3-3zeta, a member of the 14-3-3 protein family. Here, we report that ASK1 is capable of binding to other isoforms of 14-3-3, suggesting that binding ASK1 is a general property of the 14-3-3 family. In support of this notion, mutational analysis revealed that the ASK1/14-3-3 interaction was mediated by the conserved amphipathic groove of 14-3-3 with some residue selectivity. Functionally, expression of various isoforms of 14-3-3 suppressed ASK1-induced apoptosis. To understand how 14-3-3 controls the ASK1 activity, we examined intracellular localization of ASK1 upon 14-3-3 co-expression. We found that 14-3-3 co-expression is correlated with the translocation of ASK1 from the cytoplasm to a perinuclear localization, likely the ER compartment. Consistent with this notion, ASK1(S967A), a 14-3-3 binding defective mutant of ASK, showed no change in intracellular distribution upon 14-3-3 co-expression. These data support a model that 14-3-3 proteins regulate the proapoptotic function of ASK1 in part by controlling its subcellular distribution.     

Runaway cell death, but not basal disease resistance, in lsd1 is SA- and NIM1/NPR1-dependent

LSD1 was defined as a negative regulator of plant cell death and basal disease resistance based on its null mutant phenotypes. We addressed the relationship between lsd1-mediated runaway cell death and signaling components required for systemic acquired resistance (SAR), namely salicylic acid (SA) accumulation and NIM1/NPR1. We present two important findings. First, SA accumulation and NIM1/NPR1 are required for lsd1-mediated runaway cell death following pathogen infection or application of chemicals that mimic SA action. This implies that lsd1-dependent cell death occurs 'downstream' of the accumulation of SA. As SA application triggers runaway cell death in lsd1 but not wild-type plants, we infer that LSD1 negatively regulates an SA-dependent signal leading to cell death. Thus SA is both a trigger and a required mediator of lsd1 runaway cell death. Second, neither SA accumulation nor NIM1/NPR1 function is required for the basal resistance operating in lsd1. Therefore LSD1 negatively regulates a basal defense pathway that can act upstream or independently of both NIM1/NPR1 function and SA accumulation following avirulent or virulent pathogen challenge. Our data, together with results from other studies, point to the existence of an SA-dependent 'signal potentiation loop' controlling HR. Continued escalation of signaling in the absence of LSD1 leads to runaway cell death. We propose that LSD1 is a key negative regulator of this signal potentiation.     

Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1) in denbinobin-induced apoptosis in human lung adenocarcinoma (A549) cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN), two antioxidants (N-acetyl-L-cysteine (NAC) and glutathione (GSH)), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125), and an activator protein-1 (AP-1) inhibitor (curcumin). Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS) production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.     

BAG-1 inhibits p53-induced but not apoptin-induced apoptosis

BAG-1 has been identified as a Bcl-2-binding protein that inhibits apoptosis, either alone or in co-operation with Bcl-2. Here we show that BAG-1 inhibits p53- induced apoptosis in the human tumour cell line Saos-2. In contrast, BAG-1 was unable to inhibit the p53-independent pathway induced by apoptin, an apoptosis-inducing protein derived from chicken anaemia virus. Whereas BAG-1 seemed to co-operate with Bcl-2 to repress p53-induced apoptosis, co-expression of these proteins had no inhibitory effect on apoptin-induced apoptosis. Moreover, Bcl-2, and to some extent also BAG-1, paradoxically enhanced the apoptotic activity of apoptin. These results demonstrate that p53 and apoptin induce apoptosis through independent pathways, which are differentially regulated by BAG-1 and Bcl-2.     

The role of apoptosis signal-regulating kinase 1 in lymphotoxin-beta receptor-mediated cell death

LIGHT (homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytes) is a member of the tumor necrosis factor superfamily that can interact with lymphotoxin-beta receptor (LTbetaR), herpes virus entry mediator, and decoy receptor (DcR3). In our previous study, we showed that LIGHT is able to induce cell death via the non-death domain containing receptor LTbetaR to activate both caspase-dependent and caspase-independent pathway. In this study, a LIGHT mutein, LIGHT-R228E, was shown to exhibit similar binding specificity as wild type LIGHT to LTbetaR, but lose the ability to interact with herpes virus entry mediator. By using both LIGHT-R228E and agonistic anti-LTbetaR monoclonal antibody, we found that signaling triggered by LTbetaR alone is sufficient to activate both caspase-dependent and caspase-independent pathways. Cross-linking of LTbetaR is able to recruit TRAF3 and TRAF5 to activate ASK1, whereas its activity is inhibited by free radical scavenger carboxyfullerenes. The activation of ASK1 is independent of caspase-3 activation, and kinase-inactive ASK1-KE mutant can inhibit LTbetaR-mediated cell death. This suggests that ASK1 is one of the factors involved in the caspase-independent pathway of LTbetaR-induced cell death.     

