JNK inhibition reduces apoptosis and neovascularization in a murine model of age-related macular degeneration.

PubMed

Du, Hongjun; Sun, Xufang; Guma, Monica; Luo, Jing; Ouyang, Hong; Zhang, Xiaohui; Zeng, Jing; Quach, John; Nguyen, Duy H; Shaw, Peter X; Karin, Michael; Zhang, Kang

2013-02-05

Age-related macular degeneration (AMD) is the leading cause of registered blindness among the elderly and affects over 30 million people worldwide. It is well established that oxidative stress, inflammation, and apoptosis play critical roles in pathogenesis of AMD. In advanced wet AMD, although, most of the severe vision loss is due to bleeding and exudation of choroidal neovascularization (CNV), and it is well known that vascular endothelial growth factor (VEGF) plays a pivotal role in the growth of the abnormal blood vessels. VEGF suppression therapy improves visual acuity in AMD patients. However, there are unresolved issues, including safety and cost. Here we show that mice lacking c-Jun N-terminal kinase 1 (JNK1) exhibit decreased inflammation, reduced CNV, lower levels of choroidal VEGF, and impaired choroidal macrophage recruitment in a murine model of wet AMD (laser-induced CNV). Interestingly, we also detected a substantial reduction in choroidal apoptosis of JNK1-deficient mice. Intravitreal injection of a pan-caspase inhibitor reduced neovascularization in the laser-induced CNV model, suggesting that apoptosis plays a role in laser-induced pathological angiogenesis. Intravitreal injection of a specific JNK inhibitor decreased choroidal VEGF expression and reduced pathological CNV. These results suggest that JNK1 plays a key role in linking oxidative stress, inflammation, macrophage recruitment apoptosis, and VEGF production in wet AMD and pharmacological JNK inhibition offers a unique and alternative avenue for prevention and treatment of AMD.

JNK pathway activation is controlled by Tao/TAOK3 to modulate ethanol sensitivity.

PubMed

Kapfhamer, David; King, Ian; Zou, Mimi E; Lim, Jana P; Heberlein, Ulrike; Wolf, Fred W

2012-01-01

Neuronal signal transduction by the JNK MAP kinase pathway is altered by a broad array of stimuli including exposure to the widely abused drug ethanol, but the behavioral relevance and the regulation of JNK signaling is unclear. Here we demonstrate that JNK signaling functions downstream of the Sterile20 kinase family gene tao/Taok3 to regulate the behavioral effects of acute ethanol exposure in both the fruit fly Drosophila and mice. In flies tao is required in neurons to promote sensitivity to the locomotor stimulant effects of acute ethanol exposure and to establish specific brain structures. Reduced expression of key JNK pathway genes substantially rescued the structural and behavioral phenotypes of tao mutants. Decreasing and increasing JNK pathway activity resulted in increased and decreased sensitivity to the locomotor stimulant properties of acute ethanol exposure, respectively. Further, JNK expression in a limited pattern of neurons that included brain regions implicated in ethanol responses was sufficient to restore normal behavior. Mice heterozygous for a disrupted allele of the homologous Taok3 gene (Taok3Gt) were resistant to the acute sedative effects of ethanol. JNK activity was constitutively increased in brains of Taok3Gt/+ mice, and acute induction of phospho-JNK in brain tissue by ethanol was occluded in Taok3Gt/+ mice. Finally, acute administration of a JNK inhibitor conferred resistance to the sedative effects of ethanol in wild-type but not Taok3Gt/+ mice. Taken together, these data support a role of a TAO/TAOK3-JNK neuronal signaling pathway in regulating sensitivity to acute ethanol exposure in flies and in mice.

Activation of the Jnk signaling pathway by a dual-specificity phosphatase, JSP-1

PubMed Central

Shen, Yu; Luche, Ralf; Wei, Bo; Gordon, Marcia L.; Diltz, Curtis D.; Tonks, Nicholas K.

2001-01-01

The mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli, such as growth factors, hormones, and cytokines, and to a wide variety of environmental stresses. The MAPKs, which are stimulated by phosphorylation of a TXY motif in their activation loop, are components of signal transduction cascades in which sequential activation of protein kinases culminates in their activation and their subsequent phosphorylation of various effector proteins that mediate the physiological response. MAPKs are also subject to dephosphorylation and inactivation, both by enzymes that recognize the residues of the TXY motif independently and by dual specificity phosphatases, which dephosphroylate both Tyr and Ser/Thr residues. We report the identification and characterization of a novel dual specificity phosphatase. Contrary to expectation, this broadly expressed enzyme did not inactivate MAPKs in transient cotransfection assays but instead displayed the capacity to function as a selective activator of the MAPK Jnk, hence the name, Jnk Stimulatory Phosphatase-1 (JSP-1). This study illustrates a new aspect of the regulation of MAPK-dependent signal transduction and raises the possibility that JSP-1 may offer a different perspective to the study of various inflammatory and proliferative disorders associated with dysfunctional Jnk signaling. PMID:11717427

Activation of the Jnk signaling pathway by a dual-specificity phosphatase, JSP-1.

PubMed

Shen, Y; Luche, R; Wei, B; Gordon, M L; Diltz, C D; Tonks, N K

2001-11-20

The mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli, such as growth factors, hormones, and cytokines, and to a wide variety of environmental stresses. The MAPKs, which are stimulated by phosphorylation of a TXY motif in their activation loop, are components of signal transduction cascades in which sequential activation of protein kinases culminates in their activation and their subsequent phosphorylation of various effector proteins that mediate the physiological response. MAPKs are also subject to dephosphorylation and inactivation, both by enzymes that recognize the residues of the TXY motif independently and by dual specificity phosphatases, which dephosphroylate both Tyr and Ser/Thr residues. We report the identification and characterization of a novel dual specificity phosphatase. Contrary to expectation, this broadly expressed enzyme did not inactivate MAPKs in transient cotransfection assays but instead displayed the capacity to function as a selective activator of the MAPK Jnk, hence the name, Jnk Stimulatory Phosphatase-1 (JSP-1). This study illustrates a new aspect of the regulation of MAPK-dependent signal transduction and raises the possibility that JSP-1 may offer a different perspective to the study of various inflammatory and proliferative disorders associated with dysfunctional Jnk signaling.

The Proteasome Inhibitor Bortezomib Enhances ATRA-Induced Differentiation of Neuroblastoma Cells via the JNK Mitogen-Activated Protein Kinase Pathway

PubMed Central

Luo, Peihua; Lin, Meili; Li, Lin; Yang, Bo; He, Qiaojun

2011-01-01

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Differentiated human NBs are associated with better outcome and lower stage; induction of differentiation is considered to be therapeutically advantageous. All-trans retinoic acid (ATRA) has been shown to induce the differentiation of neuroblastoma (NB) cell lines. The proteasome inhibitor bortezomib inhibits cell growth and angiogenesis in NBs. Here, we investigated the synergistic effect between bortezomib and ATRA in inducing NB cell differentiation in different NB cell lines. Bortezomib combined with ATRA had a significantly enhanced antiproliferative effect. This inhibition was characterized by a synergistic increase in neuronal differentiation. At the same time, the combination therapy showed little neuronal toxicity which was assessed in primary cultures of rat cerebellar granule cells by the MTT assay, PI staining. The combination of bortezomib and ATRA triggered increased differentiation through the activation of proteins, including RARα, RARβ, RARγ, p-JNK and p21, compared with ATRA treatment alone. Using JNK inhibitor SP600125 to block JNK-dependent activity, the combination therapy-induced neuronal differentiation was partially attenuated. In addition, p21 shRNA had no effect on the combination therapy-induced neuronal differentiation. The in vivo antitumor activities were examined in human NB cell xenografts and GFP-labeled human NB cell xenografts. Treatment of human NB cell CHP126-bearing nude mice with ATRA plus bortezomib resulted in more significant tumor growth inhibition than mice treated with either drug alone. These findings provide the rationale for the development of a new therapeutic strategy for NB based on the pharmacological combination of ATRA and bortezomib. PMID:22087283

Protective effect of resveratrol against nigrostriatal pathway injury in striatum via JNK pathway.

PubMed

Li, Dan; Liu, Nan; Zhao, Liang; Tong, Lei; Kawano, Hitoshi; Yan, Hong-Jing; Li, Hong-Peng

2017-01-01

Nigrostriatal pathway injury is one of the traumatic brain injury models that usually lead to neurological dysfunction or neuron necrosis. Resveratrol-induced benefits have recently been demonstrated in several models of neuronal degeneration diseases. However, the protective properties of resveratrol against neurodegeneration have not been explored definitely. Thus, we employ the nigrostriatal pathway injury model to mimic the insults on the brain. Resveratrol decreased the p-ERK expression and increased the p-JNK expression compared to the DMSO group, but not alter the p38 MAPK proteins around the lesion site by Western blot. Prior to the injury, mice were infused with resveratrol intracerebroventricularly with or without JNK-IN-8, a specific c-JNK pathway inhibitor for JNK1, JNK2 and JNK4. The study assessed modified improved neurological function score (mNSS) and beam/walking test, the level of inflammatory cytokines IL-1β, IL-6 and TNF-α, and striatal expression of Bax and Bcl-2 proteins associated with neuronal apoptosis. The results revealed that resveratrol exerted a neuroprotective effect as shown by the improved mNSS and beam latency, anti-inflammatory effects as indicated by the decreased level of IL-1β, TNF-α and IL-6. Furthermore, resveratrol up-regulated the protein expression of p-JNK and Bcl-2, down-regulated the expression of Bax and the number of Fluoro-Jade C (FJC) positive neurons. However, these advantages of resveratrol were abolished by JNK-IN-8 treatment. Overall, we demonstrated that resveratrol treatment attenuates the nigrostriatal pathway injury-induced neuronal apoptosis and inflammation via activation of c-JNK signaling. Copyright © 2016 Elsevier B.V. All rights reserved.

Febuxostat, an Inhibitor of Xanthine Oxidase, Suppresses Lipopolysaccharide-Induced MCP-1 Production via MAPK Phosphatase-1-Mediated Inactivation of JNK

PubMed Central

Nomura, Johji; Busso, Nathalie; Ives, Annette; Tsujimoto, Syunsuke; Tamura, Mizuho; So, Alexander; Yamanaka, Yoshihiro

2013-01-01

Excess reactive oxygen species (ROS) formation can trigger various pathological conditions such as inflammation, in which xanthine oxidase (XO) is one major enzymatic source of ROS. Although XO has been reported to play essential roles in inflammatory conditions, the molecular mechanisms underlying the involvement of XO in inflammatory pathways remain unclear. Febuxostat, a selective and potent inhibitor of XO, effectively inhibits not only the generation of uric acid but also the formation of ROS. In this study, therefore, we examined the effects of febuxostat on lipopolysaccharide (LPS)-mediated inflammatory responses. Here we show that febuxostat suppresses LPS-induced MCP-1 production and mRNA expression via activating MAPK phosphatase-1 (MKP-1) which, in turn, leads to dephosphorylation and inactivation of JNK in macrophages. Moreover, these effects of febuxostat are mediated by inhibiting XO-mediated intracellular ROS production. Taken together, our data suggest that XO mediates LPS-induced phosphorylation of JNK through ROS production and MKP-1 inactivation, leading to MCP-1 production in macrophages. These studies may bring new insights into the novel role of XO in regulating inflammatory process through MAPK phosphatase, and demonstrate the potential use of XO inhibitor in modulating the inflammatory processes. PMID:24086554

Molybdenum induces pancreatic β-cell dysfunction and apoptosis via interdependent of JNK and AMPK activation-regulated mitochondria-dependent and ER stress-triggered pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Tsung-Yuan; Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung 402, Taiwan; Yen, Cheng-Chieh

2016-03-01

Molybdenum (Mo), a well-known toxic environmental and industrial pollutant, causes adverse health effects and diseases in humans and has received attention as a potential risk factor for DM. However, the roles of Mo in the mechanisms of the toxicological effects in pancreatic β-cells are mostly unclear. In this study, the results revealed dysfunction of insulin secretion and apoptosis in the pancreatic β-cell-derived RIN-m5F cells and the isolated mouse islets in response to Mo. These effects were accompanied by a mitochondria-dependent apoptotic signals including a decreased in the MMP, an increase in cytochrome c release, and the activation of caspase cascadesmore » and PARP. In addition, ER stress was triggered as indicated by several key molecules of the UPR. Furthermore, exposure to Mo induced the activation of ERK1/2, JNK, AMPKα, and GSK3-α/β. Pretreatment with specific pharmacological inhibitors (in RIN-m5F cells and isolated mouse islets) of JNK (SP600125) and AMPK (Compound C) or transfection with si-RNAs (in RIN-m5F cells) specific to JNK and AMPKα effectively prevented the Mo-induced apoptosis and related signals, but inhibitors of ERK1/2 and GSK3-α/β (PD98059 and LiCl, respectively) did not reverse the Mo-induced effects. Additionally, both the inhibitors and specific si-RNAs could suppress the Mo-induced phosphorylation of JNK and AMPKα each other. Taken together, these results suggest that Mo exerts its cytotoxicity on pancreatic β-cells by inducing dysfunction and apoptosis via interdependent JNK and AMPK activation downstream-regulated mitochondrial-dependent and ER stress-triggered apoptosis pathways. - Highlights: • Molybdenum (Mo) induces pancreatic β-cell dysfunction and apoptosis. • Mo causes β-cell death via mitochondria-dependent caspase cascades signals. • ER stress-triggered apoptotic pathway also regulates Mo-induced β-cell death. • Interdependent of JNK and AMPK activation involves in Mo-induced β-cell apoptosis.« less

Targeting p53 via JNK pathway: a novel role of RITA for apoptotic signaling in multiple myeloma.

PubMed

Saha, Manujendra N; Jiang, Hua; Yang, Yijun; Zhu, Xiaoyun; Wang, Xiaoming; Schimmer, Aaron D; Qiu, Lugui; Chang, Hong

2012-01-01

The low frequency of p53 alterations e.g., mutations/deletions (∼10%) in multiple myeloma (MM) makes this tumor type an ideal candidate for p53-targeted therapies. RITA is a small molecule which can induce apoptosis in tumor cells by activating the p53 pathway. We previously showed that RITA strongly activates p53 while selectively inhibiting growth of MM cells without inducing genotoxicity, indicating its potential as a drug lead for p53-targeted therapy in MM. However, the molecular mechanisms underlying the pro-apoptotic effect of RITA are largely undefined. Gene expression analysis by microarray identified a significant number of differentially expressed genes associated with stress response including c-Jun N-terminal kinase (JNK) signaling pathway. By Western blot analysis we further confirmed that RITA induced activation of p53 in conjunction with up-regulation of phosphorylated ASK-1, MKK-4 and c-Jun. These results suggest that RITA induced the activation of JNK signaling. Chromatin immunoprecipitation (ChIP) analysis showed that activated c-Jun binds to the activator protein-1 (AP-1) binding site of the p53 promoter region. Disruption of the JNK signal pathway by small interfering RNA (siRNA) against JNK or JNK specific inhibitor, SP-600125 inhibited the activation of p53 and attenuated apoptosis induced by RITA in myeloma cells carrying wild type p53. On the other hand, p53 transcriptional inhibitor, PFT-α or p53 siRNA not only inhibited the activation of p53 transcriptional targets but also blocked the activation of c-Jun suggesting the presence of a positive feedback loop between p53 and JNK. In addition, RITA in combination with dexamethasone, known as a JNK activator, displays synergistic cytotoxic responses in MM cell lines and patient samples. Our study unveils a previously undescribed mechanism of RITA-induced p53-mediated apoptosis through JNK signaling pathway and provides the rationale for combination of p53 activating drugs with JNK

Targeting p53 via JNK Pathway: A Novel Role of RITA for Apoptotic Signaling in Multiple Myeloma

PubMed Central

Saha, Manujendra N.; Jiang, Hua; Yang, Yijun; Zhu, Xiaoyun; Wang, Xiaoming; Schimmer, Aaron D.; Qiu, Lugui; Chang, Hong

2012-01-01

The low frequency of p53 alterations e.g., mutations/deletions (∼10%) in multiple myeloma (MM) makes this tumor type an ideal candidate for p53-targeted therapies. RITA is a small molecule which can induce apoptosis in tumor cells by activating the p53 pathway. We previously showed that RITA strongly activates p53 while selectively inhibiting growth of MM cells without inducing genotoxicity, indicating its potential as a drug lead for p53-targeted therapy in MM. However, the molecular mechanisms underlying the pro-apoptotic effect of RITA are largely undefined. Gene expression analysis by microarray identified a significant number of differentially expressed genes associated with stress response including c-Jun N-terminal kinase (JNK) signaling pathway. By Western blot analysis we further confirmed that RITA induced activation of p53 in conjunction with up-regulation of phosphorylated ASK-1, MKK-4 and c-Jun. These results suggest that RITA induced the activation of JNK signaling. Chromatin immunoprecipitation (ChIP) analysis showed that activated c-Jun binds to the activator protein-1 (AP-1) binding site of the p53 promoter region. Disruption of the JNK signal pathway by small interfering RNA (siRNA) against JNK or JNK specific inhibitor, SP-600125 inhibited the activation of p53 and attenuated apoptosis induced by RITA in myeloma cells carrying wild type p53. On the other hand, p53 transcriptional inhibitor, PFT-α or p53 siRNA not only inhibited the activation of p53 transcriptional targets but also blocked the activation of c-Jun suggesting the presence of a positive feedback loop between p53 and JNK. In addition, RITA in combination with dexamethasone, known as a JNK activator, displays synergistic cytotoxic responses in MM cell lines and patient samples. Our study unveils a previously undescribed mechanism of RITA-induced p53-mediated apoptosis through JNK signaling pathway and provides the rationale for combination of p53 activating drugs with JNK

Cypermethrin Induces Macrophages Death through Cell Cycle Arrest and Oxidative Stress-Mediated JNK/ERK Signaling Regulated Apoptosis

PubMed Central

Huang, Fang; Liu, Qiaoyun; Xie, Shujun; Xu, Jian; Huang, Bo; Wu, Yihua; Xia, Dajing

2016-01-01

Cypermethrin is one of the most highly effective synthetic pyrethroid insecticides. The toxicity of cypermethrin to the reproductive and nervous systems has been well studied. However, little is known about the toxic effect of cypermethrin on immune cells such as macrophages. Here, we investigated the cytotoxicity of cypermethrin on macrophages and the underlying molecular mechanisms. We found that cypermethrin reduced cell viability and induced apoptosis in RAW 264.7 cells. Cypermethrin also increased reactive oxygen species (ROS) production and DNA damage in a dose-dependent manner. Moreover, cypermethrin-induced G1 cell cycle arrest was associated with an enhanced expression of p21, wild-type p53, and down-regulation of cyclin D1, cyclin E and CDK4. In addition, cypermethrin treatment activated MAPK signal pathways by inducing c-Jun N-terminal kinase (JNK) and extracellular regulated protein kinases 1/2 ERK1/2 phosphorylation, and increased the cleaved poly ADP-ribose polymerase (PARP). Further, pretreatment with antioxidant N-acetylcysteine (NAC) effectively abrogated cypermethrin-induced cell cytotoxicity, G1 cell cycle arrest, DNA damage, PARP activity, and JNK and ERK1/2 activation. The specific JNK inhibitor (SP600125) and ERK1/2 inhibitor (PD98059) effectively reversed the phosphorylation level of JNK and ERK1/2, and attenuated the apoptosis. Taken together, these data suggested that cypermethrin caused immune cell death via inducing cell cycle arrest and apoptosis regulated by ROS-mediated JNK/ERK pathway. PMID:27322250

Rotenone-induced death of RGC-5 cells is caspase independent, involves the JNK and p38 pathways and is attenuated by specific green tea flavonoids.

PubMed

Kamalden, T A; Ji, D; Osborne, N N

2012-05-01

The aim of the present studies was to characterise cell death following inhibition of mitochondrial complex I with rotenone in a transformed cell line (RGC-5 cells) and to examine the neuroprotective properties of the flavonoids genistein, epigallocatechin gallate (EGCG), epicatechin (EC) and baicalin. Rotenone-induced cell death of RGC-5 cells results in a generation of reactive oxygen species, a breakdown of DNA, the translocation of membrane phosphatidylserine, an up-regulation of haemoxygenase-1 and is unaffected by necrostatin-1 (inhibitor of necroptosis), z-VAD-fmk (pan caspase inhibitor) or NU1025 (PARP inhibitor) but attenuated with SP600125 (JNK inhibitor). Rotenone-induced toxicity of RGC-5 cells also caused an activation of mitogen-activated kinases indicated by an up-regulation and translocation into mitochondria of p-c-Jun, pJNK and pp38. Exposure of RGC-5 cells to rotenone does not affect apoptosis inducing factor or significantly stimulate caspase-3 activity. EGCG and EC both significantly blunt rotenone toxicity of RGC-5 cells at concentrations of 50 μM while genistein and baicalin were without effect. Significantly, genistein is approximately 20 times less efficacious than EGCG (IC(50) 2.5 μM) and EC (IC(50) 1.5 μM) at inhibiting sodium nitroprusside-induced lipid peroxidation. These studies show that rotenone toxicity of RGC-5 cells is neither necroptosis nor caspase-dependent apoptosis but involves the activation of mitogen-activated kinases and is inhibited by a JNK inhibitor, EGCG and EC. Genistein attenuates lipid peroxidation less efficaciously than EC and EGCG and does not affect rotenone toxicity of RGC-5 cells.

Autophagy blockade sensitizes the anticancer activity of CA-4 via JNK-Bcl-2 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Yangling; Luo, Peihua; Wang, Jincheng

Combretastatin A-4 (CA-4) has already entered clinical trials of solid tumors over ten years. However, the limited anticancer activity and dose-dependent toxicity restrict its clinical application. Here, we offered convincing evidence that CA-4 induced autophagy in various cancer cells, which was demonstrated by acridine orange staining of intracellular acidic vesicles, the degradation of p62, the conversion of LC3-I to LC3-II and GFP-LC3 punctate fluorescence. Interestingly, CA-4-mediated apoptotic cell death was further potentiated by pretreatment with autophagy inhibitors (3-methyladenine and bafilomycin A1) or small interfering RNAs against the autophagic genes (Atg5 and Beclin 1). The enhanced anticancer activity of CA-4 andmore » 3-MA was further confirmed in the SGC-7901 xenograft tumor model. These findings suggested that CA-4-elicited autophagic response played a protective role that impeded the eventual cell death while autophagy inhibition was expected to improve chemotherapeutic efficacy of CA-4. Meanwhile, CA-4 treatment led to phosphorylation/activation of JNK and JNK-dependent phosphorylation of Bcl-2. Importantly, JNK inhibitor or JNK siRNA inhibited autophagy but promoted CA-4-induced apoptosis, indicating a key requirement of JNK-Bcl-2 pathway in the activation of autophagy by CA-4. We also identified that pretreatment of Bcl-2 inhibitor (ABT-737) could significantly enhance anticancer activity of CA-4 due to inhibition of autophagy. Taken together, our data suggested that the JNK-Bcl-2 pathway was considered as the critical regulator of CA-4-induced protective autophagy and a potential drug target for chemotherapeutic combination. - Highlights: • Autophagy inhibition could be a potential for combretastatin A-4 antitumor efficacy. • The JNK-Bcl-2 pathway plays a critical role in CA-4-induced autophagy. • ABT-737 enhances CA-4 anticancer activity due to inhibition of autophagy.« less

JNK-1 Inhibition Leads to Antitumor Activity in Ovarian Cancer

PubMed Central

Vivas-Mejia, Pablo; Benito, Juliana Maria; Fernandez, Ariel; Han, Hee-Dong; Mangala, Lingegowda; Rodriguez-Aguayo, Cristian; Chavez-Reyes, Arturo; Lin, Yvonne G.; Nick, Alpa M.; Stone, Rebecca L.; Kim, Hye Sun; Claret, Francois-Xavier; Bornmann, William; Hennessy, Bryan TJ.; Sanguino, Angela; Peng, Zhengong; Sood, Anil K.; Lopez-Berestein, Gabriel

2011-01-01

Purpose To demonstrate the functional, clinical and biological significance of JNK-1 in ovarian carcinoma. Experimental Design Analysis of the impact of JNK on 116 epithelial ovarian cancers was conducted. The role of JNK in vitro and in experimental models of ovarian cancer was assessed. We studied the role of WBZ_4, a novel JNK inhibitor redesigned from imatinib based on targeting wrapping defects, in cell lines and in experimental models of ovarian cancer. Results We found a significant association of pJNK with progression free survival in the 116 epithelial ovarian cancers obtained at primary debulking therapy. WBZ_4 led to cell growth inhibition and increased apoptosis in a dose dependent fashion in four ovarian cancer cell lines. In vivo, while imatinib had no effect on tumor growth, WBZ_4 inhibited tumor growth in orthotopic murine models of ovarian cancer. The anti-tumor effect was further increased in combination with docetaxel. Silencing of JNK-1 with systemically administered siRNA led to significantly reduced tumor weights as compared to non-silencing siRNA controls, indicating that indeed the antitumor effects observed were due to JNK-1 inhibition. Conclusions These studies identify JNK-1 as an attractive therapeutic target in ovarian carcinoma and that the re-designed WBZ_4 compound should be considered for further clinical development. PMID:20028751

ROS-dependent activation of JNK converts p53 into an efficient inhibitor of oncogenes leading to robust apoptosis

PubMed Central

Shi, Y; Nikulenkov, F; Zawacka-Pankau, J; Li, H; Gabdoulline, R; Xu, J; Eriksson, S; Hedström, E; Issaeva, N; Kel, A; Arnér, E S J; Selivanova, G

2014-01-01

Rescue of the p53 tumor suppressor is an attractive cancer therapy approach. However, pharmacologically activated p53 can induce diverse responses ranging from cell death to growth arrest and DNA repair, which limits the efficient application of p53-reactivating drugs in clinic. Elucidation of the molecular mechanisms defining the biological outcome upon p53 activation remains a grand challenge in the p53 field. Here, we report that concurrent pharmacological activation of p53 and inhibition of thioredoxin reductase followed by generation of reactive oxygen species (ROS), result in the synthetic lethality in cancer cells. ROS promote the activation of c-Jun N-terminal kinase (JNK) and DNA damage response, which establishes a positive feedback loop with p53. This converts the p53-induced growth arrest/senescence to apoptosis. We identified several survival oncogenes inhibited by p53 in JNK-dependent manner, including Mcl1, PI3K, eIF4E, as well as p53 inhibitors Wip1 and MdmX. Further, we show that Wip1 is one of the crucial executors downstream of JNK whose ablation confers the enhanced and sustained p53 transcriptional response contributing to cell death. Our study provides novel insights for manipulating p53 response in a controlled way. Further, our results may enable new pharmacological strategy to exploit abnormally high ROS level, often linked with higher aggressiveness in cancer, to selectively kill cancer cells upon pharmacological reactivation of p53. PMID:24413150

Involvement of the Tyr kinase/JNK pathway in carbachol-induced bronchial smooth muscle contraction in the rat.

PubMed

Sakai, Hiroyasu; Watanabe, Yu; Honda, Mai; Tsuiki, Rika; Ueda, Yusuke; Nagai, Yuki; Narita, Minoru; Misawa, Miwa; Chiba, Yoshihiko

2013-05-01

Tyrosine (Tyr) kinases and mitogen-activated protein kinases have been thought to participate in the contractile response in various smooth muscles. The aim of the current study was to investigate the involvement of the Tyr kinase pathway in the contraction of bronchial smooth muscle. Ring preparations of bronchi isolated from rats were suspended in an organ bath. Isometric contraction of circular smooth muscle was measured. Immunoblotting was used to examine the phosphorylation of c-Jun N-terminal kinasess (JNKs) in bronchial smooth muscle. To examine the role of mitogen-activated protein kinase(s) in bronchial smooth muscle contraction, the effects of MPAK inhibitors were investigated in this study. The contraction induced by carbachol (CCh) was significantly inhibited by pretreatment with selective Tyr kinase inhibitors (genistein and ST638, n = 6, respectively), and a JNK inhibitor (SP600125, n = 6). The contractions induced by high K depolarization (n = 4), orthovanadate (a potent Tyr phosphatase inhibitor) and sodium fluoride (a G protein activator; NaF) were also significantly inhibited by selective Tyr kinase inhibitors and a JNK inhibitor (n = 4, respectively). However, the contraction induced by calyculin-A was not affected by SP600125. On the other hand, JNKs were phosphorylated by CCh (2.2 ± 0,4 [mean±SEM] fold increase). The JNK phosphorylation induced by CCh was significantly inhibited by SP600125 (n = 4). These findings suggest that the Tyr kinase/JNK pathway may play a role in bronchial smooth muscle contraction. Strategies to inhibit JNK activation may represent a novel therapeutic approach for diseases involving airway obstruction, such as asthma and chronic obstructive pulmonary disease.

Rhodanine derivatives as inhibitors of JSP-1.

PubMed

Cutshall, Neil S; O'Day, Christine; Prezhdo, Marina

2005-07-15

Dual-specificity phosphatases (DSPs) are a subclass within the protein tyrosine phosphatase family (PTPs). A series of rhodanine-based inhibitors was synthesized and shown to be novel, potent, and selective inhibitors against the DSP family member JNK-stimulating phosphatase-1 (JSP-1). Compounds of this class may be useful for the treatment of inflammatory and proliferative disorders.

JNK3-Mediated Apoptotic Cell Death in Primary Dopaminergic Neurons

PubMed Central

Choi, Won-Seok; Klintworth, Heather M.; Xia, Zhengui

2012-01-01

Investigation of mechanisms responsible for dopaminergic neuron death is critical for understanding the pathogenesis of Parkinson’s disease, yet this is often quite challenging technically. Here, we describe detailed methods for culturing primary mesencephalic dopaminergic neurons and examining the activation of c-Jun N-terminal protein Kinase (JNK) in these cultures. We utilized immunocytochemistry and computerized analysis to quantify the number of surviving dopaminergic neurons and JNK activation in dopaminergic neurons. TUNEL staining was used to quantify apoptotic cell death. siRNA was used to specifically inhibit JNK3, the neural specific isoform of JNK. Our data implicate the activation of JNK3 in rotenone-induced dopaminergic neuron apoptosis. PMID:21815073

Expression and proliferation profiles of PKC, JNK and p38MAPK in physiologically stretched human bladder smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wazir, Romel; Luo, De-Yi; Dai, Yi

2013-08-30

Highlights: •Stretch induces proliferation in human bladder smooth muscle cells (HBSMC). •5% Equibiaxial elongation produces maximum proliferation. •Physiologic stretch decreases apoptotic cell death. •PKC is involved in functional modulation of bladder. •JNK and p38 are not involved in proliferating HBSMC. -- Abstract: Objective: To determine protein kinase C (PKC), c-Jun NH2-Terminal Kinase (JNK) and P38 mitogen-activated protein kinases (p38MAPK) expression levels and effects of their respective inhibitors on proliferation of human bladder smooth muscle cells (HBSMCs) when physiologically stretched in vitro. Materials and methods: HBSMCs were grown on silicone membrane and stretch was applied under varying conditions; (equibiaxial elongation: 2.5%,more » 5%, 10%, 15%, 20%, 25%), (frequency: 0.05, 0.1, 0.2, 0.5, 1 Hz). Optimal physiological stretch was established by assessing proliferation with 5-Bromo-2-deoxyuridine (BrdU) assay and flow cytometry. PKC, JNK and p38 expression levels were analyzed by Western blot. Specificity was maintained by employing specific inhibitors; (GF109203X for PKC, SP600125 for JNK and SB203580 for p38MAPK), in some experiments. Results: Optimum proliferation was observed at 5% equibiaxial stretch (BrdU: 0.837 ± 0.026 (control) to 1.462 ± 0.023)%, (P < 0.05) and apoptotic cell death rate decreased from 16.4 ± 0.21% (control) to 4.5 ± 0.13% (P < 0.05) applied at 0.1 Hz. Expression of PKC was upregulated with slight increase in JNK and no change in p38MAPK after application of stretch. Inhibition had effects on proliferation (1.075 ± 0.024, P < 0.05 GF109203X); (1.418 ± 0.021, P > 0.05 SP600125) and (1.461 ± 0.01, P > 0.05 SB203580). These findings show that mechanical stretch can promote magnitude-dependent proliferative modulation through PKC and possibly JNK but not via p38MAPK in hBSMCs.« less

JNK1 regulates histone acetylation in trigeminal neurons following chemical stimulation

PubMed Central

Wu, Jing; Zhang, Xuan; Nauta, Haring J; Lin, Qing; Li, Junfa; Fang, Li

2008-01-01

Trigeminal nerve fibers in nasal and oral cavities are sensitive to various environmental hazardous stimuli, which trigger many neurotoxic problems such as chronic migraine headache and trigeminal irritated disorders. However, the role of JNK kinase cascade and its epigenetic modulation of histone remodeling in trigeminal ganglion (TG) neurons activated by environmental neurotoxins remains unknown. Here we investigated the role of JNK/c-Jun cascade in the regulation of acetylation of H3 histone in TG neurons following in vitro stimulation by a neuro-inflammatory agent, mustard oil (MO). We found that MO stimulation elicited JNK/c-Jun pathway significantly by enhancing phospho-JNK1, phospho-c-Jun expression, and c-Jun activity, which were correlated with an elevated acetylated H3 histone in TG neurons. However, increases in phospho-c-Jun and c-Jun activity were significantly blocked by a JNK inhibitor, SP600125. We also found that altered H3 histone remodeling, assessed by H3 acetylation in triggered TG neurons, was reduced by SP600125. The study suggests that the activated JNK signaling in regulation of histone remodeling may contribute to neuro-epigentic changes in peripheral sensory neurons following environmental neurotoxic exposure. PMID:18822271

JNK and NADPH Oxidase Involved in Fluoride-Induced Oxidative Stress in BV-2 Microglia Cells

PubMed Central

Yan, Ling; Liu, Shengnan; Wang, Chen; Wang, Fei; Song, Yingli; Yan, Nan; Xi, Shuhua; Liu, Ziyou; Sun, Guifan

2013-01-01

Excessive fluoride may cause central nervous system (CNS) dysfunction, and oxidative stress is a recognized mode of action of fluoride toxicity. In CNS, activated microglial cells can release more reactive oxygen species (ROS), and NADPH oxidase (NOX) is the major enzyme for the production of extracellular superoxide in microglia. ROS have been characterized as an important secondary messenger and modulator for various mammalian intracellular signaling pathways, including the MAPK pathways. In this study we examined ROS production and TNF-α, IL-1β inflammatory cytokines releasing, and the expression of MAPKs in BV-2 microglia cells treated with fluoride. We found that fluoride increased JNK phosphorylation level of BV-2 cells and pretreatment with JNK inhibitor SP600125 markedly reduced the levels of intracellular O2 ·− and NO. NOX inhibitor apocynin and iNOS inhibitor SMT dramatically decreased NaF-induced ROS and NO generations, respectively. Antioxidant melatonin (MEL) resulted in a reduction in JNK phosphorylation in fluoride-stimulated BV-2 microglia. The results confirmed that NOX and iNOS played an important role in fluoride inducing oxidative stress and NO production and JNK took part in the oxidative stress induced by fluoride and meanwhile also could be activated by ROS in fluoride-treated BV-2 cells. PMID:24072958

Identification of Chemical Inhibitors of β-Catenin-Driven Liver Tumorigenesis in Zebrafish

PubMed Central

Evason, Kimberley J.; Francisco, Macrina T.; Juric, Vladislava; Balakrishnan, Sanjeev; Lopez Pazmino, Maria del Pilar; Gordan, John D.; Kakar, Sanjay; Spitsbergen, Jan; Goga, Andrei; Stainier, Didier Y. R.

2015-01-01

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers. The search for targeted treatments has been hampered by the lack of relevant animal models for the genetically diverse subsets of HCC, including the 20-40% of HCCs that are defined by activating mutations in the gene encoding β-catenin. To address this chemotherapeutic challenge, we created and characterized transgenic zebrafish expressing hepatocyte-specific activated β-catenin. By 2 months post fertilization (mpf), 33% of transgenic zebrafish developed HCC in their livers, and 78% and 80% of transgenic zebrafish showed HCC at 6 and 12 mpf, respectively. As expected for a malignant process, transgenic zebrafish showed significantly decreased mean adult survival compared to non-transgenic control siblings. Using this novel transgenic model, we screened for druggable pathways that mediate β-catenin-induced liver growth and identified two c-Jun N-terminal kinase (JNK) inhibitors and two antidepressants (one tricyclic antidepressant, amitriptyline, and one selective serotonin reuptake inhibitor) that suppressed this phenotype. We further found that activated β-catenin was associated with JNK pathway hyperactivation in zebrafish and in human HCC. In zebrafish larvae, JNK inhibition decreased liver size specifically in the presence of activated β-catenin. The β-catenin-specific growth-inhibitory effect of targeting JNK was conserved in human liver cancer cells. Our other class of hits, antidepressants, has been used in patient treatment for decades, raising the exciting possibility that these drugs could potentially be repurposed for cancer treatment. In support of this proposal, we found that amitriptyline decreased tumor burden in a mouse HCC model. Our studies implicate JNK inhibitors and antidepressants as potential therapeutics for β-catenin-induced liver tumors. PMID:26134322

Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion.

PubMed

Xu, Jie; Qin, Xinghua; Cai, Xiaoqing; Yang, Lu; Xing, Yuan; Li, Jun; Zhang, Lihua; Tang, Ying; Liu, Jiankang; Zhang, Xing; Gao, Feng

2015-02-01

c-Jun N-terminal kinase (JNK) is a stress-activated mitogen-activated protein kinase that plays a central role in initiating apoptosis in disease conditions. Recent studies have shown that mitochondrial JNK signaling is partly responsible for ischemic myocardial dysfunction; however, the underlying mechanism remains unclear. Here we report for the first time that activation of mitochondrial JNK, rather than JNK localization on mitochondria, induces autophagy and apoptosis and aggravates myocardial ischemia/reperfusion injury. Myocardial ischemia/reperfusion induced a dominant increase of mitochondrial JNK phosphorylation, while JNK mitochondrial localization was reduced. Treatment with Tat-SabKIM1, a retro-inverso peptide which blocks JNK interaction with mitochondria, decreased mitochondrial JNK activation without affecting JNK mitochondrial localization following reperfusion. Tat-SabKIM1 treatment reduced Bcl2-regulated autophagy, cytochrome c-mediated apoptosis and myocardial infarct size. Notably, selective inhibition of mitochondrial JNK activation using Tat-SabKIM1 produced a similar infarct size-reducing effect as inhibiting universal JNK activation with JNK inhibitor SP600125. Moreover, insulin-treated animals exhibited significantly dampened mitochondrial JNK activation accompanied by reduced infarct size and diminished autophagy and apoptosis following reperfusion. Taken together, these findings demonstrate that mitochondrial JNK activation, rather than JNK mitochondrial localization, induces autophagy and apoptosis and exacerbates myocardial ischemia/reperfusion injury. Insulin selectively inhibits mitochondrial JNK activation, contributing to insulin cardioprotection against myocardial ischemic/reperfusion injury. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

Actinobacillus actinomycetemcomitans Y4 capsular polysaccharide induces IL-1β mRNA expression through the JNK pathway in differentiated THP-1 cells

PubMed Central

Iwata, T; Mitani, A; Ishihara, Y; Tanaka, S; Yamamoto, G; Kikuchi, T; Naganawa, T; Matsumura, Y; Suga, T; Koide, M; Sobue, T; Suzuki, T; Noguchi, T

2005-01-01

Capsular polysaccharide from Actinobacillus actinomycetemcomitans Y4 (Y4 CP) induces bone resorption in a mouse organ culture system and osteoclast formation in mouse bone marrow cultures, as reported in previous studies. We also found that Y4 CP inhibits the release of interleukin (IL)-6 and IL-8 from human gingival fibroblast (HGF). Thus Y4 CP induces various responses in localized tissue and leads to the secretion of several cytokines. However, the effects of Y4 CP on human monocytes/macrophages are still unclear. In this study, THP-1 cells, which are a human monocytic cell line, were stimulated with Y4 CP, and we measured gene expression in inflammatory cytokine and signal transduction pathways. IL-1β and tumour necrosis factor (TNF)-α mRNA were induced from Y4 CP-treated THP-1 cells. IL-1β mRNA expression was increased according to the dose of Y4 CP, and in a time-dependent manner. IL-1β mRNA expression induced by Y4 CP (100 µg/ml) was approximately 7- to 10-fold greater than that in the control by real-time PCR analysis. Furthermore, neither PD98059, a specific inhibitor of extracellular signal-regulated kinase nor SB203580, a specific inhibitor of p38 kinase prevented the IL-1β expression induced by Y4 CP. However, JNK Inhibitor II, a specific inhibitor of c-Jun N-terminal kinase (JNK) prevented the IL-1β mRNA expression induced by Y4 CP in a concentration-dependent manner. These results indicate that Y4 CP-mediated JNK pathways play an important role in the regulation of IL-1β mRNA. Therefore, Y4 CP-transduced signals for IL-1β induction in the antibacterial action of macrophages may provide a therapeutic strategy for periodontitis. PMID:15996190

PKCalpha-mediated ERK, JNK and p38 activation regulates the myogenic program in human rhabdomyosarcoma cells.

PubMed

Mauro, Annunziata; Ciccarelli, Carmela; De Cesaris, Paola; Scoglio, Arianna; Bouché, Marina; Molinaro, Mario; Aquino, Angelo; Zani, Bianca Maria

2002-09-15

We have previously suggested that PKCalpha has a role in 12-O-Tetradecanoylphorbol-13-acetate (TPA)-mediated growth arrest and myogenic differentiation in human embryonal rhabdomyosarcoma cells (RD). Here, by monitoring the signalling pathways triggered by TPA, we demonstrate that PKCalpha mediates these effects by inducing transient activation of c-Jun N-terminal protein kinases (JNKs) and sustained activation of both p38 kinase and extracellular signal-regulated kinases (ERKs) (all referred to as MAPKs). Activation of MAPKs following ectopic expression of constitutively active PKCalpha, but not its dominant-negative form, is also demonstrated. We investigated the selective contribution of MAPKs to growth arrest and myogenic differentiation by monitoring the activation of MAPK pathways, as well as by dissecting MAPK pathways using MEK1/2 inhibitor (UO126), p38 inhibitor (SB203580) and JNK and p38 agonist (anisomycin) treatments. Growth-arresting signals are triggered either by transient and sustained JNK activation (by TPA and anisomycin, respectively) or by preventing both ERK and JNK activation (UO126) and are maintained, rather than induced, by p38. We therefore suggest a key role for JNK in controlling ERK-mediated mitogenic activity. Notably, sarcomeric myosin expression is induced by both TPA and UO126 but is abrogated by the p38 inhibitor. This finding indicates a pivotal role for p38 in controlling the myogenic program. Anisomycin persistently activates p38 and JNKs but prevents myosin expression induced by TPA. In accordance with this negative role, reactivation of JNKs by anisomycin, in UO126-pre-treated cells, also prevents myosin expression. This indicates that, unlike the transient JNK activation that occurs in the TPA-mediated myogenic process, long-lasting JNK activation supports the growth-arrest state but antagonises p38-mediated myosin expression. Lastly, our results with the MEK inhibitor suggest a key role of the ERK pathway in regulating

c-Jun N-terminal kinase inhibitors: a patent review (2010 - 2014).

PubMed

Gehringer, Matthias; Muth, Felix; Koch, Pierre; Laufer, Stefan A

2015-01-01

c-Jun N-terminal kinases (JNKs) are involved in the emergence and progression of diverse pathologies such as neurodegenerative, cardiovascular and metabolic disorders as well as inflammation and cancer. In recent years, several highly selective pan-JNK inhibitors have been characterized and three chemical entities targeting JNKs have been investigated in clinical trials. This review summarizes patents claiming inhibitors of all JNK isoforms published between 2010 and 2014. Although primarily focusing on the patent literature, relevant peer-reviewed publications related to the covered patents have also been included. Moreover, key patents claiming novel applications of previously published chemical entities are reviewed. The article highlights a total of 28 patents from nine pharmaceutical companies and academic research groups. Although some selective pan-JNK inhibitors with reasonable in vivo profiles are now available, little is known about the isoform selectivity required for each particular indication and the development of isoform-selective JNK inhibitors still represents a challenge in JNK drug discovery. Moreover, isoform-selective tool compounds are a prerequisite to a comprehensive understanding of the biology of each JNK isoform. Potential approaches towards such compounds include the design of type-II and type-I(1)/2 binders, which are absent in the current JNK inhibitor portfolios, as well as the design of novel allosteric inhibitors. Furthermore, covalent inhibition, which already led to the first high-quality probe for JNKs, might be further exploited for gaining selectivity and in vivo efficacy. With regard to a potential therapeutic application, the recently proposed concept of covalent reversible inhibitors is expected to be attractive.

Arachidonic acid induces macrophage cell cycle arrest through the JNK signaling pathway.

PubMed

Shen, Ziying; Ma, Yunqing; Ji, Zhonghao; Hao, Yang; Yan, Xuan; Zhong, Yuan; Tang, Xiaochun; Ren, Wenzhi

2018-02-09

Arachidonic acid (AA) has potent pro-apoptotic effects on cancer cells at a low concentration and on macrophages at a very high concentration. However, the effects of AA on the macrophage cell cycle and related signaling pathways have not been fully investigated. Herein we aim to observe the effect of AA on macrophages cell cycle. AA exposure reduced the viability and number of macrophages in a dose- and time-dependent manner. The reduction in RAW264.7 cell viability was not caused by apoptosis, as indicated by caspase-3 and activated caspase-3 detection. Further research illustrated that AA exposure induced RAW264.7 cell cycle arrested at S phase, and some cell cycle-regulated proteins were altered accordingly. Moreover, JNK signaling was stimulated by AA, and the stimulation was partially reversed by a JNK signaling inhibitor in accordance with cell cycle-related factors. In addition, nuclear and total Foxo1/3a and phosphorylated Foxo1/3a were elevated by AA in a dose- and time-dependent manner, and this elevation was suppressed by the JNK signaling inhibitor. Our study demonstrated that AA inhibits macrophage viability by inducing S phase cell cycle arrest. The JNK signaling pathway and the downstream FoxO transcription factors are involved in AA-induced RAW264.7 cell cycle arrest.

Dexmedetomidine-induced Contraction Involves Phosphorylation of Caldesmon by JNK in Endothelium-denuded Rat Aortas

PubMed Central

Baik, Jiseok; Ok, Seong-Ho; Cho, Hyunhoo; Yu, Jongsun; Kim, Woochan; Nam, In-Koo; Choi, Mun-Jeoung; Lee, Heon-Keun; Sohn, Ju-Tae

2014-01-01

Caldesmon, an inhibitory actin binding protein, binds to actin and inhibits actin-myosin interactions, whereas caldesmon phosphorylation reverses the inhibitory effect of caldesmon on actin-myosin interactions, potentially leading to enhanced contraction. The goal of this study was to investigate the cellular signaling pathway responsible for caldesmon phosphorylation, which is involved in the regulation of the contraction induced by dexmedetomidine (DMT), an alpha-2 adrenoceptor agonist, in endothelium-denuded rat aortas. SP600125 (a c-Jun NH2-terminal kinase [JNK] inhibitor) dose-response curves were generated in aortas that were pre-contracted with DMT or phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator. Dose-response curves to the PKC inhibitor chelerythrine were generated in rat aortas pre-contracted with DMT. The effects of SP600125 and rauwolscine (an alpha-2 adrenoceptor inhibitor) on DMT-induced caldesmon phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) were investigated by western blot analysis. PDBu-induced caldesmon and DMT-induced PKC phosphorylation in rat aortic VSMCs was investigated by western blot analysis. The effects of GF109203X (a PKC inhibitor) on DMT- or PDBu-induced JNK phosphorylation in VSMCs were assessed. SP600125 resulted in the relaxation of aortas that were pre-contracted with DMT or PDBu, whereas rauwolscine attenuated DMT-induced contraction. Chelerythrine resulted in the vasodilation of aortas pre-contracted with DMT. SP600125 and rauwolscine inhibited DMT-induced caldesmon phosphorylation. Additionally, PDBu induced caldesmon phosphorylation, and GF109203X attenuated the JNK phosphorylation induced by DMT or PDBu. DMT induced PKC phosphorylation in rat aortic VSMCs. These results suggest that alpha-2 adrenoceptor-mediated, DMT-induced contraction involves caldesmon phosphorylation that is mediated by JNK phosphorylation by PKC. PMID:25332685

A liver full of JNK: signaling in regulation of cell function and disease pathogenesis, and clinical approaches.

PubMed

Seki, Ekihiro; Brenner, David A; Karin, Michael

2012-08-01

c-Jun-N-terminal kinase (JNK) is a mitogen-activated protein kinase family member that is activated by diverse stimuli, including cytokines (such as tumor necrosis factor and interleukin-1), reactive oxygen species (ROS), pathogens, toxins, drugs, endoplasmic reticulum stress, free fatty acids, and metabolic changes. Upon activation, JNK induces multiple biologic events through the transcription factor activator protein-1 and transcription-independent control of effector molecules. JNK isozymes regulate cell death and survival, differentiation, proliferation, ROS accumulation, metabolism, insulin signaling, and carcinogenesis in the liver. The biologic functions of JNK are isoform, cell type, and context dependent. Recent studies using genetically engineered mice showed that loss or hyperactivation of the JNK pathway contributes to the development of inflammation, fibrosis, cancer growth, and metabolic diseases that include obesity, hepatic steatosis, and insulin resistance. We review the functions and pathways of JNK in liver physiology and pathology and discuss findings from preclinical studies with JNK inhibitors. Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

Human amniotic epithelial stem cells promote wound healing by facilitating migration and proliferation of keratinocytes via ERK, JNK and AKT signaling pathways.

PubMed

Zhao, Bin; Liu, Jia-Qi; Zheng, Zhao; Zhang, Jun; Wang, Shu-Yue; Han, Shi-Chao; Zhou, Qin; Guan, Hao; Li, Chao; Su, Lin-Lin; Hu, Da-Hai

2016-07-01

Wound healing is a highly orchestrated physiological process consisting in a complex interaction of cellular and biochemical events. Human amniotic epithelial stem cells (HAESCs) have been shown to be an attractive resource for wound healing because they are primitive stem cells. However, the exact effects of amnion-derived stem cells on the migration or proliferation of keratinocytes and their potential mechanism are not fully understood. We have found that HAESCs accelerate the migration of keratinocytes and induce a remarkable increase in the activity of phospho-ERK, phospho-JNK, and phospho-AKT, the blockade of which by their specific inhibitors significantly inhibits migration induced by HAESC-conditioned medium (CM). Furthermore, the co-culture of keratinocytes with HAESCs up-regulates the expression levels of cell proliferation proteins Cyclin D1, Cyclin D3 and Mdm2. In vivo animal experiments have shown that HAESC-CM improves wound healing, whereas blockade with ERK, JNK and AKT inhibitors significantly impairs wound healing. Taken together, these results reveal, for the first time, that HAESCs promote wound healing by facilitating the migration and proliferation of keratinocytes via ERK, JNK and AKT signaling pathways and might be a potential therapy in skin wound healing.

The novel JNK inhibitor AS602801 inhibits cancer stem cells in vitro and in vivo.

PubMed

Okada, Masashi; Kuramoto, Kenta; Takeda, Hiroyuki; Watarai, Hikaru; Sakaki, Hirotsugu; Seino, Shizuka; Seino, Manabu; Suzuki, Shuhei; Kitanaka, Chifumi

2016-05-10

A phase 2 clinical trial investigating the efficacy and safety of AS602801, a newly developed JNK inhibitor, in the treatment of inflammatory endometriosis is complete. We are now examining whether AS602801 acts against human cancer cells in vitro and in vivo. In vitro, AS602801 exhibited cytotoxicity against both serum-cultured non-stem cancer cells and cancer stem cells derived from human pancreatic cancer, non-small cell lung cancer, ovarian cancer and glioblastoma at concentrations that did not decrease the viability of normal human fibroblasts. AS602801 also inhibited the self-renewal and tumor-initiating capacity of cancer stem cells surviving AS602801 treatment. Cancer stem cells in established xenograft tumors were reduced by systemic administration of AS602801 at a dose and schedule that did not adversely affect the health of the tumor-bearing mice. These findings suggest AS602801 is a promising anti-cancer stem cell agent, and further investigation of the utility of AS602801 in the treatment of cancer seems warranted.

Sphingosylphosphorylcholine promotes the differentiation of resident Sca-1 positive cardiac stem cells to cardiomyocytes through lipid raft/JNK/STAT3 and β-catenin signaling pathways.

PubMed

Li, Wenjing; Liu, Honghong; Liu, Pingping; Yin, Deling; Zhang, Shangli; Zhao, Jing

2016-07-01

Resident cardiac Sca-1-positive (+) stem cells may differentiate into cardiomyocytes to improve the function of damaged hearts. However, little is known about the inducers and molecular mechanisms underlying the myogenic conversion of Sca-1(+) stem cells. Here we report that sphingosylphosphorylcholine (SPC), a naturally occurring bioactive lipid, induces the myogenic conversion of Sca-1(+) stem cells, as evidenced by the increased expression of cardiac transcription factors (Nkx2.5 and GATA4), structural proteins (cardiac Troponin T), transcriptional enhancer (Mef2c) and GATA4 nucleus translocation. First, SPC activated JNK and STAT3, and the JNK inhibitor SP600125 or STAT3 inhibitor stattic impaired the SPC-induced expression of cardiac transcription factors and GATA4 nucleus translocation, which suggests that JNK and STAT3 participated in SPC-promoted cardiac differentiation. Moreover, STAT3 activation was inhibited by SP600125, whereas JNK was inhibited by β-cyclodextrin as a lipid raft breaker, which indicates a lipid raft/JNK/STAT3 pathway involved in SPC-induced myogenic transition. β-Catenin, degraded by activated GSK3β, was inhibited by SPC. Furthermore, GSK3β inhibitors weakened but the β-catenin inhibitor promoted SPC-induced differentiation. We found no crosstalk between the lipid raft/JNK/STAT3 and β-catenin pathway. Our study describes a lipid, SPC, as an endogenic inducer of myogenic conversion in Sca-1(+) stem cells with low toxicity and high efficiency for uptake. Copyright © 2016 Elsevier B.V. All rights reserved.

Regulation of B7.1 costimulatory molecule is mediated by the IFN regulatory factor-7 through the activation of JNK in lipopolysaccharide-stimulated human monocytic cells.

PubMed

Lim, Wilfred; Gee, Katrina; Mishra, Sasmita; Kumar, Ashok

2005-11-01

The engagement of CD28 or CTLA-4 with B7.1 provides the essential second costimulatory signal that regulates the development of immune responses, including T cell activation, differentiation, and induction of peripheral tolerance. The signaling molecules and the transcription factors involved in B7.1 regulation are poorly understood. In this study we investigated the role of MAPKs in the regulation of LPS-induced B7.1 expression in human monocytes and the promonocytic THP-1 cells. Our results show that LPS-induced B7.1 expression in monocytic cells did not involve the activation of either p38 or ERKs. Using the JNK-specific inhibitor SP600125, small interfering RNAs specific for JNK1 and JNK2, and agents such as dexamethasone that inhibit JNK activation, we determined that LPS-induced B7.1 expression was regulated by JNK MAPK in both monocytes and THP-1 cells. In addition, we identified a distinct B7.1-responsive element corresponding to the IFN regulatory factor-7 (IRF-7) binding site in the B7.1 promoter responsible for the regulation of LPS-induced B7.1 transcription. Furthermore, SP600125 and dexamethasone inhibited LPS-induced IRF-7 activity. Taken together, these results suggest that LPS-induced B7.1 transcription in human monocytic cells may be regulated by JNK-mediated activation of the IRF-7 transcription factor.

HCV upregulates Bim through the ROS/JNK signalling pathway, leading to Bax-mediated apoptosis.

PubMed

Deng, Lin; Chen, Ming; Tanaka, Motofumi; Ku, Yonson; Itoh, Tomoo; Shoji, Ikuo; Hotta, Hak

2015-09-01

We previously reported that hepatitis C virus (HCV) infection induces Bax-triggered, mitochondrion-mediated apoptosis by using the HCV J6/JFH1 strain and Huh-7.5 cells. However, it was still unclear how HCV-induced Bax activation. In this study, we showed that the HCV-induced activation and mitochondrial accumulation of Bax were significantly attenuated by treatment with a general antioxidant, N-acetyl cysteine (NAC), or a specific c-Jun N-terminal kinase (JNK) inhibitor, SP600125, with the result suggesting that the reactive oxygen species (ROS)/JNK signalling pathway is upstream of Bax activation in HCV-induced apoptosis. We also demonstrated that HCV infection transcriptionally activated the gene for the pro-apoptotic protein Bim and the protein expression of three major splice variants of Bim (BimEL, BimL and BimS). The HCV-induced increase in the Bim mRNA and protein levels was significantly counteracted by treatment with NAC or SP600125, suggesting that the ROS/JNK signalling pathway is involved in Bim upregulation. Moreover, HCV infection led to a marked accumulation of Bim on the mitochondria to facilitate its interaction with Bax. On the other hand, downregulation of Bim by siRNA (small interfering RNA) significantly prevented HCV-mediated activation of Bax and caspase 3. Taken together, these observations suggest that HCV-induced ROS/JNK signalling transcriptionally activates Bim expression, which leads to Bax activation and apoptosis induction.

Lead optimization toward proof-of-concept tools for Huntington's disease within a 4-(1H-pyrazol-4-yl)pyrimidine class of pan-JNK inhibitors.

PubMed

Wityak, John; McGee, Kevin F; Conlon, Michael P; Song, Ren Hua; Duffy, Bryan C; Clayton, Brent; Lynch, Michael; Wang, Gwen; Freeman, Emily; Haber, James; Kitchen, Douglas B; Manning, David D; Ismail, Jiffry; Khmelnitsky, Yuri; Michels, Peter; Webster, Jeff; Irigoyen, Macarena; Luche, Michele; Hultman, Monica; Bai, Mei; Kuok, IokTeng D; Newell, Ryan; Lamers, Marieke; Leonard, Philip; Yates, Dawn; Matthews, Kim; Ongeri, Lynette; Clifton, Steve; Mead, Tania; Deupree, Susan; Wheelan, Pat; Lyons, Kathy; Wilson, Claire; Kiselyov, Alex; Toledo-Sherman, Leticia; Beconi, Maria; Muñoz-Sanjuan, Ignacio; Bard, Jonathan; Dominguez, Celia

2015-04-09

Through medicinal chemistry lead optimization studies focused on calculated properties and guided by X-ray crystallography and computational modeling, potent pan-JNK inhibitors were identified that showed submicromolar activity in a cellular assay. Using in vitro ADME profiling data, 9t was identified as possessing favorable permeability and a low potential for efflux, but it was rapidly cleared in liver microsomal incubations. In a mouse pharmacokinetics study, compound 9t was brain-penetrant after oral dosing, but exposure was limited by high plasma clearance. Brain exposure at a level expected to support modulation of a pharmacodynamic marker in mouse was achieved when the compound was coadministered with the pan-cytochrome P450 inhibitor 1-aminobenzotriazole.

Nutlin-3 induces HO-1 expression by activating JNK in a transcription-independent manner of p53.

PubMed

Choe, Yun-Jeong; Lee, Sun-Young; Ko, Kyung Won; Shin, Seok Joon; Kim, Ho-Shik

2014-03-01

A recent study reported that p53 can induce HO-1 by directly binding to the putative p53 responsive element in the HO-1 promoter. In this study, we report that nutlin-3, a small molecule antagonist of HDM2, induces the transcription of HO-1 in a transcription-independent manner of p53. Nutlin-3 induced HO-1 expression at the level of transcription in human cancer cells such as U2OS and RKO cells. This induction of HO-1 did not occur in SAOS cells in which p53 was mutated and was prevented by knocking down the p53 protein using p53 siRNA transfection, but not by PFT-α, an inhibitor of the transcriptional activity of p53. Accompanying HO-1 expression, nutlin-3 stimulated the accumulation of ROS and the phosphorylation of MAPKs such as JNK, p38 MAPK and ERK1/2. Nutlin-3-induced HO-1 expression was suppressed by TEMPO, a ROS scavenger, and chemical inhibitors of JNK and p38 MAPK but not ERK1/2. In addition, nutlin‑3-induced phosphorylation of JNK but not p38 MAPK was inhibited by TEMPO. Notably, the levels of nutlin-3-induced ROS were correlated with the mitochondrial translocation of p53 and this induction was prevented by PFT-μ, an inhibitor of the mitochondrial translocation of p53. Consistent with the effect of the ROS scavenger and MAPK inhibitors, PFT-μ reduced HO-1 expression and the phosphorylation of JNK induced by nutlin-3. In the experiments of analyzing cell death, the knockdown of HO-1 augmented nutlin-3-induced apoptosis. Collectively, these results suggest that nutlin-3 induces HO-1 expression via the activation of both JNK which is dependent on ROS generated by p53 translocated to the mitochondria and p38 MAPK which appears to be stimulated by a ROS-independent mechanism, and this HO-1 induction may inhibit nutlin-3-induced apoptosis, constituting a negative feedback loop of p53-induced apoptosis.

Resveratrol Inhibits the Epidermal Growth Factor-Induced Migration of Osteoblasts: the Suppression of SAPK/JNK and Akt.

PubMed

Kawabata, Tetsu; Tokuda, Haruhiko; Fujita, Kazuhiko; Kainuma, Shingo; Sakai, Go; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Otsuka, Takanobu

2017-01-01

Resveratrol is a polyphenol enriched in the skins of grapes and berries, that shows various beneficial effects for human health. In the present study, we investigated the mechanism behind the epidermal growth factor (EGF)-induced migration of osteoblast-like MC3T3-E1 cells, and the effect of resveratrol on this cell migration. The cell migration was examined using Boyden chamber, and phosphorylation of each kinase was analyzed by Western blotting. The EGF-induced migration was suppressed by PD98059, an inhibitor of MEK1/2, as well as SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of SAPK/JNK, and deguelin, an inhibitor of Akt. In contrast, rapamycin, an inhibitor of upstream kinase of p70 S6 kinase, and fasudil, an inhibitor of Rho-kinase, hardly affected the migration. Resveratrol significantly reduced the EGF-induced migration in a dose-dependent manner. SRT1720, an SIRT1 activator, suppressed the migration by EGF. In addition, resveratrol markedly attenuated the EGF-induced phosphorylation of SAPK/JNK and Akt without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. The phosphorylation of SAPK/JNK and Akt induced by EGF was down-regulated by SRT1720. Our results strongly suggest that resveratrol reduces the EGF-stimulated migration of osteoblasts via suppression of SAPK and Akt, and that the inhibitory effect of resveratrol is mediated in part via SIRT1. © 2017 The Author(s). Published by S. Karger AG, Basel.

Antitumor effects of the flavone chalcone: inhibition of invasion and migration through the FAK/JNK signaling pathway in human gastric adenocarcinoma AGS cells.

PubMed

Lin, Su-Hsuan; Shih, Yuan-Wei

2014-06-01

Chalcones (benzylideneacetophenone) are cancer-preventive food components found in a human diet rich in fruits and vegetables. In this study, we first report the chemopreventive effect of chalcone in human gastric adenocarcinoma cell lines: AGS. The results showed that chalcone could inhibit the abilities of the adhesion, invasion, and migration by cell-matrix adhesion assay, Boyden chamber invasion/migration assay, and wound-healing assay. Molecular data showed that the effect of chalcone in AGS cells might be mediated via sustained inactivation of the phosphorylation of focal adhesion kinase (FAK) and c-Jun N-terminal kinase 1 and 2 (JNK1/2) signal involved in the downregulation of the expressions of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). Next, chalcone-treated AGS cells showed tremendous decrease in the phosphorylation and degradation of inhibitor of kappaBα (IκBα), the nuclear level of NF-κB, and the binding ability of NF-κB to NF-κB response element. Furthermore, treating FAK small interfering RNA (FAK siRNA) and specific inhibitor for JNK (SP600125) to AGS cells could reduce the phosphorylation of JNK1/2 and the activity of MMP-2 and MMP-9. Our results revealed that chalcone significantly inhibited the metastatic ability of AGS cells by reducing MMP-2 and MMP-9 expressions concomitantly with a marked reduction on cell invasion and migration through suppressing and JNK signaling pathways. We suggest that chalcone may offer the application in clinical medicine.

