Activation Of Apoptosis Signal-regulating Kinase Research Articles

Apoptosis signal-regulating kinase 1 exhibits oncogenic activity in pancreatic cancer.

Pancreatic cancer has an extremely grim prognosis, with an overall 5-year survival rate less than 5%, as a result of its rapid metastasis and late diagnosis. To combat this disease, it is crucial to better understand the molecular mechanisms that contribute to its pathogenesis. Herein, we report that apoptosis signal-regulating kinase 1 (ASK1) is overexpressed in pancreatic cancer tissues and that its expression correlates with the histological grade of pancreatic cancer. The expression of ASK1 is also elevated in pancreatic cancer cell lines at both protein and mRNA levels. In addition, ASK1 promotes the proliferation and stimulates the tumorigenic capacity of pancreatic cancer cells. These functions of ASK1 are abrogated by pharmacological inhibition of its kinase activity or by introduction of a kinase-dead mutation, suggesting that the kinase activity of ASK1 is required for its role in pancreatic cancer. However, the alteration of ASK1 expression or activity does not significantly affect the migration or invasion of pancreatic cancer cells. Collectively, these findings reveal a critical role for ASK1 in the development of pancreatic cancer and have important implications for the diagnosis and treatment of this malignancy.

Structural Insight into the 14-3-3 Protein-dependent Inhibition of Protein Kinase ASK1 (Apoptosis Signal-regulating kinase 1)

Apoptosis signal-regulating kinase 1 (ASK1, also known as MAP3K5), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, regulates diverse physiological processes. The activity of ASK1 is triggered by various stress stimuli and is involved in the pathogenesis of cancer, neurodegeneration, inflammation, and diabetes. ASK1 forms a high molecular mass complex whose activity is, under non-stress conditions, suppressed through interaction with thioredoxin and the scaffolding protein 14-3-3. The 14-3-3 protein binds to the phosphorylated Ser-966 motif downstream of the ASK1 kinase domain. The role of 14-3-3 in the inhibition of ASK1 has yet to be elucidated. In this study we performed structural analysis of the complex between the ASK1 kinase domain phosphorylated at Ser-966 (pASK1-CD) and the 14-3-3ζ protein. Small angle x-ray scattering (SAXS) measurements and chemical cross-linking revealed that the pASK1-CD·14-3-3ζ complex is dynamic and conformationally heterogeneous. In addition, structural analysis coupled with the results of phosphorus NMR and time-resolved tryptophan fluorescence measurements suggest that 14-3-3ζ interacts with the kinase domain of ASK1 in close proximity to its active site, thus indicating this interaction might block its accessibility and/or affect its conformation.

Hypoxia-inducible factor-1α mediates the toll-like receptor 4 signaling pathway leading to anti-tumor effects in human hepatocellular carcinoma cells under hypoxic conditions.

Hypoxia-inducible factor-1α (HIF-1α) and toll-like receptor 4 (TLR4) are involved in numerous mechanisms of cancer biology, including cell proliferation and survival; however the interaction of the two factors under hypoxic conditions remains unclear. The present study investigated the in vitro mechanism that results in the suppression of tumor cell growth and cellular functions when HIF-1α is silenced. In the present study, the human hepatocellular carcinoma HepG2 cell line was transfected with short hairpin RNA (shRNA) against HIF-1α and cultured under hypoxic conditions (1% O2 for 24 h). The expression of HIF-1α and various growth factors, including epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2), were examined using quantitative polymerase chain reaction and immunoblotting. Tumor growth was measured using a Cell Counting Kit-8 assay and tumor activity was measured using tumor cell invasion and migration assays. Lipopolysaccharide and TAK-242 were used to activate and inhibit TLR4, respectively, to observe the role of TLR4 in the HIF-1α silenced tumor cells. The expression of TLR4 signaling pathway associates, including myeloid differentiation primary response gene 88 (MyD88), apoptosis signal-regulating kinase 1 (ASK1), p38 mitogen-activated protein kinases and HIF-1α, were analyzed by western blot assay. Under hypoxic conditions, silencing of HIF-1α expression suppressed tumor cell growth and regulated the expression of tumor growth-associated genes, including EGF, HGF, VEGF and FG2. Suppression of tumor cell invasion and migration was also observed in the HIF-1α silenced HepG2 cell line. In addition, TLR4 was identified to be involved in HIF-1α and MyD88 accumulation, and activation of ASK1 and p38 were demonstrated to be critical for TLR4-mediated HIF-1α pathway. In conclusion, silencing of HIF-1α expression may induce anti-tumor effects under hypoxic conditions in HepG2 cells via the TLR4 mediated pathway, suggesting that the HIF-1α/TLR4 signaling cohort may act as a novel therapeutic target for the treatment of hepatocellular cancer.

