c-Jun N-terminal Kinase Inhibitor Research Articles

Poly(ε-caprolactone) Scaffolds Doped with c-Jun N-terminal Kinase Inhibitors Modulate Phagocyte Activation.

The modulation of phagocyte responses is essential for successful performance of biomaterials in order to prevent negative outcomes associated with inflammation. Herein, we developed electrospun poly(ε-caprolactone) (PCL) scaffolds doped with the novel potent c-Jun N-terminal kinase (JNK) inhibitors 11H-indeno[1,2-b]quinoxalin-11-one oxime (IQ-1) and 11H-indeno[1,2-b]quinoxalin-11-one O-(O-ethylcarboxymethyl) oxime(IQ-1E) as a promising approach for modulating phagocyte activation. Optimized electrospinning parameters allowed us to produce microfiber composite materials with suitable mechanical properties. We found that embedded compounds were bound to the polymer matrix via hydrophobic interactions and released in two steps, with release mostly controlled by Fickian diffusion. The fabricated scaffolds doped with active compounds IQ-1 and IQ-1E effectively inhibited phagocyte inflammatory responses. For example, they suppressed human neutrophil activation by the biomaterials, as indicated by decreased neutrophil reactive oxygen species (ROS) production and Ca2+ mobilization. In addition, they inhibited lipopolysaccharide (LPS)-induced NF-κB/AP-1 reporter activity in THP-1Blue cells and interleukin (IL)-6 production in MonoMac-6 cells without affecting cell viability. These effects were attributed to the released compounds rather than cell-surface interactions. Therefore, our study demonstrates that doping tissue engineering scaffolds with novel JNK inhibitors represents a powerful tool for preventing adverse immune responses to biomaterials as well as serves as a platform for drug delivery.

JNK Signaling Pathway Mediates Acetaminophen-Induced Hepatotoxicity Accompanied by Changes of Glutathione S-Transferase A1 Content and Expression.

Acetaminophen (APAP) is an analgesic–antipyretic drug and widely used in clinics. Its overdose may cause serious liver damage. Here, we examined the mechanistic role of c-Jun N-terminal kinase (JNK) signaling pathway in liver injury induced by different doses of APAP. Male mice were treated with APAP (150 and 175 mg·kg−1), and meanwhile JNK inhibitor SP600125 was used to interfere APAP-induced liver damage. The results showed that JNK signaling pathway was activated by APAP in a dose-dependent manner. C-Jun N-terminal kinase inhibitor decreased JNK and c-Jun activation significantly (P < 0.01) at 175 mg·kg−1 APAP dose, and phosphorylation levels of upstream proteins of JNK were also decreased markedly (P < 0.05). In addition, serum aminotransferases activities and hepatic oxidative stress increased in a dose-dependent manner with APAP treatment, but the levels of aminotransferases and oxidative stress decreased in mice treated with JNK inhibitor, which implied that JNK inhibition ameliorated APAP-induced liver damage. It was observed that apoptosis was increased in APAP-induced liver injury, and SP600125 can attenuate apoptosis through the inhibition of JNK phosphorylation. Meanwhile, glutathione S-transferases A1 (GSTA1) content in serum was enhanced, while GSTA1 content and expression in liver reduced significantly with administration of APAP (150 and 175 mg·kg−1). After inhibiting JNK, GSTA1 content in serum decreased significantly (P < 0.01); meanwhile, GSTA1 content and expression in liver enhanced. These findings suggested that JNK signaling pathway mediated APAP-induced hepatic injury, which was accompanied by varying GSTA1 content and expression in liver and serum.