Genetic inhibition of JNK3 ameliorates spinal muscular atrophy.

PubMed

Genabai, Naresh K; Ahmad, Saif; Zhang, Zhanying; Jiang, Xiaoting; Gabaldon, Cynthia A; Gangwani, Laxman

2015-12-15

Mutation of the Survival Motor Neuron 1 (SMN1) gene causes spinal muscular atrophy (SMA), an autosomal recessive neurodegenerative disorder that occurs in early childhood. Degeneration of spinal motor neurons caused by SMN deficiency results in progressive muscle atrophy and death in SMA. The molecular mechanism underlying neurodegeneration in SMA is unknown. No treatment is available to prevent neurodegeneration and reduce the burden of illness in SMA. We report that the c-Jun NH2-terminal kinase (JNK) signaling pathway mediates neurodegeneration in SMA. The neuron-specific isoform JNK3 is required for neuron degeneration caused by SMN deficiency. JNK3 deficiency reduces degeneration of cultured neurons caused by low levels of SMN. Genetic inhibition of JNK pathway in vivo by Jnk3 knockout results in amelioration of SMA phenotype. JNK3 deficiency prevents the loss of spinal cord motor neurons, reduces muscle degeneration, improves muscle fiber thickness and muscle growth, improves motor function and overall growth and increases lifespan of mice with SMA that shows a systemic rescue of phenotype by a SMN-independent mechanism. JNK3 represents a potential (non-SMN) therapeutic target for the treatment of SMA. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Amyloid-β Reduces Exosome Release from Astrocytes by Enhancing JNK Phosphorylation.

PubMed

Abdullah, Mohammad; Takase, Hiroshi; Nunome, Mari; Enomoto, Hiroyuki; Ito, Jin-Ichi; Gong, Jian-Sheng; Michikawa, Makoto

2016-07-02

Exosomes are small extracellular vesicles secreted by variety of cell types such as neurons, astrocytes, and oligodendrocytes. It is suggested that exosomes play essential role in the maintenance of the neuronal functions and also in the clearance of amyloid-β (Aβ) from the brain. Aβ is well known to cause neuronal cell death, whereas little is known about its effect on astrocytes. In this study, we examined the effect of Aβ on release of exosomes from astrocytes in culture. We analyzed release of exosomes and apoE, both of which are known to remove/clear Aβ from the brain, in the culture medium of astrocytes. We found that exosome and apoE-HDL were successfully separated by density gradient ultracentrifugation demonstrated by distribution of their specific markers, flotillin and HSP90, and cholesterol, and morphological analysis using electron microscopy. Exosome release was significantly reduced by Aβ1-42 treatment in cultured astrocytes accompanied by an increased JNK phosphorylation. Whereas, apoE-HDL release remained unchanged. A JNK inhibitor restored the decreased levels of exosome release induced by Aβ treatment to levels similar to those of control, suggesting that Aβ1-42 inhibits exosome release via stimulation of JNK signal pathway. Because exosomes are shown to remove Aβ in the brain, our findings suggest that increased Aβ levels in the brain may impair the exosome-mediated Aβ clearance pathway.

Inhibition of adrenergic human prostate smooth muscle contraction by the inhibitors of c-Jun N-terminal kinase, SP600125 and BI-78D3.

PubMed

Strittmatter, F; Walther, S; Gratzke, C; Göttinger, J; Beckmann, C; Roosen, A; Schlenker, B; Hedlund, P; Andersson, K E; Stief, C G; Hennenberg, M

2012-07-01

BACKGROUND AND PURPOSE α(1) -Adrenoceptor-induced contraction of prostate smooth muscle is mediated by calcium- and Rho kinase-dependent mechanisms. In addition, other mechanisms, such as activation of c-jun N-terminal kinase (JNK) may be involved. Here, we investigated whether JNK participates in α(1)-adrenoceptor-induced contraction of human prostate smooth muscle. EXPERIMENTAL APPROACH Prostate tissue was obtained from patients undergoing radical prostatectomy. Effects of the JNK inhibitors SP600125 (50 µM) and BI-78D3 (30 µM) on contractions induced by phenylephrine, noradrenaline and electric field stimulation (EFS) were studied in myographic measurements. JNK activation by noradrenaline (30 µM) and phenylephrine (10 µM), and the effects of JNK inhibitors of c-Jun phosphorylation were assessed by Western blot analyses with phospho-specific antibodies. Expression of JNK was studied by immunohistochemistry and fluorescence double staining. KEY RESULTS The JNK inhibitors SP600125 and BI-78D3 reduced phenylephrine- and noradrenaline-induced contractions of human prostate strips. In addition, SP600125 reduced EFS-induced contraction of prostate strips. Stimulation of prostate tissue with noradrenaline or phenylephrine in vitro resulted in activation of JNK. Incubation of prostate tissue with SP600125 or BI-78D3 reduced the phosphorylation state of c-Jun. Immunohistochemical staining demonstrated the expression of JNK in smooth muscle cells of human prostate tissue. Fluorescence staining showed that α(1A)-adrenoceptors and JNK are expressed in the same cells. CONCLUSIONS AND IMPLICATIONS Activation of JNK is involved in α(1)-adrenoceptor-induced prostate smooth muscle contraction. Models of α(1)-adrenoceptor-mediated prostate smooth muscle contraction should include this JNK-dependent mechanism. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

β3-adrenergic receptor activation induces TGFβ1 expression in cardiomyocytes via the PKG/JNK/c-Jun pathway.

PubMed

Xu, Zhongcheng; Wu, Jimin; Xin, Junzhou; Feng, Yenan; Hu, Guomin; Shen, Jing; Li, Mingzhe; Zhang, Youyi; Xiao, Han; Wang, Li

2018-06-05

In heart failure, the expression of cardiac β 3 -adrenergic receptors (β 3 -ARs) increases. However, the precise role of β 3 -AR signaling within cardiomyocytes remains unclear. Transforming growth factor β1 (TGFβ1) is a crucial cytokine mediating the cardiac remodeling that plays a causal role in the progression of heart failure. Here, we set out to determine the effect of β 3 -AR activation on TGFβ1 expression in rat cardiomyocytes and examine the underlying mechanism. The selective β 3 -AR agonist BRL37344 induced an increase in TGFβ1 expression and the phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun in β 3 -AR-overexpressing cardiomyocytes. Those effects of BRL37344 were suppressed by a β 3 -AR antagonist. Moreover, the inhibition of JNK and c-Jun activity by a JNK inhibitor and c-Jun siRNA blocked the increase in TGFβ1 expression upon β 3 -AR activation. A protein kinase G (PKG) inhibitor also attenuated β 3 -AR-agonist-induced TGFβ1 expression and the phosphorylation of JNK and c-Jun. In conclusion, the β 3 -AR activation in cardiomyocytes increases the expression of TGFβ1 via the PKG/JNK/c-Jun pathway. These results help us further understand the role of β 3 -AR signaling in heart failure. Copyright © 2018 Elsevier Inc. All rights reserved.

Inhibitor of apoptosis signal-regulating kinase 1 protects against acetaminophen-induced liver injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Yuchao; Ramachandran, Anup; Breckenridge, David G.

Metabolic activation and oxidant stress are key events in the pathophysiology of acetaminophen (APAP) hepatotoxicity. The initial mitochondrial oxidative stress triggered by protein adduct formation is amplified by c-jun-N-terminal kinase (JNK), resulting in mitochondrial dysfunction and ultimately cell necrosis. Apoptosis signal-regulating kinase 1 (ASK1) is considered the link between oxidant stress and JNK activation. The objective of the current study was to assess the efficacy and mechanism of action of the small-molecule ASK1 inhibitor GS-459679 in a murine model of APAP hepatotoxicity. APAP (300 mg/kg) caused extensive glutathione depletion, JNK activation and translocation to the mitochondria, oxidant stress and livermore » injury as indicated by plasma ALT activities and area of necrosis over a 24 h observation period. Pretreatment with 30 mg/kg of GS-459679 almost completely prevented JNK activation, oxidant stress and injury without affecting the metabolic activation of APAP. To evaluate the therapeutic potential of GS-459679, mice were treated with APAP and then with the inhibitor. Given 1.5 h after APAP, GS-459679 was still protective, which was paralleled by reduced JNK activation and p-JNK translocation to mitochondria. However, GS-459679 treatment was not more effective than N-acetylcysteine, and the combination of GS-459679 and N-acetylcysteine exhibited similar efficacy as N-acetylcysteine monotherapy, suggesting that GS-459769 and N-acetylcysteine affect the same pathway. Importantly, inhibition of ASK1 did not impair liver regeneration as indicated by PCNA staining. In conclusion, the ASK1 inhibitor GS-459679 protected against APAP toxicity by attenuating JNK activation and oxidant stress in mice and may have therapeutic potential for APAP overdose patients. - Highlights: • Two ASK1 inhibitors protected against acetaminophen-induced liver injury. • The ASK1 inhibitors protect when used as pre- or post-treatment. • Protection by ASK1

c-Jun N-terminal kinase 3 (JNK3) Mediates Paraquat- and Rotenone-Induced Dopaminergic Neuron Death

PubMed Central

Choi, Won Seok; Abel, Glen; Klintworth, Heather; Flavell, Richard A.; Xia, Zhengui

2011-01-01

Mechanistic studies underlying dopaminergic neuron death may identify new drug targets for the treatment of Parkinson disease (PD). Epidemiological studies have linked pesticide exposure to increased risk for sporadic PD. Here, we investigated the role of c-Jun N-terminal kinase 3 (JNK3), a neural-specific JNK isoform, in dopaminergic neuron death induced by the pesticides rotenone and paraquat. The role of JNK3 was evaluated using RNA silencing and gene deletion to block JNK3 signaling. Using an antibody that recognizes all isoforms of activated JNKs, we found that paraquat and rotenone stimulate JNK phosphorylation in primary cultured dopaminergic neurons. In cultured neurons transfected with Jnk3-specific siRNA and in neurons from Jnk3−/− mice, JNK phosphorylation was nearly abolished, suggesting that JNK3 is the main JNK isoform activated in dopaminergic neurons by these pesticides. Paraquat- and rotenone-induced death of dopaminergic neurons was also significantly reduced by Jnk3 siRNA or Jnk3 gene deletion and deletion of the Jnk3 gene completely attenuated paraquat-induced dopaminergic neuron death and motor-deficits in vivo. Our data identify JNK3 as a common and critical mediator of dopaminergic neuron death induced by paraquat and rotenone, suggesting that it is a potential drug target for PD treatment. PMID:20418776

Fucoidan/FGF-2 induces angiogenesis through JNK- and p38-mediated activation of AKT/MMP-2 signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Beom Su; Bonecell Biotech Inc., 77, Dunsan-dong, Seo-gu, Daejeon 302-830; Park, Ji-Yun

2014-08-08

-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, and AKT. MMP-2 activation was also significantly increased. Specific inhibitors of p38 (SB203580) and JNK (SP600125) inhibited tube formation and wound healing, while an ERK inhibitor (PD98059) did not. MMP-2 activation and AKT phosphorylation were also attenuated and associated with the suppression of p38 and JNK phosphorylation, but not with that of ERK. These results indicate that fucoidan, in the presence of FGF-2, induces angiogenesis through AKT/MMP-2 signalling by activating p38 and JNK. These findings provide basic molecular information on the effect of fucoidan on angiogenesis in the presence of FGF-2.« less

JNK1 controls adult hippocampal neurogenesis and imposes cell-autonomous control of anxiety behaviour from the neurogenic niche

PubMed Central

Mohammad, H; Marchisella, F; Ortega-Martinez, S; Hollos, P; Eerola, K; Komulainen, E; Kulesskaya, N; Freemantle, E; Fagerholm, V; Savontous, E; Rauvala, H; Peterson, B D; van Praag, H; Coffey, E T

2018-01-01

Promoting adult hippocampal neurogenesis is expected to induce neuroplastic changes that improve mood and alleviate anxiety. However, the underlying mechanisms remain largely unknown and the hypothesis itself is controversial. Here we show that mice lacking Jnk1, or c-Jun N-terminal kinase (JNK) inhibitor-treated mice, display increased neurogenesis in adult hippocampus characterized by enhanced cell proliferation and survival, and increased maturation in the ventral region. Correspondingly, anxiety behaviour is reduced in a battery of tests, except when neurogenesis is prevented by AraC treatment. Using engineered retroviruses, we show that exclusive inhibition of JNK in adult-born granule cells alleviates anxiety and reduces depressive-like behaviour. These data validate the neurogenesis hypothesis of anxiety. Moreover, they establish a causal role for JNK in the hippocampal neurogenic niche and anxiety behaviour, and advocate targeting of JNK as an avenue for novel therapies against affective disorders. PMID:27843149

Nobiletin inhibits human osteosarcoma cells metastasis by blocking ERK and JNK-mediated MMPs expression

PubMed Central

Cheng, Hsin-Lin; Hsieh, Ming-Ju; Yang, Jia-Sin; Lin, Chiao-Wen; Lue, Ko-Haung; Lu, Ko-Hsiu; Yang, Shun-Fa

2016-01-01

Nobiletin, a polymethoxyflavone, has a few pharmacological activities, including anti-inflammation and anti-cancer effects. However, its effect on human osteosarcoma progression remains uninvestigated. Therefore, we examined the effectiveness of nobiletin against cellular metastasis of human osteosarcoma and the underlying mechanisms. Nobiletin, up to 100 μM without cytotoxicity, significantly decreased motility, migration and invasion as well as enzymatic activities, protein levels and mRNA expressions of matrix metalloproteinase (MMP)-2 and MMP-9 in U2OS and HOS cells. In addition to inhibition of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), the inhibitory effect of nobiletin on the DNA-binding activity of the transcription factor nuclear factor-kappa B (NF-κB), cAMP response element-binding protein (CREB), and specificity protein 1 (SP-1) in U2OS and HOS cells. Co-treatment with ERK and JNK inhibitors and nobiletin further reduced U2OS cells migration and invasion. These results indicated that nobiletin inhibits human osteosarcoma U2OS and HOS cells motility, migration and invasion by down-regulating MMP-2 and MMP-9 expressions via ERK and JNK pathways and through the inactivation of downstream NF-κB, CREB, and SP-1. Nobiletin has the potential to serve as an anti-metastatic agent for treating osteosarcoma. PMID:27144433

A Quantitative RNAi Screen for JNK Modifiers Identifies Pvr as a Novel Regulator of Drosophila Immune Signaling

PubMed Central

Bond, David; Foley, Edan

2009-01-01

Drosophila melanogaster responds to gram-negative bacterial challenges through the IMD pathway, a signal transduction cassette that is driven by the coordinated activities of JNK, NF-κB and caspase modules. While many modifiers of NF-κB activity were identified in cell culture and in vivo assays, the regulatory apparatus that determines JNK inputs into the IMD pathway is relatively unexplored. In this manuscript, we present the first quantitative screen of the entire genome of Drosophila for novel regulators of JNK activity in the IMD pathway. We identified a large number of gene products that negatively or positively impact on JNK activation in the IMD pathway. In particular, we identified the Pvr receptor tyrosine kinase as a potent inhibitor of JNK activation. In a series of in vivo and cell culture assays, we demonstrated that activation of the IMD pathway drives JNK-dependent expression of the Pvr ligands, Pvf2 and Pvf3, which in turn act through the Pvr/ERK MAP kinase pathway to attenuate the JNK and NF-κB arms of the IMD pathway. Our data illuminate a poorly understood arm of a critical and evolutionarily conserved innate immune response. Furthermore, given the pleiotropic involvement of JNK in eukaryotic cell biology, we believe that many of the novel regulators identified in this screen are of interest beyond immune signaling. PMID:19893628

TRX-ASK1-JNK signaling regulation of cell density-dependent cytotoxicity in cigarette smoke-exposed human bronchial epithelial cells.

PubMed

Lee, Yong Chan; Chuang, Chun-Yu; Lee, Pak-Kei; Lee, Jin-Soo; Harper, Richart W; Buckpitt, Alan B; Wu, Reen; Oslund, Karen

2008-05-01

Cigarette smoke is a major environmental air pollutant that injures airway epithelium and incites subsequent diseases including chronic obstructive pulmonary disease. The lesion that smoke induces in airway epithelium is still incompletely understood. Using a LIVE/DEAD cytotoxicity assay, we observed that subconfluent cultures of bronchial epithelial cells derived from both human and monkey airway tissues and an immortalized normal human bronchial epithelial cell line (HBE1) were more susceptible to injury by cigarette smoke extract (CSE) and by direct cigarette smoke exposure than cells in confluent cultures. Scraping confluent cultures also caused an enhanced cell injury predominately in the leading edge of the scraped confluent cultures by CSE. Cellular ATP levels in both subconfluent and confluent cultures were drastically reduced after CSE exposure. In contrast, GSH levels were significantly reduced only in subconfluent cultures exposed to smoke and not in confluent cultures. Western blot analysis demonstrated ERK activation in both confluent and subconfluent cultures after CSE. However, activation of apoptosis signal-regulating kinase 1 (ASK1), JNK, and p38 were demonstrated only in subconfluent cultures and not in confluent cultures after CSE. Using short interfering RNA (siRNA) to JNK1 and JNK2 and a JNK inhibitor, we attenuated CSE-mediated cell death in subconfluent cultures but not with an inhibitor of the p38 pathway. Using the tetracycline (Tet)-on inducible approach, overexpression of thioredoxin (TRX) attenuated CSE-mediated cell death and JNK activation in subconfluent cultures. These results suggest that the TRX-ASK1-JNK pathway may play a critical role in mediating cell density-dependent CSE cytotoxicity.

3-MCPD 1-Palmitate Induced Tubular Cell Apoptosis In Vivo via JNK/p53 Pathways

PubMed Central

Liu, Man; Huang, Guoren; Wang, Thomas T.Y.; Sun, Xiangjun; Yu, Liangli (Lucy)

2016-01-01

Fatty acid esters of 3-chloro-1, 2-propanediol (3-MCPD esters) are a group of processing induced food contaminants with nephrotoxicity but the molecular mechanism(s) remains unclear. This study investigated whether and how the JNK/p53 pathway may play a role in the nephrotoxic effect of 3-MCPD esters using 3-MCPD 1-palmitate (MPE) as a probe compound in Sprague Dawley rats. Microarray analysis of the kidney from the Sprague Dawley rats treated with MPE, using Gene Ontology categories and KEGG pathways, revealed that MPE altered mRNA expressions of the genes involved in the mitogen-activated protein kinase (JNK and ERK), p53, and apoptotic signal transduction pathways. The changes in the mRNA expressions were confirmed by qRT-PCR and Western blot analyses and were consistent with the induction of tubular cell apoptosis as determined by histopathological, TUNEL, and immunohistochemistry analyses in the kidneys of the Sprague Dawley rats. Additionally, p53 knockout attenuated the apoptosis, and the apoptosis-related protein bax expression and cleaved caspase-3 activation induced by MPE in the p53 knockout C57BL/6 mice, whereas JNK inhibitor SP600125 but not ERK inhibitor U0126 inhibited MPE-induced apoptosis, supporting the conclusion that JNK/p53 might play a critical role in the tubular cell apoptosis induced by MPE and other 3-MCPD fatty acid esters. PMID:27008853

Domain Specificity of MAP3K Family Members, MLK and Tak1, for JNK Signaling in Drosophila

PubMed Central

Stronach, Beth; Lennox, Ashley L.; Garlena, Rebecca A.

2014-01-01

A highly diverse set of protein kinases functions as early responders in the mitogen- and stress-activated protein kinase (MAPK/SAPK) signaling pathways. For instance, humans possess 14 MAPK kinase kinases (MAP3Ks) that activate Jun kinase (JNK) signaling downstream. A major challenge is to decipher the selective and redundant functions of these upstream MAP3Ks. Taking advantage of the relative simplicity of Drosophila melanogaster as a model system, we assessed MAP3K signaling specificity in several JNK-dependent processes during development and stress response. Our approach was to generate molecular chimeras between two MAP3K family members, the mixed lineage kinase, Slpr, and the TGF-β activated kinase, Tak1, which share 32% amino acid identity across the kinase domain but otherwise differ in sequence and domain structure, and then test the contributions of various domains for protein localization, complementation of mutants, and activation of signaling. We found that overexpression of the wild-type kinases stimulated JNK signaling in alternate contexts, so cells were capable of responding to both MAP3Ks, but with distinct outcomes. Relative to wild-type, the catalytic domain swaps compensated weakly or not at all, despite having a shared substrate, the JNK kinase Hep. Tak1 C-terminal domain-containing constructs were inhibitory in Tak1 signaling contexts, including tumor necrosis factor-dependent cell death and innate immune signaling; however, depressing antimicrobial gene expression did not necessarily cause phenotypic susceptibility to infection. These same constructs were neutral in the context of Slpr-dependent developmental signaling, reflecting differential subcellular protein localization and by inference, point of activation. Altogether, our findings suggest that the selective deployment of a particular MAP3K can be attributed in part to its inherent sequence differences, cellular localization, and binding partner availability. PMID:24429281

Sodium fluoride induces apoptosis in mouse embryonic stem cells through ROS-dependent and caspase- and JNK-mediated pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nguyen Ngoc, Tam Dan; Son, Young-Ok; Lim, Shin-Saeng

2012-03-15

Sodium fluoride (NaF) is used as a source of fluoride ions in diverse applications. Fluoride salt is an effective prophylactic for dental caries and is an essential element required for bone health. However, fluoride is known to cause cytotoxicity in a concentration-dependent manner. Further, no information is available on the effects of NaF on mouse embryonic stem cells (mESCs). We investigated the mode of cell death induced by NaF and the mechanisms involved. NaF treatment greater than 1 mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G{sub 2}/M phase. The addition of NaFmore » induced cell death mainly by apoptosis rather than necrosis. Catalase (CAT) treatment significantly inhibited the NaF-mediated cell death and also suppressed the NaF-mediated increase in phospho-c-Jun N-terminal kinase (p-JNK) levels. Pre-treatment with SP600125 or z-VAD-fmk significantly attenuated the NaF-mediated reduction in cell viability. In contrast, intracellular free calcium chelator, but not of sodium or calcium ion channel blockers, facilitated NaF-induced toxicity in the cells. A JNK specific inhibitor (SP600125) prevented the NaF-induced increase in growth arrest and the DNA damage-inducible protein 45α. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-α or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1 mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways. -- Highlights: ► The mode of NaF-induced cell death and the mechanisms involved were examined. ► NaF induced mainly apoptotic death of mouse embryonic stem cells (mESCs). ► NaF induced mitochondrial-mediated and caspase-dependent apoptosis. ► JNK- and p53-mediated pathways are involved in NaF-mediated apoptosis in the cells. ► ROS are the up-stream effector in NaF-mediated activation of JNK and p53

Neuroprotective Effects of the Absence of JNK1 or JNK3 Isoforms on Kainic Acid-Induced Temporal Lobe Epilepsy-Like Symptoms.

PubMed

de Lemos, Luisa; Junyent, Felix; Camins, Antoni; Castro-Torres, Rubén Darío; Folch, Jaume; Olloquequi, Jordi; Beas-Zarate, Carlos; Verdaguer, Ester; Auladell, Carme

2018-05-01

The activation of c-Jun-N-terminal kinases (JNK) pathway has been largely associated with the pathogenesis and the neuronal death that occur in neurodegenerative diseases. Altogether, this justifies why JNKs have become a focus of screens for new therapeutic strategies. The aim of the present study was to identify the role of the different JNK isoforms (JNK1, JNK2, and JNK3) in apoptosis and inflammation after induction of brain damage. To address this aim, we induced excitotoxicity in wild-type and JNK knockout mice (jnk1 -/- , jnk2 -/- , and jnk3 -/- ) via an intraperitoneal injection of kainic acid, an agonist of glutamic-kainate-receptors, that induce status epilepticus.Each group of animals was divided into two treatments: a single intraperitoneal dose of saline solution, used as a control, and a single intraperitoneal dose (30 mg/kg) of kainic acid. Our results reported a significant decrease in neuronal degeneration in the hippocampus of jnk1 -/- and jnk3 -/- mice after kainic acid treatment, together with reduced or unaltered expression of several apoptotic genes compared to WT treated mice. In addition, both jnk1 -/- and jnk3 -/- mice exhibited a reduction in glial reactivity, as shown by the lower expression of inflammatory genes and a reduction of JNK phosphorylation. In addition, in jnk3 -/- mice, the c-Jun phosphorylation was also diminished.Collectively, these findings provide compelling evidence that the absence of JNK1 or JNK3 isoforms confers neuroprotection against neuronal damage induced by KA and evidence, for the first time, the implication of JNK1 in excitotoxicity. Accordingly, JNK1 and/or JNK3 are promising targets for the prevention of cell death and inflammation during epileptogenesis.

TGF-beta and HGF transmit the signals through JNK-dependent Smad2/3 phosphorylation at the linker regions.

PubMed

Mori, Shigeo; Matsuzaki, Koichi; Yoshida, Katsunori; Furukawa, Fukiko; Tahashi, Yoshiya; Yamagata, Hideo; Sekimoto, Go; Seki, Toshihito; Matsui, Hirofumi; Nishizawa, Mikio; Fujisawa, Jun-ichi; Okazaki, Kazuichi

2004-09-23

Although hepatocyte growth factor (HGF) can act synergistically or antagonistically with transforming growth factor-beta (TGF-beta) signaling, molecular mechanism of their crosstalk remains unknown. Using antibodies which selectively distinguished receptor-regulated Smads (R-Smads) phosphorylated at linker regions from those at C-terminal regions, we herein showed that either HGF or TGF-beta treatment of normal stomach-origin cells activated the JNK pathway, thereafter inducing endogenous R-Smads phosphorylation at linker regions. However, the phosphorylation at their C-terminal regions was not induced by HGF treatment. The activated JNK could directly phosphorylate R-Smads in vitro at the same sites that were phosphorylated in response to TGF-beta or HGF in vivo. Thus, the linker regions of R-Smads were the common phosphorylation sites for HGF and TGF-beta signaling pathways. The phosphorylation induced by simultaneous treatment with HGF and TGF-beta allowed R-Smads to associate with Smad4 and to translocate into the nucleus. JNK pathway involved HGF and TGF-beta-mediated infiltration potency since a JNK inhibitor SP600125 caused the reduction of invasive capacity induced by HGF and TGF-beta signals. Moreover, a combined treatment with HGF and TGF-beta led to a potent increase in plasminogen activator inhibitor type 1 transcriptional activity through Smad3 phosphorylation at the linker region. In contrast, HGF treatment reduced TGF-beta-dependent activation of p15INK4B promoter, in which Smad3 phosphorylation at the C-terminal region was involved. In conclusion, HGF and TGF-beta transmit the signals through JNK-mediated R-Smads phosphorylation at linker regions.

Syk-mediated tyrosine phosphorylation of mule promotes TNF-induced JNK activation and cell death.

PubMed

Lee, C K; Yang, Y; Chen, C; Liu, J

2016-04-14

The transcription factor Miz1 negatively regulates TNF-induced JNK activation and cell death by suppressing TRAF2 K63-polyubiquitination; upon TNF stimulation, the suppression is relieved by Mule/ARF-BP1-mediated Miz1 ubiquitination and subsequent degradation. It is not known how Mule is activated by TNF. Here we report that TNF activates Mule by inducing the dissociation of Mule from its inhibitor ARF. ARF binds to and thereby inhibits the E3 ligase activity of Mule in the steady state. TNF induces tyrosine phosphorylation of Mule, which subsequently dissociates from ARF and becomes activated. Inhibition of Mule phosphorylation by silencing of the Spleen Tyrosine Kinase (Syk) prevents its dissociation from ARF, thereby inhibiting Mule E3 ligase activity and TNF-induced JNK activation and cell death. Our data provides a missing link in TNF signaling pathway that leads to JNK activation and cell death.

Protective Role of Taurine against Arsenic-Induced Mitochondria-Dependent Hepatic Apoptosis via the Inhibition of PKCδ-JNK Pathway

PubMed Central

Das, Joydeep; Ghosh, Jyotirmoy; Manna, Prasenjit; Sil, Parames C.

2010-01-01

Background Oxidative stress-mediated hepatotoxic effect of arsenic (As) is mainly due to the depletion of glutathione (GSH) in liver. Taurine, on the other hand, enhances intracellular production of GSH. Little is known about the mechanism of the beneficial role of taurine in As-induced hepatic pathophysiology. Therefore, in the present study we investigated its beneficial role in As-induced hepatic cell death via mitochondria-mediated pathway. Methodology/Principal Findings Rats were exposed to NaAsO2 (2 mg/kg body weight for 6 months) and the hepatic tissue was used for oxidative stress measurements. In addition, the pathophysiologic effect of NaAsO2 (10 µM) on hepatocytes was evaluated by determining cell viability, mitochondrial membrane potential and ROS generation. As caused mitochondrial injury by increased oxidative stress and reciprocal regulation of Bcl-2, Bcl-xL/Bad, Bax, Bim in association with increased level of Apaf-1, activation of caspase 9/3, cleavage of PARP protein and ultimately led to apoptotic cell death. In addition, As markedly increased JNK and p38 phosphorylation with minimal disturbance of ERK. Pre-exposure of hepatocytes to a JNK inhibitor SP600125 prevented As-induced caspase-3 activation, ROS production and loss in cell viability. Pre-exposure of hepatocytes to a p38 inhibitor SB2035, on the other hand, had practically no effect on these events. Besides, As activated PKCδ and pre-treatment of hepatocytes with its inhibitor, rottlerin, suppressed the activation of JNK indicating that PKCδ is involved in As-induced JNK activation and mitochondrial dependent apoptosis. Oral administration of taurine (50 mg/kg body weight for 2 weeks) both pre and post to NaAsO2 exposure or incubation of the hepatocytes with taurine (25 mM) were found to be effective in counteracting As-induced oxidative stress and apoptosis. Conclusions/Significance Results indicate that taurine treatment improved As-induced hepatic damages by inhibiting PKCδ-JNK

Modulation of iridovirus-induced apoptosis by endocytosis, early expression, JNK, and apical caspase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chitnis, Nilesh S.; D'Costa, Susan M.; Paul, Eric R.

Chilo iridescent virus (CIV) is the type species for the family Iridoviridae, which are large, isometric, cytoplasmic dsDNA viruses. We examined the mechanism of apoptosis induction by CIV. High CIV doses (CIV{sub XS}; 400 {mu}g/ml), UV-irradiated virus (CIV{sub UV}; 10 {mu}g/ml) and CVPE (CIV protein extract; 10 {mu}g/ml) induced apoptosis in 60% of treated Choristoneura fumiferana (IPRI-CF-124T) cells. Normal doses of infectious CIV (10 {mu}g/ml) induced apoptosis in only 10% of C. fumiferana (CF) cells. Apoptosis was inhibited by Z-IETD-FMK, an apical caspase inhibitor, indicating that CIV-induced apoptosis requires caspase activity. The putative caspase in CF cells was designated Cf-caspase-i.more » CIV{sub UV} or CVPE enhanced Cf-caspase-i activity by 80% at 24 h relative to mock-treated cells. Since the MAP kinase pathway induces or inhibits apoptosis depending on the context, we used JNK inhibitor SP600125 and demonstrated drastic suppression of CVPE-induced apoptosis. Thus, the JNK signaling pathway is significant for apoptosis in this system. Virus interaction with the cell surface was not sufficient for apoptosis since CIV{sub UV} particles bound to polysterene beads failed to induce apoptosis. Endocytosis inhibitors (bafilomycin or ammonium chloride) negated apoptosis induction by CIV{sub UV}, CIV{sub XS} or CVPE indicating that entry through this mode is required. Given the weak apoptotic response to infectious CIV, we postulated that viral gene expression inhibited apoptosis. CIV infection of cells pretreated with cycloheximide induced apoptosis in 69% of the cells compared to 10% in normal infections. Furthermore, blocking viral DNA replication with aphidicolin or phosphonoacetic acid suppressed apoptosis and Cf-caspase-i activity, indicating that early viral expression is necessary for inhibition of apoptosis, and de novo synthesis of viral proteins is not required for induction. We show for the first time that, in a member of the family

Identification and Characterization of a Novel Class of c-Jun N-terminal Kinase Inhibitors

PubMed Central

Schepetkin, Igor A.; Kirpotina, Liliya N.; Khlebnikov, Andrei I.; Hanks, Tracey S.; Kochetkova, Irina; Pascual, David W.; Jutila, Mark A.

2012-01-01

In efforts to identify novel small molecules with anti-inflammatory properties, we discovered a unique series of tetracyclic indenoquinoxaline derivatives that inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 activation. Compound IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) was found to be a potent, noncytotoxic inhibitor of pro-inflammatory cytokine [interleukin (IL)-1α, IL-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α, interferon-γ, and granulocyte-macrophage colony-stimulating factor] and nitric oxide production by human and murine monocyte/macrophages. Three additional potent inhibitors of cytokine production were identified through further screening of IQ-1 analogs. The sodium salt of IQ-1 inhibited LPS-induced TNF-α and IL-6 production in MonoMac-6 cells with IC50 values of 0.25 and 0.61 μM, respectively. Screening of 131 protein kinases revealed that derivative IQ-3 [11H-indeno[1,2-b]quinoxalin-11-one-O-(2-furoyl)oxime]was a specific inhibitor of the c-Jun N-terminal kinase (JNK) family, with preference for JNK3. This compound, as well as IQ-1 and three additional oxime indenoquinoxalines, were found to be high-affinity JNK inhibitors with nanomolar binding affinity and ability to inhibit c-Jun phosphorylation. Furthermore, docking studies showed that hydrogen bonding interactions of the active indenoquinoxalines with Asn152, Gln155, and Met149 of JNK3 played an important role in enzyme binding activity. Finally, we showed that the sodium salt of IQ-1 had favorable pharmacokinetics and inhibited the ovalbumin-induced CD4+ T-cell immune response in a murine delayed-type hypersensitivity model in vivo. We conclude that compounds with an indenoquinoxaline nucleus can serve as specific small-molecule modulators for mechanistic studies of JNKs as well as a potential leads for the development of anti-inflammatory drugs. PMID:22434859

Activation of ERK and JNK signaling pathways by mycotoxin citrinin in human cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, C.-H.; Yu, F.-Y.; Wang, L.-T.

2009-06-15

Mycotoxin citrinin (CTN) is commonly found in foods and feeds that are contaminated/inoculated with Penicillium, Aspergillus and Monascus species. The exposure of human embryonic kidney (HEK293) and HeLa cells to CTN resulted in a dose-dependent increase in the phosphorylation of two major mitogen-activated protein kinases (MAPKs), ERK1/2 and JNK. In HEK293 cultures, the administering of CTN increased both the mRNA and protein levels of egr-1, c-fos and c-jun genes; additionally, the ERK1/2 pathway contributed to the upregulation of Egr-1 and c-Fos protein expression. CTN treatment also induced the transcription activity of Egr-1 and AP-1 proteins, as evidenced by luciferase reportermore » assays. Bioinformatic analyses indicated two genes Gadd45{beta} and MMP3 have Egr-1 and AP-1 response elements in their promoters, respectively. Furthermore, co-exposure of HEK293 cells to CTN and MAPK pathway inhibitors demonstrated that CTN increased the levels of Gadd45{beta} mRNA through ERK1/2 signaling pathway and up-regulated the MMP3 transcripts majorly via JNK pathway. Finally, CTN-triggered caspase 3 activity was significantly reduced in the presence of MAPK inhibitors. Our results suggest that CTN positively regulates ERK1/2 and JNK pathways as well as their downstream effectors in human cells; activated MAPK pathways are also involved in CTN-induced apoptosis.« less

Proteasome inhibitor MG132 induces selective apoptosis in glioblastoma cells through inhibition of PI3K/Akt and NFkappaB pathways, mitochondrial dysfunction, and activation of p38-JNK1/2 signaling.

PubMed

Zanotto-Filho, Alfeu; Braganhol, Elizandra; Battastini, Ana Maria Oliveira; Moreira, José Cláudio Fonseca

2012-12-01

Proteasome inhibitors are emerging as a new class of anticancer agents. In this work, we examined the mechanisms underlying cytotoxicity, selectivity and adjuvant potential of the proteasome inhibitor MG132 in a panel of glioblastoma (GBM) cells (U138MG, C6, U87 and U373) and in normal astrocytes. MG132 markedly inhibited GBM cells growth irrespective of the p53 or PTEN mutational status of the cells whereas astrocytic viability was not affected, suggesting a selective toxicity of MG132 to cancerous glial cells. Mechanistically, MG132 arrested cells in G2/M phase of the cell cycle and increased p21(WAF1) protein immunocontent. Following cell arrest, cells become apoptotic as shown by annexin-V binding, caspase-3 activation, chromatin condensation and formation of sub-G1 apoptotic cells. MG132 promoted mitochondrial depolarization and decreased the mitochondrial antiapoptotic protein bcl-xL; it also induced activation of JNK and p38, and inhibition of NFkappaB and PI3K/Akt survival pathways. Pre-treatment of GBMs with the mitochondrial permeability transition pore inhibitor, bongkrekic acid, or pharmacological inhibitors of JNK1/2 and p38, SP600125 and SB203580, attenuated MG132-induced cell death. Besides its apoptotic effect alone, MG132 also enhanced the antiglioma effect of the chemotherapeutics cisplatin, taxol and doxorubicin in C6 and U138MG cells, indicating an adjuvant/chemosensitizer potential. In summary, MG132 exerted profound and selective toxicity in GBMs, being a potential agent for further testing in animal models of the disease.

Blockage of JNK pathway enhances arsenic trioxide-induced apoptosis in human keratinocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, H.-S., E-mail: huanghs@mail.ncku.edu.t; Liu, Z.-M.; Hong, D.-Y.

2010-04-15

Arsenic is well known as a carcinogen predisposing humans to some severe diseases and also as an effective medicine for treating acute promyelocytic leukemia, syphilis, and psoriasis. Multiple active mechanisms, including cell cycle arrest and apoptosis, have been proposed in therapy; however, the opposing effects of arsenic remain controversial. Our previous study found that arsenic trioxide (ATO)-induced activation of p21{sup WAF1/CIP1} (p21) led to A431 cell death through the antagonistic effects of the signaling of ERK1/2 and JNK1. In the current study, the inhibitory effects of JNK1 on ATO-induced p21 expression were explored. Over-expression of JNK1 in A431 cells couldmore » inhibit p21 expression, which was associated with HDAC1 and TGIF. Using the GST pull-down assay and fluorescence resonance energy transfer analysis, N-terminal domain (amino acids 1-108) of TGIF, critical to its binding with c-Jun, was found. Using reporter assays, requirement of the C-terminal domain (amino acids 138-272) of TGIF to suppress ATO-induced p21 expression was observed. Thus, the domains of TGIF that carried out its inhibitory effects on p21 were identified. Finally, treatment with JNK inhibitor SP600125 could enhance ATO-induced apoptosis of HaCaT keratinocytes by using flow cytometry.« less

Fluoride induces oxidative damage and SIRT1/autophagy through ROS-mediated JNK signaling.

PubMed

Suzuki, Maiko; Bandoski, Cheryl; Bartlett, John D

2015-12-01

Fluoride is an effective caries prophylactic, but at high doses can also be an environmental health hazard. Acute or chronic exposure to high fluoride doses can result in dental enamel and skeletal and soft tissue fluorosis. Dental fluorosis is manifested as mottled, discolored, porous enamel that is susceptible to dental caries. Fluoride induces cell stress, including endoplasmic reticulum stress and oxidative stress, which leads to impairment of ameloblasts responsible for dental enamel formation. Recently we reported that fluoride activates SIRT1 and autophagy as an adaptive response to protect cells from stress. However, it still remains unclear how SIRT1/autophagy is regulated in dental fluorosis. In this study, we demonstrate that fluoride exposure generates reactive oxygen species (ROS) and the resulting oxidative damage is counteracted by SIRT1/autophagy induction through c-Jun N-terminal kinase (JNK) signaling in ameloblasts. In the mouse-ameloblast-derived cell line LS8, fluoride induced ROS, mitochondrial damage including cytochrome-c release, up-regulation of UCP2, attenuation of ATP synthesis, and H2AX phosphorylation (γH2AX), which is a marker of DNA damage. We evaluated the effects of the ROS inhibitor N-acetylcysteine (NAC) and the JNK inhibitor SP600125 on fluoride-induced SIRT1/autophagy activation. NAC decreased fluoride-induced ROS generation and attenuated JNK and c-Jun phosphorylation. NAC decreased SIRT1 phosphorylation and formation of the autophagy marker LC3II, which resulted in an increase in the apoptosis mediators γH2AX and cleaved/activated caspase-3. SP600125 attenuated fluoride-induced SIRT1 phosphorylation, indicating that fluoride activates SIRT1/autophagy via the ROS-mediated JNK pathway. In enamel organs from rats or mice treated with 50, 100, or 125 ppm fluoride for 6 weeks, cytochrome-c release and the DNA damage markers 8-oxoguanine, p-ATM, and γH2AX were increased compared to those in controls (0 ppm fluoride). These

Fluoride induces oxidative damage and SIRT1/autophagy through ROS-mediated JNK signaling

PubMed Central

Suzuki, Maiko; Bandoski, Cheryl; Bartlett, John D.

2015-01-01

Fluoride is an effective caries prophylactic, but at high doses can also be an environmental health hazard. Acute or chronic exposure to high fluoride doses can result in dental enamel and skeletal and soft tissue fluorosis. Dental fluorosis is manifested as mottled, discolored, porous enamel that is susceptible to dental caries. Fluoride induces cell stress, including endoplasmic reticulum stress and oxidative stress, which leads to impairment of ameloblasts responsible for dental enamel formation. Recently we reported that fluoride activates SIRT1 and autophagy as an adaptive response to protect cells from stress. However, it still remains unclear how SIRT1/autophagy is regulated in dental fluorosis. In this study, we demonstrate that fluoride exposure generates reactive oxygen species (ROS) and the resulting oxidative damage is counteracted by SIRT1/autophagy induction through c-Jun N-terminal kinase (JNK) signaling in ameloblasts. In the mouse-ameloblast-derived cell line LS8, fluoride induced ROS, mitochondrial damage including cytochrome-c release, up-regulation of UCP2, attenuation of ATP synthesis, and H2AX phosphorylation (γH2AX), which is a marker of DNA damage. We evaluated the effects of the ROS inhibitor N-acetylcysteine (NAC) and the JNK inhibitor SP600125 on fluoride-induced SIRT1/autophagy activation. NAC decreased fluoride-induced ROS generation and attenuated JNK and c-Jun phosphorylation. NAC decreased SIRT1 phosphorylation and formation of the autophagy marker LC3II, which resulted in an increase in the apoptosis mediators γH2AX and cleaved/activated caspase-3. SP600125 attenuated fluoride-induced SIRT1 phosphorylation, indicating that fluoride activates SIRT1/autophagy via the ROS-mediated JNK pathway. In enamel organs from rats or mice treated with 50, 100, or 125 ppm fluoride for 6 weeks, cytochrome-c release and the DNA damage markers 8-oxoguanine, p-ATM, and γH2AX were increased compared to those in controls (0 ppm fluoride). These

High-Dose Fluoride Impairs the Properties of Human Embryonic Stem Cells via JNK Signaling.

PubMed

Fu, Xin; Xie, Fang-Nan; Dong, Ping; Li, Qiu-Chen; Yu, Guang-Yan; Xiao, Ran

2016-01-01

Fluoride is a ubiquitous natural substance that is often used in dental products to prevent dental caries. The biphasic actions of fluoride imply that excessive systemic exposure to fluoride can cause harmful effects on embryonic development in both animal models and humans. However, insufficient information is available on the effects of fluoride on human embryonic stem cells (hESCs), which is a novel in vitro humanized model for analyzing the embryotoxicities of chemical compounds. Therefore, we investigated the effects of sodium fluoride (NaF) on the proliferation, differentiation and viability of H9 hESCs. For the first time, we showed that 1 mM NaF did not significantly affect the proliferation of hESCs but did disturb the gene expression patterns of hESCs during embryoid body (EB) differentiation. Higher doses of NaF (2 mM and above) markedly decreased the viability and proliferation of hESCs. The mode and underlying mechanism of high-dose NaF-induced cell death were further investigated by assessing the sub-cellular morphology, mitochondrial membrane potential (MMP), caspase activities, cellular reactive oxygen species (ROS) levels and activation of mitogen-activated protein kinases (MAPKs). High-dose NaF caused the death of hESCs via apoptosis in a caspase-mediated but ROS-independent pathway, coupled with an increase in the phospho-c-Jun N-terminal kinase (p-JNK) levels. Pretreatment with a p-JNK-specific inhibitor (SP600125) could effectively protect hESCs from NaF-induced cell death in a concentration- and time-dependent manner. These findings suggest that NaF might interfere with early human embryogenesis by disturbing the specification of the three germ layers as well as osteogenic lineage commitment and that high-dose NaF could cause apoptosis through a JNK-dependent pathway in hESCs.

Ras promotes cell survival by antagonizing both JNK and Hid signals in the Drosophila eye.

PubMed

Wu, Yue; Zhuang, Yuan; Han, Min; Xu, Tian; Deng, Kejing

2009-10-20

Programmed cell death, or apoptosis, is a fundamental physiological process during normal development or in pathological conditions. The activation of apoptosis can be elicited by numerous signalling pathways. Ras is known to mediate anti-apoptotic signals by inhibiting Hid activity in the Drosophila eye. Here we report the isolation of a new loss-of-function ras allele, rasKP, which causes excessive apoptosis in the Drosophila eye. This new function is likely to be mediated through the JNK pathway since the inhibition of JNK signalling can significantly suppress rasKP-induced apoptosis, whereas the removal of hid only weakly suppresses the phenotype. Furthermore, the reduction of JNK signalling together with the expression of the baculovirus caspase inhibitor p35, which blocks Hid activity, strongly suppresses the rasKP cell death. In addition, we find a strong correlation between rasKP-induced apoptosis in the eye disc and the activation of JNK signalling. In the Drosophila eye, Ras may protect cells from apoptosis by inhibiting both JNK and Hid activities. Surprisingly, reducing Ras activity in the wing, however, does not cause apoptosis but rather affects cell and organ size. Thus, in addition to its requirement for cell viability, Ras appears to mediate different biological roles depending on the developmental context and on the level of its expression.

JNK-induced MCP-1 production in spinal cord astrocytes contributes to central sensitization and neuropathic pain

PubMed Central

Gao, Yong-Jing; Zhang, Ling; Samad, Omar Abdel; Suter, Marc R.; Yasuhiko, Kawasaki; Xu, Zhen-Zhong; Park, Jong-Yeon; Lind, Anne-Li; Ma, Qiufu; Ji, Ru-Rong

2009-01-01

Our previous study showed that activation of c-jun-N-terminal kinase (JNK) in spinal astrocytes plays an important role in neuropathic pain sensitization. We further investigated how JNK regulates neuropathic pain. In cultured astrocytes, TNF-α transiently activated JNK via TNF receptor-1. Cytokine array indicated that the chemokine CCL2/MCP-1 (monocyte chemoattractant protein-1) was strongly induced by the TNF-α/JNK pathway. MCP-1 upregulation by TNF-α was dose-dependently inhibited by the JNK inhibitors SP600125 and D-JNKI-1. Spinal injection of TNF-α produced JNK-dependent pain hypersensitivity and MCP-1 upregulation in the spinal cord. Further, spinal nerve ligation (SNL) induced persistent neuropathic pain and MCP-1 upregulation in the spinal cord, and both were suppressed by D-JNKI-1. Remarkably, MCP-1 was primarily induced in spinal cord astrocytes after SNL. Spinal administration of MCP-1 neutralizing antibody attenuated neuropathic pain. Conversely, spinal application of MCP-1 induced heat hyperalgesia and phosphorylation of extracellular signal-regulated kinase (ERK) in superficial spinal cord dorsal horn neurons, indicative of central sensitization (hyperactivity of dorsal horn neurons). Patch clamp recordings in lamina II neurons of isolated spinal cord slices showed that MCP-1 not only enhanced spontaneous excitatory synaptic currents (sEPSCs) but also potentiated NMDA- and AMPA-induced currents. Finally, the MCP-1 receptor CCR2 was expressed in neurons and some non-neuronal cells in the spinal cord. Taken together, we have revealed a previously unknown mechanism of MCP-1 induction and action. MCP-1 induction in astrocytes following JNK activation contributes to central sensitization and neuropathic pain facilitation by enhancing excitatory synaptic transmission. Inhibition of the JNK/MCP-1 pathway may provide a new therapy for neuropathic pain management. PMID:19339605

JNK-induced MCP-1 production in spinal cord astrocytes contributes to central sensitization and neuropathic pain.

PubMed

Gao, Yong-Jing; Zhang, Ling; Samad, Omar Abdel; Suter, Marc R; Yasuhiko, Kawasaki; Xu, Zhen-Zhong; Park, Jong-Yeon; Lind, Anne-Li; Ma, Qiufu; Ji, Ru-Rong

2009-04-01

Our previous study showed that activation of c-jun-N-terminal kinase (JNK) in spinal astrocytes plays an important role in neuropathic pain sensitization. We further investigated how JNK regulates neuropathic pain. In cultured astrocytes, tumor necrosis factor alpha (TNF-alpha) transiently activated JNK via TNF receptor-1. Cytokine array indicated that the chemokine CCL2/MCP-1 (monocyte chemoattractant protein-1) was strongly induced by the TNF-alpha/JNK pathway. MCP-1 upregulation by TNF-alpha was dose dependently inhibited by the JNK inhibitors SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one) and D-JNKI-1. Spinal injection of TNF-alpha produced JNK-dependent pain hypersensitivity and MCP-1 upregulation in the spinal cord. Furthermore, spinal nerve ligation (SNL) induced persistent neuropathic pain and MCP-1 upregulation in the spinal cord, and both were suppressed by D-JNKI-1. Remarkably, MCP-1 was primarily induced in spinal cord astrocytes after SNL. Spinal administration of MCP-1 neutralizing antibody attenuated neuropathic pain. Conversely, spinal application of MCP-1 induced heat hyperalgesia and phosphorylation of extracellular signal-regulated kinase in superficial spinal cord dorsal horn neurons, indicative of central sensitization (hyperactivity of dorsal horn neurons). Patch-clamp recordings in lamina II neurons of isolated spinal cord slices showed that MCP-1 not only enhanced spontaneous EPSCs but also potentiated NMDA- and AMPA-induced currents. Finally, the MCP-1 receptor CCR2 was expressed in neurons and some non-neuronal cells in the spinal cord. Together, we have revealed a previously unknown mechanism of MCP-1 induction and action. MCP-1 induction in astrocytes after JNK activation contributes to central sensitization and neuropathic pain facilitation by enhancing excitatory synaptic transmission. Inhibition of the JNK/MCP-1 pathway may provide a new therapy for neuropathic pain management.

Leflunomide or A77 1726 protect from acetaminophen-induced cell injury through inhibition of JNK-mediated mitochondrial permeability transition in immortalized human hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Latchoumycandane, Calivarathan; Seah, Quee Ming; Tan, Rachel C.H.

2006-11-15

Leflunomide, a disease-modifying anti-rheumatic drug, protects against T-cell-mediated liver injury by poorly understood mechanisms. The active metabolite of leflunomide, A77 1726 (teriflunomide) has been shown to inhibit stress-activated protein kinases (JNK pathway), which are key regulators of mitochondria-mediated cell death. Therefore, we hypothesized that leflunomide may protect from drugs that induce the mitochondrial permeability transition (mPT) by blocking the JNK signaling pathway. To this end, we exposed cultured immortalized human hepatocytes (HC-04) to the standard protoxicant drug acetaminophen (APAP), which induces CsA-sensitive mPT-mediated cell death. We determined the effects of leflunomide on the extent of APAP-induced hepatocyte injury and themore » upstream JNK-mediated mitochondrial signaling pathways. We found that leflunomide or A77 1726 concentration-dependently protected hepatocytes from APAP (1 mM)-induced mitochondrial permeabilization and lethal cell injury. This was not due to proximal inhibition of CYP-catalyzed APAP bioactivation to its thiol-reactive metabolite. Instead, we demonstrate that leflunomide (20 {mu}M) inhibited the APAP-induced early (3 h) activation (phosphorylation) of JNK1/2, thus inhibiting phosphorylation of the anti-apoptotic protein Bcl-2 and preventing P-Bcl-2-mediated induction of the mPT. This greatly attenuated mitochondrial cytochrome c release, which we used as a marker for mitochondrial permeabilization. The specific JNK2 inhibitor SP600125 similarly protected from APAP-induced cell death. In conclusion, these findings are consistent with our hypothesis that leflunomide protects from protoxicant-induced hepatocyte injury by inhibiting JNK signaling and preventing mPT induction.« less

Interleukin-1 Acts via the JNK-2 Signaling Pathway to Induce Aggrecan Degradation by Human Chondrocytes.

PubMed

Ismail, Heba M; Yamamoto, Kazuhiro; Vincent, Tonia L; Nagase, Hideaki; Troeberg, Linda; Saklatvala, Jeremy

2015-07-01

Aggrecan enables articular cartilage to bear load and resist compression. Aggrecan loss occurs early in osteoarthritis and rheumatoid arthritis and can be induced by inflammatory cytokines such as interleukin-1 (IL-1). IL-1 induces cleavage of specific aggrecans characteristic of the ADAMTS proteinases. The aim of this study was to identify the intracellular signaling pathways by which IL-1 causes aggrecan degradation by human chondrocytes and to investigate how aggrecanase activity is controlled by chondrocytes. We developed a cell-based assay combining small interfering RNA (siRNA)-induced knockdown with aggrecan degradation assays. Human articular chondrocytes were overlaid with bovine aggrecan after transfection with siRNAs against molecules of the IL-1 signaling pathway. After IL-1 stimulation, released aggrecan fragments were detected with AGEG and ARGS neoepitope antibodies. Aggrecanase activity and tissue inhibitor of metalloproteinases 3 levels were measured by enzyme-linked immunosorbent assay. Low-density lipoprotein receptor-related protein 1 (LRP-1) shedding was analyzed by Western blotting. ADAMTS-5 is a major aggrecanase in human chondrocytes, regulating aggrecan degradation in response to IL-1. The tumor necrosis factor receptor-associated 6 (TRAF-6)/transforming growth factor β-activated kinase 1 (TAK-1)/MKK-4 signaling axis is essential for IL-1-induced aggrecan degradation, while NF-κB is not. Of the 3 MAPKs (ERK, p38, and JNK), only JNK-2 showed a significant role in aggrecan degradation. Chondrocytes constitutively secreted aggrecanase, which was continuously endocytosed by LRP-1, keeping the extracellular level of aggrecanase low. IL-1 induced aggrecanase activity in the medium in a JNK-2-dependent manner, possibly by reducing aggrecanase endocytosis, because IL-1 caused JNK-2-dependent shedding of LRP-1. The signaling axis TRAF-6/TAK-1/MKK-4/JNK-2 mediates IL-1-induced aggrecanolysis. The level of aggrecanase is controlled by its

The Anti-Apoptotic and Cardioprotective Effects of Salvianolic Acid A on Rat Cardiomyocytes following Ischemia/Reperfusion by DUSP-Mediated Regulation of the ERK1/2/JNK Pathway

PubMed Central

Chen, Qiuping; Zhu, Shasha; Liu, Yang; Pan, Defeng; Chen, Xiaohu; Li, Dongye

2014-01-01

The purpose of this study was to observe the effects of salvianolic acid A (SAA) pretreatment on the myocardium during ischemia/reperfusion (I/R) and to illuminate the interrelationships among dual specificity protein phosphatase (DUSP) 2/4/16, ERK1/2 and JNK pathways during myocardial I/R, with the ultimate goal of elucidating how SAA exerts cardioprotection against I/R injury (IRI). Wistar rats were divided into the following six groups: control group (CON), I/R group, SAA+I/R group, ERK1/2 inhibitor PD098059+I/R group (PD+I/R), PD+SAA+I/R group, and JNK inhibitor SP600125+I/R group (SP+I/R). The cardioprotective effects of SAA on the myocardium during I/R were investigated with a Langendorff device. Heart rate (HR), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), maximum rate of ventricular pressure rise and fall (±dp/dtmax), myocardial infarction areas (MIA), lactate dehydrogenase (LDH), and cardiomyocytes apoptosis were monitored. To determine the crosstalk betwee JNK and ERK1/2 via DUSP2/4/16 with SAA pretreatment, siRNA-DUSP2/4/16 were performed. The expression levels of Bcl-2, Bax, caspase 3, p-JNK, p-ERK1/2 and DUSP2/4/16 in cardiomyocytes were assayed by Western blot. Our results showed that LDH, MIA and cell apoptosis were decreased, and various parameters of heart function were improved by SAA pretreatment and SP application. In the I/R group, the expression levels of p-ERK1/2 and DUSP4/16 were not significantly different compared with the CON group, however, the protein expression levels of p-ERK1/2, Bcl-2 and DUSP4/16 were higher, while p-JNK, Bax, caspase 3 and DUSP2 levels were reduced among the SAA+I/R, PD+SAA+I/R and SP+I/R groups. The above indices were not significantly different between the SAA+I/R and SP+I/R groups. Compared with the SAA+I/R group, p-ERK1/2 was increased and p-JNK was decreased in the SAA+si-DUSP2+I/R, however, p-ERK was downregulated and p-JNK was upregulated in SAA+si-DUSP4+I

Gremlin 2 promotes differentiation of embryonic stem cells to atrial fate by activation of the JNK signaling pathway

PubMed Central

Tanwar, Vineeta; Bylund, Jeffery B.; Hu, Jianyong; Yan, Jingbo; Walthall, Joel M.; Mukherjee, Amrita; Heaton, William H.; Wang, Wen-Der; Potet, Franck; Rai, Meena; Kupershmidt, Sabina; Knapik, Ela W.; Hatzopoulos, Antonis K.

2014-01-01

The Bone Morphogenetic Protein antagonist Gremlin 2 (Grem2) is required for atrial differentiation and establishment of cardiac rhythm during embryonic development. A human Grem2 variant has been associated with familial atrial fibrillation, suggesting that abnormal Grem2 activity causes arrhythmias. However, it is not known how Grem2 integrates into signaling pathways to direct atrial cardiomyocyte differentiation. Here, we demonstrate that Grem2 expression is induced concurrently with the emergence of cardiovascular progenitor cells during differentiation of mouse embryonic (ES) stem cells. Grem2 exposure enhances the cardiogenic potential of ES cells by ~20–120 fold, preferentially inducing genes expressed in atrial myocytes such as Myl7, Nppa and Sarcolipin. We show that Grem2 acts upstream to upregulate pro-atrial transcriptional factors CoupTFII and Hey1 and downregulate atrial fate repressors Irx4 and Hey2. The molecular phenotype of Grem2-induced atrial cardiomyocytes was further supported by induction of ion channels encoded by Kcnj3, Kcnj5, and Cacna1D genes and establishment of atrial-like action potentials shown by electrophysiological recordings. We show that promotion of atrial-like cardiomyocyte is specific to the Gremlin subfamily of BMP antagonists. Grem2 pro-atrial differentiation activity is conveyed by non-canonical BMP signaling through phosphorylation of JNK and can be reversed by specific JNK inhibitors, but not by dorsomorphin, an inhibitor of canonical BMP signaling. Taken together, our data provide novel mechanistic insights into atrial cardiomyocyte differentiation from pluripotent stem cells and will assist the development of future approaches to study and treat arrhythmias. PMID:24648383

Endophilin-1 regulates blood-brain barrier permeability via EGFR-JNK signaling pathway.

PubMed

Chen, Lin; Liu, Wenjing; Wang, Ping; Xue, Yixue; Su, Qingjie; Zeng, Chaosheng; Shang, Xiuli

2015-05-05

Endophilin-1 (Endo1), a multifunctional protein, is essential for synaptic vesicle endocytosis. However, the role and mechanism of endophilin-1 in blood-brain barrier (BBB) function are still unclear. This study was performed to determine whether endophilin-1 regulated BBB permeability via the EGFR-JNK signaling pathway. In the present study, we found that endophilin-1 over-expression in human cerebral microvascular endothelial cell (hCMEC/D3) increased BBB permeability and meanwhile reduced the expression levels of epidermal growth factor receptor (EGFR), phosphorylated c-Jun N-terminal kinase (p-JNK). While endophilin-1 knockdown led to the contrary results. After JNK inhibitor SP600125 was administered to the endophilin-1 silenced hCMEC/D3 cells, the transendothelial electrical resistance (TEER) value was decreased and the permeability coefficient values to 4kDa and 40kDa FITC-dextran were increased. Results observed by Transmission electron microscopy (TEM) showed that tight junctions (TJs) were opened. Moreover, immunofluorescence and Western blot assays revealed the discontinuous distribution of TJ-associated proteins ZO-1, occludin on cell-cell boundaries and a significant decrease in protein expressing levels. Therefore, these results indicated that endophilin-1 positively regulated BBB permeability via the EGFR-JNK signaling pathway in hCMEC/D3 cells, which would provide an experimental basis for further research on endophilin-1 mediated the opening of BBB. Copyright © 2015 Elsevier B.V. All rights reserved.

Reactive oxygen species mediate nitric oxide production through ERK/JNK MAPK signaling in HAPI microglia after PFOS exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Cheng; Nie, Xiaoke; Zhang, Yan

2015-10-15

Perfluorooctane sulfonate (PFOS), an emerging persistent contaminant that is commonly encountered during daily life, has been shown to exert toxic effects on the central nervous system (CNS). However, the molecular mechanisms underlying the neurotoxicity of PFOS remain largely unknown. It has been widely acknowledged that the inflammatory mediators released by hyper-activated microglia play vital roles in the pathogenesis of various neurological diseases. In the present study, we examined the impact of PFOS exposure on microglial activation and the release of proinflammatory mediators, including nitric oxide (NO) and reactive oxidative species (ROS). We found that PFOS exposure led to concentration-dependent NOmore » and ROS production by rat HAPI microglia. We also discovered that there was rapid activation of the ERK/JNK MAPK signaling pathway in the HAPI microglia following PFOS treatment. Moreover, the PFOS-induced iNOS expression and NO production were attenuated after the inhibition of ERK or JNK MAPK by their corresponding inhibitors, PD98059 and SP600125. Interestingly, NAC, a ROS inhibitor, blocked iNOS expression, NO production, and activation of ERK and JNK MAPKs, which suggested that PFOS-mediated microglial NO production occurs via a ROS/ERK/JNK MAPK signaling pathway. Finally, by exposing SH-SY5Y cells to PFOS-treated microglia-conditioned medium, we demonstrated that NO was responsible for PFOS-mediated neuronal apoptosis. - Highlights: • PFOS exposure induced expression of iNOS and production of NO in HAPI microglia. • PFOS induced the production of ROS in HAPI microglia. • ERK/JNK MAPK pathways were activated following PFOS exposure in HAPI microglia. • NO released by HAPI microglia participated in the apoptosis of SH-SY5Y cells.« less

Endotoxin-activated microglia injure brain derived endothelial cells via NF-κB, JAK-STAT and JNK stress kinase pathways

PubMed Central

2011-01-01

Background We previously showed that microglia damage blood brain barrier (BBB) components following ischemic brain insults, but the underlying mechanism(s) is/are not well known. Recent work has established the contribution of toll-like receptor 4 (TLR4) activation to several brain pathologies including ischemia, neurodegeneration and sepsis. The present study established the requirement of microglia for lipopolysaccharide (LPS) mediated endothelial cell death, and explored pathways involved in this toxicity. LPS is a classic TLR4 agonist, and is used here to model aspects of brain conditions where TLR4 stimulation occurs. Methods/Results In monocultures, LPS induced death in microglia, but not brain derived endothelial cells (EC). However, LPS increased EC death when cocultured with microglia. LPS led to nitric oxide (NO) and inducible NO synthase (iNOS) induction in microglia, but not in EC. Inhibiting microglial activation by blocking iNOS and other generators of NO or blocking reactive oxygen species (ROS) also prevented injury in these cocultures. To assess the signaling pathway(s) involved, inhibitors of several downstream TLR-4 activated pathways were studied. Inhibitors of NF-κB, JAK-STAT and JNK/SAPK decreased microglial activation and prevented cell death, although the effect of blocking JNK/SAPK was rather modest. Inhibitors of PI3K, ERK, and p38 MAPK had no effect. Conclusions We show that LPS-activated microglia promote BBB disruption through injury to endothelial cells, and the specific blockade of JAK-STAT, NF-κB may prove to be especially useful anti-inflammatory strategies to confer cerebrovascular protection. PMID:21385378

Vitamin K2 Induces Mitochondria-Related Apoptosis in Human Bladder Cancer Cells via ROS and JNK/p38 MAPK Signal Pathways

PubMed Central

Duan, Fengsen; Yu, Yuejin; Guan, Rijian; Xu, Zhiliang; Liang, Huageng; Hong, Ling

2016-01-01

The effects of vitamin K2 on apoptosis in a variety of cancer cells have been well established in previous studies. However, the apoptotic effect of vitamin K2 on bladder cancer cells has not been evaluated. The aim of this study is to examine the apoptotic activity of Vitamin K2 in bladder cancer cells and investigate the underlying mechanism. In this study, Vitamin K2 induced apoptosis in bladder cancer cells through mitochondria pathway including loss of mitochondria membrane potential, cytochrome C release and caspase-3 cascade. Furthermore, the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK was detected in Vitamin K2-treated cells and both SP600125 (an inhibitor of JNK) and SB203580 (an inhibitor of p38 MAPK) completely abolished the Vitamin K2-induced apoptosis and loss of mitochondria membrane potential. Moreover, the generation of reactive oxygen species (ROS) was detected in bladder cancer cells, upon treatment of vitamin K2 and the anti-oxidant N-acetyl cysteine (NAC) almost blocked the Vitamin K2-triggered apoptosis, loss of mitochondria membrane potential and activation of JNK and p38 MAPK. Taken together, these findings revealed that Vitamin K2 induces apoptosis in bladder cancer cells via ROS-mediated JNK/p38 MAPK and Mitochondrial pathways. PMID:27570977

Vitamin K2 Induces Mitochondria-Related Apoptosis in Human Bladder Cancer Cells via ROS and JNK/p38 MAPK Signal Pathways.