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.

Glycogen synthase kinase-3β antagonizes ROS-induced hepatocellular carcinoma cell death through suppression of the apoptosis signal-regulating kinase 1.

Glycogen synthase kinase-3β (GSK-3β), a multifunctional kinase, is an important regulator of cancer cell survival. Apoptosis signal-regulating kinase 1 (ASK1) is also a key factor for controlling several cellular events including the cell cycle, senescence, and apoptosis, in response to reactive oxygen species (ROS). The role of GSK-3β regulating the activity and protein level of ASK1 in the cancer cells remains largely unexplored. In this study, we showed that GSK-3β inhibits ROS-induced hepatocellular carcinoma cell death by suppressing ASK1. We first found that ectopic expression of GSK-3β suppressed hydrogen peroxide (H2O2)-induced cell death in HepG2 cells and knockdown of endogenous GSK-3β expression exhibited opposite effects. Moreover, GSK-3β expression clearly inhibited H2O2-induced phosphorylation of ASK1 in HepG2 cells, in association with a decrease in ASK1 protein level. Further exploration revealed that GSK-3β induced ubiquitination and proteasome-dependent degradation of ASK1 via inhibition of ubiquitin-specific protease USP9X. Our results thus suggest that GSK-3β is a key factor involved in ASK1 activation and ROS-induced cell death.

Indolin-2-one compounds targeting thioredoxin reductase as potential anticancer drug leads

Several compounds bearing the indolinone chemical scaffold are known to possess anticancer properties. For example, the tyrosine kinase inhibitor sunitinib is an arylideneindolin-2-one compound. The chemical versatility associated with structural modifications of indolinone compounds underlies the potential to discover additional derivatives possessing anticancer properties. Previously synthesized 3-(2-oxoethylidene)indolin-2-one compounds, also known as supercinnamaldehyde (SCA) compounds in reference to the parent compound 1 [1-methyl-3(2-oxopropylidene)indolin-2-one], bear a nitrogen-linked α,β-unsaturated carbonyl (Michael acceptor) moiety. Here we found that analogs bearing N-substituents, in particular compound 4 and 5 carrying an N-butyl and N-benzyl substituent, respectively, were strongly cytotoxic towards human HCT 116 colorectal and MCF-7 breast carcinoma cells. These compounds also displayed strong thioredoxin reductase (TrxR) inhibitory activity that was likely attributed to the electrophilicity of the Michael acceptor moiety. Their selectivity towards cellular TrxR inhibition over related antioxidant enzymes glutathione reductase (GR), thioredoxin (Trx) and glutathione peroxidase (GPx) was mediated through targeting of the selenocysteine (Sec) residue in the highly accessible C-terminal active site of TrxR. TrxR inhibition mediated by indolin-2-one compounds led to cellular Trx oxidation, increased oxidative stress and activation of apoptosis signal-regulating kinase 1 (ASK1). These events also led to activation of p38 and JNK mitogen-activated protein kinase (MAPK) signaling pathways, and cell death with apoptotic features of PARP cleavage and caspase 3 activation. In conclusion, these results suggest that indolin-2-one-based compounds specifically targeting TrxR may serve as novel drug leads for anticancer therapy.

