Apoptosis Signal-regulating Kinase Research Articles

DKK3 ameliorates neuropathic pain via inhibiting ASK-1/JNK/p-38-mediated microglia polarization and neuroinflammation

BackgroundNeuropathic pain is a common and severely disabling state that affects millions of people worldwide. Microglial activation in the spinal cord plays a critical role in the pathogenesis of neuropathic pain. However, the mechanisms underlying spinal microglial activation during neuropathic pain remain incompletely understood. Here, we investigated the role of Dickkopf (DKK) 3 and its interplay with microglial activation in the spinal cord in neuropathic pain.MethodsIn this study, we investigated the effects of intrathecal injection of recombinant DKK3 (rDKK3) on mechanical allodynia and microglial activation in the spinal cord after spared nerve injury (SNI) in rats by western blot (WB), immunofluorescence (IF), quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA).ResultsWe found that SNI induced a significant decrease in the levels of DKK3, Kremen-1 and Dishevelled-1 (DVL-1) and up-regulated the expression of phosphorylated apoptosis signal-regulating kinase 1 (p-ASK1), phosphorylated c-JUN N-terminal kinase (p-JNK), phosphorylated p38 (p-p38) in the spinal cord. Moreover, our results showed that exogenous intrathecal administration of rDKK3 inhibited expression of p-ASK1, p-JNK, p-p38, promoted the transformation of microglia from M1 type to M2 type, and decreased the production of pro-inflammatory cytokines compared to the rats of SNI + Vehicle. However, these effects were reversed by intrathecal administration of Kremen-1 siRNA or Dishevelled-1 (DVL-1) siRNA.ConclusionsThese results suggest that DKK3 ameliorates neuropathic pain via inhibiting ASK-1/JNK/p-38-mediated microglia polarization and neuroinflammation, at least partly, by the Kremen-1 and DVL-1 pathways.

Agomelatine rescues lipopolysaccharide-induced neural injury and depression-like behaviors via suppression of the Gαi-2-PKA-ASK1 signaling pathway

BackgroundAgomelatine has been shown to be effective in the treatment of depression, but the molecular mechanisms underlying its antidepressant effects have yet to be elucidated. Identification of these molecular mechanisms would not only offer new insights into the basis for depression but also provide the foundation for the development of novel treatments for this disorder.MethodsIntraperitoneal injection of LPS was used to induce depression-like behaviors in rats. The interactions of the 5-HT2C reporter and Gαi-2 were verified by immunoprecipitation or immunofluorescence assay. Inflammatory related proteins, autophagy related proteins and apoptosis markers were verified by immunoblotting or immunofluorescence assay. Finally, electron microscopy analysis was used to observe the synapse and ultrastructural pathology.ResultsHere, we found that the capacity for agomelatine to ameliorate depression and anxiety in a lipopolysaccharide (LPS)-induced rat model of depression was associated with an alleviation of neuroinflammation, abnormal autophagy and neuronal apoptosis as well as the promotion of neurogenesis in the hippocampal dentate gyrus (DG) region of these rats. We also found that the 5-HT2C receptor is coupled with G alphai (2) (Gαi-2) protein within hippocampal neurons and, agomelatine, acting as a 5-HT2C receptor antagonist, can up-regulate activity of the Gαi-2-cAMP-PKA pathway. Such events then suppress activation of the apoptosis signal-regulating kinase 1 (ASK1) pathway, a member of the mitogen-activated protein kinase (MAPK) family involved in pathological processes of many diseases.ConclusionTaken together, these results suggest that agomelatine plays a neuroprotective role in regulating neuroinflammation, autophagy disorder and apoptosis in this LPS-induced rat model of depression, effects which are associated with the display of antidepressant behaviors. These findings provide evidence for some of the potential mechanisms for the antidepressant effects of agomelatine.Graphical

ASK1 is a novel molecular target for preventing aminoglycoside-induced hair cell death