PubMed

Duan, Fengsen; Yu, Yuejin; Guan, Rijian; Xu, Zhiliang; Liang, Huageng; Hong, Ling

2016-01-01

The effects of vitamin K2 on apoptosis in a variety of cancer cells have been well established in previous studies. However, the apoptotic effect of vitamin K2 on bladder cancer cells has not been evaluated. The aim of this study is to examine the apoptotic activity of Vitamin K2 in bladder cancer cells and investigate the underlying mechanism. In this study, Vitamin K2 induced apoptosis in bladder cancer cells through mitochondria pathway including loss of mitochondria membrane potential, cytochrome C release and caspase-3 cascade. Furthermore, the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK was detected in Vitamin K2-treated cells and both SP600125 (an inhibitor of JNK) and SB203580 (an inhibitor of p38 MAPK) completely abolished the Vitamin K2-induced apoptosis and loss of mitochondria membrane potential. Moreover, the generation of reactive oxygen species (ROS) was detected in bladder cancer cells, upon treatment of vitamin K2 and the anti-oxidant N-acetyl cysteine (NAC) almost blocked the Vitamin K2-triggered apoptosis, loss of mitochondria membrane potential and activation of JNK and p38 MAPK. Taken together, these findings revealed that Vitamin K2 induces apoptosis in bladder cancer cells via ROS-mediated JNK/p38 MAPK and Mitochondrial pathways.

Ciclopirox induces autophagy through reactive oxygen species-mediated activation of JNK signaling pathway

PubMed Central

Zhou, Hongyu; Shen, Tao; Shang, Chaowei; Luo, Yan; Liu, Lei; Yan, Juming; Li, Yan; Huang, Shile

2014-01-01

Ciclopirox olamine (CPX), a fungicide, has been demonstrated as a potential anticancer agent. However, the underlying anticancer mechanism is not well understood. Here, we found that CPX induced autophagy in human rhabdomyosarcoma (Rh30 and RD) cells. It appeared that CPX-induced autophagy was attributed to induction of reactive oxygen species (ROS), as N-acetyl-L-cysteine (NAC), a ROS scavenger and antioxidant, prevented this process. Furthermore, we observed that CPX induced activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 MAPK, which was also blocked by NAC. However, only inhibition of JNK (with SP600125) or expression of dominant negative c-Jun partially prevented CPX-induced autophagy, indicating that ROS-mediated activation of JNK signaling pathway contributed to CPX-induced autophagy. Of interest, inhibition of autophagy by chloroquine (CQ) enhanced CPX-induced cell death, indicating that CPX-induced autophagy plays a pro-survival role in human rhabdomyosarcoma cells. Our finding suggests that the combination with autophagy inhibitors may be a novel strategy in potentiating the anticancer activity of CPX for treatment of rhabdomyosarcoma. PMID:25294812

Curcumol suppresses RANKL-induced osteoclast formation by attenuating the JNK signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Mingxiang, E-mail: yu.mingxiang@zs-hospital.sh.cn; Chen, Xianying; Lv, Chaoyang

Highlights: • Curcumol suppresses osteoclasts differentiation in vitro. • Curcumol impairs JNK/AP-1 signaling pathway. • Curcumol may be used for treating osteoclast related diseases. - Abstract: Osteoclasts, derived from hemopoietic progenitors of the monocyte/macrophage lineage, have a unique role in bone resorption, and are considered a potential therapeutic target in the treatment of such pathologic bone diseases as osteoporosis, rheumatoid arthritis, and periodontitis. In the present study, we demonstrate that curcumol, one of the major components of the essential oil of Rhizoma Curcumae, exhibits an inhibitory effect on receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclast differentiation with bothmore » bone marrow-derived macrophages and RAW264.7 cells in a dose-dependent manner. In addition, RANKL-induced mRNA expression of osteoclast-specific genes, such as tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K, is prominently reduced in the presence of curcumol. Furthermore, the molecular mechanism of action was investigated, and curcumol inhibited osteoclastogenesis by specifically impairing RANKL-induced c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) signaling, which was further identified in rescue studies by means of anisomycin, a JNK signaling-specific activator. Taken together, these findings suggest that curcumol suppresses RANKL-induced osteoclast differentiation through the JNK/AP-1 signaling pathway, and may be useful as a therapeutic treatment for bone resorption-associated diseases.« less

Synthesis and optimization of thiadiazole derivatives as a novel class of substrate competitive c-Jun N-terminal kinase inhibitors

PubMed Central

De, Surya K.; Chen, Vida; Stebbins, John L.; Chen, Li-Hsing; Cellitti, Jason F.; Machleidt, Thomas; Barile, Elisa; Riel-Mehan, Megan; Dahl, Russell; Yang, Li; Emdadi, Aras; Murphy, Ria; Pellecchia, Maurizio

2009-01-01

A series of thiadiazole derivatives has been designed as potential allosteric, substrate competitive inhibitors of the protein kinase JNK. We report on the synthesis, characterization and evaluation of a series of compounds that resulted in the identification of potent and selective JNK inhibitors targeting its JIP-1 docking site. PMID:20045647

The mechanism by which MEK/ERK regulates JNK and p38 activity in polyamine depleted IEC-6 cells during apoptosis

PubMed Central

Bavaria, Mitul N.; Jin, Shi; Ray, Ramesh M.; Johnson, Leonard R.

2014-01-01

Polyamine-depletion inhibited apoptosis by activating ERK1/2, while, preventing JNK1/2 activation. MKP-1 knockdown by SiRNA increased ERK1/2, JNK1/2, and p38 phosphorylation and apoptosis. Therefore, we predicted that polyamines might regulate MKP1 via MEK/ERK and thereby apoptosis. We examined the role of MEK/ERK in the regulation of MKP1 and JNK, and p38 activities and apoptosis. Inhibition of MKP-1 activity with a pharmacological inhibitor, sanguinarine (SA), increased JNK1/2, p38, and ERK1/2 activities without causing apoptosis. However, pre-activation of these kinases by SA significantly increased camptothecin (CPT)-induced apoptosis suggesting different roles for MAPKs during survival and apoptosis. Inhibition of MEK1 activity prevented the expression of MKP-1 protein and augmented CPT-induced apoptosis, which correlated with increased activities of JNK1/2, caspases, and DNA fragmentation. Polyamine depleted cells had higher levels of MKP-1 protein and decreased JNK1/2 activity and apoptosis. Inhibition of MEK1 prevented MKP-1 expression and increased JNK1/2 and apoptosis. Phospho-JNK1/2, phospho-ERK2, MKP-1, and the catalytic subunit of protein phosphatase 2A (PP2Ac) formed a complex in response to TNF/CPT. Inactivation of PP2Ac had no effect on the association of MKP-1 and JNK1. However, inhibition of MKP-1 activity decreased the formation of the MKP-1, PP2Ac and JNK complex. Following inhibition by SA, MKP-1 localized in the cytoplasm, while basal and CPT-induced MKP-1 remained in the nuclear fraction. These results suggest that nuclear MKP-1 translocates to the cytoplasm, binds phosphorylated JNK and p38 resulting in dephosphorylation and decreased activity. Thus, MEK/ERK activity controls the levels of MKP-1 and, thereby, regulates JNK activity in polyamine-depleted cells. PMID:24253595

Gefitinib enhances human colon cancer cells to TRAIL-induced apoptosis of via autophagy- and JNK-mediated death receptors upregulation.

PubMed

Chen, Lei; Meng, Yue; Guo, Xiaoqing; Sheng, Xiaotong; Tai, Guihua; Zhang, Fenglei; Cheng, Hairong; Zhou, Yifa

2016-11-01

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a potent cancer cell-specific apoptosis-inducing cytokine with little toxicity to most normal cells. Here, we report that gefitinib and TRAIL in combination produce a potent synergistic effect on TRAIL-sensitive human colon cancer HCT116 cells and an additive effect on TRAIL-resistant HT-29 cells. Interestingly, gefitinib increases the expression of cell surface receptors DR4 and DR5, possibly explaining the synergistic effect. Knockdown of DR4 and DR5 by siRNA significantly decreases gefitinib- and TRAIL-mediated cell apoptosis, supporting this idea. Because the inhibition of gefitinib-induced autophagy by 3-MA significantly decreases DR4 and DR5 upregulation, as well as reduces gefitinib- and TRAIL-induced apoptosis, we conclude that death receptor upregulation is autophagy mediated. Furthermore, our results indicate that death receptor expression may also be regulated by JNK activation, because pre-treatment of cells with JNK inhibitor SP600125 significantly decreases gefitinib-induced death receptor upregulation. Interestingly, SP600125 also inhibits the expression CHOP, yet CHOP has no impact on death receptor expressions. We also find here that phosphorylation of Akt and ERK might also be required for TRAIL sensitization. In summary, our results indicate that gefitinib effectively enhances TRAIL-induced apoptosis, likely via autophagy and JNK- mediated death receptor expression and phosphorylation of Akt and ERK.

Jellyfish collagen stimulates production of TNF-α and IL-6 by J774.1 cells through activation of NF-κB and JNK via TLR4 signaling pathway.

PubMed

Putra, Agus Budiawan Naro; Nishi, Kosuke; Shiraishi, Ryusuke; Doi, Mikiharu; Sugahara, Takuya

2014-03-01

We previously reported that jellyfish collagen stimulates both the acquired and innate immune responses. In the acquired immune response, jellyfish collagen enhanced immunoglobulin production by lymphocytes in vitro and in vivo. Meanwhile, in the innate immune response jellyfish collagen promoted cytokine production and phagocytotic activity of macrophages. The facts that jellyfish collagen plays several potential roles in stimulating cytokine production by macrophages have further attracted us to uncover its mechanisms. We herein describe that the cytokine production-stimulating activity of jellyfish collagen was canceled by a Toll-like receptor 4 (TLR4) inhibitor. Moreover, jellyfish collagen stimulated phosphorylation of inhibitor of κBα (IκBα), promoted the translocation of nucleus factor-κB (NF-κB), and activated c-Jun N-terminal kinase (JNK). A JNK inhibitor also abrogated the cytokine production-stimulating activity of jellyfish collagen. These results suggest that jellyfish collagen may facilitate cytokine production by macrophages through activation of NF-κB and JNK via the TLR4 signaling pathways. Copyright © 2013 Elsevier Ltd. All rights reserved.

UVC-induced apoptosis in Dubca cells is independent of JNK activation and p53{sup Ser-15} phosphorylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chathoth, Shahanas; Thayyullathil, Faisal; Hago, Abdulkader

2009-06-12

Ultraviolet C (UVC) irradiation in mammalian cell lines activates a complex signaling network that leads to apoptosis. By using Dubca cells as a model system, we report the presence of a UVC-induced apoptotic pathway that is independent of c-Jun N-terminal kinases (JNKs) activation and p53 phosphorylation at Ser{sup 15}. Irradiation of Dubca cells with UVC results in a rapid JNK activation and phosphorylation of its downstream target c-Jun, as well as, phosphorylation of activating transcription factor 2 (ATF2). Pre-treatment with JNK inhibitor, SP600125, inhibited UVC-induced c-Jun phosphorylation without preventing UVC-induced apoptosis. Similarly, inhibition of UVC-induced p53 phosphorylation did not preventmore » Dubca cell apoptosis, suggesting that p53{sup Ser-15} phosphorylation is not associated with UVC-induced apoptosis signaling. The pan-caspase inhibitor z-VAD-fmk inhibited UVC-induced PARP cleavage, DNA fragmentation, and ultimately apoptosis of Dubca cells. Altogether, our study clearly indicates that UVC-induced apoptosis is independent of JNK and p53 activation in Dubca cells, rather, it is mediated through a caspase dependent pathway. Our findings are not in line with the ascribed critical role for JNKs activation, and downstream phosphorylation of targets such as c-Jun and ATF2 in UVC-induced apoptosis.« less

Both internalization and AIP1 association are required for tumor necrosis factor receptor 2-mediated JNK signaling.

PubMed

Ji, Weidong; Li, Yonghao; Wan, Ting; Wang, Jing; Zhang, Haifeng; Chen, Hong; Min, Wang

2012-09-01

The proinflammtory cytokine tumor necrosis factor (TNF), primarily via TNF receptor 1 (TNFR1), induces nuclear factor-κB (NF-κB)-dependent cell survival, and c-Jun N-terminal kinase (JNK) and caspase-dependent cell death, regulating vascular endothelial cell (EC) activation and apoptosis. However, signaling by the second receptor, TNFR2, is poorly understood. The goal of this study was to dissect how TNFR2 mediates NF-κB and JNK signaling in vascular EC, and its relevance to in vivo EC function. We show that TNFR2 contributes to TNF-induced NF-κB and JNK signaling in EC as TNFR2 deletion or knockdown reduces the TNF responses. To dissect the critical domains of TNFR2 that mediate the TNF responses, we examine the activity of TNFR2 mutant with a specific deletion of the TNFR2 intracellular region, which contains conserved domain I, domain II, domain III, and 2 TNFR-associated factor-2-binding sites. Deletion analyses indicate that different sequences on TNFR2 have distinct roles in NF-κB and JNK activation. Specifically, deletion of the TNFR-associated factor-2-binding sites (TNFR2-59) diminishes the TNFR2-mediated NF-κB, but not JNK activation; whereas, deletion of domain II or domain III blunts TNFR2-mediated JNK but not NF-κB activation. Interestingly, we find that the TNFR-associated factor-2-binding sites ensure TNFR2 on the plasma membrane, but the di-leucine LL motif within the domain II and aa338-355 within the domain III are required for TNFR2 internalization as well as TNFR2-dependent JNK signaling. Moreover, domain III of TNFR2 is responsible for association with ASK1-interacting protein-1, a signaling adaptor critical for TNF-induced JNK signaling. While TNFR2 containing the TNFR-associated factor-2-binding sites prevents EC cell death, a specific activation of JNK without NF-κB activation by TNFR2-59 strongly induces caspase activation and EC apoptosis. Our data reveal that both internalization and ASK1-interacting protein-1 association are

Dihydroartemisinin induces endothelial cell anoikis through the activation of the JNK signaling pathway

PubMed Central

Zhang, Jiao; Guo, Ling; Zhou, Xia; Dong, Fengyun; Li, Liqun; Cheng, Zuowang; Xu, Yinghua; Liang, Jiyong; Xie, Qi; Liu, Ju

2016-01-01

Angiogenesis is required for the growth and metastasis of solid tumors. The anti-malarial agent dihydroartemisinin (DHA) demonstrates potent anti-angiogenic activity, but the underlying molecular mechanisms are not yet fully understood. During the process of angiogenesis, endothelial cells migrating from existing capillaries may undergo programmed cell death after detaching from the extracellular matrix, a process that is defined as anchorage-dependent apoptosis or anoikis. In the present study, DHA-induced cell death was compared in human umbilical vein endothelial cells (HUVECs) cultured in suspension and attached to culture plates. In suspended HUVECs, the cell viability was decreased and apoptosis was increased with the treatment of 50 µM DHA for 5 h, while the same treatment did not affect the attached HUVECs. In addition, 50 µM DHA increased the phosphorylation of c-Jun N-terminal kinase (JNK) in suspended HUVECs, but not in attached HUVECs, for up to 5 h of treatment. The JNK inhibitor, SP600125, reversed DHA-induced cell death in suspended HUVECs, suggesting that the JNK pathway may mediate DHA-induced endothelial cell anoikis. The data from the present study indicates a novel mechanism for understanding the anti-angiogenic effects of DHA, which may be used as a component for chemotherapy. PMID:27602117

Anti-Inflammatory Effects and Joint Protection in Collagen-Induced Arthritis after Treatment with IQ-1S, a Selective c-Jun N-Terminal Kinase Inhibitor

PubMed Central

Schepetkin, Igor A.; Kirpotina, Liliya N.; Hammaker, Deepa; Kochetkova, Irina; Khlebnikov, Andrei I.; Lyakhov, Sergey A.; Firestein, Gary S.

2015-01-01

c-Jun N-terminal kinases (JNKs) participate in many physiologic and pathologic processes, including inflammatory diseases. We recently synthesized the sodium salt of IQ-1S (11H-indeno[1,2-b]quinoxalin-11-one oxime) and demonstrated that it is a high-affinity JNK inhibitor and inhibits murine delayed-type hypersensitivity. Here we show that IQ-1S is highly specific for JNK and that its neutral form is the most abundant species at physiologic pH. Molecular docking of the IQ-1S syn isomer into the JNK1 binding site gave the best pose, which corresponded to the position of cocrystallized JNK inhibitor SP600125 (1,9-pyrazoloanthrone). Evaluation of the therapeutic potential of IQ-1S showed that it inhibited matrix metalloproteinase 1 and 3 gene expression induced by interleukin-1β in human fibroblast-like synoviocytes and significantly attenuated development of murine collagen-induced arthritis (CIA). Treatment with IQ-1S either before or after induction of CIA resulted in decreased clinical scores, and joint sections from IQ-1S–treated CIA mice exhibited only mild signs of inflammation and minimal cartilage loss compared with those from control mice. Collagen II–specific antibody responses were also reduced by IQ-1S treatment. By contrast, the inactive ketone derivative 11H-indeno[1,2-b]quinoxalin-11-one had no effect on CIA clinical scores or collagen II–specific antibody titers. IQ-1S treatment also suppressed proinflammatory cytokine and chemokine levels in joints and lymph node cells. Finally, treatment with IQ-1S increased the number of Foxp3+CD4+CD25+ regulatory T cells in lymph nodes. Thus, IQ-1S can reduce inflammation and cartilage loss associated with CIA and can serve as a small-molecule modulator for mechanistic studies of JNK function in rheumatoid arthritis. PMID:25784649

Eicosapentaenoic acid (EPA) induced apoptosis in HepG2 cells through ROS–Ca{sup 2+}–JNK mitochondrial pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yuanyuan; Han, Lirong; Qi, Wentao

Highlights: • EPA evoked ROS formation, [Ca{sup 2+}]{sub c} accumulation, the opening of MPTP and the phosphorylation of JNK. • EPA-induced [Ca{sup 2+}]{sub c} elevation was depended on production of ROS. • EPA-induced ROS generation, [Ca{sup 2+}]{sub c} increase, and JNK activated caused MPTP opening. • The apoptosis induced by EPA was related to release of cytochrome C through the MPTP. • EPA induced HepG2 cells apoptosis through ROS–Ca{sup 2+}–JNK mitochondrial pathways. - Abstract: Eicosapentaenoic acid (EPA), a well-known dietary n−3 PUFAS, has been considered to inhibit proliferation of tumor cells. However, the molecular mechanism related to EPA-induced liver cancermore » cells apoptosis has not been reported. In this study, we investigated the effect of EPA on HepG2 cells proliferation and apoptosis mechanism through mitochondrial pathways. EPA inhibited proliferation of HepG2 cells in a dose-dependent manner and had no significant effect on the cell viability of humor normal liver L-02 cells. It was found that EPA initially evoked ROS formation, leading to [Ca{sup 2+}]{sub c} accumulation and the mitochondrial permeability transition pore (MPTP) opening; EPA-induced HepG2 cells apoptosis was inhibited by N-acetylcysteine (NAC, an inhibitor of ROS), 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, a chelator of calcium) and CsA (inhibitor of MPTP). The relationship between ROS production, the increase of cytoplasmic Ca and MPTP opening was detected. It seems that ROS may act as an upstream regulator of EPA-induced [Ca{sup 2+}]{sub c} generation, moreover, generation of ROS, overload of mitochondrial [Ca{sup 2+}]{sub c}, and JNK activated cause the opening of MPTP. Western blotting results showed that EPA elevated the phosphorylation status of JNK, processes associated with the ROS generation. Simultaneously, the apoptosis induced by EPA was related to release of cytochrome C from mitochondria to cytoplasm through

Induction apoptosis of luteolin in human hepatoma HepG2 cells involving mitochondria translocation of Bax/Bak and activation of JNK

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, H.-J.; Wang, C.-J.; Kuo, H.-C.

2005-03-01

Since hepatocellular carcinoma remains a major challenging clinical problem in many parts of the world including Eastern Asia and Southern Africa, it is imperative to develop more effective chemopreventive and chemotherapy agents. Herein, we present an investigation regarding the anticancer potential of luteolin, a natural flavonoid, and the mechanism of its action in human hepatoma HepG2 cells. Using DNA fragmentation assay and nuclear staining assay, it showed that luteolin induced apoptosis of HepG2 cells. Luteolin induced the cytosolic release of cytochrome c and activated CPP32. We found that Bax and Bak translocated to mitochondria apparently, whereas Fas ligand (FasL) wasmore » unchanged after a treatment with luteolin for 3 h. In addition, it showed that c-Jun NH{sub 2}-terminal kinase (JNK) was activated after the treatment of luteolin for 3-12 h. Further investigation showed that a specific JNK inhibitor, SP600125, reduced the activation of CPP 32, the mitochondrial translocation of Bax, as well as the cytosolic release of cytochrome c that induced by luteolin. Finally, the apoptosis induced by luteolin was suppressed by a pretreatment with SP600125 via evaluating annexin V-FITC binding assay. These data suggest that luteolin induced apoptosis via mechanisms involving mitochondria translocation of Bax/Bak and activation of JNK.« less

Phloretin induces apoptosis of non-small cell lung carcinoma A549 cells via JNK1/2 and p38 MAPK pathways.

PubMed

Min, Jie; Huang, Kenan; Tang, Hua; Ding, Xinyu; Qi, Chen; Qin, Xiong; Xu, Zhifei

2015-12-01

Phloretin (Ph) existing in apples, pears and various vegetables is known to have antitumor activities in several cancer cell lines. However, little is known about its effect on human lung cancer cells. The aim of the present study was to see whether Ph could induce apoptosis of non-small cell lung cancer (NSCLC) cells, and explore the possible underlying mechanism of action. We found that Ph markedly induced cell apoptosis of NSCLC cell line A549, and inhibited the migration of A549 cells in a dose-dependent manner. The expression level of BAX, cleaved caspase-3 and -9, and degraded form of PARP was increased and Bcl-2 was decreased after Ph treatment. In addition, the phosphorylation of P38 MAPK, ERK1/2 and JNK1/2 was increased in a dose‑dependent manner in parallel with Ph treatment. Inhibition of P38 MAPK and JNK1/2 by specific inhibitors significantly abolished the Ph-induced activation of the caspase-3 and -9. In vivo tumor-suppression assay further indicated that Ph (20 mg/kg) displayed a more significant inhibitory effect on A549 xenografts in tumor growth. All these findings indicate that Ph is able to inhibit NSCLC A549 cell growth by inducing apoptosis through P38 MAPK and JNK1/2 pathways, and therefore may prove to be an adjuvant to the treatment of NSCLC.

JNK1 and JNK3 play a significant role in both neuronal apoptosis and necrosis. Evaluation based on in vitro approach using tert-butylhydroperoxide induced oxidative stress in neuro-2A cells and perturbation through 3-aminobenzamide.

PubMed

Muthaiah, Vijaya Prakash Krishnan; Michael, Felicia Mary; Palaniappan, Tamilselvi; Rajan, Sridhar Skylab; Chandrasekar, Kirubhanand; Venkatachalam, Sankar

2017-06-01

In spinal cord injury (SCI), oxidative stress in the penumbra of the injury site is a characteristic feature. The predominance of necrosis over apoptosis in the ensuing delayed cell death results in progressive waves of necrosis affecting neighboring cells and thus exaggerates the severity of the lesion. Necrosis has been classified into subtypes based on the active molecular players and parthanatos is one among them, which is characterized by the over activation of PARP1 as the pre-mitochondrial event that triggers necrosis. Parthanatos being the necrosis mode reported in SCI, we intended to study the molecular players in the elusive pre-mitochondrial events of PARP1 over activation using an in vitro model. tert-Butylhydroperoxide (tBuOOH) was reported to induce oxidative stress in various cell types including Neuro-2A cells. Using a tailored protocol, a predominantly PARP1 mediated necrotic mode of cell death was obtained in Neuro-2A cells using tBuOOH. By perturbing the progress of necrosis using 3-amniobenzamide, a known PARP1 inhibitor, it was found that JNK1 and JNK3 but not JNK2 were involved in pre-mitochondrial stages of PARP1 mediated cell death. Given that JNK1 and JNK3 play a role in apoptosis also, they may serve as common targets to counter both apoptosis and necrosis. The in vitro model used in the present study may be useful in delineating molecular mechanisms in necrosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Long Non-Coding RNA CASC2 Improves Diabetic Nephropathy by Inhibiting JNK Pathway.

PubMed

Yang, Huihui; Kan, Quan E; Su, Yong; Man, Hua

2018-06-11

It's known that long non-coding RNA CASC2 overexpression inhibit the JNK pathway in some disease models, while JNK pathway activation exacerbates diabetic nephropathy. Therefore we speculate that long non-coding RNA CASC2 can improve diabetic nephropathy by inhibiting JNK pathway. Thus, our study was carried out to investigate the involvement of CASC2 in diabetic nephropathy. We found that serum level of CASC2 was significantly lower in diabetic nephropathy patients than in normal people, and serum level of CASC2 showed no significant correlations with age, gender, alcohol consumption and smoking habits, but was correlated with course of disease. ROC curve analysis showed that serum level of CASC2 could be used to accurately predict diabetic nephropathy. Diabetes mellitus has many complications. This study also included a series of complications of diabetes, such as diabetic retinopathy, diabetic ketoacidosis, diabetic foot infections and diabetic cardiopathy, while serum level of CASC2 was specifically reduced in diabetic nephropathy. CASC2 expression level decreased, while JNK1 phosphorylation level increased in mouse podocyte cells treated with high glucose. CASC2 overexpression inhibited apoptosis of podocyte cells and reduced phosphorylation level of JNK1. We conclude that long non-coding RNA CASC2 may improve diabetic nephropathy by inhibiting JNK pathway. © Georg Thieme Verlag KG Stuttgart · New York.

Linking JNK Activity to the DNA Damage Response

PubMed Central

Picco, Vincent

2013-01-01

The activity of c-Jun N-terminal kinase (JNK) was initially described as ultraviolet- and oncogene-induced kinase activity on c-Jun. Shortly after this initial discovery, JNK activation was reported for a wider variety of DNA-damaging agents, including γ-irradiation and chemotherapeutic compounds. As the DNA damage response mechanisms were progressively uncovered, the mechanisms governing the activation of JNK upon genotoxic stresses became better understood. In particular, a recent set of papers links the physical breakage in DNA, the activation of the transcription factor NF-κB, the secretion of TNF-α, and an autocrine activation of the JNK pathway. In this review, we will focus on the pathway that is initiated by a physical break in the DNA helix, leading to JNK activation and the resultant cellular consequences. The implications of these findings will be discussed in the context of cancer therapy with DNA-damaging agents. PMID:24349633

Gadd45a, the gene induced by the mood stabilizer valproic acid, regulates neurite outgrowth through JNK and the substrate paxillin in N1E-115 neuroblastoma cells.

PubMed

Yamauchi, Junji; Miyamoto, Yuki; Murabe, Mayu; Fujiwara, Yoko; Sanbe, Atsushi; Fujita, Yuko; Murase, Shoko; Tanoue, Akito

2007-05-15

Valproic acid (VPA), a mood stabilizer and anticonvulsant, has a variety of neurotrophic functions; however, less is known about how VPA regulates neurite outgrowth. Here, using N1E-115 neuroblastoma cells as the model, we show that VPA upregulates Gadd45a to trigger activation of the downstream JNK cascade controlling neurite outgrowth. VPA induces the phosphorylation of c-Jun N-terminal kinase (JNK) and the substrate paxillin, while VPA induction of neurite outgrowth is inhibited by JNK inhibitors (SP600125 and the small JNK-binding peptide) or a paxillin construct harboring a Ser 178-to-Ala mutation at the JNK phosphorylation. Transfection of Gadd45a, acting through the effector MEKK4, leads to the phosphorylation of the JNK cascade. Conversely, knockdown of Gadd45a with siRNA reduces the effect of VPA. Taken together, these results suggest that upregulation of Gadd45a explains one of the mechanisms whereby VPA induces the neurotrophic effect, providing a new role of Gadd45a in neurite outgrowth.

Bornyl caffeate induces apoptosis in human breast cancer MCF-7 cells via the ROS- and JNK-mediated pathways

PubMed Central

Yang, Chuan-bin; Pei, Wei-jing; Zhao, Jia; Cheng, Yuan-yuan; Zheng, Xiao-hui; Rong, Jian-hui

2014-01-01

Aim: To investigate the effects of bornyl caffeate discovered in several species of plant on human breast cancer cells in vitro and the underlying mechanisms. Methods: Human breast cancer cell line MCF-7 and other tumor cell lines (T47D, HepG2, HeLa, and PC12) were tested. Cell viability was determined using MTT assay, and apoptosis was defined by monitoring the morphology of the nuclei and staining with Annexin V-FITC. Mitochondrial membrane potential (MMP) was measured using JC-1 under fluorescence microscopy. Intracellular reactive oxygen species (ROS) were assessed by flow cytometry. The expression of apoptosis-associated proteins was determined by Western blotting analysis. Results: Bornyl caffeate (10, 25, and 50 μmol/L) suppressed the viability of MCF-7 cells in dose- and time-dependent manners, but neither caffeic acid nor borneol showed cytotoxicity at a concentration of 50 μmol/L. Bornyl caffeate also exerted cytotoxicity to HepG2, Hela, T47D, and PC12 cells. Bornyl caffeate dose-dependently induced apoptosis of MCF-7 cells, increased the expression of Bax and decreased the expression of Bcl-xl, resulting in the disruption of MMP and subsequent activation of caspase-3. Moreover, bornyl caffeate triggered the formation of ROS and activated p38 and c-Jun JNK. In MCF-7 cells, the cytotoxicity of bornyl caffeate was significantly attenuated by SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), z-VAD (pan-caspase inhibitor) or the thiol antioxidant L-NAC. Conclusion: Bornyl caffeate exerts non-selective cytotoxicity against cancer cells of different origin in vitro. The compound induces apoptosis in human breast cancer MCF-7 cells via the ROS- and JNK-mediated pathways. PMID:24335836

A synthetic diosgenin primary amine derivative attenuates LPS-stimulated inflammation via inhibition of NF-κB and JNK MAPK signaling in microglial BV2 cells.

PubMed

Cai, Bangrong; Seong, Kyung-Joo; Bae, Sun-Woong; Chun, Changju; Kim, Won-Jae; Jung, Ji-Yeon

2018-06-08

Diosgenin, a precursor of steroid hormones in plants, is known to exhibit diverse pharmacological activities including anti-inflammatory properties. In this study, (3β, 25R)‑spirost‑5‑en‑3‑oxyl (2‑((2((2‑aminoethyl)amino)ethyl)amino)ethyl) carbamate (DGP), a new synthetic diosgenin derivative incorporating primary amine was used to investigate its anti-inflammatory effects and underlying mechanisms of action in lipopolysaccharide (LPS)-stimulated microglial BV2 cells. Pretreatment with DGP resulted in significant inhibition of nitric oxide (NO) synthesis, and down-regulation of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated microglial BV2 cells. In addition, DGP decreased the production of reactive oxygen species (ROS) and pro-inflammatory cytokines such as interleukin (IL)-6, IL-1β, and tumor necrosis factor alpha (TNF-α). The inhibitory effects of DGP on these inflammatory mediators in LPS-stimulated microglial BV2 cells were regulated by NF-κB signaling through blocking p65 nuclear translocation and NF-κB p65/DNA binding activity. DGP also blocked the phosphorylation of c-Jun amino-terminal kinase (JNK), but not p38 kinase or extracellular signal-regulated kinases (ERK). The NF-κB inhibitor JSH-23 and JNK-specific inhibitor SP600125 significantly decreased NO production and IL-6 release in LPS-stimulated BV2 cells, respectively. The overall results demonstrate that DGP has anti-inflammatory effects on LPS-stimulated BV2 cells via inhibition of NF-κB and JNK activation, suggesting that DGP is a potential prophylactic agent in various neurodegenerative disorders. Copyright © 2018. Published by Elsevier B.V.

Cav-1 promotes atherosclerosis by activating JNK-associated signaling.

PubMed

Wang, Dong-Xia; Pan, Yong-Quan; Liu, Bing; Dai, Li

2018-05-07

The objective of the study is to calculate the role and underlying the molecular mechanisms of caveolin-1 (Cav-1) in atherosclerosis (AS). Cav-1 was mainly expressed in the endothelial cells of atherosclerotic lesions in both human patients and apolipoprotein E deficient (ApoE -/- ) mice. Cav-1 deficiency (Cav-1 -/- ) attenuated high-fat diet (HFD)-induced atherosclerotic lesions in ApoE -/- mice, supported by the reduced aortic plaques. Cav-1 -/- reduced the macrophage content and decreased the release of inflammation-related cytokines or chemokine in serum or abdominal aortas, accompanied with the inactivation of inhibitor κB kinase κ (IKKβ)/p65/IκBα signaling pathway. Also, the activity of mitogen-activated protein kinases 7/c-Jun-N-terminal kinase (MKK7/JNK) signaling was decreased by Cav-1 -/- . In addition, oxidative stress induced by HFD in ApoE -/- mice was alleviated by Cav-1 -/- . In response to HFD, Cav-1 -/- markedly reduced triglyceride (TG), total cholesterol (TC), low-density lipoprotein-cholesterol (LDLC) and very low-density lipoprotein-cholesterol (VLDLC) in serum of HFD-fed ApoE -/- mice, whereas enhanced high-density lipoprotein-cholesterol (HDLC) contents. Consistent with these findings, haematoxylin and eosin (H&E) and Oil Red O staining showed fewer lipid droplets in the liver of Cav-1-deficient mice. Further, real time-quantitative PCR (RT-qPCR) analysis indicated that Cav-1 -/- alleviated dyslipidemia both in liver and abdominal aortas of ApoE -/- mice fed with HFD. Cav-1 inhibition-induced attenuation of inflammatory response, oxidative stress and dyslipidemia were confirmed in vitro using mouse vascular smooth muscle cells (VSMCs) treated with ox-LDL. Surprisingly, the processes regulated by Cav-1-knockdown could be abolished through promoting JNK activation in ox-LDL-treated VSMCs. In conclusion, Cav-1 expression could promote HFD-induced AS in a JNK-dependent manner. Copyright © 2018. Published by Elsevier Inc.

JNK1/2-dependent phosphorylation of angulin-1/LSR is required for the exclusive localization of angulin-1/LSR and tricellulin at tricellular contacts in EpH4 epithelial sheet.

PubMed

Nakatsu, Daiki; Kano, Fumi; Taguchi, Yuki; Sugawara, Taichi; Nishizono, Takashi; Nishikawa, Kiyotaka; Oda, Yukako; Furuse, Mikio; Murata, Masayuki

2014-07-01

Tricellular tight junctions (tTJs) are specialized structural variants of tight junctions within tricellular contacts of an epithelial sheet and comprise several transmembrane proteins including lipolysis-stimulated lipoprotein receptor (angulin-1/LSR) and tricellulin. To elucidate the mechanism of its formation, we carried out stepwise screening of kinase inhibitors followed by RNAi screening to identify kinases that regulate intracellular localization of angulin-1/LSR to the tTJs using a fluorescence image-based screen. We found that the activity of JNK1 and JNK2, but not JNK3, was required for the exclusive localization of angulin-1/LSR at the tTJs. Based on a bioinformatics approach, we estimated the potential phosphorylation site of angulin-1/LSR by JNK1 to be serine 288 and experimentally confirmed that JNK1 directly phosphorylates angulin-1/LSR at this site. We found that JNK2 was also involved in the phosphorylation of angulin-1/LSR. Furthermore, GFP-tagged angulin-1/LSR(S288A), in which serine 288 was substituted by alanine, was observed to be dispersed to bicellular junctions, indicating that phosphorylation of Ser288 is crucial for the exclusive localization of angulin-1/LSR and tricellulin at tTJs. Our fluorescence image-based screening for kinases inhibitor or siRNAs combined with the phosphorylation site prediction could become a versatile and useful tool to elucidate the mechanisms underlying the maintenance of tTJs regulated by kinase networks. © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

Drosophila MOF regulates DIAP1 and induces apoptosis in a JNK dependent pathway.

PubMed

Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Ramaiah, M Janaki; Koteswara Rao, G; Bag, Indira; Bhadra, Utpal; Pal-Bhadra, Manika

2016-03-01

Histone modulations have been implicated in various cellular and developmental processes where in Drosophila Mof is involved in acetylation of H4K16. Reduction in the size of larval imaginal discs is observed in the null mutants of mof with increased apoptosis. Deficiency involving Hid, Reaper and Grim [H99] alleviated mof (RNAi) induced apoptosis in the eye discs. mof (RNAi) induced apoptosis leads to activation of caspases which is suppressed by over expression of caspase inhibitors like P35 and Diap1 clearly depicting the role of caspases in programmed cell death. Also apoptosis induced by knockdown of mof is rescued by JNK mutants of bsk and tak1 indicating the role of JNK in mof (RNAi) induced apoptosis. The adult eye ablation phenotype produced by ectopic expression of Hid, Rpr and Grim, was restored by over expression of Mof. Accumulation of Mof at the Diap1 promoter 800 bp upstream of the transcription start site in wild type larvae is significantly higher (up to twofolds) compared to mof (1) mutants. This enrichment coincides with modification of histone H4K16Ac indicating an induction of direct transcriptional up regulation of Diap1 by Mof. Based on these results we propose that apoptosis triggered by mof (RNAi) proceeds through a caspase-dependent and JNK mediated pathway.

Crosstalk between Fas and JNK determines lymphocyte apoptosis after ionizing radiation.

PubMed

Praveen, Koganti; Saxena, Nandita

2013-06-01

Radiation simultaneously activate Fas and JNK pathway in lymphocytes but their precise interaction is not clearly understood. Activation of Fas pathway is required for radiation induced apoptosis, however induction of JNK pathway may or may not contribute in apoptosis. Here we report that Fas, Fas associated death domain and total JNK are activated in a dose- and time-dependent radiation exposure. A biphasic pattern of phospho-JNK was found at lower doses (1 and 2 Gy), however at higher doses of radiation phospho-JNK was continuously activated. Interestingly, Fas ligand expression remained biphasic at all the doses of radiation. Our results suggest that the Fas pathway is the major player in radiation-induced apoptosis, with JNK playing a contributory role. We also observed that Fas ligand expression by radiation is dependent on JNK activation. We also propose that radiation activates JNK pathway, but sustained activation is required for maximal induction of apoptosis at later times. Our findings define a mechanism for crosstalk between JNK and Fas pathway in radiation-induced apoptosis, which may lead to the development of new therapeutic strategies.

Electroacupuncture attenuates mechanical allodynia by suppressing the spinal JNK1/2 pathway in a rat model of inflammatory pain.

PubMed

Du, Jun-Ying; Fang, Jian-Qiao; Liang, Yi; Fang, Jun-Fan

2014-09-01

Electroacupuncture (EA) has a substantial analgesic effect on inflammatory pain induced by complete Freund's adjuvant (CFA). The activation of the c-Jun N-terminal kinase 1/2 (JNK1/2) signal transduction pathway in the spinal cord is associated with inflammatory pain. However, the relationship between EA's analgesic effect and the JNK1/2 signal transduction pathway in the inflammatory pain remain unclear. In the present study, we used the established rat model of CFA-induced inflammatory pain to investigate the role of the spinal JNK1/2 pathway in EA-mediated analgesia. We observed a decrease in paw withdrawal thresholds and an increase in paw edema at 1 and 3 days after injecting CFA into the right hindpaw. CFA, 3 days after injection, upregulated expression of phospho-c-Jun N-terminal kinase1/2 (p-JNK1/2) protein and its downstream targets, the transcriptional regulators p-c-Jun and activator protein-1 (AP-1), as well as cyclooxygenase-2 (COX-2) and the transient receptor potential vanilloid 1 (TRPV1). EA significantly alleviated CFA-induced inflammatory pain. In addition, EA reduced p-JNK1/2 protein levels and COX-2 mRNA expressions, a degree of down-regulated p-c-Jun protein level and AP-1 DNA binding activity in the spinal dorsal horn of CFA-administered animals, but it had no effect on TRPV1 mRNA expression. Furthermore, EA and the JNK inhibitor SP600125 synergistically inhibited CFA-induced hyperalgesia and suppressed the COX-2 mRNA expression in the spinal dorsal horn. Our findings indicate that EA alleviates inflammatory pain behavior, at least in part, by reducing COX-2 expression in the spinal cord via the JNK1/2 signaling pathway. Inactivation of the spinal JNK1/2 signal transduction pathway maybe the potential mechanism of EA's antinociception in the inflammatory pain model. Copyright © 2014 Elsevier Inc. All rights reserved.

Acute ethanol exposure-induced autophagy-mediated cardiac injury via activation of the ROS-JNK-Bcl-2 pathway.

PubMed

Zhu, Zhongxin; Huang, Yewei; Lv, Lingchun; Tao, Youli; Shao, Minglong; Zhao, Congcong; Xue, Mei; Sun, Jia; Niu, Chao; Wang, Yang; Kim, Sunam; Cong, Weitao; Mao, Wei; Jin, Litai

2018-02-01

Binge drinking is associated with increased cardiac autophagy, and often triggers heart injury. Given the essential role of autophagy in various cardiac diseases, this study was designed to investigate the role of autophagy in ethanol-induced cardiac injury and the underlying mechanism. Our study showed that ethanol exposure enhanced the levels of LC3-II and LC3-II positive puncta and promoted cardiomyocyte apoptosis in vivo and in vitro. In addition, we found that ethanol induced autophagy and cardiac injury largely via the sequential triggering of reactive oxygen species (ROS) accumulation, activation of c-Jun NH2-terminal kinase (JNK), phosphorylation of Bcl-2, and dissociation of the Beclin 1/Bcl-2 complex. By contrast, inhibition of ethanol-induced autophagic flux with pharmacologic agents in the hearts of mice and cultured cells significantly alleviated ethanol-induced cardiomyocyte apoptosis and heart injury. Elimination of ROS with the antioxidant N-acetyl cysteine (NAC) or inhibition of JNK with the JNK inhibitor SP600125 reduced ethanol-induced autophagy and subsequent autophagy-mediated apoptosis. Moreover, metallothionein (MT), which can scavenge reactive oxygen and nitrogen species, also attenuated ethanol-induced autophagy and cell apoptosis in MT-TG mice. In conclusion, our findings suggest that acute ethanol exposure induced autophagy-mediated heart toxicity and injury mainly through the ROS-JNK-Bcl-2 signaling pathway. © 2017 Wiley Periodicals, Inc.

β1 integrin- and JNK-dependent tumor growth upon hypofractionated radiation.

PubMed

Sayeed, Aejaz; Lu, Huimin; Liu, Qin; Deming, David; Duffy, Alexander; McCue, Peter; Dicker, Adam P; Davis, Roger J; Gabrilovich, Dmitry; Rodeck, Ulrich; Altieri, Dario C; Languino, Lucia R

2016-08-16

Radiation therapy is an effective cancer treatment modality although tumors invariably become resistant. Using the transgenic adenocarcinoma of mouse prostate (TRAMP) model system, we report that a hypofractionated radiation schedule (10 Gy/day for 5 consecutive days) effectively blocks prostate tumor growth in wild type (β1wt /TRAMP) mice as well as in mice carrying a conditional ablation of β1 integrins in the prostatic epithelium (β1pc-/- /TRAMP). Since JNK is known to be suppressed by β1 integrins and mediates radiation-induced apoptosis, we tested the effect of SP600125, an inhibitor of c-Jun amino-terminal kinase (JNK) in the TRAMP model system. Our results show that SP600125 negates the effect of radiation on tumor growth in β1pc-/- /TRAMP mice and leads to invasive adenocarcinoma. These effects are associated with increased focal adhesion kinase (FAK) expression and phosphorylation in prostate tumors in β1pc-/- /TRAMP mice. In marked contrast, radiation-induced tumor growth suppression, FAK expression and phosphorylation are not altered by SP600125 treatment of β1wt /TRAMP mice. Furthermore, we have reported earlier that abrogation of insulin-like growth factor receptor (IGF-IR) in prostate cancer cells enhances the sensitivity to radiation. Here we further explore the β1/IGF-IR crosstalk and report that β1 integrins promote cell proliferation partly by enhancing the expression of IGF-IR. In conclusion, we demonstrate that β1 integrin-mediated inhibition of JNK signaling modulates tumor growth rate upon hypofractionated radiation.

PI3-K/Akt/JNK/NF-κB is essential for MMP-9 expression and outgrowth in human limbal epithelial cells on intact amniotic membrane.

PubMed

Cheng, Ching-Yi; Hsieh, Hsi-Lung; Hsiao, Li-Der; Yang, Chuen-Mao

2012-07-01

Matrix metalloproteinase-9 (MMP-9) plays an important role in the outgrowth of expanded human limbal epithelial cells on intact amniotic membranes (AM). The mechanisms of MMP-9 expression and cell outgrowth remain unknown. Here, we demonstrated that MMP-9 is preferentially expressed at the leading edge of limbal epithelial outgrowth. Treatment with the inhibitors of PI3-K (LY294002), Akt (SH-5), MEK1/2 (U0126), and JNK1/2 (SP600125) attenuated the outgrowth area, indicating that PI3-K/Akt, p42/p44 MAPK, and JNK1/2 are involved in the outgrowth of intact AM-expanded limbal epithelial cells. However, MMP-9 expression at both transcriptional and translational levels was attenuated by treatment with SP600125, LY294002, or SH-5, not by U0126 and SB202190, suggesting that JNK1/2 and PI3-K/Akt participate in MMP-9 expression. Moreover, NF-κB phosphorylation and nuclear translocation was especially noted at the leading edge, which was attenuated by treatment with SP600125 or LY294002. Helenalin, a selective NF-κB inhibitor, reduced both the limbal epithelial outgrowth and MMP-9 expression. Finally, the data reveal that PI3-K/Akt is an upstream component of the JNK1/2 pathway in MMP-9 expression. Thus, both MAPKs and PI3-K/Akt are required for limbal epithelial outgrowth on intact AM, only the PI3-K/Akt/JNK is essential for MMP-9 expression mediated through activation of transcriptional factor NF-κB in this model. Copyright © 2012 Elsevier B.V. All rights reserved.

Inhibitors of stress-activated protein/mitogen-activated protein kinase pathways.

PubMed

Malemud, Charles J

2007-06-01

The importance of stress-activated protein/mitogen-activated protein kinase (SAP/MAPK) pathway signalling (involving c-Jun-N-terminal kinase [JNK], extracellular signal-regulated kinase [ERK] and p38 kinase) in normal cellular proliferation, differentiation and programmed cell death has led to significant recent advances in our understanding of the role of SAP/MAPK signaling in inflammatory disorders such as arthritis and cardiovascular disease, cancer, and pulmonary and neurogenerative diseases. The discovery that several natural products such as resveratrol, tangeretin and ligustilide non-specifically inhibit SAP/MAPK signalling in vitro should now be logically extended to studies designed to determine how agents in these natural products regulate SAP/MAPK pathways in animal models of disease. A new generation of small-molecule SAP/MAPK inhibitors that demonstrate increasing specificity for each of the JNK, ERK and p38 kinase isoforms has shown promise in animal studies and could eventually prove effective for treating human diseases. Several of these compounds are already being tested in human subjects to assess their oral bioavailability, pharmacokinetics and toxicity.

Tumor necrosis factor-{alpha} induces MMP-9 expression via p42/p44 MAPK, JNK, and nuclear factor-{kappa}B in A549 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, C.-C.; Tseng, Hsiao-Wei; Hsieh, Hsi-Lung

2008-06-15

Matrix metalloproteinases (MMPs), in particular MMP-9, have been shown to be induced by cytokines including tumor necrosis factor-{alpha} (TNF-{alpha}) and contributes to airway inflammation. However, the mechanisms underlying MMP-9 expression induced by TNF-{alpha} in human A549 cells remain unclear. Here, we showed that TNF-{alpha} induced production of MMP-9 protein and mRNA is determined by zymographic, Western blotting, RT-PCR and ELISA assay, which were attenuated by inhibitors of MEK1/2 (U0126), JNK (SP600125), and NF-{kappa}B (helenalin), and transfection with dominant negative mutants of ERK2 ({delta}ERK) and JNK ({delta}JNK), and siRNAs for MEK1, p42 and JNK2. TNF-{alpha}-stimulated phosphorylation of p42/p44 MAPK and JNKmore » were attenuated by pretreatment with the inhibitors U0126 and SP600125 or transfection with dominant negative mutants of {delta}ERK and {delta}JNK. Furthermore, the involvement of NF-{kappa}B in TNF-{alpha}-induced MMP-9 production was consistent with that TNF-{alpha}-stimulated degradation of I{kappa}B-{alpha} and translocation of NF-{kappa}B into the nucleus which were blocked by helenalin, but not by U0126 and SP600125, revealed by immunofluorescence staining. The regulation of MMP-9 gene transcription by MAPKs and NF-{kappa}B was further confirmed by gene luciferase activity assay. MMP-9 promoter activity was enhanced by TNF-{alpha} in A549 cells transfected with wild-type MMP-9-Luc, which was inhibited by helenalin, U0126, or SP600125. In contrast, TNF-{alpha}-stimulated MMP-9 luciferase activity was totally lost in cells transfected with mutant-NF-{kappa}B MMP-9-luc. Moreover, pretreatment with actinomycin D and cycloheximide attenuated TNF-{alpha}-induced MMP-9 expression. These results suggest that in A549 cells, phosphorylation of p42/p44 MAPK, JNK, and transactivation of NF-{kappa}B are essential for TNF-{alpha}-induced MMP-9 gene expression.« less

Glabridin induces apoptosis and cell cycle arrest in oral cancer cells through the JNK1/2 signaling pathway.

PubMed

Chen, Chang-Tai; Chen, Yi-Tzu; Hsieh, Yi-Hsien; Weng, Chia-Jui; Yeh, Jung-Chun; Yang, Shun-Fa; Lin, Chiao-Wen; Yang, Jia-Sin

2018-06-01

Glabridin, a flavonoid extracted from licorice (Glycyrrhiza glabra), possesses various biological properties, including anticancer activities. However, the effect of glabridin on oral cancer cell apoptosis and the underlying molecular mechanisms has not been elucidated. In this study, we demonstrated that glabridin treatment significantly inhibits cell proliferation in human oral cancer SCC-9 and SAS cell lines. Flow cytometric assays demonstrated that glabridin induced several features of apoptosis, such as sub-G1 phase cell increase and phosphatidylserine externalization. Furthermore, glabridin induced apoptosis dose-dependently in SCC-9 cells through caspase-3, -8, and -9 activation and poly (ADP-ribose) polymerase cleavage. Moreover, glabridin increased the phosphorylation of the extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase (JNK) pathways in a dose-dependent manner. Moreover, the inhibition of the JNK1/2 inhibitor significantly reversed the glabridin-induced activation of the caspase pathway. In conclusion, our findings suggest that glabridin induces oral cancer cell apoptosis through the JNK1/2 pathway and is a potential therapeutic agent for oral cancer. © 2018 Wiley Periodicals, Inc.

Phloretin induces apoptosis of non-small cell lung carcinoma A549 cells via JNK1/2 and p38 MAPK pathways

PubMed Central

MIN, JIE; LI, XU; HUANG, KENAN; TANG, HUA; DING, XINYU; QI, CHEN; QIN, XIONG; XU, ZHIFEI

2015-01-01

Phloretin (Ph) existing in apples, pears and various vegetables is known to have antitumor activities in several cancer cell lines. However, little is known about its effect on human lung cancer cells. The aim of the present study was to see whether Ph could induce apoptosis of non-small cell lung cancer (NSCLC) cells, and explore the possible underlying mechanism of action. We found that Ph markedly induced cell apoptosis of NSCLC cell line A549, and inhibited the migration of A549 cells in a dose-dependent manner. The expression level of BAX, cleaved caspase-3 and -9, and degraded form of PARP was increased and Bcl-2 was decreased after Ph treatment. In addition, the phosphorylation of P38 MAPK, ERK1/2 and JNK1/2 was increased in a dose-dependent manner in parallel with Ph treatment. Inhibition of P38 MAPK and JNK1/2 by specific inhibitors significantly abolished the Ph-induced activation of the caspase-3 and -9. In vivo tumor-suppression assay further indicated that Ph (20 mg/kg) displayed a more significant inhibitory effect on A549 xenografts in tumor growth. All these findings indicate that Ph is able to inhibit NSCLC A549 cell growth by inducing apoptosis through P38 MAPK and JNK1/2 pathways, and therefore may prove to be an adjuvant to the treatment of NSCLC. PMID:26503828

Inhibiting Src family tyrosine kinase activity blocks glutamate signalling to ERK1/2 and Akt/PKB but not JNK in cultured striatal neurones.

PubMed

Crossthwaite, Andrew J; Valli, Haseeb; Williams, Robert J

2004-03-01

Glutamate receptor activation of mitogen-activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+-influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor-induced phosphorylation of ERK1/2 and Akt/PKB in a concentration-dependent manner, and reduced cAMP response element-binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase-mediated phosphorylation and PI 3-kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3-kinase regulatory subunit p85. This revealed a Src-dependent phosphorylation of a focal adhesion kinase (FAK)-p85 complex on glutamate stimulation. Demonstrating that PI3-kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3-kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+-dependent phosphorylation of c-Jun N-terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor-dependent JNK1/2 phosphorylation, suggesting that Src family kinase-dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3-kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.

Involvement of p38 MAPK- and JNK-modulated expression of Bcl-2 and Bax in Naja nigricollis CMS-9-induced apoptosis of human leukemia K562 cells.

PubMed

Chen, Ying-Jung; Liu, Wen-Hsin; Kao, Pei-Hsiu; Wang, Jeh-Jeng; Chang, Long-Sen

2010-06-15

CMS-9, a phospholipase A(2) (PLA(2)) isolated from Naja nigricollis venom, induced apoptosis of human leukemia K562 cells, characterized by mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Moreover, an increase in intracellular Ca2+ concentration and the production of reactive oxygen species (ROS) was noted. Pretreatment with BAPTA-AM (Ca2+ chelator) and N-acetylcysteine (NAC, ROS scavenger) proved that Ca2+ was an upstream event in inducing ROS generation. Upon exposure to CMS-9, activation of p38 MAPK and JNK was observed in K562 cells. BAPTA-AM or NAC abrogated CMS-9-elicited p38 MAPK and JNK activation, and rescued viability of CMS-9-treated K562 cells. SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) suppressed CMS-9-induced dissipation of mitochondrial membrane potential, Bcl-2 down-regulation, Bax up-regulation and increased mitochondrial translocation of Bax. Inactivation of PLA(2) activity reduced drastically the cytotoxicity of CMS-9, and a combination of lysophosphatidylcholine and stearic acid mimicked the cytotoxic effects of CMS-9. Taken together, our data suggest that CMS-9-induced apoptosis of K562 cells is catalytic activity-dependent and is mediated through mitochondria-mediated death pathway triggered by Ca2+/ROS-evoked p38 MAPK and JNK activation. 2010 Elsevier Ltd. All rights reserved.

PLCγ2 promotes apoptosis while inhibits proliferation in rat hepatocytes through PKCD/JNK MAPK and PKCD/p38 MAPK signalling.

PubMed

Chen, Xiaoguang; Lv, Qiongxia; Ma, Jun; Liu, Yumei

2018-02-11

The PLCG2 (PLCγ2) gene is a member of PLC gene family encoding transmembrane signalling enzymes involved in various biological processes including cell proliferation and apoptosis. Our earlier study indicated that PLCγ2 may be involved in the termination of regeneration of the liver which is mainly composed of hepatocytes, but its exact biological function and molecular mechanism in liver regeneration termination remains unclear. This study aims to examine the role of PLCγ2 in the growth of hepatocytes. A recombinant adenovirus expressing PLCγ2 was used to infect primary rat hepatocytes. PLCγ2 mRNA and protein levels were detected by qRT-PCR and Western blot. The subcellular location of PLCγ2 protein was tested by an immunofluorescence assay. The proliferation of hepatocytes was measured by MTT assay. The cell cycle and apoptosis were analysed by flow cytometry. Caspase-3, -8 and -9 activities were measured by a spectrophotometry method. Phosphorylation levels of PKCD, JNK and p38 in the infected cells were detected by Western blot. The possible mechanism underlying the role of PLCγ2 in hepatocyte growth was also explored by adding a signalling pathway inhibitor. Hepatocyte proliferation was dramatically reduced, while cell apoptosis was remarkably increased. The results demonstrated that PLCγ2 increased the phosphorylation of PKCD, p38 and JNK in rat hepatocytes. After PKCD activity was inhibited by the inhibitor Go 6983, the levels of both p-p38 and p-JNK MAPKs significantly decreased, and PLCγ2-induced cell proliferation inhibition and cell apoptosis were obviously reversed. This study showed that PLCγ2 regulates hepatocyte growth through PKCD-dependently activating p38 MAPK and JNK MAPK pathways; this result was experimentally based on the further exploration of the effect of PLCγ2 on hepatocyte growth in vivo. © 2018 John Wiley & Sons Ltd.

Complete inhibition of anisomycin and UV radiation but not cytokine induced JNK and p38 activation by an aryl-substituted dihydropyrrolopyrazole quinoline and mixed lineage kinase 7 small interfering RNA.

PubMed

Wang, Xushan; Mader, Mary M; Toth, John E; Yu, Xiaohong; Jin, Najia; Campbell, Robert M; Smallwood, Jeffrey K; Christe, Michael E; Chatterjee, Arindam; Goodson, Theodore; Vlahos, Chris J; Matter, William F; Bloem, Laura J

2005-05-13

Mixed lineage kinase 7 (MLK7) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the pro-apoptotic signaling pathways p38 and JNK. A library of potential kinase inhibitors was screened, and a series of dihydropyrrolopyrazole quinolines was identified as highly potent inhibitors of MLK7 in vitro catalytic activity. Of this series, an aryl-substituted dihydropyrrolopyrazole quinoline (DHP-2) demonstrated an IC50 of 70 nM for inhibition of pJNK formation in COS-7 cell MLK7/JNK co-transfection assays. In stimulated cells, DHP-2 at 200 nM or MLK7 small interfering RNA completely blocked anisomycin and UV induced but had no effect on interleukin-1beta or tumor necrosis factor-alpha-induced p38 and JNK activation. Additionally, the compound blocked anisomycin and UV-induced apoptosis in COS-7 cells. Heart tissue homogenates from MLK7 transgenic mice treated with DHP-2 at 30 mg/kg had reduced JNK and p38 activation with no apparent effect on ERK activation, demonstrating that this compound can be used to block MLK7-driven MAPK pathway activation in vivo. Taken together, these data demonstrate that MLK7 is the MAPKKK required for modulation of the stress-activated MAPKs downstream of anisomycin and UV stimulation and that DHP-2 can be used to block MLK7 pathway activation in cells as well as in vivo.

Glutamine deprivation stimulates mTOR-JNK-dependent chemokine secretion

PubMed Central

Shanware, Naval P.; Bray, Kevin; Eng, Christina H.; Wang, Fang; Follettie, Maximillian; Myers, Jeremy; Fantin, Valeria R.; Abraham, Robert T.

2014-01-01

The non-essential amino acid, glutamine, exerts pleiotropic effects on cell metabolism, signalling and stress resistance. Here we demonstrate that short-term glutamine restriction triggers an endoplasmic reticulum (ER) stress response that leads to production of the pro-inflammatory chemokine, interleukin-8 (IL-8). Glutamine deprivation-induced ER stress triggers colocalization of autophagosomes, lysosomes and the Golgi into a subcellular structure whose integrity is essential for IL-8 secretion. The stimulatory effect of glutamine restriction on IL-8 production is attributable to depletion of tricarboxylic acid cycle intermediates. The protein kinase, mTOR, is also colocalized with the lysosomal membrane clusters induced by glutamine deprivation, and inhibition of mTORC1 activity abolishes both endomembrane reorganization and IL-8 secretion. Activated mTORC1 elicits IL8 gene expression via the activation of an IRE1-JNK signalling cascade. Treatment of cells with a glutaminase inhibitor phenocopies glutamine restriction, suggesting that these results will be relevant to the clinical development of glutamine metabolism inhibitors as anticancer agents. PMID:25254627

Anti-Inflammatory Effects and Joint Protection in Collagen-Induced Arthritis after Treatment with IQ-1S, a Selective c-Jun N-Terminal Kinase Inhibitor.

PubMed

Schepetkin, Igor A; Kirpotina, Liliya N; Hammaker, Deepa; Kochetkova, Irina; Khlebnikov, Andrei I; Lyakhov, Sergey A; Firestein, Gary S; Quinn, Mark T

2015-06-01

c-Jun N-terminal kinases (JNKs) participate in many physiologic and pathologic processes, including inflammatory diseases. We recently synthesized the sodium salt of IQ-1S (11H-indeno[1,2-b]quinoxalin-11-one oxime) and demonstrated that it is a high-affinity JNK inhibitor and inhibits murine delayed-type hypersensitivity. Here we show that IQ-1S is highly specific for JNK and that its neutral form is the most abundant species at physiologic pH. Molecular docking of the IQ-1S syn isomer into the JNK1 binding site gave the best pose, which corresponded to the position of cocrystallized JNK inhibitor SP600125 (1,9-pyrazoloanthrone). Evaluation of the therapeutic potential of IQ-1S showed that it inhibited matrix metalloproteinase 1 and 3 gene expression induced by interleukin-1β in human fibroblast-like synoviocytes and significantly attenuated development of murine collagen-induced arthritis (CIA). Treatment with IQ-1S either before or after induction of CIA resulted in decreased clinical scores, and joint sections from IQ-1S-treated CIA mice exhibited only mild signs of inflammation and minimal cartilage loss compared with those from control mice. Collagen II-specific antibody responses were also reduced by IQ-1S treatment. By contrast, the inactive ketone derivative 11H-indeno[1,2-b]quinoxalin-11-one had no effect on CIA clinical scores or collagen II-specific antibody titers. IQ-1S treatment also suppressed proinflammatory cytokine and chemokine levels in joints and lymph node cells. Finally, treatment with IQ-1S increased the number of Foxp3(+)CD4(+)CD25(+) regulatory T cells in lymph nodes. Thus, IQ-1S can reduce inflammation and cartilage loss associated with CIA and can serve as a small-molecule modulator for mechanistic studies of JNK function in rheumatoid arthritis. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Inhibition of JNK by pi class of glutathione S-transferase through PKA/CREB pathway is associated with carnosic acid protection against 6-hydroxydopamine-induced apoptosis.

