Apoptosis Signal-regulating Kinase Research Articles

ASK1/p38‑mediated NLRP3 inflammasome signaling pathway contributes to aberrant retinal angiogenesis in diabetic retinopathy.

Diabetic retinopathy (DR) is the leading cause of blindness among the working-age population in several countries. Despite the available treatments, some patients are diagnosed at the late stages of the disease when treatment is more difficult. Hence, it is crucial that novel targets are identified in order to improve the clinical therapy of DR. In the present study, an animal model of DR and a cell model using primary human retinal microvascular endothelial cells exposed to high glucose were constructed to examine the association between apoptosis signal-regulating kinase 1 (ASK1)/p38 and NLR family pyrin domain containing 3 (NLRP3) in DR. The results revealed that DR induced inflammatory response and micro-vascular cell proliferation. NLRP3 contributed to DR-mediated inflammatory development and progression, which promoted the expression of inflammatory-related cytokines. In addition, NLRP3 promoted the tube formation of retinal microvascular endothelial cells and angiogenesis. Moreover, further research indicated that the NLRP3-mediated aberrant retinal angiogenesis in DR was regulated by ASK1 and p38. It was thus suggested that ASK1/p38 may be novel target for the treatment of DR.

FGF21 Induced by the ASK1-p38 Pathway Promotes Mechanical Cell Competition by Attracting Cells

Cell competition is a social cellular phenomenon in which unfit cells are selectively eliminated to maintain tissue homeostasis.1-3 Recent studies have revealed that mechanical forces induce competitive cell-cell interactions in Drosophila.4-6 This mechanical cell competition has also been reported to play an important role in mammalian cells, using Madin-Darby canine kidney (MDCK) cells depleted of a polarity regulator Scribble in a tetracycline-inducible manner (scribKD cells).7scribKD cells are hypersensitive to crowding due to the lower homeostatic density than wild-type (WT) cells,7,8 and in the context of cell competition, scribKD cells are compacted and eliminated by WT cells.7-10 Although p38 and p53 are involved in this process,7,10 the molecular mechanism by which WT cells recognize and mechanically eliminate scribKD cells remains unclear. Here, we report that scribKD cells secrete fibroblast growth factor 21 (FGF21) to drive cell competition. Knockdown of FGF21 in scribKD cells or loss of FGFR1 in WT cells suppresses cell competition, suggesting that WT cells recognize scribKD cells through FGF21. FGF21-containing culture medium of scribKD cells activates cell motility. Moreover, FGF21 promotes the compression and elimination of scribKD cells by attracting surrounding WT cells. We also demonstrate that activation of the apoptosis signal-regulating kinase 1 (ASK1)-p38 pathway in scribKD cells induces FGF21 to drive cell competition. Our findings reveal a mechanism whereby WT cells mechanically eliminate scribKD cells and propose a new function for FGF21 in cell-cell communication.

Tiao Geng decoction inhibits tributyltin chloride-induced GT1-7 neuronal apoptosis through ASK1/MKK7/JNK signaling pathway

Ethnopharmacological relevanceTiao Geng (TG) decoction is a Chinese herbal medicine extract that has been utilized for the treatment of menopausal symptoms for a history of over 30 years. In our previous study, we suggest that TG decoction possibly exerts an anti-apoptotic effect on hypothalamic neurons of ovariectomized rats via the ASK1/MKK7/JNK pathway. Tributyltin chloride (TBTC) causes oxidative damage and induces apoptosis of primary hypothalamic neurons in rats. Aim of the studyThe present work aimed to explore the inhibition of TG decoction on TBTC-induced GT1-7 cell apoptosis and its possible molecular mechanism. Materials and methodsThe GT1-7 cell line was exposed to TG decoction at diverse doses (31.25, 62.5, 125 μg/mL) for 24 h and later with TBTC (1 mg/L) for 1 h, with 17β-E2 (100 nM) treatment being the positive control. Then, CCK8 assay was conducted to evaluate cell viability, while flow cytometric analysis was conducted to examine the apoptosis level. Related pathways and differentially expressed proteins were identified by tandem mass tag (TMT)-based quantitative phosphoproteomics. qRT-PCR was carried out to examine mRNA levels of Bax and B-cell lymphoma-2 (Bcl-2). Western blotting was performed to detect the levels of Bax, Bcl-2, c-Jun, c-Jun N-terminal kinase (JNK), Caspase-3 (Casp3), Mitogen-activated protein kinase kinase 7 (MKK7), and apoptosis signal-regulating kinase 1 (ASK1) .Finally, cells were pretreated with SP600125, an inhibitor of JNK, later the expression of JNK and Casp3 was measured. ResultsApplication of TG decoction mitigated the GT1-7 cell apoptosis and injury caused by TBTC; besides, it inhibited the activation of the ASK1/MKK7/JNK pathway. Moreover, Bcl-2/Bax ratio became higher, and the MKK7, ASK1, Casp3 and c-Jun levels were inhibited. Besides, TG decoction combined with SP600125 (the JNK inhibitor) more significantly inhibited GT1-7 cell apoptosis caused by TBTC. ConclusionAs discovered from the experiment in this study, TG decoction has a neuroprotective effect, which is achieved through inhibiting the ASK1/MKK7/JNK signal transduction pathway to reduce GT1-7 cell apoptosis.