Aminoglycoside antibiotics are lifesaving medicines, crucial for the treatment of chronic or drug resistant infections. However, aminoglycosides are toxic to the sensory hair cells in the inner ear. As a result, aminoglycoside-treated individuals can develop permanent hearing loss and vestibular impairment. There is considerable evidence that reactive oxygen species (ROS) production and the subsequent phosphorylation of c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (P38) drives apoptosis in aminoglycoside-treated hair cells. However, treatment strategies that directly inhibit ROS, JNK, or P38 are limited by the importance of these molecules for normal cellular function. Alternatively, the upstream regulator apoptosis signal-regulating kinase 1 (ASK1/MAP3K5) is a key mediator of ROS-induced JNK and P38 activation under pathologic but not homeostatic conditions. We investigated ASK1 as a mediator of drug-induced hair cell death using cochlear explants from Ask1 knockout mice, demonstrating that Ask1 deficiency attenuates neomycin-induced hair cell death. We then evaluated pharmacological inhibition of ASK1 with GS-444217 as a potential otoprotective therapy. GS-444217 significantly attenuated hair cell death in neomycin-treated explants but did not impact aminoglycoside efficacy against P. aeruginosa in the broth dilution test. Overall, we provide significant pre-clinical evidence that ASK1 inhibition represents a novel strategy for preventing aminoglycoside ototoxicity.Key messagesASK1 is an upstream, redox-sensitive regulator of P38 and JNK, which are known mediators of hair cell death.Ask1 knockout does not affect hair cell development in vivo, but significantly reduces aminoglycoside-induced hair cell death in vitro.A small-molecule inhibitor of ASK1 attenuates neomycin-induced hair cell death, and does not impact antibiotic efficacy in vitro.ASK1 may be a novel molecular target for preventing aminoglycoside-induced hearing loss.

Phosphoproteomics identifies pathways underlying the role of receptor-interaction protein kinase 3 in alcohol-associated liver disease and uncovers apoptosis signal-regulating kinase 1 as a target.

Receptor‐interaction protein kinase 3 (RIP3), a critical determinant of the necroptotic pathway of programmed cell death, contributes to injury in murine models of alcohol‐associated liver disease (ALD); however, the underlying mechanisms are unknown. We investigated the effect of chronic ethanol feeding on the hepatic phosphoproteome in C57BL/6 and RIP3‐deficient (Rip3 −/−) mice, focusing on death receptor (DR) signaling pathways. C57BL/6 and Rip3 −/− mice were fed an ethanol‐containing liquid diet or pair‐fed control diet. A label‐free mass spectrometry‐based approach identified differentially phosphorylated proteins that were mapped to pathways affected by ethanol and Rip3 genotype. Identified targets were validated in both the murine model of ALD and in liver tissue from patients with alcohol‐associated hepatitis (AH) and healthy controls. Chronic ethanol dysregulated hepatic tumor necrosis factor‐induced DR signaling pathways. Of particular importance, chronic ethanol feeding to C57BL/6 mice decreased the phosphorylation of apoptosis signal‐regulating kinase 1 (ASK1) at serine (S)1036/S1040 (S1029/S1033 human), sites linked with the inhibition of ASK1 death‐promoting activity. This decrease in phosphorylation of inhibitory sites was muted in Rip3 −/− mice. Decreased phosphorylation at S1033 was also lower in liver of patients with severe AH compared to healthy controls, and phosphorylation at the ASK1 activation site (threonine [Thr]‐838) was increased in patients with AH. The net impact of these changes in phosphorylation of ASK1 was associated with increased phosphorylation of p38, a downstream target of ASK1, in patients with AH and C57BL/6 but not Rip3 −/− mice. Similarly, chronic ethanol feeding affected the c‐Jun N‐terminal kinase pathway in C57BL/6 but not Rip3 −/− mice. Taken together, our data indicate that changes in inhibitory phosphorylation of ASK1 are an important target in ALD and suggest the involvement of noncanonical functions of Rip3 in ALD.

Major Vault Protein Prevents Atherosclerotic Plaque Destabilization by Suppressing Macrophage ASK1-JNK Signaling.

Macrophages are implicated in atherosclerotic plaque instability by inflammation and degradation of extracellular matrix. However, the regulatory mechanisms driving these macrophage-associated processes are not well understood. Here, we aimed to identify the plaque destabilization-associated cytokines and signaling pathways in macrophages. The atherosclerotic models of myeloid-specific MVP (major vault protein) knockout mice and control mice were generated. Atherosclerotic instability, macrophage inflammatory signaling, and active cytokines released by macrophages were examined in vivo and in vitro by using cellular and molecular biological approaches. MVP deficiency in myeloid cells exacerbated murine plaque instability by increasing production of both MMP (matrix metallopeptidase)-9 and proinflammatory cytokines in artery wall. Mechanistically, expression of MMP-9 was mediated via ASK1 (apoptosis signal-regulating kinase 1)-MKK-4 (mitogen-activated protein kinase kinase 4)-JNK (c-Jun N-terminal kinase) signaling in macrophages. MVP and its α-helical domain could bind with ASK1 and inhibit its dimerization and phosphorylation. A 62 amino acid peptide (MVP-[686-747]) in the α-helical domain of MVP showed a crucial role in preventing macrophage MMP-9 production and plaque instability. MVP may act as an inhibitor for ASK1-JNK signaling-mediated MMP-9 production in macrophages and, thereby, attenuate unstable plaque formation. Our findings suggest that suppression of macrophage ASK1-JNK signaling may be a useful strategy antagonizing atherosclerotic diseases.

