Liver ASK1 protects from non-alcoholic fatty liver disease and fibrosis.

Non-alcoholic fatty liver disease (NAFLD) is strongly associated with obesity and may progress to non-alcoholic steatohepatitis (NASH) and liver fibrosis. The deficit of pharmacological therapies for the latter mainly results from an incomplete understanding of involved pathological mechanisms. Herein, we identify apoptosis signal-regulating kinase 1 (ASK1) as a suppressor of NASH and fibrosis formation. High-fat diet-fed and aged chow-fed liver-specific ASK1-knockout mice develop a higher degree of hepatic steatosis, inflammation, and fibrosis compared to controls. In addition, pharmacological inhibition of ASK1 increased hepatic lipid accumulation in wild-type mice. In line, liver-specific ASK1 overexpression protected mice from the development of high-fat diet-induced hepatic steatosis and carbon tetrachloride-induced fibrosis. Mechanistically, ASK1 depletion blunts autophagy, thereby enhancing lipid droplet accumulation and liver fibrosis. In human livers of lean and obese subjects, ASK1 expression correlated negatively with liver fat content and NASH scores, but positively with markers for autophagy. Taken together, ASK1 may be a novel therapeutic target to tackle NAFLD and liver fibrosis.

Role of ASK1/p38 Cascade in a Mouse Model of Alzheimer's Disease and Brain Aging.

To examine the role of ASK1 in Alzheimer's disease (AD), we generated 5XFAD mice deficient in ASK1 and investigated the characteristics of old 5XFAD and wild-type mice with ASK1 deficiency. ASK1 deficiency improved cognitive function in 24-month-old 5XFAD mice, which was associated with the reduction of phosphorylated p38. Thus, ASK1/p38 cascade seems to play some role in the pathogenesis of AD in mice. In 24-month-old wild-type mice, ASK1 deficiency increased cerebral vasoreactivity to acetazolamide and significantly reduced brain soluble Aß, which were also associated with the reduction of phosphorylated p38. Thus, ASK1/p38 cascade may contribute to brain aging of wild-type mice. Collectively, our present results provided the evidence suggesting the involvement of ASK1/p38 cascade in AD and brain aging.

Ask1 gene deletion blocks maternal diabetes-induced endoplasmic reticulum stress in the developing embryo by disrupting the unfolded protein response signalosome.

Apoptosis signal-regulating kinase 1 (ASK1) is activated by various stresses. The link between ASK1 activation and endoplasmic reticulum (ER) stress, two causal events in diabetic embryopathy, has not been determined. We sought to investigate whether ASK1 is involved in the unfolded protein response (UPR) that leads to ER stress. Deleting Ask1 abrogated diabetes-induced UPR by suppressing phosphorylation of inositol-requiring enzyme 1α (IRE1α), and double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) blocked the mitochondrial translocation of proapoptotic Bcl-2 members and ER stress. ASK1 participated in the IRE1α signalosome, and removing ASK1 abrogated the proapoptotic kinase activity of IRE1α. Ask1 deletion suppressed diabetes-induced IRE1α endoriboneclease activities, which led to X-box binding protein 1 mRNA cleavage, an ER stress marker, decreased expression of microRNAs, and increased expression of a miR-17 target, thioredoxin-interacting protein (Txnip), a thioredoxin binding protein, which enhanced ASK1 activation by disrupting the thioredoxin-ASK1 complexes. ASK1 is essential for the assembly and function of the IRE1α signalosome, which forms a positive feedback loop with ASK1 through Txnip. ASK1 knockdown in C17.2 neural stem cells diminished high glucose- or tunicamycin-induced IRE1α activation, which further supports our hypothesis that ASK1 plays a causal role in diabetes-induced ER stress and apoptosis.

[Inhibition of stress-responsive signaling pathway prevents neural cell death following optic nerve injury].