Dysregulated Immune Responses by ASK1 Deficiency Alter Epithelial Progenitor Cell Fate and Accelerate Metaplasia Development during H. pylori Infection.

The mechanism of H. pylori-induced atrophy and metaplasia has not been fully understood. Here, we demonstrate the novel role of Apoptosis signal-regulating kinase 1 (ASK1) and downstream MAPKs as a regulator of host immune responses and epithelial maintenance against H. pylori infection. ASK1 gene deficiency resulted in enhanced inflammation with numerous inflammatory cells including Gr-1+CD11b+ myeloid-derived suppressor cells (MDSCs) recruited into the infected stomach. Increase of IL-1β release from apoptotic macrophages and enhancement of TH1-polarized immune responses caused STAT1 and NF-κB activation in epithelial cells in ASK1 knockout mice. Dysregulated immune and epithelial activation in ASK1 knockout mice led to dramatic expansion of gastric progenitor cells and massive metaplasia development. Bone marrow transplantation experiments revealed that ASK1 in inflammatory cells is critical for inducing immune disorder and metaplastic changes in epithelium, while ASK1 in epithelial cells regulates cell proliferation in stem/progenitor zone without changes in inflammation and differentiation. These results suggest that H. pylori-induced immune cells may regulate epithelial homeostasis and cell fate as an inflammatory niche via ASK1 signaling.

Role of cardiac drugs and flavonoids on the IRE1–JNK pathway as revealed by re-ranked molecular docking scores, MM/PBSA and umbrella sampling

One of the important causes of cardiac dysfunction is the triggering of apoptosis through the IRE1–JNK signaling pathway due to excessive ER stress (endoplasmic reticulum stress). Although there are various studies on beneficial or harmful side effects of cardiac drugs, knowledge about the molecular mechanism of their interactions on this pathway is very limited. In this study, we investigated interactions of statins, ace inhibitors, antiarrhythmic drugs and flavonoids in IRE1, ASK1(apoptosis signal-regulating kinase 1) and JNK1 at an atomic level in comparison with their well-known inhibitors. The rank of scores obtained from four different docking algorithms (Autodock 4, Autodock Vina, iGEMDOCK and GOLD) were combined so that they could be compared with each other and evaluated together. According to combined results, the most potent compound for each compound group was selected for molecular dynamics simulations, MM/PBSA (molecular mechanics/Poisson–Boltzmann surface area) and umbrella sampling calculations. We observed that the statin group drugs had the best affinity by interacting with ASK1 and JNK1 by having a similar effect with their inhibitors, and atorvastatin and pitavastatin came to the fore. Norizalpinine from the flavonoid group had a strong binding interaction with IRE1, and amiodarone from the antiarrhythmic drug group had high binding affinities with IRE1, ASK1 and JNK1. Our study has shown that atorvastatin, pitavastatin, norizalpinine and amiodarone may have a role in preventing cardiac dysfunctions caused by ER stress and may shed light on further in vitro and in vivo research. Communicated by Ramaswamy H. Sarma

Milk Fat Globule-Epidermal Growth Factor-Factor 8 Improves Hepatic Steatosis and Inflammation.

Milk fat globule-epidermal growth factor-factor 8 (MFGE8) has been shown to be a critical extracellular molecule that mediates apoptotic signaling in the pathological process of nonalcoholic fatty liver disease (NAFLD). MFGE8 is abundantly expressed in hepatocytes, but its function in the pathogenesis of NAFLD has not been characterized. In our current study, hepatic MFGE8 showed a protective role in the pathogenesis of NAFLD. Hepatic MFGE8 deletion largely exacerbated lipid accumulation and inflammatory responses in the liver in response to overnutrition. Mechanistically, intercellular MFGE8 was shown to directly bind to apoptosis signal-regulating kinase 1 (ASK1) and to inhibit its dimerization and phosphorylation under a normal diet. However, under metabolic challenges, decreased cytoplasmic MFGE8 facilitated the dimerization and phosphorylation of ASK1 and subsequent mitogen-activated protein kinase signaling in hepatocytes. Hepatic MFGE8 is an endogenous inhibitor that halts the progression of hepatic steatosis and inflammation. Metabolic challenge-induced loss of intracellular MFGE8 facilitates ASK1 dimerization and phosphorylation. Therefore, maintaining hepatic MFGE8 levels may serve as an alternative strategy for the treatment of NAFLD.