Lys29-linkage of ASK1 by Skp1-Cullin 1-Fbxo21 ubiquitin ligase complex is required for antiviral innate response.

Protein ubiquitination regulated by ubiquitin ligases plays important roles in innate immunity. However, key regulators of ubiquitination during innate response and roles of new types of ubiquitination (apart from Lys48- and Lys63-linkage) in control of innate signaling have not been clearly understood. Here we report that F-box only protein Fbxo21, a functionally unknown component of SCF (Skp1-Cul1-F-box protein) complex, facilitates Lys29-linkage and activation of ASK1 (apoptosis signal-regulating kinase 1), and promotes type I interferon production upon viral infection. Fbxo21 deficiency in mice cells impairs virus-induced Lys29-linkage and activation of ASK1, attenuates c-Jun N-terminal kinase (JNK) and p38 signaling pathway, and decreases the production of proinflammatory cytokines and type I interferon, resulting in reduced antiviral innate response and enhanced virus replication. Therefore Fbxo21 is required for ASK1 activation via Lys29-linkage of ASK1 during antiviral innate response, providing mechanistic insights into non-proteolytic roles of SCF complex in innate immune response.

EM23, A Natural Sesquiterpene Lactone from Elephantopus mollis, Induces Apoptosis in Human Myeloid Leukemia Cells through Thioredoxin- and Reactive Oxygen Species-Mediated Signaling Pathways.

Elephantopus mollis (EM) is a traditional herbal medicine with multiple pharmacological activities. However, the efficacy of EM in treating human leukemia is currently unknown. In the current study, we report that EM23, a natural sesquiterpene lactone isolated from EM, inhibits the proliferation of human chronic myeloid leukemia (CML) K562 cells and acute myeloid leukemia (AML) HL-60 cells by inducing apoptosis. Translocation of membrane-associated phospholipid phosphatidylserines, changes in cell morphology, activation of caspases, and cleavage of PARP were concomitant with this inhibition. The involvement of the mitochondrial pathway in EM23-mediated apoptosis was suggested by observed disruptions in mitochondrial membrane potential. Mechanistic studies indicated that EM23 caused a marked increase in the level of reactive oxygen species (ROS). Pretreatment with N-acetyl-L-cysteine, a ROS scavenger, almost fully reversed EM23-mediated apoptosis. In EM23-treated cells, the expression levels of thioredoxin (Trx) and thioredoxinreductase (TrxR), two components of the Trx system involved in maintaining cellular redox homeostasis, were significantly down-regulated. Concomitantly, Trx regulated the activation of apoptosis signal-regulating kinase 1 (ASK1) and its downstream regulatory targets, the p38, JNK, and ERK MAPKs. EM23-mediated activation of ASK1/MAPKs was significantly inhibited in the presence of NAC. Furthermore, tumor necrosis factor alpha (TNF-α)-mediated activation of nuclear factor-κB (NF-κB) was suppressed by EM23, as suggested by the observed blockage of p65 nuclear translocation, phosphorylation, and reversion of IκBα degradation following EM23 treatment. Taken together, these results provide important insights into the anticancer activities of the EM component EM23 against human CML K562 cells and AML HL-60 cells.

DKK3 expression in hepatocytes defines susceptibility to liver steatosis and obesity