Optic nerve injury (ONI) induces retinal ganglion cell (RGC) death and optic nerve atrophy that lead to visual loss. Apoptosis signal-regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen-activated protein kinase kinase kinase (MAPKKK) and plays an important role in stress-induced RGC apoptosis. In this study, we found that ONI-induced p38 MAPK activation and RGC loss were suppressed in ASK1-deficient mice. Sequential in vivo retinal imaging revealed that post-ONI treatment with a p38 MAPK inhibitor into the eyeball was effective for RGC protection. ONI-induced monocyte chemotactic protein-1 (MCP-1) production in RGCs and microglial accumulation around RGCs were suppressed in ASK1-deficient mice. In addition, the productions of tumor necrosis factor (TNF) and inducible nitric oxide synthase (iNOS) in microglia were decreased when the ASK1-p38 MAPK pathway was blocked by inhibitor of ASK1 or p38 MAPK. ONI-induced expression of TNF and iNOS in the retina were absent in ASK1-deficient mice. These results suggest that ASK1 activation in both neural and glial cells is involved in neural cell death, and that pharmacological interruption of ASK1-p38 MAPK pathways could be beneficial in the treatment of ONI.

ASK1 promotes the contact hypersensitivity response through IL-17 production.

Contact hypersensitivity (CHS) is a form of delayed-type hypersensitivity triggered by the response to reactive haptens (sensitization) and subsequent challenge (elicitation). Here, we show that ASK1 promotes CHS and that suppression of ASK1 during the elicitation phase is sufficient to attenuate CHS. ASK1 knockout (KO) mice exhibited impaired 2,4-dinitrofluorobenzene (DNFB)-induced CHS. The suppression of ASK1 activity during the elicitation phase through a chemical genetic approach or a specific inhibitory compound significantly reduced the CHS response to a level similar to that observed in ASK1 KO mice. The reduced response was concomitant with the strong inhibition of production of IL-17, a cytokine that plays an important role in CHS and other inflammatory diseases, from sensitized lymph node cells. These results suggest that ASK1 is relevant to the overall CHS response during the elicitation phase and that ASK1 may be a promising therapeutic target for allergic contact dermatitis and other IL-17-related inflammatory diseases.

Apoptosis signal-regulating kinase 1 is a novel target molecule for cognitive impairment induced by chronic cerebral hypoperfusion.

OBJECTIVE: There are currently no specific strategies for the treatment or prevention of vascular dementia. White matter lesions, a common pathology in cerebral small vessel disease, are a major cause of vascular dementia. We investigated whether apoptosis signal-regulating kinase 1 (ASK1) might be a key molecule in cerebral hypoperfusion, associated with blood-brain barrier breakdown and white matter lesions. APPROACH AND RESULTS: A mouse model of cognitive impairment was developed by inducing chronic cerebral hypoperfusion in white matter including the corpus callosum via bilateral common carotid artery stenosis (BCAS) surgery. BCAS-induced white matter lesions caused cognitive decline in C57BL/6J (wild-type) mice but not in ASK1-deficient (ASK1(-/-)) mice. Phosphorylated ASK1 increased in wild-type mouse brains, and phosphorylated p38 and tumor necrosis factor-α expression increased in corpus callosum cerebral endothelial cells after BCAS in wild-type mice but not in ASK1(-/-) mice. BCAS decreased claudin-5 expression and disrupted blood-brain barrier in the corpus callosum of wild-type but not ASK1(-/-) mice. Cerebral nitrotyrosine was increased in wild-type and ASK1(-/-) BCAS mice. Cerebral phosphorylated ASK1 did not increase in wild-type mice treated with NADPH-oxidase inhibitor. A p38 inhibitor and NADPH-oxidase inhibitor mimicked the protective effect of ASK1 deficiency against cognitive impairment. Specific ASK1 inhibitor prevented cognitive decline in BCAS mice. In vitro oxygen-glucose deprivation and tumor necrosis factor-α stimulation caused the disruption of endothelial tight junctions from wild-type mice but not ASK1(-/-) mice. CONCLUSIONS: Oxidative stress-ASK1-p38 cascade plays a role in the pathogenesis of cognitive impairment, through blood-brain barrier breakdown via the disruption of endothelial tight junctions. ASK1 might be a promising therapeutic target for chronic cerebral hypoperfusion-induced cognitive impairment.

Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays a crucial role in stress-induced apoptosis. Recently, we have reported that suppressed macrophage apoptosis in ASK1 and apolipoprotein E double-knockout mice accelerates atheromatous plaques in the hyperlipidemia-induced atherosclerotic model. However, the pathogenic role of smooth muscle cell (SMC) apoptosis in atherosclerosis still remains unclear. We investigated neointimal remodeling in ligated carotid arteries of ASK1-deficient mice (ASK1(-/-)) for 3 weeks. ASK1(-/-) mice had significantly more suppressed intimal formation, inversely manifesting as potential anti-atherogenic aspects of ASK1 deficiency, characterized by fewer SMCs and less collagen synthesis; and fewer apoptotic SMCs, infiltrating T lymphocytes, and microvessels, associated with decreased apoptosis of luminal endothelial cells, compared with those of wild-type mice. Injured arteries of ASK1(-/-) mice also showed significantly down-regulated expression of pro-apoptotic markers, adhesion molecules, and pro-inflammatory signaling factors. Moreover, tumor necrosis factor-α-induced apoptosis was markedly suppressed in cultured aortic SMCs from ASK1(-/-) mice. These findings suggest that ASK1 accelerates mechanical injury-induced vascular remodeling with activated SMC migration via increased neovascularization and/or enhanced SMC and endothelial cell apoptosis. ASK1 expression, especially in the SMCs, might be crucial, and reciprocally responsible for various pro-atherogenic functions, and SMC apoptosis seems to be detrimental in this model.

Apoptosis signal-regulating kinase 1 mediates MPTP toxicity and regulates glial activation.

Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase 3 family, is activated by oxidative stress. The death-signaling pathway mediated by ASK1 is inhibited by DJ-1, which is linked to recessively inherited Parkinson's disease (PD). Considering that DJ-1 deficiency exacerbates the toxicity of the mitochondrial complex I inhibitor 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we sought to investigate the direct role and mechanism of ASK1 in MPTP-induced dopamine neuron toxicity. In the present study, we found that MPTP administration to wild-type mice activates ASK1 in the midbrain. In ASK1 null mice, MPTP-induced motor impairment was less profound, and striatal dopamine content and nigral dopamine neuron counts were relatively preserved compared to wild-type littermates. Further, microglia and astrocyte activation seen in wild-type mice challenged with MPTP was markedly attenuated in ASK⁻/⁻ mice. These data suggest that ASK1 is a key player in MPTP-induced glial activation linking oxidative stress with neuroinflammation, two well recognized pathogenetic factors in PD. These findings demonstrate that ASK1 is an important effector of MPTP-induced toxicity and suggest that inhibiting this kinase is a plausible therapeutic strategy for protecting dopamine neurons in PD.

Levodopa activates apoptosis signaling kinase 1 (ASK1) and promotes apoptosis in a neuronal model: implications for the treatment of Parkinson's disease.

Oxidative stress is implicated in the etiology of Parkinson's disease (PD), the second most common neurodegenerative disease. PD is treated with chronic administration of l-3,4-dihydroxyphenylalanine (levodopa, L-DOPA), and typically, increasing doses are used during progression of the disease. Paradoxically, L-DOPA is a pro-oxidant and induces cell death in cellular models of PD through disruption of sulfhydryl homeostasis involving loss of the thiol-disulfide oxidoreductase functions of the glutaredoxin (Grx1) and thioredoxin (Trx1) enzyme systems [Sabens, E. A., Distler, A. M., and Mieyal, J. J. (2010) Biochemistry 49 (12), 2715-2724]. Considering this loss of both Grx1 and Trx1 activities upon L-DOPA treatment, we sought to elucidate the mechanism(s) of L-DOPA-induced apoptosis. In other contexts, both the NFκB (nuclear factor κB) pathway and the ASK1 (apoptosis signaling kinase 1) pathway have been shown to be regulated by both Grx1 and Trx1, and both pathways have been implicated in cell death signaling in model systems of PD. Moreover, mixed lineage kinase (MLK) has been considered as a potential therapeutic target for PD. Using SHSY5Y cells as model dopaminergic neurons, we found that NFκB activity was not altered by L-DOPA treatment, and the selective MLK inhibitor (CEP-1347) did not protect the cells from L-DOPA. In contrast, ASK1 was activated with L-DOPA treatment as indicated by phosphorylation of its downstream mitogen-activated protein kinases (MAPK), p38 and JNK. Chemical inhibition of either p38 or JNK provided protection from L-DOPA-induced apoptosis. Moreover, direct knockdown of ASK1 protected from L-DOPA-induced neuronal cell death. These results identify ASK1 as the main pro-apoptotic pathway activated in response to L-DOPA treatment, implicating it as a potential target for adjunct therapy in PD.

Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.

OBJECTIVE: The pathogenic role of macrophage apoptosis in atherosclerosis is still debatable, but it is considered to be a suppressor of plaque progression in early stages but a promoter of plaque necrosis in advanced stages. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays a pivotal role in stress-induced apoptosis. In the current study, we investigated the functions of ASK1 in hyperlipidemia-induced atherosclerosis. METHODS AND RESULTS: We generated ASK1 and apolipoprotein E (apoE) double-knockout mice (ASK1(-/-)/apoE(-/-)) and analyzed atherosclerosis in ASK1(-/-)/apoE(-/-) mice fed a high-cholesterol diet for 12 weeks. ASK1(-/-)/apoE(-/-) mice had accelerated hyperlipidemia-induced atherosclerosis, which was characterized by less apoptosis of macrophages and fewer necrotic areas, and more macrophages and elastolysis compared with apoE(-/-) mice. Bone marrow transplantation from ASK1(-/-) or wild-type to apoE(-/-) mice confirmed the above observation that the recipient mice of ASK1(-/-) donors had more pronounced hyperlipidemia-induced atherosclerosis than recipient mice of wild-type donors. CONCLUSIONS: These findings suggest that ASK1 suppresses hyperlipidemia-induced atherosclerosis via increased macrophage apoptosis and that ASK1 may cause pronounced plaque vulnerability via necrotic core development.

ASK1 deficiency attenuates neural cell death in GLAST-deficient mice, a model of normal tension glaucoma.

Apoptosis signal-regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen-activated protein kinase (MAPK) kinase kinase and has an important role in stress-induced retinal ganglion cell (RGC) apoptosis. In the mammalian retina, glutamate/aspartate transporter (GLAST) is a major glutamate transporter, and the loss of GLAST leads to optic nerve degeneration similar to normal tension glaucoma (NTG). In GLAST⁻(/)⁻ mice, the glutathione level in the retina is decreased, suggesting the involvement of oxidative stress in NTG pathogenesis. To test this hypothesis, we examined the histology and visual function of GLAST(+/)⁻:ASK1⁻(/)⁻ and GLAST⁻(/)⁻:ASK1⁻(/)⁻ mice by multifocal electroretinograms. ASK1 deficiency protected RGCs and decreased the number of degenerating axons in the optic nerve. Consistent with this finding, visual function was significantly improved in GLAST(+/)⁻:ASK1⁻(/)⁻ and GLAST⁻(/)⁻:ASK1⁻(/)⁻ mice compared with GLAST(+/)⁻ and GLAST⁻(/)⁻ mice, respectively. The loss of ASK1 had no effects on the production of glutathione or malondialdehyde in the retina or on the intraocular pressure. Tumor necrosis factor (TNF)-induced activation of p38 MAPK and the production of inducible nitric oxide synthase were suppressed in ASK1-deficient Müller glial cells. In addition, TNF-induced cell death was suppressed in ASK1-deficient RGCs. These results suggest that ASK1 activation is involved in NTG-like pathology in both neural and glial cells and that interrupting ASK1-dependent pathways could be beneficial in the treatment of glaucoma, including NTG.