Loss of the spectraplakin gene Short stop induces a DNA damage response in Drosophila epithelia

Epithelia are an eminent tissue type and a common driver of tumorigenesis, requiring continual precision in cell division to maintain tissue structure and genome integrity. Mitotic defects often trigger apoptosis, impairing cell viability as a tradeoff for tumor suppression. Identifying conditions that lead to cell death and understanding the mechanisms behind this response are therefore of considerable importance. Here we investigated how epithelia of the Drosophila wing disc respond to loss of Short stop (Shot), a cytoskeletal crosslinking spectraplakin protein that we previously found to control mitotic spindle assembly and chromosome dynamics. In contrast to other known spindle-regulating genes, Shot knockdown induces apoptosis in the absence of Jun kinase (JNK) activation, but instead leads to elevated levels of active p38 kinase. Shot loss leads to double-strand break (DSB) DNA damage, and the apoptotic response is exacerbated by concomitant loss of p53. DSB accumulation is increased by suppression of the spindle assembly checkpoint, suggesting this effect results from chromosome damage during error-prone mitoses. Consistent with DSB induction, we found that the DNA damage and stress response genes, Growth arrest and DNA damage (GADD45) and Apoptosis signal-regulating kinase 1 (Ask1), are transcriptionally upregulated as part of the shot-induced apoptotic response. Finally, co-depletion of Shot and GADD45 induced significantly higher rates of chromosome segregation errors in cultured cells and suppressed shot-induced mitotic arrest. Our results demonstrate that epithelia are capable of mounting molecularly distinct responses to loss of different spindle-associated genes and underscore the importance of proper cytoskeletal organization in tissue homeostasis.

ASK1 Enhances Angiotensin II-Induced Liver Fibrosis In Vitro by Mediating Endoplasmic Reticulum Stress-Dependent Exosomes.

Background Apoptosis signal-regulating kinase 1 (ASK1) has been reported to induce fibrotic signaling in the setting of oxidative stress. However, the role of ASK1 and its mechanism of action in angiotensin II- (Ang II-) induced liver fibrosis remain largely unknown. Methods Human hepatic LX-2 stellate cells were treated with Ang II alone or cotreated with Ang II plus an ASK1 inhibitor (GS-4997) or siRNA-targeting ASK1. Immunofluorescent staining, real-time PCR, and western blotting were used to determine the expressionof α-SMA, Col I, and Col III expression. Cell viability was assessed by the CCK-8 assay. The concentrations of IL-1β, IL-18, and TNF-α in conditioned medium were determined by ELISA. The levels of intracellular ROS in LX-2 cells were analyzed using a ROS assay kit. Exosome size was determined by electron microscopy. Results Ang II markedly increased the expression of extracellular matrix (ECM) proteins (α-SMA, Col I, and Col III) and proinflammatory cytokines (IL-1β, IL-18, and TNF-α). Ang II also increased the expression of endoplasmic reticulum stress (ERS) markers (GRP78, p-PERK, and CHOP) and p-ASK1. Results also showed that pretreatment with GS-4997 or siRNA could abolish all the abovementioned effects on LX-2 cells. Furthermore, we found that exosome release caused by ASK1-mediated ERS was involved in the activation of LX-2 cells by Ang II. The activation of LX-2 cells could be blocked by treating the exosomes with annexin. Conclusions In summary, we found that ASK1 mediates Ang II-activated ERS in HSCs and the subsequent activation of HSCs, suggesting a promising strategy for treating liver fibrosis.

Apelin‑36 protects against lipopolysaccharide‑induced acute lung injury by inhibiting the ASK1/MAPK signaling pathway.