PubMed

Lin, Chia-Yuan; Fu, Ru-Huei; Chou, Ruey-Hwang; Chen, Jing-Hsien; Wu, Chi-Rei; Chang, Shu-Wei; Tsai, Chia-Wen

2017-05-01

Pi class of glutathione S-transferase (GST) is known to suppress c-Jun N-terminal kinase (JNK)-related apoptosis through protein-protein interactions. Moreover, signaling by PKA/cAMP response element binding protein (CREB) is necessary for GSTP up-regulation. This study explored whether carnosic acid (CA) from rosemary prevents 6-hydroxydopamine (6-OHDA)-induced neurotoxicity by inhibition of JNK through GSTP via PKA/CREB signaling. Results indicated that the GSTP protein was increased in SH-SY5Y cells treated with CA for 18 and 24 h. However, CA had no significant effect on alpha or mu class of GST. Treatment of CA increased the induction of p-PKAα, nuclear p-CREB, and CRE-DNA binding activity. These effects of CA were attenuated in cells pretreated with the PKA inhibitor H89. CA pretreatment suppressed 6-OHDA-induced apoptosis by inhibition of JNK phosphorylation, poly(ADP)-ribose polymerase cleavage, and nuclear condensation. Pretreatment with H89 and GSTP siRNA attenuated the ability of CA to reverse 6-OHDA-induced apoptosis. By use of immunoprecipitation with JNK antibody to examine the interaction of GSTP-JNK with CA, we showed that CA pretreatment increased the immunoprecipitation of GSTP after 6-OHDA treatment, which suggests that CA promoted the interaction between GSTP and JNK. CA prevents 6-OHDA-induced apoptosis via inhibition of JNK by GSTP through the PKA/CREB pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

Puerarin reduces apoptosis in rat hippocampal neurons culturea in high glucose medium by modulating the p38 mitogen activated protein kinase and c-Jun N-terminal kinase signaling pathways.

PubMed

Xu, Xiaohan; Wang, Jingbo; Zhang, Hong; Tian, Guoqing; Liu, Yuqin

2016-02-01

To investigate the neuroprotective etfect of puerarin on rat hippocampal neurons cultured in high glucose medium, and to examine the role of the p38 mitogen activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) signaling pathways in this effect. Primary cultures of hippocampal neurons were prepared from newborn Sprague Dawley rats. Neuron-specific enolase immunocytochemistry was used to identify neurons. The neurons were cultured with normal medium (control group) or with high-glucose medium (high-glucose group), and puerarin (puerarin group), a p38 MAPK inhibitor (SB239063; p38 MAPK inhibitor group) or a JNK inhibitor (SP600125; JNK inhibitor group) were added. After 72 h of treatment, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was performed to detect apoptosis, and western blotting was used to assess protein levels of p-p38, p38, p-JNK and JNK. In the high-glucose group, the neuronal apoptosis rate and the p-p38/p38 and p-JNK/JNK ratios were higher than in the control group. The p38 MAPK and JNK inhibitors prevented this increase in the apoptosis rate. The apoptosis rates in the puerarin group, the p38 MAPK inhibitor group and the JNK inhibitor group were significantly decreased compared with the high-glucose group. Moreover, protein levels of p-p38 and p-JNK were significantly reduced, and the p-p38/p38 and p-JNK/JNK ratios were decreased in the puerarin group compared with the high-glucose group. In addition, compared with the high-glucose group, p-p38 levels and the p-p38/p38 ratio were reduced in the p38 MAPK inhibitor group, and p-JNK levels and the p-JNK/JNK ratio were decreased in the JNK inhibitor group. Puerarin attenuates neuronal apoptosis induced by high glucose by reducing the phosphorylation of p38 and JNK.

Eicosapentaenoic acid (EPA) induced apoptosis in HepG2 cells through ROS-Ca(2+)-JNK mitochondrial pathways.

PubMed

Zhang, Yuanyuan; Han, Lirong; Qi, Wentao; Cheng, Dai; Ma, Xiaolei; Hou, Lihua; Cao, Xiaohong; Wang, Chunling

2015-01-24

Eicosapentaenoic acid (EPA), a well-known dietary n-3 PUFAS, has been considered to inhibit proliferation of tumor cells. However, the molecular mechanism related to EPA-induced liver cancer cells apoptosis has not been reported. In this study, we investigated the effect of EPA on HepG2 cells proliferation and apoptosis mechanism through mitochondrial pathways. EPA inhibited proliferation of HepG2 cells in a dose-dependent manner and had no significant effect on the cell viability of humor normal liver L-02 cells. It was found that EPA initially evoked ROS formation, leading to [Ca(2+)]c accumulation and the mitochondrial permeability transition pore (MPTP) opening; EPA-induced HepG2 cells apoptosis was inhibited by N-acetylcysteine (NAC, an inhibitor of ROS), 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM, a chelator of calcium) and CsA (inhibitor of MPTP). The relationship between ROS production, the increase of cytoplasmic Ca and MPTP opening was detected. It seems that ROS may act as an upstream regulator of EPA-induced [Ca(2+)]c generation, moreover, generation of ROS, overload of mitochondrial [Ca(2+)]c, and JNK activated cause the opening of MPTP. Western blotting results showed that EPA elevated the phosphorylation status of JNK, processes associated with the ROS generation. Simultaneously, the apoptosis induced by EPA was related to release of cytochrome C from mitochondria to cytoplasm through the MPTP and activation of caspase-9 and caspase-3. These results suggest that EPA induces apoptosis through ROS-Ca(2+)-JNK mitochondrial pathways. Copyright © 2014 Elsevier Inc. All rights reserved.

Discovery of potent and selective rhodanine type IKKβ inhibitors by hit-to-lead strategy.

PubMed

Song, Hyeseung; Lee, Yun Suk; Roh, Eun Joo; Seo, Jae Hong; Oh, Kwang-Seok; Lee, Byung Ho; Han, Hogyu; Shin, Kye Jung

2012-09-01

Regulation of NF-κB activation through the inhibition of IKKβ has been identified as a promising target for the treatment of inflammatory and autoimmune disease such as rheumatoid arthritis. In order to develop novel IKKβ inhibitors, we performed high throughput screening toward around 8000 library compounds, and identified a hit compound containing rhodanine moiety. We modified the structure of hit compound to obtain potent and selective IKKβ inhibitors. Throughout hit-to-lead studies, we have discovered optimized compounds which possess blocking effect toward NF-κB activation and TNFα production in cell as well as inhibition activity against IKKβ. Among them, compound 3q showed the potent inhibitory activity against IKKβ, and excellent selectivity over other kinases such as p38α, p38β, JNK1, JNK2, and JNK3 as well as IKKα. Copyright © 2012 Elsevier Ltd. All rights reserved.

LPS-Induced Low-Grade Inflammation Increases Hypothalamic JNK Expression and Causes Central Insulin Resistance Irrespective of Body Weight Changes.

PubMed

Rorato, Rodrigo; Borges, Beatriz de Carvalho; Uchoa, Ernane Torres; Antunes-Rodrigues, José; Elias, Carol Fuzeti; Elias, Lucila Leico Kagohara

2017-07-04

Metabolic endotoxemia contributes to low-grade inflammation in obesity, which causes insulin resistance due to the activation of intracellular proinflammatory pathways, such as the c-Jun N-terminal Kinase (JNK) cascade in the hypothalamus and other tissues. However, it remains unclear whether the proinflammatory process precedes insulin resistance or it appears because of the development of obesity. Hypothalamic low-grade inflammation was induced by prolonged lipopolysaccharide (LPS) exposure to investigate if central insulin resistance is induced by an inflammatory stimulus regardless of obesity. Male Wistar rats were treated with single (1 LPS) or repeated injections (6 LPS) of LPS (100 μg/kg, IP) to evaluate the phosphorylation of the insulin receptor substrate-1 (IRS1), Protein kinase B (AKT), and JNK in the hypothalamus. Single LPS increased the expression of pIRS1, pAKT, and pJNK, whereas the repeated LPS treatment failed to recruit pIRS1 and pAKT. The 6 LPS treated rats showed increased total JNK and pJNK. The 6 LPS rats became unresponsive to the hypophagic effect induced by central insulin administration (12 μM/5 μL, ICV). Prolonged exposure to LPS (24 h) impaired the insulin-induced AKT phosphorylation and the translocation of the transcription factor forkhead box protein O1 (FoxO1) from the nucleus to the cytoplasm of the cultured hypothalamic GT1-7 cells. Central administration of the JNK inhibitor (20 μM/5 μL, ICV) restored the ability of insulin to phosphorylate IRS1 and AKT in 6 LPS rats. The present data suggest that an increased JNK activity in the hypothalamus underlies the development of insulin resistance during prolonged exposure to endotoxins. Our study reveals that weight gain is not mandatory for the development of hypothalamic insulin resistance and the blockade of proinflammatory pathways could be useful for restoring the insulin signaling during prolonged low-grade inflammation as seen in obesity.

DISTINCT FUNCTIONS OF JNK AND C-JUN IN OXIDANT-INDUCED HEPATOCYTE DEATH

PubMed Central

Amir, Muhammad; Liu, Kun; Zhao, Enpeng; Czaja, Mark J.

2013-01-01

Overactivation of c-Jun N-terminal kinase (JNK)/c-Jun signaling is a central mechanism of hepatocyte injury and death including that from oxidative stress. However, the functions of JNK and c-Jun are still unclear, and this pathway also inhibits hepatocyte death. Previous studies of menadione-induced oxidant stress demonstrated that toxicity resulted from sustained JNK/c-Jun activation as death was blocked by the c-Jun dominant negative TAM67. To further delineate the function of JNK/c-Jun signaling in hepatocyte injury from oxidant stress, the effects of direct JNK inhibition on menadione-induced death were examined. In contrast to the inhibitory effect of TAM67, pharmacological JNK inhibition by SP600125 sensitized the rat hepatocyte cell line RALA255-10G to death from menadione. SP600125 similarly sensitized mouse primary hepatocytes to menadione toxicity. Death from SP600125/menadione was c-Jun dependent as it was blocked by TAM67, but independent of c-Jun phosphorylation. Death occurred by apoptosis and necrosis and activation of the mitochondrial death pathway. Short hairpin RNA knockdowns of total JNK or JNK2 sensitized to death from menadione, whereas a jnk1 knockdown was protective. Jnk2 null mouse primary hepatocytes were also sensitized to menadione death. JNK inhibition magnified decreases in cellular ATP content and β-oxidation induced by menadione. This effect mediated cell death as chemical inhibition of β-oxidation also sensitized cells to death from menadione, and supplementation with the β-oxidation substrate oleate blocked death. Components of the JNK/c-Jun signaling pathway have opposing functions in hepatocyte oxidant stress with JNK2 mediating resistance to cell death and c-Jun promoting death. PMID:22644775

Amyloid precursor protein modulates Nav1.6 sodium channel currents through a Go-coupled JNK pathway.

PubMed

Li, Shao; Wang, Xi; Ma, Quan-Hong; Yang, Wu-Lin; Zhang, Xiao-Gang; Dawe, Gavin S; Xiao, Zhi-Cheng

2016-12-23

Amyloid precursor protein (APP), commonly associated with Alzheimer's disease, also marks axonal degeneration. In the recent studies, we demonstrated that APP aggregated at nodes of Ranvier (NORs) in myelinated central nervous system (CNS) axons and interacted with Nav1.6. However, the physiological function of APP remains unknown. In this study, we described reduced sodium current densities in APP knockout hippocampal neurons. Coexpression of APP or its intracellular domains containing a VTPEER motif with Na v 1.6 sodium channels in Xenopus oocytes resulted in an increase in peak sodium currents, which was enhanced by constitutively active Go mutant and blocked by a dominant negative mutant. JNK and CDK5 inhibitor attenuated increases in Nav1.6 sodium currents induced by overexpression of APP. Nav1.6 sodium currents were increased by APPT668E (mutant Thr to Glu) and decreased by T668A (mutant Thr to ALa) mutant, respectively. The cell surface expression of Nav1.6 sodium channels in the white matter of spinal cord and the spinal conduction velocity is decreased in APP, p35 and JNK3 knockout mice. Therefore, APP modulates Nav1.6 sodium channels through a Go-coupled JNK pathway, which is dependent on phosphorylation of APP at Thr668.

Amyloid precursor protein modulates Nav1.6 sodium channel currents through a Go-coupled JNK pathway

PubMed Central

Li, Shao; Wang, Xi; Ma, Quan-Hong; Yang, Wu-lin; Zhang, Xiao-Gang; Dawe, Gavin S.; Xiao, Zhi-Cheng

2016-01-01

Amyloid precursor protein (APP), commonly associated with Alzheimer’s disease, also marks axonal degeneration. In the recent studies, we demonstrated that APP aggregated at nodes of Ranvier (NORs) in myelinated central nervous system (CNS) axons and interacted with Nav1.6. However, the physiological function of APP remains unknown. In this study, we described reduced sodium current densities in APP knockout hippocampal neurons. Coexpression of APP or its intracellular domains containing a VTPEER motif with Nav1.6 sodium channels in Xenopus oocytes resulted in an increase in peak sodium currents, which was enhanced by constitutively active Go mutant and blocked by a dominant negative mutant. JNK and CDK5 inhibitor attenuated increases in Nav1.6 sodium currents induced by overexpression of APP. Nav1.6 sodium currents were increased by APPT668E (mutant Thr to Glu) and decreased by T668A (mutant Thr to ALa) mutant, respectively. The cell surface expression of Nav1.6 sodium channels in the white matter of spinal cord and the spinal conduction velocity is decreased in APP, p35 and JNK3 knockout mice. Therefore, APP modulates Nav1.6 sodium channels through a Go-coupled JNK pathway, which is dependent on phosphorylation of APP at Thr668. PMID:28008944

Activation of the HMGB1-RAGE axis upregulates TH expression in dopaminergic neurons via JNK phosphorylation.

PubMed

Kim, Soo Jeong; Ryu, Min Jeong; Han, Jeongsu; Jang, Yunseon; Kim, Jungim; Lee, Min Joung; Ryu, Ilhwan; Ju, Xianshu; Oh, Eungseok; Chung, Woosuk; Kweon, Gi Ryang; Heo, Jun Young

2017-11-04

The derangement of tyrosine hydroxylase (TH) activity reduces dopamine synthesis and is implicated in the pathogenesis of Parkinson's disease. However, the extracellular modulator and intracellular regulatory mechanisms of TH have yet to be identified. Recently, high-mobility group box 1 (HMGB1) was reported to be actively secreted from glial cells and is regarded as a mediator of dopaminergic neuronal loss. However, the mechanism for how HMGB1 affects TH expression, particularly through the receptor for advanced glycation endproducts (RAGE), has not yet been investigated. We found that recombinant HMGB1 (rHMGB1) upregulates TH mRNA expression via simultaneous activation of JNK phosphorylation, and this induction of TH expression is blocked by inhibitors of RAGE and JNK. To investigate how TH expression levels change through the HMGB1-RAGE axis as a result of MPP + toxicity, we co-treated SN4741 dopaminergic cells with MPP + and rHMGB1. rHMGB1 blocked the reduction of TH mRNA following MPP + treatment without altering cell survival rates. Our results suggest that HMGB1 upregulates TH expression to maintain dopaminergic neuronal function via activating RAGE, which is dependent on JNK phosphorylation. Copyright © 2017 Elsevier Inc. All rights reserved.

JIP1-Mediated JNK Activation Negatively Regulates Synaptic Plasticity and Spatial Memory.

PubMed

Morel, Caroline; Sherrin, Tessi; Kennedy, Norman J; Forest, Kelly H; Avcioglu Barutcu, Seda; Robles, Michael; Carpenter-Hyland, Ezekiel; Alfulaij, Naghum; Standen, Claire L; Nichols, Robert A; Benveniste, Morris; Davis, Roger J; Todorovic, Cedomir

2018-04-11

The c-Jun N-terminal kinase (JNK) signal transduction pathway is implicated in learning and memory. Here, we examined the role of JNK activation mediated by the JNK-interacting protein 1 (JIP1) scaffold protein. We compared male wild-type mice with a mouse model harboring a point mutation in the Jip1 gene that selectively blocks JIP1-mediated JNK activation. These male mutant mice exhibited increased NMDAR currents, increased NMDAR-mediated gene expression, and a lower threshold for induction of hippocampal long-term potentiation. The JIP1 mutant mice also displayed improved hippocampus-dependent spatial memory and enhanced associative fear conditioning. These results were confirmed using a second JIP1 mutant mouse model that suppresses JNK activity. Together, these observations establish that JIP1-mediated JNK activation contributes to the regulation of hippocampus-dependent, NMDAR-mediated synaptic plasticity and learning. SIGNIFICANCE STATEMENT The results of this study demonstrate that c-Jun N-terminal kinase (JNK) activation induced by the JNK-interacting protein 1 (JIP1) scaffold protein negatively regulates the threshold for induction of long-term synaptic plasticity through the NMDA-type glutamate receptor. This change in plasticity threshold influences learning. Indeed, mice with defects in JIP1-mediated JNK activation display enhanced memory in hippocampus-dependent tasks, such as contextual fear conditioning and Morris water maze, indicating that JIP1-JNK constrains spatial memory. This study identifies JIP1-mediated JNK activation as a novel molecular pathway that negatively regulates NMDAR-dependent synaptic plasticity and memory. Copyright © 2018 the authors 0270-6474/18/383708-21$15.00/0.

Calcium/Ask1/MKK7/JNK2/c-Src signalling cascade mediates disruption of intestinal epithelial tight junctions by dextran sulfate sodium.

PubMed

Samak, Geetha; Chaudhry, Kamaljit K; Gangwar, Ruchika; Narayanan, Damodaran; Jaggar, Jonathan H; Rao, RadhaKrishna

2015-02-01

Disruption of intestinal epithelial tight junctions is an important event in the pathogenesis of ulcerative colitis. Dextran sodium sulfate (DSS) induces colitis in mice with symptoms similar to ulcerative colitis. However, the mechanism of DSS-induced colitis is unknown. We investigated the mechanism of DSS-induced disruption of intestinal epithelial tight junctions and barrier dysfunction in Caco-2 cell monolayers in vitro and mouse colon in vivo. DSS treatment resulted in disruption of tight junctions, adherens junctions and actin cytoskeleton leading to barrier dysfunction in Caco-2 cell monolayers. DSS induced a rapid activation of c-Jun N-terminal kinase (JNK), and the inhibition or knockdown of JNK2 attenuated DSS-induced tight junction disruption and barrier dysfunction. In mice, DSS administration for 4 days caused redistribution of tight junction and adherens junction proteins from the epithelial junctions, which was blocked by JNK inhibitor. In Caco-2 cell monolayers, DSS increased intracellular Ca(2+) concentration, and depletion of intracellular Ca(2+) by 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM) or thapsigargin attenuated DSS-induced JNK activation, tight junction disruption and barrier dysfunction. Knockdown of apoptosis signal-regulated kinase 1 (Ask1) or MKK7 blocked DSS-induced tight junction disruption and barrier dysfunction. DSS activated c-Src by a Ca2+ and JNK-dependent mechanism. Inhibition of Src kinase activity or knockdown of c-Src blocked DSS-induced tight junction disruption and barrier dysfunction. DSS increased tyrosine phosphorylation of occludin, zonula occludens-1 (ZO-1), E-cadherin and β-catenin. SP600125 abrogated DSS-induced tyrosine phosphorylation of junctional proteins. Recombinant JNK2 induced threonine phosphorylation and auto-phosphorylation of c-Src. The present study demonstrates that Ca(2+)/Ask1/MKK7/JNK2/cSrc signalling cascade mediates DSS-induced tight

Function-specific intracellular signaling pathways downstream of heparin-binding EGF-like growth factor utilized by human trophoblasts.

PubMed

Jessmon, Philip; Kilburn, Brian A; Romero, Roberto; Leach, Richard E; Armant, D Randall

2010-05-01

Heparin-binding EGF-like growth factor (HBEGF) is expressed by trophoblast cells throughout gestation. First-trimester cytotrophoblast cells are protected from hypoxia-induced apoptosis because of the accumulation of HBEGF through a posttranscriptional autocrine mechanism. Exogenous application of HBEGF is cytoprotective in a hypoxia/reoxygenation (H/R) injury model and initiates trophoblast extravillous differentiation to an invasive phenotype. The downstream signaling pathways induced by HBEGF that mediate these various cellular activities were identified using two human first-trimester cytotrophoblast cell lines, HTR-8/SVneo and SW.71, with similar results. Recombinant HBEGF (1 nM) induced transient phosphorylation of MAPK3/1 (ERK), MAPK14 (p38), and AKT within 15 min and JNK after 1-2 h. To determine which downstream pathways regulate the various functions of HBEGF, cells were treated with specific inhibitors of the ERK upstream regulator MEK (U0126), the AKT upstream regulator phosphoinositide-3 (PI3)-kinase (LY294002), MAPK14 (SB203580), and JNK (SP600125), as well as with inactive structural analogues. Only SB203580 specifically prevented HBEGF-mediated rescue during H/R, while each inhibitor attenuated HBEGF-stimulated cell migration. Accumulation of HBEGF at reduced oxygen was blocked only by a combination of U0126, SB203580, and SP600125. We conclude that HBEGF advances trophoblast extravillous differentiation through coordinate activation of PI3 kinase, ERK, MAPK14, and JNK, while only MAPK14 is required for its antiapoptotic activity. Additionally, hypoxia induces an autocrine increase in HBEGF protein levels through MAPK14, JNK or ERK. These experiments reveal a complexity of the intracellular signaling circuitry that regulates trophoblast functions critical for implantation and placentation.

Downregulation of Ras C-terminal processing by JNK inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mouri, Wataru; Department of Neurosurgery, Yamagata University School of Medicine, Yamagata 990-9585; Biology Division, National Cancer Center Research Institute, Tokyo 104-0045

2008-06-27

After translation, Ras proteins undergo a series of modifications at their C-termini. This post-translational C-terminal processing is essential for Ras to become functional, but it remains unknown whether and how Ras C-terminal processing is regulated. Here we show that the C-terminal processing and subsequent plasma membrane localization of H-Ras as well as the activation of the downstream signaling pathways by H-Ras are prevented by JNK inhibition. Conversely, JNK activation by ultraviolet irradiation resulted in promotion of C-terminal processing of H-Ras. Furthermore, increased cell density promoted C-terminal processing of H-Ras most likely through an autocrine/paracrine mechanism, which was also blocked undermore » JNK-inhibited condition. Ras C-terminal processing was sensitive to JNK inhibition in the case of H- and N-Ras but not K-Ras, and in a variety of cell types. Thus, our results suggest for the first time that Ras C-terminal processing is a regulated mechanism in which JNK is involved.« less

TSG-6 secreted by human umbilical cord-MSCs attenuates severe burn-induced excessive inflammation via inhibiting activations of P38 and JNK signaling.

PubMed

Liu, Lingying; Song, Huifeng; Duan, Hongjie; Chai, Jiake; Yang, Jing; Li, Xiao; Yu, Yonghui; Zhang, Xulong; Hu, Xiaohong; Xiao, Mengjing; Feng, Rui; Yin, Huinan; Hu, Quan; Yang, Longlong; Du, Jundong; Li, Tianran

2016-07-22

The hMSCs have become a promising approach for inflammation treatment in acute phase. Our previous study has demonstrated that human umbilical cord-MSCs could alleviate the inflammatory reaction of severely burned wound. In this study, we further investigated the potential role and mechanism of the MSCs on severe burn-induced excessive inflammation. Wistar rats were randomly divided into following groups: Sham, Burn, Burn+MSCs, Burn+MAPKs inhibitors, and Burn, Burn+MSCs, Burn+Vehicle, Burn+siTSG-6, Burn+rhTSG-6 in the both experiments. It was found that MSCs could only down-regulate P38 and JNK signaling, but had no effect on ERK in peritoneal macrophages of severe burn rats. Furthermore, suppression of P38 and JNK activations significantly reduced the excessive inflammation induced by severe burn. TSG-6 was secreted by MSCs using different inflammatory mediators. TSG-6 from MSCs and recombinant human (rh)TSG-6 all significantly reduced activations of P38 and JNK signaling induced by severe burn and then attenuated excessive inflammations. On the contrary, knockdown TSG-6 in the cells significantly increased phosphorylation of P38 and JNK signaling and reduced therapeutic effect of the MSCs on excessive inflammation. Taken together, this study suggested TSG-6 from MSCs attenuated severe burn-induced excessive inflammation via inhibiting activation of P38 and JNK signaling.

Cleome rutidosperma and Euphorbia thymifolia Suppress Inflammatory Response via Upregulation of Phase II Enzymes and Modulation of NF-κB and JNK Activation in LPS-Stimulated BV2 Microglia

PubMed Central

Ding, Hsiou-Yu; Wu, Pei-Shan; Wu, Ming-Jiuan

2016-01-01

Cleome rutidosperma DC. and Euphorbia thymifolia L. are herbal medicines used in traditional Indian and Chinese medicine to treat various illnesses. Reports document that they have antioxidant and anti-inflammatory activities; nonetheless, the molecular mechanisms involved in their anti-inflammatory actions have not yet been elucidated. The anti-neuroinflammatory activities and underlying mechanisms of ethanol extracts of Cleome rutidosperma (CR) and Euphorbia thymifolia (ET) were studied using lipopolysaccharide (LPS)-stimulated microglial cell line BV2. The morphology changes and production of pro-inflammatory mediators were assayed. Gene expression of inflammatory genes such as inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, interleukin (IL)-1β, and CC chemokine ligand (CCL)-2, as well as phase II enzymes such as heme oxygenase (HO)-1, the modifier subunit of glutamate cysteine ligase (GCLM) and NAD(P)H quinone dehydrogenase 1 (NQO1), were further investigated using reverse transcription quantitative-PCR (RT-Q-PCR) and Western blotting. The effects of CR and ET on mitogen activated protein kinases (MAPKs) and nuclear factor (NF)-κB signaling pathways were examined using Western blotting and specific inhibitors. CR and ET suppressed BV2 activation, down-regulated iNOS and COX-2 expression and inhibited nitric oxide (NO) overproduction without affecting cell viability. They reduced LPS-mediated tumor necrosis factor (TNF) and IL-6 production, attenuated IL-1β and CCL2 expression, but upregulated HO-1, GCLM and NQO1 expression. They also inhibited p65 NF-κB phosphorylation and modulated Jun-N terminal kinase (JNK) activation in BV2 cells. SP600125, the JNK inhibitor, significantly augmented the anti-IL-6 activity of ET. NF-κB inhibitor, Bay 11-7082, enhanced the anti-IL-6 effects of both CR and ET. Znpp, a competitive inhibitor of HO-1, attenuated the anti-NO effects of CR and ET. Our results show that CR and ET exhibit anti

The adaptor protein Cindr regulates JNK activity to maintain epithelial sheet integrity.

PubMed

Yasin, Hannah W R; van Rensburg, Samuel H; Feiler, Christina E; Johnson, Ruth I

2016-02-15

Epithelia are essential barrier tissues that must be appropriately maintained for their correct function. To achieve this a plethora of protein interactions regulate epithelial cell number, structure and adhesion, and differentiation. Here we show that Cindr (the Drosophila Cin85 and Cd2ap ortholog) is required to maintain epithelial integrity. Reducing Cindr triggered cell delamination and movement. Most delaminating cells died. These behaviors were consistent with JNK activation previously associated with loss of epithelial integrity in response to ectopic oncogene activity. We confirmed a novel interaction between Cindr and Drosophila JNK (dJNK), which when perturbed caused inappropriate JNK signaling. Genetically reducing JNK signaling activity suppressed the effects of reducing Cindr. Furthermore, ectopic JNK signaling phenocopied loss of Cindr and was partially rescued by concomitant cindr over-expression. Thus, correct Cindr-dJNK stoichiometry is essential to maintain epithelial integrity and disturbing this balance may contribute to the pathogenesis of disease states, including cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Function-Specific Intracellular Signaling Pathways Downstream of Heparin-Binding EGF-Like Growth Factor Utilized by Human Trophoblasts1

PubMed Central

Jessmon, Philip; Kilburn, Brian A.; Romero, Roberto; Leach, Richard E.; Armant, D. Randall

2010-01-01

Heparin-binding EGF-like growth factor (HBEGF) is expressed by trophoblast cells throughout gestation. First-trimester cytotrophoblast cells are protected from hypoxia-induced apoptosis because of the accumulation of HBEGF through a posttranscriptional autocrine mechanism. Exogenous application of HBEGF is cytoprotective in a hypoxia/reoxygenation (H/R) injury model and initiates trophoblast extravillous differentiation to an invasive phenotype. The downstream signaling pathways induced by HBEGF that mediate these various cellular activities were identified using two human first-trimester cytotrophoblast cell lines, HTR-8/SVneo and SW.71, with similar results. Recombinant HBEGF (1 nM) induced transient phosphorylation of MAPK3/1 (ERK), MAPK14 (p38), and AKT within 15 min and JNK after 1–2 h. To determine which downstream pathways regulate the various functions of HBEGF, cells were treated with specific inhibitors of the ERK upstream regulator MEK (U0126), the AKT upstream regulator phosphoinositide-3 (PI3)-kinase (LY294002), MAPK14 (SB203580), and JNK (SP600125), as well as with inactive structural analogues. Only SB203580 specifically prevented HBEGF-mediated rescue during H/R, while each inhibitor attenuated HBEGF-stimulated cell migration. Accumulation of HBEGF at reduced oxygen was blocked only by a combination of U0126, SB203580, and SP600125. We conclude that HBEGF advances trophoblast extravillous differentiation through coordinate activation of PI3 kinase, ERK, MAPK14, and JNK, while only MAPK14 is required for its antiapoptotic activity. Additionally, hypoxia induces an autocrine increase in HBEGF protein levels through MAPK14, JNK or ERK. These experiments reveal a complexity of the intracellular signaling circuitry that regulates trophoblast functions critical for implantation and placentation. PMID:20130271

Cytosolic calcium mediates RIP1/RIP3 complex-dependent necroptosis through JNK activation and mitochondrial ROS production in human colon cancer cells.

PubMed

Sun, Wen; Wu, Xiaxia; Gao, Hongwei; Yu, Jie; Zhao, Wenwen; Lu, Jin-Jian; Wang, Jinhua; Du, Guanhua; Chen, Xiuping

2017-07-01

Necroptosis is a form of programmed necrosis mediated by signaling complexes with receptor-interacting protein 1 (RIP1) and RIP3 kinases as the main mediators. However, the underlying execution pathways of this phenomenon have yet to be elucidated in detail. In this study, a RIP1/RIP3 complex was formed in 2-methoxy-6-acetyl-7-methyljuglone (MAM)-treated HCT116 and HT29 colon cancer cells. With this formation, mitochondrial reactive oxygen species (ROS) levels increased, mitochondrial depolarization occurred, and ATP concentrations decreased. This process was identified as necroptosis. This finding was confirmed by experiments showing that MAM-induced cell death was attenuated by the pharmacological or genetic blockage of necroptosis signaling, including RIP1 inhibitor necrostatin-1s (Nec-1s) and siRNA-mediated gene silencing of RIP1 and RIP3, but was unaffected by caspase inhibitor z-vad-fmk or necrosis inhibitor 2-(1H-Indol-3-yl)-3-pentylamino-maleimide (IM54). Transmission electron microscopy (TEM) analysis further revealed the ultrastructural features of MAM-induced necroptosis. MAM-induced RIP1/RIP3 complex triggered necroptosis through cytosolic calcium (Ca 2+ ) accumulation and sustained c-Jun N-terminal kinase (JNK) activation. Both calcium chelator BAPTA-AM and JNK inhibitor SP600125 could attenuate necroptotic features, including mitochondrial ROS elevation, mitochondrial depolarization, and ATP depletion. 2-thenoyltrifluoroacetone (TTFA), which is a mitochondrial complex II inhibitor, was found to effectively reverse both MAM induced mitochondrial ROS generation and cell death, indicating the complex II was the ROS-producing site. The essential role of mitochondrial ROS was confirmed by the protective effect of overexpression of manganese superoxide dismutase (MnSOD). MAM-induced necroptosis was independent of TNFα, p53, MLKL, and lysosomal membrane permeabilization. In summary, our study demonstrated that RIP1/RIP3 complex-triggered cytosolic calcium

Cannabinoid Receptor 2 Suppresses Leukocyte Inflammatory Migration by Modulating the JNK/c-Jun/Alox5 Pathway*

PubMed Central

Liu, Yi-Jie; Fan, Hong-Bo; Jin, Yi; Ren, Chun-Guang; Jia, Xiao-E; Wang, Lei; Chen, Yi; Dong, Mei; Zhu, Kang-Yong; Dong, Zhi-Wei; Ye, Bai-Xin; Zhong, Zhong; Deng, Min; Liu, Ting Xi; Ren, Ruibao

2013-01-01

Inflammatory migration of immune cells is involved in many human diseases. Identification of molecular pathways and modulators controlling inflammatory migration could lead to therapeutic strategies for treating human inflammation-associated diseases. The role of cannabinoid receptor type 2 (Cnr2) in regulating immune function had been widely investigated, but the mechanism is not fully understood. Through a chemical genetic screen using a zebrafish model for leukocyte migration, we found that both an agonist of the Cnr2 and inhibitor of the 5-lipoxygenase (Alox5, encoded by alox5) inhibit leukocyte migration in response to acute injury. These agents have a similar effect on migration of human myeloid cells. Consistent with these results, we found that inactivation of Cnr2 by zinc finger nuclease-mediated mutagenesis enhances leukocyte migration, while inactivation of Alox5 blocks leukocyte migration. Further investigation indicates that there is a signaling link between Cnr2 and Alox5 and that alox5 is a target of c-Jun. Cnr2 activation down-regulates alox5 expression by suppressing the JNK/c-Jun activation. These studies demonstrate that Cnr2, JNK, and Alox5 constitute a pathway regulating leukocyte migration. The cooperative effect between the Cnr2 agonist and Alox5 inhibitor also provides a potential therapeutic strategy for treating human inflammation-associated diseases. PMID:23539630

A ginseng metabolite, compound K, induces autophagy and apoptosis via generation of reactive oxygen species and activation of JNK in human colon cancer cells

PubMed Central

Kim, A D; Kang, K A; Kim, H S; Kim, D H; Choi, Y H; Lee, S J; Kim, H S; Hyun, J W

2013-01-01

Compound K (20-O-(β-D-glucopyranosyl)-20(S)-protopanaxadiol) is an active metabolite of ginsenosides and induces apoptosis in various types of cancer cells. This study investigated the role of autophagy in compound K-induced cell death of human HCT-116 colon cancer cells. Compound K activated an autophagy pathway characterized by the accumulation of vesicles, the increased positive acridine orange-stained cells, the accumulation of LC3-II, and the elevation of autophagic flux. Whereas blockade of compound K-induced autophagy by 3-methyladenein and bafilomycin A1 significantly increased cell viability. In addition, compound K augmented the time-dependent expression of the autophagy-related proteins Atg5, Atg6, and Atg7. However, knockdown of Atg5, Atg6, and Atg7 markedly inhibited the detrimental impact of compound K on LC3-II accumulation and cell vitality. Compound K-provoked autophagy was also linked to the generation of intracellular reactive oxygen species (ROS); both of these processes were mitigated by the pre-treatment of cells with the antioxidant N-acetylcysteine. Moreover, compound K activated the c-Jun NH2-terminal kinase (JNK) signaling pathway, whereas downregulation of JNK by its specific inhibitor SP600125 or by small interfering RNA against JNK attenuated autophagy-mediated cell death in response to compound K. Compound K also provoked apoptosis, as evidenced by an increased number of apoptotic bodies and sub-G1 hypodiploid cells, enhanced activation of caspase-3 and caspase-9, and modulation of Bcl-2 and Bcl-2-associated X protein expression. Notably, compound K-stimulated autophagy as well as apoptosis was induced by disrupting the interaction between Atg6 and Bcl-2. Taken together, these results indicate that the induction of autophagy and apoptosis by compound K is mediated through ROS generation and JNK activation in human colon cancer cells. PMID:23907464

Magnolol reduced TNF-α-induced vascular cell adhesion molecule-1 expression in endothelial cells via JNK/p38 and NF-κB signaling pathways.

PubMed

Liang, Chan-Jung; Lee, Chiang-Wen; Sung, Hsin-Ching; Chen, Yung-Hsiang; Wang, Shu-Huei; Wu, Pei-Jhen; Chiang, Yao-Chang; Tsai, Jaw-Shiun; Wu, Chau-Chung; Li, Chi-Yuan; Chen, Yuh-Lien

2014-01-01

Expression of cell adhesion molecules by the endothelium and the attachment of leukocytes to these cells play major roles in inflammation and cardiovascular disorders. Magnolol, a major active component of Magnolia officinalis, has antioxidative and anti-inflammatory properties. In the present study, the effects of magnolol on the expression of vascular cell adhesion molecule-1 (VCAM-1) in human aortic endothelial cells (HAECs) and the related mechanisms were investigated. TNF-α induced VCAM-1 protein expression and mRNA stability were significantly decreased in HAECs pre-treated with magnolol. Magnolol significantly reduced the phosphorylation of ERK, JNK, and p38 in TNF-α-treated HAECs. The decrease in VCAM-1 expression in response to TNF-α treatment was affected by JNK and p38 inhibitors, not by an ERK inhibitor. Magnolol also attenuates NF-κB activation and the translocation of HuR (an RNA binding protein) in TNF-α-stimulated HAECs. The VCAM-1 expression was weaker in the aortas of TNF-α-treated apo-E deficient mice with magnolol treatment. These data demonstrate that magnolol inhibits TNF-α-induced JNK/p38 phosphorylation, HuR translocation, NF-κB activation, and thereby suppresses VCAM-1 expression resulting in reduced leukocyte adhesion. Taken together, these results suggest that magnolol has an anti-inflammatory property and may play an important role in the prevention of atherosclerosis and inflammatory responses.

Lymphomagenic CARD11/BCL10/MALT1 signaling drives malignant B-cell proliferation via cooperative NF-κB and JNK activation.

PubMed

Knies, Nathalie; Alankus, Begüm; Weilemann, Andre; Tzankov, Alexandar; Brunner, Kristina; Ruff, Tanja; Kremer, Marcus; Keller, Ulrich B; Lenz, Georg; Ruland, Jürgen

2015-12-29

The aggressive activated B cell-like subtype of diffuse large B-cell lymphoma is characterized by aberrant B-cell receptor (BCR) signaling and constitutive nuclear factor kappa-B (NF-κB) activation, which is required for tumor cell survival. BCR-induced NF-κB activation requires caspase recruitment domain-containing protein 11 (CARD11), and CARD11 gain-of-function mutations are recurrently detected in human diffuse large B-cell lymphoma (DLBCL). To investigate the consequences of dysregulated CARD11 signaling in vivo, we generated mice that conditionally express the human DLBCL-derived CARD11(L225LI) mutant. Surprisingly, CARD11(L225LI) was sufficient to trigger aggressive B-cell lymphoproliferation, leading to early postnatal lethality. CARD11(L225LI) constitutively associated with B-cell CLL/lymphoma 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) to simultaneously activate the NF-κB and c-Jun N-terminal kinase (JNK) signaling cascades. Genetic deficiencies of either BCL10 or MALT1 completely rescued the phenotype, and pharmacological inhibition of JNK was, similar to NF-κB blockage, toxic to autonomously proliferating CARD11(L225LI)-expressing B cells. Moreover, constitutive JNK activity was observed in primary human activated B cell-like (ABC)-DLBCL specimens, and human ABC-DLBCL cells were also sensitive to JNK inhibitors. Thus, our results demonstrate that enforced activation of CARD11/BCL10/MALT1 signaling is sufficient to drive transformed B-cell expansion in vivo and identify the JNK pathway as a therapeutic target for ABC-DLBCL.

EF1A1/HSC70 Cooperatively Suppress Brain Endothelial Cell Apoptosis via Regulating JNK Activity.

PubMed

Liu, Ying; Jiang, Shu; Yang, Peng-Yuan; Zhang, Yue-Fan; Li, Tie-Jun; Rui, Yao-Cheng

2016-10-01

In our previous study, eEF1A1 was identified to be a new target for protecting brain ischemia injury, but the mechanism remains largely unknown. In this study, we screened the downstream cellular protein molecules interacted with eEF1A1 and found mechanism of eEF1A1 in brain ischemia protection. Through co-immunoprecipitation and mass spectrometry for searching the interaction of proteins with eEF1A1 in bEnd3 cells, HSC70 was identified to be a binding protein of eEF1A1, which was further validated by Western blot and immunofluorescence. eEF1A1 or HSC70 knockdown, respectively, increased OGD-induced apoptosis of brain vascular endothelial cells, which was detected by Annexin V-FITC/PI staining. HSC70 or eEF1A1 knockdown enhances phosphorylated JNK, phosphorylation of c-JUN (Ser63, Ser73), cleaved caspase-9, and cleaved caspase-3 expression, which could be rescued by JNK inhibitor. In summary, our data suggest that the presence of chaperone forms of interaction between eEF1A1 and HSC70 in brain vascular endothelial cells, eEF1A1 and HSC70 can play a protective role in the process of ischemic stroke by inhibiting the JNK signaling pathway activation. © 2016 John Wiley & Sons Ltd.

The MAP kinase JNK2 mediates cigarette smoke-induced arterial thrombosis.

PubMed

Breitenstein, Alexander; Stämpfli, Simon F; Reiner, Martin F; Shi, Yi; Keller, Stephan; Akhmedov, Alexander; Schaub Clerigué, Ariane; Spescha, Remo D; Beer, Hans-Jürg; Lüscher, Thomas F; Tanner, Felix C; Camici, Giovanni G

2017-01-05

Despite public awareness of its deleterious effects, smoking remains a major cause of death. Indeed, it is a risk factor for atherothrombotic complications and in line with this, the introduction of smoking ban in public areas reduced smoking-associated cardiovascular complications. Nonetheless, smoking remains a major concern, and molecular mechanisms by which it causes cardiovascular disease are not known. Peripheral blood monocytes from healthy smokers displayed increased JNK2 and tissue factor (TF) gene expression compared to non-smokers (n=15, p<0.05). Similarly, human aortic endothelial cells exposed to cigarette smoke total particulate matter (CS-TPM) revealed increased TF expression mediated by JNK2 (n=4; p<0.05). Wild-type and JNK2 -/- mice were exposed to cigarette smoke for two weeks after which arterial thrombosis was investigated. Wild-type mice exposed to smoke displayed reduced time to thrombotic arterial occlusion (n=8; p<0.05) and increased tissue factor activity (n=7; p<0.05) as compared to wild-type controls (n=6), while JNK2 -/- mice exposed to smoke maintained an unaltered thrombotic potential (n=8; p=NS) and tissue factor activity (n=8) comparable to that of JNK2 -/- and wild-type controls (n=6; p=NS). Smoking caused an increased production of reactive oxygen species (ROS) in wild-type but not in JNK2 -/- mice (n=7; p<0.05 for wild-type mice and n=5-6; p=NS for JNK2 -/- mice). In conclusion, the MAP kinase JNK2 mediates cigarette smoke-induced TF activation, arterial thrombosis and ROS production. These results underscore a major role of JNK2 in smoke-mediated thrombus formation and may offer an attractive target to prevent smoke-related thrombosis in those subjects which do not manage quitting.

c-Jun localizes to the nucleus independent of its phosphorylation by and interaction with JNK and vice versa promotes nuclear accumulation of JNK

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schreck, Ilona; Al-Rawi, Marco; Mingot, Jose-Manuel

2011-04-22

Highlights: {yields} HSP70, Ku70 and 80 as well as importin 8 are novel interactors of c-Jun. {yields} Nuclear accumulation of c-Jun does not require its functions as a transcription factor. {yields} Nuclear accumulation of c-Jun does not require the interaction with its kinase JNK. {yields} Nuclear accumulation of JNK is regulated by interaction with c-Jun. -- Abstract: In order to activate gene expression, transcription factors such as c-Jun have to reside in the nucleus. The abundance of c-Jun in the nucleus correlates with the activity of its target genes. As a consequence of excessive c-Jun activation, cells undergo apoptosis ormore » changes in differentiation whereas decreased c-Jun function can reduce proliferation. In the present study we addressed how nuclear accumulation of the transcription factor c-Jun is regulated. First, we analyzed which functions of c-Jun are required for efficient nuclear accumulation. Mutants of c-Jun deficient in dimerization or DNA-binding show no defect in nuclear transport. Furthermore, c-Jun import into the nucleus of living cells occurred when the c-Jun phosphorylation sites were mutated as well in cells that lack the major c-Jun kinase, JNK, suggesting that c-Jun transport into the nucleus does not require JNK signaling. Conversely, however, binding of c-Jun seemed to enhance nuclear accumulation of JNK. In order to identify proteins that might be relevant for the nuclear translocation of c-Jun we searched for novel binding partners by a proteomic approach. In addition to the heat shock protein HSP70 and the DNA damage repair factors Ku70 and 80, we isolated human importin 8 as a novel interactor of c-Jun. Interaction of Imp 8 with c-Jun in human cells was confirmed by co-immunoprecipitation experiments. Nuclear accumulation of c-Jun does not require its functions as a transcription factor or the interaction with its kinase JNK. Interestingly, nuclear accumulation of JNK is regulated by interaction with c-Jun. Unraveling

Role of JNK isoforms in the kainic acid experimental model of epilepsy and neurodegeneration.

PubMed

Auladell, Carme; de Lemos, Luisa; Verdaguer, Ester; Ettcheto, Miren; Busquets, Oriol; Lazarowski, Alberto; Beas-Zarate, Carlos; Olloquequi, Jordi; Folch, Jaume; Camins, Antoni

2017-01-01

Chemoconvulsants that induce status epilepticus in rodents have been widely used over the past decades due to their capacity to reproduce with high similarity neuropathological and electroencephalographic features observed in patients with temporal lobe epilepsy (TLE). Kainic acid  is one of the most used chemoconvulsants in experimental models. KA administration mainly induces neuronal loss in the hippocampus. We focused the present review inthe c-Jun N-terminal kinase-signaling pathway (JNK), since it has been shown to play a key role in the process of neuronal death following KA activation. Among the three isoforms of JNK (JNK1, JNK2, JNK3), JNK3 is widely localized in the majority of areas of the hippocampus, whereas JNK1 levels are located exclusively in the CA3 and CA4 areas and in dentate gyrus. Disruption of the gene encoding JNK3 in mice renders neuroprotection to KA, since these animals showed a reduction in seizure activity and a diminution in hippocampal neuronal apoptosis. In light of this, JNK3 could be a promising subcellular target for future therapeutic interventions in epilepsy.

TNF{alpha} acting on TNFR1 promotes breast cancer growth via p42/P44 MAPK, JNK, Akt and NF-{kappa}B-dependent pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rivas, Martin A.; Carnevale, Romina P.; Proietti, Cecilia J.

2008-02-01

Tumor necrosis factor {alpha} (TNF{alpha}) enhances proliferation of chemically-induced mammary tumors and of T47D human cell line through not fully understood pathways. Here, we explored the intracellular signaling pathways triggered by TNF{alpha}, the participation of TNF{alpha} receptor (TNFR) 1 and TNFR2 and the molecular mechanism leading to breast cancer growth. We demonstrate that TNF{alpha} induced proliferation of C4HD murine mammary tumor cells and of T47D cells through the activation of p42/p44 MAPK, JNK, PI3-K/Akt pathways and nuclear factor-kappaB (NF-{kappa}B) transcriptional activation. A TNF{alpha}-specific mutein selectively binding to TNFR1 induced p42/p44 MAPK, JNK, Akt activation, NF-{kappa}B transcriptional activation and cell proliferation,more » just like wild-type TNF{alpha}, while a mutein selective for TNFR2 induced only p42/p44 MAPK activation. Interestingly, blockage of TNFR1 or TNFR2 with specific antibodies was enough to impair TNF{alpha} signaling and biological effect. Moreover, in vivo TNF{alpha} administration supported C4HD tumor growth. We also demonstrated, for the first time, that injection of a selective inhibitor of NF-{kappa}B activity, Bay 11-7082, resulted in regression of TNF{alpha}-promoted tumor. Bay 11-7082 blocked TNF{alpha} capacity to induce cell proliferation and up-regulation of cyclin D1 and of Bcl-x{sub L}in vivo and in vitro. Our results reveal evidence for TNF{alpha} as a breast tumor promoter, and provide novel data for a future therapeutic approach using TNF{alpha} antagonists and NF-{kappa}B pharmacological inhibitors in established breast cancer treatment.« less

Fluid Shear Stress-Induced JNK Activity Leads to Actin Remodeling for Cell Alignment

PubMed Central

Mengistu, Meron; Brotzman, Hannah; Ghadiali, Samir; Lowe-Krentz, Linda

2012-01-01

Fluid shear stress (FSS) exerted on endothelial cell surfaces induces actin cytoskeleton remodeling through mechanotransduction. This study was designed to determine whether FSS activates Jun N-terminal kinase (JNK), to examine the spatial and temporal distribution of active JNK relative to the actin cytoskeleton in endothelial cells exposed to different FSS conditions, and to evaluate the effects of active JNK on actin realignment. Exposure to 15 and 20 dyn/cm2 FSS induced higher activity levels of JNK than the lower 2 and 4 dyn/cm2 flow conditions. At the higher FSS treatments, JNK activity increased with increasing exposure time, peaking 30 minutes after flow onset with an 8-fold activity increase compared to cells in static culture. FSS-induced phospho-JNK co-localized with actin filaments at cell peripheries, as well as with stress fibers. Pharmacologically blocking JNK activity altered FSS-induced actin structure and distribution as a response to FSS. Our results indicate that FSS-induced actin remodeling occurs in three phases, and that JNK plays a role in at least one, suggesting that this kinase activity is involved in mechanotransduction from the apical surface to the actin cytoskeleton in endothelial cells. PMID:20626006

Stathmin Mediates Hepatocyte Resistance to Death from Oxidative Stress by down Regulating JNK

PubMed Central

Zhao, Enpeng; Amir, Muhammad; Lin, Yu; Czaja, Mark J.

2014-01-01

Stathmin 1 performs a critical function in cell proliferation by regulating microtubule polymerization. This proliferative function is thought to explain the frequent overexpression of stathmin in human cancer and its correlation with a bad prognosis. Whether stathmin also functions in cell death pathways is unclear. Stathmin regulates microtubules in part by binding free tubulin, a process inhibited by stathmin phosphorylation from kinases including c-Jun N-terminal kinase (JNK). The involvement of JNK activation both in stathmin phosphorylation, and in hepatocellular resistance to oxidative stress, led to an examination of the role of stathmin/JNK crosstalk in oxidant-induced hepatocyte death. Oxidative stress from menadione-generated superoxide induced JNK-dependent stathmin phosphorylation at Ser-16, Ser-25 and Ser-38 in hepatocytes. A stathmin knockdown sensitized hepatocytes to both apoptotic and necrotic cell death from menadione without altering levels of oxidant generation. The absence of stathmin during oxidative stress led to JNK overactivation that was the mechanism of cell death as a concomitant knockdown of JNK1 or JNK2 blocked death. Hepatocyte death from JNK overactivation was mediated by the effects of JNK on mitochondria. Mitochondrial outer membrane permeabilization occurred in stathmin knockdown cells at low concentrations of menadione that triggered apoptosis, whereas mitochondrial β-oxidation and ATP homeostasis were compromised at higher, necrotic menadione concentrations. Stathmin therefore mediates hepatocyte resistance to death from oxidative stress by down regulating JNK and maintaining mitochondrial integrity. These findings demonstrate a new mechanism by which stathmin promotes cell survival and potentially tumor growth. PMID:25285524

Hippo signaling promotes JNK-dependent cell migration.

PubMed

Ma, Xianjue; Wang, Hongxiang; Ji, Jiansong; Xu, Wenyan; Sun, Yihao; Li, Wenzhe; Zhang, Xiaoping; Chen, Juxiang; Xue, Lei

2017-02-21

Overwhelming studies show that dysregulation of the Hippo pathway is positively correlated with cell proliferation, growth, and tumorigenesis. Paradoxically, the detailed molecular roles of the Hippo pathway in cell invasion remain debatable. Using a Drosophila invasion model in wing epithelium, we show herein that activated Hippo signaling promotes cell invasion and epithelial-mesenchymal transition through JNK, as inhibition of JNK signaling dramatically blocked Hippo pathway activation-induced matrix metalloproteinase 1 expression and cell invasion. Furthermore, we identify bantam -Rox8 modules as essential components downstream of Yorkie in mediating JNK-dependent cell invasion. Finally, we confirm that YAP (Yes-associated protein) expression negatively regulates TIA1 (Rox8 ortholog) expression and cell invasion in human cancer cells. Together, these findings provide molecular insights into Hippo pathway-mediated cell invasion and also raise a noteworthy concern in therapeutic interventions of Hippo-related cancers, as simply inhibiting Yorkie or YAP activity might paradoxically accelerate cell invasion and metastasis.

Increased level of apoptosis in rat brains and SH-SY5Y cells exposed to excessive fluoride--a mechanism connected with activating JNK phosphorylation.

PubMed

Liu, Yan-Jie; Guan, Zhi-Zhong; Gao, Qin; Pei, Jin-Jing

2011-07-28

In order to reveal the mechanism of the brain injury induced by chronic fluorosis, the levels of apoptosis and c-Jun N-terminal kinases (JNK) in brains of rats and SH-SY5Y cells exposed to different concentrations of sodium fluoride (NaF) were detected. The dental fluorosis and fluoride contents in blood, urine and bones of rats were measured to evaluate the exhibition of fluorosis. The apoptotic death rate was measured by flow cytometry and the expression of JNK at protein level by Western blotting. The results showed that as compared with controls, the apoptotic death rate was obviously increased in brains of the rats exposed to high-fluoride (50ppm) for 6 months with a concentration dependent manner, but no significant change for 3 months. In SH-SY5Y cells treated with high concentration (50ppm) of fluoride, the increased apoptotic death rate was obviously observed as compared to controls. In addition, the expressions of phospho-JNK at protein level were raised by 20.5% and 107.6%, respectively, in brains of the rats exposed to low-fluoride (5ppm) and high-fluoride for 6 months; while no significant changes were found between the rats exposed to fluoride and the controls for 3 months. The protein level of phospho-JNK was also increased in SH-SY5Y cells exposed to high-fluoride. There were no changes of total-JNK both in the rats and in the SH-SY5Y cells exposed to excessive fluoride as compared to controls. When SH-SY5Y cells were singly treated with SP600125, an inhibitor of phospho-JNK, the decreased expression of phospho-JNK, but no apoptosis, was detected. Interestingly, after JNK phosphorylation in the cultured cells was inhibited by SP600125, the treatment with high-fluoride did not induce the increase of apoptosis. In addition, there was a positive correlation between the expression of phospho-JNK and the apoptotic death rate in rat brains or SH-SY5Y cells treated with high-fluoride. The results indicated that exposure to excessive fluoride resulted in

The tumor promoter arsenite stimulates AP-1 activity by inhibiting a JNK phosphatase.

PubMed Central

Cavigelli, M; Li, W W; Lin, A; Su, B; Yoshioka, K; Karin, M

1996-01-01

Trivalent arsenic (As3+) is highly carcinogenic, but devoid of known mutagenic activity. Therefore, it is likely to act as a tumor promoter. To understand the molecular basis for the tumor-promoting activity of As3+, we examined its effect on transcription factor AP-1, whose activity is stimulated by several other tumor promoters. We found that As3+, but not As5+, which is toxic but not carcinogenic, is a potent stimulator of AP-1 transcriptional activity and an efficient inducer of c-fos and c-jun gene expression. Induction of c-jun and c-fos transcription by As3+ correlates with activation of Jun kinases (JNKs) and p38/Mpk2, which phosphorylate transcription factors that activate these immediate early genes. No effect on ERK activity was observed. As5+, on the other hand, had a negligible effect on JNK or p38/Mpk2 activity. Biochemical analysis and co-transfection experiments strongly suggest that the primary mechanism by which As3+ stimulates JNK activity involves the inhibition of a constitutive dual-specificity JNK phosphatase. This phosphatase activity appears to be responsible for maintaining low basal JNK activity in non-stimulated cells and its inhibition may lead to tumor promotion through induction of proto-oncogenes such as c-jun and c-fos, and stimulation of AP-1 activity. The same phosphatase may also regulate p38/Mpk2 activity. Images PMID:8947050

Anti-proliferation of triple-negative breast cancer cells with physagulide P: ROS/JNK signaling pathway induces apoptosis and autophagic cell death

PubMed Central

Gao, Cai-Yun; Ma, Ting; Zhang, Hao; Zhou, Miao-Miao; Yang, Yan-Wei; Yang, Lei; Kong, Ling-Yi

2017-01-01

Physagulide P (PP), a new natural compound, was isolated from Physalis angulate L. in our laboratory. In this study, we demonstrated that PP potently suppressed cell proliferation by inducing G2/M phase arrest in MDA-MB-231 and MDA-MB-468 cells. Moreover, PP provoked apoptosis by decreasing the mitochondrial membrane potential and elevating the Bax/Bcl-2 protein expression ratio. The caspase inhibitor Z-VAD-FMK partly restore cell viability, suggesting that apoptosis plays as an important role in the anti-proliferative effect of PP. PP-treated cells also underwent autophagy, as evidenced by the formation of autophagosomes and the accumulation of LC3BII. Furthermore, the knockdown of LC3B reduced PP-induced cytotoxicity, indicating that autophagy played an anticancer effect. PP also induced the generation of reactive oxygen species (ROS) and resulted in c-Jun N-terminal kinases (JNK) activation. Accordingly, JNK siRNA significantly attenuated PP-triggered apoptosis and autophagy, and ROS scavengers almost completely reverse this apoptosis and autophagy. The ROS scavenger also blocked PP-induced G2/M phase arrest and the phosphorylation of JNK. Our results revealed that PP induced G2/M phase arrest, apoptosis and autophagy via the ROS/JNK signaling pathway in MDA-MB-231 and MDA-MB-468 cells. Therefore, PP is a promising candidate for the development of antitumor drugs for the treatment of triple-negative breast cancer. PMID:28969050

A Low-Level Carbon Dioxide Laser Promotes Fibroblast Proliferation and Migration through Activation of Akt, ERK, and JNK

PubMed Central

Shingyochi, Yoshiaki; Kanazawa, Shigeyuki; Tajima, Satoshi; Tanaka, Rica; Mizuno, Hiroshi; Tobita, Morikuni

2017-01-01

Background Low-level laser therapy (LLLT) with various types of lasers promotes fibroblast proliferation and migration during the process of wound healing. Although LLLT with a carbon dioxide (CO2) laser was also reported to promote wound healing, the underlying mechanisms at the cellular level have not been previously described. Herein, we investigated the effect of LLLT with a CO2 laser on fibroblast proliferation and migration. Materials and Methods Cultured human dermal fibroblasts were prepared. MTS and cell migration assays were performed with fibroblasts after LLLT with a CO2 laser at various doses (0.1, 0.5, 1.0, 2.0, or 5.0 J/cm2) to observe the effects of LLLT with a CO2 laser on the proliferation and migration of fibroblasts. The non-irradiated group served as the control. Moreover, western blot analysis was performed using fibroblasts after LLLT with a CO2 laser to analyze changes in the activities of Akt, extracellular signal-regulated kinase (ERK), and Jun N-terminal kinase (JNK), which are signaling molecules associated with cell proliferation and migration. Finally, the MTS assay, a cell migration assay, and western blot analysis were performed using fibroblasts treated with inhibitors of Akt, ERK, or JNK before LLLT with a CO2 laser. Results In MTS and cell migration assays, fibroblast proliferation and migration were promoted after LLLT with a CO2 laser at 1.0 J/cm2. Western blot analysis revealed that Akt, ERK, and JNK activities were promoted in fibroblasts after LLLT with a CO2 laser at 1.0 J/cm2. Moreover, inhibition of Akt, ERK, or JNK significantly blocked fibroblast proliferation and migration. Conclusions These findings suggested that LLLT with a CO2 laser would accelerate wound healing by promoting the proliferation and migration of fibroblasts. Activation of Akt, ERK, and JNK was essential for CO2 laser-induced proliferation and migration of fibroblasts. PMID:28045948

Calcineurin inhibitors recruit protein kinases JAK2 and JNK, TLR signaling and the UPR to activate NF-κB-mediated inflammatory responses in kidney tubular cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

González-Guerrero, Cristian, E-mail: cristian.gonzalez@fjd.es; Ocaña-Salceda, Carlos, E-mail: carlos.ocana@fjd.es; Berzal, Sergio, E-mail: sberzal@fjd.es

The calcineurin inhibitors (CNIs) cyclosporine (CsA) and tacrolimus are key drugs in current immunosuppressive regimes for solid organ transplantation. However, they are nephrotoxic and promote death and profibrotic responses in tubular cells. Moreover, renal inflammation is observed in CNI nephrotoxicity but the mechanisms are poorly understood. We have now studied molecular pathways leading to inflammation elicited by the CNIs in cultured and kidney tubular cells. Both CsA and tacrolimus elicited a proinflammatory response in tubular cells as evidenced by a transcriptomics approach. Transcriptomics also suggested several potential pathways leading to expression of proinflammatory genes. Validation and functional studies disclosed thatmore » in tubular cells, CNIs activated protein kinases such as the JAK2/STAT3 and TAK1/JNK/AP-1 pathways, TLR4/Myd88/IRAK signaling and the Unfolded Protein Response (UPR) to promote NF-κB activation and proinflammatory gene expression. CNIs also activated an Nrf2/HO-1-dependent compensatory response and the Nrf2 activator sulforaphane inhibited JAK2 and JNK activation and inflammation. A murine model of CsA nephrotoxicity corroborated activation of the proinflammatory pathways identified in cell cultures. Human CNIs nephrotoxicity was also associated with NF-κB, STAT3 and IRE1α activation. In conclusion, CNIs recruit several intracellular pathways leading to previously non-described proinflammatory actions in renal tubular cells. Identification of these pathways provides novel clues for therapeutic intervention to limit CNIs nephrotoxicity. - Highlights: • Molecular mechanisms modulating CNI renal inflammation were investigated. • Kinases, immune receptors and ER stress mediate the inflammatory response to CNIs. • Several intracellular pathways activate NF-κB in CNIs-treated tubular cells. • A NF-κB-dependent cytokine profile characterizes CNIs-induced inflammation. • CNI nephrotoxicity was associated to

Spatiotemporal regulation of ERK2 by dual specificity phosphatases.

PubMed

Caunt, Christopher J; Armstrong, Stephen P; Rivers, Caroline A; Norman, Michael R; McArdle, Craig A

2008-09-26

Although many stimuli activate extracellular signal-regulated kinases 1 and 2 (ERK1/2), the kinetics and compartmentalization of ERK1/2 signals are stimulus-dependent and dictate physiological consequences. ERKs can be inactivated by dual specificity phosphatases (DUSPs), notably the MAPK phosphatases (MKPs) and atypical DUSPs, that can both dephosphorylate and scaffold ERK1/2. Using a cell imaging model (based on knockdown of endogenous ERKs and add-back of wild-type or mutated ERK2-GFP reporters), we explored possible effects of DUSPs on responses to transient or sustained ERK2 activators (epidermal growth factor and phorbol 12,13-dibutyrate, respectively). For both stimuli, a D319N mutation (which impairs DUSP binding) increased ERK2 activity and reduced nuclear accumulation. These stimuli also increased mRNA levels for eight DUSPs. In a short inhibitory RNA screen, 12 of 16 DUSPs influenced ERK2 responses. These effects were evident among nuclear inducible MKP, cytoplasmic ERK MKP, JNK/p38 MKP, and atypical DUSP subtypes and, with the exception of the nuclear inducible MKPs, were paralleled by corresponding changes in Egr-1 luciferase activation. Simultaneous removal of all JNK/p38 MKPs or nuclear inducible MKPs revealed them as positive and negative regulators of ERK2 signaling, respectively. The effects of JNK/p38 MKP short inhibitory RNAs were not dependent on protein neosynthesis but were reversed in the presence of JNK and p38 kinase inhibitors, indicating DUSP-mediated cross-talk between MAPK pathways. Overall, our data reveal that a large number of DUSPs influence ERK2 signaling. Together with the known tissue-specific expression of DUSPs and the importance of ERK1/2 in cell regulation, our data support the potential value of DUSPs as targets for drug therapy.