Non-cell autonomous modulation of tyrosine hydroxylase by HMGB1 released from astrocytes in an acute MPTP-induced Parkinsonian mouse model

High-mobility group box 1 (HMGB1) is actively secreted from inflammatory cells and acts via a non-cell-autonomous mechanism to play an important role in mediating cell proliferation and migration. The HMGB1-RAGE (receptor for advanced glycation end products) axis upregulates tyrosine hydroxylase (TH) expression in response to extracellular insults in dopaminergic neurons in vitro, but little is known about HMGB1 in modulation of dopaminergic neurons in vivo. Here, using immunohistochemistry, we show that HMGB1 and RAGE expression are higher in the nigral area of MPTP (methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-treated mice, a toxin-induced Parkinsonian mouse model, compared with saline-treated controls. HMGB1 was predominantly localized to astrocytes and may affect neighboring dopaminergic neurons in the MPTP mouse model, owing to co-localization of RAGE in these TH-positive cells. In addition, MPTP induced a decrease in TH expression, an effect that was potentiated by inhibition of c-Jun N-terminal kinase (JNK) or RAGE. Moreover, stereotaxic injection of recombinant HMGB1 attenuated the MPTP-induced reduction of TH in a Parkinsonian mouse model. Collectively, our results suggest that an increase of HMGB1, released from astrocytes, upregulates TH expression in an acute MPTP-induced Parkinsonian mouse model, thereby maintaining dopaminergic neuronal functions.

Neuroprotective properties of the C-Jun N-terminal kinase (JNK) inhibitor in hypoxic hypoxia

The aim of the study was to reveal the influence of the JNK inhibitor on the induction of disturbances in the psychoneurological status of experimental animals in the modeling of posthypoxic encephalopathy and to reveal the mechanisms of its action related to the functioning of the neural stem cells of the brain. Materials and methods. The experiments were performed on 64 male outbred mice. Posthypoxic encephalopathy was modeled in non-native mice with hypoxia of the hermetic volume. The JNK inhibitor was administered to mice subcutaneously at a dose of 15 mg/kg once before hypoxic exposure. We studied the neuropsychiatric status, the content of neuronal stem cells in the subventricular zone of the brain of experimental animals, and the direct effect of the JNK inhibitor on intact neural stem cells in vitro. Results. The expressed cerebroprotective action of the pharmacological agent was revealed, which consisted of normalizing the indices of orientation and exploratory behavior and conditioned activity in experimental animals. These effects developed against a background of a significant increase in the content of neural stem cells in the subventricular zone of the brain. In the experiments in vitro, a direct stimulating effect of the JNK inhibitor on neural stem cells was found. Conclusions. The obtained results showed a neuroprotective action of the JNK inhibitor. At the same time, the prevention and compensation of the development of disturbances in the activity of the central nervous system is based on the preservation of the ability of the nerve tissue to repair andassociated with the functioning of resident neural stem cells.

Xiaoyaosan Produces Antidepressant Effects in Rats via the JNK Signaling Pathway

Background: Xiaoyaosan (XYS) has achieved definite curative effects in clinic. However, the mechanism is not clear. Previous studies of our team indicated XYS improved anxiety-like behaviors through inhibiting c-Jun N-terminal kinase (JNK) signaling pathway of hippocampus. Objectives: In the study, we explored whether the JNK signaling pathway is involved in the mechanism of XYS treating depression. Method: Forty-eight rats were divided randomly into 4 groups (n = 12): the control group (deionized water, p.o.), the model group (deionized water, p.o.), the fluoxetine group (2.08 mg/kg/day, p.o.), and the XYS group (3.9 g/kg/day, p.o.). All rats except for the control group were given continuous 21 days of chronic immobilization stress (CIS; 3 h/day). On day 29, the body weights and the behavioral tests, including the novelty suppressed feeding test, the open field test, and the elevated plus maze test, were measured. On day 30, all the rats were sacrificed, and three indices of the JNK signaling pathway were tested by Western blot. Results: The body weight and behavioral tests of all groups indicated that 21 days of CIS induced depression-like behaviors. After 21 days of treatment with fluoxetine and XYS, changes were seen in body weight, behaviors, and JNK, phosphorylated JNK (P-JNK), and phosphorylated c-Jun (P-c-Jun) levels in the hippocampus. Conclusions: XYS ameliorated the depression-like behaviors, potentially through affecting the JNK signaling pathway in the hippocampus.

