Apoptosis Signal-regulating Kinase Research Articles

Selonsertib Alleviates the Progression of Rat Osteoarthritis: An in vitro and in vivo Study.

Osteoarthritis (OA) is a prevalent degenerative joint disease. Its development is highly associated with inflammatory response and apoptosis in chondrocytes. Selonsertib (Ser), the inhibitor of Apoptosis Signal-regulated kinase-1 (ASK1), has exhibited multiple therapeutic effects in several diseases. However, the exact role of Ser in OA remains unclear. Herein, we investigated the anti-arthritic effects as well as the potential mechanism of Ser on rat OA. Our results showed that Ser could markedly prevent the IL-1β-induced inflammatory reaction, cartilage degradation and cell apoptosis in rat chondrocytes. Meanwhile, the ASK1/P38/JNK and NFκB pathways were involved in the protective roles of Ser. Furthermore, intra-articular injection of Ser could significantly alleviate the surgery induced cartilage damage in rat OA model. In conclusion, our work provided insights into the therapeutic potential of Ser in OA, indicating that Ser might serve as a new avenue in OA treatment.

Abstract 2029: The ER stress protein IRE1a regulates intracellular calcium, ROS, and the UV damage response through the IP3R inhibitor CIB1

Abstract UV induced DNA damage is the primary driver of human skin cancer. UV can also cause endoplasmic reticulum (ER) stress, but the role of unfolded protein response (UPR) in the cellular response to UV exposure is not clear. Inositol-requiring enzyme 1α (IRE1α) is one of the key regulators of the UPR but its function in the UV response has not been resolved. Here we show that IRE1α, is transiently dephosphorylated and its RNase activity downregulated by UVB resulting in reduced XBP1 splicing. Keratinocytes with either IRE1α knockout or knockdown have elevated cytosolic calcium and ROS resulting in increased apoptosis after UV. Pharmacological inhibition of the ER inositol triphosphate receptor (IP3R) or phospholipase C (PLC) restored normal cytosolic calcium and ROS levels to cells lacking IRE1α. While loss of IRE1α did not alter the frequency of cells with UV-induced cyclo-pyrimidine dimers (CPD), it significantly reduced the percent of γH2AX positive keratinocytes 6 hours after UVB irradiation. This percent of CPD positive cells and the level of γH2AX 6 hours after UV suggesting a defect in the DNA repair response. Pharmacological inhibition of IRE1α RNase or Xbp1 knockdown did not phenocopy IRE1α loss, but overexpression of IRE1α in IRE1α knockdown cells restored γH2AX expression and reduced keratinocyte apoptosis. IRE1α overexpression also enhanced the survival of keratinocytes deficient in p53 function after UV irradiation. Additionally, mice with an epidermal deletion of IRE1α showed more rapid loss of UV-induced CPD positive keratinocytes compared to wildtype mice, primarily through accelerated sloughing of UV damaged cells from the skin. Knockdown of Calcium and integrin binding protein 1 (CIB1), which binds and inhibits the IP3R, elevated cytosolic calcium in keratinocytes and suppressed UV-induced γH2AX. Knockdown of Apoptosis signal-regulating kinase 1 (ASK1), which can interact with IRE1α through TRAF2 as well as CIB1, restored normal levels of UV-induced γH2AX to IRE1α knockdown keratinocytes. Together these results show that IRE1α is essential for maintaining normal cellular calcium and ROS levels and the UV-induced repair response, and suggest a model where IRE1a activation state drives CIB1 binding either to the IP3R or ASK1 to regulate ER calcium efflux, ROS and DNA repair responses following UV. Citation Format: Jeongin Son, Saie Mogre, Adam Glick. The ER stress protein IRE1a regulates intracellular calcium, ROS, and the UV damage response through the IP3R inhibitor CIB1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2029.