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

PubMed Central

Zeke, András; Misheva, Mariya

2016-01-01

SUMMARY The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. PMID:27466283

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships.

PubMed

Zeke, András; Misheva, Mariya; Reményi, Attila; Bogoyevitch, Marie A

2016-09-01

The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55γ as a Stress-Sensitive Inhibitor of c-SRC

PubMed Central

Eichhorn, Pieter J. A; Creyghton, Menno P; Wilhelmsen, Kevin; van Dam, Hans; Bernards, René

2007-01-01

Protein Phosphatase type 2A (PP2A) represents a family of holoenzyme complexes with diverse biological activities. Specific holoenzyme complexes are thought to be deregulated during oncogenic transformation and oncogene-induced signaling. Since most studies on the role of this phosphatase family have relied on the use of generic PP2A inhibitors, the contribution of individual PP2A holoenzyme complexes in PP2A-controlled signaling pathways is largely unclear. To gain insight into this, we have constructed a set of shRNA vectors targeting the individual PP2A regulatory subunits for suppression by RNA interference. Here, we identify PR55γ and PR55δ as inhibitors of c-Jun NH2-terminal kinase (JNK) activation by UV irradiation. We show that PR55γ binds c-SRC and modulates the phosphorylation of serine 12 of c-SRC, a residue we demonstrate to be required for JNK activation by c-SRC. We also find that the physical interaction between PR55γ and c-SRC is sensitive to UV irradiation. Our data reveal a novel mechanism of c-SRC regulation whereby in response to stress c-SRC activity is regulated, at least in part, through loss of the interaction with its inhibitor, PR55γ. PMID:18069897

Blocking autophagy enhances the apoptotic effect of 18β-glycyrrhetinic acid on human sarcoma cells via endoplasmic reticulum stress and JNK activation.

PubMed

Shen, Shuying; Zhou, Menglu; Huang, Kangmao; Wu, Yizheng; Ma, Yan; Wang, Jiying; Ma, Jianjun; Fan, Shunwu

2017-09-21

Sarcoma, a rare form of cancer, is unlike the much more common carcinomas as it occurs in a distinct type of tissue. The potent antitumor effects of 18β-glycyrrhetinic acid (GA), a novel naturally derived agent, have been demonstrated in various cancers. However, the effect of GA on human sarcoma, and the underlying mechanisms, remain to be elucidated. In the current study, we show that GA inhibits sarcoma cell proliferation by inducing G0/G1-phase arrest. Exposure to GA resulted in the activation of caspase-3, -8, and -9, indicating that GA induced apoptosis through both extrinsic and intrinsic pathways. In addition, the autophagy pathway, characterized by the conversion of LC3-I to LC3- II, was activated, resulting in increased Beclin-1 protein levels, decreased p62 expression, and stimulation of autophagic flux. The present findings showed that GA stimulated autophagy by inducing endoplasmic reticulum (ER) stress via the IRE1-JNK pathway. Our data supported the prosurvival role of GA-induced autophagy when the autophagy pathway was blocked with specific chemical inhibitors. Finally, GA markedly reduced sarcoma growth, with little organ-related toxicity, in vivo. The present results suggest that the combination of GA with a specific autophagy inhibitor represents a promising therapeutic approach for the treatment of sarcoma.

Inhibition of c-Jun N-terminal kinase sensitizes tumor cells to flavonoid-induced apoptosis through down-regulation of JunD

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kook, Sung-Ho; Research Center of Bioactive Materials, Chonbuk National University, Chonju 561-756; Son, Young-Ok

Reduction of susceptibility to apoptosis signals is a crucial step in carcinogenesis. Therefore, sensitization of tumor cells to apoptosis is a promising therapeutic strategy. c-Jun NH{sub 2}-terminal kinase (JNK) has been implicated in stress-induced apoptosis. However, many studies also emphasize the role of JNK on cell survival, although its mechanisms are not completely understood. Previously, we found that inhibition of JNK activity promotes flavonoid-mediated apoptosis of human osteosarcoma cells. We thus determined whether inhibition of JNK sensitizes tumor cells to a bioflavonoid-induced apoptosis, and whether this effect of JNK is a general effect. As the results, quercetin and genistein asmore » well as a flavonoid fraction induced apoptosis of tumor cells, which was further accelerated by specific JNK inhibitor, SP600125 or by small interfering RNA specific to JNK1/2. This effect was specific to types of cells because it was further apparent in tumorigenic cell lines. Inhibition of JNK by SP600125 also reduced flavonoid-stimulated nuclear induction of JunD which was known to have protective role in apoptosis, whereas JNK inhibition alone had little effect on apoptosis. The flavonoid-induced apoptosis of tumor cells was significantly enhanced by transfecting them with antisense JunD oligonucleotides. These results suggest that inhibition of JNK facilitates flavonoid-induced apoptosis through down-regulation of JunD, which is further sensitive to tumor cells. Therefore, combination with a specific JNK inhibitor further enhances the anti-cancer and chemopreventive potential of bio-flavonoids.« less

PDE5 Inhibitors Enhance Celecoxib Killing in Multiple Tumor Types

PubMed Central

BOOTH, LAURENCE; ROBERTS, JANE L.; CRUICKSHANKS, NICHOLA; TAVALLAI, SEYEDMEHRAD; WEBB, TIMOTHY; SAMUEL, PETER; CONLEY, ADAM; BINION, BRITTANY; YOUNG, HAROLD F.; POKLEPOVIC, ANDREW; SPIEGEL, SARAH; DENT, PAUL

2015-01-01

The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with a clinically relevant NSAID, celecoxib, to kill tumor cells. Celecoxib and PDE5 inhibitors interacted in a greater than additive fashion to kill multiple tumor cell types. Celecoxib and sildenafil killed ex vivo primary human glioma cells as well as their associated activated microglia. Knock down of PDE5 recapitulated the effects of PDE5 inhibitor treatment; the nitric oxide synthase inhibitor L-NAME suppressed drug combination toxicity. The effects of celecoxib were COX2 independent. Over-expression of c-FLIP-s or knock down of CD95/FADD significantly reduced killing by the drug combination. CD95 activation was dependent on nitric oxide and ceramide signaling. CD95 signaling activated the JNK pathway and inhibition of JNK suppressed cell killing. The drug combination inactivated mTOR and increased the levels of autophagy and knock down of Beclin1 or ATG5 strongly suppressed killing by the drug combination. The drug combination caused an ER stress response; knock down of IRE1α/XBP1 enhanced killing whereas knock down of eIF2α/ATF4/CHOP suppressed killing. Sildenafil and celecoxib treatment suppressed the growth of mammary tumors in vivo. Collectively our data demonstrate that clinically achievable concentrations of celecoxib and sildenafil have the potential to be a new therapeutic approach for cancer. PMID:25303541

Protocatechuic Acid from Alpinia oxyphylla Induces Schwann Cell Migration via ERK1/2, JNK and p38 Activation.

PubMed

Ju, Da-Tong; Kuo, Wei-Wen; Ho, Tsung-Jung; Paul, Catherine Reena; Kuo, Chia-Hua; Viswanadha, Vijaya Padma; Lin, Chien-Chung; Chen, Yueh-Sheng; Chang, Yung-Ming; Huang, Chih-Yang

2015-01-01

Alpinia oxyphylla MIQ (Alpinate Oxyphyllae Fructus, AOF) is an important traditional Chinese medicinal herb whose fruits is widely used to prepare tonics and is used as an aphrodisiac, anti salivary, anti diuretic and nerve-protective agent. Protocatechuic acid (PCA), a simple phenolic compound was isolated from the kernels of AOF. This study investigated the role of PCA in promoting neural regeneration and the underlying molecular mechanisms. Nerve regeneration is a complex physiological response that takes place after injury. Schwann cells play a crucial role in the endogenous repair of peripheral nerves due to their ability to proliferate and migrate. The role of PCA in Schwann cell migration was determined by assessing the induced migration potential of RSC96 Schwann cells. PCA induced changes in the expression of proteins of three MAPK pathways, as determined using Western blot analysis. In order to determine the roles of MAPK (ERK1/2, JNK, and p38) pathways in PCA-induced matrix-degrading proteolytic enzyme (PAs and MMP2/9) production, the expression of several MAPK-associated proteins was analyzed after siRNA-mediated inhibition assays. Treatment with PCA-induced ERK1/2, JNK, and p38 phosphorylation that activated the downstream expression of PAs and MMPs. PCA-stimulated ERK1/2, JNK and p38 phosphorylation was attenuated by individual pretreatment with siRNAs or MAPK inhibitors (U0126, SP600125, and SB203580), resulting in the inhibition of migration and the uPA-related signal pathway. Taken together, our data suggest that PCA extract regulate the MAPK (ERK1/2, JNK, and p38)/PA (uPA, tPA)/MMP (MMP2, MMP9) mediated regeneration and migration signaling pathways in Schwann cells. Therefore, PCA plays a major role in Schwann cell migration and the regeneration of damaged peripheral nerve.

JNK signaling mediates EPHA2-dependent tumor cell proliferation, motility, and cancer stem cell-like properties in non-small cell lung cancer

PubMed Central

Song, Wenqiang; Ma, Yufang; Wang, Jialiang; Brantley-Sieders, Dana; Chen, Jin

2014-01-01

Recent genome-wide analyses in human lung cancer revealed that EPHA2 receptor tyrosine kinase is overexpressed in non-small cell lung cancer (NSCLC), and high levels of EPHA2 correlate with poor clinical outcome. However, the mechanistic basis for EPHA2-mediated tumor promotion in lung cancer remains poorly understood. Here we show that the JNK/c-JUN signaling mediates EPHA2-dependent tumor cell proliferation and motility. A screen of phospho-kinase arrays revealed a decrease in phospho-c-JUN levels in EPHA2 knockdown cells. Knockdown of EPHA2 inhibited p-JNK and p-c-JUN levels in approximately 50% of NSCLC lines tested. Treatment of parental cells with SP600125, a JNK inhibitor, recapitulated defects in EPHA2-deficient tumor cells; whereas constitutively activated JNK mutants were sufficient to rescue phenotypes. Knockdown of EPHA2 also inhibited tumor formation and progression in xenograft animal models in vivo. Furthermore, we investigated the role of EPHA2 in cancer stem-like cells. RNAi-mediated depletion of EPHA2 in multiple NSCLC lines decreased the ALDH positive cancer stem-like population and tumor spheroid formation in suspension. Depletion of EPHA2 in sorted ALDH positive populations markedly inhibited tumorigenicity in nude mice. Furthermore, analysis of a human lung cancer tissue microarray revealed a significant, positive association between EPHA2 and ALDH expression, indicating an important role for EPHA2 in human lung cancer stem-like cells. Collectively, these studies revealed a critical role of JNK signaling in EPHA2-dependent lung cancer cell proliferation and motility and a role for EPHA2 in cancer stem-like cell function, providing evidence for EPHA2 as a potential therapeutic target in NSCLC. PMID:24607842

Design and synthesis of the first generation of novel potent, selective, and in vivo active (benzothiazol-2-yl)acetonitrile inhibitors of the c-Jun N-terminal kinase.

PubMed

Gaillard, Pascale; Jeanclaude-Etter, Isabelle; Ardissone, Vittoria; Arkinstall, Steve; Cambet, Yves; Camps, Montserrat; Chabert, Christian; Church, Dennis; Cirillo, Rocco; Gretener, Denise; Halazy, Serge; Nichols, Anthony; Szyndralewiez, Cedric; Vitte, Pierre-Alain; Gotteland, Jean-Pierre

2005-07-14

Several lines of evidence support the hypothesis that c-Jun N-terminal kinase (JNKs) plays a critical role in a wide range of diseases including cell death (apoptosis)-related disorders (neurodegenerative diseases, brain, heart, and renal ischemia, epilepsy) and inflammatory disorders (multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases). Screening of our internal compound collection for inhibitors of JNK3 led to the identification of (benzothiazol-2-yl)acetonitrile derivatives as potent and selective JNK1, -2, -3 inhibitors. Starting from initial hit 1 (AS007149), the chemistry and initial structure-activity relationship (SAR) of this novel and unique kinase inhibitor template were explored. Investigation of the SAR rapidly revealed that the benzothiazol-2-ylacetonitrile pyrimidine core was crucial to retain a good level of potency on rat JNK3. Therefore, compound 6 was further optimized by exploring a number of distal combinations in place of the chlorine atom. This led to the observation that the presence of an aromatic group, two carbons away from the aminopyrimidine moiety and bearing substituents conferring hydrogen bond acceptor (HBA) properties, could improve the potency. Further improvements to the biological and biopharmaceutical profile of the most promising compounds were performed, resulting in the discovery of compound 59 (AS601245). The in vitro and in vivo anti-inflammatory potential of this new JNK inhibitor was investigated and found to demonstrate efficacy per oral route in an experimental model of rheumatoid arthritis (RA).

CARFILZOMIB INTERACTS SYNERGISTICALY WITH HISTONE DEACETYLASE INHIBITORS IN MANTLE CELL LYMPHOMA CELLS IN VITRO AND IN VIVO

PubMed Central

Dasmahapatra, Girija; Lembersky, Dmitry; Son, Minkyeong P.; Attkisson, Elisa; Dent, Paul; Fisher, Richard. I.; Friedberg, Jonathan W.; Grant, Steven

2011-01-01

Interactions between the proteasome inhibitor carfilzomib and the HDAC inhibitors vorinostat and SNDX-275 were examined in mantle cell lymphoma (MCL) cells in vitro and in vivo. Co-administration of very low, marginally toxic carfilzomib concentrations (e.g., 3–4 nM) with minimally lethal vorinostat or SNDX-275 concentrations induced sharp increases in mitochondrial injury and apoptosis in multiple MCL cell lines and primary MCL cells. Enhanced lethalitly was associated with JNK1/2 activation, increased DNA damage (induction of λH2A.X), and ERK1/2 and AKT1/2 inactivation. Co-administration of carfilzomib and HDACIs induced a marked increase in ROS generation, and G2M arrest. Significantly, the free radical scavenger TBAP blocked carfilzomib/HDACI-mediated ROS generation, λH2A.X formation, JNK1/2 activation, and lethality. Genetic (shRNA) knock down of JNK1/2 significantly attenuated carfilzomib/HDACI-induced apoptosis, but did not prevent ROS generation or DNA damage. Carfilzomib/HDACI regimens were also active against bortezomib-resistant MCL cells. Finally, carfilzomib/vorinostat co-administrationo resulted in a pronounced reduction in tumor growth compared to single agent treatment in a MCL xenograft model associated with enhanced apoptosis, λH2A.X formation, and JNK activation. Collectively, these findings suggest that carfilzomib/HDACI regimens warrants attention in MCL. PMID:21750224

JNK1 induces hedgehog signaling from stellate cells to accelerate liver regeneration in mice.

PubMed

Langiewicz, Magda; Graf, Rolf; Humar, Bostjan; Clavien, Pierre A

2018-04-28

To improve outcomes of two-staged hepatectomies for large/multiple liver tumors, portal vein ligation (PVL) has been combined with parenchymal transection (associating liver partition and portal vein ligation for staged hepatectomy [coined ALPPS]) to greatly accelerate liver regeneration. In a novel ALPPS mouse model, we have reported paracrine Indian hedgehog (IHH) signaling from stellate cells as an early contributor to augmented regeneration. Here, we sought to identify upstream regulators of IHH. ALPPS in mice was compared against PVL and additional control surgeries. Potential IHH regulators were identified through in silico mining of transcriptomic data. c-Jun N-terminal kinase (JNK1 [Mapk8]) activity was reduced through SP600125 to evaluate its effects on IHH signaling. Recombinant IHH was injected after JNK1 diminution to substantiate their relationship during accelerated liver regeneration. Transcriptomic analysis linked Ihh to Mapk8. JNK1 upregulation after ALPPS was validated and preceded the IHH peak. On immunofluorescence, JNK1 and IHH co-localized in alpha-smooth muscle actin-positive non-parenchymal cells. Inhibition of JNK1 prior to ALPPS surgery reduced liver weight gain to PVL levels and was accompanied by downregulation of hepatocellular proliferation and the IHH-GLI1-CCND1 axis. In JNK1-inhibited mice, recombinant IHH restored ALPPS-like acceleration of regeneration and re-elevated JNK1 activity, suggesting the presence of a positive IHH-JNK1 feedback loop. JNK1-mediated induction of IHH paracrine signaling from hepatic stellate cells is essential for accelerated regeneration of parenchymal mass. The JNK1-IHH axis is a mechanism unique to ALPPS surgery and may point to therapeutic alternatives for patients with insufficient regenerative capacity. Associating liver partition and portal vein ligation for staged hepatectomy (so called ALPPS), is a new two-staged approach to hepatectomy, which induces an unprecedented acceleration of liver

HER2-induced metastasis is mediated by AKT/JNK/EMT signaling pathway in gastric cancer

PubMed Central

Choi, Yiseul; Ko, Young San; Park, Jinju; Choi, Youngsun; Kim, Younghoon; Pyo, Jung-Soo; Jang, Bo Gun; Hwang, Douk Ho; Kim, Woo Ho; Lee, Byung Lan

2016-01-01

AIM To investigated the relationships between HER2, c-Jun N-terminal kinase (JNK) and protein kinase B (AKT) with respect to metastatic potential of HER2-positive gastric cancer (GC) cells. METHODS Immunohistochemistry was performed on tissue array slides containing 423 human GC specimens. Using HER2-positve GC cell lines SNU-216 and NCI-N87, HER2 expression was silenced by RNA interference, and the activations of JNK and AKT were suppressed by SP600125 and LY294002, respectively. Transwell assay, Western blot, semi-quantitative reverse transcription-polymerase chain reaction and immunofluorescence staining were used in cell culture experiments. RESULTS In GC specimens, HER2, JNK, and AKT activations were positively correlated with each other. In vitro analysis revealed a positive regulatory feedback loop between HER2 and JNK in GC cell lines and the role of JNK as a downstream effector of AKT in the HER2/AKT signaling pathway. JNK inhibition suppressed migratory capacity through reversing EMT and dual inhibition of JNK and AKT induced a more profound effect on cancer cell motility. CONCLUSION HER2, JNK and AKT in human GC specimens are positively associated with each other. JNK and AKT, downstream effectors of HER2, co-operatively contribute to the metastatic potential of HER2-positive GC cells. Thus, targeting of these two molecules in combination with HER2 downregulation may be a good approach to combat HER2-positive GC. PMID:27895401

Transforming growth factor-β1 induces expression of human coagulation factor XII via Smad3 and JNK signaling pathways in human lung fibroblasts.

PubMed

Jablonska, Ewa; Markart, Philipp; Zakrzewicz, Dariusz; Preissner, Klaus T; Wygrecka, Malgorzata

2010-04-09

Coagulation factor XII (FXII) is a liver-derived serine protease involved in fibrinolysis, coagulation, and inflammation. The regulation of FXII expression is largely unknown. Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine that has been linked to several pathological processes, including tissue fibrosis by modulating procoagulant and fibrinolytic activities. This study investigated whether TGF-beta1 may regulate FXII expression in human lung fibroblasts. Treatment of human lung fibroblasts with TGF-beta1 resulted in a time-dependent increase in FXII production, activation of p44/42, p38, JNK, and Akt, and phosphorylation and translocation into the nucleus of Smad3. However, TGF-beta1-induced FXII expression was repressed only by the JNK inhibitor and JNK and Smad3 antisense oligonucleotides but not by MEK, p38, or phosphoinositide 3-kinase blockers. JNK inhibition had no effect on TGF-beta1-induced Smad3 phosphorylation, association with Smad4, and its translocation into the nucleus but strongly suppressed Smad3-DNA complex formation. FXII promoter analysis revealed that the -299/+1 region was sufficient for TGF-beta1 to induce FXII expression. Sequence analysis of this region detected a potential Smad-binding element at position -272/-269 (SBE-(-272/-269)). Chromatin immunoprecipitation and streptavidin pulldown assays demonstrated TGF-beta1-dependent Smad3 binding to SBE-(-272/-269). Mutation or deletion of SBE-(-272/-269) substantially reduced TGF-beta1-mediated activation of the FXII promoter. Clinical relevance was demonstrated by elevated FXII levels and its co-localization with fibroblasts in the lungs of patients with acute respiratory distress syndrome. Our results show that JNK/Smad3 pathway plays a critical role in TGF-beta1-induced FXII expression in human lung fibroblasts and implicate its possible involvement in pathological conditions characterized by elevated TGF-beta1 levels.

A Protein Scaffold Coordinates SRC-Mediated JNK Activation in Response to Metabolic Stress.

PubMed

Kant, Shashi; Standen, Claire L; Morel, Caroline; Jung, Dae Young; Kim, Jason K; Swat, Wojciech; Flavell, Richard A; Davis, Roger J

2017-09-19

Obesity is a major risk factor for the development of metabolic syndrome and type 2 diabetes. How obesity contributes to metabolic syndrome is unclear. Free fatty acid (FFA) activation of a non-receptor tyrosine kinase (SRC)-dependent cJun NH 2 -terminal kinase (JNK) signaling pathway is implicated in this process. However, the mechanism that mediates SRC-dependent JNK activation is unclear. Here, we identify a role for the scaffold protein JIP1 in SRC-dependent JNK activation. SRC phosphorylation of JIP1 creates phosphotyrosine interaction motifs that bind the SH2 domains of SRC and the guanine nucleotide exchange factor VAV. These interactions are required for SRC-induced activation of VAV and the subsequent engagement of a JIP1-tethered JNK signaling module. The JIP1 scaffold protein, therefore, plays a dual role in FFA signaling by coordinating upstream SRC functions together with downstream effector signaling by the JNK pathway. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Endothelial Dysfunction in Human Diabetes is mediated by Wnt5a-JNK Signaling

PubMed Central

Bretón-Romero, Rosa; Feng, Bihua; Holbrook, Monika; Farb, Melissa G.; Fetterman, Jessica L.; Linder, Erika A.; Berk, Brittany D.; Masaki, Nobuyuki; Weisbrod, Robert M.; Inagaki, Elica; Gokce, Noyan; Fuster, Jose J.; Walsh, Kenneth; Hamburg, Naomi M.

2016-01-01

Objectives Endothelial dysfunction is linked to insulin resistance, inflammatory activation and increased cardiovascular risk in diabetes mellitus; however the mechanisms remain incompletely understood. Recent studies have identified pro-inflammatory signaling of Wnt5a through JNK as a regulator of metabolic dysfunction with potential relevance to vascular function. We sought to gain evidence that increased activation of Wnt5a-JNK signaling contributes to impaired endothelial function in patients with diabetes mellitus. Approach We measured flow-mediated dilation of the brachial artery and characterized freshly isolated endothelial cells by protein expression, eNOS activation, and nitric oxide production in from 85 subjects with Type 2 diabetes mellitus (n=42) and age- and sex-matched non-diabetic controls (n=43) and in human aortic endothelial cells treated with Wnt5a. Results Endothelial cells from patients with diabetes displayed 1.3-fold higher Wnt5a levels (P=0.01) along with 1.4-fold higher JNK activation (P<0.01) without a difference in total JNK levels. Higher JNK activation was associated with lower flow-mediated dilation, consistent with endothelial dysfunction (r=0.53, P=0.02). Inhibition of Wnt5a and JNK signaling restored insulin and A23187-mediated eNOS activation and improved nitric oxide production in endothelial cells from patients with diabetes. In endothelial cells from non-diabetic controls, rWnt5a treatment inhibited eNOS activation replicating the diabetic endothelial phenotype. In HAECs, Wnt5a-induced impairment of eNOS activation and nitric oxide production was reversed by Wnt5a and JNK inhibition. Conclusions Our findings demonstrate that non-canonical Wnt5a signaling and JNK activity contributes to vascular insulin resistance and endothelial dysfunction and may represent a novel therapeutic opportunity to protect the vasculature in patients with diabetes. PMID:26800561

Endothelial Dysfunction in Human Diabetes Is Mediated by Wnt5a-JNK Signaling.

PubMed

Bretón-Romero, Rosa; Feng, Bihua; Holbrook, Monika; Farb, Melissa G; Fetterman, Jessica L; Linder, Erika A; Berk, Brittany D; Masaki, Nobuyuki; Weisbrod, Robert M; Inagaki, Elica; Gokce, Noyan; Fuster, Jose J; Walsh, Kenneth; Hamburg, Naomi M

2016-03-01

Endothelial dysfunction is linked to insulin resistance, inflammatory activation, and increased cardiovascular risk in diabetes mellitus; however, the mechanisms remain incompletely understood. Recent studies have identified proinflammatory signaling of wingless-type family member (Wnt) 5a through c-jun N-terminal kinase (JNK) as a regulator of metabolic dysfunction with potential relevance to vascular function. We sought to gain evidence that increased activation of Wnt5a-JNK signaling contributes to impaired endothelial function in patients with diabetes mellitus. We measured flow-mediated dilation of the brachial artery and characterized freshly isolated endothelial cells by protein expression, eNOS activation, and nitric oxide production in 85 subjects with type 2 diabetes mellitus (n=42) and age- and sex-matched nondiabetic controls (n=43) and in human aortic endothelial cells treated with Wnt5a. Endothelial cells from patients with diabetes mellitus displayed 1.3-fold higher Wnt5a levels (P=0.01) along with 1.4-fold higher JNK activation (P<0.01) without a difference in total JNK levels. Higher JNK activation was associated with lower flow-mediated dilation, consistent with endothelial dysfunction (r=0.53, P=0.02). Inhibition of Wnt5a and JNK signaling restored insulin and A23187-mediated eNOS activation and improved nitric oxide production in endothelial cells from patients with diabetes mellitus. In endothelial cells from nondiabetic controls, rWnt5a treatment inhibited eNOS activation replicating the diabetic endothelial phenotype. In human aortic endothelial cells, Wnt5a-induced impairment of eNOS activation and nitric oxide production was reversed by Wnt5a and JNK inhibition. Our findings demonstrate that noncanonical Wnt5a signaling and JNK activity contribute to vascular insulin resistance and endothelial dysfunction and may represent a novel therapeutic opportunity to protect the vasculature in patients with diabetes mellitus. © 2016 American Heart

ATF3 mediates the inhibitory action of TNF-α on osteoblast differentiation through the JNK signaling pathway.

PubMed

Jeong, Byung-Chul

2018-05-15

Tumor necrosis factor (TNF)-α, which is a proinflammatory cytokine, inhibits osteoblast differentiation under diverse inflammatory conditions. Activating transcription factor 3 (ATF3), which is a member of the ATF/cAMP response element-binding protein family of transcription factors, has been implicated in the regulation of cell proliferation and differentiation. However, the precise interactions between ATF3 and the TNF-α signaling pathway in the regulation of osteoblast differentiation remain unclear. In this study, we examined the role of ATF3 in the TNF-α-mediated inhibition of osteoblast differentiation and investigated the signaling pathways involved. The treatment of cells with TNF-α downregulated osteogenic markers, but significantly upregulated the expression of Atf3. The inhibition of Atf3 by small interfering RNAs rescued osteogenesis, which was inhibited by TNF-α. Conversely, the enforced expression of Atf3 enhanced the TNF-α-mediated inhibition of osteoblast differentiation, as revealed by the measurement of osteogenic markers and alkaline phosphatase staining. Mechanistically, TNF-α-induced Atf3 expression was significantly suppressed by the inhibition of the c-Jun N-terminal kinase (JNK) pathway. Furthermore, the overexpression of Atf3 did not affect the rescue effect that inhibiting TNF-α expression using a JNK inhibitor had on alkaline phosphatase activity and mineralization. Taken together, these results indicate that ATF3 mediates the inhibitory action of TNF-α on osteoblast differentiation and that the TNF-α-activated JNK pathway is responsible for the induction of Atf3 expression. Copyright © 2018 Elsevier Inc. All rights reserved.

[Effects of several inhibitors of intracellular signaling on production of cytokines and signal proteins in RAW 264.7 cells cultivated with low dose ammonium].

PubMed

Novoselova, E G; Parfeniuk, S B; Glushkova, O V; Khrenov, M O; Novoselova, T V; Lunin, S M; Fesenko, E E

2012-01-01

Effects of four inhibitors of NF-kappaB, SAPK/JNK and TLR4 signaling, namely, inhibitor XII, SP600125, CLI-095 and Oxpapc on a macrophage response to low dose ammonium were studied in RAW 264.7 cells. Low dose ammonium induced pro-inflammatory response in cells as judged from enhanced production of TNF-alpha, IF-gamma, and IL-6, and by activation of signal cascades. The increase in production of cytokines, namely TNF, IFN, and IL-6, demonstrated that low-dose ammonium induced a pro-inflammatory cellular response. In addition, an activation of NF-kappaB and SAPK/JNK cascades, as well as enhancement of TLR4 expression was shown. Each of used inhibitors reduced to a variable degree the pro-inflammatory response of RAW 264.7 cells on chemical toxin by decreasing cytokine production. The inhibitor of NF-kappaB cascade, IKK Inhibitor XII, was more effective, and not only prevented the development of pro-inflammatory response induced by ammonium, but also decreased cytokine production below control values. The inhibitor of extra cellular domains of TLR2 and TLR4 (OxPAPC) had almost the same anti-inflammatory effect, and an addition of the inhibitor of JNK cascade (SP600125) to cell culture practically neutralized effect of ammonium ions by decreasing cytokine production to control level. Inhibitory analysis showed that activation of RAW 264.7 cells induced by chemical toxin coincide incompletely with intracellular signaling pathways that were early determined regarding macrophage's response to toxin from gram-negative bacteria. Nevertheless, application of the inhibitors defended RAW 264.7 from toxic effect of the low dose ammonium.

Novel mechanism of JNK pathway activation by adenoviral E1A

PubMed Central

Morrison, Helen; Pospelova, Tatiana V.; Pospelov, Valery A.; Herrlich, Peter

2014-01-01

The adenoviral oncoprotein E1A influences cellular regulation by interacting with a number of cellular proteins. In collaboration with complementary oncogenes, E1A fully transforms primary cells. As part of this action, E1A inhibits transcription of c-Jun:Fos target genes while promoting that of c-Jun:ATF2-dependent genes including jun. Both c-Jun and ATF2 are hyperphosphorylated in response to E1A. In the current study, E1A was fused with the ligand binding domain of the estrogen receptor (E1A-ER) to monitor the immediate effect of E1A activation. With this approach we now show that E1A activates c-Jun N-terminal kinase (JNK), the upstream kinases MKK4 and MKK7, as well as the small GTPase Rac1. Activation of the JNK pathway requires the N-terminal domain of E1A, and, importantly, is independent of transcription. In addition, it requires the presence of ERM proteins. Downregulation of signaling components upstream of JNK inhibits E1A-dependent JNK/c-Jun activation. Taking these findings together, we show that E1A activates the JNK/c-Jun signaling pathway upstream of Rac1 in a transcription-independent manner, demonstrating a novel mechanism of E1A action. PMID:24742962

The nucleolus as a stress sensor: JNK2 inactivates the transcription factor TIF-IA and down-regulates rRNA synthesis.

PubMed

Mayer, Christine; Bierhoff, Holger; Grummt, Ingrid

2005-04-15

Cells respond to a variety of extracellular and intracellular forms of stress by down-regulating rRNA synthesis. We have investigated the mechanism underlying stress-dependent inhibition of RNA polymerase I (Pol I) transcription and show that the Pol I-specific transcription factor TIF-IA is inactivated upon stress. Inactivation is due to phosphorylation of TIF-IA by c-Jun N-terminal kinase (JNK) at a single threonine residue (Thr 200). Phosphorylation at Thr 200 impairs the interaction of TIF-IA with Pol I and the TBP-containing factor TIF-IB/SL1, thereby abrogating initiation complex formation. Moreover, TIF-IA is translocated from the nucleolus into the nucleoplasm. Substitution of Thr 200 by valine as well as knock-out of Jnk2 prevent inactivation and translocation of TIF-IA, leading to stress-resistance of Pol I transcription. Our data identify TIF-IA as a downstream target of the JNK pathway and suggest a critical role of JNK2 to protect rRNA synthesis against the harmful consequences of cellular stress.

Protective Effects of Astaxanthin on ConA-Induced Autoimmune Hepatitis by the JNK/p-JNK Pathway-Mediated Inhibition of Autophagy and Apoptosis

PubMed Central

Liu, Tong; Wang, Junshan; Dai, Weiqi; Wang, Fan; Zheng, Yuanyuan; Chen, Kan; Li, Sainan; Abudumijiti, Huerxidan; Zhou, Zheng; Wang, Jianrong; Lu, Wenxia; Zhu, Rong; Yang, Jing; Zhang, Huawei; Yin, Qin; Wang, Chengfen; Zhou, Yuqing; Lu, Jie; Zhou, Yingqun; Guo, Chuanyong

2015-01-01

Objective Astaxanthin, a potent antioxidant, exhibits a wide range of biological activities, including antioxidant, atherosclerosis and antitumor activities. However, its effect on concanavalin A (ConA)-induced autoimmune hepatitis remains unclear. The aim of this study was to investigate the protective effects of astaxanthin on ConA-induced hepatitis in mice, and to elucidate the mechanisms of regulation. Materials and Methods Autoimmune hepatitis was induced in in Balb/C mice using ConA (25 mg/kg), and astaxanthin was orally administered daily at two doses (20 mg/kg and 40 mg/kg) for 14 days before ConA injection. Levels of serum liver enzymes and the histopathology of inflammatory cytokines and other maker proteins were determined at three time points (2, 8 and 24 h). Primary hepatocytes were pretreated with astaxanthin (80 μM) in vitro 24 h before stimulation with TNF-α (10 ng/ml). The apoptosis rate and related protein expression were determined 24 h after the administration of TNF-α. Results Astaxanthin attenuated serum liver enzymes and pathological damage by reducing the release of inflammatory factors. It performed anti-apoptotic effects via the descending phosphorylation of Bcl-2 through the down-regulation of the JNK/p-JNK pathway. Conclusion This research firstly expounded that astaxanthin reduced immune liver injury in ConA-induced autoimmune hepatitis. The mode of action appears to be downregulation of JNK/p-JNK-mediated apoptosis and autophagy. PMID:25761053

Persistency of use of COX-2-specific inhibitors and non-specific non-steroidal anti-inflammatory drugs (NSAIDs) in Quebec.

PubMed

Moride, Y; Ducruet, T; Rochon, S; Lavoie, F

2003-11-01

The effectiveness of pharmacological therapies is dependent in part on patient persistency with the prescribed therapeutic regimen. In the case of non-specific non-steroidal anti-inflammatory drugs (NSAIDs), effectiveness is often compromised by undesirable side-effects, poor compliance or discontinuation of therapy. While patterns of utilization of non-specific NSAIDs have been investigated, few data are available on the patterns of persistency for cyclooxygenase (COX)-2-specific inhibitors. This study used a provincial health-care system database in Quebec, Canada, to determine the duration of treatment in new users of COX-2-specific inhibitors and non-specific NSAIDs over the first 3 months of treatment, and to characterize the factors associated with treatment persistency. Results demonstrate that the median duration of treatment was longer among patients initially prescribed COX-2-specific inhibitors (30 days and 23 days for celecoxib and rofecoxib respectively) than in those prescribed non-selective NSAIDs (10 days). Although the percentage of patients remaining on COX-2-specific drugs declined over the course of treatment, few patients on either celecoxib or rofecoxib switched drugs, either to the other COX-2-specific inhibitor or to non-specific NSAIDs. Factors associated with persistent drug use were: COX-2-specific inhibitors, age, and the use of gastroprotective agents either at treatment initiation or during follow-up. Dosage, chronic disease score and prescriber's specialty were only marginally associated with persistency. Prior use of gastroprotective agents was associated with lower persistency. Although the limitations of this study, which included lack of information on the indication for the prescription and the reason for switch or discontinuation, preclude definite conclusions regarding patterns of use of these drugs, the data suggest that the use of COX-2-specific inhibitors may result in increased persistency with treatment.

Preparation of 6-substituted quinoxaline JSP-1 inhibitors by microwave accelerated nucleophilic substitution.

PubMed

Zhang, Li; Qiu, Beiying; Li, Xin; Wang, Xin; Li, Jingya; Zhang, Yongliang; Liu, Jian; Li, Jia; Shen, Jingkang

2006-12-21

A small library of 6-aminoquinoxalines has been prepared by nucleophilic substitution of 6-fluoroquinoxaline with amines and nitrogen-containing heterocycles under computer-controlled microwave irradiation. Some compounds were found to be potent inhibitors of JNK Stimulatory Phosphatase-1 (JSP-1) in an in vitro biological assay.

Increased levels of cerebrospinal fluid JNK3 associated with amyloid pathology: links to cognitive decline

PubMed Central

Gourmaud, Sarah; Paquet, Claire; Dumurgier, Julien; Pace, Clarisse; Bouras, Constantin; Gray, Françoise; Laplanche, Jean-Louis; Meurs, Eliane F.; Mouton-Liger, François; Hugon, Jacques

2015-01-01

Background Alzheimer disease is characterized by cognitive decline, senile plaques of β-amyloid (Aβ) peptides, neurofibrillary tangles composed of hyperphosphorylated τ proteins and neuronal loss. Aβ and τ are useful markers in the cerebrospinal fluid (CSF). C-Jun N-terminal kinases (JNKs) are serine-threonine protein kinases activated by phosphorylation and involved in neuronal death. Methods In this study, Western blots, enzyme-linked immunosorbent assay and histological approaches were used to assess the concentrations of Aβ, τ and JNK isoforms in postmortem brain tissue samples (10 Alzheimer disease and 10 control) and in CSF samples from 30 living patients with Alzheimer disease and 27 controls with neurologic disease excluding Alzheimer disease. Patients with Alzheimer disease were followed for 1–3 years and assessed using Mini–Mental State Examination scores. Results The biochemical and morphological results showed a significant increase of JNK3 and phosphorylated JNK levels in patients with Alzheimer disease, and JNK3 levels correlated with Aβ42 levels. Confocal microscopy revealed that JNK3 was associated with Aβ in senile plaques. The JNK3 levels in the CSF were significantly elevated in patients with Alzheimer disease and correlated statistically with the rate of cognitive decline in a mixed linear model. Limitations The study involved different samples grouped into 3 small cohorts. Evaluation of JNK3 in CSF was possible only with immunoblot analysis. Conclusion We found that JNK3 levels are increased in brain tissue and CSF from patients with Alzheimer disease. The finding that increased JNK3 levels in CSF could reflect the rate of cognitive decline is new and merits further investigation. PMID:25455349

Specific inhibitors of mammalian DNA polymerase species.

PubMed

Mizushina, Yoshiyuki

2009-06-01

In screening of selective inhibitors of eukaryotic DNA polymerases (pols) for 15 years, more than 100 inhibitors have been discovered from natural and chemical sources. Some compounds selectively inhibit the activities of mammalian pols, and in particular, dehydroaltenusin and curcumin derivatives, such as monoacetyl-curcumin, were found to be specific inhibitors of pol alpha and pol lambda, respectively. Dehydroaltenusin was isolated from a fungus (Alternaria tennuis), and this compound inhibited cell proliferation of human cancer cell lines by arresting the cells at the S-phase, and was effective in suppressing the growth on nude mice of solid tumors of human cervical cancer cell line HeLa. Curcumin derivatives had anti-12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammatory activity with the same tendency as pol lambda inhibitory activity. These compounds might be useful not only as "molecular probes" for pol research, but also as biomedical and chemotherapeutic drugs for anti-cancer or anti-inflammation.

Diet-induced obesity mediated by the JNK/DIO2 signal transduction pathway

PubMed Central

Vernia, Santiago; Cavanagh-Kyros, Julie; Barrett, Tamera; Jung, Dae Young; Kim, Jason K.; Davis, Roger J.

2013-01-01

The cJun N-terminal kinase (JNK) signaling pathway is a key mediator of metabolic stress responses caused by consuming a high-fat diet, including the development of obesity. To test the role of JNK, we examined diet-induced obesity in mice with targeted ablation of Jnk genes in the anterior pituitary gland. These mice exhibited an increase in the pituitary expression of thyroid-stimulating hormone (TSH), an increase in the blood concentration of thyroid hormone (T4), increased energy expenditure, and markedly reduced obesity compared with control mice. The increased amount of pituitary TSH was caused by reduced expression of type 2 iodothyronine deiodinase (Dio2), a gene that is required for T4-mediated negative feedback regulation of TSH expression. These data establish a molecular mechanism that accounts for the regulation of energy expenditure and the development of obesity by the JNK signaling pathway. PMID:24186979

AIP1 recruits phosphatase PP2A to ASK1 in tumor necrosis factor-induced ASK1-JNK activation.

PubMed

Min, Wang; Lin, Yan; Tang, Shibo; Yu, Luyang; Zhang, Haifeng; Wan, Ting; Luhn, Tricia; Fu, Haian; Chen, Hong

2008-04-11

Previously we have shown that AIP1 (apoptosis signal-regulating kinase [ASK]1-interacting protein 1), a novel member of the Ras-GAP protein family, facilitates dephosphorylation of ASK1 at pSer967 and subsequently 14-3-3 release from ASK1, leading to enhanced ASK1-JNK signaling. However, the phosphatase(s) responsible for ASK1 dephosphorylation at pSer967 has not been identified. In the present study, we identified protein phosphatase (PP)2A as a potential phosphatase in vascular endothelial cells (ECs). Tumor necrosis factor (TNF)-induced dephosphorylation of ASK1 pSer967 in ECs was blocked by PP2A inhibitor okadaic acid. Overexpression of PP2A catalytic subunit induced dephosphorylation of ASK1 pSer967 and activation of c-Jun N-terminal kinase (JNK). In contrast, a catalytic inactive form of PP2A or PP2A small interfering RNA blunted TNF-induced dephosphorylation of ASK1 pSer967 and activation of JNK without effects on NF-kappaB activation. Whereas AIP1, via its C2 domain, binds to ASK1, PP2A binds to the GAP domain of AIP1. Endogenous AIP1-PP2A complex can be detected in the resting state, and TNF induces a complex formation of AIP1-PP2A with ASK1. Furthermore, TNF-induced association of PP2A with ASK1 was diminished in AIP1-knockdown ECs, suggesting a critical role of AIP1 in recruiting PP2A to ASK1. TNF-signaling molecules TRAF2 and RIP1, known to be in complex with AIP1 and activate AIP1 by phosphorylating AIP1 at Ser604, are critical for TNF-induced ASK1 dephosphorylation. Finally, PP2A and AIP1 cooperatively induce activation of ASK1-JNK signaling and EC apoptosis, as demonstrated by both overexpression and small interfering RNA knockdown approaches. Taken together, our data support a critical role of PP2A-AIP1 complex in TNF-induced activation of ASK1-JNK apoptotic signaling.

Non-nucleoside reverse transcriptase inhibitor efavirenz increases monolayer permeability of human coronary artery endothelial cells

PubMed Central

Jamaluddin, Md Saha; Lin, Peter H.; Yao, Qizhi; Chen, Changyi

2009-01-01

Highly active antiretroviral therapy (HAART) is often associated with endothelial dysfunction and cardiovascular complications. In this study, we determined whether HIV non-nucleoside reverse transcriptase inhibitor efavirenz (EFV) could increase endothelial permeability. Human coronary artery endothelial cells (HCAECs) were treated with EFV (1, 5 and 10 µg/ml) and endothelial permeability was determined by a transwell system with a fluorescence-labeled dextran tracer. HCAECs treated with EFV showed a significant increase of endothelial permeability in a concentration-dependent manner. With real time PCR analysis, EFV significantly reduced the mRNA levels of tight junction proteins claudin-1, occludin, zonula occluden-1 and junctional adhesion molecule-1 compared with controls (P < 0.05). Protein levels of these tight junction molecules were also reduced substantially in the EFV-treated cells by western blot and flow cytometry analyses. In addition, EFV also increased superoxide anion production with dihydroethidium and cellular glutathione assays, while it decreased mitochondrial membrane potential with JC-staining. Antioxidants (ginkgolide B and MnTBAP) effectively blocked EFV-induced endothelial permeability and mitochondrial dysfunction. Furthermore, EFV increased the phosphorylation of MAPK JNK and IκBα, thereby increasing NFκB translocation to the nucleus. Chemical JNK inhibitor and dominant negative mutant JNK and IkBa adenoviruses effectively blocked the effects of EFV on HCAECs. Thus, EFV increases endothelial permeability which may be due to the decrease of tight junction proteins and the increase of superoxide anion. JNK and NFκB activation may be directly involved in the signal transduction pathway of EFV action in HCAECs. PMID:19674747

Vitamin K3-2,3-epoxide induction of apoptosis with activation of ROS-dependent ERK and JNK protein phosphorylation in human glioma cells.

PubMed

Wu, Jender; Chien, Chih-Chiang; Yang, Liang-Yo; Huang, Guan-Cheng; Cheng, Min-Chi; Lin, Che-Tong; Shen, Shing-Chuan; Chen, Yen-Chou

2011-08-15

2-Methyl-1,4-naphthoquinone (menadione or vitamin K3; EPO) and K3-2,3-epoxide (EPO1), but not vitamin K3-3-OH (EPO2), exhibited cytotoxicity that caused DNA fragmentation and chromatin condensation in U87 and C6 cells. EPO1 showed more-potent cytotoxicity than EPO, and the IC(50) values of EPO and EPO1 in U87 cells were 37.5 and 15.7μM, respectively. Activation of caspase 3 enzyme activity with cleavage of caspase 3 protein was detected in EPO1-treated U87 and C6 cells, and the addition of the caspase 3 peptidyl inhibitor, DEVD-FMK, reduced the cytotoxic effect of EPO1. An increase in the intracellular ROS level by EPO1 was observed in the DCHF-DA analysis, and EPO1-induced apoptosis and caspase 3 protein cleavage were prevented by adding the antioxidant, N-acetyl-cysteine (NAC), with decreased ROS production elicited by EPO1. Activation of ERK and JNK, but not p38, via phosphorylation induction was identified in EPO1- but not EPO- or EPO2-treated U87 and C6 cells, and this was blocked by adding NAC. However, the ERK inhibitor, PD98059, and the JNK inhibitor, SP600125, showed no effect on EPO1-induced cytotoxicity in either cell type. Our findings demonstrate that 2,3-epoxide substitution significantly potentiates the apoptotic effect of vitamin K3 via stimulating ROS production, which may be useful in the chemotherapy of glioblastoma cells. Copyright © 2011. Published by Elsevier Ireland Ltd.

Induction of c-fos expression through JNK-mediated TCF/Elk-1 phosphorylation.

PubMed Central

Cavigelli, M; Dolfi, F; Claret, F X; Karin, M

1995-01-01

Growth factors induce c-fos transcription by stimulating phosphorylation of transcription factor TCF/Elk-1, which binds to the serum response element (SRE). Under such conditions Elk-1 could be phosphorylated by the mitogen-activated protein kinases (MAPKs) ERK1 and ERK2. However, c-fos transcription and SRE activity are also induced by stimuli, such as UV irradiation and activation of the protein kinase MEKK1, that cause only an insignificant increase in ERK1/2 activity. However, both of these stimuli strongly activate two other MAPKs, JNK1 and JNK2, and stimulate Elk-1 transcriptional activity and phosphorylation. We find that the JNKs are the predominant Elk-1 activation domain kinases in extracts of UV-irradiated cells and that immunopurified JNK1/2 phosphorylate Elk-1 on the same major sites recognized by ERK1/2, that potentiate its transcriptional activity. Finally, we show that UV irradiation, but not serum or phorbol esters, stimulate translocation of JNK1 to the nucleus. As Elk-1 is most likely phosphorylated while bound to the c-fos promoter, these results suggest that UV irradiation and MEKK1 activation stimulate TCF/Elk-1 activity through JNK activation, while growth factors induce c-fos through ERK activation. Images PMID:8846788

Specific inhibition of c-Jun N-terminal kinase delays preterm labour and reduces mortality

PubMed Central

Pirianov, Grisha; MacIntyre, David A; Lee, Yun; Waddington, Simon N; Terzidou, Vasso; Mehmet, Huseyin; Bennett, Phillip R

2015-01-01

Preterm labour (PTL) is commonly associated with infection and/or inflammation. Lipopolysaccharide (LPS) from different bacteria can be used to independently or mutually activate Jun N-terminal kinase (JNK)/AP1- or NF-κB-driven inflammatory pathways that lead to PTL. Previous studies using Salmonella abortus LPS, which activates both JNK/AP-1 and NF-κB, showed that selective inhibition of NF-κB delays labour and improves pup outcome. Where labour is induced using Escherichia coli LPS (O111), which upregulates JNK/AP-1 but not NF-κB, inhibition of JNK/AP-1 activation also delays labour. In this study, to determine the potential role of JNK as a therapeutic target in PTL, we investigated the specific contribution of JNK signalling to S. Abortus LPS-induced PTL in mice. Intrauterine administration of S. Abortus LPS to pregnant mice resulted in the activation of JNK in the maternal uterus and fetal brain, upregulation of pro-inflammatory proteins COX-2, CXCL1, and CCL2, phosphorylation of cPLA2 in myometrium, and induction of PTL. Specific inhibition of JNK by co-administration of specific D-JNK inhibitory peptide (D-JNKI) delayed LPS-induced preterm delivery and reduced fetal mortality. This is associated with inhibition of myometrial cPLA2 phosphorylation and proinflammatory proteins synthesis. In addition, we report that D-JNKI inhibits the activation of JNK/JNK3 and caspase-3, which are important mediators of neural cell death in the neonatal brain. Our data demonstrate that specific inhibition of TLR4-activated JNK signalling pathways has potential as a therapeutic approach in the management of infection/inflammation-associated PTL and prevention of the associated detrimental effects to the neonatal brain. PMID:26183892

ROS regulation of axonal mitochondrial transport is mediated by Ca2+ and JNK in Drosophila

PubMed Central

Liao, Pin-Chao; Tandarich, Lauren C.

2017-01-01

Mitochondria perform critical functions including aerobic ATP production and calcium (Ca2+) homeostasis, but are also a major source of reactive oxygen species (ROS) production. To maintain cellular function and survival in neurons, mitochondria are transported along axons, and accumulate in regions with high demand for their functions. Oxidative stress and abnormal mitochondrial axonal transport are associated with neurodegenerative disorders. However, we know little about the connection between these two. Using the Drosophila third instar larval nervous system as the in vivo model, we found that ROS inhibited mitochondrial axonal transport more specifically, primarily due to reduced flux and velocity, but did not affect transport of other organelles. To understand the mechanisms underlying these effects, we examined Ca2+ levels and the JNK (c-Jun N-terminal Kinase) pathway, which have been shown to regulate mitochondrial transport and general fast axonal transport, respectively. We found that elevated ROS increased Ca2+ levels, and that experimental reduction of Ca2+ to physiological levels rescued ROS-induced defects in mitochondrial transport in primary neuron cell cultures. In addition, in vivo activation of the JNK pathway reduced mitochondrial flux and velocities, while JNK knockdown partially rescued ROS-induced defects in the anterograde direction. We conclude that ROS have the capacity to regulate mitochondrial traffic, and that Ca2+ and JNK signaling play roles in mediating these effects. In addition to transport defects, ROS produces imbalances in mitochondrial fission-fusion and metabolic state, indicating that mitochondrial transport, fission-fusion steady state, and metabolic state are closely interrelated in the response to ROS. PMID:28542430

A sestrin-dependent Erk/Jnk/p38 MAPK activation complex inhibits immunity during ageing

PubMed Central

Lanna, Alessio; Gomes, Daniel C O; Muller-Durovic, Bojana; McDonnell, Thomas; Escors, David; Gilroy, Derek W; Lee, Jun Hee; Karin, Michael; Akbar, Arne N

2016-01-01

Mitogen activated protein kinases (MAPKs) including Erk, Jnk and p38 regulate diverse cellular functions, and are thought to be controlled by independent upstream activation cascades. Here we show that the sestrins bind to and co-ordinate simultaneous Erk, Jnk and p38 MAPK activation in T lymphocytes within a new immune-inhibitory complex (sestrin-MAPK Activation Complex; sMAC). Whereas sestrin ablation resulted in broad reconstitution of immune function in stressed T cells, inhibition of individual MAPKs only allowed partial functional recovery. T cells from old humans and mice were more likely to form the sMAC, and disruption of this complex restored antigen-specific functional responses in these cells. Correspondingly, sestrin deficiency or simultaneous inhibition of all three MAPKs enhanced vaccine responsiveness in old mice. Thus, disruption of sMAC provides a foundation for rejuvenating immunity during ageing. PMID:28114291

PARP1-mediated necrosis is dependent on parallel JNK and Ca2+/calpain pathways

PubMed Central

Douglas, Diana L.; Baines, Christopher P.

2014-01-01

ABSTRACT Poly(ADP-ribose) polymerase-1 (PARP1) is a nuclear enzyme that can trigger caspase-independent necrosis. Two main mechanisms for this have been proposed: one involving RIP1 and JNK kinases and mitochondrial permeability transition (MPT), the other involving calpain-mediated activation of Bax and mitochondrial release of apoptosis-inducing factor (AIF). However, whether these two mechanisms represent distinct pathways for PARP1-induced necrosis, or whether they are simply different components of the same pathway has yet to be tested. Mouse embryonic fibroblasts (MEFs) were treated with either N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or β-Lapachone, resulting in PARP1-dependent necrosis. This was associated with increases in calpain activity, JNK activation and AIF translocation. JNK inhibition significantly reduced MNNG- and β-Lapachone-induced JNK activation, AIF translocation, and necrosis, but not calpain activation. In contrast, inhibition of calpain either by Ca2+ chelation or knockdown attenuated necrosis, but did not affect JNK activation or AIF translocation. To our surprise, genetic and/or pharmacological inhibition of RIP1, AIF, Bax and the MPT pore failed to abrogate MNNG- and β-Lapachone-induced necrosis. In conclusion, although JNK and calpain both contribute to PARP1-induced necrosis, they do so via parallel mechanisms. PMID:25052090

Samsoeum, a traditional herbal medicine, elicits apoptotic and autophagic cell death by inhibiting Akt/mTOR and activating the JNK pathway in cancer cells

PubMed Central

2013-01-01

Background Samsoeum (SSE), a traditional herbal formula, has been widely used to treat cough, fever, congestion, and emesis for centuries. Recent studies have demonstrated that SSE retains potent pharmacological efficiency in anti-allergic and anti-inflammatory reactions. However, the anti-cancer activity of SSE and its underlying mechanisms have not been studied. Thus, the present study was designed to determine the effect of SSE on cell death and elucidate its detailed mechanism. Methods Following SSE treatment, cell growth and cell death were measured using an MTT assay and trypan blue exclusion assay, respectively. Cell cycle arrest and YO-PRO-1 uptake were assayed using flow cytometry, and LC3 redistribution was observed using confocal microscope. The mechanisms of anti-cancer effect of SSE were investigated through western blot analysis. Results We initially found that SSE caused dose- and time-dependent cell death in cancer cells but not in normal primary hepatocytes. In addition, during early SSE treatment (6–12 h), cells were arrested in G2/M phase concomitant with up-regulation of p21 and p27 and down-regulation of cyclin D1 and cyclin B1, followed by an increase in apoptotic YO-PRO-1 (+) cells. SSE also induced autophagy via up-regulation of Beclin-1 expression, conversion of microtubule-associated protein light chain 3 (LC3) I to LC3-II, and re-distribution of LC3, indicating autophagosome formation. Moreover, the level of B-cell lymphoma 2 (Bcl-2), which is critical for cross-talk between apoptosis and autophagy, was significantly reduced in SSE-treated cells. Phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) was increased, followed by suppression of the protein kinase B/mammalian target of rapamycin (Akt/mTOR) pathway, and phosphorylation of mitogen-activated protein kinases (MAPKs) in response to SSE treatment. In particular, among MAPKs inhibitors, only the c-Jun N-terminal kinase (JNK)-specific inhibitor SP600125 nearly

The Core Molecular Machinery Used for Engulfment of Apoptotic Cells Regulates the JNK Pathway Mediating Axon Regeneration in Caenorhabditis elegans.

PubMed

Pastuhov, Strahil Iv; Fujiki, Kota; Tsuge, Anna; Asai, Kazuma; Ishikawa, Sho; Hirose, Kazuya; Matsumoto, Kunihiro; Hisamoto, Naoki

2016-09-14

The mechanisms that govern the ability of specific neurons to regenerate their axons after injury are not well understood. In Caenorhabditis elegans, the initiation of axon regeneration is positively regulated by the JNK-MAPK pathway. In this study, we identify two components functioning upstream of the JNK pathway: the Ste20-related protein kinase MAX-2 and the Rac-type GTPase CED-10. CED-10, when bound by GTP, interacts with MAX-2 and functions as its upstream regulator in axon regeneration. CED-10, in turn, is activated by axon injury via signals initiated from the integrin α-subunit INA-1 and the nonreceptor tyrosine kinase SRC-1 and transmitted via the signaling module CED-2/CrkII-CED-5/Dock180-CED-12/ELMO. This module is also known to regulate the engulfment of apoptotic cells during development. Our findings thus reveal that the molecular machinery used for engulfment of apoptotic cells also promotes axon regeneration through activation of the JNK pathway. The molecular mechanisms of axon regeneration after injury remain poorly understood. In Caenorhabditis elegans, the initiation of axon regeneration is positively regulated by the JNK-MAPK pathway. In this study, we show that integrin, Rac-GTPase, and several other molecules, all of which are known to regulate engulfment of apoptotic cells during development, also regulate axon regeneration. This signaling module activates the JNK-MAPK cascade via MAX-2, a PAK-like protein kinase that binds Rac. Our findings thus reveal that the molecular machinery used for engulfment of apoptotic cells also promotes axon regeneration through activation of the JNK pathway. Copyright © 2016 the authors 0270-6474/16/369710-12$15.00/0.

JNK Controls the Onset of Mitosis in Planarian Stem Cells and Triggers Apoptotic Cell Death Required for Regeneration and Remodeling

PubMed Central

Almuedo-Castillo, María; Crespo, Xenia; Seebeck, Florian; Bartscherer, Kerstin; Salò, Emili; Adell, Teresa

2014-01-01

Regeneration of lost tissues depends on the precise interpretation of molecular signals that control and coordinate the onset of proliferation, cellular differentiation and cell death. However, the nature of those molecular signals and the mechanisms that integrate the cellular responses remain largely unknown. The planarian flatworm is a unique model in which regeneration and tissue renewal can be comprehensively studied in vivo. The presence of a population of adult pluripotent stem cells combined with the ability to decode signaling after wounding enable planarians to regenerate a complete, correctly proportioned animal within a few days after any kind of amputation, and to adapt their size to nutritional changes without compromising functionality. Here, we demonstrate that the stress-activated c-jun–NH2–kinase (JNK) links wound-induced apoptosis to the stem cell response during planarian regeneration. We show that JNK modulates the expression of wound-related genes, triggers apoptosis and attenuates the onset of mitosis in stem cells specifically after tissue loss. Furthermore, in pre-existing body regions, JNK activity is required to establish a positive balance between cell death and stem cell proliferation to enable tissue renewal, remodeling and the maintenance of proportionality. During homeostatic degrowth, JNK RNAi blocks apoptosis, resulting in impaired organ remodeling and rescaling. Our findings indicate that JNK-dependent apoptotic cell death is crucial to coordinate tissue renewal and remodeling required to regenerate and to maintain a correctly proportioned animal. Hence, JNK might act as a hub, translating wound signals into apoptotic cell death, controlled stem cell proliferation and differentiation, all of which are required to coordinate regeneration and tissue renewal. PMID:24922054

Metformin and pioglitazone combination therapy ameliorate polycystic ovary syndrome through AMPK/PI3K/JNK pathway

PubMed Central

Wu, Yuanyuan; Li, Pengfen; Zhang, Dan; Sun, Yingpu

2018-01-01

Polycystic ovary syndrome (PCOS) is a common gynecological endocrine disorder, which results in health problems such as menstrual disorders, hyperandrogenism and persistent anovulation. Hyperandrogenism and insulin resistance are the basic characteristics of PCOS. To investigate the combined effect of metformin and pioglitazone on POCS and the potential mechanisms, a rat model of PCOS was established by intramuscular injection of estradiol valerate (EV). The effect of metformin and pioglitazone monotherapy or combination therapy in control rats and PCOS rats was evaluated, involving the testosterone level, follicular development and insulin resistance. The potential mechanism for the therapeutic effect of metformin and pioglitazone on POCS was explored through using three inhibitors of the 5′adenosine monophosphate-activated protein kinase (AMPK)/phosphoinositide-3 kinase (PI3K)/c-Jun N-terminal kinase (JNK) pathway (Compound C, Wortmannin and SP600125). The results showed that EV-induced PCOS rats demonstrated hyperandrogenemia, hyperinsulinemia and follicular dysplasia. Metformin or pioglitazone monotherapy significantly suppressed the high level of testosterone, reduced the raised percentage of cystic follicles and primary follicles, promoted the number of early antral follicles, and markedly decreased the high concentration of fasting insulin and homeostatic model assessment for insulin resistance index in PCOS rats. In addition, metformin and pioglitazone combination therapy demonstrated greater efficacy than its individual components. Furthermore, individual or joint treatment with metformin and pioglitazone affected the phosphorylation level of JNK in PCOS rats. Compound C and Wortmannin eliminated the effect of metformin and pioglitazone combination therapy on improving the follicular growth in PCOS rats, whereas SP600125 treatment enhanced this combination therapy effect. These data suggested that metformin and pioglitazone combination therapy

In vitro metabolism of a novel JNK inhibitor tanzisertib: interspecies differences in oxido-reduction and characterization of enzymes involved in metabolism.

PubMed

Atsriku, Christian; Hoffmann, Matthew; Moghaddam, Mehran; Kumar, Gondi; Surapaneni, Sekhar

2015-01-01

1. In vitro metabolism of Tanzisertib [(1S,4R)-4-(9-((S)tetrahydrofuran-3-yl)-8-(2,4,6-trifluorophenylamino)-9H-purin-2-ylamino) cyclohexanol], a potent, selective c-Jun amino-terminal kinase (JNK) inhibitor, was investigated in mouse, rat, rabbit, dog, monkey and human hepatocytes over 4 h. The extent of metabolism of [(14)C]tanzisertib was variable, with <10% metabolized in dog and human, <20% metabolized in rabbit and monkey and >75% metabolized in rat and mouse. Primary metabolic pathways in human and dog hepatocytes, were direct glucuronidation and oxidation of cyclohexanol to a keto metabolite, which was subsequently reduced to parent or cis-isomer, followed by glucuronidation. Rat and mouse produced oxidative metabolites and cis-isomer, including direct glucuronides and sulfates of tanzisertib and cis-isomer. 2. Enzymology of oxido-reductive pathways revealed that human aldo-keto reductases AKR1C1, 1C2, 1C3 and 1C4 were responsible for oxido-reduction of tanzisertib, CC-418424 and keto tanzisertib. Characterizations of enzyme kinetics revealed that AKR1C4 had a high affinity for reduction of keto tanzisertib to tanzisertib compared to other isoforms. These results demonstrate unique stereoselectivity of the reductive properties documented by human AKR1C enzymes for the same substrate. 3. Characterization of UGT isoenzymes in glucuronidation of tanzisertib and CC-418424 revealed that, tanzisertib glucuronide was catalyzed by: UGT1A1, 1A4, 1A10 and 2B4, while CC-418424 glucuronidation was catalyzed by UGT2B4 and 2B7.

