Isao Naguro

Requirement of Reactive Oxygen Species-dependent Activation of ASK1-p38 MAPK Pathway for Extracellular ATP-induced Apoptosis in Macrophage

Extracellular ATP, an autocrine or paracrine intercellular transmitter, is known to induce apoptosis in macrophages. However, the precise signaling mechanisms of ATP-induced apoptosis remain to be elucidated. Here we showed that activation of p38 mitogen-activated protein kinase (MAPK) plays a critical role in ATP-induced apoptosis. p38 activation and apoptosis in macrophages were induced by ATP. ATP-induced apoptosis was mediated in part by production of reactive oxygen species (ROS) derived from NOX2/gp91phox, a component of the NADPH oxidase complex expressed in macrophages and neutrophils. Furthermore, ATP-induced ROS generation, p38 activation, and apoptosis were almost completely inhibited by selective P2X7 receptor antagonists. We also found that ATP-induced apoptosis were diminished in ASK1-deficient macrophages accompanied by the lack of p38 activation. These results demonstrate that ROS-mediated activation of the ASK1-p38 MAPK pathway downstream of P2X7 receptor is required for ATP-induced apoptosis in macrophages.

The roles of ASK family proteins in stress responses and diseases

Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein kinase kinase kinase family, which activates c-Jun N-terminal kinase and p38 in response to a diverse array of stresses such as oxidative stress, endoplasmic reticulum stress and calcium influx. In the past decade, various regulatory mechanisms of ASK1 have been elucidated, including its oxidative stress-dependent activation. Recently, it has emerged that ASK family proteins play key roles in cancer, cardiovascular diseases and neurodegenerative diseases. In this review, we summarize the recent findings on ASK family proteins and their implications in various diseases.

ASK1 and ASK2 differentially regulate the counteracting roles of apoptosis and inflammation in tumorigenesis

Apoptosis and inflammation generally exert opposite effects on tumorigenesis: apoptosis serves as a barrier to tumour initiation, whereas inflammation promotes tumorigenesis. Although both events are induced by various common stressors, relatively little is known about the stress‐induced signalling pathways regulating these events in tumorigenesis. Here, we show that stress‐activated MAP3Ks, ASK1 and ASK2, which are involved in cellular responses to various stressors such as reactive oxygen species, differentially regulate the initiation and promotion of tumorigenesis. ASK2 in cooperation with ASK1 functioned as a tumour suppressor by exerting proapoptotic activity in epithelial cells, which was consistent with the reduction in ASK2 expression in human cancer cells and tissues. In contrast, ASK1‐dependent cytokine production in inflammatory cells promoted tumorigenesis. Our findings suggest that ASK1 and ASK2 are critically involved in tumorigenesis by differentially regulating apoptosis and inflammation.

Apoptosis Signal-regulating Kinase (ASK) 2 Functions as a Mitogen-activated Protein Kinase Kinase Kinase in a Heteromeric Complex with ASK1

Apoptosis signal-regulating kinase (ASK) 1 is a mitogen-activated protein kinase kinase kinase (MAP3K) in the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase pathways that play multiple important roles in cytokine and stress responses. Here we show that ASK2, a highly related serine/threonine kinase to ASK1, also functions as a MAP3K only in a heteromeric complex with ASK1. We found that endogenous ASK2 was constitutively degraded in ASK1-deficient cells, suggesting that ASK1 is required for the stability of ASK2. ASK2 in a heteromeric complex with a kinase-negative mutant of ASK1 (ASK1-KN) effectively activated MAP2K and was more competent to respond to oxidative stress than ASK2 alone. Knockdown of ASK2 revealed that ASK2 was required for oxidative stress-induced JNK activation. These results suggest that ASK2 forms a functional MAP3K complex with ASK1, in which ASK1 supports the stability and the active configuration of ASK2. Moreover, ASK2 was found to activate ASK1 by direct phosphorylation, suggesting that ASK1 and ASK2 in a heteromeric complex facilitate their activities to each other by distinct mechanisms. Such a formation of functional heteromeric complex between different MAP3Ks may be advantageous for cells to cope with a wide variety of stimuli by fine regulation of cellular responses.

Stress-Activated MAP Kinase Cascades in Cellular Senescence

In response to progressive telomere shortening in successive cell divisions, normal somatic cells withdraw from the cell cycle and exhibit irreversible growth arrest. This state, called cellular senescence, is induced not only by telomere shortening but also by various physico-chemical stressors that induce DNA damage and chromatin disruption as well as by strong mitogenic signals. Because senescent cells never re-enter the cell cycle, cellular senescence appears to prevent malignant transformation of damaged cells and thus contributes to tumor suppression. On the other hand, excess accumulation of senescent cells attenuates the integrity and normal function of tissues, leading to age-related diseases. In addition to the well-established roles of p53 and pRB in cellular senescence, recent evidence suggests that stress-activated mitogen-activated protein kinase (MAPK) cascades that converge on c-Jun N-terminal kinases (JNKs) and p38 MAPKs also play important roles in the regulation of cellular senescence. In this review, we focus on signaling that regulates stress-induced cellular senescence, with special focus on the JNK and p38 MAPK cascades.

Activation mechanisms of ASK1 in response to various stresses and its significance in intracellular signaling.

Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein kinase kinase kinase family. ASK1 activates c-jun N-terminal kinase (JNK) and p38 in response to various stimuli such as oxidative stress, endoplasmic reticulum stress, infection and calcium influx. Under these stress conditions, ASK1 plays important roles in intracellular signaling pathways and biological functions. Diverse proteins are known to interact with ASK1 and regulate the activity of ASK1. However, activation mechanisms of ASK1 and ASK1-binding proteins which regulate the activity of ASK1 have not been completely understood. In this review, we focus on the recent findings on ASK1 and update the regulatory mechanisms of ASK1 activity.