Dickkopf-3 (DKK3), a protein belonging to the DKK family, has been extensively investigated in the context of cancer, including liver cancer. However, the role of DKK3 in hepatic steatosis and related metabolic disorders remains largely unexplored. We detected the expression of DKK3 in the fatty livers of NAFLD patients and of obese mice and investigated the function of DKK3 in hepatic steatosis and related metabolic disorders by using hepatocyte-specific DKK3 deficiency or overexpression obese mice induced by high fat diet (HFD) or genetic defect (ob/ob). The molecular mechanisms underlying DKK3-regulated hepatic steatosis were further explored and verified in mice. DKK3 expression was significantly decreased in the livers of NAFLD patients and of obese mice as well as in cultured hepatocytes stimulated with palmitate. Further investigation indicated that specific overexpression of DKK3 in hepatocytes enhanced insulin sensitivity and glucose tolerance, reduced the inflammatory response, and ameliorated the imbalance of lipid metabolism in response to HFD or genetic defects. In contrast, DKK3 deficiency in hepatocytes led to an almost complete reversal of these pathologies. Mechanistically, DKK3 combined with Apoptosis signal-regulating kinase 1 (ASK1) under palmitate stimulation, and thus inhibited the activation of the downstream P38/JNK pathway. Importantly, dominant-negative ASK1 blocked the accelerated effects of DKK3 deficiency, while the constitutively active form of ASK1 overcame the inhibitory effects of DKK3 overexpression on HFD-induced metabolic disorders in vivo. DKK3 functions as a negative regulator of insulin resistance, hepatic steatosis, and associated inflammatory responses, which depends on its inhibitory regulation of ASK1 activity. DKK3 expression is decreased in the non-alcoholic fatty liver of humans and mice. Adding DKK3 expression alleviates fatty liver in mice by inhibiting ASK1 activity.

Coordinate Activation of Redox-Dependent ASK1/TGF-β Signaling by a Multiprotein Complex (MPK38, ASK1, SMADs, ZPR9, and TRX) Improves Glucose and Lipid Metabolism in Mice.

To explore the molecular connections between redox-dependent apoptosis signal-regulating kinase 1 (ASK1) and transforming growth factor-β (TGF-β) signaling pathways and to examine the physiological processes in which coordinated regulation of these two signaling pathways plays a critical role. We provide evidence that the ASK1 and TGF-β signaling pathways are interconnected by a multiprotein complex harboring murine protein serine-threonine kinase 38 (MPK38), ASK1, Sma- and Mad-related proteins (SMADs), zinc-finger-like protein 9 (ZPR9), and thioredoxin (TRX) and demonstrate that the activation of either ASK1 or TGF-β activity is sufficient to activate both the redox-dependent ASK1 and TGF-β signaling pathways. Physiologically, the restoration of the downregulated activation levels of ASK1 and TGF-β signaling in genetically and diet-induced obese mice by adenoviral delivery of SMAD3 or ZPR9 results in the amelioration of adiposity, hyperglycemia, hyperlipidemia, and impaired ketogenesis. Our data suggest that the multiprotein complex linking ASK1 and TGF-β signaling pathways may be a potential target for redox-mediated metabolic complications.

Mycobacterium avium MAV2052 protein induces apoptosis in murine macrophage cells through Toll-like receptor 4.

Mycobacterium avium and its sonic extracts induce apoptosis in macrophages. However, little is known about the M. avium components regulating macrophage apoptosis. In this study, using multidimensional fractionation, we identified MAV2052 protein, which induced macrophage apoptosis in M. avium culture filtrates. The recombinant MAV2052 induced macrophage apoptosis in a caspase-dependent manner. The loss of mitochondrial transmembrane potential (ΔΨm), mitochondrial translocation of Bax, and release of cytochrome c from mitochondria were observed in macrophages treated with MAV2052. Further, reactive oxygen species (ROS) production was required for the apoptosis induced by MAV2052. In addition, ROS and mitogen-activated protein kinases were involved in MAV2052-mediated TNF-α and IL-6 production. ROS-mediated activation of apoptosis signal-regulating kinase 1 (ASK1)-JNK pathway was a major signaling pathway for MAV2052-induced apoptosis. Moreover, MAV2052 bound to Toll-like receptor (TLR) 4 molecule and MAV2052-induced ROS production, ΔΨm loss, and apoptosis were all significantly reduced in TLR4(-/-) macrophages. Altogether, our results suggest that MAV2052 induces apoptotic cell death through TLR4 dependent ROS production and JNK pathway in murine macrophages.