Geldanamycin attenuates 3-nitropropionic acid-induced apoptosis and JNK activation through the expression of HSP 70 in striatal cells

PubMed Central

CHOI, YONG-JOON; KIM, NAM HO; LIM, MAN SUP; LEE, HEE JAE; KIM, SUNG SOO; CHUN, WANJOO

2014-01-01

Although selective striatal cell death is a characteristic hallmark in the pathogenesis of Huntington’s disease (HD), the underlying mechanism of striatal susceptibility remains to be clarified. Heat shock proteins (HSPs) have been reported to suppress the aggregate formation of mutant huntingtin and concurrent striatal cell death. In a previous study, we observed that heat shock transcription factor 1 (HSF1), a major transcription factor of HSPs, significantly attenuated 3-nitropropionic acid (3NP)-induced reactive oxygen species (ROS) production and apoptosis through the expression of HSP 70 in striatal cells. To investigate the differential roles of HSPs in 3NP-induced striatal cell death, the effect of geldanamycin (GA), an HSP 90 inhibitor, was examined in 3NP-stimulated striatal cells. GA significantly attenuated 3NP-induced striatal apoptosis and ROS production with an increased expression of HSP 70. Triptolide (TL), an HSP 70 inhibitor, abolished GA-mediated protective effects in 3NP-stimulated striatal cells. To understand the underlying mechanism by which GA-mediated HSP 70 protects striatal cells against 3NP stimulation, the involvement of various signaling pathways was examined. GA significantly attenuated 3NP-induced c-Jun N-terminal kinase (JNK) phosphorylation and subsequent c-Jun phosphorylation in striatal cells. Taken together, the present study demonstrated that GA exhibits protective properties against 3NP-induced apoptosis and JNK activation via the induction of HSP 70 in striatal cells, suggesting that expression of HSP 70 may be a valuable therapeutic target in the treatment of HD. PMID:24756698

Crystal structure of human dual specificity phosphatase, JNK stimulatory phosphatase-1, at 1.5 A resolution.

PubMed

Yokota, Takehiro; Nara, Yukinori; Kashima, Akiko; Matsubara, Keiko; Misawa, Satoru; Kato, Ryohei; Sugio, Shigetoshi

2007-02-01

Human JNK stimulatory phosphatase-1 (JSP-1) is a novel member of dual specificity phosphatases. A C-terminus truncated JSP-1 was expressed in Escherichia coli and was crystallized using the sitting-drop vapor diffusion method. Thin-plate crystals obtained at 278 K belong to a monoclinic space group, C2, with unit-cell parameters a = 84.0 A, b = 49.3 A, c = 47.3 A, and beta = 119.5 degrees , and diffract up to 1.5 A resolution at 100 K. The structure of JSP-1 has a single compact (alpha/beta) domain, which consists of six alpha-helices and five beta-strands, and shows a conserved structural scaffold in regard to both DSPs and PTPs. A cleft formed by a PTP-loop at the active site is very shallow, and is occupied by one sulfonate compound, MES, at the bottom. In the binary complex structure of JSP-1 with MES, the conformations of three important segments in regard to the catalytic mechanism are not similar to those in PTP1B. JSP-1 has no loop corresponding to the Lys120-loop of PTP1B, and tryptophan residue corresponding to the substrate-stacking in PTP1B is substituted by alanine residue in JSP-1. Copyright 2006 Wiley-Liss, Inc.

Resveratrol Ameliorates Palmitate-Induced Inflammation in Skeletal Muscle Cells by Attenuating Oxidative Stress and JNK/NF-κB Pathway in a SIRT1-Independent Mechanism.

PubMed

Sadeghi, Asie; Seyyed Ebrahimi, Shadi Sadat; Golestani, Abolfazl; Meshkani, Reza

2017-09-01

Resveratrol has been shown to exert anti-inflammatory and anti-oxidant effects in a variety of cell types, however, its role in prevention of inflammatory responses mediated by palmitate in skeletal muscle cells remains unexplored. In the present study, we investigated the effects of resveratrol on palmitate-induced inflammation and elucidated the underlying mechanisms in skeletal muscle cells. The results showed that palmitate significantly enhanced TNF-α and IL-6 mRNA expression and protein secretion from C2C12 cells at 12, 24, and 36 h treatments. Increased expression of cytokines was accompanied by an enhanced phosphorylation of JNK, P38, ERK1/2, and IKKα/IKKβ. In addition, JNK and P38 inhibitors could significantly attenuate palmitate-induced mRNA expression of TNF-α and IL-6, respectively, whereas NF-κB inhibitor reduced the expression of both cytokines in palmitate-treated cells. Resveratrol pretreatment significantly prevented palmitate-induced TNF-α and IL-6 mRNA expression and protein secretion in C2C12 cells. Importantly, pre-treatment of the cells with resveratrol completely abrogated the phosphorylation of ERK1/2, JNK, and IKKα/IKKβ in palmitate treated cells. The protection from palmitate-induced inflammation by resveratrol was accompanied by a decrease in the generation of reactive oxygen species (ROS). N-acetyl cysteine (NAC), a known scavenger of ROS, could protect palmitate-induced expression of TNF-α and IL-6. Furthermore, inhibition of SIRT1 by shRNA or sirtinol demonstrated that the anti-inflammatory effect of resveratrol in muscle cells is mediated through a SIRT1-independent mechanism. Taken together, these findings suggest that resveratrol may represent a promising therapy for prevention of inflammation in skeletal muscle cells. J. Cell. Biochem. 118: 2654-2663, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Drosophila myeloid leukemia factor acts with DREF to activate the JNK signaling pathway

PubMed Central

Yanai, H; Yoshioka, Y; Yoshida, H; Nakao, Y; Plessis, A; Yamaguchi, M

2014-01-01

Drosophila myelodysplasia/myeloid leukemia factor (dMLF), a homolog of human MLF1, oncogene was first identified by yeast two-hybrid screen using the DNA replication-related element-binding factor (DREF) as bait. DREF is a transcription factor that regulates proliferation-related genes in Drosophila. It is known that overexpression of dMLF in the wing imaginal discs through the engrailed-GAL4 driver causes an atrophied wing phenotype associated with the induction of apoptosis. However, the precise mechanisms involved have yet to be clarified. Here, we found the atrophied phenotype to be suppressed by loss-of-function mutation of Drosophila Jun N-terminal kinase (JNK), basket (bsk). Overexpression of dMLF induced ectopic JNK activation in the wing disc monitored with the puckered-lacZ reporter line, resulting in induction of apoptosis. The DREF-binding consensus DRE sequence could be shown to exist in the bsk promoter. Chromatin immunoprecipitation assays in S2 cells with anti-dMLF IgG and quantitative real-time PCR revealed that dMLF binds specifically to the bsk promoter region containing the DRE sequence. Furthermore, using a transient luciferase expression assay, we provide evidence that knockdown of dMLF reduced bsk gene promoter activity in S2 cells. Finally, we show that dMLF interacts with DREF in vivo. Altogether, these data indicate that dMLF acts with DREF to stimulate the bsk promoter and consequently activates the JNK pathway to promote apoptosis. PMID:24752236

Drosophila myeloid leukemia factor acts with DREF to activate the JNK signaling pathway.

PubMed

Yanai, H; Yoshioka, Y; Yoshida, H; Nakao, Y; Plessis, A; Yamaguchi, M

2014-04-21

Drosophila myelodysplasia/myeloid leukemia factor (dMLF), a homolog of human MLF1, oncogene was first identified by yeast two-hybrid screen using the DNA replication-related element-binding factor (DREF) as bait. DREF is a transcription factor that regulates proliferation-related genes in Drosophila. It is known that overexpression of dMLF in the wing imaginal discs through the engrailed-GAL4 driver causes an atrophied wing phenotype associated with the induction of apoptosis. However, the precise mechanisms involved have yet to be clarified. Here, we found the atrophied phenotype to be suppressed by loss-of-function mutation of Drosophila Jun N-terminal kinase (JNK), basket (bsk). Overexpression of dMLF induced ectopic JNK activation in the wing disc monitored with the puckered-lacZ reporter line, resulting in induction of apoptosis. The DREF-binding consensus DRE sequence could be shown to exist in the bsk promoter. Chromatin immunoprecipitation assays in S2 cells with anti-dMLF IgG and quantitative real-time PCR revealed that dMLF binds specifically to the bsk promoter region containing the DRE sequence. Furthermore, using a transient luciferase expression assay, we provide evidence that knockdown of dMLF reduced bsk gene promoter activity in S2 cells. Finally, we show that dMLF interacts with DREF in vivo. Altogether, these data indicate that dMLF acts with DREF to stimulate the bsk promoter and consequently activates the JNK pathway to promote apoptosis.

JNK-Interacting Protein 3 Mediates the Retrograde Transport of Activated c-Jun N-Terminal Kinase and Lysosomes

PubMed Central

Drerup, Catherine M.; Nechiporuk, Alex V.

2013-01-01

Retrograde axonal transport requires an intricate interaction between the dynein motor and its cargo. What mediates this interaction is largely unknown. Using forward genetics and a novel in vivo imaging approach, we identified JNK-interacting protein 3 (Jip3) as a direct mediator of dynein-based retrograde transport of activated (phosphorylated) c-Jun N-terminal Kinase (JNK) and lysosomes. Zebrafish jip3 mutants (jip3nl7) displayed large axon terminal swellings that contained high levels of activated JNK and lysosomes, but not other retrograde cargos such as late endosomes and autophagosomes. Using in vivo analysis of axonal transport, we demonstrated that the terminal accumulations of activated JNK and lysosomes were due to a decreased frequency of retrograde movement of these cargos in jip3nl7, whereas anterograde transport was largely unaffected. Through rescue experiments with Jip3 engineered to lack the JNK binding domain and exogenous expression of constitutively active JNK, we further showed that loss of Jip3–JNK interaction underlies deficits in pJNK retrograde transport, which subsequently caused axon terminal swellings but not lysosome accumulation. Lysosome accumulation, rather, resulted from loss of lysosome association with dynein light intermediate chain (dynein accessory protein) in jip3nl7, as demonstrated by our co-transport analyses. Thus, our results demonstrate that Jip3 is necessary for the retrograde transport of two distinct cargos, active JNK and lysosomes. Furthermore, our data provide strong evidence that Jip3 in fact serves as an adapter protein linking these cargos to dynein. PMID:23468645

bFGF Regulates PI3-Kinase-Rac1-JNK Pathway and Promotes Fibroblast Migration in Wound Healing

PubMed Central

Kanazawa, Shigeyuki; Fujiwara, Toshihiro; Matsuzaki, Shinsuke; Shingaki, Kenta; Taniguchi, Manabu; Miyata, Shingo; Tohyama, Masaya; Sakai, Yasuo; Yano, Kenji; Hosokawa, Ko; Kubo, Tateki

2010-01-01

Fibroblast proliferation and migration play important roles in wound healing. bFGF is known to promote both fibroblast proliferation and migration during the process of wound healing. However, the signal transduction of bFGF-induced fibroblast migration is still unclear, because bFGF can affect both proliferation and migration. Herein, we investigated the effect of bFGF on fibroblast migration regardless of its effect on fibroblast proliferation. We noticed involvement of the small GTPases of the Rho family, PI3-kinase, and JNK. bFGF activated RhoA, Rac1, PI3-kinase, and JNK in cultured fibroblasts. Inhibition of RhoA did not block bFGF-induced fibroblast migration, whereas inhibition of Rac1, PI3-kinase, or JNK blocked the fibroblast migration significantly. PI3-kinase-inhibited cells down-regulated the activities of Rac1 and JNK, and Rac1-inhibited cells down-regulated JNK activity, suggesting that PI3-kinase is upstream of Rac1 and that JNK is downstream of Rac1. Thus, we concluded that PI3-kinase, Rac1, and JNK were essential for bFGF-induced fibroblast migration, which is a novel pathway of bFGF-induced cell migration. PMID:20808927

MKP-7, a JNK phosphatase, blocks ERK-dependent gene activation by anchoring phosphorylated ERK in the cytoplasm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Masuda, Kouhei; Katagiri, Chiaki; Division of Biochemical Oncology and Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo

2010-03-05

MAPK phosphatase-7 (MKP-7) was identified as a JNK-specific phosphatase. However, despite its high specificity for JNK, MKP-7 interacts also with ERK. We previously showed that as a physiological consequence of their interaction, activated ERK phosphorylates MKP-7 at Ser-446, and stabilizing MKP-7. In the present study, we analyzed MKP-7 function in activation of ERK. A time-course experiment showed that both MKP-7 and its phosphatase-dead mutant prolonged mitogen-induced ERK phosphorylation, suggesting that MKP-7 functions as a scaffold for ERK. An important immunohistological finding was that nuclear translocation of phospho-ERK following PMA stimulation was blocked by co-expressed MKP-7 and, moreover, that phospho-ERK co-localizedmore » with MKP-7 in the cytoplasm. Reporter gene analysis indicated that MKP-7 blocks ERK-mediated transcription. Overall, our data indicate that MKP-7 down-regulates ERK-dependent gene expression by blocking nuclear accumulation of phospho-ERK.« less

Geldanamycin attenuates 3‑nitropropionic acid‑induced apoptosis and JNK activation through the expression of HSP 70 in striatal cells.

PubMed

Choi, Yong-Joon; Kim, Nam Ho; Lim, Man Sup; Lee, Hee Jae; Kim, Sung Soo; Chun, Wanjoo

2014-07-01

Although selective striatal cell death is a characteristic hallmark in the pathogenesis of Huntington's disease (HD), the underlying mechanism of striatal susceptibility remains to be clarified. Heat shock proteins (HSPs) have been reported to suppress the aggregate formation of mutant huntingtin and concurrent striatal cell death. In a previous study, we observed that heat shock transcription factor 1 (HSF1), a major transcription factor of HSPs, significantly attenuated 3‑nitropropionic acid (3NP)‑induced reactive oxygen species (ROS) production and apoptosis through the expression of HSP 70 in striatal cells. To investigate the differential roles of HSPs in 3NP‑induced striatal cell death, the effect of geldanamycin (GA), an HSP 90 inhibitor, was examined in 3NP‑stimulated striatal cells. GA significantly attenuated 3NP‑induced striatal apoptosis and ROS production with an increased expression of HSP 70. Triptolide (TL), an HSP 70 inhibitor, abolished GA‑mediated protective effects in 3NP‑stimulated striatal cells. To understand the underlying mechanism by which GA‑mediated HSP 70 protects striatal cells against 3NP stimulation, the involvement of various signaling pathways was examined. GA significantly attenuated 3NP‑induced c‑Jun N‑terminal kinase (JNK) phosphorylation and subsequent c‑Jun phosphorylation in striatal cells. Taken together, the present study demonstrated that GA exhibits protective properties against 3NP‑induced apoptosis and JNK activation via the induction of HSP 70 in striatal cells, suggesting that expression of HSP 70 may be a valuable therapeutic target in the treatment of HD.

Propofol protects hippocampal neurons from apoptosis in ischemic brain injury by increasing GLT-1 expression and inhibiting the activation of NMDAR via the JNK/Akt signaling pathway.

PubMed

Gong, Hong-Yan; Zheng, Fang; Zhang, Chao; Chen, Xi-Yan; Liu, Jing-Jing; Yue, Xiu-Qin

2016-09-01

Ischemic brain injury (IBI) can cause nerve injury and is a leading cause of morbidity and mortality worldwide. The neuroprotective effects of propofol against IBI have been previously demonstrated. However, the neuroprotective effects of propofol on hippocampal neurons are not yet entirely clear. In the present study, models of IBI were established in hypoxia-exposed hippocampal neuronal cells. Cell viability assay and apoptosis assay were performed to examine the neuroprotective effects of propofol on hippocampal neurons in IBI. A significant decrease in cell viability and a significant increase in cell apoptosis were observed in the IBI group compared with the control group, accompanied by a decrease in glial glutamate transporter-1 (GLT‑1) expression as determined by RT-qPCR and western blot analysis. The effects of IBI were reversed by propofol treatment. The siRNA-mediated knockdown of GLT‑1 in the hypoxia-exposed hippocampal neuronal cells led to an increase in cell apoptosis, Jun N-terminal kinase (JNK) activation and N-methyl-D‑aspartate (NMDA) receptor (NR1 and NR2B) activation, as well as to a decrease in cell viability and a decrease in Akt activation. The effects of RNA interference-mediated GLT‑1 gene silencing on cell viability, JNK activation, NMDAR activation, cell apoptosis and Akt activation in the hippocampal neuronal cells were slightly reversed by propofol treatment. The JNK agonist, anisomycin, and the Akt inhibitor, LY294002, both significantly blocked the effects of propofol on hippocampal neuronal cell viability and apoptosis in IBI. The decrease in JNK activation and the increase in Akt activation caused by GLT‑1 overexpression were reversed by NMDA. Collectively, our findings suggest that propofol treatment protects hippocampal neurons against IBI by enhancing GLT‑1 expression and inhibiting the activation of NMDAR via the JNK/Akt signaling pathway.

The Inhibitor Ko143 Is Not Specific for ABCG2.

PubMed

Weidner, Lora D; Zoghbi, Sami S; Lu, Shuiyu; Shukla, Suneet; Ambudkar, Suresh V; Pike, Victor W; Mulder, Jan; Gottesman, Michael M; Innis, Robert B; Hall, Matthew D

2015-09-01

Imaging ATP-binding cassette (ABC) transporter activity in vivo with positron emission tomography requires both a substrate and a transporter inhibitor. However, for ABCG2, there is no inhibitor proven to be specific to that transporter alone at the blood-brain barrier. Ko143 [[(3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1',2':1,6]pyrido[3,4- b]indole-3-propanoic acid 1,1-dimethylethyl ester], a nontoxic analog of fungal toxin fumitremorgin C, is a potent inhibitor of ABCG2, although its specificity in mouse and human systems is unclear. This study examined the selectivity of Ko143 using human embryonic kidney cell lines transfected with ABCG2, ABCB1, or ABCC1 in several in vitro assays. The stability of Ko143 in rat plasma was measured using high performance liquid chromatography. Our results show that, in addition to being a potent inhibitor of ABCG2, at higher concentrations (≥1 μM) Ko143 also has an effect on the transport activity of both ABCB1 and ABCC1. Furthermore, Ko143 was found to be unstable in rat plasma. These findings indicate that Ko143 lacks specificity for ABCG2 and this should be taken into consideration when using Ko143 for both in vitro and in vivo experiments. U.S. Government work not protected by U.S. copyright.

Mu opioid receptor stimulation activates c-Jun N-terminal kinase 2 by distinct arrestin-dependent and independent mechanisms.

PubMed

Kuhar, Jamie Rose; Bedini, Andrea; Melief, Erica J; Chiu, Yen-Chen; Striegel, Heather N; Chavkin, Charles

2015-09-01

G protein-coupled receptor desensitization is typically mediated by receptor phosphorylation by G protein-coupled receptor kinase (GRK) and subsequent arrestin binding; morphine, however, was previously found to activate a c-Jun N-terminal kinase (JNK)-dependent, GRK/arrestin-independent pathway to produce mu opioid receptor (MOR) inactivation in spinally-mediated, acute anti-nociceptive responses [Melief et al.] [1]. In the current study, we determined that JNK2 was also required for centrally-mediated analgesic tolerance to morphine using the hotplate assay. We compared JNK activation by morphine and fentanyl in JNK1(-/-), JNK2(-/-), JNK3(-/-), and GRK3(-/-) mice and found that both compounds specifically activate JNK2 in vivo; however, fentanyl activation of JNK2 was GRK3-dependent, whereas morphine activation of JNK2 was GRK3-independent. In MOR-GFP expressing HEK293 cells, treatment with either arrestin siRNA, the Src family kinase inhibitor PP2, or the protein kinase C (PKC) inhibitor Gö6976 indicated that morphine activated JNK2 through an arrestin-independent Src- and PKC-dependent mechanism, whereas fentanyl activated JNK2 through a Src-GRK3/arrestin-2-dependent and PKC-independent mechanism. This study resolves distinct ligand-directed mechanisms of JNK activation by mu opioid agonists and understanding ligand-directed signaling at MOR may improve opioid therapeutics. Copyright © 2015 Elsevier Inc. All rights reserved.

Phosphorylation of Glutathione S-Transferase P1 (GSTP1) by Epidermal Growth Factor Receptor (EGFR) Promotes Formation of the GSTP1-c-Jun N-terminal kinase (JNK) Complex and Suppresses JNK Downstream Signaling and Apoptosis in Brain Tumor Cells*

PubMed Central

Okamura, Tatsunori; Antoun, Gamil; Keir, Stephen T.; Friedman, Henry; Bigner, Darell D.; Ali-Osman, Francis

2015-01-01

Under normal physiologic conditions, the glutathione S-transferase P1 (GSTP1) protein exists intracellularly as a dimer in reversible equilibrium with its monomeric subunits. In the latter form, GSTP1 binds to the mitogen-activated protein kinase, JNK, and inhibits JNK downstream signaling. In tumor cells, which frequently are characterized by constitutively high GSTP1 expression, GSTP1 undergoes phosphorylation by epidermal growth factor receptor (EGFR) at tyrosine residues 3, 7, and 198. Here we report on the effect of this EGFR-dependent GSTP1 tyrosine phosphorylation on the interaction of GSTP1 with JNK, on the regulation of JNK downstream signaling by GSTP1, and on tumor cell survival. Using in vitro and in vivo growing human brain tumors, we show that tyrosine phosphorylation shifts the GSTP1 dimer-monomer equilibrium to the monomeric state and facilitates the formation of the GSTP1-JNK complex, in which JNK is functionally inhibited. Targeted mutagenesis and functional analysis demonstrated that the increased GSTP1 binding to JNK results from phosphorylation of the GSTP1 C-terminal Tyr-198 by EGFR and is associated with a >2.5-fold decrease in JNK downstream signaling and a significant suppression of both spontaneous and drug-induced apoptosis in the tumor cells. The findings define a novel mechanism of regulatory control of JNK signaling that is mediated by the EGFR/GSTP1 cross-talk and provides a survival advantage for tumors with activated EGFR and high GSTP1 expression. The results lay the foundation for a novel strategy of dual EGFR/GSTP1 for treating EGFR+ve, GSTP1 expressing GBMs. PMID:26429914

Phosphorylation of Glutathione S-Transferase P1 (GSTP1) by Epidermal Growth Factor Receptor (EGFR) Promotes Formation of the GSTP1-c-Jun N-terminal kinase (JNK) Complex and Suppresses JNK Downstream Signaling and Apoptosis in Brain Tumor Cells.

PubMed

Okamura, Tatsunori; Antoun, Gamil; Keir, Stephen T; Friedman, Henry; Bigner, Darell D; Ali-Osman, Francis

2015-12-25

Under normal physiologic conditions, the glutathione S-transferase P1 (GSTP1) protein exists intracellularly as a dimer in reversible equilibrium with its monomeric subunits. In the latter form, GSTP1 binds to the mitogen-activated protein kinase, JNK, and inhibits JNK downstream signaling. In tumor cells, which frequently are characterized by constitutively high GSTP1 expression, GSTP1 undergoes phosphorylation by epidermal growth factor receptor (EGFR) at tyrosine residues 3, 7, and 198. Here we report on the effect of this EGFR-dependent GSTP1 tyrosine phosphorylation on the interaction of GSTP1 with JNK, on the regulation of JNK downstream signaling by GSTP1, and on tumor cell survival. Using in vitro and in vivo growing human brain tumors, we show that tyrosine phosphorylation shifts the GSTP1 dimer-monomer equilibrium to the monomeric state and facilitates the formation of the GSTP1-JNK complex, in which JNK is functionally inhibited. Targeted mutagenesis and functional analysis demonstrated that the increased GSTP1 binding to JNK results from phosphorylation of the GSTP1 C-terminal Tyr-198 by EGFR and is associated with a >2.5-fold decrease in JNK downstream signaling and a significant suppression of both spontaneous and drug-induced apoptosis in the tumor cells. The findings define a novel mechanism of regulatory control of JNK signaling that is mediated by the EGFR/GSTP1 cross-talk and provides a survival advantage for tumors with activated EGFR and high GSTP1 expression. The results lay the foundation for a novel strategy of dual EGFR/GSTP1 for treating EGFR+ve, GSTP1 expressing GBMs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A novel dual NO-donating oxime and c-Jun N-terminal kinase inhibitor protects against cerebral ischemia-reperfusion injury in mice.

PubMed

Atochin, Dmitriy N; Schepetkin, Igor A; Khlebnikov, Andrei I; Seledtsov, Victor I; Swanson, Helen; Quinn, Mark T; Huang, Paul L

2016-04-08

The c-Jun N-terminal kinase (JNK) has been shown to be an important regulator of neuronal cell death. Previously, we synthesized the sodium salt of 11H-indeno[1,2-b]quinoxalin-11-one (IQ-1S) and demonstrated that it was a high-affinity inhibitor of the JNK family. In the present work, we found that IQ-1S could release nitric oxide (NO) during its enzymatic metabolism by liver microsomes. Moreover, serum nitrite/nitrate concentration in mice increased after intraperitoneal injection of IQ-1S. Because of these dual actions as JNK inhibitor and NO-donor, the therapeutic potential of IQ-1S was evaluated in an animal stroke model. We subjected wild-type C57BL6 mice to focal ischemia (30min) with subsequent reperfusion (48h). Mice were treated with IQ-1S (25mg/kg) suspended in 10% solutol or with vehicle alone 30min before and 24h after middle cerebral artery (MCA) occlusion (MCAO). Using laser-Doppler flowmetry, we monitored cerebral blood flow (CBF) above the MCA during 30min of MCAO provoked by a filament and during the first 30min of subsequent reperfusion. In mice treated with IQ-1S, ischemic and reperfusion values of CBF were not different from vehicle-treated mice. However, IQ-1S treated mice demonstrated markedly reduced neurological deficit and infarct volumes as compared with vehicle-treated mice after 48h of reperfusion. Our results indicate that the novel JNK inhibitor releases NO during its oxidoreductive bioconversion and improves stroke outcome in a mouse model of cerebral reperfusion. We conclude that IQ-1S is a promising dual functional agent for the treatment of cerebral ischemia and reperfusion injury. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A Novel Dual NO-donating Oxime and c-Jun N-terminal Kinase Inhibitor Protects Against Cerebral Ischemia–Reperfusion Injury in Mice

PubMed Central

Atochin, Dmitriy N.; Schepetkin, Igor A.; Khlebnikov, Andrei I.; Seledtsov, Victor I.; Swanson, Helen; Quinn, Mark T.; Huang, Paul L.

2017-01-01

The c-Jun N-terminal kinase (JNK) has been shown to be an important regulator of neuronal cell death. Previously, we synthesized the sodium salt of 11H-indeno[1,2-b]quinoxalin-11-one (IQ-1S) and demonstrated that it was a high-affinity inhibitor of the JNK family. In the present work, we found that IQ-1S could release nitric oxide (NO) during its enzymatic metabolism by liver microsomes. Moreover, serum nitrite/nitrate concentration in mice increased after intraperitoneal injection of IQ-1S. Because of these dual actions as JNK inhibitor and NO-donor, the therapeutic potential of IQ-1S was evaluated in an animal stroke model. We subjected wild-type C57BL6 mice to focal ischemia (30 minutes) with subsequent reperfusion (48 hours). Mice were treated with IQ-1S (25 mg/kg) suspended in 10% solutol or with vehicle alone 30 minutes before and 24 hours after middle cerebral artery MCA) occlusion (MCAO). Using laser-Doppler flowmetry, we monitored cerebral blood flow (CBF) above the MCA during 30 minutes of MCAO provoked by a filament and during the first 30 minutes of subsequent reperfusion. In mice treated with IQ-1S, ischemic and reperfusion values of CBF were not different from vehicle-treated mice. However, IQ-1S treated mice demonstrated markedly reduced neurological deficit and infarct volumes as compared with vehicle-treated mice after 48 hours of reperfusion. Our results indicate that the novel JNK inhibitor releases NO during its oxidoreductive bioconversion and improves stroke outcome in a mouse model of cerebral reperfusion. We conclude that IQ-1S is a promising dual functional agent for the treatment of cerebral ischemia and reperfusion injury. PMID:26923672

WNT5A-JNK regulation of vascular insulin resistance in human obesity.

PubMed

Farb, Melissa G; Karki, Shakun; Park, Song-Young; Saggese, Samantha M; Carmine, Brian; Hess, Donald T; Apovian, Caroline; Fetterman, Jessica L; Bretón-Romero, Rosa; Hamburg, Naomi M; Fuster, José J; Zuriaga, María A; Walsh, Kenneth; Gokce, Noyan

2016-12-01

Obesity is associated with the development of vascular insulin resistance; however, pathophysiological mechanisms are poorly understood. We sought to investigate the role of WNT5A-JNK in the regulation of insulin-mediated vasodilator responses in human adipose tissue arterioles prone to endothelial dysfunction. In 43 severely obese (BMI 44±11 kg/m 2 ) and five metabolically normal non-obese (BMI 26±2 kg/m 2 ) subjects, we isolated arterioles from subcutaneous and visceral fat during planned surgeries. Using videomicroscopy, we examined insulin-mediated, endothelium-dependent vasodilator responses and characterized adipose tissue gene and protein expression using real-time polymerase chain reaction and Western blot analyses. Immunofluorescence was used to quantify endothelial nitric oxide synthase (eNOS) phosphorylation. Insulin-mediated vasodilation was markedly impaired in visceral compared to subcutaneous vessels from obese subjects (p<0.001), but preserved in non-obese individuals. Visceral adiposity was associated with increased JNK activation and elevated expression of WNT5A and its non-canonical receptors, which correlated negatively with insulin signaling. Pharmacological JNK antagonism with SP600125 markedly improved insulin-mediated vasodilation by sixfold (p<0.001), while endothelial cells exposed to recombinant WNT5A developed insulin resistance and impaired eNOS phosphorylation (p<0.05). We observed profound vascular insulin resistance in the visceral adipose tissue arterioles of obese subjects that was associated with up-regulated WNT5A-JNK signaling and impaired endothelial eNOS activation. Pharmacological JNK antagonism markedly improved vascular endothelial function, and may represent a potential therapeutic target in obesity-related vascular disease. © The Author(s) 2016.

WNT5A-JNK regulation of vascular insulin resistance in human obesity

PubMed Central

Farb, Melissa G; Karki, Shakun; Park, Song-Young; Saggese, Samantha M; Carmine, Brian; Hess, Donald T; Apovian, Caroline; Fetterman, Jessica L; Bretón-Romero, Rosa; Hamburg, Naomi M; Fuster, José J; Zuriaga, María A; Walsh, Kenneth; Gokce, Noyan

2017-01-01

Obesity is associated with the development of vascular insulin resistance; however, pathophysiological mechanisms are poorly understood. We sought to investigate the role of WNT5A-JNK in the regulation of insulin-mediated vasodilator responses in human adipose tissue arterioles prone to endothelial dysfunction. In 43 severely obese (BMI 44±11 kg/m2) and five metabolically normal non-obese (BMI 26±2 kg/m2) subjects, we isolated arterioles from subcutaneous and visceral fat during planned surgeries. Using videomicroscopy, we examined insulin-mediated, endothelium-dependent vasodilator responses and characterized adipose tissue gene and protein expression using real-time polymerase chain reaction and Western blot analyses. Immunofluorescence was used to quantify endothelial nitric oxide synthase (eNOS) phosphorylation. Insulin-mediated vasodilation was markedly impaired in visceral compared to subcutaneous vessels from obese subjects (p<0.001), but preserved in non-obese individuals. Visceral adiposity was associated with increased JNK activation and elevated expression of WNT5A and its non-canonical receptors, which correlated negatively with insulin signaling. Pharmacological JNK antagonism with SP600125 markedly improved insulin-mediated vasodilation by sixfold (p<0.001), while endothelial cells exposed to recombinant WNT5A developed insulin resistance and impaired eNOS phosphorylation (p<0.05). We observed profound vascular insulin resistance in the visceral adipose tissue arterioles of obese subjects that was associated with up-regulated WNT5A-JNK signaling and impaired endothelial eNOS activation. Pharmacological JNK antagonism markedly improved vascular endothelial function, and may represent a potential therapeutic target in obesity-related vascular disease. PMID:27688298

JNK1 inhibition by Licochalcone A leads to neuronal protection against excitotoxic insults derived of kainic acid.

PubMed

Busquets, Oriol; Ettcheto, Miren; Verdaguer, Ester; Castro-Torres, Ruben D; Auladell, Carme; Beas-Zarate, Carlos; Folch, Jaume; Camins, Antoni

2018-03-15

The mitogen-activated protein kinase family (MAPK) is an important group of enzymes involved in cellular responses to diverse external stimuli. One of the members of this family is the c-Jun-N-terminal kinase (JNK). The activation of the JNK pathway has been largely associated with the pathogenesis that occurs in epilepsy and neurodegeneration. Kainic acid (KA) administration in rodents is an experimental approach that induces status epilepticus (SE) and replicates many of the phenomenological features of human temporal lobe epilepsy (TLE). Recent studies in our group have evidenced that the absence of the JNK1 gene has neuroprotective effects against the damage induced by KA, as it occurs with the absence of JNK3. The aim of the present study was to analyse whether the pharmacological inhibition of JNK1 by Licochalcone A (Lic-A) had similar effects and if it may be considered as a new molecule for the treatment of SE. In order to achieve this objective, animals were pre-treated with Lic-A and posteriorly administered with KA as a model for TLE. In addition, a comparative study with KA was performed between wild type pre-treated with Lic-A and single knock-out transgenic mice for the Jnk1 -/- gene. Our results showed that JNK1 inhibition by Lic-A, previous to KA administration, caused a reduction in the convulsive pattern. Furthermore, it reduced phosphorylation levels of the JNK, as well as its activity. In addition, Lic-A prevented hippocampal neuronal degeneration, increased pro-survival anti-apoptotic mechanisms, reduced pro-apoptotic biomarkers, decreased cellular stress and neuroinflammatory processes. Thus, our results suggest that inhibition of the JNK1 by Lic-A has neuroprotective effects and that; it could be a new potential approach for the treatment of SE and neurodegeneration. Copyright © 2017 Elsevier Ltd. All rights reserved.

c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis

DTIC Science & Technology

2014-10-01

AWARD NUMBER: W81XWH-12-1-0431 TITLE: “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis ” PRINCIPAL INVESTIGATOR...TITLE AND SUBTITLE “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Scelerosis” 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH

Blockade of the spinal BDNF-activated JNK pathway prevents the development of antiretroviral-induced neuropathic pain.

PubMed

Sanna, Maria Domenica; Ghelardini, Carla; Galeotti, Nicoletta

2016-06-01

Although antiretroviral agents have been used successfully in suppressing viral production, they have also been associated with a number of side effects. The antiretroviral toxic neuropathy induces debilitating and extremely difficult to treat pain syndromes that often lead to discontinuation of antiretroviral therapy. Due to the critical need for the identification of novel therapeutic targets to improve antiretroviral neuropathic pain management, we investigated the role of the JNK signalling pathway in the mechanism of antiretroviral painful neuropathy. Mice were exposed to zalcitabine (2',3'-dideoxycytidine, ddC) and stavudine (2',3'-didehydro-3'-deoxythymidine, d4T) that induced a persistent mechanical allodynia and a transient cold allodynia. Treatment with the JNK blocker SP600125 before antiretroviral administration abolished mechanical hypersensitivity with no effect on thermal response. A robust spinal JNK overphosphorylation was observed on post-injection day 1 and 3, along with a JNK-dependent increase in p-c-Jun and ATF3 protein levels. Co-immunoprecipitation experiments showed the presence of a heterodimeric complex between ATF3 and c-Jun indicating that these transcription factors can act together to regulate transcription through heterodimerization. A rise in BDNF and caspase-3 protein levels was detected on day 1 and BDNF sequestration prevented both caspase-3 and p-JNK increase. These data suggest that BDNF plays a role in the early stages of ddC-induced allodynia by promoting apoptotic events and the activation of a hypernociceptive JNK-mediated pathway. We illustrated the activation of a BDNF-mediated JNK pathway involved in the early events responsible for the promotion of neuropathic pain, leading to a better knowledge of the mechanisms involved in the antiretroviral neuropathy. JNK blockade prevents antiretroviral-induced pain hypersensitivity. This may represent a potential prophylactic treatment of neuropathic pain to improve antiretroviral

Synthesis and evaluation of phosphorus containing, specific CDK9/CycT1 inhibitors.

PubMed

Németh, Gábor; Greff, Zoltán; Sipos, Anna; Varga, Zoltán; Székely, Rita; Sebestyén, Mónika; Jászay, Zsuzsa; Béni, Szabolcs; Nemes, Zoltán; Pirat, Jean-Luc; Volle, Jean-Noël; Virieux, David; Gyuris, Ágnes; Kelemenics, Katalin; Ay, Eva; Minarovits, Janos; Szathmary, Susan; Kéri, György; Orfi, László

2014-05-22

Although there is a significant effort in the design of a selective CDK9/CycT1 inhibitor, no compound has been proven to be a specific inhibitor of this kinase so far. The aim of this research was to develop novel and selective phosphorus containing CDK9/CycT1 inhibitors. Molecules bearing phosphonamidate, phosphonate, and phosphinate moieties were synthesized. Prepared compounds were evaluated in an enzymatic CDK9/CycT1 assay. The most potent molecules were tested in cell-based toxicity and HIV proliferation assays. Selectivity of shortlisted compounds against CDKs and other kinases was tested. The best compound was shown to be a highly specific, ATP-competitive inhibitor of CDK9/CycT1 with antiviral activity.

Sirtuin 3 enhanced drug sensitivity of human hepatoma cells through glutathione S-transferase pi 1/JNK signaling pathway

PubMed Central

Cai, Xue-Fei; Zhang, Wen-Lu; Ren, Ji-Hua; Zhou, Li; Chen, Xiang; Chen, Ke; Li, Wan-Yu; Liu, Bo; Yang, Qiu-Xia; Cheng, Sheng-Tao; Huang, Li-Xia; Huang, Ai-Long; Chen, Juan

2016-01-01

SIRT3, a class III histone deacetylase, has been implicated in various cancers as a novel therapeutic target. In hepatocellular carcinoma (HCC), we previously reported that SIRT3 induced cell apoptosis by regulating GSK-3β/Bax signaling pathway. Downregulation of SIRT3 in HCC cells facilitates tumor cell survival. In this study, we found that chemotherapeutic agents (doxorubicin, cisplatin and epirubicin) and sorafenib treatment downregulated SIRT3 mRNA and protein levels in three HCC cell lines. MTS assay found that SIRT3 overexpression sensitized liver cancer cells to chemotherapeutic agents and sorafenib in SMMC-7721, Huh-7 and PLC/PRF/5 cell lines. Moreover, SIRT3 overexpression promoted chemotherapeutic agents-induced or sorafenib-induced apoptosis as evidenced by flow cytometry, enhanced PARP cleavage and enhanced Caspase-9 cleavage in three HCC cells. In contrast, SIRT3 silencing increased drug resistance of HCC cells to chemotherapeutic agents. Mechanistic study found that SIRT3 downregulated the mRNA and protein levels of glutathione S-transferase pi 1 (GSTP1), which is a member of phase II detoxification enzymes families involved in metabolizing for chemotherapeutic agents. Moreover, SIRT3 decreased the amount of GSTP1 that was associated with JNK, which finally contributed the activation of JNK activity and activation of downstream target c-Jun and Bim. Importantly, GSTP1 overexpression or JNK inhibitor abolished SIRT3-induced apoptosis in HCC cells exposed to chemotherapeutic agents. Finally, there was a negative correlation between SIRT3 expression and GSTP1 expression in human HCC tissues. Together, our findings revealed SIRT3 could enhance the drug sensitivity of HCC cells to an array of chemotherapeutic agents. SIRT3 may serve as a potential target for improving the chemosensitivity of HCC patients. PMID:27367026

Sirtuin 3 enhanced drug sensitivity of human hepatoma cells through glutathione S-transferase pi 1/JNK signaling pathway.

PubMed

Tao, Na-Na; Zhou, Hong-Zhong; Tang, Hua; Cai, Xue-Fei; Zhang, Wen-Lu; Ren, Ji-Hua; Zhou, Li; Chen, Xiang; Chen, Ke; Li, Wan-Yu; Liu, Bo; Yang, Qiu-Xia; Cheng, Sheng-Tao; Huang, Li-Xia; Huang, Ai-Long; Chen, Juan

2016-08-02

SIRT3, a class III histone deacetylase, has been implicated in various cancers as a novel therapeutic target. In hepatocellular carcinoma (HCC), we previously reported that SIRT3 induced cell apoptosis by regulating GSK-3β/Bax signaling pathway. Downregulation of SIRT3 in HCC cells facilitates tumor cell survival. In this study, we found that chemotherapeutic agents (doxorubicin, cisplatin and epirubicin) and sorafenib treatment downregulated SIRT3 mRNA and protein levels in three HCC cell lines. MTS assay found that SIRT3 overexpression sensitized liver cancer cells to chemotherapeutic agents and sorafenib in SMMC-7721, Huh-7 and PLC/PRF/5 cell lines. Moreover, SIRT3 overexpression promoted chemotherapeutic agents-induced or sorafenib-induced apoptosis as evidenced by flow cytometry, enhanced PARP cleavage and enhanced Caspase-9 cleavage in three HCC cells. In contrast, SIRT3 silencing increased drug resistance of HCC cells to chemotherapeutic agents. Mechanistic study found that SIRT3 downregulated the mRNA and protein levels of glutathione S-transferase pi 1 (GSTP1), which is a member of phase II detoxification enzymes families involved in metabolizing for chemotherapeutic agents. Moreover, SIRT3 decreased the amount of GSTP1 that was associated with JNK, which finally contributed the activation of JNK activity and activation of downstream target c-Jun and Bim. Importantly, GSTP1 overexpression or JNK inhibitor abolished SIRT3-induced apoptosis in HCC cells exposed to chemotherapeutic agents. Finally, there was a negative correlation between SIRT3 expression and GSTP1 expression in human HCC tissues. Together, our findings revealed SIRT3 could enhance the drug sensitivity of HCC cells to an array of chemotherapeutic agents. SIRT3 may serve as a potential target for improving the chemosensitivity of HCC patients.

Aminopyridine-based c-Jun N-terminal kinase inhibitors with cellular activity and minimal cross-kinase activity.

PubMed

Szczepankiewicz, Bruce G; Kosogof, Christi; Nelson, Lissa T J; Liu, Gang; Liu, Bo; Zhao, Hongyu; Serby, Michael D; Xin, Zhili; Liu, Mei; Gum, Rebecca J; Haasch, Deanna L; Wang, Sanyi; Clampit, Jill E; Johnson, Eric F; Lubben, Thomas H; Stashko, Michael A; Olejniczak, Edward T; Sun, Chaohong; Dorwin, Sarah A; Haskins, Kristi; Abad-Zapatero, Cele; Fry, Elizabeth H; Hutchins, Charles W; Sham, Hing L; Rondinone, Cristina M; Trevillyan, James M

2006-06-15

The c-Jun N-terminal kinases (JNK-1, -2, and -3) are members of the mitogen activated protein (MAP) kinase family of enzymes. They are activated in response to certain cytokines, as well as by cellular stresses including chemotoxins, peroxides, and irradiation. They have been implicated in the pathology of a variety of different diseases with an inflammatory component including asthma, stroke, Alzheimer's disease, and type 2 diabetes mellitus. In this work, high-throughput screening identified a JNK inhibitor with an excellent kinase selectivity profile. Using X-ray crystallography and biochemical screening to guide our lead optimization, we prepared compounds with inhibitory potencies in the low-double-digit nanomolar range, activity in whole cells, and pharmacokinetics suitable for in vivo use. The new compounds were over 1,000-fold selective for JNK-1 and -2 over other MAP kinases including ERK2, p38alpha, and p38delta and showed little inhibitory activity against a panel of 74 kinases.

Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele

Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites onmore » the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes

Iodine-131 treatment of thyroid cancer cells leads to suppression of cell proliferation followed by induction of cell apoptosis and cell cycle arrest by regulation of B-cell translocation gene 2-mediated JNK/NF-κB pathways.

PubMed

Zhao, L M; Pang, A X

2017-01-16

Iodine-131 (131I) is widely used for the treatment of thyroid-related diseases. This study aimed to investigate the expression of p53 and BTG2 genes following 131I therapy in thyroid cancer cell line SW579 and the possible underlying mechanism. SW579 human thyroid squamous carcinoma cells were cultured and treated with 131I. They were then assessed for 131I uptake, cell viability, apoptosis, cell cycle arrest, p53 expression, and BTG2 gene expression. SW579 cells were transfected with BTG2 siRNA, p53 siRNA and siNC and were then examined for the same aforementioned parameters. When treated with a JNK inhibitor of SP600125 and 131I or with a NF-κB inhibitor of BMS-345541 and 131I, non-transfected SW579 cells were assessed in JNK/NFκB pathways. It was observed that 131I significantly inhibited cell proliferation, promoted cell apoptosis and cell cycle arrest. Both BTG2 and p53 expression were enhanced in a dose-dependent manner. An increase in cell viability by up-regulation in Bcl2 gene, a decrease in apoptosis by enhanced CDK2 gene expression and a decrease in cell cycle arrest at G0/G1 phase were also observed in SW579 cell lines transfected with silenced BTG2 gene. When treated with SP600125 and 131I, the non-transfected SW579 cell lines significantly inhibited JNK pathway, NF-κB pathway and the expression of BTG2. However, when treated with BMS-345541 and 131I, only the NF-κB pathway was suppressed. 131I suppressed cell proliferation, induced cell apoptosis, and promoted cell cycle arrest of thyroid cancer cells by up-regulating B-cell translocation gene 2-mediated activation of JNK/NF-κB pathways.

Inhibition of spinal astrocytic c-Jun N-terminal kinase (JNK) activation correlates with the analgesic effects of ketamine in neuropathic pain

PubMed Central

2011-01-01

Background We have previously reported that inhibition of astrocytic activation contributes to the analgesic effects of intrathecal ketamine on spinal nerve ligation (SNL)-induced neuropathic pain. However, the underlying mechanisms are still unclear. c-Jun N-terminal kinase (JNK), a member of mitogen-activated protein kinase (MAPK) family, has been reported to be critical for spinal astrocytic activation and neuropathic pain development after SNL. Ketamine can decrease lipopolysaccharide (LPS)-induced phosphorylated JNK (pJNK) expression and could thus exert its anti-inflammatory effect. We hypothesized that inhibition of astrocytic JNK activation might be involved in the suppressive effect of ketamine on SNL-induced spinal astrocytic activation. Methods Immunofluorescence histochemical staining was used to detect SNL-induced spinal pJNK expression and localization. The effects of ketamine on SNL-induced mechanical allodynia were confirmed by behavioral testing. Immunofluorescence histochemistry and Western blot were used to quantify the SNL-induced spinal pJNK expression after ketamine administration. Results The present study showed that SNL induced ipsilateral pJNK up-regulation in astrocytes but not microglia or neurons within the spinal dorsal horn. Intrathecal ketamine relieved SNL-induced mechanical allodynia without interfering with motor performance. Additionally, intrathecal administration of ketamine attenuated SNL-induced spinal astrocytic JNK activation in a dose-dependent manner, but not JNK protein expression. Conclusions The present results suggest that inhibition of JNK activation may be involved in the suppressive effects of ketamine on SNL-induced spinal astrocyte activation. Therefore, inhibition of spinal JNK activation may be involved in the analgesic effects of ketamine on SNL-induced neuropathic pain. PMID:21255465

Improvement of liver injury and survival by JNK2 and iNOS deficiency in liver transplants from cardiac death mice.

PubMed

Liu, Qinlong; Rehman, Hasibur; Krishnasamy, Yasodha; Schnellmann, Rick G; Lemasters, John J; Zhong, Zhi

2015-07-01

Inclusion of liver grafts from cardiac death donors (CDD) would increase the availability of donor livers but is hampered by a higher risk of primary non-function. Here, we seek to determine mechanisms that contribute to primary non-function of liver grafts from CDD with the goal to develop strategies for improved function and outcome, focusing on c-Jun-N-terminal kinase (JNK) activation and mitochondrial depolarization, two known mediators of graft failure. Livers explanted from wild-type, inducible nitric oxide synthase knockout (iNOS(-/-)), JNK1(-/-) or JNK2(-/-) mice after 45-min aorta clamping were implanted into wild-type recipients. Mitochondrial depolarization was detected by intravital confocal microscopy in living recipients. After transplantation of wild-type CDD livers, graft iNOS expression and 3-nitrotyrosine adducts increased, but hepatic endothelial NOS expression was unchanged. Graft injury and dysfunction were substantially higher in CDD grafts than in non-CDD grafts. iNOS deficiency and inhibition attenuated injury and improved function and survival of CDD grafts. JNK1/2 and apoptosis signal-regulating kinase-1 activation increased markedly in wild-type CDD grafts, which was blunted by iNOS deficiency. JNK inhibition and JNK2 deficiency, but not JNK1 deficiency, decreased injury and improved function and survival of CDD grafts. Mitochondrial depolarization and binding of phospho-JNK2 to Sab, a mitochondrial protein linked to the mitochondrial permeability transition, were higher in CDD than in non-CDD grafts. iNOS deficiency, JNK inhibition and JNK2 deficiency all decreased mitochondrial depolarization and blunted ATP depletion in CDD grafts. JNK inhibition and deficiency did not decrease 3-nitrotyrosine adducts in CDD grafts. The iNOS-JNK2-Sab pathway promotes CDD graft failure via increased mitochondrial depolarization, and is an attractive target to improve liver function and survival in CDD liver transplantation recipients. Copyright © 2015

Cone arrestin binding to JNK3 and Mdm2: conformational preference and localization of interaction sites

PubMed Central

Song, Xiufeng; Gurevich, Eugenia V.; Gurevich, Vsevolod V.

2008-01-01

Arrestins are multi-functional regulators of G protein-coupled receptors. Receptor-bound arrestins interact with >30 remarkably diverse proteins and redirect the signaling to G protein-independent pathways. The functions of free arrestins are poorly understood, and the interaction sites of the non-receptor arrestin partners are largely unknown. In this study, we show that cone arrestin, the least studied member of the family, binds c-Jun N-terminal kinase (JNK3) and Mdm2 and regulates their subcellular distribution. Using arrestin mutants with increased or reduced structural flexibility, we demonstrate that arrestin in all conformations binds JNK3 comparably, whereas Mdm2 preferentially binds cone arrestin ‘frozen’ in the basal state. To localize the interaction sites, we expressed separate N- and C-domains of cone and rod arrestins and found that individual domains bind JNK3 and remove it from the nucleus as efficiently as full-length proteins. Thus, the arrestin binding site for JNK3 includes elements in both domains with the affinity of partial sites on individual domains sufficient for JNK3 relocalization. N-domain of rod arrestin binds Mdm2, which localizes its main interaction site to this region. Comparable binding of JNK3 and Mdm2 to four arrestin subtypes allowed us to identify conserved residues likely involved in these interactions. PMID:17680991

PPARδ inhibits UVB-induced secretion of MMP-1 through MKP-7-mediated suppression of JNK signaling.

PubMed

Ham, Sun A; Kang, Eun S; Lee, Hanna; Hwang, Jung S; Yoo, Taesik; Paek, Kyung S; Park, Chankyu; Kim, Jin-Hoi; Lim, Dae-Seog; Seo, Han G

2013-11-01

In the present study, we investigated the role of peroxisome proliferator-activated receptor (PPAR) δ in modulating matrix-degrading metalloproteinases and other mechanisms underlying photoaging processes in the skin. In human dermal fibroblasts (HDFs), activation of PPARδ by its specific ligand GW501516 markedly attenuated UVB-induced secretion of matrix metalloproteinase (MMP)-1, concomitant with decreased generation of reactive oxygen species. These effects were significantly reduced in the presence of PPARδ small interfering RNA and GSK0660. Furthermore, c-Jun N-terminal kinase (JNK), but not p38 or extracellular signal-regulated kinase, mediated PPARδ-dependent inhibition of MMP-1 secretion in HDFs exposed to UVB. PPARδ-mediated messenger RNA stabilization of mitogen-activated protein kinase phosphatase (MKP)-7 was responsible for the GW501516-mediated inhibition of JNK signaling. Inhibition of UVB-induced secretion of MMP-1 by PPARδ was associated with the restoration of types I and III collagen to levels approaching those in cells not exposed to UVB. Finally, in HR-1 hairless mice exposed to UVB, administration of GW501516 significantly reduced wrinkle formation and skin thickness, downregulated MMP-1 and JNK phosphorylation, and restored the levels of MKP-7, types I and III collagen. These results suggest that PPARδ-mediated inhibition of MMP-1 secretion prevents some effects of photoaging and maintains the integrity of skin by inhibiting the degradation of the collagenous extracellular matrix.

A dual-specificity isoform of the protein kinase inhibitor PKI produced by alternate gene splicing.

PubMed

Kumar, Priyadarsini; Walsh, Donal A

2002-03-15

We have previously shown that the protein kinase inhibitor beta (PKIbeta) form of the cAMP-dependent protein kinase inhibitor exists in multiple isoforms, some of which are specific inhibitors of the cAMP-dependent protein kinase, whereas others also inhibit the cGMP-dependent enzyme [Kumar, Van Patten and Walsh (1997), J. Biol. Chem. 272, 20011-20020]. We have now demonstrated that the switch from a cAMP-dependent protein kinase (PKA)-specific inhibitor to one with dual specificity arises as a consequence of alternate gene splicing. We have confirmed using bacterially produced pure protein that a single inhibitor species has dual specificity for both PKA and cGMP-dependent protein kinase (PKG), inhibiting each with very high and closely similar inhibitory potencies. The gene splicing converted a protein with 70 amino acids into one of 109 amino acids, and did not change the inhibitory potency to PKA, but changed it from a protein that had no detectable PKG inhibitory activity to one that now inhibited PKG in the nanomolar range.

Histone-like DNA binding protein of Streptococcus intermedius induces the expression of pro-inflammatory cytokines in human monocytes via activation of ERK1/2 and JNK pathways.

PubMed

Liu, Dali; Yumoto, Hiromichi; Hirota, Katsuhiko; Murakami, Keiji; Takahashi, Kanako; Hirao, Kouji; Matsuo, Takashi; Ohkura, Kazuto; Nagamune, Hideaki; Miyake, Yoichiro

2008-01-01

Streptococcus intermedius is a commensal associated with serious, deep-seated purulent infections in major organs, such as the brain and liver. Histone-like DNA binding protein (HLP) is an accessory architectural protein in a variety of bacterial cellular processes. In this study, we investigated the mechanisms of pro-inflammatory cytokine inductions in THP-1 cells by stimulation with recombinant HLP of S. intermedius (rSi-HLP). rSi-HLP stimulation-induced production of pro-inflammatory cytokines (IL-8, IL-1 beta and TNF-alpha) occurred in a time- and dose-dependent manner. In contrast with the heat-stable activity of DNA binding, the induction activity of rSi-HLP was heat-unstable. In subsequent studies, rSi-HLP acted cooperatively with lipoteichoic acid, the synthetic Toll-like receptor 2 agonist, Pam3CSK4, and the cytosolic nucleotide binding oligomerization domain 2 receptor agonist, muramyldipeptide. Furthermore, Western blot and blocking assays with specific inhibitors showed that rSi-HLP stimulation induced the activation of cell signal transduction pathways, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). In addition to its physiological role in bacterial growth through DNA binding, these results indicate that Si-HLP can trigger a cascade of events that induce pro-inflammatory responses via ERK1/2 and JNK signal pathways, and suggest that bacterial HLP may contribute to the activation of host innate immunity during bacterial infection.

Nitric oxide balances osteoblast and adipocyte lineage differentiation via the JNK/MAPK signaling pathway in periodontal ligament stem cells.

PubMed

Yang, Shan; Guo, Lijia; Su, Yingying; Wen, Jing; Du, Juan; Li, Xiaoyan; Liu, Yitong; Feng, Jie; Xie, Yongmei; Bai, Yuxing; Wang, Hao; Liu, Yi

2018-05-02

Critical tissues that undergo regeneration in periodontal tissue are of mesenchymal origin; thus, investigating the regulatory mechanisms underlying the fate of periodontal ligament stem cells could be beneficial for application in periodontal tissue regeneration. Nitric oxide (NO) regulates many biological processes in developing embryos and adult stem cells. The present study was designed to investigate the effects of NO on the function of human periodontal ligament stem cells (PDLSCs) as well as to elucidate the underlying molecular mechanisms. Immunofluorescent staining and flow cytometry were used for stem cell identification. Western blot, reverse transcription polymerase chain reaction (RT-PCR), immunofluorescent staining, and flow cytometry were used to examine the expression of NO-synthesizing enzymes. The proliferative capacity of PDLSCs was determined by EdU assays. The osteogenic potential of PDLSCs was tested using alkaline phosphatase (ALP) staining, Alizarin Red staining, and calcium concentration detection. Oil Red O staining was used to analyze the adipogenic ability. Western blot, RT-PCR, and staining were used to examine the signaling pathway. Human PDLSCs expressed both inducible NO synthase (iNOS) and endothelial NO synthase (eNOS) and produced NO. Blocking the generation of NO with the NOS inhibitor L-N G -monomethyl arginine (L-NMMA) had no influence on PDLSC proliferation and apoptosis but significantly attenuated the osteogenic differentiation capacity and stimulated the adipogenic differentiation capacity of PDLSCs. Increasing the physiological level of NO with NO donor sodium nitroprusside (SNP) significantly promoted the osteogenic differentiation capacity but reduced the adipogenic differentiation capacity of PDLSCs. NO balances the osteoblast and adipocyte lineage differentiation in periodontal ligament stem cells via the c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase (MAPK) signaling pathway. NO is essential for

Protective effect of Ginkgo biloba leaves extract, EGb761, on endotoxin-induced acute lung injury via a JNK- and Akt-dependent NFκB pathway.

PubMed

Lee, Chien-Ying; Yang, Jiann-Jou; Lee, Shiuan-Shinn; Chen, Chun-Jung; Huang, Yi-Chun; Huang, Kuang-Hua; Kuan, Yu-Hsiang

2014-07-09

Acute lung injury (ALI) is a clinical syndrome mainly caused by Gram-negative bacteria which is still in need of an effective therapeutic medicine. EGb761, an extract of Ginkgo biloba leaves, has several bioeffects including anti-inflammation, cardioprotection, neuroprotection, and free radical scavenging. Preadministration of EGb761 inhibited lipopolysaccharide (LPS)-induced histopathological changes and exchange of arterial blood gas. In addition, LPS-induced expression of proinflammatory mediators, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, macrophage inflammatory protein (MIP)-2, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), were suppressed by EGb761. The activation of nuclear factor (NF)κB, a transcription factor of proinflammatory mediators, and phosphorylation of IκB, an inhibitor of NFκB, were also reduced by EGb761. Furthermore, we found the inhibitory concentration of EGb761 on phosphorylation of JNK and Akt was less than those of ERK and p38 MAPK. In conclusion, EGb761 is a potential protective agent for ALI, possibly via downregulating the JNK- and Akt-dependent NFκB activation pathway.

Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer.

PubMed

Berger, Stephanie; Procko, Erik; Margineantu, Daciana; Lee, Erinna F; Shen, Betty W; Zelter, Alex; Silva, Daniel-Adriano; Chawla, Kusum; Herold, Marco J; Garnier, Jean-Marc; Johnson, Richard; MacCoss, Michael J; Lessene, Guillaume; Davis, Trisha N; Stayton, Patrick S; Stoddard, Barry L; Fairlie, W Douglas; Hockenbery, David M; Baker, David

2016-11-02

Many cancers overexpress one or more of the six human pro-survival BCL2 family proteins to evade apoptosis. To determine which BCL2 protein or proteins block apoptosis in different cancers, we computationally designed three-helix bundle protein inhibitors specific for each BCL2 pro-survival protein. Following in vitro optimization, each inhibitor binds its target with high picomolar to low nanomolar affinity and at least 300-fold specificity. Expression of the designed inhibitors in human cancer cell lines revealed unique dependencies on BCL2 proteins for survival which could not be inferred from other BCL2 profiling methods. Our results show that designed inhibitors can be generated for each member of a closely-knit protein family to probe the importance of specific protein-protein interactions in complex biological processes.

Opposing roles of p38 and JNK in a Drosophila model of TDP-43 proteinopathy reveal oxidative stress and innate immunity as pathogenic components of neurodegeneration.

PubMed

Zhan, Lihong; Xie, Qijing; Tibbetts, Randal S

2015-02-01

Pathological aggregation and mutation of the 43-kDa TAR DNA-binding protein (TDP-43) are strongly implicated in the pathogenesis amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 neurotoxicity has been extensively modeled in mice, zebrafish, Caenorhabditis elegans and Drosophila, where selective expression of TDP-43 in motoneurons led to paralysis and premature lethality. Through a genetic screen aimed to identify genetic modifiers of TDP-43, we found that the Drosophila dual leucine kinase Wallenda (Wnd) and its downstream kinases JNK and p38 influenced TDP-43 neurotoxicity. Reducing Wnd gene dosage or overexpressing its antagonist highwire partially rescued TDP-43-associated premature lethality. Downstream of Wnd, the JNK and p38 kinases played opposing roles in TDP-43-associated neurodegeneration. LOF alleles of the p38b gene as well as p38 inhibitors diminished TDP-43-associated premature lethality, whereas p38b GOF caused phenotypic worsening. In stark contrast, disruptive alleles of Basket (Bsk), the Drosophila homologue of JNK, exacerbated longevity shortening, whereas overexpression of Bsk extended lifespan. Among possible mechanisms, we found motoneuron-directed expression of TDP-43 elicited oxidative stress and innate immune gene activation that were exacerbated by p38 GOF and Bsk LOF, respectively. A key pathologic role for innate immunity in TDP-43-associated neurodegeneration was further supported by the finding that genetic suppression of the Toll/Dif and Imd/Relish inflammatory pathways dramatically extended lifespan of TDP-43 transgenic flies. We propose that oxidative stress and neuroinflammation are intrinsic components of TDP-43-associated neurodegeneration and that the balance between cytoprotective JNK and cytotoxic p38 signaling dictates phenotypic outcome to TDP-43 expression in Drosophila. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of known inhibitors.

PubMed

Iglesias, Jose; Lamontagne, Julien; Erb, Heidi; Gezzar, Sari; Zhao, Shangang; Joly, Erik; Truong, Vouy Linh; Skorey, Kathryn; Crane, Sheldon; Madiraju, S R Murthy; Prentki, Marc

2016-01-01

Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/β-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of known inhibitors

PubMed Central

Iglesias, Jose; Lamontagne, Julien; Erb, Heidi; Gezzar, Sari; Zhao, Shangang; Joly, Erik; Truong, Vouy Linh; Skorey, Kathryn; Crane, Sheldon; Madiraju, S. R. Murthy; Prentki, Marc

2016-01-01

Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/β-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors. PMID:26423520

PEGylated DX-1000: pharmacokinetics and antineoplastic activity of a specific plasmin inhibitor.

PubMed

Devy, Laetitia; Rabbani, Shafaat A; Stochl, Mark; Ruskowski, Mary; Mackie, Ian; Naa, Laurent; Toews, Mark; van Gool, Reinoud; Chen, Jie; Ley, Art; Ladner, Robert C; Dransfield, Daniel T; Henderikx, Paula

2007-11-01

Novel inhibitors of the urokinase-mediated plasminogen (plg) activation system are potentially of great clinical benefit as anticancer treatments. Using phage display, we identified DX-1000 a tissue factor pathway inhibitor-derived Kunitz domain protein which is a specific high-affinity inhibitor of plasmin (pln) (K(i) = 99 pM). When tested in vitro, DX-1000 blocks plasmin-mediated pro-matrix metalloproteinase-9 (proMMP-9) activation on cells and dose-dependently inhibits tube formation, while not significantly affecting hemostasis and coagulation. However, this low-molecular weight protein inhibitor ( approximately 7 kDa) exhibits rapid plasma clearance in mice and rabbits, limiting its potential clinical use in chronic diseases. After site-specific PEGylation, DX-1000 retains its activity and exhibits a decreased plasma clearance. This PEGylated derivative is effective in vitro, as well as potent in inhibiting tumor growth of green fluorescent protein (GFP)-labeled MDA-MB-231 cells. 4PEG-DX-1000 treatment causes a significant reduction of urokinase-type plasminogen activator (uPA) and plasminogen expressions, a reduction of tumor proliferation, and vascularization. 4PEG-DX-1000 treatment significantly decreases the level of active mitogen-activated protein kinase (MAPK) in the primary tumors and reduces metastasis incidence. Together, our results demonstrate the potential value of plasmin inhibitors as therapeutic agents for blocking breast cancer growth and metastasis.