Lipopolysaccharide inhibits GPR120 expression in macrophages via Toll-like receptor 4 and p38 MAPK activation.

Free fatty acid receptor G protein-coupled receptor 120 (GPR120) is highly expressed in macrophages and was reported to inhibit lipopolysaccharide (LPS)-stimulated cytokine expression. Under inflammation, macrophages exhibit striking functional changes, but changes in GPR120 expression and signaling are not known. In this study, the effects of LPS treatment on macrophage GPR120 expression and activation were investigated. The results showed that LPS inhibited GPR120 expression in mouse macrophage cell line Ana-1 cells. Moreover, LPS treatment inhibited GPR120 expression in mouse alveolar macrophages both in vitro and in vivo. The inhibitory effect of LPS on GPR120 expression was blocked by Toll-like receptor 4 (TLR4) inhibitor TAK242 and p38 mitogen-activated protein kinase inhibitor LY222820, but not by ERK1/2 inhibitor U0126 and c-Jun N-terminal kinase inhibitor SP600125. LPS-induced inhibition of GPR120 expression was not attenuated by GPR120 agonists TUG891 and GW9508. TUG891 inhibited the phagocytosis of alveolar macrophages, and LPS treatment counteracted the effects of TUG891 on phagocytosis. These results indicate that pretreatment with LPS inhibits GPR120 expression and activation in macrophages. It is suggested that LPS-induced inhibition of GPR120 expression is areaction enhancingthe LPS-induced pro-inflammatory response of macrophages.

Interleukin-1β promotes interleulin-6 expression via ERK1/2 signaling pathway in canine dermal fibroblasts.

Interleukin-6 (IL-6) is a pleiotropic cytokine involved in the regulation of the immune response and inflammation. In this study, we investigated effect of the proinflammatory cytokine interleukin-1β (IL-1β) on IL-6 expression in canine dermal fibroblasts. IL-1β induced IL-6 mRNA expression and protein release in a time- and dose-dependent manner. When cells were treated with inhibitors of mitogen-activated protein kinases (MAPKs), the extracellular signal-regulated kinase (ERK) inhibitor FR180240 inhibited IL-1β-induced IL-6 mRNA expression, but not SP600125 or SKF86002, which are c-Jun N-terminal kinase (JNK) and p38 MAPK inhibitors, respectively. In cells treated with U0126, an inhibitor of MAPK/ERK kinase (MEK), which activates ERK, IL-1β-induced IL-6 mRNA expression was also inhibited. IL-1β stimulated ERK1/2 phosphorylation. In cells transfected with ERK1 and ERK2 isoform siRNAs, IL-1β-induced IL-6 mRNA expression was reduced. These observations suggest that IL-1β induces IL-6 expression via ERK1/2 signaling pathway in canine dermal fibroblasts.

Docosahexaenoic acid ameliorates traumatic brain injury involving JNK-mediated Tau phosphorylation signaling

Hyperphosphorylation of Tau has been found in patients with traumatic brain injury (TBI). Inhibition of c-Jun N-terminal kinases (JNKs) improves neurological function by suppressing Tau phosphorylation. By inhibiting JNK, docosahexaenoic acid (DHA) protects against cognitive decline in a mouse model of Alzheimer’s disease (AD). We hypothesize that DHA protects against neuronal damage and behavioral deficits by inhibition of JNK mediated Tau phosphorylation. We induced TBI in mice and examined the phosphorylation status of JNK and Tau. We treated TBI and sham operated mice with DHA, and investigated the effects of DHA on JNK and Tau phosphorylation, hippocampal long term potentiation (LTP), learning and memory, and motor function by Western blot analysis, electrophysiology recording and behavioral assessments including Morris water maze test, beam-balance test, beam-walk test and rotarod test. We found that TBI induction lead to increased phosphorylation of JNK and Tau. DHA suppressed TBI induced JNK and Tau hyperphosphorylation, rescued TBI mediated hippocampal LTP deficits and hippocampus dependent learning and memory dysfunction, and improved motor function. Inhibition of JNK and Tau phosphorylation by DHA may represent a potential therapeutic strategy for TBI induced neurological dysfunction and Tauopathy.