MiR-17 Downregulation by High Glucose Stabilizes Thioredoxin-Interacting Protein and Removes Thioredoxin Inhibition on ASK1 Leading to Apoptosis.

Pregestational diabetes significantly increases the risk of neural tube defects (NTDs). Maternal diabetes activates an Apoptosis Signal-regulating Kinase 1 (ASK1)-initiated pathway, which triggers neural stem cell apoptosis of the developing neuroepithelium leading to NTD formation. How high glucose of diabetes activates ASK1 is still unclear. In this study, we investigated the mechanism underlying high glucose-induced ASK1 activation. High glucose suppressed miR-17 expression, which led to an increase in its target gene Txnip (Thioredoxin-interacting protein). High glucose-increased Txnip enhanced its binding to the ASK1 inhibitor, thioredoxin (Trx), and thereby sequestered Trx from the Trx-ASK1 complex. High glucose-induced ASK1 activation and consequent apoptosis were abrogated by either the miR-17 mimic or Txnip siRNA knockdown. In contrast, the miR-17 inhibitor or Txnip ectopic overexpression mimicked the stimulative effect of high glucose on ASK1 and apoptosis. Thus, our study demonstrated that miR-17 repression mediates the pro-apoptotic effect of high glucose, and revealed a new mechanism underlying ASK1 activation, in which decreased miR-17 removes Trx inhibition on ASK1 through Txnip.

Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway

1,2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE) induces both necrosis/necroptosis and apoptosis of NCI-H28 malignant pleural mesothelioma (MPM) cells. The present study was conducted to understand the mechanism for DAPE-induced apoptosis of NCI-H28 cells. DAPE induced caspase-independent apoptosis of NCI-H28 malignant pleural mesothelioma (MPM) cells, and the effect of DAPE was prevented by antioxidants or an inhibitor of NADPH oxidase (NOX). DAPE generated reactive oxygen species (ROS) and inhibited activity of thioredoxin (Trx) reductase (TrxR). DAPE decreased an association of apoptosis signal-regulating kinase 1 (ASK1) with thioredoxin (Trx), thereby releasing ASK1. DAPE activated p38 mitogen-activated protein kinase (MAPK), which was inhibited by an antioxidant or knocking-down ASK1. In addition, DAPE-induced NCI-H28 cell death was also prevented by knocking-down ASK1. Taken together, the results of the present study indicate that DAPE stimulates NOX-mediated ROS production and suppresses TrxR activity, resulting in the decrease of reduced Trx and the dissociation of ASK1 from a complex with Trx, allowing sequential activation of ASK1 and p38 MAPK, to induce apoptosis of NCI-H28 MPM cells.

Berberine attenuates CCN2-induced IL-1β expression and prevents cartilage degradation in a rat model of osteoarthritis

Connective tissue growth factor (CTGF; also known as CCN2) is an inflammatory mediator that is abundantly expressed in osteoarthritis (OA). Interleukin-1β (IL-1β) plays a pivotal role in OA pathogenesis. Berberine exhibits an anti-inflammatory effect, but the mechanisms by which it modulates CCN2-induced IL-1β expression in OA synovial fibroblasts (OASFs) remain unknown. We showed that CCN2-induced IL-1β expression is mediated by the activation of αvβ3/αvβ5 integrin-dependent reactive oxygen species (ROS) generation, and subsequent activation of apoptosis signal-regulating kinase 1 (ASK1), p38/JNK, and nuclear factor-κB (NF-κB) signaling pathways. This IL-1β expression in OASFs is attenuated by N-acetylcysteine (NAC), inhibitors of ASK1, p38, or JNK, or treatment with berberine. Furthermore, berberine also reverses cartilage damage in an experimental model of collagenase-induced OA (CIOA). We observed that CCN2 increased IL-1β expression via αvβ3/αvβ5 integrins, ROS, and ASK1, p38/JNK, and NF-κB signaling pathways. Berberine was found to inhibit these signaling components in OASFs in vitro and prevent cartilage degradation in vivo. We suggest a novel therapeutic strategy of using berberine for managing OA.