SOD1 as a Molecular Switch for Initiating the Homeostatic ER Stress Response under Zinc Deficiency

Zinc is an essential trace element, and impaired zinc homeostasis is implicated in the pathogenesis of various human diseases. However, the mechanisms cells use to respond to zinc deficiency are poorly understood. We previously reported that amyotrophic lateral sclerosis (ALS)-linked pathogenic mutants of SOD1 cause chronic endoplasmic reticulum (ER) stress through specific interactions with Derlin-1, which is a component of the ER-associated degradation machinery. Moreover, we recently demonstrated that this interaction is common to ALS-linked SOD1 mutants, and wild-type SOD1 (SOD1(WT)) comprises a masked Derlin-1 binding region (DBR). Here, we found that, under zinc-deficient conditions, SOD1(WT) adopts a mutant-like conformation that exposes the DBR and induces the homeostatic ER stress response, including the inhibition of protein synthesis and induction of a zinc transporter. We conclude that SOD1 has a function as a molecular switch that activates the ER stress response, which plays an important role in cellular homeostasis under zinc-deficient conditions.

Apoptosis signal-regulating kinase 1 as a therapeutic target

Introduction: All organisms are ordinarily exposed to various stresses. It is important for organisms to possess appropriate stress response mechanisms and to maintain homeostasis because the disruption of a stress response system can cause various diseases. Apoptosis signal-regulating kinase 1 (ASK1) is one of the stress-responsive MAP3Ks. ASK1 plays an important role in the response to reactive oxygen species (ROS), endoplasmic reticulum stress and pro-inflammatory cytokines, and it is involved in the pathogenesis of various diseases. Areas covered: In this review, the authors describe recent literature concerning the intricate and elaborate regulation system of ASK1, the function of ASK1 during a cellular stress response and the involvement of ASK1 in many diseases, including cancer, neurodegenerative diseases, infections, diabetes and cardiovascular diseases. Expert opinion: In certain disease conditions, ASK1 plays a protective role, whereas ASK1 can exacerbate the pathology of other diseases. Although ASK1 is involved in various diseases, there is no therapy or drug that targets ASK1 for use in a clinical setting. Recently, ASK1 inhibitors (K811 and MSC2032964A) have emerged, and their therapeutic potentials have been tested in vivo. ASK1 is currently receiving considerable attention as a new therapeutic target.

A novel monoclonal antibody reveals a conformational alteration shared by amyotrophic lateral sclerosis-linked SOD1 mutants

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by the selective loss of upper and lower motoneurons. Although >100 different Cu, Zn superoxide dismutase (SOD1) mutations have been identified in ALS patients, it remains controversial whether all of them are disease‐causative mutations. Therefore, it is necessary to develop molecular mechanism‐based diagnosis and treatment of ALS caused by SOD1 mutations.

Roquin-2 Promotes Ubiquitin-Mediated Degradation of ASK1 to Regulate Stress Responses

Roquin-2 promotes degradation of the kinase ASK1 to mediate an appropriate response to oxidative stress and infection. ASK Under Stress Cells produce reactive oxygen species (ROS) to defend against infection, but sustained ROS signaling such as that associated with oxidative stress can lead to cell death. ROS activates the protein kinase ASK1, which promotes phosphorylation of downstream stress-responsive proteins. However, prolonged ASK1 activity can lead to cell death, and ROS-induced degradation of ASK1 by the ubiquitin-proteasome pathway is required to prevent cell death. Maruyama et al. identified Roquin-2 in a screen in human cultured cells of E3 ligases that promote ASK1 degradation. Roquin-2 promoted the ubiquitination of ROS-activated ASK1, and loss of Roquin-2 increased the abundance of ASK1, sustained the activation of downstream proteins, and reduced viability in human cancer cells exposed to exogenous ROS. In worms, genetic ablation of the Caenorhabditis elegans homolog of Roquin-2 increased the abundance of ASK1 and delayed the death of worms after exposure to lethal bacteria. Knocking down ASK1 in Roquin-2–deficient worms increased susceptibility to death due to bacterial infection. Thus, the ability of Roquin-2 to control ASK1 activity is an evolutionarily conserved module involved in oxidative signaling responses. Apoptosis signal–regulating kinase 1 (ASK1, also known as MAP3K5) mediates reactive oxygen species (ROS)–induced cell death. When activated by ROS, ASK1 ultimately becomes ubiquitinated and degraded by the proteasome, a process that is antagonized by the ubiquitin-specific protease USP9X. Using a functional siRNA (small interfering RNA) screen in HeLa cells, we identified Roquin-2 (also called RC3H2) as an E3 ubiquitin ligase required for ROS-induced ubiquitination and degradation of ASK1. In cells treated with H2O2, knockdown of Roquin-2 promoted sustained activation of ASK1 and the downstream stress-responsive kinases JNK (c-Jun amino-terminal kinase) and p38 MAPK (mitogen-activated protein kinase), and led to cell death. The nematode Caenorhabditis elegans produces ROS as a defense mechanism in response to bacterial infection. In C. elegans, mutation of the gene encoding the Roquin-2 ortholog RLE-1 promoted accumulation of the activated form of the ASK1 ortholog NSY-1 and conferred resistance to infection by the bacteria Pseudomonas aeruginosa. Thus, these data suggest that degradation of ASK1 mediated by Roquin-2 is an evolutionarily conserved mechanism required for the appropriate regulation of stress responses, including pathogen resistance and cell death.