Anthocyanin attenuates oxygen-glucose deprivation/reperfusion-induced apoptosis of PC12 cells via inactivation of ASK1/JNK/p38 signaling pathway

Purpose: To investigate whether the cytoprotective effect of anthocyanin (Anc) on oxygen-glucose deprivation/reperfusion (OGD/R)-induced cell injury is related to apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK)/p38 signaling pathway.
 Methods: PC12 cells were pre-treated with various concentrations of Anc (10, 50, and 100 μg/mL) in OGD/R-induced cell injury model. The 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di-phenytetrazoliumromide (MTT) assay was used to assess cell viability. Cell apoptosis was measured by lactic acid dehydrogenase (LDH) release assay and flow cytometry. Western blot was employed to determine the protein expressions of BCL-2, BAX, caspase-3, p-ASK1 (Thr845), p-JNK, and p-p38.
 Results: The results indicate that Anc increased the viability of PC12 cells after OGD/R exposure (p < 0.05), and also efficiently rescued OGD/R-induced apoptosis (p < 0.05). Mechanistic studies showed that these protective roles of Anc are related to the inhibition of ASK1/JNK/p38 signaling pathway.
 Conclusion: The results indicate Anc protects against OGD/R-induced cell injury by enhancing cell viability and inhibiting cell apoptosis. The underlying mechanism of action is partly via inactivation of ASK1/JNK/p38 signaling pathway. Thus, Anc has promise as a potential natural agent to prevent and treat cerebral ischemia-reperfusion injury.

Novel Anti-inflammatory and Anti-fibrotic Agents for Diabetic Kidney Disease-From Bench to Bedside.

Chronic low-grade inflammation, now coined by the new paradigm as "metaflammation" or "metainflammation", has been linked to chronic kidney disease and its progression. In diabetes, altered metabolism denotes factors associated with the metabolic syndrome and hyperglycemia, among others. The interplay among hyperglycemia, oxidative stress, and inflammation in the pathogenesis of diabetic kidney disease (DKD) has been broadly explored. Identification of mediators of inflammatory processes involving macrophage infiltration, production of inflammasomes, release of cytokines, and activation of pertinent signaling pathways including mitogen-activated protein kinase, Jun N-terminal kinase, Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway (JAK/STAT), and apoptosis signal-regulating kinase 1 signaling mechanisms have enabled the development of therapeutic agents for DKD. This review describes the evidence supporting the contribution of the inflammatory response and fibrotic changes and focuses on selected, novel, promising drugs as well as repurposed drugs that have made it to phase 2, 3, or 4 of clinical trials in adults with type 2 diabetes mellitus and their potential to become an important part of our armamentarium to improve the management of DKD. Importantly, drugs that solely target inflammatory processes may be insufficient to fully optimize care of patients with DKD because of the complex nature of the disease.

ASK1 Activation in Platelets By Oxidized LDL Is Independent of CD36

Oxidized low-density lipoprotein (oxLDL) is a major contributor to platelet hyper-activation in hyperlipidemic conditions. OxLDL induces a prothrombotic phenotype by sensitizing the platelets for activation. Several receptors have been identified for oxLDL on platelets, but majority of the studies were focused on CD36. OxLDL binding to CD36 results in activation of multiple signaling molecules including Src family kinases (SFKs), Vav 1&3, JNK2, ERK5, and reactive oxygen species (ROS). The mechanism(s) by which oxLDL sensitizes platelets for activation is poorly understood. We have recently identified Apoptosis Signal Regulating Kinase 1 (ASK1), a MAP kinase kinase kinase, in platelets. Here, we show that in human platelets ASK1 is activated by oxLDL as determined by its activation-specific phosphorylation on T845. ASK1 activation was evident with oxLDL concentrations as low as 10μg/ml, with maximal activation observed with 100μg/ml. In contrast, native LDL (nLDL, 10-200μg/ml) failed to stimulate ASK1 activation. The activation of ASK1 was rapid peaking at 1 minute and returning to base line by 5 minutes. To determine if ROS is responsible for the activation of ASK1, we pretreated platelets with MnTMPyP, a ROS scavenger. MnTMPyP failed to inhibit the activation of ASK1 induced by oxLDL. To determine the mechanism by which oxLDL induces ASK1 activation, we pretreated platelets with 10μM each of pharmacological inhibitors, PP2 for SFKs, Y27632 for Rho kinase, bisindolylmaleimide for PKC, LY294002 for PI3 Kinase, U73122 for PLC, and 25μM of BAPTA-AM to chelate calcium. We found that platelets pretreated with U73122 and BAPTA-AM abolished oxLDL induced ASK1 activation, whereas others had no effect suggesting that ASK1 is activated in a calcium-dependent manner. To investigate the role of CD36 in oxLDL induced ASK1 activation, we used FA6-152, a function-blocking antibody for human CD36, and murine CD36nullplatelets. Interestingly, either blocking or lack of CD36 had no effect on oxLDL-induced ASK1/p38 activation suggesting that CD36 is not involved in oxLDL induced ASK1 activation. To determine if ASK1 play a role in oxLDL-induced sensitization of platelets for activation, we treated platelets with GS-4997, a specific ASK1 inhibitor, before stimulating them with ADP and oxLDL. ADP (10µM) or oxLDL (100µg/ml), showed no aggregation, but combination of oxLDL and ADP showed robust aggregation of vehicle-treated platelets. However, pretreatment of platelets with GS-4997 significantly inhibited oxLDL-induced potentiation of ADP-induced platelet aggregation suggesting that ASK1 plays a role in platelet activation under hyperlipidemic conditions. DisclosuresNo relevant conflicts of interest to declare.