Differential caspase activity in the cortex and striatum with chronic infusion of 3-nitropropionic acid

Systemic administration of 3-nitropropionic acid (3-NP) facilitates the development of select striatal lesions, and some reports provide clues about this pathology. In this study, we investigated the relationship between reduced levels of brain-derived neurotrophic factor (BDNF) in lesioned brain regions and caspase activity, as well as involvement of apoptosis signal-regulating kinase 1 (ASK1) in caspase activation. We analyzed apoptotic cell death, BDNF distribution, caspase-3 activity, caspase-6 activity, ASK1 expression level, and active ASK1 in the cortex and striatum. There were different levels and distributions of these factors within each sub-region. Caspase-6 activity was reduced with down-regulation of ASK1 in the cortex. BDNF protein levels did not decrease in the cortex, but there was replenishment of severely reduced BDNF in the striatum. The present study suggests that an increase in ASK1 in the damaged cortex is related to caspase-6 activation and is involved in cortical depletion of BDNF in the striatum. Furthermore, with systemic infusion of 3-NP, differential expression of ASK1 in the cortex and striatum suggests that this kinase may modulate caspase activation and striatal degeneration.

Cobalt Protoporphyrin Upregulates Cyclooxygenase-2 Expression Through a Heme Oxygenase-Independent Mechanism.

Cobalt protoporphyrin (CoPP) is a potent HO-1 inducer and generally known to be an antioxidant in various cell types. Little is known about the CoPP-induced cyclooxygenase-2 (COX-2) expression and its downstream signaling in microglial cells. In current study, CoPP caused concentration- and time-dependent increases in COX-2 expression in microglial cells. Furthermore, activation of apoptosis signal-regulating kinase (ASK) 1/MAP kinase involved in CoPP-induced COX-2 expression in microglia. CoPP also induced P2X7 receptor activation, and treatment of P2X7 inhibitors effectively reduced CoPP-induced COX-2 expression. Protein inhibitor of activated STAT (PIAS) 1 is reported to be involved in modulating anti-inflammatory response through negative regulation of transcription factors. Interestingly, treatment with CoPP markedly induced PIAS1 degradation which is regulated by PI3K, Akt, and glycogen synthase kinase 3α/β (GSK3α/β) signaling pathways. These results suggest that CoPP induces COX-2 expression through activating P2X7 receptors and ASK1/MAP kinases as well as PIAS1 degradation signaling pathways. Our study provides a new insight into the regulatory effect of CoPP on neuroinflammation in microglial cells.

MPTP activates ASK1–p38 MAPK signaling pathway through TNF-dependent Trx1 oxidation in parkinsonism mouse model

Activation of apoptosis signal-regulating kinase 1 (ASK1)–p38 MAPK death signaling cascade is implicated in the death of dopaminergic neurons in substantia nigra in Parkinson's disease (PD). We investigated upstream activators of ASK1 using an MPTP mouse model of parkinsonism and assessed the temporal cascade of death signaling in ventral midbrain (VMB) and striatum (ST). MPTP selectively activated ASK1 and downstream p38 MAPK in a time-dependent manner in VMB alone. This occurred through selective protein thiol oxidation of the redox-sensitive thiol disulfide oxidoreductase, thioredoxin (Trx1), resulting in release of its inhibitory association with ASK1, while glutathione-S-transferase µ 1 (GSTM1) remained in reduced form in association with ASK1. Levels of tumor necrosis factor (TNF), a known activator of ASK1, increased early after MPTP in VMB. Protein covariation network analysis (PCNA) using protein states as nodes revealed TNF to be an important node regulating the ASK1 signaling cascade. In confirmation, blocking MPTP-mediated TNF signaling through intrathecal administration of TNF-neutralizing antibody prevented Trx1 oxidation and downstream ASK1–p38 MAPK activation. Averting an early increase in TNF, which leads to protein thiol oxidation resulting in activation of ASK1–p38 signaling, may be critical for neuroprotection in PD. Importantly, network analysis can help in understanding the cause/effect relationship within protein networks in complex disease states.

