MicroRNA-532-3p Regulates Pro-Inflammatory Human THP-1 Macrophages by Targeting ASK1/p38 MAPK Pathway.

Inflammation is a complex biological process which alters the normal physiological function of the immune system resulting in an abnormal microenvironment that leads to several clinical complications. The process of inflammation is mediated through various intracellular signaling factors inside the cells. Apoptosis signal-regulating kinase 1 (ASK1) is an inflammation-derived kinase that controls the activation of other family of kinases such as p38 mitogen-activated protein kinases (p38 MAPKs), which mediates various the inflammatory processes. In this study, we cultured THP-1 macrophage cells to undergo inflammatory proliferation with LPS (1 µg/ml) and TNFα (10 ng/ml) stimulation. Initial in silico analysis was utilized to predict novel microRNAs (miRNAs) that target ASK1 signaling and its expression levels in LPS and TNFα stimulated THP-1 cells were estimated. Among the miRNAs, miR-532-3p showcased the highest binding affinity towards ASK1 kinase. We witnessed that transient transfection of miR-532-3p diminished the levels of ASK1 and downstream phosphorylation/translocation of p38 MAPK. Furthermore, direct targeting of ASK1 resulted in regulation of uncontrolled release of cytokines (TNFα, IL-6, and IL-23) and chemokines (GM-CSF and MIP-2α). Overall, we suggest that miR-532-3p attenuates the pro-inflammatory nature of macrophages by targeting ASK1/p38 MAPK signaling pathway and can be used as a molecular intervention for treating inflammatory diseases.

Inhibition of Apoptosis Signal-Regulating Kinase 1 Attenuates Myocyte Hypertrophy and Fibroblast Collagen Synthesis.

BACKGROUND: Cardiac remodelling is a dynamic process whereby structural and functional changes occur within the heart in response to injury or inflammation. Recent studies have demonstrated reactive oxygen species sensitive MAPK, apoptosis signal-regulating kinase 1 (ASK1) plays a critical role in cardiac remodelling. This study aims to determine the effectiveness of small molecule ASK1 inhibitors on these processes and their therapeutic potential. METHODS: Neonatal rat cardiac fibroblasts (NCF) were pre-treated with ASK1 inhibitors, G2261818A (G226) and G2358939A (G235), for 2hours before stimulated with 100nM angiotensin II (AngII), 10µM indoxyl sulphate (IS) or 10ng/ml transforming growth factor ß1 (TGFß1) for 48hours. Neonatal rat cardiac myocytes (NCM) were pre-treated with G226 and G235 for 2hours before being stimulated with 100nM AngII for 60hours, 10µM IS, 10ng/ml interleukin 1ß (IL-1ß) or tumour necrosis factor α (TNFα) for 48hours. 3H-proline and 3H-leucine incorporation was used to assess collagen turnover and hypertrophy, respectively. Pro-fibrotic, pro-hypertrophic and THP-1 inflammatory cytokine gene expressions were determined by RT-PCR. RESULTS: Both G226 and G235 dose-dependently attenuated AngII-, IS-, IL-1ß- and TNFα-stimulated NCM hypertrophy and hypertrophic gene expression, IS-, AngII- and TGFß1-stimulated NCF collagen synthesis and AngII- and TGFß1-stimulated pro-fibrotic gene expression. Inhibition of ASK1 by G226 and G235 inhibited lipopolysaccharides-stimulated inflammatory cytokine gene expression in THP-1 cells. CONCLUSIONS: Selective ASK1 inhibition confers anti-hypertrophic and anti-fibrotic effects in cardiac cells, and anti-inflammation in monocytic cells. ASK1 inhibitors may represent novel therapeutic agents to alleviate cardiac remodelling post cardiac injury where hypertrophy, fibrosis and inflammation play critical roles.

The Elevated ASK1 Expression Inhibits Proliferation and Invasion in Gastric Cancer HGC-27 Cells.

