ASK family kinases mediate cellular stress and redox signaling to circadian clock.

Daily rhythms of behaviors and physiologies are generated by the circadian clock, which is composed of clock genes and the encoded proteins forming transcriptional/translational feedback loops (TTFLs). The circadian clock is a self-sustained oscillator and flexibly responds to various time cues to synchronize with environmental 24-h cycles. However, the key molecule that transmits cellular stress to the circadian clockwork is unknown. Here we identified apoptosis signal-regulating kinase (ASK), a member of the MAPKKK family, as an essential mediator determining the circadian period and phase of cultured cells in response to osmotic changes of the medium. The physiological impact of ASK signaling was demonstrated by a response of the clock to changes in intracellular redox states. Intriguingly, the TTFLs drive rhythmic expression of Ask genes, indicating ASK-mediated association of the TTFLs with intracellular redox. In behavioral analysis, Ask1, Ask2, and Ask3 triple-KO mice exhibited compromised light responses of the circadian period and phase in their activity rhythms. LC-MS/MS-based proteomic analysis identified a series of ASK-dependent and osmotic stress-responsive phosphorylations of proteins, among which CLOCK, a key component of the molecular clockwork, was phosphorylated at Thr843 or Ser845 in the carboxyl-terminal region. These findings reveal the ASK-dependent stress response as an underlying mechanism of circadian clock flexibility.

Structural Insights Support Targeting ASK1 Kinase for Therapeutic Interventions.

Apoptosis signal-regulating kinase (ASK) 1, a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, modulates diverse responses to oxidative and endoplasmic reticulum (ER) stress and calcium influx. As a crucial cellular stress sensor, ASK1 activates c-Jun N-terminal kinases (JNKs) and p38 MAPKs. Their excessive and sustained activation leads to cell death, inflammation and fibrosis in various tissues and is implicated in the development of many neurological disorders, such as Alzheimer's, Parkinson's and Huntington disease and amyotrophic lateral sclerosis, in addition to cardiovascular diseases, diabetes and cancer. However, currently available inhibitors of JNK and p38 kinases either lack efficacy or have undesirable side effects. Therefore, targeted inhibition of their upstream activator, ASK1, stands out as a promising therapeutic strategy for treating such severe pathological conditions. This review summarizes recent structural findings on ASK1 regulation and its role in various diseases, highlighting prospects for ASK1 inhibition in the treatment of these pathologies.

Apoptosis signal-regulating kinase (ASK-1) controls ovarian cell functions.

The involvement of the apoptosis signal-regulating kinase 1 (ASK1)-related signalling pathway in the control of reproduction is unknown. This study aimed to investigate the role of ASK-1 in the control of basic ovarian functions (proliferation, apoptosis and hormone release) and its response to ovarian hormonal regulators (leptin and FSH). We compared the accumulation of ASK-1, proliferation marker proliferating cell nuclear antigen (PCNA), apoptosis marker Bax and apoptosis and proliferation regulating transcription factor p53 and the release of progesterone (P4), oxytocin (OT), insulin-like growth factor I (IGF-I) and prostaglandins F (PGF) and E (PGE) using cultured porcine ovarian granulosa cells transfected with ASK-1 cDNA and cultured with leptin or FSH. This study is the first to demonstrate that ASK-1 does not affect cell apoptosis and viability in ovarian cells, but promotes cell proliferation, suppresses p53, alters the release of ovarian hormones (P4, OT, IGF-I, PGF and PGE) and defines their response to the upstream hormonal regulators leptin and FSH. Therefore, ASK-1 can be considered a new and important regulator of multiple ovarian functions.

Redox-Inactive Peptide Disrupting Trx1-Ask1 Interaction for Selective Activation of Stress Signaling.