Berberine induces non-small cell lung cancer apoptosis via the activation of the ROS/ASK1/JNK pathway.

BackgroundNon-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. Berberine (BBR), an isoquinoline alkaloid, is commonly used in traditional Chinese medicine. Previous studies have shown that BBR has a potential anti-tumor effect. However, the mechanisms of BBR on mitochondrial function in anti-lung cancer remain unknown. The aim of this study was to explore mitochondrial function in anti-tumor mechanisms of BBR in NSCLC.MethodsThe NSCLCs were cultured and treated with various doses (40, 80, 120 µg/mL) of BBR for 24 and 48 h. Cell viability was evaluated using Cell Counting Kit-8 (CCK-8). Cell apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected by flow cytometry. Relative protein expression was examined by western blot and immunohistochemical (IHC) analysis.ResultsBBR potently suppressed NSCLC cells growth by inducing apoptosis in a dose-and time-dependent manner. BBR induced apoptosis in NSCLC cells as evidenced by caspase-3 cleavage, cytochrome c release, and mitochondrial membrane depolarization. BBR-induced, dose-dependent induction of apoptosis was accompanied by sustained phosphorylation of c-jun-NH2-kinase (JNK) and the JNK inhibitor (SP600125) significantly suppressed BBR-induced apoptosis, N-acetyl cysteine (NAC), a ROS scavenger, was sufficient to both suppress apoptosis signal-regulating kinase 1 (ASK1) and JNK activation and disrupt apoptotic induction.ConclusionsThe results suggest that BBR induces apoptosis of NSCLC cells via ROS-mediated ASK1/JNK activation and the mitochondrial pathway.

Probing the Interaction of Selonsertib with Human Serum Albumin: In silico and In vitro Approaches.

Selonsertib, the most recently developed selective inhibitor of apoptosis signal-regulating kinase 1. We elucidated the binding characteristics, mechanism of interaction, and dynamic behaviors of selonsertib with human serum albumin (HSA), a major circulatory transport protein. Different biophysical approaches (fluorescence quenching and isothermal titration calorimetry (ITC) were combined with various in silico techniques to examine the binding of selonsertib to HSA. Molecular docking results, analysis of molecular dynamics trajectories, and essential dynamics investigations indicated the stable binding of selonsertib to HSA. Further in vitro studies were performed to validate the observed interaction. ITC results confirmed the robust binding and high affinity of selonsertib and HSA. Likewise, the fluorescence quenching results highlighted the binding affinity of selonsertib and HSA. Collectively, our findings offer deeper insight into the binding mechanism of selonsertib and HSA, emphasizing the selonsertib-mediated structural changes within HSA, along with a comprehensive rationale for the biological transport and accumulation of selonsertib in the blood plasma. Therefore, considering the bioavailability and effectiveness of selonsertib, assessing the interactions of this inhibitor with carrier proteins is crucial to elucidate its biological processes at the molecular level. This evidence carries the considerable scientific potential for future drug design.

MTOR-AKT Signaling in Cellular Clock Resetting Triggered by Osmotic Stress.

Aims: The circadian clock oscillates in a cell-autonomous manner with a period of ∼24 h, and the phase is regulated by various time cues such as light and temperature through multiple clock input pathways. We previously found that osmotic and oxidative stress strongly affected the circadian period and phase of cellular rhythms, and triple knockout of apoptosis signal-regulating kinase (ASK) family members, Ask1, Ask2, and Ask3, abolished the phase shift (clock resetting) induced by hyperosmotic pulse treatment. We aimed at exploring a key molecule(s) and signaling events in the clock input pathway dependent on ASK kinases. Results: The phase shift of the cellular clock induced by the hyperosmotic pulse treatment was significantly reduced by combined deficiencies of the clock(-related) genes, Dec1, Dec2, and E4 promoter-binding protein 4 (also known as Nfil3) (E4bp4). In addition, liquid chromatography mass/mass spectrometry (LC-MS/MS)-based proteomic analysis identified hyperosmotic pulse-induced phosphorylation of circadian locomotor output cycles caput (CLOCK) Ser845 in an AKT-dependent manner. We found that AKT kinase was phosphorylated at Ser473 (i.e., activated) in response to the hyperosmotic pulse experiments. Inhibition of mechanistic target of rapamycin (mTOR) kinase by Torin 1 treatment completely abolished the AKT activation, suppressed the phosphorylation of CLOCK Ser845, and blocked the clock resetting induced by the hyperosmotic pulse treatment. Innovation and Conclusions: We conclude that mTOR-AKT signaling is indispensable for the CLOCK Ser845 phosphorylation, which correlates with the clock resetting induced by the hyperosmotic pulse treatment. Immediate early induction of the clock(-related) genes and CLOCK carboxyl-terminal (C-terminal) region containing Ser845 also play important roles in the clock input pathway through redox-sensitive ASK kinases. Antioxid. Redox Signal. 37, 631-646.