ASK1 modulates the expression of microRNA Let7A in microglia under high glucose in vitro condition.

Hyperglycemia results in oxidative stress and leads to neuronal apoptosis in the brain. Diabetes studies show that microglia participate in the progression of neuropathogenesis through their involvement in inflammation in vivo and in vitro. In high-glucose-induced inflammation, apoptosis signal regulating kinase 1 (ASK1) triggers the release of apoptosis cytokines and apoptotic gene expression. MicroRNA-Let7A (miR-Let7A) is reported to be a regulator of inflammation. In the present study, we investigated whether miR-Let7A regulates the function of microglia by controlling ASK1 in response to high-glucose-induced oxidative stress. We performed reverse transcription (RT) polymerase chain reaction, Taqman assay, real-time polymerase chain reaction, and immunocytochemistry to confirm the alteration of microglia function. Our results show that miR-Let7A is associated with the activation of ASK1 and the expression of anti-inflammatory cytokine (interleukin (IL)-10) and Mycs (c-Myc and N-Myc). Thus, the relationship between Let-7A and ASK1 could be a novel target for enhancing the beneficial function of microglia in central nervous system (CNS) disorders.

Cell cycle-dependent Cdc25C phosphatase determines cell survival by regulating apoptosis signal-regulating kinase 1

Cdc25C (cell division cycle 25C) phosphatase triggers entry into mitosis in the cell cycle by dephosphorylating cyclin B-Cdk1. Cdc25C exhibits basal phosphatase activity during interphase and then becomes activated at the G2/M transition after hyperphosphorylation on multiple sites and dissociation from 14-3-3. Although the role of Cdc25C in mitosis has been extensively studied, its function in interphase remains elusive. Here, we show that during interphase Cdc25C suppresses apoptosis signal-regulating kinase 1 (ASK1), a member of mitogen-activated protein (MAP) kinase kinase kinase family that mediates apoptosis. Cdc25C phosphatase dephosphorylates phospho-Thr-838 in the activation loop of ASK1 in vitro and in interphase cells. In addition, knockdown of Cdc25C increases the activity of ASK1 and ASK1 downstream targets in interphase cells, and overexpression of Cdc25C inhibits ASK1-mediated apoptosis, suggesting that Cdc25C binds to and negatively regulates ASK1. Furthermore, we showed that ASK1 kinase activity correlated with Cdc25C activation during mitotic arrest and enhanced ASK1 activity in the presence of activated Cdc25C resulted from the weak association between ASK1 and Cdc25C. In cells synchronized in mitosis following nocodazole treatment, phosphorylation of Thr-838 in the activation loop of ASK1 increased. Compared with hypophosphorylated Cdc25C, which exhibited basal phosphatase activity in interphase, hyperphosphorylated Cdc25C exhibited enhanced phosphatase activity during mitotic arrest, but had significantly reduced affinity to ASK1, suggesting that enhanced ASK1 activity in mitosis was due to reduced binding of hyperphosphorylated Cdc25C to ASK1. These findings suggest that Cdc25C negatively regulates proapoptotic ASK1 in a cell cycle-dependent manner and may play a role in G2/M checkpoint-mediated apoptosis.