Apoptosis Signal-Regulating Kinase 1 (ASK1) Is a Novel Regulator of Heparin-Induced Thrombocytopenia and Thrombosis in Mice

Heparin-induced thrombocytopenia and thrombosis (HITT) is a life-threatening (20-30% mortality risk) disease in which IgG antibodies against the heparin-PF4 complex bind and activate platelets via FcγRIIA. Our laboratory has identified that the Apoptosis Signal-Regulating Kinase (ASK1), a MAP3K, is present in both human and murine platelets and potentiates many platelet functions. Given that ASK1 regulates platelet function, and that platelets are known to play a major role in the pathogenesis of HITT, we hypothesized that ASK1 is a novel regulator of HITT.To establish if ASK1 is activated downstream of FcγRIIA, we first stimulated washed human platelets with anti-CD9 (700ng/mL). Anti-CD9 induces the activation of FcγRIIA. We found that anti-CD9 induced a robust activation of ASK1, as measured by phosphorylation of ASK1 Thr845 (a marker of ASK1's kinase activity). ASK1 exclusively activates p38 in platelets; therefore we also measured phosphorylation of p38 a marker of ASK1's signaling activity. We found that anti-CD9 also induced a robust phosphorylation of p38.To determine the role ASK1 plays in platelet-FcγRIIA signaling and HITT, we crossed Ask1-/- mice to FcγRIIA+/+(hFcR) mice. We found that genetic ablation of Ask1 did not have any effect on anti-CD9-induced activation of PLCγ2 or Syk in hFcR/ Ask1-/- (KO) platelets compared to hFcR/ Ask1+/+ (WT). However, loss of Ask1 did result in the complete absence of p38 activation in KO platelets following activation of FcγRIIA by anti-CD9 (500ng/mL). Further, we observed that anti-CD9-induced integrin αIIbβ3 activation, α-granule secretion, and platelet aggregation were all significantly attenuated in KO platelets (P<0.01). These in vitro results strongly suggested that ASK1 plays a prominent role in FcγRIIA-mediated platelet activation.To further investigate the role of ASK1 in HITT pathogenesis, WT and KO mice were subjected to an in vivo model of HITT by injecting anti-CD9 IgG. Platelet counts were measured in samples of whole blood collected at various time points post-injection, and compared to resting platelet counts taken before HITT was initiated. We found that KO mice were significantly (P<0.01) protected from thrombocytopenia compared to WT mice when injected with 250μg/kg anti-CD9. However, when injected with 500μg/kg anti-CD9, both WT and KO mice displayed the same level of thrombocytopenia. Despite not protecting from thrombocytopenia, when observed for signs of shock, we found that KO mice were significantly (P<0.01) protected from anti-CD9 induced shock compared to WT mice regardless of the dose.Taken together these in vitro and in vivo data strongly suggest that ASK1 regulates FcγRIIA-mediated platelet activation, and that ASK1 plays a key role in the pathogenesis of HITT. Furthermore, these data highlight the much-overlooked ASK1/p38 signaling pathway as key mediator of platelet activation during HITT pathogenesis. ASK1 may be a potential target for the development of novel drugs to treat HITT. DisclosuresNo relevant conflicts of interest to declare.