This study aimed to evaluate the effects and mechanism of action of ASK1 gene on the growth and migration of gastric cancer (GC) cells. Total RNA was extracted from the gastric cell lines and GC tissues. The expression level of ASK1, and the association between ASK1 expression and clinicopathological characteristics was assessed by real-time polymerase chain reaction. The effects of ASK1 on the proliferation of HGC-27 cells were assessed by the CCK-8 assay. In addition, the effects of ASK1 on the migration of HGC-27 cells were analyzed by the migration assay using transwell chambers. The expression levels of signaling proteins related to cell migration were detected by Western blotting. Although no significant differences were observed in the expression levels of ASK1 between the GC tissue samples and the normal tissue samples (P = 0.241), ASK1 expression correlated with tumor lymph node metastasis (P = 0.008). Furthermore, ASK1 inhibited proliferation and migration of HGC-27 cells. The increase in the expression of ASK1 in HGC-27 cells induced the activation of the JNK and p38 signaling pathways. The findings demonstrated that increased ASK1 expression level inhibited migration and proliferation of HGC-27 gastric cancer cells, whereas the possible mechanism of action may be attributed to the activation of the JNK and p38 signaling pathways. Anat Rec, 301:1815-1819, 2018. © 2018 Wiley Periodicals, Inc.

Knockdown of MiR-20a Enhances Sensitivity of Colorectal Cancer Cells to Cisplatin by Increasing ASK1 Expression.

BACKGROUND/AIMS: Platinum-based chemotherapy is one of the most important strategies for treatment of colorectal cancer. To improve the therapeutic efficiency, adjuvant drugs were sought to sensitize colorectal cancer cells to platinum-based agents such as cisplatin. As previous research has shown that miRNAs are associated with chemosensitivity, we aimed to alter miRNA regulation in colorectal cancer cells to increase their chemosensitivity. METHODS: MTT assays were performed to determine the viability of HT29, SW480, and LoVo cells. Quantitative real time polymerase chain reaction (qRT-PCR) was performed to examine the expression of miR-20a in these cell lines. Regulation of the miR-20a/ASK1 axis was confirmed by western blotting and luciferase reporter assays. After treatment with miR-20a inhibitor (anti-miR-20a) and cisplatin, production of reactive oxygen species (ROS), mitochondrial membrane potential, and apoptosis were measured by flow cytometry. Activation of ASK1, Bcl-xl, JNK, and caspase-9, -7, and -3 was detected by western blotting. RESULTS: miR-20a was overexpressed in colorectal cancer cell lines. Furthermore, knockdown of miR-20a increased the sensitivity of colorectal cancer cells to cisplatin treatment in vitro and in vivo. We demonstrated that the ASK1 gene was the target of miR-20a, and knockdown of miR-20a increased the expression of ASK1 in colorectal cancer cells. As cisplatin treatment induced production of ROS, knockdown of miR-20a enhanced ROS signaling through promoting the phosphorylation of ASK1. Phosphorylation of JNK and the subsequent mitochondrial apoptosis were triggered by the combination of cisplatin and anti-miR-20a. CONCLUSIONS: Knockdown of miR-20a enhanced sensitivity of colorectal cancer cells to cisplatin through the ROS/ASK1/JNK pathway.

MiR17 improves insulin sensitivity through inhibiting expression of ASK1 and anti-inflammation of macrophages.

OBJECTIVES: MicroRNAs (miRNAs) are involved in the pathological progression of various disease including type 2 diabetes (T2D). Chronic inflammation in adipose tissue is a cause of insulin resistance and T2D. MiR-17 palys an anti-inflammatory role in many biological processes. We hypothesized that miR-17 suppressed inflammatory macrophage that is related to insulin resistance in patients with T2D. METHODS: Macrophage migration and secretion of inflammatory cytokines including TNF-α, IL-6 and IL-1ß were detected through transwell migration assay and enzyme-linked immunosorbent assay, respectively. Insulin-stimulated glucose uptake was tested by the radioactivity of tritium-labeled glucose in 3T3-L1 adipocytes. Dual luciferase reporter gene assay was employed to evaluate the interaction between miR-17 and 3'UTR of ASK1. RESULTS: Our results showed that miR-17 inhibited macrophage infiltration and secretion of TNF-α, IL-6 and IL-1ß. Moreover, insulin-stimulated glucose uptake of 3T3-L1 was suppressed by treatment with LPS-induced macrophage conditioned media (CM), whereas the opposite effect was showed after treatment with the CM of macrophages transfected with miR-17. Furthermore, we found that miR-17 directly prevented expression of ASK1 by binding to its 3'UTR. CONCLUSION: miR-17 improved inflammation-induced insulin resistance by suppressing ASK1 expression in macrophages. These results indicated that miR-17 had an anti-diabetic acitivity by its anti-inflammation effect on macrophage.