Thioredoxin 1 (Trx1) and glutaredoxin 1 (Grx1) are two ubiquitous redox enzymes that are central for redox homeostasis but also are implicated in many other processes, including stress sensing, inflammation, and apoptosis. In addition to their enzymatic redox activity, a growing body of evidence shows that Trx1 and Grx1 play regulatory roles via protein-protein interactions with specific proteins, including Ask1. The currently available inhibitors of Trx1 and Grx1 are thiol-reactive electrophiles or disulfides that may suffer from low selectivity because of their thiol reactivity. In this report, we used a phage peptide library to identify a 7-mer peptide, 2GTP1, that binds to both Trx1 and Grx1. We further showed that a cell-permeable derivative of 2GTP1, TAT-2GTP1, disrupts the Trx1-Ask1 interaction, which induces Ask1 phosphorylation with subsequent activation of JNK, stabilization of p53, and reduced viability of cancer cells. Notably, as opposed to a disulfide-derived Trx1 inhibitor (PX-12), TAT-2GTP1 was selective for activating the Ask1 pathway without affecting other stress signaling pathways, such as endoplasmic reticulum stress and AMPK activation. Overall, 2GTP1 will serve as a useful probe for investigating protein interactions of Trx1.

Caspase recruitment domain 6 protects against hepatic ischemia/reperfusion injury by suppressing ASK1.

BACKGROUND & AIMS: The hepatic injury caused by ischemia/reperfusion (I/R) insult is predominantly determined by the complex interplay of sterile inflammation and liver cell death. Caspase recruitment domain family member 6 (CARD6) was initially shown to play important roles in NF-κB activation. In our preliminary studies, CARD6 downregulation was closely related to hepatic I/R injury in liver transplantation patients and mouse models. Thus, we hypothesized that CARD6 protects against hepatic I/R injury and investigated the underlying molecular mechanisms. METHODS: A partial hepatic I/R operation was performed in hepatocyte-specific Card6 knockout mice (HKO), Card6 transgenic mice with CARD6 overexpression specifically in hepatocytes (HTG), and the corresponding control mice. Hepatic histology, serum aminotransferases, inflammatory cytokines/chemokines, cell death, and inflammatory signaling were examined to assess liver damage. The molecular mechanisms of CARD6 function were explored in vivo and in vitro. RESULTS: Liver injury was alleviated in Card6-HTG mice compared with control mice as shown by decreased cell death, lower serum aminotransferase levels, and reduced inflammation and infiltration, whereas Card6-HKO mice had the opposite phenotype. Mechanistically, phosphorylation of ASK1 and its downstream effectors JNK and p38 were increased in the livers of Card6-HKO mice but repressed in those of Card6-HTG mice. Furthermore, ASK1 knockdown normalized the effect of CARD6 deficiency on the activation of NF-κB, JNK and p38, while ASK1 overexpression abrogated the suppressive effect of CARD6. CARD6 was also shown to interact with ASK1. Mutant CARD6 that lacked the ability to interact with ASK1 could not inhibit ASK1 and failed to protect against hepatic I/R injury. CONCLUSIONS: CARD6 is a novel protective factor against hepatic I/R injury that suppresses inflammation and liver cell death by inhibiting the ASK1 signaling pathway. LAY SUMMARY: The protein CARD6 plays an important role during the process of liver blood flow restriction (ischemia) and restoration (reperfusion). By suppressing the activity of ASK1, CARD6 can protect against hepatocyte injury. Targeting CARD6 is a potential strategy for prevention and treatment of ischemia/reperfusion injury.

BET Bromodomain inhibition promotes De-repression of TXNIP and activation of ASK1-MAPK pathway in acute myeloid leukemia.

BACKGROUND: Targeted therapy has always been the focus in developing therapeutic approaches in cancer, especially in the treatment of acute myeloid leukemia (AML). A new small molecular inhibitor, JQ1, targeting BRD4, which recognizes the acetylated lysine residues, has been shown to induce cell cycle arrest in different cancers by inhibiting MYC oncogene. However, the downstream signaling of MYC inhibition induced by BET inhibitor is not well understood. METHODS: In this study, we explored the more mechanisms of JQ1-induced cell death in acute myeloid lukemia and downstream signaling of JQ1. RESULTS: We found that JQ1 is able to reactivate the tumor suppressor gene, TXNIP, and induces apoptosis through the ASK1-MAPK pathway. Further studies confirmed that MYC could repress the expression of TXNIP through the miR-17-92 cluster. CONCLUSIONS: These findings provide novel insight on how BET inhibitor can induce apoptosis in AML, and further support the development of BET inhibitors as a promising therapeutic strategy against AML.