The Molecular Structure and Role of Humanin in Neural and Skeletal Diseases, and in Tissue Regeneration.

Humanin (HN) belongs to a member of mitochondrial-derived peptides (MDPs) which are encoded by mitochondrial genes. HN shares sequence homology with thirteen HN-like proteins, named MTRNR2L1 to MTRNR2L13, which encompass 24–28 amino acid residues in length. HN mediates mitochondrial status and cell survival by acting via an intracellular mechanism, or as a secreted factor via extracellular signals. Intracellularly, it binds Bcl2-associated X protein (BAX), Bim and tBid, and IGFBP3 to inhibit caspase activity and cell apoptosis. When released from cells as a secreted peptide, HN interacts with G protein-coupled formyl peptide receptor-like 1 (FPRL1/2) to mediate apoptosis signal-regulating kinase (ASK) and c-Jun N-terminal kinase (JNK) signalling pathways. Additionally, it interacts with CNTFR-α/gp130/WSX-1 trimeric receptors to induce JAK2/STA3 signalling cascades. HN also binds soluble extracellular proteins such as VSTM2L and IGFBP3 to modulate cytoprotection. It is reported that HN plays a role in neuronal disorders such as Alzheimer’s disease, as well as in diabetes mellitus, infertility, and cardiac diseases. Its roles in the skeletal system are emerging, where it appears to be involved with the regulation of osteoclasts, osteoblasts, and chondrocytes. Understanding the molecular structure and role of HN in neural and skeletal diseases is vital to the application of HN in tissue regeneration.

Exploring molecular targets in diabetic kidney disease.

Diabetic kidney disease is the leading cause of end-stage kidney disease, and it remains a major challenge. Many factors, such as glomerular hyperfiltration, oxidative stress, inflammation, hypoxia, and epigenetics, are associated with the progression of diabetic kidney disease; however, the whole mechanism is not yet completely understood. No specific treatment for diabetic kidney disease has been established, so new approaches are being explored extensively. Sodium-glucose cotransporter 2 inhibitors have shown renoprotective effects in several human clinical trials. Glucagon-like peptide 1 receptor agonists and mineralocorticoid receptor antagonists have been reported to be effective in diabetic kidney disease, and novel therapeutic candidates are also being examined. In the TSUBAKI trial, a nuclear factor erythroid 2-related factor 2 activator, bardoxolone methyl, improved the glomerular filtration rate of diabetic kidney disease patients. Similarly, new agents that act in the oxidative stress and inflammation pathways are of major interest, such as pentoxifylline, apoptosis signal-regulating kinase-1 inhibitors, C-C chemokine receptor 2 inhibitors, and Janus kinase-1/2 inhibitors. Endothelin-1 receptor A antagonists and soluble guanylate cyclase stimulators are also expected to affect renal hemodynamics. Some preclinical studies suggest that hypoxia-inducible factor prolyl hydroxylase inhibitors, which influence multiple inflammations and oxidative stress pathways, reduce albuminuria in diabetic kidney disease. Advanced glycation end-product inhibitors and treatments related to epigenetics have also shown promise as potential diabetic kidney disease treatments in preclinical studies. The discovery of new targets could provide new therapeutic options for overcoming diabetic kidney disease.

Hepatotoxicity-Related Oxidative Modifications of Thioredoxin 1/Peroxiredoxin 1 Induced by Different Cadmium-Based Quantum Dots.