Apelin-36 is able to mediate a range of effects on various diseases, and is upregulated in lipopolysaccharide (LPS)-induced acute lung injury (ALI). However, to the best of our knowledge, whether apelin-36 is able to regulate LPS-induced ALI has yet to be investigated. The present study aimed to investigate the role of apelin-36 in LPS-induced ALI, and the putative underlying mechanisms. Rats were assigned to one of four treatment groups: The Control group, apelin-36 group, LPS group and LPS + apelin-36 group. At 4 h after intratracheal instillation of LPS (5 mg/kg), rats were intraperitoneally treated with 10 nmol/kg apelin-36. Subsequently, pathological manifestations and the extent of inflammation and apoptosis of the lung tissues were assessed. Untransfected and apoptosis signal-regulating kinase 1 (ASK1)-overexpressing Beas-2B cells were treated with LPS in the absence or presence of apelin-36, and subsequently the levels of inflammation and apoptosis were assessed. The results obtained showed that the level of apelin-36 was increased in the bronchoalveolar lavage fluid (BALF) of LPS-treated rats. Co-treatment with apelin-36 alleviated LPS-induced lung injury and pulmonary edema, reduced the levels of pro-inflammatory cytokines, including interleukin-6, monocyte chemoattractant protein-1 and tumor necrosis factor-α, in BALF, and inhibited apoptosis in the lung tissues. The presence of apelin-36 also blocked the activation of LPS-induced ASK1, p38, c-Jun N-terminal kinase and extracellular signal-regulated kinase in lung tissues. In vitro studies performed with Beas-2B cells showed that the addition of apelin-36 led to an increase in the cell viability of LPS-induced Beas-2B cells in a concentration-dependent manner. Additionally, co-treatment with 1 µM apelin-36 prevented LPS-induced inflammation and apoptosis. However, overexpression of ASK1 significantly reversed the inhibitory effects of apelin-36 on LPS-induced inflammation and apoptosis. Taken together, the results of the present study demonstrated that apelin-36 was able to protect against LPS-induced lung injury both in vivo and in vitro, and these actions may be dependent on inhibition of the ASK1/mitogen-activated protein kinase signaling pathway.

Apoptosis signal‐regulating kinase 1 regulates immune‐mediated thrombocytopenia, thrombosis, and systemic shock

BackgroundImmune complexes (ICs) bind to and activate platelets via FcγRIIA, causing patients to experience thrombocytopenia, as well as an increased risk of forming occlusive thrombi. Although platelets have been shown to mediate IC‐induced pathologies, the mechanisms involved have yet to be fully elucidated. We identified that apoptosis signal‐regulating kinase 1 (ASK1) is present in both human and mouse platelets and potentiates many platelet functions. ObjectivesHere we set out to study ASK1's role in regulating IC‐mediated platelet functions in vitro and IC‐induced pathologies using an in vivo mouse model. MethodsUsing human platelets treated with an ASK1‐specific inhibitor and platelets from FCGR2A/Ask1‐/‐ transgenic mice, we examined various platelet functions induced by model ICs in vitro and in vivo. ResultsWe found that ASK1 was activated in human platelets following cross‐linking of FcγRIIA using either anti‐hCD9 or IV.3 + goat‐anti‐mouse. Although genetic deletion or inhibition of ASK1 significantly attenuated anti‐CD9‐induced platelet aggregation, activation of the canonical FcγRIIA signaling targets Syk and PLCγ2 was unaffected. We further found that anti‐mCD9‐induced cPla2 phosphorylation and TxA2 generation is delayed in Ask1 null transgenic mouse platelets leading to diminished δ‐granule secretion. In vivo, absence of Ask1 protected FCGR2A transgenic mice from thrombocytopenia, thrombosis, and systemic shock following injection of anti‐mCD9. In whole blood microfluidics, platelet adhesion and thrombus formation on fibrinogen was enhanced by Ask1. ConclusionsThese findings suggest that ASK1 inhibition may be a potential target for the treatment of IC‐induced shock and other immune‐mediated thrombotic disorders.

Inhibition of apoptosis signal-regulating kinase 1 mitigates the pathogenesis of human immunodeficiency virus-associated nephropathy.

Chronic kidney disease (CKD) is a common cause of morbidity and mortality in human immunodeficiency virus (HIV)-positive individuals. Among the HIV-related kidney diseases, HIV-associated nephropathy (HIVAN) is a rapidly progressive renal disease characterized by collapsing focal glomerulosclerosis (GS), microcystic tubular dilation, interstitial inflammation and fibrosis. Although the incidence of end-stage renal disease due to HIVAN has dramatically decreased with the widespread use of antiretroviral therapy, the prevalence of CKD continues to increase in HIV-positive individuals. Recent studies have highlighted the role of apoptosis signal-regulating kinase 1 (ASK1) in driving kidney disease progression through the activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase and selective ASK-1 inhibitor GS-444217 was recently shown to reduce kidney injury and disease progression in various experimental models. Therefore we examined the efficacy of ASK1 antagonism by GS-444217 in the attenuation of HIVAN in Tg26 mice. GS-444217-supplemented rodent chow was administered in Tg26 mice at 4 weeks of age when mild GS and proteinuria were already established. After 6 weeks of treatment, the kidney function assessment and histological analyses were performed and compared between age- and gender-matched control Tg26 and GS-444217-treated Tg26 mice. GS-444217 attenuated the development of GS, podocyte loss, tubular injury, interstitial inflammation and renal fibrosis in Tg26 mice. These improvements were accompanied by a marked reduction in albuminuria and improved renal function. Taken together, GS-4442217 attenuated the full spectrum of HIVAN pathology in Tg26 mice. ASK1 signaling cascade is central to the development of HIVAN in Tg26 mice. Our results suggest that the select inhibition of ASK1 could be a potential adjunctive therapy for the treatment of HIVAN.