Ubiquitin-like sequence in ASK1 plays critical roles in the recognition and stabilization by USP9X and oxidative stress-induced cell death.

Ubiquitination is an important posttranslational modification that regulates various cellular processes, including signal transduction. However, physiological roles of ubiquitination in the regulation of MAPK pathways are poorly understood. Here, we identified the deubiquitinating enzyme USP9X as a binding partner of ASK1 that mediates oxidative stress-induced cell death through activation of the JNK and p38 MAPK pathways. In the recognition of ubiquitin by deubiquitinating enzymes, the importance of a tandem glycine-glycine sequence in the ubiquitin C terminus has been suggested. Interestingly, ASK1 contains six amino acids identical to the ubiquitin C terminus (LRLRGG), and the GG sequence of ASK1 was required for the USP9X-ASK1 interaction. We also found that USP9X interacted with oxidative stress-activated ASK1 and prevented it from undergoing ubiquitin-dependent degradation. In USP9X-deficient cells, oxidative stress-induced JNK activation and subsequent cell death were reduced. These results demonstrate that USP9X-dependent stabilization of activated ASK1 plays a crucial role in oxidative stress-induced cell death.

Targeting hepatic TRAF1-ASK1 signaling to improve inflammation, insulin resistance, and hepatic steatosis.

BACKGROUND & AIMS: Tumor necrosis factor receptor-associated factor 1 (TRAF1) is an important adapter protein that is largely implicated in molecular events regulating immunity/inflammation and cell death. Although inflammation is closely related to and forms a vicious circle with insulin dysfunction and hepatic lipid accumulation, the role of TRAF1 in hepatic steatosis and the related metabolic disorders remains unclear. METHODS: The participation of TRAF1 in the initiation and progression of hepatic steatosis was evaluated in high fat diet (HFD)-induced and genetic obesity. Mice with global TRAF1 knockout or liver-specific TRAF1 overexpression were employed to investigate the role of TRAF1 in insulin resistance, inflammation, and hepatic steatosis based on various phenotypic examinations. Molecular mechanisms underlying TRAF1-regulated hepatic steatosis were further explored in vivo and in vitro. RESULTS: TRAF1 expression was significantly upregulated in the livers of NAFLD patients and obese mice and in palmitate-treated hepatocytes. In response to HFD administration or in ob/ob mice, TRAF1 deficiency was hepatoprotective, whereas the overexpression of TRAF1 in hepatocytes contributed to the pathological development of insulin resistance, inflammatory response and hepatic steatosis. Mechanistically, hepatocyte TRAF1 promotes hepatic steatosis through enhancing the activation of ASK1-mediated P38/JNK cascades, as evidenced by the fact that ASK1 inhibition abolished the exacerbated effect of TRAF1 on insulin dysfunction, inflammation, and hepatic lipid accumulation. CONCLUSIONS: TRAF1 functions as a positive regulator of insulin resistance, inflammation, and hepatic steatosis dependent on the activation of ASK1-P38/JNK axis.

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.

Synergistic toxic interactions between CYP2E1, LPS/TNFα, and JNK/p38 MAP kinase and their implications in alcohol-induced liver injury.