Psoralen Inhibited Apoptosis of Osteoporotic Osteoblasts by Modulating IRE1-ASK1-JNK Pathway.

Osteoporosis is a common disease causing fracture in older populations. Abnormal apoptosis of osteoblasts contributes to the genesis of osteoporosis. Inhibiting apoptosis of osteoblasts provides a promising strategy to prevent osteoporosis. The proliferation of osteoblasts isolated from osteoporotic patients or healthy subjects was determined by MTT assay. Apoptosis was determined by Annexin V/PI assay. Protein expression was measured by western blot. The proliferation of osteoblasts isolated from osteoporotic patients was inhibited and the apoptosis level of these cells was higher than the osteoblasts from healthy subjects. Incubation with psoralen or estradiol significantly enhanced the proliferation and decreased the apoptosis level of osteoporotic osteoblasts. Western blot demonstrated that psoralen or estradiol treatment downregulated the expression of IRE1, p-ASK, p-JNK, and Bax. Meanwhile, expression of Bcl-2 was upregulated. Pretreatment by IRE1 agonist tunicamycin or JNK agonist anisomycin attenuated the effect of psoralen on osteoporotic osteoblasts. Psoralen inhibited apoptosis of osteoporotic osteoblasts by regulating IRE1-ASK1-JNK pathway.

Purification, auto-activation and kinetic characterization of apoptosis signal-regulating kinase I.

Apoptosis signal-regulating kinase I (ASK1) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the downstream MAP kinase kinases (MKKs) from two MAP kinase cascades: c-Jun N-terminal kinase (JNK) and p38. The essential physiological functions of ASK1 have attracted extensive attention. However, our understanding of the molecular mechanisms of ASK1, including the activation mechanism of ASK1 and the catalytic mechanism of ASK1-mediated MKK phosphorylation, remain unclear. The lack of purified ASK1 protein has hindered the elucidation of ASK1-initiated signal transduction mechanisms. Here, we report a one-step chromatography method for the expression and purification of functional full-length ASK1 from Escherichia coli. The purified ASK1 demonstrates auto-phosphorylation activity. The kinase activity of auto-phosphorylated ASK1 (pASK1) was also evaluated on two MKK substrates, MKK4 and 7, from the JNK cascades. Our results show that MKK7 can be phosphorylated by pASK1 more effectively than MKK4. The steady-state kinetic analysis demonstrates that MKK7 is a better ASK1 substrate than MKK4. These observations are further confirmed by direct pull-down assays which shows ASK1 binds MKK7 significantly stronger than MKK4. Furthermore, robust phospho-tyrosine signal is observed in MKK4 phosphorylation by pASK1 in addition to the phospho-serine and phospho-threonine. This study provides novel mechanistic and kinetic insights into the ASK1-initiated MAPK signal transduction via highly controlled reconstructed protein systems.

Production of recombinant human apoptosis signal-regulating kinase 1 (ASK1) in Escherichia coli.

Apoptosis signal-regulating kinase 1 (ASK1) is a mediator of the MAPK signaling cascade, which regulates different cellular processes including apoptosis, cell survival, and differentiation. The increased activity of ASK1 is associated with a number of human diseases and this protein kinase is considered as promising therapeutic target. In the present study, the kinase domain of human ASK1 was expressed in Escherichia coli (E. coli) in soluble form. The expression level of ASK1 was around 0.3-0.47 g per 1 L after using auto-induction protocol or IPTG induction. A one-step on column method for the efficient purification of recombinant ASK1 was performed. Our approach yields sufficient amount of recombinant ASK1, which can be used for inhibitor screening assays and different crystallographic studies.

Curcumin enhances the antitumor effect of ABT-737 via activation of the ROS-ASK1-JNK pathway in hepatocellular carcinoma cells.