Apoptosis signal-regulating kinase 1 regulates colitis and colitis-associated tumorigenesis by the innate immune responses.

BACKGROUND & AIMS: Mitogen-activated protein kinase (MAPK) signaling pathways regulate multiple cellular functions and are implicated in the pathogenesis of inflammatory bowel disease and colitis-associated cancer (CAC). Apoptosis signal-regulating kinase 1 (ASK1) is a MAPK kinase kinase; little is known about the role of ASK1 in colonic disease. We assessed the involvement of ASK1 in the development of intestinal inflammation and CAC. METHODS: Dextran sodium sulfate (DSS) or Citrobacter rodentium was used to induce colitis in wild-type (WT) and ASK1 knock-out (ASK1(-/-)) mice; CAC was induced by azoxymethane injection followed by repeated intake of DSS by the mice. Primary macrophages were isolated from WT and ASK1(-/-) mice and used to investigate the involvement of ASK1 in innate immune responses. Bone marrow chimeric mice were used to study the contribution of myeloid cells to colitis activity. RESULTS: ASK1 deficiency increased susceptibility to colonic inflammation in both models of colitis. In vitro, ASK1(-/-) macrophages were impaired in their ability to kill bacteria and had increased susceptibility to bacterial-induced apoptosis, because p38 was inactivated. Expression of antiapoptotic genes was greatly reduced in ASK1(-/-) macrophages. WT mice given transplants of ASK1(-/-) mouse-derived bone marrow cells developed more severe DSS-induced colitis than mice with WT-derived bone marrow cells. In the CAC model, ASK1(-/-) mice developed more numerous and larger tumors than WT mice through increased colonic inflammation. CONCLUSIONS: ASK1 controls the development of intestinal inflammation and CAC through the regulation of innate immunity.

Hyperactivity in novel environment with increased dopamine and impaired novelty preference in apoptosis signal-regulating kinase 1 (ASK1)-deficient mice.

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein (MAP) kinase kinase kinase family member, which induces apoptosis in various cells through JNK and p38 MAP kinase cascades. In addition to apoptosis signaling, a number of recent in vitro studies have suggested that ASK1 may play roles in neural function. However, the behavioral significance of ASK1 has remained unclear. Here, we subjected ASK1 (-/-) mice to a battery of behavioral tests and found that they displayed temporary hyperactivity in an open-field test. Activities in the familiar field were normal, indicating that the hyperactivity observed was specific to the novel environment. ASK1 (-/-) mice also exhibited impairment of novelty preference 24h after training and superior performance on the rotarod test. Brain tissue contents of dopamine and 4-dihydroxyphenylacetic acid (DOPAC) were elevated in ASK1 (-/-) mice. Our findings thus demonstrate novel behavioral functions of ASK1, including regulation of locomotor activity, novelty preference, and motor coordination with dopaminergic transmission.

Apoptosis signal-regulating kinase-1 is involved in vascular endothelial and cardiac remodeling caused by nitric oxide deficiency.

Long-term treatment with N(omega)-nitro-l-arginine methylester (l-NAME), an NO synthase inhibitor, induces hypertension and cardiovascular injury. However, its precise mechanism is unknown. Using apoptosis signal-regulating kinase-1 (ASK1)-deficient mice, we investigated the role of ASK1 in cardiovascular injury caused by l-NAME treatment. l-NAME was orally administered to ASK1-deficient and C57BL/6J (wild) mice for 8 weeks. l-NAME treatment increased blood pressure of wild and ASK1-deficient mice to a similar extent, indicating no role of ASK1 in NO-deficient hypertension. l-NAME treatment significantly impaired acetylcholine-induced carotid arterial relaxation in wild mice (P<0.01), being associated with the decreased endothelial NO synthase (eNOS) activity (P<0.01) and the increased disruption of eNOS dimer (P<0.01), whereas these changes by l-NAME were substantially attenuated in ASK1-deficient mice. Thus, ASK1 is involved in the impairment of vascular endothelial function by reducing eNOS activity and disrupting eNOS dimer. l-NAME treatment increased vascular reduced nicotinamide-adenine dinucleotide phosphate oxidase activity and superoxide in wild mice to a greater extent than in ASK1 deficient mice. l-NAME treatment in wild mice caused cardiac hypertrophy, myocyte apoptosis, macrophage infiltration, coronary arterial remodeling, interstitial fibrosis, and the expression of monocyte chemoattractant protein-1 and transforming growth factor-beta1, whereas these cardiac changes by l-NAME were absent in ASK1-deficient mice. Cardiac reduced nicotinamide-adenine dinucleotide phosphate oxidase activation and superoxide elevation by l-NAME were much less in ASK1-deficient mice than in wild mice. Our work provided the first evidence that ASK1 is implicated in vascular endothelial dysfunction and cardiovascular remodeling induced by NO deficiency by regulating eNOS and reduced nicotinamide-adenine dinucleotide phosphate oxidase.