The mechanisms by which alcohol causes cell injury are not clear. Many pathways have been suggested to play a role in how alcohol induces oxidative stress. Considerable attention has been given to alcohol-elevated production of lipopolysaccharide (LPS) and TNFα and to alcohol induction of CYP2E1. These two pathways are not exclusive of each other; however, associations and interactions between them, especially in vivo, have not been extensively evaluated. We have shown that increased oxidative stress from induction of CYP2E1 in vivo sensitizes hepatocytes to LPS and TNFα toxicity and that oxidative stress, activation of p38 and JNK MAP kinases, and mitochondrial dysfunction are downstream mediators of this CYP2E1-LPS/TNFα potentiated hepatotoxicity. This Review will summarize studies showing potentiated interactions between these two risk factors in promoting liver injury and the mechanisms involved including activation of the mitogen-activated kinase kinase kinase ASK-1 as a result of CYP2E1-derived reactive oxygen intermediates promoting dissociation of the inhibitory thioredoxin from ASK-1. This activation of ASK-1 is followed by activation of the mitogen-activated kinase kinases MKK3/MKK6 and MKK4/MMK7 and subsequently p38 and JNK MAP kinases. Synergistic toxicity occurs between CYP2E1 and the JNK1 but not the JNK2 isoform as JNK1 knockout mice are completely protected against CYP2E1 plus TNFα toxicity, elevated oxidative stress, and mitochondrial dysfunction. We hypothesize that similar interactions occur as a result of ethanol induction of CYP2E1 and TNFα.

ASK1/p38 signaling in renal tubular epithelial cells promotes renal fibrosis in the mouse obstructed kidney.

Stress-activated kinases p38 MAPK and JNK promote renal fibrosis; however, how the pathways by which these kinases are activated in kidney disease remain poorly defined. Apoptosis signal-regulating kinase 1 (ASK1/MAPKKK5) is a member of the MAPKKK family that can induce activation of p38 and JNK. The present study examined whether ASK1 induces p38/JNK activation and renal fibrosis in unilateral ureteric obstruction (UUO) using wild-type (WT) and Ask1-deficient (Ask1(-/-)) mice. Basal p38 and JNK activation in WT kidneys was increased three- to fivefold in day 7 UUO mice in association with renal fibrosis. In contrast, there was no increase in p38 activation in Ask1(-/-) UUO mice, whereas JNK activation was only partially increased. The progressive increase in kidney collagen (hydroxyproline) content seen on days 7 and 12 of UUO in WT mice was significantly reduced in Ask1(-/-) UUO mice in association with reduced α-smooth muscle actin-positive myofibroblast accumulation. However, cultured WT and Ask1(-/-) renal fibroblasts showed equivalent proliferation and matrix production, indicating that ASK1 acts indirectly on fibroblasts. Tubular epithelial cells are the main site of p38 activation in the obstructed kidney. Angiotensin II and H2O2, but not IL-1 or lipopolysaccharide, induced p38 activation and upregulation of transforming growth factor-ß1, platelet-derived growth factor-B, and monocyte chemoattractant protein-1 production was suppressed in Ask1(-/-) tubular epithelial cells. In addition, macrophage accumulation was significantly inhibited in Ask1(-/-) UUO mice. In conclusion, ASK1 is an important upstream activator of p38 and JNK signaling in the obstructed kidney, and ASK1 is a potential therapeutic target in renal fibrosis.

ASK1 promotes apoptosis of normal and malignant plasma cells.

Although the overproduction of immunoglobulins by short-lived plasma cells accompanying an immune response links with their apoptosis, how long-lived plasma cells adapt to ensure their longevity in this context is obscure. Here, we show that apoptosis signal-regulating kinase 1 (ASK1) contributes to apoptosis of plasma cells because ASK1 activity was induced during differentiation of short-lived plasma cells, and, when produced by ASK1-deficient mice, these cells survived better than those of control mice. Moreover, antigen-specific long-lived plasma cells generated by immunization accumulated in ASK1-deficient mice, suggesting ASK1 also plays a negative role in survival of long-lived plasma cells. In malignant plasma cells, ASK1 transcription was directly suppressed by B lymphocyte-induced maturation protein-1 (Blimp-1). The expression of ASK1 and Blimp-1 showed an inverse correlation between normal human mature B cells and bone marrow plasma cells from patients with multiple myeloma (MM). Suppression of ASK1 is crucial for cell survival because its enforced expression in MM cells caused apoptosis in vitro and lowered MM load in a xenograft animal model; furthermore, alteration of ASK1 activity affected MM cell survival. Our findings indicate a novel mechanism underlying the regulation of survival in normal and malignant plasma cells by ASK1.

[ASK1-MAP kinase signaling pathway as a therapeutic target for human diseases].