Proteasome dysfunction under compromised redox metabolism dictates liver injury in NASH through ASK1/PPARγ binodal complementary modules

Incidence of hepatotoxicity following acute drug-induced proteasomal inhibition and development of chronic proteasome dysfunction in obesity and insulin resistance underscores the crucial importance of hepatic protein homeostasis albeit with an elusive molecular basis and therapeutic opportunities. Apart from lipotoxicity and endoplasmic reticulum (ER) stress, herein we report that hepatocytes are highly susceptible to proteasome-associated metabolic stress attune to altered redox homeostasis. Bortezomib-induced proteasomal inhibition caused severe hepatocellular injury independent of ER stress via proapoptotic Apoptosis Signal-regulating Kinase 1 (ASK1)- c-Jun N-terminal kinase (JNK1)- p38 signaling concomitant with inadequate peroxisome proliferator-activated receptor γ (PPARγ)- Nuclear factor erythroid 2-related factor 2 (Nrf2) -driven antioxidant response. Although inhibition of ASK1 rescued acute hepatotoxicity, hepatic depletion of PPARγ or its physiological activator pigment epithelium-derived factor (PEDF) further aggravated liver injury even under ASK1 inhibition, emphasizing that endogenous PPARγ driven antioxidant activity serves as a prerequisite for the favorable therapeutic outcome of ASK1 inhibition. Consequently, ASK1 inhibitor selonsertib and PPARγ agonist pioglitazone in pharmacological synergism ameliorated bortezomib-induced hepatotoxicity and significantly prolonged survival duration in mice. Moreover, we showed that proteasome dysfunction is associated with ASK1 activation and insufficient PPARγ/Nrf2-driven antioxidative response in a subset of human nonalcoholic steatohepatitis (NASH) patients and the preclinical NASH model. The latter remains highly responsive to the drug combination marked by revamped proteasomal activity and alleviated hallmarks of NASH such as steatosis, fibrosis, and hepatocellular death. We thus uncovered a pharmacologically amenable interdependent binodal molecular circuit underlying hepatic proteasomal dysfunction and associated oxidative injury.

Thioredoxin-1 Is a Target to Attenuate Alzheimer-Like Pathology in Diabetic Encephalopathy by Alleviating Endoplasmic Reticulum Stress and Oxidative Stress.

Varying degrees of central nervous system neuropathy induced by diabetes mellitus (DM) contribute to a cognitive disorder known as diabetic encephalopathy (DE), which is also one of the independent risk factors for Alzheimer’s disease (AD). Endoplasmic reticulum stress (ERS) plays a critical role in the occurrence and development of DE and AD. However, its molecular mechanism remains largely unknown. This study aims to investigate whether thioredoxin-1 (Trx-1) could alleviate DE and AD through ERS, oxidative stress (OS) and apoptosis signaling pathways. Mice were randomly divided into a wild-type group (WT-NC), a streptozotocin (STZ)-treated DM group (WT-DM), a Trx-1-TG group (TG-NC) and a Trx-1-TG DM group (TG-DM). Diabetic animals showed an increase in the time spent in the target quadrant and the number of platform crossings as well as AD-like behavior in the water maze experiment. The immunocontent of the AD-related protein Tau and the levels of cell apoptosis, β-amyloid (Aβ) plaque formation and neuronal degeneration in the hippocampus of the diabetic group were increased. Some key factors associated with ERS, such as protein disulfide isomerase (PDI), glucose-regulated protein 78 (GRP78), inositol-requiring enzyme 1α (IRE1α), tumor necrosis factor receptor-associated factor 2 (TRAF2), apoptosis signal-regulating kinase-1 (ASK1), c-Jun N-terminal kinase (JNK), protein kinase RNA (PKR)-like ER kinase (PERK), and C/EBP homologous protein (CHOP), were upregulated, and other factors related to anti-oxidant stress, such as nuclear factor erythroid 2-related factor (Nrf2), were downregulated in the DM group. Moreover, DM caused an increase in the immunocontents of caspase-3 and caspase-12. However, these changes were reversed in the Trx-1-tg DM group. Therefore, we conclude that Trx-1 might be a key factor in alleviating DE and AD by regulating ERS and oxidative stress response, thus preventing apoptosis.