Elaidic Acid Potentiates Extracellular ATP-Induced Apoptosis via the P2X7-ROS-ASK1-p38 Axis in Microglial Cell Lines.

trans-Fatty acids (TFAs) are unsaturated fatty acids with at least one carbon-carbon double bond in trans configuration. TFA consumption has been epidemiologically associated with neurodegenerative diseases (NDs) including Alzheimer's disease. However, the underlying mechanisms of TFA-related NDs remain unknown. Here, we show a novel microglial signaling pathway that induces inflammation and cell death, which is dramatically enhanced by elaidic acid (EA), the most abundant TFA derived from food. We found that extracellular ATP, one of the damage-associated molecular patterns (DAMPs) leaked from injured cells, induced activation of the apoptosis signal-regulating kinase 1 (ASK1)-p38 pathway, which is one of the major stress-responsive mitogen-activated protein (MAP) kinase signaling pathways, and subsequent caspase-3 cleavage and DNA ladder formation (hallmarks of apoptosis) in mouse microglial cell lines including BV2 and MG6 cells. Furthermore, we found that in these microglial cell lines, EA, but not its cis isomer oleic acid, facilitated extracellular ATP-induced ASK1/p38 activation and apoptosis, which was suppressed by pharmacological inhibition of either p38, reactive oxygen species (ROS) generation, P2X purinoceptor 7 (P2X7), or Ca2+/calmodulin-dependent kinase II (CaMKII). These results demonstrate that in microglial cells, extracellular ATP induces activation of the ASK1-p38 MAP kinase pathway and ultimately apoptosis downstream of P2X7 receptor and ROS generation, and that EA promotes ATP-induced apoptosis through CaMKII-dependent hyperactivation of the ASK1-p38 pathway, in the same manner as in macrophages. Our study may provide an insight into the pathogenesis of NDs associated with TFAs.

In silico and in vitro investigations on the protein–protein interactions of glutathione S-transferases with mitogen-activated protein kinase 8 and apoptosis signal-regulating kinase 1

Cytosolic glutathione S-transferase (GST) enzymes participate in several cellular processes in addition to facilitating glutathione conjugation reactions that eliminate endogenous and exogenous toxic compounds, especially electrophiles. GSTs are thought to interact with various kinases, resulting in the modulation of apoptotic processes and cellular proliferation. The present research used a combination of in silico and in vitro studies to investigate protein–protein interactions between the seven most abundant cytosolic GSTs—GST alpha-1 (GST-A1), GST alpha-2 (GST-A2), GST mu-1 (GST-M1), GST mu-2 (GST-M2), GST mu-5 (GST-M5), GST theta-1 (GST-T1) and GST pi-1 (GST-P1)—and Mitogen-activated protein kinase 8 (MAPK8) and Apoptosis signal-regulating kinase 1 (ASK1). MAPK8 and ASK1 were chosen as this study’s protein interaction partners because of their predominant role in electrophile or cytokine-induced stress-mediated apoptosis, inflammation and fibrosis. The highest degree of sequence homology or sequence similarity was observed in two GST subgroups: the GST-A1, GST-A2 and GST-P1 isoforms constituted subgroup1; the GST-M1, GST-M2 and GST-M5 isoforms constituted subgroup 2. The GST-T1 isoform diverged from these isoforms. In silico investigations revealed that GST-M1 showed a significantly higher binding affinity to MAPK8, and its complex was more structurally stable than the other isoforms, in the order GST-M1 > GST-M5 > GST-P1 > GST-A2 > GST-A1 > GST-M2 > GST-T1. Similarly, GST-A1, GST-P1 and GST-T1 actively interacted with ASK1, and their structural stability was also better, in the order GST-T1 > GST-A1 > GST-P1 > GST-A2 > GST-M5 > GST-M1 > GST-M2. To validate in silico results, we performed in vitro crosslinking and mass spectroscopy experiments. Results indicated that GST-M1 interacted with GST-T1 to form heterodimers and confirmed the predicted interaction between GST-M1 and MAPK8. Communicated by Ramaswamy H. Sarma

Natriuretic Peptides and MicroRNAs 155 And 425 in Human Heart Failure: Biomarker Roles and Pathophysiologic Regulation