IL-35 alleviates inflammation progression in a rat model of diabetic neuropathic pain via inhibition of JNK signaling

BackgroundEmerging evidence has demonstrated that inflammation is involved in the occurrence and development of diabetic neuropathic pain (DNP). The anti-inflammatory property of interleukin (IL)-35 makes it a promising candidate to block the pain perception. The present study was undertaken to investigate whether IL-35 could attenuate DNP in streptozotocin (STZ)-induced rat model and its potential mechanism.MethodsThe rat model of DNP was established by a single STZ injection followed by measurements of fasting blood glucose and insulin. Fourteen days after STZ injection, DNP rats were intrathecally injected with IL-35, c-Jun N-terminal kinase (JNK) inhibitor or activator or dimethylsulfoxide (DMSO) as vehicle control, respectively. The mechanical allodynia was assayed to evaluate the therapeutic effect of IL-35. In mechanism study, the serum and protein levels of inflammatory cytokines using ELISA and western blotting and the activation of JNK signaling were further evaluated by quantitative reverse transcription PCR (qRT-PCR). Histopathologic changes were evaluated by Nissl staining. Apoptosis was examined using TUNEL staining.ResultsDNP rats exhibited increased fasting blood glucose and insulin levels and reduced insulin sensitivity index (ISI). Intrathecal injection of IL-35 reduced accumulation of pro-inflammatory cytokines in the spinal cord of DNP rats. Furthermore, IL-35 displayed anti-inflammatory and anti-apoptotic effects via inhibition of JNK pathway.ConclusionIL-35 treatment mitigated DNP via downregulating JNK signaling pathway.

β-Conglycinin-Induced Intestinal Porcine Epithelial Cell Damage via the Nuclear Factor κB/Mitogen-Activated Protein Kinase Signaling Pathway

Soybean allergy is a serious health risk to humans and animals; β-conglycinin is the primary antigenic protein in soybean. Intestinal porcine epithelial (IPEC-J2) cells were used as an in vitro physiological model of the intestinal epithelium to study the effects of different concentrations of soybean antigen protein β-conglycinin to identify the involved signaling pathways. The cells were divided into eight groups and either untreated or treated with different concentrations of β-conglycinin, pyrrolidine dithiocarbamate (PDTC), Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), SP600125, and SB202190 either alone or in combination. The cells were incubated with 1, 5, and 10 mg·mL-1 β-conglycinin or 5 mg·mL-1 β-conglycinin and 1 μmol·L-1 nuclear factor κB (NF-κB) inhibitor (PDTC), inducible nitric oxide synthase inhibitor (l-NAME), c-Jun N-terminal kinase (JNK) inhibitor (SP600125), and p38 inhibitor (SB202190) for 24 h, separately; controls were left untreated. The mRNA, protein, and phosphorylation levels of NF-κB, p38, and JNK were higher in the treated groups than in the control group. β-Conglycinin decreased tight junction distribution, destroyed the cytoskeleton of IPEC-J2 cells, and caused cell death. After the addition of the inhibitors, β-conglycinin-induced IPEC-J2 cell damage was significantly reduced. β-Conglycinin caused damage to IPEC-J2 cells via the mitogen-activated protein kinase/NF-κB signaling pathway. The results of this study are crucial for exploring the mechanisms underlying allergic reactions caused by soybean antigen proteins.