The hepatotoxicity of cadmium-based quantum dots (Cd-QDs) has become the focus with their extensive applications in biomedicine. Previous reports have demonstrated that high oxidative stress and consequent redox imbalance play critical roles in their toxicity mechanisms. Intracellular antioxidant proteins, such as thioredoxin 1 (Trx1) and peroxiredoxin 1 (Prx1), could regulate redox homeostasis through thiol-disulfide exchange. Herein, we hypothesized that the excessive reactive oxygen species (ROS) induced by Cd-QD exposure affects the functions of Trx1 or Prx1, which further causes abnormal apoptosis of liver cells and hepatotoxicity. Thereby, three types of Cd-QDs, CdS, CdSe, and CdTe QDs, were selected for conducting an intensive study. Under the same conditions, the H2O2 level in the CdTe QD group was much higher than that of CdS or CdSe QDs, and it also corresponded to the higher hepatotoxicity. Mass spectrometry (MS) results show that excessive H2O2 leads to sulfonation modification (-SO3H) at the active sites of Trx1 (Cys32 and Cys35) and Prx1 (Cys52 and Cys173). The irreversible oxidative modifications broke their cross-linking with the apoptosis signal-regulating kinase 1 (ASK1), resulting in the release and activation of ASK1, and activation of the downstream JNK/p38 signaling finally promoted liver cell apoptosis. These results highlight the key effect of the high oxidative stress, which caused irreversible oxidative modifications of Trx1 and Prx1 in the mechanisms involved in Cd-QD-induced hepatotoxicity. This work provides a new perspective on the hepatotoxicity mechanisms of Cd-QDs and helps design safe and reliable Cd-containing nanoplatforms.

Hexavalent chromium induces hepatocyte apoptosis via regulation of apoptosis signal-regulating kinase 1/c-Jun amino-terminal kinase signaling.

With the spread of hexavalent chromium (Cr(VI)) contamination, Cr(VI)-induced hepatotoxicity has attracted increasing attention in recent years. To date, however, the exact mechanism of Cr(VI) toxicity remains unclear. In this study, we investigated the role of apoptosis signal-regulating kinase 1 (ASK1)/c-Jun amino-terminal kinase (JNK) in Cr(VI)-induced hepatic toxicity and the possible related mechanisms. AML-12 hepatocyte cell-lines were treated with 0, 1, 4, and 16 μmol/Lof Cr(VI) with or without GS-444271 (an ASK1 inhibitor). Adult male mice were administered with 0, 2, 8, and 32 mg/kg body mass (BM)/day of Cr(VI) for 5 days. The level of hepatocyte apoptosis/proliferation, generation of reactive oxygen species (ROS), and expression levels of mRNAs and proteins related to ASK1/JNK and nuclear factor-E2-related factor 2 (Nrf2) signaling were assessed. Results showed that high Cr(VI) exposure induced hepatocyte apoptosis and liver injury by generation of ROS and down-regulation of Nrf2 signaling. In addition, ASK1/JNK signaling activity was upregulated in the Cr(VI)-treated group. Furthermore, GS-444217 treatment significantly rescued Cr(VI)-induced hepatocyte apoptosis and liver dysfunction in vitro and in vivo by down-regulation of ASK1/JNK signaling. Thus, ASK1/JNK signaling appears to play an important role in Cr(VI)-induced hepatocyte apoptosis and liver injury. This study should help improve our understanding of the mechanism of Cr(VI)-induced liver injury and provide support for future investigations on liver disease therapy.

ASK1 Regulates Bleomycin-induced Pulmonary Fibrosis.

Pulmonary fibrosis (PF) is an abnormal remodeling of cellular composition and extracellular matrix that results in histological and functional alterations in the lungs. Apoptosis signal-regulating kinase-1 (ASK1) is a member of the mitogen-activated protein (MAP) kinase family that is activated by oxidative stress and promotes inflammation and apoptosis. Here we show that bleomycin-induced PF is reduced in Ask1 knockout mice (Ask1-/-) compared with wild-type (WT) mice, with improved survival and histological and functional parameters restored to basal levels. In WT mice, bleomycin caused activation of ASK1, p38, and extracellular signal-regulated kinase 1/2 (ERK1/2) in lung tissue, as well as changes in redox indicators (thioredoxin and heme-oxygenase-1), collagen content, and epithelial-mesenchymal transition markers (EMTs). These changes were largely restored toward untreated WT control levels in bleomycin-treated Ask1-/- mice. We further investigated whether treatment of WT mice with an ASK1 inhibitor, selonsertib (GS-4997), during the fibrotic phase would attenuate the development of PF. We found that pharmacological inhibition of ASK1 reduced activation of ASK1, p38, and ERK1/2 and promoted the restoration of redox and EMT indicators, as well as improvements in histological parameters. Our results suggest that ASK1 plays a central role in the development of bleomycin-induced PF in mice via p38 and ERK1/2 signaling. Together, these data indicate a possible therapeutic target for PF that involves an ASK1/p38/ERK1/2 axis.