Introduction MicroRNAs (miRNAs) have recently emerged as important molecular entities in heart failure (HF) pathophysiology and diagnosis. To date, miR-155 and miR-425 have been implicated in the inhibition of the cardiac natriuretic peptide (NP) ANP gene with their role on BNP undefined. In HF, adrenergic and neurohormonal pathways are dysregulated. Our goal was to advance our understanding of the regulation of these two miRNAs and their predictive value in human HF. Hypothesis We hypothesized that miR-155 and -425 levels are reduced in HF compared to normal subjects. We also investigated the molecular mechanisms of NPs expression regulated by the miR-425 and -155. Methods Normal (healthy, NM, n=100) and Acute HF (AHF, n=74) cohorts were studied. Two confirmatory cohorts were also recruited: NM (n=20) and AHF (n=28). Plasma NT-proBNP was measured. Plasma miR-155 and -425 expression were quantified with RT-PCR. In left ventricular myocardium (LV) from NM or HF (n=5 each), Nppa (ANP gene), Nppb (BNP gene), miR-155, and -425 expressions were measured. In vitro, human cardiomyocytes (HCM) were treated with vehicle (Veh), 10-4 M catecholamine isoproterenol (ISO) and 10-6 M cytokine endothelin (ET) which are known to be activated in HF, mimics of miR-155 or -425, followed by gene expression assessment. Data expressed as means±SEM. * p Results MiR-155 and miR-425 were significantly lower in AHF patients (miR-155: NM: 1.8±0.29, HF: 0.13±0.02*), (miR-425: NM: 0.15±0.03, HF: 0.01±0.003*) than NM. The area under the curve (AUC) of the two miRNAs are 0.86 and 0.87 in AHF cohorts respectively (vs NT-proBNP 0.98). MiR-425 negatively correlates with NT-proBNP in AHF cohorts (r=-0.33*). Downregulation of both miRNAs were validated in a second AHF cohort. Similarly, in HF LV tissues, HF displayed lower miR-155 and miR-425 levels than NM group, while NP gene and protein expression were increased. In vitro, treatment with ISO or ET in HCM reduced miR-155, miR-425 levels and significantly elevated Nppa and Nppb levels. Furthermore in HCM, treatment with mimic of miR-425 reduced Nppa (Veh: 1.0±0.03, miR-425 mimic: 0.7±0.02*) and Nppb levels (Veh: 1.0±0.03, miR-425 mimic: 0.5±0.01*). We also demonstrated that apoptosis signal-regulating kinase 1 (ASK1) and Cofilin 2, which have been reported to enhance Nppa and Nppb expression, was also reduced by miR-425. Conclusion MiR-155 and miR-425 are reduced in HF, which may be one of the mechanisms contributing to an increase in circulating ANP and BNP. MiR-425 also displays a negative correlation with NT-proBNP. In vitro, cardiac stress hormones known to be activated in HF downregulate these 2 miRNAs and increase NP gene expression which provides further insight into mechanisms of NP activation in HF. The findings for the first time implicate the pathway of stress hormones/miR-425/NP production in human HF simulated in vitro and further our understanding of NP activation in HF. These studies also support biomarker value for miR-155 and miR-425 and the compensatory nature of this decline in HF.

Redox Regulation of Cardiac ASK1 (Apoptosis Signal-Regulating Kinase 1) Controls p38-MAPK (Mitogen-Activated Protein Kinase) and Orchestrates Cardiac Remodeling to Hypertension.

Systemic hypertension increases cardiac workload causing cardiomyocyte hypertrophy and increased cardiac fibrosis. An underlying feature is increased production of reactive oxygen species. Redox-sensitive ASK1 (apoptosis signal-regulating kinase 1) activates stress-regulated protein kinases (p38-MAPK [mitogen-activated protein kinases] and JNKs [c-Jun N-terminal kinases]) and promotes fibrosis in various tissues. Here, we determined the specificity of ASK1 signaling in the heart, with the hypothesis that ASK1 inhibitors may be used to manage fibrosis in hypertensive heart disease. Using immunoblotting, we established that moderate levels of H2O2 activate ASK1 in neonatal rat cardiomyocytes and perfused rat hearts. ASK1 was activated during ischemia in adult rat hearts, but not on reperfusion, consistent with activation by moderate (not high) reactive oxygen species levels. In contrast, IL (interleukin)-1β activated an alternative kinase, TAK1 (transforming growth factor-activated kinase 1). ASK1 was not activated by IL1β in cardiomyocytes and activation in perfused hearts was due to increased reactive oxygen species. Selonsertib (ASK1 inhibitor) prevented activation of p38-MAPKs (but not JNKs) by oxidative stresses in cultured cardiomyocytes and perfused hearts. In vivo (C57Bl/6J mice with osmotic minipumps for drug delivery), selonsertib (4 mg/[kg·d]) alone did not affect cardiac function/dimensions (assessed by echocardiography). However, it suppressed hypertension-induced cardiac hypertrophy resulting from angiotensin II (0.8 mg/[kg·d], 7d), with inhibition of Nppa/Nppb mRNA upregulation, reduced cardiomyocyte hypertrophy and, notably, significant reductions in interstitial and perivascular fibrosis. Our data identify a specific reactive oxygen species→ASK1→p38-MAPK pathway in the heart and establish that ASK1 inhibitors protect the heart from hypertension-induced cardiac remodeling. Thus, targeting the ASK1→p38-MAPK nexus has potential therapeutic viability as a treatment for hypertensive heart disease.