Activation of JNK and IRE1 is critically involved in tanshinone I-induced p62 dependent autophagy in malignant pleural mesothelioma cells: implication of p62 UBA domain

PubMed Central

Yoon, Sangwook; Won, Gunho; Kim, Chang Geun; Jung, Ji Hoon; Kim, Sung-Hoon

2017-01-01

The aim of present study is to elucidate autophagic mechanism of tanshinone I (Tan I) in H28 and H2452 mesothelioma cells. Herein, Tan I exerted cytotoxicity with autophagic features of autophagy protein 5 (ATG5)/ microtubule-associated protein 1A/1B-light chain 3II (LC3 II) activation, p62/sequestosome 1 (SQSTM1) accumulation and increased number of LC3II punctae, acridine orange-stained cells and autophagic vacuoles. However, 3-methyladenine (3MA) and NH4Cl increased cytotoxicity in Tan I treated H28 cells. Furthermore, autophagy flux was enhanced in Tan I-treated H28 cells transfected by RFP-GFP-LC3 constructs, with colocalization of GFP-LC3 punctae with LAMP1 or Lysotracker. Interestingly, C-terminal UBA domain is required for Tan 1 induced aggregation of p62 in H28 cells. Notably, Tan I upregulated CCAAT-enhancer-binding protein homologous protein (CHOP), inositol-requiring protein-1 (IRE1) and p-c-Jun N-terminal kinase (p-JNK), but silencing of IRE1 or p62 and JNK inhibitor SP600125 blocked the LC3II accumulation in Tan I-treated H28 cells. Overall, these findings demonstrate that Tan I exerts antitumor activity through a compromise between apoptosis and p62/SQSTM1-dependent autophagy via activation of JNK and IRE 1 in malignant mesothelioma cells. PMID:28212571

Activation of JNK and IRE1 is critically involved in tanshinone I-induced p62 dependent autophagy in malignant pleural mesothelioma cells: implication of p62 UBA domain.

PubMed

Lee, Jihyun; Sohn, Eun Jung; Yoon, Sangwook; Won, Gunho; Kim, Chang Geun; Jung, Ji Hoon; Kim, Sung-Hoon

2017-04-11

The aim of present study is to elucidate autophagic mechanism of tanshinone I (Tan I) in H28 and H2452 mesothelioma cells. Herein, Tan I exerted cytotoxicity with autophagic features of autophagy protein 5 (ATG5)/ microtubule-associated protein 1A/1B-light chain 3II (LC3 II) activation, p62/sequestosome 1 (SQSTM1) accumulation and increased number of LC3II punctae, acridine orange-stained cells and autophagic vacuoles. However, 3-methyladenine (3MA) and NH4Cl increased cytotoxicity in Tan I treated H28 cells. Furthermore, autophagy flux was enhanced in Tan I-treated H28 cells transfected by RFP-GFP-LC3 constructs, with colocalization of GFP-LC3 punctae with LAMP1 or Lysotracker. Interestingly, C-terminal UBA domain is required for Tan 1 induced aggregation of p62 in H28 cells. Notably, Tan I upregulated CCAAT-enhancer-binding protein homologous protein (CHOP), inositol-requiring protein-1 (IRE1) and p-c-Jun N-terminal kinase (p-JNK), but silencing of IRE1 or p62 and JNK inhibitor SP600125 blocked the LC3II accumulation in Tan I-treated H28 cells. Overall, these findings demonstrate that Tan I exerts antitumor activity through a compromise between apoptosis and p62/SQSTM1-dependent autophagy via activation of JNK and IRE 1 in malignant mesothelioma cells.

Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer

PubMed Central

Berger, Stephanie; Procko, Erik; Margineantu, Daciana; Lee, Erinna F; Shen, Betty W; Zelter, Alex; Silva, Daniel-Adriano; Chawla, Kusum; Herold, Marco J; Garnier, Jean-Marc; Johnson, Richard; MacCoss, Michael J; Lessene, Guillaume; Davis, Trisha N; Stayton, Patrick S; Stoddard, Barry L; Fairlie, W Douglas; Hockenbery, David M; Baker, David

2016-01-01

Many cancers overexpress one or more of the six human pro-survival BCL2 family proteins to evade apoptosis. To determine which BCL2 protein or proteins block apoptosis in different cancers, we computationally designed three-helix bundle protein inhibitors specific for each BCL2 pro-survival protein. Following in vitro optimization, each inhibitor binds its target with high picomolar to low nanomolar affinity and at least 300-fold specificity. Expression of the designed inhibitors in human cancer cell lines revealed unique dependencies on BCL2 proteins for survival which could not be inferred from other BCL2 profiling methods. Our results show that designed inhibitors can be generated for each member of a closely-knit protein family to probe the importance of specific protein-protein interactions in complex biological processes. DOI: http://dx.doi.org/10.7554/eLife.20352.001 PMID:27805565

Ectopic expression of H2AX protein promotes TrkA-induced cell death via modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jung, Eun Joo; Kim, Deok Ryong, E-mail: drkim@gnu.ac.kr

2011-01-21

Research highlights: {yields} We established TrkA-inducible U2OS cells stably expressing GFP-H2AX proteins. {yields} GFP-H2AX was colocalized with TrkA in the cytoplasm. {yields} {gamma}H2AX production was significantly increased upon activation of TrkA and suppressed by TrkA inhibitor or JNK inhibitor. {yields} Ectopic expression of H2AX promoted TrkA-mediated cell death through the modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage. -- Abstract: We previously reported that TrkA overexpression causes accumulation of {gamma}H2AX proteins in the cytoplasm, subsequently leading to massive cell death in U2OS cells. To further investigate how cytoplasmic H2AX is associated with TrkA-induced cell death, we establishedmore » TrkA-inducible cells stably expressing GFP-tagged H2AX. We found that TrkA co-localizes with ectopically expressed GFP-H2AX proteins in the cytoplasm, especially at the juxta-nuclear membranes, which supports our previous results about a functional connection between TrkA and {gamma}H2AX in TrkA-induced cell death. {gamma}H2AX production from GFP-H2AX proteins was significantly increased when TrkA was overexpressed. Moreover, ectopic expression of H2AX activated TrkA-mediated signal pathways via up-regulation of TrkA tyrosine-490 phosphorylation. In addition, suppression of TrkA tyrosine-490 phosphorylation under a certain condition was removed by ectopic expression of H2AX, indicating a functional role of H2AX in the maintenance of TrkA activity. Indeed, TrkA-induced cell death was highly elevated by ectopic H2AX expression, and it was further accelerated by DNA damage via JNK activation. These all results suggest that cytoplasmic H2AX could play an important role in TrkA-mediated cell death by modulating TrkA upon DNA damage.« less

MarvelD3 couples tight junctions to the MEKK1–JNK pathway to regulate cell behavior and survival

PubMed Central

Steed, Emily; Elbediwy, Ahmed; Vacca, Barbara; Dupasquier, Sébastien; Hemkemeyer, Sandra A.; Suddason, Tesha; Costa, Ana C.; Beaudry, Jean-Bernard; Zihni, Ceniz; Gallagher, Ewen; Pierreux, Christophe E.

2014-01-01

MarvelD3 is a transmembrane component of tight junctions, but there is little evidence for a direct involvement in the junctional permeability barrier. Tight junctions also regulate signaling mechanisms that guide cell proliferation; however, the transmembrane components that link the junction to such signaling pathways are not well understood. In this paper, we show that MarvelD3 is a dynamic junctional regulator of the MEKK1–c-Jun NH2-terminal kinase (JNK) pathway. Loss of MarvelD3 expression in differentiating Caco-2 cells resulted in increased cell migration and proliferation, whereas reexpression in a metastatic tumor cell line inhibited migration, proliferation, and in vivo tumor formation. Expression levels of MarvelD3 inversely correlated with JNK activity, as MarvelD3 recruited MEKK1 to junctions, leading to down-regulation of JNK phosphorylation and inhibition of JNK-regulated transcriptional mechanisms. Interplay between MarvelD3 internalization and JNK activation tuned activation of MEKK1 during osmotic stress, leading to junction dissociation and cell death in MarvelD3-depleted cells. MarvelD3 thus couples tight junctions to the MEKK1–JNK pathway to regulate cell behavior and survival. PMID:24567356

The effect of menadione on glutathione S-transferase A1 (GSTA1): c-Jun N-terminal kinase (JNK) complex dissociation in human colonic adenocarcinoma Caco-2 cells.

PubMed

Adnan, Humaira; Antenos, Monica; Kirby, Gordon M

2012-10-02

Glutathione S-transferases (GSTs) act as modulators of mitogen-activated protein kinase signal transduction pathways via a mechanism involving protein-protein interactions. We have demonstrated that GSTA1 forms complexes with JNK and modifies JNK activation during cellular stress, but the factors that influence complex association and dissociation are unknown. We hypothesized that menadione causes dissociation of GSTA1-JNK complexes, activates JNK, and the consequences of menadione exposure depend on GSTA1 expression. We demonstrate that menadione causes GSTA1-JNK dissociation and JNK activation in preconfluent Caco-2 cells, whereas postconfluent cells are resistant to this effect. Moreover, preconfluent cells are more sensitive than postconfluent cells to menadione-induced cytotoxicity. Activation of JNK is transient since removal of menadione causes GSTA1 to re-associate with JNK reducing cytotoxicity. Over-expression and knockdown of GSTA1 did not alter JNK activation by menadione or sensitivity to menadione-induced cytotoxicity. These results indicate that GSTA1-JNK complex integrity does not affect the ability of menadione to activate JNK. N-acetyl cysteine prevents GSH depletion and blocks menadione-induced complex dissociation, JNK activation and inhibits menadione-induced cytotoxicity. JNK activation and inhibits menadione-induced cytotoxicity. The data suggest that the mechanism of menadione-induced JNK activation involves the production of reactive oxygen species, likely superoxide anion, and intracellular GSH levels play an important role in preventing GSTA1-JNK complex dissociation, subsequent JNK activation and induction of cytotoxicity. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

[Curcumin alleviates early brain injury following subarachnoid hemorrhage in rats by inhibiting JNK/c-Jun signal pathway].

PubMed

Li, Xia; Zhu, Ji

2018-03-01

Objective To investigate the inhibitory effect of curcumin on early brain injury following subarachnoid hemorrhage (SAH) by inhibiting JNK/ c-Jun signal pathway. Methods Sixty adult male SD rats were randomly divided into four groups: sham operation group (sham group), SAH group, SAH group treated with 100 mg/(kg.d) curcumin and SAH group treated with 200 mg/(kg.d) curcumin, with 15 rats in each group. Endovascular puncture was used to induce SAH model. Nissl staining was used to test whether neurons were broken. TUNEL staining was used to detect apoptosis. Immunohistochemistry was used to investigate the expression of caspase-3. Western blot analysis was used to detect the expressions of p-JNK, JNK, p-c-Jun, c-Jun, and caspase-3. Results Nissl staining indicated the decrease of Nissl bodies in SAH group, but increase of Nissl bodies in SAH group treated with curcumin. TUNEL staining showed that there were more apoptotic neurons in SAH group compared with sham group, while apoptotic neurons decreased after the treatment with curcumin, more obviously in the group treated with 200 mg/(kg.d) curcumin. The expressions of p-JNK, JNK, p-c-Jun, c-Jun, and caspase-3 were up-regulated in SAH group compared with sham group. However, the expressions of those proteins were down-regulated after the treatment with curcumin, especially by higher-dose curcumin treatment. Conclusion Curcumin might suppress early brain injury after SAH by inhibiting JNK/c-Jun signal pathway and neuron apoptosis.

A Novel c-Jun N-terminal Kinase (JNK) Signaling Complex Involved in Neuronal Migration during Brain Development.

PubMed

Zhang, Feng; Yu, Jingwen; Yang, Tao; Xu, Dan; Chi, Zhixia; Xia, Yanheng; Xu, Zhiheng

2016-05-27

Disturbance of neuronal migration may cause various neurological disorders. Both the transforming growth factor-β (TGF-β) signaling and microcephaly-associated protein WDR62 are important for neuronal migration during brain development; however, the underlying molecular mechanisms involved remain unclear. We show here that knock-out or knockdown of Tak1 (TGFβ-activated kinase 1) and Jnk2 (c-Jun N-terminal kinase 2) perturbs neuronal migration during cortical development and that the migration defects incurred by knock-out and/or knockdown of Tβr2 (type II TGF-β receptor) or Tak1 can be partially rescued by expression of TAK1 and JNK2, respectively. Furthermore, TAK1 forms a protein complex with RAC1 and two scaffold proteins of the JNK pathway, the microcephaly-associated protein WDR62 and the RAC1-interacting protein POSH (plenty of Src homology). Components of the complex coordinate with each other in the regulation of TAK1 as well as JNK activities. We suggest that unique JNK protein complexes are involved in the diversified biological and pathological functions during brain development and pathogenesis of diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of STAT1 and STAT3 Specific Inhibitors Using Comparative Virtual Screening and Docking Validation

PubMed Central

Szelag, Malgorzata; Czerwoniec, Anna; Wesoly, Joanna; Bluyssen, Hans A. R.

2015-01-01

Signal transducers and activators of transcription (STATs) facilitate action of cytokines, growth factors and pathogens. STAT activation is mediated by a highly conserved SH2 domain, which interacts with phosphotyrosine motifs for specific STAT-receptor contacts and STAT dimerization. The active dimers induce gene transcription in the nucleus by binding to a specific DNA-response element in the promoter of target genes. Abnormal activation of STAT signaling pathways is implicated in many human diseases, like cancer, inflammation and auto-immunity. Searches for STAT-targeting compounds, exploring the phosphotyrosine (pTyr)-SH2 interaction site, yielded many small molecules for STAT3 but sparsely for other STATs. However, many of these inhibitors seem not STAT3-specific, thereby questioning the present modeling and selection strategies of SH2 domain-based STAT inhibitors. We generated new 3D structure models for all human (h)STATs and developed a comparative in silico docking strategy to obtain further insight into STAT-SH2 cross-binding specificity of a selection of previously identified STAT3 inhibitors. Indeed, by primarily targeting the highly conserved pTyr-SH2 binding pocket the majority of these compounds exhibited similar binding affinity and tendency scores for all STATs. By comparative screening of a natural product library we provided initial proof for the possibility to identify STAT1 as well as STAT3-specific inhibitors, introducing the ‘STAT-comparative binding affinity value’ and ‘ligand binding pose variation’ as selection criteria. In silico screening of a multi-million clean leads (CL) compound library for binding of all STATs, likewise identified potential specific inhibitors for STAT1 and STAT3 after docking validation. Based on comparative virtual screening and docking validation, we developed a novel STAT inhibitor screening tool that allows identification of specific STAT1 and STAT3 inhibitory compounds. This could increase our

Overexpressed DNA polymerase iota regulated by JNK/c-Jun contributes to hypermutagenesis in bladder cancer.

PubMed

Yuan, Fang; Xu, Zhigang; Yang, Mingzhen; Wei, Quanfang; Zhang, Yi; Yu, Jin; Zhi, Yi; Liu, Yang; Chen, Zhiwen; Yang, Jin

2013-01-01

Human DNA polymerase iota (pol ι) possesses high error-prone DNA replication features and performs translesion DNA synthesis. It may be specialized and strictly regulated in normal mammalian cells. Dysregulation of pol ι may contribute to the acquisition of a mutator phenotype. However, there are few reports describing the transcription regulatory mechanism of pol ι, and there is controversy regarding its role in carcinogenesis. In this study, we performed the deletion and point-mutation experiment, EMSA, ChIP, RNA interference and western blot assay to prove that c-Jun activated by c-Jun N-terminal kinase (JNK) regulates the transcription of pol ι in normal and cancer cells. Xeroderma pigmentosum group C protein (XPC) and ataxia-telangiectasia mutated related protein (ATR) promote early JNK activation in response to DNA damage and consequently enhance the expression of pol ι, indicating that the novel role of JNK signal pathway is involved in DNA damage response. Furthermore, associated with elevated c-Jun activity, the overexpression of pol ι is positively correlated with the clinical tumor grade in 97 bladder cancer samples and may contribute to the hypermutagenesis. The overexpressed pol ι-involved mutagenesis is dependent on JNK/c-Jun pathway in bladder cancer cells identifying by the special mutation spectra. Our results support the conclusion that dysregulation of pol ι by JNK/c-Jun is involved in carcinogenesis and offer a novel understanding of the role of pol ι or c-Jun in mutagenesis.

Overexpressed DNA Polymerase Iota Regulated by JNK/c-Jun Contributes to Hypermutagenesis in Bladder Cancer

PubMed Central

Yuan, Fang; Xu, Zhigang; Yang, Mingzhen; Wei, Quanfang; Zhang, Yi; Yu, Jin; Zhi, Yi; Liu, Yang; Chen, Zhiwen; Yang, Jin

2013-01-01

Human DNA polymerase iota (pol ι) possesses high error-prone DNA replication features and performs translesion DNA synthesis. It may be specialized and strictly regulated in normal mammalian cells. Dysregulation of pol ι may contribute to the acquisition of a mutator phenotype. However, there are few reports describing the transcription regulatory mechanism of pol ι, and there is controversy regarding its role in carcinogenesis. In this study, we performed the deletion and point-mutation experiment, EMSA, ChIP, RNA interference and western blot assay to prove that c-Jun activated by c-Jun N-terminal kinase (JNK) regulates the transcription of pol ι in normal and cancer cells. Xeroderma pigmentosum group C protein (XPC) and ataxia-telangiectasia mutated related protein (ATR) promote early JNK activation in response to DNA damage and consequently enhance the expression of pol ι, indicating that the novel role of JNK signal pathway is involved in DNA damage response. Furthermore, associated with elevated c-Jun activity, the overexpression of pol ι is positively correlated with the clinical tumor grade in 97 bladder cancer samples and may contribute to the hypermutagenesis. The overexpressed pol ι-involved mutagenesis is dependent on JNK/c-Jun pathway in bladder cancer cells identifying by the special mutation spectra. Our results support the conclusion that dysregulation of pol ι by JNK/c-Jun is involved in carcinogenesis and offer a novel understanding of the role of pol ι or c-Jun in mutagenesis. PMID:23922701

c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis

DTIC Science & Technology

2015-03-01

1 AWARD NUMBER: W81XWH-12-1-0431 TITLE: “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis ” PRINCIPAL...TITLE AND SUBTITLE “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Scelerosis” 5a. CONTRACT NUMBER 5b. GRANT NUMBER... Lateral   Sclerosis ”   Final  Report:  Project  Period  Sept  2012-­‐Dec  2014     Personnel  List:     Feng,  Yangbo

Periostin promotes migration and osteogenic differentiation of human periodontal ligament mesenchymal stem cells via the Jun amino-terminal kinases (JNK) pathway under inflammatory conditions.

PubMed

Tang, Yi; Liu, Lin; Wang, Pei; Chen, Donglei; Wu, Ziqiang; Tang, Chunbo

2017-12-01

Mesenchymal stem cell (MSC)-mediated periodontal tissue regeneration is considered to be a promising method for periodontitis treatment. The molecular mechanism of functional regulation by MSCs remains unclear, thus limiting their application. Our previous study discovered that Periostin (POSTN) promoted the migration and osteogenic differentiation of periodontal ligament mesenchymal stem cells (PDLSCs), but it is still unclear whether POSTN is able to restore the regenerative potential of PDLSCs under inflammatory conditions. In this study, we investigated the effect of POSTN on PDLSCs under inflammatory conditions and its mechanism. PDLSCs were isolated from periodontal ligament tissue. TNF-α was used at 10 ng/mL to mimic inflammatory conditions. Lentivirus POSTN shRNA was used to knock down POSTN. Recombinant human POSTN (rhPOSTN) was used to stimulate PDLSCs. A scratch assay was used to analyse cell migration. Alkaline phosphatase (ALP) activity, Alizarin Red staining and expression of osteogenesis-related genes were used to investigate the osteogenic differentiation potential. Western blot analysis was used to detect the mitogen-activated protein kinases (MAPK) and AKT signalling pathways. After a 10 ng/mL TNF-α treatment, knockdown of POSTN impeded scratch closure, inhibited ALP activity and mineralization in vitro, and decreased expression of RUNX2, OSX, OPN and OCN in PDLSCs, while 75 ng/mL rhPOSTN significantly accelerated scratch closure, enhanced ALP activity and mineralization in vitro, and increased expression of RUNX2, OSX, OPN and OCN. In addition, knockdown of POSTN inhibited expression of phosphorylated c-Jun N-terminal kinase (p-JNK), while 75 ng/mL rhPOSTN increased expression of p-JNK in PDLSCs with TNF-α treatment. Furthermore, inhibition of JNK by its inhibitor SP600125 dramatically blocked POSTN-enhanced scratch closure, ALP activity and mineralization in PDLSCs. Our results revealed that POSTN might promote the migration and

SCF/C-Kit/JNK/AP-1 Signaling Pathway Promotes Claudin-3 Expression in Colonic Epithelium and Colorectal Carcinoma.

PubMed

Wang, Yaxi; Sun, Tingyi; Sun, Haimei; Yang, Shu; Li, Dandan; Zhou, Deshan

2017-04-06

Claudin-3 is a major protein of tight junctions (TJs) in the intestinal epithelium and is critical for maintaining cell-cell adhesion, barrier function, and epithelium polarity. Recent studies have shown high claudin-3 levels in several solid tumors, but the regulation mechanism of claudin-3 expression remains poorly understood. In the present study, colorectal cancer (CRC) tissues, HT-29 and DLD-1 CRC cell lines, CRC murine model (C57BL/6 mice) and c-kit loss-of-function mutant mice were used. We demonstrated that elevated claudin-3 levels were positively correlated with highly expressed c-kit in CRC tissues based upon analysis of protein expression. In vitro, claudin-3 expression was clearly increased in CRC cells by overexpressed c-kit or stimulated by exogenous recombinant human stem cell factor (rhSCF), while significantly decreased by the treatment with c-kit or c-Jun N-terminal kinase (JNK) inhibitors. Chromatin immunoprecipitation (ChIP) and luciferase reporter assay showed that SCF/c-kit signaling significantly promoted activator protein-1 (AP-1) binding with CLDN-3 promoter and enhanced its transcription activity. Furthermore, decreased expression of claudin-3 was obtained in the colonic epithelium from the c-Kit loss-of-function mutant mice. In conclusion, SCF/c-kit-JNK/AP-1 signaling pathway significantly promoted claudin-3 expression in colonic epithelium and CRC, which could contribute to epithelial barrier function maintenance and to CRC development.

Reversion of apoptotic resistance of TP53-mutated Burkitt lymphoma B-cells to spindle poisons by exogenous activation of JNK and p38 MAP kinases.

PubMed

Farhat, M; Poissonnier, A; Hamze, A; Ouk-Martin, C; Brion, J-D; Alami, M; Feuillard, J; Jayat-Vignoles, C

2014-05-01

Defects in apoptosis are frequently the cause of cancer emergence, as well as cellular resistance to chemotherapy. These phenotypes may be due to mutations of the tumor suppressor TP53 gene. In this study, we examined the effect of various mitotic spindle poisons, including the new isocombretastatin derivative isoNH2CA-4 (a tubulin-destabilizing molecule, considered to bind to the colchicine site by analogy with combretastatin A-4), on BL (Burkitt lymphoma) cells. We found that resistance to spindle poison-induced apoptosis could be reverted in tumor protein p53 (TP53)-mutated cells by EBV (Epstein Barr virus) infection. This reversion was due to restoration of the intrinsic apoptotic pathway, as assessed by relocation of the pro-apoptotic molecule Bax to mitochondria, loss of mitochondrial integrity and activation of the caspase cascade with PARP (poly ADP ribose polymerase) cleavage. EBV sensitized TP53-mutated BL cells to all spindle poisons tested, including vincristine and taxol, an effect that was systematically downmodulated by pretreatment of cells with inhibitors of p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases. Exogenous activation of p38 and JNK pathways by dihydrosphingosine reverted resistance of TP53-mutated BL cells to spindle poisons. Dihydrosphingosine treatment of TP53-deficient Jurkat and K562 cell lines was also able to induce cell death. We conclude that activation of p38 and JNK pathways may revert resistance of TP53-mutated cells to spindle poisons. This opens new perspectives for developing alternative therapeutic strategies when the TP53 gene is inactivated.

Propiconazole is a specific and accessible brassinosteroid (BR) biosynthesis inhibitor for Arabidopsis and maize.

PubMed

Hartwig, Thomas; Corvalan, Claudia; Best, Norman B; Budka, Joshua S; Zhu, Jia-Ying; Choe, Sunghwa; Schulz, Burkhard

2012-01-01

Brassinosteroids (BRs) are steroidal hormones that play pivotal roles during plant development. In addition to the characterization of BR deficient mutants, specific BR biosynthesis inhibitors played an essential role in the elucidation of BR function in plants. However, high costs and limited availability of common BR biosynthetic inhibitors constrain their key advantage as a species-independent tool to investigate BR function. We studied propiconazole (Pcz) as an alternative to the BR inhibitor brassinazole (Brz). Arabidopsis seedlings treated with Pcz phenocopied BR biosynthetic mutants. The steady state mRNA levels of BR, but not gibberellic acid (GA), regulated genes increased proportional to the concentrations of Pcz. Moreover, root inhibition and Pcz-induced expression of BR biosynthetic genes were rescued by 24epi-brassinolide, but not by GA(3) co-applications. Maize seedlings treated with Pcz showed impaired mesocotyl, coleoptile, and true leaf elongation. Interestingly, the genetic background strongly impacted the tissue specific sensitivity towards Pcz. Based on these findings we conclude that Pcz is a potent and specific inhibitor of BR biosynthesis and an alternative to Brz. The reduced cost and increased availability of Pcz, compared to Brz, opens new possibilities to study BR function in larger crop species.

Salmonella enteritidis Effector AvrA Stabilizes Intestinal Tight Junctions via the JNK Pathway.

PubMed

Lin, Zhijie; Zhang, Yong-Guo; Xia, Yinglin; Xu, Xiulong; Jiao, Xinan; Sun, Jun

2016-12-23

Salmonella pathogenesis studies to date have focused on Salmonella typhimurium, and the pathogenesis of a second major serotype, Salmonella enteritidis, is poorly understood. Salmonella spp. possess effector proteins that display biochemical activities and modulate host functions. Here, we generated a deletion mutant of the effector AvrA, S.E-AvrA - , and a plasmid-mediated complementary strain, S.E-AvrA - /pAvrA + (S.E-AvrA + ), in S. Enteritidis. Using in vitro and in vivo infection models, we showed that AvrA stabilizes epithelial tight junction (TJ) proteins, such as ZO-1, in human intestinal epithelial cells. Transepithelial electrical resistance was significantly higher in cells infected with S.E-AvrA + than in cells infected with S.E-AvrA - Inhibition of the JNK pathway suppresses the disassembly of TJ proteins; we found that enteritidis AvrA inhibited JNK activity in cells infected with wild type or S.E-AvrA + strains. Therefore, Enteritidis AvrA-induced ZO-1 stability is achieved via suppression of the JNK pathway. Furthermore, the S.E-AvrA - strain led to enhanced bacterial invasion, both in vitro and in vivo Taken together, our data reveal a novel role for AvrA in S. Enteritidis: Enteritidis AvrA stabilizes intestinal TJs and attenuates bacterial invasion. The manipulation of JNK activity and TJs in microbial-epithelial interactions may be a novel therapeutic approach for the treatment of infectious diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Salmonella enteritidis Effector AvrA Stabilizes Intestinal Tight Junctions via the JNK Pathway*

PubMed Central

Lin, Zhijie; Zhang, Yong-Guo; Xia, Yinglin; Xu, Xiulong; Jiao, Xinan

2016-01-01

Salmonella pathogenesis studies to date have focused on Salmonella typhimurium, and the pathogenesis of a second major serotype, Salmonella enteritidis, is poorly understood. Salmonella spp. possess effector proteins that display biochemical activities and modulate host functions. Here, we generated a deletion mutant of the effector AvrA, S.E-AvrA−, and a plasmid-mediated complementary strain, S.E-AvrA−/pAvrA+ (S.E-AvrA+), in S. Enteritidis. Using in vitro and in vivo infection models, we showed that AvrA stabilizes epithelial tight junction (TJ) proteins, such as ZO-1, in human intestinal epithelial cells. Transepithelial electrical resistance was significantly higher in cells infected with S.E-AvrA+ than in cells infected with S.E-AvrA−. Inhibition of the JNK pathway suppresses the disassembly of TJ proteins; we found that enteritidis AvrA inhibited JNK activity in cells infected with wild type or S.E-AvrA+ strains. Therefore, Enteritidis AvrA-induced ZO-1 stability is achieved via suppression of the JNK pathway. Furthermore, the S.E-AvrA− strain led to enhanced bacterial invasion, both in vitro and in vivo. Taken together, our data reveal a novel role for AvrA in S. Enteritidis: Enteritidis AvrA stabilizes intestinal TJs and attenuates bacterial invasion. The manipulation of JNK activity and TJs in microbial-epithelial interactions may be a novel therapeutic approach for the treatment of infectious diseases. PMID:27875307

Atpenins, potent and specific inhibitors of mitochondrial complex II (succinate-ubiquinone oxidoreductase)

PubMed Central

Miyadera, Hiroko; Shiomi, Kazuro; Ui, Hideaki; Yamaguchi, Yuichi; Masuma, Rokuro; Tomoda, Hiroshi; Miyoshi, Hideto; Osanai, Arihiro; Kita, Kiyoshi; Ōmura, Satoshi

2003-01-01

Enzymes in the mitochondrial respiratory chain are involved in various physiological events in addition to their essential role in the production of ATP by oxidative phosphorylation. The use of specific and potent inhibitors of complex I (NADH-ubiquinone reductase) and complex III (ubiquinol-cytochrome c reductase), such as rotenone and antimycin, respectively, has allowed determination of the role of these enzymes in physiological processes. However, unlike complexes I, III, and IV (cytochrome c oxidase), there are few potent and specific inhibitors of complex II (succinate-ubiquinone reductase) that have been described. In this article, we report that atpenins potently and specifically inhibit the succinate-ubiquinone reductase activity of mitochondrial complex II. Therefore, atpenins may be useful tools for clarifying the biochemical and structural properties of complex II, as well as for determining its physiological roles in mammalian tissues. PMID:12515859

Loss of miR-223 and JNK Signaling Contribute to Elevated Stathmin in Malignant Pleural Mesothelioma.

PubMed

Birnie, Kimberly A; Yip, Yan Y; Ng, Dominic C H; Kirschner, Michaela B; Reid, Glen; Prêle, Cecilia M; Musk, Arthur W Bill; Lee, Y C Gary; Thompson, Philip J; Mutsaers, Steven E; Badrian, Bahareh

2015-07-01

Malignant pleural mesothelioma (MPM) is often fatal, and studies have revealed that aberrant miRNAs contribute to MPM development and aggressiveness. Here, a screen of miRNAs identified reduced levels of miR-223 in MPM patient specimens. Interestingly, miR-223 targets Stathmin (STMN1), a microtubule regulator that has been associated with MPM. However, whether miR-223 regulates STMN1 in MPM and the functions of miR-223 and STMN1 in this disease are yet to be determined. STMN1 is also regulated by c-Jun N-terminal kinase (JNK) signaling, but whether this occurs in MPM and whether miR-223 plays a role are unknown. The relationship between STMN1, miR-223, and JNK was assessed using MPM cell lines, cells from pleural effusions, and MPM tissue. Evidence indicates that miR-223 is decreased in all MPM tissue compared with normal/healthy tissue. Conversely, STMN1 expression was higher in MPM cell lines when compared with primary mesothelial cell controls. Following overexpression of miR-223 in MPM cell lines, STMN1 levels were reduced, cell motility was inhibited, and tubulin acetylation induced. Knockdown of STMN1 using siRNAs led to inhibition of MPM cell proliferation and motility. Finally, miR-223 levels increased while STMN1 was reduced following the re-expression of the JNK isoforms in JNK-null murine embryonic fibroblasts, and STMN1 was reduced in MPM cell lines following the activation of JNK signaling. miR-223 regulates STMN1 in MPM, and both are in turn regulated by the JNK signaling pathway. As such, miR-223 and STMN1 play an important role in regulating MPM cell motility and may be therapeutic targets. ©2015 American Association for Cancer Research.

Pro-life role for c-Jun N-terminal kinase and p38 mitogen-activated protein kinase at rostral ventrolateral medulla in experimental brain stem death

PubMed Central

2012-01-01

, rather than Elk-1 at Ser383 in RVLM were also augmented during the pro-life phase. Furthermore, pretreatment by microinjection into the bilateral RVLM of specific JNK inhibitors, JNK inhibitor I (100 pmol) or SP600125 (5 pmol), or specific p38MAPK inhibitors, p38MAPK inhibitor III (500 pmol) or SB203580 (2 nmol), exacerbated the depressor effect and blunted the augmented life-and-death signal exhibited during the pro-life phase. On the other hand, pretreatment with the negative control for JNK or p38MAPK inhibitor, JNK inhibitor I negative control (100 pmol) or SB202474 (2 nmol), was ineffective in the vehicle-controls and Mev-treatment groups. Conclusions Our results demonstrated that activation of JNK or p38MAPK in RVLM by their upstream activators MAP2K4 or MAP2K6 plays a preferential pro-life role by sustaining the central cardiovascular regulatory machinery during experimental brain stem death via phosphorylation and activation of nuclear transcription factor ATF-2 or c-Jun. PMID:23157661

Pro-life role for c-Jun N-terminal kinase and p38 mitogen-activated protein kinase at rostral ventrolateral medulla in experimental brain stem death.

PubMed

Chang, Alice Y W

2012-11-17

Ser383 in RVLM were also augmented during the pro-life phase. Furthermore, pretreatment by microinjection into the bilateral RVLM of specific JNK inhibitors, JNK inhibitor I (100 pmol) or SP600125 (5 pmol), or specific p38MAPK inhibitors, p38MAPK inhibitor III (500 pmol) or SB203580 (2 nmol), exacerbated the depressor effect and blunted the augmented life-and-death signal exhibited during the pro-life phase. On the other hand, pretreatment with the negative control for JNK or p38MAPK inhibitor, JNK inhibitor I negative control (100 pmol) or SB202474 (2 nmol), was ineffective in the vehicle-controls and Mev-treatment groups. Our results demonstrated that activation of JNK or p38MAPK in RVLM by their upstream activators MAP2K4 or MAP2K6 plays a preferential pro-life role by sustaining the central cardiovascular regulatory machinery during experimental brain stem death via phosphorylation and activation of nuclear transcription factor ATF-2 or c-Jun.

JNK Activation Contributes to Oxidative Stress-Induced Parthanatos in Glioma Cells via Increase of Intracellular ROS Production.

PubMed

Zheng, Linjie; Wang, Chen; Luo, Tianfei; Lu, Bin; Ma, Hongxi; Zhou, Zijian; Zhu, Dong; Chi, Guangfan; Ge, Pengfei; Luo, Yinan

2017-07-01

Parthanatos is a form of PARP-1-dependent programmed cell death. The induction of parthanatos is emerging as a new strategy to kill gliomas which are the most common type of primary malignant brain tumor. Oxidative stress is thought to be a critical factor triggering parthanatos, but its underlying mechanism is poorly understood. In this study, we used glioma cell lines and H 2 O 2 to investigate the role of JNK in glioma cell parthanatos induced by oxidative stress. We found that exposure to H 2 O 2 not only induced intracellular accumulation of ROS but also resulted in glioma cell death in a concentration- and incubation time-dependent manner, which was accompanied with cytoplasmic formation of PAR polymer, expressional upregulation of PARP-1, mitochondrial depolarization, and AIF translocation to nucleus. Pharmacological inhibition of PARP-1 with 3AB or genetic knockdown of its level with siRNA rescued glioma cell death, as well as suppressed cytoplasmic accumulation of PAR polymer and nuclear translocation of AIF, which were consistent with the definition of parthanatos. Moreover, the phosphorylated level of JNK increased markedly with the extension of H 2 O 2 exposure time. Either attenuation of intracellular ROS with antioxidant NAC or inhibition of JNK phosphorylation with SP600125 or JNK siRNA could significantly prevent H 2 O 2 -induced parthanatos in glioma cells. Additionally, inhibition of JNK with SP600125 alleviated intracellular accumulation of ROS and attenuated mitochondrial generation of superoxide. Thus, we demonstrated that JNK activation contributes to glioma cell parthanatos caused by oxidative stress via increase of intracellular ROS generation.

JNK-associated scattered growth of YD-10B oral squamous carcinoma cells while maintaining the epithelial phenotype

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Gayoung; Kim, Hyun-Man

Cell scattering of epithelial carcinoma cancer cells is one of the critical event in tumorigenesis. Cells losing epithelial cohesion detach from aggregated epithelial cell masses and may migrate to fatal organs through metastasis. The present study investigated the molecular mechanism by which squamous cell carcinoma cells grow scattered at the early phase of transformation while maintaining the epithelial phenotype. We studied YD-10B cells, which are established from human oral squamous cell carcinoma, because the cells grow scattered without the development of E-cadherin junctions (ECJs) under routine culture conditions despite the high expression of functional E-cadherin. The functionality of their E-cadherinmore » was demonstrated in that YD-10B cells developed ECJs, transiently or persistently, when they were cultured on substrates coated with a low amount of fibronectin or to confluence. The phosphorylation of JNK was up-regulated in YD-10B cells compared with that in human normal oral keratinocyte cells or human squamous cell carcinoma cells, which grew aggregated along with well-organized ECJs. The suppression of JNK activity induced the aggregated growth of YD-10B cells concomitant with the development of ECJs. These results indicate for the first time that inherently up-regulated JNK activity induces the scattered growth of the oral squamous cell carcinoma cells through down-regulating the development of ECJ despite the expression of functional E-cadherin, a hallmark of the epithelial phenotype. - Highlights: • JNK dissociates YD-10B oral squamous cell carcinoma cells. • JNK suppresses the development of E-cadherin junctions of oral carcinoma cells. • Suppression of JNK activity reverses the scattered growth of oral carcinoma cells.« less

JNK1 ablation in mice confers long-term metabolic protection from diet-induced obesity at the cost of moderate skin oxidative damage.

PubMed

Becattini, Barbara; Zani, Fabio; Breasson, Ludovic; Sardi, Claudia; D'Agostino, Vito Giuseppe; Choo, Min-Kyung; Provenzani, Alessandro; Park, Jin Mo; Solinas, Giovanni

2016-09-01

Obesity and insulin resistance are associated with oxidative stress, which may be implicated in the progression of obesity-related diseases. The kinase JNK1 has emerged as a promising drug target for the treatment of obesity and type 2 diabetes. JNK1 is also a key mediator of the oxidative stress response, which can promote cell death or survival, depending on the magnitude and context of its activation. In this article, we describe a study in which the long-term effects of JNK1 inactivation on glucose homeostasis and oxidative stress in obese mice were investigated for the first time. Mice lacking JNK1 (JNK1(-/-)) were fed an obesogenic high-fat diet (HFD) for a long period. JNK1(-/-) mice fed an HFD for the long term had reduced expression of antioxidant genes in their skin, more skin oxidative damage, and increased epidermal thickness and inflammation compared with the effects in control wild-type mice. However, we also observed that the protection from obesity, adipose tissue inflammation, steatosis, and insulin resistance, conferred by JNK1 ablation, was sustained over a long period and was paralleled by decreased oxidative damage in fat and liver. We conclude that compounds targeting JNK1 activity in brain and adipose tissue, which do not accumulate in the skin, may be safer and most effective.-Becattini, B., Zani, F., Breasson, L., Sardi, C., D'Agostino, V. G., Choo, M.-K., Provenzani, A., Park, J. M., Solinas, G. JNK1 ablation in mice confers long-term metabolic protection from diet-induced obesity at the cost of moderate skin oxidative damage. © FASEB.

Taurine zinc solid dispersions enhance bile-incubated L02 cell viability and improve liver function by inhibiting ERK2 and JNK phosphorylation during cholestasis.

PubMed

Wang, Yu; Mei, Xueting; Yuan, Jingquan; Lai, Xiaofang; Xu, Donghui

2016-07-29

Dietary intakes of taurine and zinc are associated with decreased risk of liver disease. In this study, solid dispersions (SDs) of a taurine zinc complex on hepatic injury were examined in vitro using the immortalized human hepatocyte cell line L02 and in a rat model of bile duct ligation. Sham-operated and bile duct ligated Sprague-Dawley rats were treated with the vehicle alone or taurine zinc (40, 80, 160mg/kg) for 17days. Bile duct ligation significantly increased blood lipid levels, and promoted hepatocyte apoptosis, inflammation and compensatory biliary proliferation. In vitro, incubation with bile significantly reduced L02 cell viability; this effect was significantly attenuated by pretreatment with SP600125 (a JNK inhibitor) and enhanced when co-incubated with taurine zinc SDs. In vivo, administration of taurine zinc SDs decreased serum alanine aminotransferase and aspartate aminotransferase activities in a dose-dependent manner and attenuated the increases in serum total bilirubin, total cholesterol and low density lipoprotein cholesterol levels after bile duct ligation. Additionally, taurine zinc SDs downregulated the expression of interleukin-1β and inhibited the phosphorylation of Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase2 (ERK2) in the liver after bile duct ligation. Moreover, taurine zinc SDs had more potent blood lipid regulatory and anti-apoptotic effects than the physical mixture of taurine and zinc acetate. Therefore, we speculate that taurine zinc SDs protect liver function at least in part via a mechanism linked to reduce phosphorylation of JNK and ERK2, which suppresses inflammation, apoptosis and cholangiocyte proliferation during cholestasis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

JNK signalling is necessary for a Wnt- and stem cell-dependent regeneration programme

PubMed Central

Tejada-Romero, Belen; Carter, Jean-Michel; Mihaylova, Yuliana; Neumann, Bjoern; Aboobaker, A. Aziz

2015-01-01

Regeneration involves the integration of new and old tissues in the context of an adult life history. It is clear that the core conserved signalling pathways that orchestrate development also play central roles in regeneration, and further study of conserved signalling pathways is required. Here we have studied the role of the conserved JNK signalling cascade during planarian regeneration. Abrogation of JNK signalling by RNAi or pharmacological inhibition blocks posterior regeneration and animals fail to express posterior markers. While the early injury-induced expression of polarity markers is unaffected, the later stem cell-dependent phase of posterior Wnt expression is not established. This defect can be rescued by overactivation of the Hh or Wnt signalling pathway to promote posterior Wnt activity. Together, our data suggest that JNK signalling is required to establish stem cell-dependent Wnt expression after posterior injury. Given that Jun is known to be required in vertebrates for the expression of Wnt and Wnt target genes, we propose that this interaction may be conserved and is an instructive part of planarian posterior regeneration. PMID:26062938

Substrate specificity effects of lipoxygenase products and inhibitors on soybean lipoxygenase-1.

PubMed

Wecksler, Aaron T; Garcia, Natalie K; Holman, Theodore R

2009-09-15

Recently, it has been shown that lipoxygenase (LO) products affect the substrate specificity of human 15-LO. In the current paper, we demonstrate that soybean LO-1 (sLO-1) is not affected by its own products, however, inhibitors which bind the allosteric site, oleyl sulfate (OS) and palmitoleyl sulfate (PS), not only lower catalytic activity, but also change the substrate specificity, by increasing the arachidonic acid (AA)/linoleic acid (LA) ratio to 4.8 and 4.0, respectively. The fact that LO inhibitors can lower activity and also change the LO product ratio is a new concept in lipoxygenase inhibition, where the goal is to not only reduce the catalytic activity but also alter substrate selectivity towards a physiologically beneficial product.

Indazole-based potent and cell-active Mps1 kinase inhibitors: rational design from pan-kinase inhibitor anthrapyrazolone (SP600125).

PubMed

Kusakabe, Ken-ichi; Ide, Nobuyuki; Daigo, Yataro; Tachibana, Yuki; Itoh, Takeshi; Yamamoto, Takahiko; Hashizume, Hiroshi; Hato, Yoshio; Higashino, Kenichi; Okano, Yousuke; Sato, Yuji; Inoue, Makiko; Iguchi, Motofumi; Kanazawa, Takayuki; Ishioka, Yukichi; Dohi, Keiji; Kido, Yasuto; Sakamoto, Shingo; Yasuo, Kazuya; Maeda, Masahiro; Higaki, Masayo; Ueda, Kazuo; Yoshizawa, Hidenori; Baba, Yoshiyasu; Shiota, Takeshi; Murai, Hitoshi; Nakamura, Yusuke

2013-06-13

Monopolar spindle 1 (Mps1) is essential for centrosome duplication, the spindle assembly check point, and the maintenance of chromosomal instability. Mps1 is highly expressed in cancer cells, and its expression levels correlate with the histological grades of cancers. Thus, selective Mps1 inhibitors offer an attractive opportunity for the development of novel cancer therapies. To design novel Mps1 inhibitors, we utilized the pan-kinase inhibitor anthrapyrazolone (4, SP600125) and its crystal structure bound to JNK1. Our design efforts led to the identification of indazole-based lead 6 with an Mps1 IC50 value of 498 nM. Optimization of the 3- and 6-positions on the indazole core of 6 resulted in 23c with improved Mps1 activity (IC50 = 3.06 nM). Finally, application of structure-based design using the X-ray structure of 23d bound to Mps1 culminated in the discovery of 32a and 32b with improved potency for cellular Mps1 and A549 lung cancer cells. Moreover, 32a and 32b exhibited reasonable selectivities over 120 and 166 kinases, respectively.

ROCK mediates phorbol ester-induced apoptosis in prostate cancer cells via p21Cip1 up-regulation and JNK.

PubMed

Xiao, Liqing; Eto, Masumi; Kazanietz, Marcelo G

2009-10-23

It is established that androgen-dependent prostate cancer cells undergo apoptosis upon treatment with phorbol esters and related analogs, an effect primarily mediated by PKCdelta. Treatment of LNCaP prostate cancer cells with phorbol 12-myristate 13-acetate (PMA) causes a strong and sustained activation of RhoA and its downstream effector ROCK (Rho kinase) as well as the formation of stress fibers. These effects are impaired in cells subjected to PKCdelta RNA interference depletion. Functional studies revealed that expression of a dominant negative RhoA mutant or treatment with the ROCK inhibitor Y-27632 inhibits the apoptotic effect of PMA in LNCaP cells. Remarkably, the cytoskeleton inhibitors cytochalasin B and blebbistatin blocked not only PMA-induced apoptosis but also the activation of JNK, a mediator of the cell death effect by the phorbol ester. In addition, we found that up-regulation of the cell cycle inhibitor p21(Cip1) is required for PMA-induced apoptosis and that inhibitors of ROCK or the cytoskeleton organization prevent p21(Cip1) induction. Real time PCR analysis and reporter gene assay revealed that PMA induces p21(Cip1) transcriptionally in a ROCK- and cytoskeleton-dependent manner. p21(Cip1) promoter analysis revealed that PMA induction is dependent on Sp1 elements in the p21(Cip1) promoter but independent of p53. Taken together, our studies implicate ROCK-mediated up-regulation of p21(Cip1) and the cytoskeleton in PKCdelta-dependent apoptosis in prostate cancer cells.

N-Acetylcysteine Attenuates Ischemia-Reperfusion-Induced Apoptosis and Autophagy in Mouse Liver via Regulation of the ROS/JNK/Bcl-2 Pathway

PubMed Central

Xia, Yujing; Dai, Weiqi; Wang, Fan; Shen, Miao; Cheng, Ping; Wang, Junshan; Lu, Jie; Zhang, Yan; Yang, Jing; Zhu, Rong; Zhang, Huawei; Li, Jingjing; Zheng, Yuanyuan; Zhou, Yingqun; Guo, Chuanyong

2014-01-01

Background Hepatic ischemia–reperfusion injury (HIRI) remains a pivotal clinical problem after hemorrhagic shock, transplantation, and some types of toxic hepatic injury. Apoptosis and autophagy play important roles in cell death during HIRI. It is also known that N-acetylcysteine (NAC) has significant pharmacologic effects on HIRI including elimination of reactive oxygen species (ROS) and attenuation of hepatic apoptosis. However, the effects of NAC on HIRI-induced autophagy have not been reported. In this study, we evaluated the effects of NAC on autophagy and apoptosis in HIRI, and explored the possible mechanism involved. Methods A mouse model of segmental (70%) hepatic warm ischemia was adopted to determine hepatic injury. NAC (150 mg/kg), a hepatoprotection agent, was administered before surgery. We hypothesized that the mechanism of NAC may involve the ROS/JNK/Bcl-2 pathway. We evaluated the expression of JNK, P-JNK, Bcl-2, Beclin 1 and LC3 by western blotting and immunohistochemical staining. Autophagosomes were evaluated by transmission electron microscopy (TEM). Results We found that ALT, AST and pathological changes were significantly improved in the NAC group. Western blotting analysis showed that the expression levels of Beclin 1 and LC3 were significantly decreased in NAC-treated mice. In addition, JNK, p-JNK, Bax, TNF-α, NF-κB, IL2, IL6 and levels were also decreased in NAC-treated mice. Conclusion NAC can prevent HIRI-induced autophagy and apoptosis by influencing the JNK signal pathway. The mechanism is likely to involve attenuation of JNK and p-JNK via scavenged ROS, an indirect increase in Bcl-2 level, and finally an alteration in the balance of Beclin 1 and Bcl-2. PMID:25264893

Theoretical studies on beta and delta isoform-specific binding mechanisms of phosphoinositide 3-kinase inhibitors.

PubMed

Zhu, Jingyu; Pan, Peichen; Li, Youyong; Wang, Man; Li, Dan; Cao, Biyin; Mao, Xinliang; Hou, Tingjun

2014-03-04

Phosphoinositide 3-kinase (PI3K) is known to be closely related to tumorigenesis and cell proliferation, and controls a variety of cellular processes, including proliferation, growth, apoptosis, migration, metabolism, etc. The PI3K family comprises eight catalytic isoforms, which are subdivided into three classes. Recently, the discovery of inhibitors that block a single isoform of PI3K has continued to attract special attention because they may have higher selectivity for certain tumors and less toxicity for healthy cells. The PI3Kβ and PI3Kδ share fewer studies than α/γ, and therefore, in this work, the combination of molecular dynamics simulations and free energy calculations was employed to explore the binding of three isoform-specific PI3K inhibitors (COM8, IC87114, and GDC-0941) to PI3Kβ or PI3Kδ. The isoform specificities of the studied inhibitors derived from the predicted binding free energies are in good agreement with the experimental data. In addition, the key residues critical for PI3Kβ or PI3Kδ selectivity were highlighted by decomposing the binding free energies into the contributions from individual residues. It was observed that although PI3Kβ and PI3Kδ share the conserved ATP-binding pockets, individual residues do behave differently, particularly the residues critical for PI3Kβ or PI3Kδ selectivity. It can be concluded that the inhibitor specificity between PI3Kβ and PI3Kδ is determined by the additive contributions from multiple residues, not just a single one. This study provides valuable information for understanding the isoform-specific binding mechanisms of PI3K inhibitors, and should be useful for the rational design of novel and selective PI3K inhibitors.

JIP3 deficiency attenuates cardiac hypertrophy by suppression of JNK pathway.

PubMed

Ma, Qinghua; Liu, Yuxiu; Chen, Lianghua

2018-06-15

Pathological cardiac hypertrophy is a leading cause of morbidity and mortality worldwide; however, our understanding of the molecular mechanisms revealing the disease is still unclear. In the present study, we suggested that c-Jun N-terminal kinase (JNK)-interacting protein 3 (JIP3), involved in various cellular processes, played an essential role in regulating pathological cardiac hypertrophy through in vivo and in vitro studies. JIP3 was highly expressed in human hearts with hypertrophic cardiomyopathy (HCM), and in mouse hypertrophic hearts. Following, the wild type (WT) and JIP3-knockout (KO) mice subjected to aortic banding (AB) challenge were used as animal models with cardiac hypertrophy. The results showed that JIP3-KO mice after AB operation exhibited attenuated cardiac function, reduced fibrosis levels and decreased hypertrophic marker proteins, including atrial natriuretic peptides (Anp) and brain/B-type natriuretic peptides (Bnp) and β-myosin heavy chain (β-Mhc). Loss of JIP3 also ameliorated oxidative stress, inflammatory response, apoptosis and endoplasmic reticulum (ER) stress in hearts of mice after AB surgery. Consistently, the expressions of ER stress-related molecules, such as phosphorylated-α-subunit of the eukaryotic initiation factor-2 (eIF2α), glucose-regulated protein (GRP) 78 and C/-EBP homologous protein (CHOP), were markedly decreased by JIP3-deficiency in hearts of AB-operated mice. JNK and its down-streaming signal of p90rsk was highly activated by AB operation in WT mice, while being significantly reversed by JIP3-ablation. Intriguingly, the in vitro results showed that promoting JNK activation by using its activator of anisomycin enhanced AngII-stimulated ER stress, oxidative stress, apoptosis and inflammatory response in cardiomyocytes isolated from WT mice. However, JIP3-KO-attenuated these pathologies was rescued by anisomycin treatment in AngII-incubated cardiomyocytes. Together, the findings indicated that blockage of JIP3

Development of Small-molecule HIV Entry Inhibitors Specifically Targeting gp120 or gp41

PubMed Central

Lu, Lu; Yu, Fei; Cai, Lifeng; Debnath, Asim K.; Jiang, Shibo

2015-01-01

Human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein surface subunit gp120 and transmembrane subunit gp41 play important roles in HIV-1 entry, thus serving as key targets for the development of HIV-1 entry inhibitors. T20 peptide (enfuvirtide) is the first U.S. FDA-approved HIV entry inhibitor; however, its clinical application is limited by the lack of oral availability. Here, we have described the structure and function of the HIV-1 gp120 and gp41 subunits and reviewed advancements in the development of small-molecule HIV entry inhibitors specifically targeting these two Env glycoproteins. We then compared the advantages and disadvantages of different categories of HIV entry inhibitor candidates and further predicted the future trend of HIV entry inhibitor development. PMID:26324044

Proteolytic Inhibition of Salmonella enterica Serovar Typhimurium-Induced Activation of the Mitogen-Activated Protein Kinases ERK and JNK in Cultured Human Intestinal Cells

PubMed Central

Mynott, Tracey L.; Crossett, Ben; Prathalingam, S. Radhika

2002-01-01

Bromelain, a mixture of cysteine proteases from pineapple stems, blocks signaling by the mitogen-activated protein (MAP) kinases extracellular regulated kinase 1 (ERK-1) and ERK-2, inhibits inflammation, and protects against enterotoxigenic Escherichia coli infection. In this study, we examined the effect of bromelain on Salmonella enterica serovar Typhimurium infection, since an important feature of its pathogenesis is its ability to induce activation of ERK-1 and ERK-2, which leads to internalization of bacteria and induction of inflammatory responses. Our results show that bromelain dose dependently blocks serovar Typhimurium-induced ERK-1, ERK-2, and c-Jun NH2-terminal kinase (JNK) activation in Caco-2 cells. Bromelain also blocked signaling induced by carbachol and anisomycin, pharmacological MAP kinase agonists. Despite bromelain inhibition of serovar Typhimurium-induced MAP kinase signaling, it did not prevent subsequent invasion of the Caco-2 cells by serovar Typhimurium or alter serovar Typhimurium -induced decreases in resistance across Caco-2 monolayers. Surprisingly, bromelain also did not block serovar Typhimurium-induced interleukin-8 (IL-8) secretion but synergized with serovar Typhimurium to enhance IL-8 production. We also found that serovar Typhimurium does not induce ERK phosphorylation in Caco-2 cells in the absence of serum but that serovar Typhimurium-induced invasion and decreases in monolayer resistance are unaffected. Collectively, these data indicate that serovar Typhimurium-induced invasion of Caco-2 cells, changes in the resistance of epithelial cell monolayers, and IL-8 production can occur independently of the ERK and JNK signaling pathways. Data also confirm that bromelain is a novel inhibitor of MAP kinase signaling pathways and suggest a novel role for proteases as inhibitors of signal transduction pathways in intestinal epithelial cells. PMID:11748167

Structural features, substrate specificity, kinetic properties of insect α-amylase and specificity of plant α-amylase inhibitors.

PubMed

Kaur, Rimaljeet; Kaur, Narinder; Gupta, Anil Kumar

2014-11-01

α-Amylase is an important digestive enzyme required for the optimal growth and development of insects. Several insect α-amylases had been purified and their physical and chemical properties were characterized. Insect α-amylases of different orders display variability in structure, properties and substrate specificity. Such diverse properties of amylases could be due to different feeding habits and gut environment of insects. In this review, structural features and properties of several insect α-amylases were compared. This could be helpful in exploring the diversity in characteristics of α-amylase between the members of the same class (insecta). Properties like pH optima are reflected in enzyme structural features. In plants, α-amylase inhibitors (α-AIs) occur as part of natural defense mechanisms against pests by interfering in their digestion process and thus could also provide access to new pest management strategies. AIs are quite specific in their action; therefore, these could be employed according to their effectiveness against target amylases. Potential of transgenics with α-AIs has also been discussed for insect resistance and controlling infestation. The differences in structural features of insect α-amylases provided reasons for their efficient functioning at different pH and the specificity towards various substrates. Various proteinaceous and non-proteinaceous inhibitors discussed could be helpful in controlling pest infestation. In depth detailed studies are required on proteinaceous α-AI-α-amylase interaction at different pH's as well as the insect proteinase action on these inhibitors before selecting the α-AI for making transgenics resistant to particular insect. Copyright © 2014 Elsevier Inc. All rights reserved.

Glycyrrhetinic acid inhibits ICAM-1 expression via blocking JNK and NF-κB pathways in TNF-α-activated endothelial cells

PubMed Central

Chang, Ying-ling; Chen, Chien-lin; Kuo, Chao-Lin; Chen, Bor-chyuan; You, Jyh-sheng

2010-01-01

Aim: To investigate the effects of glycyrrhetinic acid (GA), an active component extracted from the root of Glycyrrhizae glabra, on the expression of intercellular adhesion molecule-1 (ICAM-1) in tumor necrosis factor-α (TNF-α)-activated human umbilical vein endothelial cells (HUVEC). Methods: ICAM-1 mRNA and protein levels were detected using RT-PCR and cell enzyme-linked immunosorbent assays. The adherence of human monocytic THP-1 cells labeled with [3H]thymidine to HUVEC was determined by counting radioactivity with a scintillation counter. The activation of mitogen-activated protein kinases as well as the degradation of IκB and nuclear factor-κB (NF-κB) or phospho-c-Jun in the nucleus were detected by western blots. NF-κB binding activity was detected using electrophoretic mobility shift assay. Results: GA (50 and 100 μmol/L) significantly inhibits TNF-α-induced ICAM-1 mRNA and protein expressions, as well as THP-1 cell adhesiveness in HUVEC. GA selectively inhibited TNF-α-activated signal pathway of c-Jun N-terminal kinase (JNK), without affecting extracellular signal-regulated kinase 1/2 and p38. Furthermore, GA apparently inhibited IκB/NF-κB signaling system by preventing IκB degradation, NF-κB translocation, and NF-κB/DNA binding activity. Finally, pretreatment with GA or the inhibitors of NF-κB, JNK, and p38 reduced the ICAM-1 protein expression induced by TNF-α. Conclusion: GA inhibits TNF-α-stimulated ICAM-1 expression, leading to a decrease in adherent monocytes to HUVEC. This inhibition is attributed to GA interruption of both JNK/c-Jun and IκB/NF-κB signaling pathways, which decrease activator protein-1 (AP-1) and NF-κB mediated ICAM-1 expressions. The results suggest that GA may provide a beneficial effect in treating vascular diseases associated with inflammation, such as atherosclerosis. PMID:20418897

A specific pharmacophore model of sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors.

PubMed

Tang, Chunlei; Zhu, Xiaoyun; Huang, Dandan; Zan, Xin; Yang, Baowei; Li, Ying; Du, Xiaoyong; Qian, Hai; Huang, Wenlong

2012-06-01

Sodium-dependent glucose co-transporter 2 (SGLT2) plays a pivotal role in maintaining glucose equilibrium in the human body, emerging as one of the most promising targets for the treatment of diabetes mellitus type 2. Pharmacophore models of SGLT2 inhibitors have been generated with a training set of 25 SGLT2 inhibitors using Discovery Studio V2.1. The best hypothesis (Hypo1(SGLT2)) contains one hydrogen bond donor, five excluded volumes, one ring aromatic and three hydrophobic features, and has a correlation coefficient of 0.955, cost difference of 68.76, RMSD of 0.85. This model was validated by test set, Fischer randomization test and decoy set methods. The specificity of Hypo1(SGLT2) was evaluated. The pharmacophore features of Hypo1(SGLT2) were different from the best pharmacophore model (Hypo1(SGLT1)) of SGLT1 inhibitors we developed. Moreover, Hypo1(SGLT2) could effectively distinguish selective inhibitors of SGLT2 from those of SGLT1. These results indicate that a highly predictive and specific pharmacophore model of SGLT2 inhibitors has been successfully obtained. Then Hypo1(SGLT2) was used as a 3D query to screen databases including NCI and Maybridge for identifying new inhibitors of SGLT2. The hit compounds were subsequently subjected to filtering by Lipinski's rule of five. And several compounds selected from the top ranked hits have been suggested for further experimental assay studies.

Alteration of Substrate and Inhibitor Specificity of Feline Immunodeficiency Virus Protease

PubMed Central

Lin, Ying-Chuan; Beck, Zachary; Lee, Taekyu; Le, Van-Duc; Morris, Garrett M.; Olson, Arthur J.; Wong, Chi-Huey; Elder, John H.

2000-01-01

Feline immunodeficiency virus (FIV) protease is structurally very similar to human immunodeficiency virus (HIV) protease but exhibits distinct substrate and inhibitor specificities. We performed mutagenesis of subsite residues of FIV protease in order to define interactions that dictate this specificity. The I37V, N55M, M56I, V59I, and Q99V mutants yielded full activity. The I37V, N55M, V59I, and Q99V mutants showed a significant increase in activity against the HIV-1 reverse transcriptase/integrase and P2/nucleocapsid junction peptides compared with wild-type (wt) FIV protease. The I37V, V59I, and Q99V mutants also showed an increase in activity against two rapidly cleaved peptides selected by cleavage of a phage display library with HIV-1 protease. Mutations at Q54K, I98P, and L101I dramatically reduced activity. Mutants containing a I35D or I57G substitution showed no activity against either FIV or HIV substrates. FIV proteases all failed to cut HIV-1 matrix/capsid, P1/P6, P6/protease, and protease/reverse transcriptase junctions, indicating that none of the substitutions were sufficient to change the specificity completely. The I37V, N55M, M56I, V59I, and Q99V mutants, compared with wt FIV protease, all showed inhibitor specificity more similar to that of HIV-1 protease. The data also suggest that FIV protease prefers a hydrophobic P2/P2′ residue like Val over Asn or Glu, which are utilized by HIV-1 protease, and that S2/S2′ might play a critical role in distinguishing FIV and HIV-1 protease by specificity. The findings extend our observations regarding the interactions involved in substrate binding and aid in the development of broad-based inhibitors. PMID:10775609

A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA

PubMed Central

Schaenzer, Adam J.; Wlodarchak, Nathan; Drewry, David H.; Zuercher, William J.; Rose, Warren E.; Striker, Rob; Sauer, John-Demian

2017-01-01

Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial Penicillin-binding-protein And Serine/Threonine kinase-Associated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition. PMID:28821610

A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA.

PubMed

Schaenzer, Adam J; Wlodarchak, Nathan; Drewry, David H; Zuercher, William J; Rose, Warren E; Striker, Rob; Sauer, John-Demian

2017-10-13

Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial P enicillin-binding-protein A nd S erine/ T hreonine kinase- A ssociated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition.

Genetic Correction of SOD1 Mutant iPSCs Reveals ERK and JNK Activated AP1 as a Driver of Neurodegeneration in Amyotrophic Lateral Sclerosis.