ASK1, a Novel Regulator of Thrombosis, Is Activated Downstream of G-Protein Coupledreceptors in Human Platelets

In the event of vascular injury, platelets rapidly adhere to sub-endothelial matrix proteins such as collagen and Von Willebrand factor and activate to form a stable hemostatic platelet plug. Defects in the molecular mechanisms dictating platelet plug formation are responsible for numerous thrombotic disorders. Elucidating the signaling pathways and molecular mechanisms of platelet activation is paramount to the development of safer and more effective anti-thrombotic drugs. While it is known that MAP-Kinase signaling participates in platelet activation, it is unknown how MAP-Kinase signaling specifically mediates platelet activation. Our laboratory has identified the presence and activation of a MAP-Kinase Kinase Kinase known as Apoptosis Signal Regulating Kinase 1 (ASK1). We have demonstrated using an ASK1 knockout mouse model that ablation of ASK1 leads to a significantly increased (p = .0003) time of vessel occlusion associated with unstable thrombus formation following a carotid artery injury induced by 10% FeCl3. Furthermore, ASK1 knockout mice display protection from pulmonary thromboembolism induced by an intravenous injection of collagen and epinephrine. In order to determine the kinetics of ASK1 activation by physiological agonists, washed human platelets (4 x 108 platelets/mL) were treated with 0.1 U/mL of thrombin for 30”, 1’, 3’, 5’, and 8’. Robust activation of ASK1 by thrombin occurred as early as 30 seconds up until 5 min, after which ASK1 activation decreased sharply. Platelets treated with 100 µM of PAR1 (SFLLRN) or PAR4 (AYPGKF) peptides resulted in strong ASK1 activation, suggesting that both the PAR1 and PAR4 receptors lead to ASK1 activation. Inhibition of Src family kinases by PP2 or PI3K by wortmannin or Rho kinase by Y-27632 had no effect on thrombin-induced ASK1 activation. However, inhibition of PLC-β2, a mediator of platelet activation downstream of the PAR1/4 receptors, strongly inhibited ASK1 activation by thrombin. We next determined whether TxA2 generation was responsible for ASK1 activation by thrombin. Washed platelets were pre-treated with 1 mM aspirin to block TxA2 generation, followed by treatment with 0.1 U/mL of thrombin. It was found that blocking TxA2 generation eliminated ASK1 activation by thrombin at 30” and 1’, but not at a later time point, suggesting there may be an additional pathway contributing to ASK1 activation. The observation that TxA2 generation contributes to ASK1 activation by thrombin seemed to correlate with the finding that treatment of platelets with 1 µM of the TxA2 mimetic U46619, which activates the TP-α receptor, could also activate ASK1. We also determined whether ADP released from dense granules, which would activate the P2Y1 and P2Y12 receptors, leads to ASK1 activation. To test this, washed platelets were pre-treated with 1 U/mL of apyrase to hydrolyze secreted ADP. It was found that apyrase treatment completely eliminates ASK1 activation by thrombin, suggesting a strong dependency of thrombin-induced ASK1 activation on ADP release from dense granules. To further investigate this possibility, washed platelets were pre-treated with 50 µM of the P2Y1 antagonist MRS2179 or P2Y12 antagonist 2-MeSAMP, followed by treatment with 0.1 U/mL of thrombin. Antagonism of the P2Y12 receptor and not P2Y1 receptor severely diminished ASK1 activation by thrombin. This indicates that ASK1 activation by thrombin is also dependent on ADP released from dense granules and subsequent activation of the P2Y12 receptor. Surprisingly, collagen, a strong activator of platelets, was unable to activate ASK1 in washed platelets at a concentration of 2 µg/mL. Similarly, 2 µM epinephrine treatment also had no effect. However, when washed platelets were treated with 2 µg/mL collagen and 2 µM epinephrine together, a strong ASK1 activation was observed (p=.0012). This suggests the existence of a novel mechanism for ASK1 activation by simultaneous stimulation of the collagen receptors GPVI/α2β1 and epinephrine receptor α2A. The finding that ASK1 activation occurs downstream of TP-α, P2Y12, and possibly α2A receptors highlights the importance of ASK1 in regulation of these G-Protein Coupled Receptors in platelet activation. In conclusion, our data indicates ASK1 to be a key mediator in platelet activation and represents a novel target for anti-thrombotic drug therapy. DisclosuresNo relevant conflicts of interest to declare.