Apoptosis signal-regulating kinase 1 regulates the expression of caspase-11

Caspase-11 is an inducible caspase involved in the regulation of cell death and inflammation. In the present study, we examined whether apoptosis signal-regulating kinase 1 (Ask1)-mediated signaling pathway is involved in the expression of caspase-11 induced by lipopolysaccharide (LPS). We found that the induction of caspase-11 was suppressed by the inhibitors of NADPH oxidase (Nox) or knockdown of Nox4 that acts downstream of toll-like receptor 4 and generates Ask1-activating reactive oxygen species. Overexpression of dominant negative tumor necrosis factor receptor associate factor 6 also suppressed the induction of caspase-11. Importantly, knockdown or dominant negative form of Ask1 suppressed the induction of caspase-11 following LPS stimulation. Taken together, our results show that Ask1 regulates the expression of caspase-11 following LPS stimulation.     

Mitochondrial calcium uniporter protein MCU is involved in oxidative stress-induced cell death

Mitochondrial calcium uniporter (MCU) is a conserved Ca²⁺ transporter at mitochondrial in eukaryotic cells. However, the role of MCU protein in oxidative stressinduced cell death remains unclear. Here, we showed that ectopically expressed MCU is mitochondrial localized in both HeLa and primary cerebellar granule neurons (CGNs). Knockdown of endogenous MCU decreases mitochondrial Ca²⁺ uptake following histamine stimulation and attenuates cell death induced by oxidative stress in both HeLa cells and CGNs. We also found MCU interacts with VDAC1 and mediates VDAC1 overexpression-induced cell death in CGNs. This finding demonstrates that MCU-VDAC1 complex regulates mitochondrial Ca²⁺ uptake and oxidative stress-induced apoptosis, which might represent therapeutic targets for oxidative stress related diseases.     

Apoptosis signal-regulating kinase (ASK)-1 mediates apoptosis through activation of JNK1 following engagement of membrane immunoglobulin

Engagement of membrane immunoglobulin (mIg) on WEHI-231 mouse B lymphoma cells results in growth arrest at the G1 phase of the cell cycle, followed by a reduction of mitochondrial membrane potential (ΔΨm) and apoptosis. WEHI-231 cells resemble immature B cells in terms of the cell surface phenotype and sensitivity to mIg engagement. However, the molecular mechanisms underlying mIg-induced loss of ΔΨm and apoptosis have not yet been established. In this study, we show that apoptosis signal-regulating kinase 1 (ASK1)-c-Jun N-terminal kinase 1 (JNK1) signaling pathway participates in mIg-induced apoptosis through the generation of reactive oxygen species (ROS). Stimulation of WEHI-231 cells with anti-IgM induces phosphorylation and subsequent activation of ASK1, leading to JNK activation. Anti-IgM stimulation immediately (5 min) induces hydrogen peroxide (H₂O₂) production with a substantial increase during later time points (36-48 h), accompanied by loss of ΔΨm and an increase in cells with sub-G1 DNA content. The anti-IgM-induced late-phase H₂O₂ production, loss of ΔΨm, and increase in the sub-G1 fraction were all reduced substantially in WEHI-231 cells overexpressing a dominant-negative form of ASK1, compared with control vector alone, but enhanced substantially in cells overexpressing a constitutively active form of ASK1. These mIg-mediated events were also partially abrogated by ROS scavenger N-acetyl-l-cysteine (NAC). Taken together, these results suggest that mIg engagement induces H₂O₂ production leading to activation of ASK1-JNK1 pathway, creating a feedback amplification loop of ROS-ASK/JNK that leads to loss of ΔΨm and finally apoptosis.     

A novel function of peroxiredoxin 1 (Prx-1) in apoptosis signal-regulating kinase 1 (ASK1)-mediated signaling pathway

We report a novel function of peroxiredoxin-1 (Prx-1) in the ASK1-mediated signaling pathway. Prx-1 interacts with ASK1 via the thioredoxin-binding domain of ASK1 and this interaction is highly inducible by H2O2. However, catalytic mutants of Prx1, C52A, C173A, and C52A/C173A, could not undergo H2O2 inducible interactions, indicating that the redox-sensitive catalytic activity of Prx-1 is required for the interaction with ASK1. Prx-1 overexpression inhibited the activation of ASK1, and resulted in the inhibition of downstream signaling cascades such as the MKK3/6 and p38 pathway. In Prx-1 knockdown cells, ASK1, p38, and JNK were quickly activated, leading to apoptosis in response to H2O2. These findings suggest a negative role of Prx-1 in ASK1-induced apoptosis.