Regulatory Mechanism of MicroRNA-9 / Long Non-Coding RNA XIST Expression on Mouse Macrophage RAW264.7 Apoptosis Induced by Oxidized Low Density Lipoprotein

ABSTRACT It aims to analyze the influential mechanism of microRNA-9 (miR-9) and long non-coding RNA XIST (lncRNA XIST) expression on the proliferation and apoptosis of macrophages induced by oxidized-low density lipoprotein (ox-LDL). Firstly, lncRNA XIST overexpression vector was constructed, and then RAW264.7 cells were used as the research object. Methylthiazolyl tetrazolium (MTT) method, flow cytometry, and Western blot were used to detect and compare the differences of cell proliferation, apoptosis, and the expression levels of apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase (JNK), matrix metalloproteinase-9 (MMP-9), and B-cell lymphoma-2 (Bcl-2) after ox-LDL induction and transfection of miR-9 mimic, miR-9 inhibitor and XIST expression vector, respectively. The results showed that lncRNA XIST overexpression vector was successfully constructed and transfected into cells, wh5ich can inhibit the expression level of miR-9. Compared with the normal control group, ox-LDL can inhibit cell proliferation, promote cell apoptosis, and increase the expression level of target protein. Moreover, transfection of XIST expression vector based on ox-LDL induction can significantly enhance the inhibition of cell proliferation, and promote cell apoptosis and the expression of target protein. Transfection of miR-9 mimic can improve the biological changes induced by ox-LDL. After co-transfection of miR-9 mimic and XIST expression vector based on ox-LDL induction, cell proliferation, apoptosis, and target protein expression level were not significantly different from those induced by ox-LDL alone. In summary, the increased expression level of miR-9 can inhibit the apoptosis of macrophages induced by ox-LDL. lncRNA XIST can positively regulate the apoptosis of macrophages induced by ox-LDL.

ASK1 signaling regulates phase-specific glial interactions during neuroinflammation

Neuroinflammation is well known to be associated with neurodegenerative diseases. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that has been implicated in neuroinflammation, but its precise cellular and molecular mechanisms remain unknown. In this study, we generated conditional knockout (CKO) mice that lack ASK1 in T cells, dendritic cells, microglia/macrophages, microglia, or astrocytes, to assess the roles of ASK1 during experimental autoimmune encephalomyelitis (EAE). We found that neuroinflammation was reduced in both the early and later stages of EAE in microglia/macrophage-specific ASK1 knockout mice, whereas only the later-stage neuroinflammation was ameliorated in astrocyte-specific ASK1 knockout mice. ASK1 deficiency in T cells and dendritic cells had no significant effects on EAE severity. Further, we found that ASK1 in microglia/macrophages induces a proinflammatory environment, which subsequently activates astrocytes to exacerbate neuroinflammation. Microglia-specific ASK1 deletion was achieved using a CX3CR1CreER system, and we found that ASK1 signaling in microglia played a major role in generating and maintaining disease. Activated astrocytes produce key inflammatory mediators, including CCL2, that further activated and recruited microglia/macrophages, in an astrocytic ASK1-dependent manner. Astrocyte-specific analysis revealed CCL2 expression was higher in the later stage compared with the early stage, suggesting a greater proinflammatory role of astrocytes in the later stage. Our findings demonstrate cell-type-specific roles of ASK1 and suggest phase-specific ASK1-dependent glial cell interactions in EAE pathophysiology. We propose glial ASK1 as a promising therapeutic target for reducing neuroinflammation.

Exploration of binding mechanism of triclosan towards cancer markers using molecular docking and molecular dynamics