MCP-induced protein 1 attenuates sepsis-induced acute lung injury by modulating macrophage polarization via the JNK/c-Myc pathway

Sepsis is a potentially fatal systemic inflammatory response syndrome caused by infection. In this study, we evaluated the effects of MCP-induced protein 1 (MCPIP1), a recently discovered inflammation-related ribonuclease, on sepsis-induced acute lung injury (ALI) and investigated the underlying mechanisms. Cecal ligation puncture and lipopolysaccharide induction were performed on Sprague-Dawley rats and RAW264.7 cells, respectively, to establish sepsis-induced ALI models. The proteasome inhibitor MG132 used as an activator of MCPIP1 overexpression, and we showed that MG132 can indeed increase the expression of MCPIP1. MCPIP1 overexpression induced by MG132 alleviated sepsis-induced pathologic changes, water content and protein leakage in the lungs, and induction of systemic inflammatory mediators, and improved the 7-day mortality rate in the model rats. We also showed that MCPIP1 p showed romoted macrophage polarization from the M1 to the M2 type in sepsis-induced ALI. Furthermore, MCPIP1-enhanced M2 polarization was inhibited by an MCPIP1-targeting small interfering RNA (siMCPIP1) in RAW264.7 cells. Further mechanistic studies showed that the promotive effect of MCPIP1 on M2 polarization was related to the inhibition of c-Jun N-terminal kinase (JNK) and its downstream transcription factor c-Myc in the in vitro model. Conversely, siMCPIP1 transfection resulted in the recovery of JNK and c-Myc expression in LPS-treated cells. Taken together, these findings indicate that MCPIP1 plays a protective role in sepsis-induced ALI by modulating macrophage polarization through inhibition of the JNK/c-Myc signaling pathway. Our study presents a potentially novel therapeutic strategy for the treatment of lung injury involving the inflammatory cascade.

Effect of a Small Selective Inhibitor of C-Jun N-Terminal Kinase on the Inducible mRNA Expression of Interleukin-6 and Interleukin-18.

BACKGROUND:The expression of many inducible genes, involved in cell growth and differentiation as cytokine genes are regulated by receptor-activated intracellular signalling pathways, including the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase pathway.AIM:We examined the involvement of the JNK signalling pathway in the regulation of the inducible interleukin-6 (IL-6) and interleukin-18 (IL-18) gene expression at the transcriptional level.METHODS:Peripheral blood mononuclear cells (PBMC) from healthy donors were stimulated with lipopolysaccharide (LPS) and C3 binding glycoprotein (C3bgp) with or without SP600125 and cultured for 6 h. After mRNA isolation, a qRT-PCR was performed.RESULTS:Regarding IL-6 and IL-18 mRNA expression, donors were divided into two groups of high and low responders. SP600125 inhibited significantly IL-6 mRNA transcription in the high responder group and did not influence the transcription level in the low responder group. Concerning IL-18 mRNA, we detect the significant effect of SP600125 on the inducible mRNA in high responder group upon C3bgp stimulation.CONCLUSION:JNK transduction pathway is involved in the production of IL-6 mRNA, after LPS and C3bgp stimulation. We suggest that the inhibition of JNK may be beneficial only for higher responding patients during the treatment of inflammatory and autoimmune diseases.

Quercetin Inhibits the Production of IL-1β-Induced Inflammatory Cytokines and Chemokines in ARPE-19 Cells via the MAPK and NF-κB Signaling Pathways.

Quercetin, a bioflavonoid derived from vegetables and fruits, exerts anti-inflammatory effects in various diseases. Our previous study revealed that quercetin could suppress the expression of matrix metalloprotease-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1) to achieve anti-inflammatory effects in tumor necrosis factor-α (TNF-α)-stimulated human retinal pigment epithelial (ARPE-19) cells. The present study explored whether quercetin can inhibit the interleukin-1β (IL-1β)-induced production of inflammatory cytokines and chemokines in ARPE-19 cells. Prior to stimulation by IL-1β, ARPE-19 cells were pretreated with quercetin at various concentrations (2.5–20 µM). The results showed that quercetin could dose-dependently decrease the mRNA and protein levels of ICAM-1, IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1). It also attenuated the adherence of the human monocytic leukemia cell line THP-1 to IL-1β-stimulated ARPE-19 cells. We also demonstrated that quercetin inhibited signaling pathways related to the inflammatory process, including phosphorylation of mitogen-activated protein kinases (MAPKs), inhibitor of nuclear factor κ-B kinase (IKK)α/β, c-Jun, cAMP response element-binding protein (CREB), activating transcription factor 2 (ATF2) and nuclear factor (NF)-κB p65, and blocked the translocation of NF-κB p65 into the nucleus. Furthermore, MAPK inhibitors including an extracellular signal-regulated kinase (ERK) 1/2 inhibitor (U0126), a p38 inhibitor (SB202190) and a c-Jun N-terminal kinase (JNK) inhibitor (SP600125) decreased the expression of soluble ICAM-1 (sICAM-1), but not ICAM-1. U0126 and SB202190 could inhibit the expression of IL-6, IL-8 and MCP-1, but SP600125 could not. An NF-κB inhibitor (Bay 11-7082) also reduced the expression of ICAM-1, sICAM-1, IL-6, IL-8 and MCP-1. Taken together, these results provide evidence that quercetin protects ARPE-19 cells from the IL-1β-stimulated increase in ICAM-1, sICAM-1, IL-6, IL-8 and MCP-1 production by blocking the activation of MAPK and NF-κB signaling pathways to ameliorate the inflammatory response.