At present, the therapeutic treatment strategies for patients with hepatocellular carcinoma (HCC) remain unsatisfactory, and novel methods are urgently required to treat this disease. Members of the B cell lymphoma (Bcl)-2 family are anti­apoptotic proteins, which are commonly expressed at high levels in certain HCC tissues and positively correlate with the treatment resistance of patients with HCC. ABT-737, an inhibitor of Bcl-2 anti-apoptotic proteins, has been demonstrated to exhibit potent antitumor effects in several types of tumor, including HCC. However, treatment with ABT-737 alone also activates certain pro-survival signaling pathways, which attenuate the antitumor validity of ABT-737. Curcumin, which is obtained from Curcuma longa, is also an antitumor potentiator in multiple types of cancer. In the present study, the synergistic effect of curcumin and ABT-737 on HCC cells was investigated for the first time, to the best of our knowledge. It was found that curcumin markedly enhanced the antitumor effects of ABT-737 on HepG2 cells, which was partially dependent on the induction of apoptosis, according to western blot analysis and flow cytometric apoptosis analysis. In addition, the sustained activation of the ROS-ASK1-c-Jun N-terminal kinase pathway may be an important mediator of the synergistic effect of curcumin and ABT-737. Collectively, these results indicated that the combination of curcumin and ABT-737 can efficaciously induce the death of HCC cells, and may offer a potential treatment strategy for patients with HCC.

Thioredoxin-2 inhibits mitochondrial reactive oxygen species generation and apoptosis stress kinase-1 activity to maintain cardiac function.

BACKGROUND: Thioredoxin 2 (Trx2) is a key mitochondrial protein that regulates cellular redox and survival by suppressing mitochondrial reactive oxygen species generation and by inhibiting apoptosis stress kinase-1 (ASK1)-dependent apoptotic signaling. To date, the role of the mitochondrial Trx2 system in heart failure pathogenesis has not been investigated. METHODS AND RESULTS: Western blot and histological analysis revealed that Trx2 protein expression levels were reduced in hearts from patients with dilated cardiomyopathy, with a concomitant increase in ASK1 phosphorylation/activity. Cardiac-specific Trx2 knockout mice develop spontaneous dilated cardiomyopathy at 1 month of age with increased heart size, reduced ventricular wall thickness, and a progressive decline in left ventricular contractile function, resulting in mortality due to heart failure by ≈4 months of age. The progressive decline in cardiac function observed in cardiac-specific Trx2 knockout mice was accompanied by the disruption of mitochondrial ultrastructure, mitochondrial membrane depolarization, increased mitochondrial reactive oxygen species generation, and reduced ATP production, correlating with increased ASK1 signaling and increased cardiomyocyte apoptosis. Chronic administration of a highly selective ASK1 inhibitor improved cardiac phenotype and reduced maladaptive left ventricular remodeling with significant reductions in oxidative stress, apoptosis, fibrosis, and cardiac failure. Cellular data from Trx2-deficient cardiomyocytes demonstrated that ASK1 inhibition reduced apoptosis and reduced mitochondrial reactive oxygen species generation. CONCLUSIONS: Our data support an essential role for mitochondrial Trx2 in preserving cardiac function by suppressing mitochondrial reactive oxygen species production and ASK1-dependent apoptosis. Inhibition of ASK1 represents a promising therapeutic strategy for the treatment of dilated cardiomyopathy and heart failure.

Development of an HTS-compatible assay for the discovery of ASK1 signalosome inhibitors using alphascreen technology.

Genetic target validation studies have demonstrated that the apoptosis signal-regulating kinase 1 (ASK1) represents an important target for the treatment of rheumatoid arthritis, cardiac diseases, and several neurodegenerative disorders. To identify small-molecule inhibitors of ASK1, we have developed a high-throughput screening-compatible, homogenous, biochemical assay using AlphaScreen technology. This novel assay design utilizes purified stress-activated ASK1 signalosome complex, and it monitors phosphorylation of its full-length native substrate, MKK6. The assay has been optimized in a 384-well format and validated by screening the Sigma LOPAC library. The results presented here demonstrate that the assay is sensitive and robust with a Z' factor value of 0.88±0.04 and a signal-to-background ratio of 11, indicating that this assay can be used to screen large chemical libraries to discover novel inhibitors of ASK1.

Unveiling the paradoxical nature of myelodysplastic syndromes (MDS): why hypercellular marrow strongly favors accelerated apoptosis.