Apoptosis signal-regulating kinase 1 (ASK1) is a serine/threonine kinase belonging to the mitogen-activated protein kinase (MAPK) kinase kinase family. ASK1 is activated in response to various stresses, such as reactive oxygen species (ROS), tumor necrosis factor (TNF) alpha, lipopolysaccharide (LPS) and endoplasmic reticulum (ER) stress, and plays pivotal roles in a wide variety of cellular responses, including cell death, differentiation and inflammation. Recent studies have shown that ASK1-MAPK signaling pathway has key roles in human diseases induced by the dysfunction of cellular stress responses. In this review, we provide an overview of the current understanding of ASK1 as a potential therapeutic target for human diseases.

Apoptosis signal-regulating kinase-1 aggravates ROS-mediated striatal degeneration in 3-nitropropionic acid-infused mice.

Apoptosis signal-regulating kinase-1 (ASK1), an early signaling element in the cell death pathway, has been suggested to participate in the pathology of neurodegenerative diseases, which may be associated with environmental factors that impact the diseases. Although it is not entirely elucidated, 3-nitropropionic acid (3-NP) provokes mitochondrial dysfunction and selectively forms striatal lesions similar to those found in Huntington's disease. The current study investigated whether ASK1 is involved in striatal pathology following chronic systemic infusion of 3-NP. The results show that ASK1 acts as a primary mediator of there active oxygen species (ROS) cell death signal cascade in the 3-NP-damaged striatal region by disrupting the positive feedback cycle. In 3-NP-infused striatal lesions, ROS increased ASK1. Superoxide dismutase transgenic (SOD-tg) mice reduced ASK1by scavenging ROS, and reduction of ASK1leads to a reduction in cell death. However, ASK1 down-regulation in 3-NP infusion mice also decreased striatal cell death without scavenging ROS. In contrast decreasing cell death by si-ASK1 treatment along with 3-NP in both SOD tg and wild-type mice (wt), cell death rebounded when ASK1 peptide was added to SOD tg mice. The present study suggests that ROS-inducing ASK1 may be an important step in the pathogenesis of 3-NP infused striatal lesions in murine brains.

Pivotal role of apoptosis signal-regulating kinase 1 in monoclonal free light chain-mediated apoptosis.

Renal failure, a major complication associated with multiple myeloma, is usually related to deposition of monoclonal immunoglobulin free light chains (FLCs) and directly contributes to morbidity and mortality in this disease. The present study focused on the cytotoxic effects of monoclonal FLCs. Human proximal tubular epithelial cells (HK-2) were examined after incubation with two human monoclonal FLCs (termed κ2 and λ3). Incubation of HK-2 cells for 24 and 48 hours with either FLCs at 1 mg/mL promoted activation of caspase-9 and caspase-3 and increased the rate of apoptosis. Because prior studies demonstrated that FLCs generated intracellular oxidative stress, our studies focused on the redox-sensitive mitogen-activated protein kinase kinase kinase known as apoptosis signal-regulating kinase 1 (ASK1). A time-dependent increase in phosphorylation of ASK1 at T845, indicating activation of this enzyme, was observed. Small interfering RNA designed to reduce ASK1 expression in HK-2 cells successfully decreased ASK1, which was confirmed by Western blot analysis. Incubation of ASK1-depleted HK-2 cells with the two FLCs prevented the increase in apoptosis while pretreating HK-2 cell with nontargeting small interfering RNA did not prevent FLCs-mediated apoptosis. The combined data demonstrate that monoclonal FLCs activated the intrinsic apoptotic pathway in renal epithelial cells by activation of ASK1.

Apoptosis signal-regulating kinase 1 inhibits hepatocarcinogenesis by controlling the tumor-suppressing function of stress-activated mitogen-activated protein kinase.