Trans-Fatty acids promote p53-dependent apoptosis triggered by cisplatin-induced DNA interstrand crosslinks via the Nox-RIP1-ASK1-MAPK pathway

trans-Fatty acids (TFAs) are food-derived fatty acids associated with various diseases including cardiovascular diseases. However, the underlying etiology is poorly understood. Here, we show a pro-apoptotic mechanism of TFAs such as elaidic acid (EA), in response to DNA interstrand crosslinks (ICLs) induced by cisplatin (CDDP). We previously reported that TFAs promote apoptosis induced by doxorubicin (Dox), a double strand break (DSB)-inducing agent, via a non-canonical apoptotic pathway independent of tumor suppressor p53 and apoptosis signal-regulating kinase (ASK1), a reactive oxygen species (ROS)-responsive kinase. However, here we found that in the case of CDDP-induced apoptosis, EA-mediated pro-apoptotic action was reversed by knockout of either p53 or ASK1, despite no increase in p53 apoptotic activity. Upon CDDP treatment, EA predominantly enhanced ROS generation, ASK1-p38/c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathway activation, and ultimately cell death, all of which were suppressed either by co-treatment of the NADPH oxidase (Nox) inhibitor Apocynin, or by knocking out its regulatory protein, receptor-interacting protein 1 (RIP1). These results demonstrate that in response to CDDP ICLs, TFAs promote p53-dependent apoptosis through the enhancement of the Nox-RIP1-ASK1-MAPK pathway activation, providing insight into the diverse pathogenetic mechanisms of TFAs according to the types of DNA damage.

Methylmercury induces neuronal cell death by inducing TNF-\u03b1 expression through the ASK1/p38 signaling pathway in microglia

We recently found that tumor necrosis factor-α (TNF-α) may be involved in neuronal cell death induced by methylmercury in the mouse brain. Here, we examined the cells involved in the induction of TNF-α expression by methylmercury in the mouse brain by in situ hybridization. TNF-α-expressing cells were found throughout the brain and were identified as microglia by immunostaining for ionized calcium binding adaptor molecule 1 (Iba1). Methylmercury induced TNF-α expression in mouse primary microglia and mouse microglial cell line BV2. Knockdown of apoptosis signal-regulating kinase 1 (ASK1), an inflammatory cytokine up-regulator that is responsible for reactive oxygen species (ROS), decreased methylmercury-induced TNF-α expression through decreased phosphorylation of p38 MAP kinase in BV2 cells. Suppression of methylmercury-induced reactive oxygen species (ROS) by antioxidant treatment largely abolished the induction of TNF-α expression and phosphorylation of p38 by methylmercury in BV2 cells. Finally, in mouse brain slices, the TNF-α antagonist (WP9QY) inhibited neuronal cell death induced by methylmercury, as did the p38 inhibitor SB203580 and liposomal clodronate (a microglia-depleting agent). These results indicate that methylmercury induces mitochondrial ROS that are involved in activation of the ASK1/p38 pathway in microglia and that this is associated with induction of TNF-α expression and neuronal cell death.

A computational analysis of the binding free energies of apoptosis signal-regulating kinase 1 inhibitors from different chemotypes

ABSTRACT Apoptosis signal-regulating kinase 1 (ASK1) has recently emerged as an attractive molecular target for pharmaceutical intervention. Several classes of small-molecular ASK1 inhibitors have been identified. In this article, we analysed the binding of small-molecular inhibitors from different chemotypes with ASK1 using molecular dynamic simulations. The umbrella sampling technique was used to calculate the binding free energy (ΔG) for these compounds with ASK1 ATP-binding pocket. According to the obtained results, umbrella sampling gave correct ranking of the binding affinities for investigated inhibitors relative to their experimental pIC50 values and can be useful for further structure-based design of ASK1 inhibitors to accelerate lead optimisation. Trial registration: ClinicalTrials.gov identifier: NCT02466516..