Protein Tyrosine Phosphatase Inhibitor, Orthovanadate, Induces Contraction &lt;i&gt;via&lt;/i&gt; Rho Kinase Activation in Mouse Thoracic Aortas

Orthovanadate (OVA), a protein tyrosine phosphatase inhibitor, induces contraction in endothelium-denuded mouse thoracic aortas. OVA-induced contraction was significantly (vs. control rings) suppressed by Rho kinase (Y-27632, 10 µM), extracellular signal-regulated kinase 1 and 2 (Erk1/2, FR180204, 10 µM), Erk1/2 kinase (MEK, PD98059, 10 µM), epidermal growth factor receptor (EGFR, AG1478, 10 µM), and Src inhibitors, and was partially suppressed by c-Jun N-terminal kinase (JNK, AS601245, 10 µM) and p38 (SB203580, 10 µM) inhibitors. However, a myosin light chain kinase inhibitor (ML-7, 10 µM) and a metalloproteinase inhibitor (TAPI-0, 10 µM) had no effect on OVA-induced contraction in mouse thoracic aortas. Phosphorylation of myosin phosphatase target subunit 1 (MYPT1) was abolished by inhibitors of Src, EGFR, MEK, Erk1/2, and Rho kinase, but not by inhibitors of JNK and p38. Erk1/2 phosphorylation by OVA was blocked by inhibitors of EGFR, Src, MEK, and Erk1/2, but not by Rho kinase inhibition. Src phosphorylation at Tyr-416 was abrogated by only Src inhibitor. EGFR phosphorylation at Tyr-1173 was suppressed by a Src inhibitor. These findings suggest that OVA induces contraction via activation of Src, EGFR, MEK, Erk1/2, and Rho kinase, leading to inactivation of myosin light chain phosphatase via MYPT1 phosphorylation.

Neurotoxic Effect of Ethanolic Extract of Propolis in the Presence of Copper Ions is Mediated through Enhanced Production of ROS and Stimulation of caspase-3/7 Activity.

Elevated amounts of copper are considered to be contributing factor in the progression of neurodegenerative diseases as they promote oxidative stress conditions. The aim of our study was to examine the effects of ethanolic extract of propolis (EEP) against copper-induced neuronal damage. In cultured P19 neuronal cells, EEP exacerbated copper-provoked neuronal cell death by increasing the generation of reactive oxygen species (ROS) and through the activation of caspase-3/7 activity. EEP augmented copper-induced up-regulation of p53 and Bax mRNA expressions. Neurotoxic effects of EEP were accompanied by a strong induction of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression and decrease in the expression of c-fos mRNA. SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK) prevented detrimental effects of EEP, whereas SP600125, an inhibitor of c-Jun N-terminal kinase (JNK), exacerbated EEP-induced neuronal cell death. Quercetin, a polyphenolic nutraceutical, which is usually present in propolis, was also able to exacerbate copper-induced neuronal death. Our data indicates a pro-oxidative and apoptotic mode of EEP action in the presence of excess copper, wherein ROS/p53/p38 interactions play an important role in death cascades. Our study also pointed out that detailed pharmacological and toxicological studies must be carried out for propolis and other dietary supplements in order to fully recognize the potential adverse effects in specific conditions.