The pathogenesis of bone marrow failure in myelodysplastic syndromes (MDS) is an unresolved mystery. MDS causes peripheral blood cytopenias and increased bone marrow cellularity. This apparent paradox has been interpreted as a sign of intramedullary destruction of a substantial portion of the developing hematopoietic cells by apoptosis. The present study aimed to delineate the exact mechanistic relationship between the bone marrow hypercellularity and the accelerated apoptosis in an N-ethyl-N-nitrosourea (ENU)-induced experimental MDS mouse model. The observations made so far clarify the quantitative and qualitative changes that occur in the bone marrow microenvironment through cell cycle analysis, especially involving the telomerase reverse transcriptase (TERT) and p53 expression patterns. The survival fate of the bone marrow cells were observed by measuring the expression level of some intracellular protein molecules like apoptosis signal-regulating kinase 1 (ASK-1), c-Jun N-terminal kinase (JNK), and cleaved caspase-3 of the extrinsic pathway toward apoptosis. We found myelodysplasia damage occurs within one or more multipotent progenitor populations resulting in uncontrolled cellular proliferation within the MDS bone marrow. Then, due to homeostatic balance, this high cellular burden is minimized by activating the apoptosis pathway. As a result, the peripheral blood suffers cellular deprivation. This study can throw some light on the mechanism of disease progression and also help to reveal the paradoxical nature of the disease.

Probucol inhibits hydrogen peroxide to induce apoptosis of vascular smooth muscle cells.

The aim of this study was to investigate the effect of the antioxidant probucol on hydrogen peroxide (H2O2)-induced vascular smooth muscle cell (VSMC) apoptosis. H2O2 (1 mmol/l) was used as the VSMC apoptosis-inducing agent, and 100, 10 and 1 µmol/l probucol concentrations were incubated with cells for 6 h. The cell apoptosis was observed using Annexin V-FITC, TUNEL and Hoechst 33258 staining methods. H2O2 promoted VSMC apoptosis, ASK-1 protein expression increased and Trx-l protein expression decreased. Probucol reduced H2O2-induced apoptosis in a dose-dependent manner; while ASK-1 protein expression decreased, Trx-1 protein expression increased. Probuco1 is capable of antagonizing the H2O2-induced apoptosis in VSMCs, which may be related to the decrease in ASK-1 protein expression and the increase in Trx-1 protein expression.

ASK1 overexpression accelerates paraquat-induced autophagy via endoplasmic reticulum stress.

Apoptosis signal-regulating kinase 1 (ASK1) is activated by various types of stress, including, endoplasmic reticulum (ER) stress. ER stress-induced ASK1 activation could play an important role both in neuronal apoptosis and an autophagic response in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease. The mechanism by which ASK1 executes apoptosis and/or autophagy under ER stress is still unclear. We have addressed this question using SH-SY5Y cells overexpressing wild-type (WT) ASK1. We show an important autophagic response and an acceleration of the paraquat (PQ)-induced autophagy with hallmarks as accumulation of autophagic vacuoles, activation of beclin-1, accumulation of LC3 II, p62 degradation, and mammalian target of rapamycin dephosphorylation. Inhibition of autophagy caused an exacerbation of the apoptosis induced by WT ASK1 overexpression with or without PQ. These data support the idea that the autophagic response could have a protector role. We found also an increase in the phosphorylation of the proteins such as IRE1 and eIF2α in response to both the overexpression of WT ASK1 and pesticide exposure. These data suggest that the WT ASK1 overexpression-induced autophagy is an event that occurs in parallel with ER stress activation. The importance of ER stress in the autophagy induced by ASK1 and/or PQ was confirmed with salubrinal, a selective inhibitor of eIF2α dephosphorylation. In conclusion, we report that PQ induces an early ER stress response that is correlated with the activation of autophagy as a protective response, which is accelerated in cells that overexpress WT ASK1. However, when the toxic stimuli remain, the cell eventually succumbs to apoptosis.

Activation of c-Jun N-terminal kinase is essential for mitochondrial membrane potential change and apoptosis induced by doxycycline in melanoma cells.