The molecule 5-chloro-2-(2,4-dichlorophenoxy) phenol is well-known as Triclosan (TCS), which is also a potential endocrine disrupting synthetic chemical. TCS exposure has been connected to the control of the human enoyl-acyl carrier protein-reductase (hER), which has been linked to a range of life threatening diseases. However, other than hER, the new protein targets for TCS that are responsible for a variety of cancers are yet unclear. The goal of this work is to investigate into the protein binding patterns of TCS and proteins from various cancer signaling pathways. Discovery Studio 4.1 was used to perform molecular docking and molecular dynamics (MD) on the protein-triclosan complex. The proteins were first screened using CHARMM-based docking with a CDOCKER energy greater than −21.40 kcal/mol. The CDOCKER energies of Fas-associated death domain (FADD), Receptor-interacting protein 1 (RIP1), F-κB-inducing kinase (NIK), c-Jun N-terminal kinase (JNK), Apoptosis signal-regulating kinase 1 (ASK1), B-cell lymphoma 2 (Bcl-2), Apoptosis-inducing factor (AIF), α-tubulin, and Actin were −20.68 kcal/mol, −26.88 kcal/mol, −23.43 kcal/mol, −22.21 kcal/mol, −20.40 kcal/mol, −21.10 kcal/mol, −20.98 kcal/mol, −24.67 kcal/mol, and −23.09 kcal/mol respectively. MD was performed on the screened proteins by standard dynamics cascade tool using CHARMM Force field. The MD results were accessed using the energy–time graph, root-mean-square deviation (RMSD), and root mean square fluctuations (RMSF). The 100 conformers of α-tubulin, NIK, FADD, and RIP1 were found to have a trend of increasing RMSD, whereas Bcl-2, ASK1, AIF, Actin, and JNK proteins had lower RMSD values. In compared to FADD, AIF, and JNK, the RMSF variations of the Bcl-2, ASK1, α-tubulin, Actin, NIK, and RIP1 residues were shown to be high. Similar patterns were seen in the energy variations, which range from 1000 kcal/mol to 2000 kcal/mol. RIP1 and Bcl-2 showed more variation in the sidechain RMSF in comparison to FADD, ASK1, AIF, Actin, α-tubulin, NIK and JNK. Thus, it can be postulated that AIF and JNK proteins of apoptosis signaling pathway are pivotal in the TCS mediated reactions.

PDK4 Decrease Neuronal Apoptosis via Inhibiting ROS-ASK1/P38 Pathway in Early Brain Injury After Subarachnoid Hemorrhage.

Aims: Metabolic disorders may play key roles in oxidative stress and neuronal apoptosis in response to early brain injury (EBI) after subarachnoid hemorrhage (SAH). Pyruvate dehydrogenase (PDH) is related to oxidative stress in EBI, and its activity obviously decreases after SAH. We discovered that only pyruvate dehydrogenase kinase 4 (PDK4) expression was obviously increased among the four PDK isozymes after SAH in preliminary experiments. Therefore, we attempted to investigate the effects and corresponding mechanisms of PDK4 on oxidative stress after SAH. Results: First, we confirmed that PDK4 overexpression promoted PDH phosphorylation, inhibited PDH activity, and changed cell metabolism after SAH. A small interfering RNA (siRNA) targeting PDK4, a lentiviral PDK4 overexpression vector, and dichloroacetic acid (DCA) were used to regulate the expression and activity of PDK4. The siRNA decreased PDH phosphorylation, promoted reactive oxygen species (ROS) production, activated the apoptosis signal-regulating kinase 1 (ASK1)/P38 pathway, and induced neuronal apoptosis. The lentivirus further attenuated PDH activity, oxidative stress, and neuronal apoptosis. DCA inhibited the activity of PDK4, but increased the expression of PDK4 due to a feedback mechanism. Inactivated PDK4 did not effectively suppress PDH activity, which increased ROS production, activated the ASK1/P38 pathway, and led to neuronal apoptosis. Innovation: This study provides new insights into the potential antioxidant and antiapoptotic effects of the PDK4-PDH axis on EBI after SAH. Conclusions: The early overexpression of PDK4 after SAH may attenuate neuronal apoptosis by reducing oxidative stress via the ROS/ASK1/P38 pathway. PDK4 may be a new potential therapeutic target to ameliorate EBI after SAH. Antioxid. Redox Signal. 36, 505-524.

Hepatocyte-specific deletion of cellular repressor of E1A-stimulated genes 1 exacerbates alcohol-induced liver injury by activating stress kinases.