Inhibiting c-Jun N-terminal kinase (JNK)-mediated apoptotic signaling pathway in PC12 cells by a polysaccharide (CCP) from Coptis chinensis against Amyloid-β (Aβ)-induced neurotoxicity

In this study, we investigated the protective effect and possible mechanism of a polysaccharide (CCP) from Coptis chinensis against Amyloid-β protein (Aβ)-induced toxicity in PC12 cells. The results showed pretreatment with CCP significantly protected PC12 cells from Aβ25–35 induced cell death, lactate dehydrogenase (LDH) release, nuclear fragmentation, mitochondrial dysfunction and cytochrome c release from mitochondria. Furthermore, CCP (100 μg/ml) significantly inhibited Aβ25–35 induced c-Jun N-terminal kinase (JNK) phosphorylation, but not influence signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (p38MAPK) pathway, and interestingly, the promoting effect of CCP on PC12 cell survival was only blocked by pre-treatment with a SP600125 (JNK inhibitor). In addition, Aβ25–35-induced increase of Bax and cleaved caspase-3, as well as decrease of Bcl-2 protein expression was markedly reversed by CCP or SP600125. Thus, our results indicate that the neuroprotective effect of CCP is associated with JNK-dependent apoptotic pathway.

Repression of hepatocyte nuclear factor 4 alpha by AP-1 underlies dyslipidemia associated with retinoic acid

All-trans retinoic acid (atRA) is used to treat certain cancers and dermatologic diseases. A common adverse effect of atRA is hypercholesterolemia; cytochrome P450 (CYP) 7A repression is suggested as a driver. However, the underlying molecular mechanisms remain unclear. We investigated CYP7A1 expression in the presence of atRA in human hepatocytes and hepatic cell lines. In HepaRG cells, atRA increased cholesterol levels dose-dependently alongside dramatic decreases in CYP7A1 expression. Lentiviral-mediated CYP7A1 overexpression reversed atRA-induced cholesterol accumulation, suggesting that CYP7A1 repression mediated cholesterol accumulation. In CYP7A1 promoter reporter assays and gene-knockdown studies, altered binding of hepatocyte nuclear factor 4 α (HNF4α) to the proximal promoter was essential for atRA-mediated CYP7A1 repression. Pharmacologic inhibition of c-Jun N-terminal kinase (JNK) and ERK pathways attenuated atRA-mediated CYP7A1 repression and cholesterol accumulation. Overexpression of AP-1 (c-Jun/c-Fos), a downstream target of JNK and ERK, repressed CYP7A1 expression. In DNA pull-down and chromatin immunoprecipitation assays, AP-1 exhibited sequence-specific binding to the proximal CYP7A1 promoter region overlapping the HNF4α binding site, and atRA increased AP-1 but decreased HNF4α recruitment to the promoter. Collectively, these results indicate that atRA activates JNK and ERK pathways and the downstream target AP-1 represses HNF4α transactivation of the CYP7A1 promoter, potentially responsible for hypercholesterolemia.

Delayed Treatment With 4-Methylpyrazole Protects Against Acetaminophen Hepatotoxicity in Mice by Inhibition of c-Jun n-Terminal Kinase.