BACKGROUND AND PURPOSE: Tetracyclines were recently found to induce tumour cell death, but the early processes involved in this cytotoxic effect remain unclear. EXPERIMENTAL APPROACH: Viability of human and mouse melanoma cells was determined by MTT assay and flow cytometry. Kinase/protein/caspase activation was measured by Western blotting and mitochondrial membrane potential (DeltaPsi(m)) was analyzed by fluorescence microscopy and flow cytometry. KEY RESULTS: Human and mouse melanoma cells were treated with doxycycline or minocycline but only doxycycline was cytotoxic. This cell death (apoptosis) in A2058 cells involved activation of caspase-3, -7 and -9 and contributed to inhibition, by doxycycline, of matrix metalloproteinase (MMP) activity and migration of these cells. Doxycycline induced intra-cellular reactive oxygen species (ROS) production, apoptosis signal-regulated kinase 1 (ASK1), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) activation at an early stage of treatment and induced mitochondrial cytochrome c release into cytosol and DeltaPsi(m) change during apoptosis. The JNK inhibitor/small interference RNA inhibited doxycycline-induced JNK activation, DeltaPsi(m) change and apoptosis, but did not affect ASK1 activation, suggesting a role of ASK1 for JNK activation in melanoma cell apoptosis. Two ROS scavengers reduced doxycycline-induced JNK and caspase activation, and apoptosis. Taken together, the results suggest the involvement of a ROS-ASK1-JNK pathway in doxycycline-induced melanoma cell apoptosis. CONCLUSIONS AND IMPLICATIONS: We have shown a promising cytotoxic effect of doxycycline on melanoma cells, have identified ROS and ASK1 as the possible initiators and have demonstrated that JNK activation is necessary for doxycycline-induced melanoma cell apoptosis.

Japanese encephalitis virus down-regulates thioredoxin and induces ROS-mediated ASK1-ERK/p38 MAPK activation in human promonocyte cells.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes severe neurological disease with high mortality. Molecular mechanisms of JEV pathogenesis such as upstream apoptotic processes and pathways are not yet completely resolved or understood. In this study, JEV replication in human promonocyte cells induced time-dependent apoptosis and activated virus dose-dependent caspases 3, 8 and 9. Proteomic analysis demonstrated up- and down-regulated (more or less than 1.5-fold) proteins in JEV-infected promonocyte cells. Biological process categorization showed processes of antioxidation, free radical removal, and sulfur redox metabolism entailed many identified up- and down-regulated proteins. Down-regulation of thioredoxin, confirmed by using Western blotting, was involved in the apoptosis process of the oxidative stress response pathway. JEV infection caused increased intracellular ROS production and activation of ASK1-ERK/p38 MAPK signaling. ERK/p38 MAPK inhibitor PD98059 treatment definitely suppressed this apoptosis. Down-regulation of thioredoxin, increased intracellular ROS, and activation of ASK1-ERK/p38 MAPK signaling all were associated with JEV-induced apoptosis. These results are suggestive of an oxidative stress-pathway as a key element of JE pathogenesis.

Phosphorylation-dependent Lys63-linked polyubiquitination of Daxx is essential for sustained TNF-{alpha}-induced ASK1 activation.

Apoptosis signal-regulating kinase 1 (ASK1) is a key regulatory kinase in the proapoptotic response to various stresses. ASK1 phosphorylation of Daxx, an ASK1 activator protein, increases Daxx accumulation in cells and further enhances ASK1 activity through a positive feedback mechanism. Here, we show that ASK1-dependent phosphorylation of Daxx induces Lys(63) (K63)-linked polyubiquitination on Lys(122) of Daxx. Polyubiquitination is dispensable for Daxx accumulation or Daxx interaction with ASK1 because mutant Daxx deficient in polyubiquitin still exhibits ASK1-dependent accumulation and interaction with cellular ASK1. However, K63-linked Daxx polyubiquitination is required for tumor necrosis factor-alpha (TNF-alpha)-induced activation of ASK1. Therefore, K63-linked polyubiquitination of Daxx functions as a molecular switch to initiate and amplify the stress kinase response in the TNF-alpha signaling pathway.

Paraquat activates the IRE1/ASK1/JNK cascade associated with apoptosis in human neuroblastoma SH-SY5Y cells.

Epidemiologic and laboratory studies suggest that paraquat can be an environmental etiologic factor in Parkinson's disease (PD). One mechanism by which paraquat may mediate cell death of dopaminergic neurons is by inducing endoplasmic reticulum (ER) stress, as suggested in a recent report. In this study, we further investigated this linkage by examining ER stress cascades. To this aim, human neuroblastoma cells (SH-SY5Y cells) were treated with paraquat and the signaling cascades through which ER stress results in apoptosis were examined. Then, it was examined whether ER stress is produced by paraquat. Paraquat increased ER stress biomarker proteins, glucose-regulated protein 78 (GRP78), ER degradation-enhancing alpha-mannosidae-like protein (EDEM), and C/EBP homologous protein (CHOP). Then, it was investigated which ER stress cascades are affected by paraquat. Paraquat activated inositol-requiring enzyme 1 (IRE1), apoptosis signal regulating kinase 1 (ASK1), and c-jun kinase (JNK). Also, paraquat activated calpain and caspase 3, but did not affect the levels of intracellular calcium and the activity of caspase 12. Finally, apoptotic DNA damage by paraquat was investigated and this damage was attenuated by salubrinal (ER stress inhibitor), thioredoxin (ASK1 inhibitor) and SP600125 (JNK inhibitor). Therefore, current data indicate that paraquat activates the IRE1/ASK1/JNK cascade associated with apoptosis in SY5Y cells.