MicroRNA-532-3p Regulates Pro-Inflammatory Human THP-1 Macrophages by Targeting ASK1/p38 MAPK Pathway.

Inflammation is a complex biological process which alters the normal physiological function of the immune system resulting in an abnormal microenvironment that leads to several clinical complications. The process of inflammation is mediated through various intracellular signaling factors inside the cells. Apoptosis signal-regulating kinase 1 (ASK1) is an inflammation-derived kinase that controls the activation of other family of kinases such as p38 mitogen-activated protein kinases (p38 MAPKs), which mediates various the inflammatory processes. In this study, we cultured THP-1 macrophage cells to undergo inflammatory proliferation with LPS (1μg/ml) and TNFα (10ng/ml) stimulation. Initial in silico analysis was utilized to predict novel microRNAs (miRNAs) that target ASK1 signaling and its expression levels in LPS and TNFαstimulated THP-1 cells were estimated. Among the miRNAs, miR-532-3p showcased the highest binding affinity towards ASK1 kinase. We witnessed that transient transfection of miR-532-3p diminished the levels of ASK1 and downstream phosphorylation/translocation of p38 MAPK. Furthermore, direct targeting of ASK1 resulted in regulation of uncontrolled release of cytokines (TNFα, IL-6, and IL-23) and chemokines (GM-CSF and MIP-2α). Overall, we suggest that miR-532-3p attenuates the pro-inflammatory nature of macrophages by targeting ASK1/p38 MAPK signaling pathway and can be used as a molecular intervention for treating inflammatory diseases.

Regulation of Cellular Stress Signaling in Bladder Ischemia.

IntroductionThe etiology of lower urinary tract symptoms in patients with non-obstructed non-neurogenic bladder remains largely unknown. Clinical studies divulged a significant correlation between reduced bladder blood flow and low bladder compliance. Animal models of bladder ischemia displayed structural modifications, characterized by loss of smooth muscle cells and accumulation of connective tissue in the bladder wall. The underlying mechanisms contributing to structural damage in bladder ischemia remain largely elusive. We previously reported that structural modifications in bladder ischemia correlate with upregulated stress proteins and cell survival signaling, suggesting the potential role of cellular stress in ischemic damage. However, stress response molecules and downstream pathways eliciting bladder damage in ischemia remain largely undetermined.MethodsUsing a rat model of bladder ischemia along with a cell culture hypoxia model, we investigated stress signaling molecules in the ischemic bladder tissues and hypoxic bladder smooth muscle cells.ResultsOur data suggest simultaneous upregulation of two major cellular stress-sensing molecules, namely apoptosis signal-regulating kinase 1 (ASK1) and caspase-3, implying degenerative insult via stress signaling pathway in bladder ischemia. Consistent with bladder ischemia, incubation of cultured human bladder smooth muscle cells at low oxygen tension increased both ASK1 and caspase-3 expression, insinuating hypoxia as an essential factor in ASK1 and caspase-3 upregulation. Gene deletion of ASK1 by ASK1 siRNA in cultured smooth muscle cells prevented caspase-3 upregulation by hypoxia, suggesting caspase-3 regulation by ASK1 under the ischemic/hypoxic conditions. Upregulation of ASK1 and caspase-3 in rat bladder ischemia and human bladder smooth muscle cell hypoxia was associated with subcellular structural modifications consistent with the initial stages of apoptotic insult.ConclusionOur data suggest that stress sensing by ASK1 and caspase-3 may contribute to subcellular structural damage and low bladder compliance. The ASK1/caspase-3 pathway may provide therapeutic targets against cellular stress and degenerative responses in bladder ischemia.

Protective Effect of Fenofibrate on Oxidative Stress-Induced Apoptosis in Retinal-Choroidal Vascular Endothelial Cells: Implication for Diabetic Retinopathy Treatment.