Rational Design, Synthesis and Evaluation of Novel C6‐Bicycloalkaneimidazole Containing Imidazo[1,2‐b]pyridazines for ASK1 Inhibition

Apoptosis signal‐regulating kinase 1 (ASK1) is a member of mitogen‐activated protein kinase kinase kinase (MAP3K) family that involves downstream phosphorylation of MAP kinases, c‐Jun N‐terminal kinases, and p38 MAP kinases. ASK1 inhibitors could possibly be beneficial for ameliorating the development and progression of diseases. Especially, ASK1 has been of interest as one of therapeutic targets for nonalcoholic fatty liver disease as the most common chronic liver diseases including simple steatosis and nonalcoholic steatohepatitis. In this manuscript, novel ASK1 inhibitor lead KTA‐29 which has an imidazo[1,2‐b]pyridazine core with novel C6‐bicycloheptaneimidazole is disclosed. With the novel imidazo[1,2‐b]pyridazine core, structure‐activity‐relationship study for ASK1 potency is described and KTA‐29 affinity toward ASK1 with molecular modeling study is explained.

MTA2 silencing attenuates the metastatic potential of cervical cancer cells by inhibiting AP1-mediated MMP12 expression via the ASK1/MEK3/p38/YB1 axis

Metastasis-associated protein 2 (MTA2) is a transcription factor that is highly associated with matrix metalloproteinase 12 (MMP12). Thus, we hypothesized that MTA2 may regulate MMP12 expression and is involved in cervical cancer metastasis. Results showed that MTA2 and MMP12 were highly expressed in cervical cancer cells, and MTA2 knockdown reduced MMP12 expression and inhibited the metastasis of cervical cancer cells in xenograft mice. MMP12 knockdown did not influence the viability of cervical cancer cells but clearly inhibited cell migration and invasion both in vitro and in vivo. MMP12 was highly expressed in cervical tumor tissues and correlated with the poor survival rate of patients with cervical cancer. Further investigations revealed that p38 mitogen-activated protein kinase (p38), mitogen-activated protein kinase kinase 3 (MEK3), and apoptosis signal-regulating kinase 1 (ASK1) were involved in MMP12 downregulation in response to MTA2 knockdown. Results also demonstrated that p38-mediated Y-box binding protein1 (YB1) phosphorylation disrupted the binding of AP1 (c-Fos/c-Jun) to the MMP12 promoter, thereby inhibiting MMP12 expression and the metastatic potential of cervical cancer cells. Collectively, targeting both MTA2 and MMP12 may be a promising strategy for the treatment of cervical cancer.

Serum deprivation-response protein induces apoptosis in hepatocellular carcinoma through ASK1-JNK/p38 MAPK pathways

Serum deprivation-response protein (SDPR), a phosphatidylserine-binding protein, which is known to have a promising role in caveolar biogenesis and morphology. However, its function in hepatocellular carcinoma (HCC) was still largely unknown. In this study, we discussed the characterization and identification of SDPR, and to present it as a novel apoptosis candidate in the incidence of HCC. We identified 81 HCC cases with lower SDPR expression in the tumor tissues with the help of qRT-PCR assay, and lower SDPR expression was potentially associated with poor prognostication. The phenotypic assays revealed that cell proliferation, invasion, and migration were profoundly connected with SDPR, both in vivo and in vitro. The data obtained from the gene set enrichment analysis (GSEA) carried out on the liver hepatocellular carcinoma (LIHC), and also The Cancer Genome Atlas (TCGA) findings indicated that SDPR was involved in apoptosis and flow cytometry experiments further confirmed this. Furthermore, we identified the interaction between SDPR and apoptosis signal-regulating kinase 1 (ASK1), which facilitated the ASK1 N-terminus-mediated dimerization and increased ASK1-mediated signaling, thereby activating the JNK/p38 mitogen-activated protein kinases (MAPKs) and finally enhanced cell apoptosis. Overall, this work identified SDPR as a tumor suppressor, because it promoted apoptosis by activating ASK1-JNK/p38 MAPK pathways in HCC.