Acetaminophen (APAP) overdose is the most common cause of hepatotoxicity and acute liver failure in the United States and many western countries. However, the only clinically approved antidote, N-acetylcysteine, has a limited therapeutic window. 4-Methylpyrazole (4MP) is an antidote for methanol and ethylene glycol poisoning, and we have recently shown that cotreatment of 4MP with APAP effectively prevents toxicity by inhibiting Cyp2E1. To evaluate if 4MP can be used therapeutically, C57BL/6J mice were treated with 300 mg/kg APAP followed by 50 mg/kg 4MP 90 min later (after the metabolism phase). In these experiments, 4MP significantly attenuated liver injury at 3, 6, and 24 h after APAP as shown by 80%-90% reduction in plasma alanine aminotransferase activities and reduced areas of necrosis. 4MP prevented c-Jun c-Jun N-terminal kinase (JNK) activation and its mitochondrial translocation, and reduced mitochondrial oxidant stress and nuclear DNA fragmentation. 4MP also prevented JNK activation in other liver injury models. Molecular docking experiments showed that 4MP can bind to the ATP binding site of JNK. These data suggest that treatment with 4MP after the metabolism phase effectively prevents APAP-induced liver injury in the clinically relevant mouse model in vivo mainly through the inhibition of JNK activation. 4MP, a drug approved for human use, is as effective as N-acetylcysteine or can be even more effective in cases of severe overdoses with prolonged metabolism (600 mg/kg). 4MP acts on alternative therapeutic targets and thus may be a novel approach to treatment of APAP overdose in patients that complements N-acetylcysteine.

JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).

Osteoblasts are versatile cells involved in multiple whole-body processes, including bone formation and immune response. Secretory amounts and patterns of osteoblast-derived proteins such as osteopontin (OPN) and osteocalcin (OCN) modulate osteoblast function. However, the regulatory mechanism of OPN and OCN expression remains unknown. Here, we demonstrate that p54/p46 c-jun N-terminal kinase (JNK) inhibition suppresses matrix mineralization and OCN expression but increases OPN expression in MC3T3-E1 cells and primary osteoblasts treated with differentiation inducers, including ascorbic acid, bone morphogenic protein-2, or fibroblast growth factor 2. Preinhibition of JNK before the onset of differentiation increased the number of osteoblasts that highly express OPN but not OCN (OPN-OBs), indicating that JNK affects OPN secretory phenotype at the early stage of osteogenic differentiation. Additionally, we identified JNK2 isoform as being critically involved in OPN-OB differentiation. Microarray analysis revealed that OPN-OBs express characteristic transcription factors, cell surface markers, and cytokines, including glycoprotein hormone α2 and endothelial cell-specific molecule 1. Moreover, we found that inhibitor of DNA binding 4 is an important regulator of OPN-OB differentiation and that dual-specificity phosphatase 16, a JNK-specific phosphatase, functions as an endogenous regulator of OPN-OB induction. OPN-OB phenotype was also observed following LPS from Porphyromonas gingivalis stimulation during osteogenic differentiation. Collectively, these results suggest that the JNK-Id4 signaling axis is crucial in the control of OPN and OCN expression during osteoblastic differentiation.-Kusuyama, J., Amir, M. S., Albertson, B. G., Bandow, K., Ohnishi, T., Nakamura, T., Noguchi, K., Shima, K., Semba, I., Matsuguchi, T. JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).

MiRNA-145-5p induces apoptosis after ischemia-reperfusion by targeting dual specificity phosphatase 6.

Disorders mainly caused by ischemia-reperfusion (I/R), including stroke and myocardial infarction, is linked to debilitating health conditions and death. Recent research indicates that microRNAs (miRNAs) mediate the process of ischemic pathology. This study investigated the effects of miR-145-5p in regulating myocardial ischemic injury. The I/R models were established in rat cardiomyocytes H9C2 and rats. Western blot analysis and quantitative polymerase chain reaction was performed to analyze protein expression. Annexin V-FITC/PI staining was conducted to evaluate cell apoptosis. The application of miR-145-5p mimics and inhibitor revealed that miR-145-5p promoted apoptosis in cardiomyocytes. Furthermore, we found that miR-145-5p directly inhibited dual specificity phosphatase 6 (DUSP6) by luciferase reporter assay. The results indicated that DUSP6 was beneficial against I/R injury through inhibiting c-Jun N-terminal kinase pathways. In conclusion, the essential roles of miR-145-5p and DUSP6 in I/R provide a novel therapeutic target to develop future intervention strategies.