Alcohol-associated liver disease (ALD) encompasses a wide range of pathologies from simple steatosis to cirrhosis and hepatocellular carcinoma and is a global health problem. Currently, there are no effective pharmacological treatments for ALD. We have previously demonstrated that aging exacerbates the pathogenesis of ALD, but the underlying mechanisms are still poorly understood. Cellular repressor of E1A-stimulated genes 1 protein (CREG1) is a recently identified small glycoprotein that has been implicated in aging process by promoting cellular senescence and activating stress kinases. Thus, the current study aimed to explore the role of aging associated CREG1 in ALD pathogenesis and CREG1 as a potential therapeutic target. Hepatic and serum CREG1 protein levels were elevated in ALD patients. Elevation of hepatic CREG1 protein and mRNA was also observed in a mouse model of Gao-binge alcohol feeding. Genetic deletion of the Creg1 gene in hepatocytes (Creg1∆hep) markedly exacerbated ethanol-induced liver injury, apoptosis, steatosis and inflammation. Compared to wild-type mice, Creg1∆hep mice had increased phosphorylation of hepatic stress kinases such as apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase (JNK) and p38 but not TGF-β-activated kinase 1 (TAK1) or extracellular signal-regulated kinase (ERK) after alcohol feeding. In vitro, ethanol treatment elevated the phosphorylation of ASK1, JNK, and p38 in mouse hepatocyte AML-12 cells. This elevation was further enhanced by CREG1 knockdown but alleviated by CREG1 overexpression. Last, treatment with an ASK1 inhibitor abolished ethanol-induced liver injury and upregulated hepatic lipogenesis, proinflammatory genes and stress kinases in Creg1∆hep mice. Taken together, our data suggest that CREG1 protects against alcoholic liver injury and inflammation by inhibiting the ASK1-JNK/p38 stress kinase pathway and that CREG1 is a potential therapeutic target for ALD.

N-acetylgalactosaminyltransferase-4 protects against hepatic ischemia/reperfusion injury by blocking apoptosis signal-regulating kinase 1 N-terminal dimerization.

Ischemia-reperfusion (I/R) injury is an inevitable complication of liver transplantation (LT) and compromises its prognosis. Glycosyltransferases have been recognized as promising targets for disease therapy, but their roles remain open for study in hepatic I/R (HIR) injury. Here, we aim to demonstrate the exact function and molecular mechanism of a glycosyltransferase, N-acetylgalactosaminyltransferase-4 (GALNT4), in HIR injury. By an RNA-sequencing data-based correlation analysis, we found a close correlation between GALNT4 expression and HIR-related molecular events in a murine model. mRNA and protein expression of GALNT4 were markedly up-regulated upon reperfusion surgery in both clinical samples from subjects who underwent LT and in a mouse model. We found that GALNT4 deficiency significantly exacerbated I/R-induced liver damage, inflammation, and cell death, whereas GALNT4 overexpression led to the opposite phenotypes. Our in-depth mechanistic exploration clarified that GALNT4 directly binds to apoptosis signal-regulating kinase 1 (ASK1) to inhibit its N-terminal dimerization and subsequent phosphorylation, leading to a robust inactivation of downstream c-Jun N-terminal kinase (JNK)/p38 and NF-κB signaling. Intriguingly, the inhibitory capacity of GALNT4 on ASK1 activation is independent of its glycosyltransferase activity. GALNT4 represents a promising therapeutic target for liver I/R injury and improves liver surgery prognosis by inactivating the ASK1-JNK/p38 signaling pathway.

Liver fibrosis: Extracellular vesicles mediated intercellular communication in perisinusoidal space.

The complex functional activity of the liver is well-orchestrated thanks to very well-structured crosstalk between all hepatic parenchymal and non-parenchymal cell populations. Paracrine interactions involving extracellular vesicles (EVs) released by various cell types in the liver are vital intercellular communication tools. In normal liver, quiescent hepatic stellate cells (HSCs) released EVs regulate cellular regeneration, survival, cell differentiation, cell migration, and immune responses. After the chronic liver injury, HSCs are activated, acquire myofibroblasts (MFBs) like phenotype and abnormally secrete extracellular matrix proteins that generate fibrous scar. To maintain the pathological status of the injured liver, activated HSCs secrete specific EVs containing profibrotic contents, which interact with hepatocytes, quiescent HSCs, liver sinusoidal endothelial cells (LSECs) and Kupffer cells in the perisinusoidal and sinusoidal spaces. The activated HSCs secrete EVs rich in miR-192, platelet-derived growth factor receptor, and apoptosis signal-regulating kinase 1 to activate quiescent HSCs via the mechanistic target of rapamycin and rho-associated protein kinase 1 signaling axis, known as "horizontal transfer process". In parallel, injured hepatocytes-derived pro-inflammatory miRNAs (miR-19a and -192) and transforming growth factor-β rich exosomes will activate toll-like receptors-9 and -3 on Kupffer cells and HSCs respectively to acquire a fibrogenic phenotype. The Kupffer cells and LSECs released exosome cargos containing miR-103-3p and sphingosine kinase 1, respectively, activate HSCs. The epidermal growth factor-rich exosomes from senescent HSCs increase the viability of hepatoma cells. Thus, perisinusoidal space is a critical microenvironment that coordinates a variety of intercellular signaling mediated by HSCs, hepatocytes, LSECs and Kupffer cells in fibrotic liver diseases.