ASK1 is activated by arsenic trioxide in leukemic cells through accumulation of reactive oxygen species and may play a negative role in induction of apoptosis.

Arsenic trioxide (ATO) is remarkably effective for treating acute promyelocytic leukemia. Here, we find that ATO treatment of NB4 and K562 leukemic cells induces activation of ASK1. ASK1 activation was induced most significantly at low concentrations of ATO, where G2/M arrest but not apoptosis was induced. On the other hand, ATO barely activated ASK1 at high concentrations, where apoptosis as well as activation of JNK and p38 was induced significantly. ATO-induced accumulation of reactive oxygen species (ROS), while the ASK1 activation was suppressed by cotreatment with an antioxidant, N-acetyl-l-cysteine. Murine embryonic fibroblasts (MEFs) from ASK1-deficient mice were more susceptible to ATO-induced apoptosis than control MEFs. Furthermore, ATO at the low concentration induced significant apoptosis in K562 cells when ASK1 was knocked down by siRNA. These results indicate that ASK1 is activated by ATO through ROS accumulation and may negatively regulate apoptosis in leukemic cells without activating p38 and JNK.

Apoptosis signal-regulating kinase 1 is involved not only in apoptosis but also in non-apoptotic cardiomyocyte death.

The molecular basis of myocardial cell death in the ischemia-reperfused heart still remains to be clarified. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays an important role in stress-induced apoptosis. We studied ASK1(-/-) mice to examine the role of ASK1 in ischemia-reperfusion injury. In the wild-type heart, ischemia-reperfusion resulted in necrotic injury, whereas infarct size was drastically reduced in the ASK1(-/-) heart. The necrotic injury was not accompanied with any evidence of apoptosis such as an increase in TUNEL-positive cells, DNA fragmentation or the activation of caspase-3. ASK1(-/-) cardiomyocytes were more resistant to H(2)O(2)- or Ca(2+)-induced apoptotic and non-apoptotic cell death compared with wild-type cells. These data suggest that ASK1 is involved in necrosis as well as apoptosis and that ASK1-dependent necrosis is likely to contribute to myocardial cell death in the ischemia-reperfused heart.

ROS-dependent activation of the TRAF6-ASK1-p38 pathway is selectively required for TLR4-mediated innate immunity.

Apoptosis signal-regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen-activated protein 3-kinase that activates both Jnk and p38 mitogen-activated protein kinases. Here we used ASK1-deficient mice to show that ASK1 was selectively required for lipopolysaccharide-induced activation of p38 but not of Jnk or the transcription factor NF-kappaB. ASK1 was required for the induction of proinflammatory cytokines dependent on Toll-like receptor 4 (TLR4) but not TLR2 or other TLRs. Consistent with this, ASK1-deficient mice were resistant to lipopolysaccharide-induced septic shock. Lipopolysaccharide induced the production of intracellular reactive oxygen species, which was required for the formation of a complex of the adaptor molecule TRAF6 and ASK1 and subsequent activation of the ASK1-p38 pathway. Our data demonstrate that the reactive oxygen species-dependent TRAF6-ASK1-p38 axis is crucial for TLR4-mediated mammalian innate immunity.