Diacylglycerol kinase η colocalizes and interacts with apoptosis signal-regulating kinase 3 in response to osmotic shock

Diacylglycerol kinase (DGK) η translocates from the cytoplasm to punctate vehicles via osmotic shock. Apoptosis signal-regulating kinase (ASK) 3 (MAP kinase kinase kinase (MAPKKK) 15) is also reported to respond to osmotic shock. Therefore, in the present study, we examined the subcellular localization of DGKη and ASK3 expressed in COS-7 cells under osmotic stress. We found that DGKη was almost completely colocalized with ASK3 in punctate structures in response to osmotic shock. In contrast, DGKδ, which is closely related to DGKη structurally, was not colocalized with ASK3, and DGKη failed to colocalize with another MAPKKK, C-Raf, even under osmotic stress. The structures in which DGKη and ASK3 localized were not stained with stress granule makers. Notably, DGKη strongly interacted with ASK3 in an osmotic shock-dependent manner. These results indicate that DGKη and ASK3 undergo osmotic shock-dependent colocalization and associate with each other in specialized structures.

Histological, immunohistochemical, and molecular investigation on the hepatotoxic effect of potassium dichromate and the ameliorating role of Persea americana mill pulp extract.

The current study has been designed to assess the role of Persea americana (P. americana) pulp extract on potassium dichromate-induced hepatotoxicity in rats. P. americana pulp extract administration improved the hepatic vascular congestion, blood extravasation, inflammatory cellular infiltration, Kupffer cell hyperplasia, and nuclear changes. It also significantly ameliorated hepatic interstitial and peri-portal fibrosis and caused retrieval of the PAS-positive reaction in the liver parenchyma and around the central vein with restoration of the glycogen granules. P. americana also significantly attenuated the immunohistochemical expression of NF-kβ p65 and its downstream inflammatory cytokines IL6 and TNFα in the liver parenchyma. The antioxidant effect of P. americana was evidenced by significant modulation of the three major components of the thioredoxin (Trx) antioxidant system, the Trx, the thioredoxin reductase (TrxR), and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase along with significant increase in the level of superoxide dismutase and glutathione, and decrease in the lipid peroxidation product malondialdehyde. P. americana pulp extract also caused significant elevation of hepatic protein phosphatase 5 with subsequent down-regulation of Apoptosis signal-regulating kinase1 (ASK1) and its downstream signaling targets MAPK kinase 4 (MKK4), p38 mitogen-activated protein kinases (p38-MAPKs), the c-JUN N-terminal kinase (JNK), and the extracellular signal-regulated kinase 1/2 (ERK 1/2). Also, In conclusion, P. americana pulp extract has anti-oxidative and anti-inflammatory effects against potassium dichromate-induced hepatotoxicity.

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.

Vanillin downregulates NNMT and attenuates NNMT‑related resistance to 5‑fluorouracil via ROS‑induced cell apoptosis in colorectal cancer cells.

Chemoresistance is the main cause of poor prognosis in colorectal cancer (CRC). Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme that is upregulated in various tumor types. It has been reported that NNMT inhibits apoptosis and enhances resistance to 5-fluorouracil (5-Fu) via inhibition of the apoptosis signal regulating kinase 1 (ASK1)-p38 MAPK pathway in CRC cells. A natural product library was screened, and it was found that vanillin, also known as 4-hydroxy-3-methoxybenzaldehyde, a plant secondary metabolite found in several essential plant oils, mainly Vanilla planifolia, Vanilla tahitensis, and Vanilla pompon, may be a promising anticancer compound targeted to NNMT. The aim of the present study was to explore the effect of vanillin on promoting apoptosis and attenuating NNMT-induced resistance to 5-Fu in CRC. Lentiviral vectors of short hairpin RNA and small interfering RNA were transfected into HT-29 cells to construct NNMT-knockdown HT-29 cell lines. Vectors containing an open reading frame of NNMT were stably transfected into SW480 cells to induce NNMT overexpression in SW480 cell lines. Vanillin was found to inhibit the mRNA and protein expression levels of NNMT following the inhibition of NNMT activity in HT-29 cell lines. Vanillin was able to reverse NNMT-induced increased cell proliferation, decreased cell apoptosis and resistance to 5-Fu by inhibiting NNMT expression. Furthermore, it increased cell apoptosis by activating the ASK1-p38 MAPK pathway, which could be inhibited by NNMT. In addition, vanillin increased cell apoptosis by promoting mitochondrial damage and reactive oxygen species. In vivo, the combination of vanillin with 5-Fu yielded a notable synergy in inhibiting tumor growth and inducing apoptosis. Considering that vanillin is an important flavor and aromatic component used in foods worldwide, vanillin is deemed to be a promising anticancer candidate by inhibiting NNMT and may attenuate NNMT-induced resistance to 5-Fu in human CRC therapy with few side effects.