UNLABELLED: The stress-activated mitogen-activated protein kinases (MAPKs), c-Jun NH2-terminal kinase (JNK), and p38 have been implicated in hepatocarcinogenesis. Although the many interrelated functions of JNK and p38 are precisely regulated by upstream signaling molecules, little is known about upstream regulators. We investigated the role of apoptosis signal-regulating kinase 1 (ASK1), a major player in the regulation of JNK and p38 activities, in hepatocarcinogenesis using a mouse hepatocellular carcinoma (HCC) model. ASK1-deficient (ASK1(-/-) ) and wildtype (WT) mice were treated with diethylnitrosamine on postnatal day 14. Strikingly, after 7 months, approximately three times as many tumors developed in ASK1(-/-) mice as in WT mice. Although JNK and p38 activation were attenuated in ASK1(-/-) HCCs relative to WT HCCs, cell proliferation was comparable in HCCs from both types of mice. On the other hand, both cancer cell apoptosis and hyperphosphorylation of BimEL, a proapoptotic Bcl-2 family member, were suppressed in the ASK1(-/-) HCCs. ASK1(-/-) mice showed remarkable resistance to Fas-induced hepatocyte apoptosis in vivo, probably because of attenuated JNK-mediated BimEL phosphorylation and mitochondrial apoptotic pathway activation. The reintroduction of ASK1 to ASK1(-/-) mouse liver using an adenoviral vector restored Fas-induced hepatocyte death and phosphorylation of JNK and BimEL. Similar findings were obtained in tumor necrosis factor alpha-induced hepatocyte apoptosis. Furthermore, ASK1 was involved in DNA damage-induced p21 up-regulation through a p38 pathway. CONCLUSION: ASK1 is involved in death receptor-mediated apoptosis and DNA-damage response by way of stress-activated MAPK in the liver, and thus acts as a tumor suppressor in hepatocarcinogenesis. This study provides new insight into the regulation of stress- activated MAPK signaling in hepatocarcinogenesis.

Inhibition of protein kinase Akt1 by apoptosis signal-regulating kinase-1 (ASK1) is involved in apoptotic inhibition of regulatory volume increase.

Most animal cell types regulate their cell volume after an osmotic volume change. The regulatory volume increase (RVI) occurs through uptake of NaCl and osmotically obliged water after osmotic shrinkage. However, apoptotic cells undergo persistent cell shrinkage without showing signs of RVI. Persistence of the apoptotic volume decrease is a prerequisite to apoptosis induction. We previously demonstrated that volume regulation is inhibited in human epithelial HeLa cells stimulated with the apoptosis inducer. Here, we studied signaling mechanisms underlying the apoptotic inhibition of RVI in HeLa cells. Hypertonic stimulation was found to induce phosphorylation of a Ser/Thr protein kinase Akt (protein kinase B). Shrinkage-induced Akt activation was essential for RVI induction because RVI was suppressed by an Akt inhibitor, expression of a dominant negative form of Akt, or small interfering RNA-mediated knockdown of Akt1 (but not Akt2). Staurosporine, tumor necrosis factor-alpha, or a Fas ligand inhibited both RVI and hypertonicity-induced Akt activation in a manner sensitive to a scavenger for reactive oxygen species (ROS). Any of apoptosis inducers also induced phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) in a ROS-dependent manner. Suppression of (ASK1) expression blocked the effects of apoptosis, in hypertonic conditions, on both RVI induction and Akt activation. Thus, it is concluded that in human epithelial cells, shrinkage-induced activation of Akt1 is involved in the RVI process and that apoptotic inhibition of RVI is caused by inhibition of Akt activation, which results from ROS-mediated activation of ASK1.

Inactivation of Ras GTPase-activating proteins promotes unrestrained activity of wild-type Ras in human liver cancer.