Apoptosis signal-regulating kinase 1 is crucial for oxidative stress-induced but not for osmotic stress-induced hepatocyte cell death.

AIMS: In this study, we investigated the involvement of apoptosis signal-regulating kinase 1 (ASK1) in oxidative stress and osmotic stress-induced hepatocyte death. MAIN METHODS: Activation of ASK1-JNK/p38 cascade and resulting cell death induced by oxidative and osmotic stress was investigated by Western immunoblot analysis and cell toxicity assay using human hepatoma cell lines, Huh7 expressing high level of ASK1 and HepG2 cells expressing low level of ASK1. Gene knock-down of ASK1 using shRNA against ASK1 was conducted using mouse hepatocyte cell line, AML12. KEY FINDINGS: Activation of ASK1-JNK/p38 cascade and cell death in Huh7 expressing high level of ASK1 was markedly induced by the oxidative stress. HepG2 expressing low level of ASK1 was resistant to oxidative stress while cell death induced by osmotic stress was comparable between Huh7 and HepG2 cells. Although the phosphorylation of ASK1 was not observed by osmotic stress, the phosphorylation of p38 and JNK and resulting cell death was induced in both cell lines. The phosphorylation of ASK1 and p38/JNK in the mouse primary hepatocyte were also increased by oxidative stress. Knock-down of ASK1 mRNA in AML12 in vitro significantly reduced oxidative stress-induced cell death, however, knock-down of ASK1 in cells did not affect the osmotic stress-induced cell death. SIGNIFICANCE: This study revealed that ASK1 regulates oxidative stress- but not osmotic stress-induced hepatocyte death, suggesting ASK1 plays a critical role in oxidative-stress induced hepatocyte death. These results raise the possibility that an ASK1 may be a promising therapeutic target for liver diseases caused by oxidative stress.

Expression of apoptosis signal-regulating kinase 1 in mouse spinal cord under chronic mechanical compression: possible involvement of the stress-activated mitogen-activated protein kinase pathways in spinal cord cell apoptosis.

STUDY DESIGN: To examine apoptosis signal cascade in neurons and oligodendrocytes under the chronic spinal cord compression of tiptoe-walking Yoshimura (TWY) mouse, which is model of progressive cervical cord compression. OBJECTIVE: To clarify the biologic mechanisms of apoptosis, which may produce destructive changes in the spinal cord under chronic mechanical compression, with a resulting irreversible neurologic deficit. SUMMARY OF BACKGROUND DATA: The stress-activated mitogen-activated protein kinase pathways including ASK1 transmitted apoptosis signals after acute spinal cord injury. Apoptosis in acute spinal cord injury induced both secondary degeneration around the site of injury and chronic demyelination. Chronic spinal cord compression showed myelin destruction, loss of axons, and oligodendrocytes in white matter, and loss of neurons in gray matter. Apoptosis associated with chronic spinal cord compression contributes to these changes. However, the biologic mechanisms of apoptosis in the spinal cord under chronic mechanical compression remain unclear. METHODS: We examined the expression of phosphorylated-apoptosis signal-regulating kinase 1 (ASK1), phosphorylated-c-Jun N-terminal kinase (JNK), phosphorylated-p38 mitogen-activated protein kinase (p38), and activated caspase-3 immunohistologically in TWY mice, an animal model of progressive cervical spinal cord compression, since the ASK1-JNK and -p38 signaling cascades participate in the signaling pathway leading to apoptosis in neural tissue and neuronal culture. RESULTS: Double immunohistochemistry for phosphorylated-ASK1, phosphorylated-JNK, phosphorylated-p38, activated-caspase3, and cell-specific markers confirmed the presence of apoptosis signals in both neurons and oligodendrocytes in compressed spinal cord cells. CONCLUSION: We found that mitogen-activated protein kinase pathways including ASK1, JNK, and p38 were activated in destructive spinal cord under chronic compression.