Design, synthesis and biological evaluation of 1H-indazole derivatives as novel ASK1 inhibitors.

Apoptosis signal-regulating kinase 1 (ASK1, MAP3K5), a member of the mitogen-activated protein kinase (MAPK) signaling pathway, is involved in cell survival, differentiation, stress response, and apoptosis. ASK1 kinase inhibition has emerged as a promising therapeutic strategy for inflammatory disease. A series of novel ASK1 inhibitors with 1H-indazole scaffold were designed, synthesized and evaluated for their ASK1 kinase activity and AP1-HEK293cell inhibitory effect. Systematic structure-activity relationship (SAR) efforts led to the discovery of promising compound 15, which showed excellent invitro ASK1 kinase activity and potent inhibitory effects on ASK1 in AP1-HEK293cells. In a tumor necrosis factor-α (TNF-α)-induced HT-29 intestinal epithelial cell model, compound 15 exhibited a significantly protective effect on cell viability comparable to that of GS-4997; moreover, compound 15 exhibited no obvious cytotoxicity against HT-29cells at concentrations up to 25μM. Mechanistic research demonstrated that compound 15 suppresses phosphorylation in the ASK1-p38/JNK signaling pathway in HT-29cells, and regulates the expression levels of apoptosis-related proteins. Altogether, these results show that compound 15 may serve as a potential candidate compound for the treatment of inflammatory bowel disease (IBD).

Functional cooperation between ASK1 and p21Waf1/Cip1 in the balance of cell-cycle arrest, cell death and tumorigenesis of stressed keratinocytes

Both CDKN1A (p21 Waf1/Cip1) and Apoptosis signal-regulating kinase 1 (ASK1) play important roles in tumorigenesis. The role of p21 Waf1/Cip1 in attenuating ASK1-induced apoptosis by various stress conditions is well established. However, how ASK1 and p21 Waf1/Cip1 functionally interact during tumorigenesis is still unclear. To address this aspect, we crossed ASK1 knockout (ASK1KO) mice with p21Waf1/Cip1 knockout (p21KO) mice to compare single and double-mutant mice. We observed that deletion of p21Waf1/Cip1 leads to increased keratinocyte proliferation but also increased cell death. This is mechanistically linked to the ASK1 axis-induced apoptosis, including p38 and PARP. Indeed, deletion of ASK1 does not alter the proliferation but decreases the apoptosis of p21KO keratinocytes. To analyze as this interaction might affect skin carcinogenesis, we investigated the response of ASK1KO and p21KO mice to DMBA/TPA-induced tumorigenesis. Here we show that while endogenous ASK1 is dispensable for skin homeostasis, ASK1KO mice are resistant to DMBA/TPA-induced tumorigenesis. However, we found that epidermis lacking both p21 and ASK1 reacquires increased sensitivity to DMBA/TPA-induced tumorigenesis. We demonstrate that apoptosis and cell-cycle progression in p21KO keratinocytes are uncoupled in the absence of ASK1. These data support the model that a critical event ensuring the balance between cell death, cell-cycle arrest, and successful divisions in keratinocytes during stress conditions is the p21-dependent ASK1 inactivation.