BACKGROUND & AIMS: Aberrant activation of the RAS pathway is ubiquitous in human hepatocarcinogenesis, but the molecular mechanisms leading to RAS induction in the absence of RAS mutations remain under-investigated. We defined the role of Ras GTPase activating proteins (GAPs) in the constitutive activity of Ras signaling during human hepatocarcinogenesis. METHODS: The mutation status of RAS genes and RAS effectors was assessed in a collection of human hepatocellular carcinomas (HCC). Levels of RAS GAPs (RASA1-4, RASAL1, nGAP, SYNGAP1, DAB2IP, and NF1) and the RASAL1 upstream inducer PITX1 were determined by real-time RT-PCR and immunoblotting. The promoter and genomic status of RASAL1, DAB2IP, NF1, and PITX1 were assessed by methylation assays and microsatellite analysis. Effects of RASAL1, DAB2IP, and PITX1 on HCC growth were evaluated by transfection and siRNA analyses of HCC cell lines. RESULTS: In the absence of Ras mutations, downregulation of at least one RAS GAP (RASAL1, DAB2IP, or NF1) was found in all HCC samples. Low levels of DAB2IP and PITX1 were detected mostly in a HCC subclass from patients with poor survival, indicating that these proteins control tumor aggressiveness. In HCC cells, reactivation of RASAL1, DAB2IP, and PITX1 inhibited proliferation and induced apoptosis, whereas their silencing increased proliferation and resistance to apoptosis. CONCLUSIONS: Selective suppression of RASAL1, DAB2IP, or NF1 RAS GAPs results in unrestrained activation of Ras signaling in the presence of wild-type RAS in HCC.

Beta-asarone attenuates beta-amyloid-induced apoptosis through the inhibition of the activation of apoptosis signal-regulating kinase 1 in SH-SY5Y cells.

Beta-amyloid (Abeta) toxicity has been postulated to initiate synaptic loss and subsequent neuronal degeneration seen in Alzheimer's disease (AD). We previously demonstrated that beta-asarone improves cognitive function by suppressing neuronal apoptosis in vivo. In this study, we assessed the neuroprotective effects of beta-asarone against the toxicity of Abeta in relation to the mitochondria-mediated cell death process, and to elucidated the role of the ASK1/MKK7/JNK and mitochondrial pathways in beta-asarone-induced neuroprotection in SH-SY5Y cells. Our results show that beta-asarone afforded protection against Abeta-induced toxicity by inhibiting apoptosis in SH-SY5Y cells. This result was also confirmed by caspase-9 and caspase-3 activity assays. Expression of p-ASK1, p-MKK7, p-JNK, Bax, Bad, and cytochrome c release decreased after pretreatment with beta-asarone in SH-SY5Y cells exposed to A1-42. Interestingly, these effects of beta-asarone against Abeta1-42 insult were enhanced by ASK1 siRNA. These findings suggest that beta-asarone prevents Abeta1-42-induced neurotoxicity through attenuating neuronal apoptosis, and might be a potential preventive or therapeutic agent for AD.

Apoptosis signal-regulating kinase 1 (ASK1) inhibition reduces endothelial cytokine production without improving permeability after toll-like receptor 4 (TLR4) challenge.

Sepsis represents a life-threatening event often mediated by the host's response to pathogens such as gram-negative organisms, which release the proinflammatory lipopolysaccharide (LPS). Within the endothelium, the mitogen-activated protein kinase (MAPK) pathway is an important driver of endothelial injury during sepsis, of which oxidant-sensitive apoptosis signal-regulating kinase 1 (ASK1) is postulated to be a critical upstream regulator. We hypothesized that ASK1 would play a key role in endothelial inflammation during bacterial challenge. Utilizing RNA sequencing data from patients and cultured human microvascular endothelial cells (HMVECs), ASK1 expression was increased in sepsis and after LPS challenge. Two ASK1 inhibitors, GS444217 and MSC2023964A, reduced cytokine production in HMVECs following LPS stimulation, but had no effect on permeability as measured by transendothelial electrical resistance and intercellular space. MAPKs are known to interact with endothelial nitric oxide synthase (eNOS) and ASK1 expression levels correlated with eNOS expression in patients with septic shock. In addition, eNOS physically interacted with ASK1, though this interaction was not altered by ASK1 inhibition, nor did inhibition alter MAPK p38 activity. Instead, among MAPKs, ASK1 inhibition only impaired LPS-induced JNK phosphorylation. The reduction in JNK activation caused by ASK1 inhibition impaired JNK-mediated cytokine production without affecting permeability. Thus, LPS triggers JNK-dependent cytokine production that requires ASK1 activation, but both its effects on permeability and activation of p38 are ASK1-independent. These data demonstrate how distinct MAPK signaling pathways regulate endothelial inflammatory outputs during acute infectious challenge.