Diabetic retinopathy (DR) is an important microvascular complication of diabetes and one of the leading causes of blindness in developed countries. Two large clinical studies showed that fenofibrate, a peroxisome proliferator-activated receptor type α (PPAR-α) agonist, reduces DR progression. We evaluated the protective effects of fenofibrate on retinal/choroidal vascular endothelial cells under oxidative stress and investigated the underlying mechanisms using RF/6A cells as the model system and paraquat (PQ) to induce oxidative stress. Pretreatment with fenofibrate suppressed reactive oxygen species (ROS) production, decreased cellular apoptosis, diminished the changes in the mitochondrial membrane potential, increased the mRNA levels of peroxiredoxin (Prx), thioredoxins (Trxs), B-cell lymphoma 2 (Bcl-2), and Bcl-xl, and reduced the level of B-cell lymphoma 2-associated X protein (Bax) in PQ-stimulated RF/6A cells. Western blot analysis revealed that fenofibrate repressed apoptosis through cytosolic and mitochondrial apoptosis signal-regulated kinase-1 (Ask)-Trx-related signaling pathways, including c-Jun amino-terminal kinase (JNK) phosphorylation, cytochrome c release, caspase 3 activation, and poly (ADP-ribose) polymerase-1 (PARP-1) cleavage. These protective effects of fenofibrate on RF/6A cells may be attributable to its anti-oxidative ability. Our research suggests that fenofibrate could serve as an effective adjunct therapy for ocular oxidative stress-related disorders, such as DR.

The role of GSTπ isoform in the cells signalling and anticancer therapy.

The glutathione S-transferases (GSTs) overexpression in hyperproliferating tumour cells resistant to chemotherapy was demonstrated. An increased GST-π activity weakens the efficacy of anti-cancer drugs by promoting their efflux from cells. This review summarises available information on the physiological role of GSTs, in particular the role of GST-π, in regulation of signalling pathways mechanisms and cellular homeostasis for understanding and explaining the basis for GST-π application as a target for anticancer therapy and implications for clinical practice. GST-π can weaken the effect of TNF receptor-associated factor 2 (TRAF2) on apoptosis signal-regulating kinase-1 by inactivation of MAP kinase pathways (c-Jun N-terminal, p38 kinases). GST-π is involved in the metabolism of endogenous lipids mediators, such as 15-detoxy-Δ12,14-prostaglandin J2 (15d-PGJ2). Reduced binding of 15d-PGJ2 to peroxisome proliferator-activated receptors accompanied by GST-π can result in the inhibition of apoptosis. GSTP1 RNA is able to increase the phosphorylation of signal transducer and activator of transcription 3, what results in negative regulation as regards transcriptional activity thereof and affects the growth factor signalling. However, the oxidation of GST-π results in inhibition of TRAF2-GST-π complexes formation and unblocks cell apoptosis. The inhibition of multidrug resistance related proteins 1 (MRP-1) promoter activity and impairment of MRP-1 function can also act as a potent non-competitive inhibitor of GST-π. GST-π is recognised as an important target in designing new anticancer drugs. These drugs are often substrates for GST-π or have the affinity with its structure, what results in weakening its activity and achieving therapeutic goal.

Melatonin Alleviates Neuroinflammation and Metabolic Disorder in DSS-Induced Depression Rats

There is a bidirectional relationship between inflammatory bowel disease (IBD) and depression/anxiety. Emerging evidences indicate that the liver may be involved in microbiota-gut-brain axis. This experiment focused on the role of melatonin in regulating the gut microbiota and explores its mechanism on dextran sulphate sodium- (DSS-) induced neuroinflammation and liver injury. Long-term DSS-treatment increased lipopolysaccharide (LPS), proinflammation cytokines IL-1β and TNF-α, and gut leak in rats, breaking blood-brain barrier and overactivated astrocytes and microglia. Ultimately, the rats showed depression-like behavior, including reduction of sucrose preference and central time in open field test and elevation of immobility time in a forced swimming test. Oral administration with melatonin alleviated neuroinflammation and depression-like behaviors. However, melatonin supplementation did not decrease the level of LPS but increase short-chain fatty acid (SCFA) production to protect DSS-induced neuroinflammation. Additionally, western blotting analysis suggested that signaling pathways farnesoid X receptor-fibroblast growth factor 15 (FXR-FGF 15) in gut and apoptosis signal-regulating kinase 1 (ASK1) in the liver overactivated in DSS-treated rats, indicating liver metabolic disorder. Supplementation with melatonin markedly inhibited the activation of these two signaling pathways and its downstream p38. As for the gut microbiota, we found that immune response- and SCFA production-related microbiota, like Lactobacillus and Clostridium significantly increased, while bile salt hydrolase activity-related microbiota, like Streptococcus and Enterococcus, significantly decreased after melatonin supplementation. These altered microbiota were consistent with the alleviation of neuroinflammation and metabolic disorder. Taken together, our findings suggest melatonin contributes to reshape gut microbiota and improves inflammatory processes in the hippocampus (HPC) and metabolic disorders in the liver of DSS rats.