PubMed

Bhinge, Akshay; Namboori, Seema C; Zhang, Xiaoyu; VanDongen, Antonius M J; Stanton, Lawrence W

2017-04-11

Although mutations in several genes with diverse functions have been known to cause amyotrophic lateral sclerosis (ALS), it is unknown to what extent causal mutations impinge on common pathways that drive motor neuron (MN)-specific neurodegeneration. In this study, we combined induced pluripotent stem cells-based disease modeling with genome engineering and deep RNA sequencing to identify pathways dysregulated by mutant SOD1 in human MNs. Gene expression profiling and pathway analysis followed by pharmacological screening identified activated ERK and JNK signaling as key drivers of neurodegeneration in mutant SOD1 MNs. The AP1 complex member JUN, an ERK/JNK downstream target, was observed to be highly expressed in MNs compared with non-MNs, providing a mechanistic insight into the specific degeneration of MNs. Importantly, investigations of mutant FUS MNs identified activated p38 and ERK, indicating that network perturbations induced by ALS-causing mutations converge partly on a few specific pathways that are drug responsive and provide immense therapeutic potential. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Cullin-4 regulates Wingless and JNK signaling-mediated cell death in the Drosophila eye

PubMed Central

Tare, Meghana; Sarkar, Ankita; Bedi, Shimpi; Kango-Singh, Madhuri; Singh, Amit

2016-01-01

In all multicellular organisms, the fundamental processes of cell proliferation and cell death are crucial for growth regulation during organogenesis. Strict regulation of cell death is important to maintain tissue homeostasis by affecting processes like regulation of cell number, and elimination of unwanted/unfit cells. The developing Drosophila eye is a versatile model to study patterning and growth, where complex signaling pathways regulate growth and cell survival. However, the molecular mechanisms underlying regulation of these processes is not fully understood. In a gain-of-function screen, we found that misexpression of cullin-4 (cul-4), an ubiquitin ligase, can rescue reduced eye mutant phenotypes. Previously, cul-4 has been shown to regulate chromatin remodeling, cell cycle and cell division. Genetic characterization of cul-4 in the developing eye revealed that loss-of-function of cul-4 exhibits a reduced eye phenotype. Analysis of twin-spots showed that in comparison with their wild-type counterparts, the cul-4 loss-of-function clones fail to survive. Here we show that cul-4 clones are eliminated by induction of cell death due to activation of caspases. Aberrant activation of signaling pathways is known to trigger cell death in the developing eye. We found that Wingless (Wg) and c-Jun-amino-terminal-(NH2)-Kinase (JNK) signaling are ectopically induced in cul-4 mutant clones, and these signals co-localize with the dying cells. Modulating levels of Wg and JNK signaling by using agonists and antagonists of these pathways demonstrated that activation of Wg and JNK signaling enhances cul-4 mutant phenotype, whereas downregulation of Wg and JNK signaling rescues the cul-4 mutant phenotypes of reduced eye. Here we present evidences to demonstrate that cul-4 is involved in restricting Wg signaling and downregulation of JNK signaling-mediated cell death during early eye development. Overall, our studies provide insights into a novel role of cul-4 in promoting cell

The Ste20 Family Kinases MAP4K4, MINK1, and TNIK Converge to Regulate Stress-Induced JNK Signaling in Neurons.

PubMed

Larhammar, Martin; Huntwork-Rodriguez, Sarah; Rudhard, York; Sengupta-Ghosh, Arundhati; Lewcock, Joseph W

2017-11-15

The c-Jun- N -terminal kinase (JNK) signaling pathway regulates nervous system development, axon regeneration, and neuronal degeneration after acute injury or in chronic neurodegenerative disease. Dual leucine zipper kinase (DLK) is required for stress-induced JNK signaling in neurons, yet the factors that initiate DLK/JNK pathway activity remain poorly defined. In the present study, we identify the Ste20 kinases MAP4K4, misshapen-like kinase 1 (MINK1 or MAP4K6) and TNIK Traf2- and Nck-interacting kinase (TNIK or MAP4K7), as upstream regulators of DLK/JNK signaling in neurons. Using a trophic factor withdrawal-based model of neurodegeneration in both male and female embryonic mouse dorsal root ganglion neurons, we show that MAP4K4, MINK1, and TNIK act redundantly to regulate DLK activation and downstream JNK-dependent phosphorylation of c-Jun in response to stress. Targeting MAP4K4, MINK1, and TNIK, but not any of these kinases individually, is sufficient to protect neurons potently from degeneration. Pharmacological inhibition of MAP4Ks blocks stabilization and phosphorylation of DLK within axons and subsequent retrograde translocation of the JNK signaling complex to the nucleus. These results position MAP4Ks as important regulators of the DLK/JNK signaling pathway. SIGNIFICANCE STATEMENT Neuronal degeneration occurs in disparate circumstances: during development to refine neuronal connections, after injury to clear damaged neurons, or pathologically during disease. The dual leucine zipper kinase (DLK)/c-Jun- N -terminal kinase (JNK) pathway represents a conserved regulator of neuronal injury signaling that drives both neurodegeneration and axon regeneration, yet little is known about the factors that initiate DLK activity. Here, we uncover a novel role for a subfamily of MAP4 kinases consisting of MAP4K4, Traf2- and Nck-interacting kinase (TNIK or MAP4K7), and misshapen-like kinase 1 (MINK1 or MAP4K6) in regulating DLK/JNK signaling in neurons. Inhibition of

Procainamide Is a Specific Inhibitor of DNA Methyltransferase 1*

PubMed Central

Lee, Byron H.; Yegnasubramanian, Srinivasan; Lin, Xiaohui; Nelson, William G.

2007-01-01

CpG island hypermethylation occurs in most cases of cancer, typically resulting in the transcriptional silencing of critical cancer genes. Procainamide has been shown to inhibit DNA methyltransferase activity and reactivate silenced gene expression in cancer cells by reversing CpG island hypermethylation. We report here that procainamide specifically inhibits the hemimethylase activity of DNA methyltransferase 1 (DNMT1), the mammalian enzyme thought to be responsible for maintaining DNA methylation patterns during replication. At micromolar concentrations, procainamide was found to be a partial competitive inhibitor of DNMT1, reducing the affinity of the enzyme for its two substrates, hemimethylated DNA and S-adenosyl-l-methionine. By doing so, procainamide significantly decreased the processivity of DNMT1 on hemimethylated DNA. Procainamide was not a potent inhibitor of the de novo methyltransferases DNMT3a and DNMT3b2. As further evidence of the specificity of procainamide for DNMT1, procainamide failed to lower genomic 5-methyl-2′-deoxycytidine levels in HCT116 colorectal cancer cells when DNMT1 was genetically deleted but significantly reduced genomic 5-methyl-2′-deoxycyti-dine content in parental HCT116 cells and in HCT116 cells where DNMT3b was genetically deleted. Because many reports have strongly linked DNMT1 with epigenetic alterations in carcinogenesis, procainamide may be a useful drug in the prevention of cancer. PMID:16230360

[Effects of inhibiting the phosphorylation of JNK by absorbed INF-γon the remodeling of nasal mucosa in allergic rhinitis rats].

PubMed

Li, Q; Chen, Y L; Ma, Y Y; Zhang, Y D; Sun, C W; You, C P

2016-07-05

Objective: To study the role of phosphorylated JNK(c-Jun N-terminal kinase) on nasal mucosa remodeling in allergic rhinitis(AR) rats and the influence of IFN-γon IL-1β,JNK and nasal mucosa remodeling. Method: According to random number table,48 Wistar rats were divided into control group(A group),AR group(B group),IFN-γgroup(C group) and triamcinolone acetonide group(D group).The rats in group B,C and D were sensitized and provocated for inducing AR by intraperitoneal injection of ovalbumin(OVA) and Al(OH)₃.Thirty minutes before intranasally challenged,rats in three groups were administrated by instillation of PBS,IFN-γand triamcinolone acetonide into nasal cavities,while the group A rats were administrated by saline solution.Ten rats in each group were selected to enter the final experiment.The density of IL-1βin serum and nasal lavage fluid were tested by ELISA.The mean absorbance (m A ) of phosphorylated JNK and c-Jun were tested by immunohistochemistry.Western Blot detected the P-JNK level in nasal tissue homogenate. Result: The density of IL-1βin serum and nasal lavage fluid in group C and group D were significantly lower than that of group B ( P <0.01).Immunohistochemistry study showed that the protein expression level of phosphorylated JNK and c-Jun of nasal mucosa were significantly increased in group B,but significantly reduced in group C and group D .The mA of phosphorylated JNK and c-Jun in group B were significantly higher than those in the group C and group D( P <0.01).The Western blot showed that the P-JNK of nasal tissue homogenate in group B was higher than that of group C and group D ( P <0.01). Conclusion: The phosphorylation of JNK played an important role in nasal mucosa remodeling.IFN-γcould inhibit the phosphorylation of JNK and reduce the nasal mucosa remodeling.The mechanisms may be achieved through down-regulation of IL-1β. Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.

Adhesion- and stress-related adaptation of glioma radiochemoresistance is circumvented by β1 integrin/JNK co-targeting.

PubMed

Vehlow, Anne; Klapproth, Erik; Storch, Katja; Dickreuter, Ellen; Seifert, Michael; Dietrich, Antje; Bütof, Rebecca; Temme, Achim; Cordes, Nils

2017-07-25

Resistance of cancer stem-like and cancer tumor bulk cells to radiochemotherapy and destructive infiltration of the brain fundamentally influence the treatment efficiency to cure of patients suffering from Glioblastoma (GBM). The interplay of adhesion and stress-related signaling and activation of bypass cascades that counteract therapeutic approaches remain to be identified in GBM cells. We here show that combined inhibition of the adhesion receptor β1 integrin and the stress-mediator c-Jun N-terminal kinase (JNK) induces radiosensitization and blocks invasion in stem-like and patient-derived GBM cultures as well as in GBM cell lines. In vivo, this treatment approach not only significantly delays tumor growth but also increases median survival of orthotopic, radiochemotherapy-treated GBM mice. Both, in vitro and in vivo, effects seen with β1 integrin/JNK co-inhibition are superior to the monotherapy. Mechanistically, the in vitro radiosensitization provoked by β1 integrin/JNK targeting is caused by defective DNA repair associated with chromatin changes, enhanced ATM phosphorylation and prolonged G2/M cell cycle arrest. Our findings identify a β1 integrin/JNK co-dependent bypass signaling for GBM therapy resistance, which might be therapeutically exploitable.

Peroxiredoxin 2 regulates PGF2α-induced corpus luteum regression in mice by inhibiting ROS-dependent JNK activation.

PubMed

Park, Sun-Ji; Kim, Jung-Hak; Kim, Tae-Shin; Lee, Sang-Rae; Park, Jeen-Woo; Lee, Seunghoon; Kim, Jin-Man; Lee, Dong-Seok

2017-07-01

Luteal regression is a natural and necessary event to regulate the reproductive process in all mammals. Prostaglandin F2α (PGF2α) is the main factor that causes functional and structural regression of the corpus luteum (CL). It is well known that PGF2α-mediated ROS generation is closely involved in luteal regression. Peroxiredoxin 2 (Prx2) as an antioxidant enzyme plays a protective role against oxidative stress-induced cell death. However, the effect of Prx2 on PGF2α-induced luteal regression has not been reported. Here, we investigated the role of Prx2 in functional and structural CL regression induced by PGF2α-mediated ROS using Prx2-deficient (-/-) mice. We found that PGF2α-induced ROS generation was significantly higher in Prx2-/- MEF cells compared with that in wild-type (WT) cells, which induced apoptosis by activating JNK-mediated apoptotic signaling pathway. Also, PGF2α treatment in the CL derived from Prx2-/- mice promoted the reduction of steroidogenic enzyme expression and the activation of JNK and caspase3. Compared to WT mice, serum progesterone levels and luteal expression of steroidogenic enzymes decreased more rapidly whereas JNK and caspase3 activations were significantly increased in Prx2-/- mice injected with PGF2α. However, the impaired steroidogenesis and PGF2α-induced JNK-dependent apoptosis were rescued by the addition of the antioxidant N-acetyl-L-cysteine (NAC). This is the first study to demonstrate that Prx2 deficiency ultimately accelerated the PGF2α-induced luteal regression through activation of the ROS-dependent JNK pathway. These findings suggest that Prx2 plays a crucial role in preventing accelerated luteal regression via inhibition of the ROS/JNK pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

Inhibition of Vascular c-Jun N-Terminal Kinase 2 Improves Obesity-Induced Endothelial Dysfunction After Roux-en-Y Gastric Bypass.

PubMed

Doytcheva, Petia; Bächler, Thomas; Tarasco, Erika; Marzolla, Vincenzo; Engeli, Michael; Pellegrini, Giovanni; Stivala, Simona; Rohrer, Lucia; Tona, Francesco; Camici, Giovanni G; Vanhoutte, Paul M; Matter, Christian M; Lutz, Thomas A; Lüscher, Thomas F; Osto, Elena

2017-11-14

Roux-en-Y gastric bypass (RYGB) reduces obesity-associated comorbidities and cardiovascular mortality. RYGB improves endothelial dysfunction, reducing c-Jun N-terminal kinase (JNK) vascular phosphorylation. JNK activation links obesity with insulin resistance and endothelial dysfunction. Herein, we examined whether JNK1 or JNK2 mediates obesity-induced endothelial dysfunction and if pharmacological JNK inhibition can mimic RYGB vascular benefits. After 7 weeks of a high-fat high-cholesterol diet, obese rats underwent RYGB or sham surgery; sham-operated ad libitum-fed rats received, for 8 days, either the control peptide D-TAT or the JNK peptide inhibitor D-JNKi-1 (20 mg/kg per day subcutaneous). JNK peptide inhibitor D-JNKi-1 treatment improved endothelial vasorelaxation in response to insulin and glucagon-like peptide-1, as observed after RYGB. Obesity increased aortic phosphorylation of JNK2, but not of JNK1. RYGB and JNK peptide inhibitor D-JNKi-1 treatment blunted aortic JNK2 phosphorylation via activation of glucagon-like peptide-1-mediated signaling. The inhibitory phosphorylation of insulin receptor substrate-1 was reduced, whereas the protein kinase B/endothelial NO synthase pathway was increased and oxidative stress was decreased, resulting in improved vascular NO bioavailability. Decreased aortic JNK2 phosphorylation after RYGB rapidly improves obesity-induced endothelial dysfunction. Pharmacological JNK inhibition mimics the endothelial protective effects of RYGB. These findings highlight the therapeutic potential of novel strategies targeting vascular JNK2 against the severe cardiovascular disease associated with obesity. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

The JNK/AP-1 pathway upregulates expression of the recycling endosome rab11a gene in B cells transformed by Theileria.

PubMed

Lizundia, Regina; Chaussepied, Marie; Naissant, Bernina; Masse, Guillemette X; Quevillon, Emmanuel; Michel, Fréderique; Monier, Solange; Weitzman, Jonathan B; Langsley, Gordon

2007-08-01

Lymphocyte transformation induced by Theileria parasites involves constitutive activation of c-Jun N-terminal kinase (JNK) and the AP-1 transcription factor. We found that JNK/AP-1 activation is associated with elevated levels of Rab11 protein in Theileria-transformed B cells. We show that AP-1 regulates rab11a promoter activity in B cells and that the induction of c-Jun activity in mouse fibroblasts also leads to increased transcription of the endogenous rab11a gene, consistent with it being an AP-1 target. Pharmacological inhibition of the JNK pathway reduced Rab11 protein levels and endosome recycling of transferrin receptor (TfR) and siRNA knockdown of JNK1 and Rab11A levels also reduced TfR surface expression. We propose a model, where activation of the JNK/AP-1 pathway during cell transformation might assure that the regulation of recycling endosomes is co-ordinated with cell-cycle progression. This might be achieved via the simultaneous upregulation of the cell cycle machinery (e.g. cyclin D1) and the recycling endosome regulators (e.g. Rab11A).

Apigenin induces apoptosis in human leukemia cells and exhibits anti-leukemic activity in vivo via inactivation of Akt and activation of JNK

PubMed Central

Budhraja, Amit; Gao, Ning; Zhang, Zhuo; Son, Young-Ok; Cheng, Senping; Wang, Xin; Ding, Songze; Hitron, Andrew; Chen, Gang; Luo, Jia; Shi, Xianglin

2015-01-01

In this study, we investigated the functional role of Akt and JNK signaling cascades in apigenin-induced apoptosis in U937 human leukemia cells and anti-leukemic activity of apigenin in vivo. Apigenin-induced apoptosis by inactivation of Akt with a concomitant activation of JNK, Mcl-1 and Bcl-2 down-regulation, cytochrome c release from mitochondria and activation of caspases. Constitutively active myristolated Akt prevented apigenin-induced JNK, caspases activation, and apoptosis. Conversely, LY294002 and a dominant negative construct of Akt potentiated apigenin-induced apoptosis in leukemia cells. Interruption of JNK pathway showed marked reduction in apigenin-induced caspases activation and apoptosis in leukemia cells. Furthermore, in vivo administration of apigenin resulted in attenuation of tumor growth in U937 xenografts accompanied inactivation of Akt and activation of JNK. Attenuation of tumor growth in U937 xenografts by apigenin raises the possibility that apigenin may have clinical implications and can be further tested for incorporating in leukemia treatment regimens. PMID:22084167

TRPM2 contributes to LPS/IFNγ-induced production of nitric oxide via the p38/JNK pathway in microglia.

PubMed

Miyake, Takahito; Shirakawa, Hisashi; Kusano, Ayaka; Sakimoto, Shinya; Konno, Masakazu; Nakagawa, Takayuki; Mori, Yasuo; Kaneko, Shuji

2014-02-07

Microglia are immune cells that maintain brain homeostasis at a resting state by surveying the environment and engulfing debris. However, in some pathological conditions, microglia can produce neurotoxic factors such as pro-inflammatory cytokines and nitric oxide (NO) that lead to neuronal degeneration. Inflammation-induced calcium (Ca(2+)) signaling is thought to underlie this abnormal activation of microglia, but the mechanisms are still obscure. We previously showed that combined application of lipopolysaccharide and interferon γ (LPS/IFNγ) induced-production of NO in microglia from wild-type (WT) mice is significantly reduced in microglia from transient receptor potential melastatin 2 (TRPM2)-knockout (KO) mice. Here, we found that LPS/IFNγ produced a late-onset Ca(2+) signaling in WT microglia, which was abolished by application of the NADPH oxidase inhibitor diphenylene iodonium (DPI) and ML-171. In addition, pharmacological blockade or gene deletion of TRPM2 channel in microglia did not show this Ca(2+) signaling. Furthermore, pharmacological manipulation and Western blotting revealed that Ca(2+) mobilization, the proline-rich tyrosine kinase 2 (Pyk2), p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH2-terminal kinase (JNK) contributed to TRPM2-mediated LPS/IFNγ-induced activation, while the extracellular signal-regulated protein kinase (ERK) did not. These results suggest that LPS/IFNγ activates TRPM2-mediated Ca(2+) signaling, which in turn increases downstream p38 MAPK and JNK signaling and results in increased NO production in microglia. Copyright © 2014 Elsevier Inc. All rights reserved.

Efavirenz and 8-hydroxyefavirenz induce cell death via a JNK- and BimEL-dependent mechanism in primary human hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bumpus, Namandje N., E-mail: nbumpus1@jhmi.edu

Chronic use of efavirenz (EFV) has been linked to incidences of hepatotoxicity in patients receiving EFV to treat HIV-1. While recent studies have demonstrated that EFV stimulates hepatic cell death a role for the metabolites of efavirenz in this process has yet to be examined. In the present study, incubation of primary human hepatocytes with synthetic 8-hydroxyEFV (8-OHEFV), which is the primary metabolite of EFV, resulted in cell death, caspase-3 activation and reactive oxygen species formation. The metabolite exerted these effects at earlier time points and using lower concentrations than were required for the parent compound. In addition, pharmacological inhibitionmore » of cytochrome P450-dependent metabolism of EFV using 1-aminobenzotriazole markedly decreased reactive oxygen species formation and cell death. Treatment of primary human hepatocytes with EFV and 8-OHEFV also stimulated phosphorylation of c-Jun N-terminal kinase (JNK) as well as phosphorylation of the JNK substrate c-Jun. Further, the mRNA and protein expression of an isoform of Bim (Bcl-2 interacting mediator of cell death) denoted as BimEL, which is proapoptotic and has been shown to be modulated by JNK, was increased. Inhibition of JNK using SP600125 prevented the EFV- and 8-OHEFV-mediated cell death. Silencing of Bim using siRNA transfected into hepatocytes also prevented cell death resulting from 8-OHEFV-treatment. These data suggest that the oxidative metabolite 8-OHEFV is a more potent inducer of hepatic cell death than the parent compound EFV. Further, activation of the JNK signaling pathway and BimEL mRNA expression appear to be required for EFV- and 8-OHEFV-mediated hepatocyte death. -- Highlights: Black-Right-Pointing-Pointer 8-Hydroxyefavirenz is a more potent stimulator of cell death than efavirenz. Black-Right-Pointing-Pointer Efavirenz and 8-hydroxyefavirenz increase JNK activity and BimEL mRNA expression. Black-Right-Pointing-Pointer JNK and Bim are required for efavirenz- and

Prolonged duration of isoflurane anesthesia impairs spatial recognition memory through the activation of JNK1/2 in the hippocampus of mice.

PubMed

Jiang, Shan; Miao, Bei; Chen, Ying

2017-05-03

Postoperative cognitive dysfunction is a frequent complication with surgery and anesthesia, and the underlying mechanism is unclear. Our aim was to investigate the effect of different durations of isoflurane anesthesia on spatial recognition memory and activation of JNK1/2 in the hippocampus of mice. In the present study, adult male mice were anesthetized with isoflurane for different durations (1.5% isoflurane for 1, 2, and 4 h). Spatial recognition memory was determined using spontaneous alternation and two-trial recognition memory in Y-maze at 24 h after anesthesia. The activation of JNK1/2 in the hippocampus was tested using western blot. Mice treated with isoflurane for 4 h showed significantly decreased spontaneous alternations and decreased exploration parameters compared with the no anesthesia group, but this was not observed in mice treated with isoflurane for 1 or 2 h. The protein levels of p-JNK1/2 in the hippocampus were significantly increased at 10 min after isoflurane anesthesia for 1, 2, and 4 h compared with no anesthesia. However, only isoflurane anesthesia for 4 h still increased JNK1/2 and p-JNK1/2 levels at 24 h after anesthesia. We concluded that prolonged duration of isoflurane anesthesia maintained the activation of JNK1/2, which led to memory impairment at 24 h after anesthesia.

Covalent cross-linking of insulin-like growth factor-1 to a specific inhibitor from human serum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ooi, G.T.; Herington, A.C.

1986-05-29

Previous studies have shown that a specific inhibitor of insulin-like growth factor (IGF) action in vitro can be isolated from normal human serum and subsequently partially purified on an IGF-affinity column. The ability of the inhibitor to bind the IGFs has now been confirmed directly using covalent cross-linking techniques. When /sup 125/I-IGF-1 was cross-linked to inhibitor using disuccinimidyl suberate, five specifically labelled bands were seen on SDS-PAGE and autoradiography. Two bands (MW 21.5 K and 25.5 K) were intensely labelled, while the remaining three (MW 37 K, 34 K and 18 K) appeared as minor bands only. Inhibitor bioactivity, followingmore » further analysis by hydrophobic interaction chromatography or Con A-Sepharose affinity chromatography, was always associated with the presence of the 21.5 K and/or 25.5 K bands.« less

Pre-equilibrium competitive library screening for tuning inhibitor association rate and specificity toward serine proteases.

PubMed

Cohen, Itay; Naftaly, Si; Ben-Zeev, Efrat; Hockla, Alexandra; Radisky, Evette S; Papo, Niv

2018-04-16

High structural and sequence similarity within protein families can pose significant challenges to the development of selective inhibitors, especially toward proteolytic enzymes. Such enzymes usually belong to large families of closely similar proteases and may also hydrolyze, with different rates, protein- or peptide-based inhibitors. To address this challenge, we employed a combinatorial yeast surface display library approach complemented with a novel pre-equilibrium, competitive screening strategy for facile assessment of the effects of multiple mutations on inhibitor association rates and binding specificity. As a proof of principle for this combined approach, we utilized this strategy to alter inhibitor/protease association rates and to tailor the selectivity of the amyloid β-protein precursor Kunitz protease inhibitor domain (APPI) for inhibition of the oncogenic protease mesotrypsin, in the presence of three competing serine proteases, anionic trypsin, cationic trypsin and kallikrein-6. We generated a variant, designated APPI P13W/M17G/I18F/F34V , with up to 30-fold greater specificity relative to the parental APPI M17G/I18F/F34V protein, and 6500- to 230 000-fold improved specificity relative to the wild-type APPI protein in the presence of the other proteases tested. A series of molecular docking simulations suggested a mechanism of interaction that supported the biochemical results. These simulations predicted that the selectivity and specificity are affected by the interaction of the mutated APPI residues with nonconserved enzyme residues located in or near the binding site. Our strategy will facilitate a better understanding of the binding landscape of multispecific proteins and will pave the way for design of new drugs and diagnostic tools targeting proteases and other proteins. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Secreted phospholipase A2 of Clonorchis sinensis activates hepatic stellate cells through a pathway involving JNK signalling.

PubMed

Wu, Yinjuan; Li, Ye; Shang, Mei; Jian, Yu; Wang, Caiqin; Bardeesi, Adham Sameer A; Li, Zhaolei; Chen, Tingjin; Zhao, Lu; Zhou, Lina; He, Ai; Huang, Yan; Lv, Zhiyue; Yu, Xinbing; Li, Xuerong

2017-03-16

Secreted phospholipase A2 (sPLA2) is a protein secreted by Clonorchis sinensis and is a component of excretory and secretory products (CsESPs). Phospholipase A2 is well known for its role in liver fibrosis and inhibition of tumour cells. The JNK signalling pathway is involved in hepatic stellate cells (HSCs) activation. Blocking JNK activity with SP600125 inhibits HSCs activation. In a previous study, the protein CssPLA2 was expressed in insoluble inclusion bodies. Therefore, it's necessary to express CssPLA2 in water-soluble form and determine whether the enzymatic activity of CssPLA2 or cell signalling pathways is involved in liver fibrosis caused by clonorchiasis. Balb/C mice were given an abdominal injection of MBP-CssPLA2. Liver sections with HE and Masson staining were observed to detect accumulation of collagen. Western blot of mouse liver was done to detect the activation of JNK signalling pathway. In vitro, HSCs were incubated with MBP-CssPLA2 to detect the activation of HSCs as well as the activation of JNK signalling pathway. The mutant of MBP-CssPLA2 without enzymatic activity was constructed and was also incubated with HSCs to check whether activation of the HSCs was related to the enzymatic activity of MBP-CssPLA2. The recombinant protein MBP-CssPLA2 was expressed soluble and of good enzymatic activity. A mutant of CssPLA2, without enzymatic activity, was also constructed. In vivo liver sections of Balb/C mice that were given an abdominal injection of 50 μg/ml MBP-CssPLA2 showed an obvious accumulation of collagen and a clear band of P-JNK1 could be seen by western blot of the liver tissue. In vitro, MBP-CssPLA2, as well as the mutant, was incubated with HSCs and it was proved that activation of HSCs was related to activation of the JNK signalling pathway instead of the enzymatic activity of MBP-CssPLA2. Activation of HSCs by CssPLA2 is related to the activation of the JNK signalling pathway instead of the enzymatic activity of CssPLA2. This finding

Anti-inflammatory effects of secondary metabolites of marine Pseudomonas sp. in human neutrophils are through inhibiting P38 MAPK, JNK, and calcium pathways.

PubMed

Yang, Shun-Chin; Sung, Ping-Jyun; Lin, Chwan-Fwu; Kuo, Jimmy; Chen, Chun-Yu; Hwang, Tsong-Long

2014-01-01

Activated neutrophils play a significant role in the pathogenesis of many inflammatory diseases. The metabolites of marine microorganisms are increasingly employed as sources for developing new drugs; however, very few marine drugs have been studied in human neutrophils. Herein, we showed that secondary metabolites of marine Pseudomonas sp. (N11) significantly inhibited superoxide anion generation and elastase release in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils, with IC50 values of 0.67±0.38 µg/ml and 0.84±0.12 µg/ml, respectively. In cell-free systems, neither superoxide anion-scavenging effect nor inhibition of elastase activity was associated with the suppressive effects of N11. N11 inhibited the phosphorylation of p38 MAP kinase and JNK, but not Erk and Akt, in FMLP-induced human neutrophils. Also, N11 dose-dependently attenuated the transient elevation of intracellular calcium concentration in activated neutrophils. In contrast, N11 failed to alter phorbol myristate acetate-induced superoxide anion generation, and the inhibitory effects of N11 were not reversed by protein kinase A inhibitor. In conclusion, the anti-inflammatory effects of N11 on superoxide anion generation and elastase release in activated human neutrophils are through inhibiting p38 MAP kinase, JNK, and calcium pathways. Our results suggest that N11 has the potential to be developed to treat neutrophil-mediated inflammatory diseases.

Nicotinamide mononucleotide inhibits JNK activation to reverse Alzheimer disease.

PubMed

Yao, Zhiwen; Yang, Wenhao; Gao, Zhiqiang; Jia, Peng

2017-04-24

Amyloid-β (Aβ) oligomers have been accepted as major neurotoxic agents in the therapy of Alzheimer's disease (AD). It has been shown that the activity of nicotinamide adenine dinucleotide (NAD+) is related with the decline of Aβ toxicity in AD. Nicotinamide mononucleotide (NMN), the important precursor of NAD+, is produced during the reaction of nicotinamide phosphoribosyl transferase (Nampt). This study aimed to figure out the potential therapeutic effects of NMN and its underlying mechanisms in APPswe/PS1dE9 (AD-Tg) mice. We found that NMN gave rise to a substantial improvement in behavioral measures of cognitive impairments compared to control AD-Tg mice. In addition, NMN treatment significantly decreased β-amyloid production, amyloid plaque burden, synaptic loss, and inflammatory responses in transgenic animals. Mechanistically, NMN effectively controlled JNK activation. Furthermore, NMN potently progressed nonamyloidogenic amyloid precursor protein (APP) and suppressed amyloidogenic APP by mediating the expression of APP cleavage secretase in AD-Tg mice. Based on our findings, it was suggested that NMN substantially decreases multiple AD-associated pathological characteristically at least partially by the inhibition of JNK activation. Copyright © 2017 Elsevier B.V. All rights reserved.

Bruton's tyrosine kinase regulates B cell antigen receptor-mediated JNK1 response through Rac1 and phospholipase C-gamma2 activation.

PubMed

Inabe, Kazunori; Miyawaki, Toshio; Longnecker, Richard; Matsukura, Hiroyoshi; Tsukada, Satoshi; Kurosaki, Tomohiro

2002-03-13

Bruton's tyrosine kinase (Btk) is essential for B cell development and B cell antigen receptor (BCR) function. Recent studies have shown that Btk plays an important role in BCR-mediated c-Jun NH(2)-terminal kinase (JNK) 1 activation; however, the mechanism by which Btk participates in the JNK1 response remains elusive. Here we show that the BCR-mediated Rac1 activation is significantly inhibited by loss of Btk, while this Rac1 activation is not affected by loss of phospholipase C-gamma2 (PLC-gamma2). Since PLC-gamma2 is also required for BCR-mediated JNK1 response, our results suggest that Btk regulates Rac1 pathway as well as PLC-gamma2 pathway, both of which contribute to the BCR-mediated JNK1 response.

Identification and activity of inhibitors of the essential nematode-specific metalloprotease DPY-31.

PubMed

France, David J; Stepek, Gillian; Houston, Douglas R; Williams, Lewis; McCormack, Gillian; Walkinshaw, Malcolm D; Page, Antony P

2015-12-15

Infection by parasitic nematodes is widespread in the developing world causing extensive morbidity and mortality. Furthermore, infection of animals is a global problem, with a substantial impact on food production. Here we identify small molecule inhibitors of a nematode-specific metalloprotease, DPY-31, using both known metalloprotease inhibitors and virtual screening. This strategy successfully identified several μM inhibitors of DPY-31 from both the human filarial nematode Brugia malayi, and the parasitic gastrointestinal nematode of sheep Teladorsagia circumcincta. Further studies using both free living and parasitic nematodes show that these inhibitors elicit the severe body morphology defect 'Dumpy' (Dpy; shorter and fatter), a predominantly non-viable phenotype consistent with mutants lacking the DPY-31 gene. Taken together, these results represent a start point in developing DPY-31 inhibition as a totally novel mechanism for treating infection by parasitic nematodes in humans and animals. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Distinct role of p38 and c-Jun N-terminal kinases in IL-10-dependent and IL-10-independent regulation of the costimulatory molecule B7.2 in lipopolysaccharide-stimulated human monocytic cells.

PubMed

Lim, Wilfred; Ma, Wei; Gee, Katrina; Aucoin, Susan; Nandan, Devki; Diaz-Mitoma, Francisco; Kozlowski, Maya; Kumar, Ashok

2002-02-15

The costimulatory molecule B7.2 (CD86) plays a vital role in immune activation and development of Th responses. The molecular mechanisms by which B7.2 expression is regulated are not understood. We investigated the role of mitogen-activated protein kinases (MAPK) in the regulation of B7.2 expression in LPS-stimulated human monocytic cells. LPS stimulation of human monocytes resulted in the down-regulation of B7.2 expression that could be abrogated by anti-IL-10 Abs. Furthermore, SB202190, a specific inhibitor of p38 MAPK, inhibited LPS-induced IL-10 production and reversed B7.2 down-regulation, suggesting that LPS-induced B7.2 down-regulation may be mediated, at least in part, via regulation of IL-10 production by p38 MAPK. In contrast to human promonocytic THP-1 cells that are refractory to the inhibitory effects of IL-10, LPS stimulation enhanced B7.2 expression. This IL-10-independent B7.2 induction was not influenced by specific inhibitors of either p38 or p42/44 MAPK. To ascertain the role of the c-Jun N-terminal kinase (JNK) MAPK, dexamethasone, an inhibitor of JNK activation, was used, which inhibited LPS-induced B7.2 expression. Transfection of THP-1 cells with a plasmid expressing a dominant-negative stress-activated protein/extracellular signal-regulated kinase kinase 1 significantly reduced LPS-induced B7.2 expression, thus confirming the involvement of JNK. To study the signaling events downstream of JNK activation, we show that dexamethasone did not inhibit LPS-induced NF-kappaB activation in THP-1 cells, suggesting that JNK may not be involved in NF-kappaB activation leading to B7.2 expression. Taken together, our results reveal the distinct involvement of p38 in IL-10-dependent, and JNK in IL-10-independent regulation of B7.2 expression in LPS-stimulated monocytic cells.

Identification of tyrosinase specific inhibitors from Xanthium strumarium fruit extract using ultrafiltration-high performance liquid chromatography.

PubMed

Wang, Zhiqiang; Hwang, Seung Hwan; Huang, Bo; Lim, Soon Sung

2015-10-01

In this study, a strategy based on ultrafiltration-high performance liquid chromatography coupled with diode array detection (UF-HPLC-DAD) was proposed for screening tyrosinase specific inhibitors in Xanthii fructus. The false negatives were distinguished by optimizing the UF-HPLC-DAD parameters to reduce the background noise; the false positives were distinguished by introducing a blocked tyrosinase in the control group for comparison. To obtain the best blocker, the competitive experiments were performed using various known ligands. Using this strategy, three competitive inhibitors (protocatechuic acid; 3,5-di-O-caffeoylquinic acid; and 1,5-di-O-caffeoylquinic acid) and one mixed-type inhibitor (chlorogenic acid) were identified. These results were verified using tyrosinase inhibition assay, kinetic analysis, and structural simulation of the complex. Our experimental results suggest that the proposed strategy could be useful for high-throughput identification of tyrosinase specific inhibitors in natural products. Copyright © 2015 Elsevier B.V. All rights reserved.

Curcumin suppresses JNK pathway to attenuate BPA-induced insulin resistance in LO2 cells.

PubMed

Geng, Shanshan; Wang, Shijia; Zhu, Weiwei; Xie, Chunfeng; Li, Xiaoting; Wu, Jieshu; Zhu, Jianyun; Jiang, Ye; Yang, Xue; Li, Yuan; Chen, Yue; Wang, Xiaoqian; Meng, Yu; Zhong, Caiyun

2018-01-01

To examine whether curcumin has protective effect on insulin resistance induced by bisphenol A (BPA) in LO2 cells and whether this effect was mediated by inhibiting the inflammatory mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) pathways. LO2 cells were stimulated with BPA in the presence or absence of curcumin for 5 days. Glucose consumption, activation of insulin signaling, MAPKs and NF-κB pathways, levels of inflammatory cytokines and MDA production were analyzed. Curcumin prevented BPA-induced reduction of glucose consumption and suppression of insulin signaling pathway, indicating curcumin alleviated BPA-triggered insulin resistance in LO2 cells. mRNA and proteins levels of TNF-α and IL-6, as well as MDA level in LO2 cells treated with BPA were decreased by curcumin. Furthermore, curcumin downregulated the activation of p38, JNK, and NF-κB pathways upon stimulation with BPA. Inhibition of JNK pathway, but not p38 nor NF-κB pathway, improved glucose consumption and insulin signaling in BPA-treated LO2 cells. Curcumin inhibits BPA-induced insulin resistance by suppressing JNK pathway. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Efficiency and specificity of RAAS inhibitors in cardiovascular diseases: how to achieve better end-organ protection?

PubMed

Nehme, Ali; Zibara, Kazem

2017-11-01

RAAS, a major pharmacological target in cardiovascular medicine, is inhibited by pharmacological classes including angiotensin converting enzyme (ACE) inhibitors (ACEIs), angiotensin-II type 1 blockers (ARBs) and aldosterone receptors antagonists, in addition to the recently introduced direct renin inhibitors (DRIs). However, currently used RAAS inhibitors still cannot achieve their desired effects and are associated with certain drawbacks, such as adverse side effects, incomplete blockage of the system and poor end-organ protection. In this review, we discuss the efficiency and specificity of the current RAAS inhibitors and propose some recommendations for achieving better treatments with better end-organ protection.

A macrophage NBR1-MEKK3 complex triggers JNK-mediated adipose-tissue inflammation in obesity

PubMed Central

Hernandez, Eloy D.; Lee, Sang Jun; Kim, Ji Young; Duran, Angeles; Linares, Juan F.; Yajima, Tomoko; Müller, Timo D.; Tschöp, Matthias H.; Smith, Steven R.; Diaz-Meco, Maria T.; Moscat, Jorge

2014-01-01

SUMMARY The c-Jun NH(2)-terminal kinase (JNK) is a critical determinant of obesity-associated inflammation and glucose intolerance. The upstream mechanisms controlling this pathway are still unknown. Here we report that the levels of the PB1 domain-containing adapter NBR1 correlated with the expression of pro-inflammatory molecules in adipose tissue from human patients with metabolic syndrome, suggesting that NBR1 plays a key role in adipose-tissue inflammation. We also show that NBR1 inactivation in the myeloid compartment impairs the function, M1 polarization and chemotactic activity of macrophages, prevents inflammation of adipose tissue, and improves glucose tolerance in obese mice. Furthermore, we demonstrate that an interaction between the PB1 domains of NBR1 and the mitogen-activated kinase kinase 3 (MEKK3) enables the formation of a signaling complex required for the activation of JNK. Together these discoveries identify an NBR1-MEKK3 complex as a key regulator of JNK signaling and adipose-tissue inflammation in obesity. PMID:25043814

Activation of c-jun N-terminal kinase upon influenza A virus (IAV) infection is independent of pathogen-related receptors but dependent on amino acid sequence variations of IAV NS1.

PubMed

Nacken, Wolfgang; Anhlan, Darisuren; Hrincius, Eike R; Mostafa, Ahmed; Wolff, Thorsten; Sadewasser, Anne; Pleschka, Stephan; Ehrhardt, Christina; Ludwig, Stephan

2014-08-01

A hallmark cell response to influenza A virus (IAV) infections is the phosphorylation and activation of c-jun N-terminal kinase (JNK). However, so far it is not fully clear which molecules are involved in the activation of JNK upon IAV infection. Here, we report that the transfection of influenza viral-RNA induces JNK in a retinoic acid-inducible gene I (RIG-I)-dependent manner. However, neither RIG-I-like receptors nor MyD88-dependent Toll-like receptors were found to be involved in the activation of JNK upon IAV infection. Viral JNK activation may be blocked by addition of cycloheximide and heat shock protein inhibitors during infection, suggesting that the expression of an IAV-encoded protein is responsible for JNK activation. Indeed, the overexpression of nonstructural protein 1 (NS1) of certain IAV subtypes activated JNK, whereas those of some other subtypes failed to activate JNK. Site-directed mutagenesis experiments using NS1 of the IAV H7N7, H5N1, and H3N2 subtypes identified the amino acid residue phenylalanine (F) at position 103 to be decisive for JNK activation. Cleavage- and polyadenylation-specific factor 30 (CPSF30), whose binding to NS1 is stabilized by the amino acids F103 and M106, is not involved in JNK activation. Conclusively, subtype-specific sequence variations in the IAV NS1 protein result in subtype-specific differences in JNK signaling upon IAV infection. Influenza A virus (IAV) infection leads to the activation or modulation of multiple signaling pathways. Here, we demonstrate for the first time that the c-jun N-terminal kinase (JNK), a long-known stress-activated mitogen-activated protein (MAP) kinase, is activated by RIG-I when cells are treated with IAV RNA. However, at the same time, nonstructural protein 1 (NS1) of IAV has an intrinsic JNK-activating property that is dependent on IAV subtype-specific amino acid variations around position 103. Our findings identify two different and independent pathways that result in the activation

Activation of c-jun N-Terminal Kinase upon Influenza A Virus (IAV) Infection Is Independent of Pathogen-Related Receptors but Dependent on Amino Acid Sequence Variations of IAV NS1

PubMed Central

Nacken, Wolfgang; Anhlan, Darisuren; Hrincius, Eike R.; Mostafa, Ahmed; Wolff, Thorsten; Sadewasser, Anne; Pleschka, Stephan; Ehrhardt, Christina

2014-01-01

ABSTRACT A hallmark cell response to influenza A virus (IAV) infections is the phosphorylation and activation of c-jun N-terminal kinase (JNK). However, so far it is not fully clear which molecules are involved in the activation of JNK upon IAV infection. Here, we report that the transfection of influenza viral-RNA induces JNK in a retinoic acid-inducible gene I (RIG-I)-dependent manner. However, neither RIG-I-like receptors nor MyD88-dependent Toll-like receptors were found to be involved in the activation of JNK upon IAV infection. Viral JNK activation may be blocked by addition of cycloheximide and heat shock protein inhibitors during infection, suggesting that the expression of an IAV-encoded protein is responsible for JNK activation. Indeed, the overexpression of nonstructural protein 1 (NS1) of certain IAV subtypes activated JNK, whereas those of some other subtypes failed to activate JNK. Site-directed mutagenesis experiments using NS1 of the IAV H7N7, H5N1, and H3N2 subtypes identified the amino acid residue phenylalanine (F) at position 103 to be decisive for JNK activation. Cleavage- and polyadenylation-specific factor 30 (CPSF30), whose binding to NS1 is stabilized by the amino acids F103 and M106, is not involved in JNK activation. Conclusively, subtype-specific sequence variations in the IAV NS1 protein result in subtype-specific differences in JNK signaling upon IAV infection. IMPORTANCE Influenza A virus (IAV) infection leads to the activation or modulation of multiple signaling pathways. Here, we demonstrate for the first time that the c-jun N-terminal kinase (JNK), a long-known stress-activated mitogen-activated protein (MAP) kinase, is activated by RIG-I when cells are treated with IAV RNA. However, at the same time, nonstructural protein 1 (NS1) of IAV has an intrinsic JNK-activating property that is dependent on IAV subtype-specific amino acid variations around position 103. Our findings identify two different and independent pathways that

bFGF Promotes the Migration of Human Dermal Fibroblasts under Diabetic Conditions through Reactive Oxygen Species Production via the PI3K/Akt-Rac1- JNK Pathways

PubMed Central

Shi, Hongxue; Cheng, Yi; Ye, Jingjing; Cai, Pingtao; Zhang, Jinjing; Li, Rui; Yang, Ying; Wang, Zhouguang; Zhang, Hongyu; Lin, Cai; Lu, Xianghong; Jiang, Liping; Hu, Aiping; Zhu, Xinbo; Zeng, Qiqiang; Fu, Xiaobing; Li, Xiaokun; Xiao, Jian

2015-01-01

Fibroblasts play a pivotal role in the process of cutaneous wound repair, whereas their migratory ability under diabetic conditions is markedly reduced. In this study, we investigated the effect of basic fibroblast growth factor (bFGF) on human dermal fibroblast migration in a high-glucose environment. bFGF significantly increased dermal fibroblast migration by increasing the percentage of fibroblasts with a high polarity index and reorganizing F-actin. A significant increase in intracellular reactive oxygen species (ROS) was observed in dermal fibroblasts under diabetic conditions following bFGF treatment. The blockage of bFGF-induced ROS production by either the ROS scavenger N-acetyl-L-cysteine (NAC) or the NADPH oxidase inhibitor diphenylene iodonium chloride (DPI) almost completely neutralized the increased migration rate of dermal fibroblasts promoted by bFGF. Akt, Rac1 and JNK were rapidly activated by bFGF in dermal fibroblasts, and bFGF-induced ROS production and promoted dermal fibroblast migration were significantly attenuated when suppressed respectively. In addition, bFGF-induced increase in ROS production was indispensable for the activation of focal adhesion kinase (FAK) and paxillin. Therefore, our data suggested that bFGF promotes the migration of human dermal fibroblasts under diabetic conditions through increased ROS production via the PI3K/Akt-Rac1-JNK pathways. PMID:26078726

Aldosterone induces clonal β-cell failure through glucocorticoid receptor

PubMed Central

Chen, Fang; Liu, Jia; Wang, Yanyang; Wu, Tijun; Shan, Wei; Zhu, Yunxia; Han, Xiao

2015-01-01

Aldosterone excess causes insulin resistance in peripheral tissues and directly impairs the function of clonal β-cell. The aim of this study was to investigate the molecular mechanisms involved in the aldosterone-induced impairment of clonal β-cells. As expected, aldosterone induced apoptosis and β-cell dysfunction, including impairment of insulin synthesis and secretion, which were reversed by Glucocorticoid receptor (GR) antagonists or GR-specific siRNA. However, mineralocorticoid receptor (MR) antagonists or MR-specific siRNA had no effect on impairment of clonal β-cells induced by aldosterone. Besides, aldosterone significantly decreased expression and activity of MafA, while activated JNK and p38 MAPK in a GR-dependent manner. In addition, JNK inhibitors (SP600125) and/or p38 inhibitors (SB203580) could abolish the effect of aldosterone on MafA expression and activity. Importantly, overexpression of JNK1 or p38 reversed the protective effect of a GR antagonist on the decrease of MafA expression and activity. Furthermore, aldosterone inhibits MafA expression at the transcriptional and post-transcriptional level through activation of JNK and p38, respectively. Consequently, overexpression of MafA increased synthesis and secretion of insulin, and decreased apoptosis in clonal β-cells exposed to aldosterone. These findings identified aldosterone as an inducer of clonal β-cell failure that operates through the GR-MAPK-MafA signaling pathway. PMID:26287126

Activation of the JNK pathway is essential for transformation by the Met oncogene.

PubMed

Rodrigues, G A; Park, M; Schlessinger, J

1997-05-15

The Met/Hepatocyte Growth Factor (HGF) receptor tyrosine kinase is oncogenically activated through a rearrangement that creates a hybrid gene Tpr-Met. The resultant chimeric p65(Tpr-Met) protein is constitutively phosphorylated on tyrosine residues in vivo and associates with a number of SH2-containing signaling molecules including the p85 subunit of PI-3 kinase and the Grb2 adaptor protein, which couples receptor tyrosine kinases to the Ras signaling pathway. Mutation of the binding site for Grb2 impairs the ability of Tpr-Met oncoprotein to transform fibroblasts, suggesting that the activation of the Ras/MAP kinase signaling pathway through Grb2 may be essential for cellular transformation. To test this hypothesis dominant-negative mutants of Grb2 with deletions of the SH3 domains were introduced into Tpr-Met transformed fibroblasts. Cells overexpressing the mutants were found to be morphologically reverted and exhibited reduced growth in soft agar. Surprisingly, the Grb2 mutants blocked activation of the JNK/SAPK but not MAP kinase activity induced by the Tpr-Met oncoprotein. Additionally, cells expressing dominant-negative Grb2 mutants had reduced PI-3-kinase activity and dominant-negative mutants of Rac1 blocked both Tpr-Met-induced transformation and activation of JNK. These experiments reveal a novel link between Met and the JNK pathway, which is essential for transformation by this oncogene.

Galectin-9 exhibits anti-myeloma activity through JNK and p38 MAP kinase pathways.

PubMed

Kobayashi, T; Kuroda, J; Ashihara, E; Oomizu, S; Terui, Y; Taniyama, A; Adachi, S; Takagi, T; Yamamoto, M; Sasaki, N; Horiike, S; Hatake, K; Yamauchi, A; Hirashima, M; Taniwaki, M

2010-04-01

Galectins constitute a family of lectins that specifically exhibit the affinity for beta-galactosides and modulate various biological events. Galectin-9 is a tandem-repeat type galectin with two carbohydrate recognition domains and has recently been shown to have an anti-proliferative effect on cancer cells. We investigated the effect of recombinant protease-resistant galectin-9 (hGal9) on multiple myeloma (MM). In vitro, hGal9 inhibited the cell proliferation of five myeloma cell lines examined, including a bortezomib-resistant subcell line, with IC(50) between 75.1 and 280.0 nM, and this effect was mediated by the induction of apoptosis with the activation of caspase-8, -9, and -3. hGal9-activated Jun NH(2)-terminal kinase (JNK) and p38 MAPK signaling pathways followed by H2AX phosphorylation. Importantly, the inhibition of either JNK or p38 MAPK partly inhibited the anti-proliferative effect of hGal9, indicating the crucial role of these pathways in the anti-MM effect of hGal9. hGal9 also induced cell death in patient-derived myeloma cells, some with poor-risk factors, such as chromosomal deletion of 13q or translocation t(4;14)(p16;q32). Finally, hGal9 potently inhibited the growth of human myeloma cells xenografted in nude mice. These suggest that hGal9 is a new therapeutic target for MM that may overcome resistance to conventional chemotherapy.

NFκB inhibitors induce cell death in glioblastomas.

PubMed

Zanotto-Filho, Alfeu; Braganhol, Elizandra; Schröder, Rafael; de Souza, Luís Henrique T; Dalmolin, Rodrigo J S; Pasquali, Matheus A Bittencourt; Gelain, Daniel Pens; Battastini, Ana Maria Oliveira; Moreira, José Cláudio Fonseca

2011-02-01

Identification of novel target pathways in glioblastoma (GBM) remains critical due to poor prognosis, inefficient therapies and recurrence associated with these tumors. In this work, we evaluated the role of nuclear-factor-kappa-B (NFκB) in the growth of GBM cells, and the potential of NFκB inhibitors as antiglioma agents. NFκB pathway was found overstimulated in GBM cell lines and in tumor specimens compared to normal astrocytes and healthy brain tissues, respectively. Treatment of a panel of established GBM cell lines (U138MG, U87, U373 and C6) with pharmacological NFκB inhibitors (BAY117082, parthenolide, MG132, curcumin and arsenic trioxide) and NFκB-p65 siRNA markedly decreased the viability of GBMs as compared to inhibitors of other signaling pathways such as MAPKs (ERK, JNK and p38), PKC, EGFR and PI3K/Akt. In addition, NFκB inhibitors presented a low toxicity to normal astrocytes, indicating selectivity to cancerous cells. In GBMs, mitochondrial dysfunction (membrane depolarization, bcl-xL downregulation and cytochrome c release) and arrest in the G2/M phase were observed at the early steps of NFκB inhibitors treatment. These events preceded sub-G1 detection, apoptotic body formation and caspase-3 activation. Also, NFκB was found overstimulated in cisplatin-resistant C6 cells, and treatment of GBMs with NFκB inhibitors overcame cisplatin resistance besides potentiating the effects of the chemotherapeutics, cisplatin and doxorubicin. These findings support NFκB as a potential target to cell death induction in GBMs, and that the NFκB inhibitors may be considered for in vivo testing on animal models and possibly on GBM therapy. Copyright © 2010 Elsevier Inc. All rights reserved.

Bis-Aryl Urea Derivatives as Potent and Selective LIM Kinase (Limk) Inhibitors

PubMed Central

Yin, Yan; Zheng, Ke; Eid, Nibal; Howard, Shannon; Jeong, Ji-Hak; Yi, Fei; Guo, Jia; Park, Chul M; Bibian, Mathieu; Wu, Weilin; Hernandez, Pamela; Park, HaJeung; Wu, Yuntao; Luo, Jun-Li; LoGrasso, Philip V.; Feng, Yangbo

2015-01-01

The discovery/optimization of bis-aryl ureas as Limk inhibitors to obtain high potency and selectivity, and appropriate pharmacokinetic properties through systematic SAR studies is reported. Docking studies supported the observed SAR. Optimized Limk inhibitors had high biochemical potency (IC50 < 25 nM), excellent selectivity against ROCK and JNK kinases (> 400-fold), potent inhibition of cofilin phosphorylation in A7r5,PC-3, and CEM-SS T cells (IC50 < 1 μM), and good in vitro and in vivo pharmacokinetic properties. In the profiling against a panel of 61 kinases, compound 18b at 1 μM inhibited only Limk1 and STK16 with ≥ 80% inhibition. Compounds 18b and 18f were highly efficient in inhibiting cell-invasion/migration in PC-3 cells. In addition, compound 18w was demonstrated to be effective on reducing intraocular pressure (IOP) on rat eyes. Taken together, these data demonstrated that we had developed a novel class of bis-aryl urea derived potent and selective Limk inhibitors. PMID:25621531

Regulation of human glioblastoma cell death by combined treatment of cannabidiol, γ-radiation and small molecule inhibitors of cell signaling pathways

PubMed Central

Ivanov, Vladimir N.; Wu, Jinhua; Hei, Tom K.

2017-01-01

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. The challenging problem in cancer treatment is to find a way to upregulate radiosensitivity of GBM while protecting neurons and neural stem/progenitor cells in the brain. The goal of the present study was upregulation of the cytotoxic effect of γ-irradiation in GBM by non-psychotropic and non-toxic cannabinoid, cannabidiol (CBD). We emphasized three main aspects of signaling mechanisms induced by CBD treatment (alone or in combination with γ-irradiation) in human GBM that govern cell death: 1) CBD significantly upregulated the active (phosphorylated) JNK1/2 and MAPK p38 levels with the subsequent downregulation of the active phospho-ERK1/2 and phospho-AKT1 levels. MAPK p38 was one of the main drivers of CBD-induced cell death, while death levels after combined treatment of CBD and radiation were dependent on both MAPK p38 and JNK. Both MAPK p38 and JNK regulate the endogenous TRAIL expression. 2) NF-κB p65-P(Ser536) was not the main target of CBD treatment and this transcription factor was found at high levels in CBD-treated GBM cells. Additional suppression of p65-P(Ser536) levels using specific small molecule inhibitors significantly increased CBD-induced apoptosis. 3) CBD treatment substantially upregulated TNF/TNFR1 and TRAIL/TRAIL-R2 signaling by modulation of both ligand and receptor levels followed by apoptosis. Our results demonstrate that radiation-induced death in GBM could be enhanced by CBD-mediated signaling in concert with its marginal effects for neural stem/progenitor cells and astrocytes. It will allow selecting efficient targets for sensitization of GBM and overcoming cancer therapy-induced severe adverse sequelae. PMID:29088769

Differentiation and upregulation of heat shock protein 70 induced by a subset of histone deacetylase inhibitors in mouse and human embryonic stem cells.

PubMed

Park, Jeong-A; Kim, Young-Eun; Seok, Hyun-Jeong; Park, Woo-Youn; Kwon, Hyung-Joo; Lee, Younghee

2011-03-01

Inhibiting histone deacetylase (HDAC) activity modulates the epigenetic status of cells, resulting in an alteration of gene expression and cellular function. Here, we investigated the effects of HDAC inhibitors on mouse embryonic stem (ES) cells. The HDAC inhibitors trichostatin A, suberoylanilide hydroxamic acid, sodium butyrate, and valproic acid induced early differentiation of mouse ES cells and triggered induction of heatshock protein (HSP)70. In contrast, class III HDAC inhibitors failed to induce differentiation or HSP70 expression. Transcriptional upregulation of HSP70 was confirmed by mRNA expression analysis, an inhibitor study, and chromatin immunoprecipitation. HSP70 induction was dependent on the SAPK/ JNK, p38, and PI3K/Akt pathways. Differentiation and induction of HSP70 by a subset of HDAC inhibitors was also examined in human ES cells, which suggests that the phenomenon generally occurs in ES cells. A better understanding of the effects of HDAC inhibitors may give more insight into their application in stem cell biology.

N-glycans of Human Protein C Inhibitor: Tissue-Specific Expression and Function

PubMed Central

Engström, Åke; Sooriyaarachchi, Sanjeewani; Ubhayasekera, Wimal; Hreinsson, Julius; Wånggren, Kjell; Clark, Gary F.; Dell, Anne; Schedin-Weiss, Sophia

2011-01-01

Protein C inhibitor (PCI) is a serpin type of serine protease inhibitor that is found in many tissues and fluids in human, including blood plasma, seminal plasma and urine. This inhibitor displays an unusually broad protease specificity compared with other serpins. Previous studies have shown that the N-glycan(s) and the NH2-terminus affect some blood-related functions of PCI. In this study, we have for the first time determined the N-glycan profile of seminal plasma PCI, by mass spectrometry. The N-glycan structures differed markedly compared with those of both blood-derived and urinary PCI, providing evidence that the N-glycans of PCI are expressed in a tissue-specific manner. The most abundant structure (m/z 2592.9) had a composition of Fuc3Hex5HexNAc4, consistent with a core fucosylated bi-antennary glycan with terminal Lewisx. A major serine protease in semen, prostate specific antigen (PSA), was used to evaluate the effects of N-glycans and the NH2-terminus on a PCI function related to the reproductive tract. Second-order rate constants for PSA inhibition by PCI were 4.3±0.2 and 4.1±0.5 M−1s−1 for the natural full-length PCI and a form lacking six amino acids at the NH2-terminus, respectively, whereas these constants were 4.8±0.1 and 29±7 M−1s−1 for the corresponding PNGase F-treated forms. The 7–8-fold higher rate constants obtained when both the N-glycans and the NH2-terminus had been removed suggest that these structures jointly affect the rate of PSA inhibition, presumably by together hindering conformational changes of PCI required to bind to the catalytic pocket of PSA. PMID:22205989

Akt mediates 17beta-estradiol and/or estrogen receptor-alpha inhibition of LPS-induced tumor necresis factor-alpha expression and myocardial cell apoptosis by suppressing the JNK1/2-NFkappaB pathway.

PubMed

Liu, Chung-Jung; Lo, Jeng-Fan; Kuo, Chia-Hua; Chu, Chun-Hsien; Chen, Li-Ming; Tsai, Fuu-Jen; Tsai, Chang-Hai; Tzang, Bor-Show; Kuo, Wei-Wen; Huang, Chih-Yang

2009-09-01

Evidence shows that women have lower tumour necrosis factor-alpha (TNF-alpha) levels and lower incidences of heart dysfunction and sepsis-related morbidity and mortality. To identify the cardioprotective effects and precise cellular/molecular mechanisms behind estrogen and estrogen receptors (ERs), we investigated the effects of 17beta-estradiol (E(2)) and estrogen receptor alpha (ERalpha) on LPS-induced apoptosis by analyzing the activation of survival and death signalling pathways in doxycycline (Dox)-inducible Tet-On/ERalpha H9c2 myocardial cells and ERalpha-transfected primary cardiomyocytes overexpressing ERalpha. We found that LPS challenge activated JNK1/2, and then induced IkappaB degradation, NFkappaB activation, TNF-alpha up-regulation and subsequent myocardial apoptotic responses. In addition, treatments involving E(2), membrane-impermeable BSA-E(2) and/or Dox, which induces ERalpha overexpression, significantly inhibited LPS-induced apoptosis by suppressing LPS-up-regulated JNK1/2 activity, IkappaB degradation, NFkappaB activation and pro-apoptotic proteins (e.g. TNF-alpha, active caspases-8, t-Bid, Bax, released cytochrome c, active caspase-9, active caspase-3) in myocardial cells. However, the cardioprotective properties of E(2), BSA-E(2) and ERalpha overexpression to inhibit LPS-induced apoptosis and promote cell survival were attenuated by applying LY294002 (PI3K inhibitor) and PI3K siRNA. These findings suggest that E(2), BSA-E(2) and ERalpha expression exert their cardioprotective effects by inhibiting JNK1/2-mediated LPS-induced TNF-alpha expression and cardiomyocyte apoptosis through activation of Akt.

Inactivation of JNK1 enhances innate IL-10 production and dampens autoimmune inflammation in the brain.

PubMed

Tran, Elise H; Azuma, Yasu-Taka; Chen, Manchuan; Weston, Claire; Davis, Roger J; Flavell, Richard A

2006-09-05

Environmental insults such as microbial pathogens can contribute to the activation of autoreactive T cells, leading to inflammation of target organs and, ultimately, autoimmune disease. Various infections have been linked to multiple sclerosis and its animal counterpart, autoimmune encephalomyelitis. The molecular process by which innate immunity triggers autoreactivity is not currently understood. By using a mouse model of multiple sclerosis, we found that the genetic loss of the MAPK, c-Jun N-terminal kinase 1 (JNK1), enhances IL-10 production, rendering innate myeloid cells unresponsive to certain microbes and less capable of generating IL-17-producing, encephalitogenic T cells. Moreover, JNK1-deficient central nervous system myeloid cells are unable to respond to effector T cell inflammatory cytokines, preventing further progression to neuroinflammation. Thus, we have identified the JNK1 signal transduction pathway in myeloid cells to be a critical component of a regulatory circuit mediating inflammatory responses in autoimmune disease. Our findings provide further insights into the pivotal MAPK-regulated network of innate and adaptive cytokines in the progression to autoimmunity.

E3 ubiquitin ligase Mule ubiquitinates Miz1 and is required for TNFalpha-induced JNK activation.

PubMed

Yang, Yi; Do, HanhChi; Tian, Xuejun; Zhang, Chaozheng; Liu, Xinyuan; Dada, Laura A; Sznajder, Jacob I; Liu, Jing

2010-07-27

The zinc finger transcription factor Miz1 is a negative regulator of TNFalpha-induced JNK activation and cell death through inhibition of TRAF2 K63-polyubiquitination in a transcription-independent manner. Upon TNFalpha stimulation, Miz1 undergoes K48-linked polyubiquitination and proteasomal degradation, thereby relieving its inhibition. However, the underling regulatory mechanism is not known. Here, we report that HECT-domain-containing Mule is the E3 ligase that catalyzes TNFalpha-induced Miz1 polyubiquitination. Mule is a Miz1-associated protein and catalyzes its K48-linked polyubiquitination. TNFalpha-induced polyubiquitination and degradation of Miz1 were inhibited by silencing of Mule and were promoted by ectopic expression of Mule. The interaction between Mule and Miz1 was promoted by TNFalpha independently of the pox virus and zinc finger domain of Miz1. Silencing of Mule stabilized Miz1, thereby suppressing TNFalpha-induced JNK activation and cell death. Thus, our study reveals a molecular mechanism by which Mule regulates TNFalpha-induced JNK activation and apoptosis by catalyzing the polyubiquitination of Miz1.

The integrin effector PINCH regulates JNK activity and epithelial migration in concert with Ras suppressor 1

PubMed Central

Kadrmas, Julie L.; Smith, Mark A.; Clark, Kathleen A.; Pronovost, Stephen M.; Muster, Nemone; Yates, John R.; Beckerle, Mary C.

2004-01-01

Cell adhesion and migration are dynamic processes requiring the coordinated action of multiple signaling pathways, but the mechanisms underlying signal integration have remained elusive. Drosophila embryonic dorsal closure (DC) requires both integrin function and c-Jun amino-terminal kinase (JNK) signaling for opposed epithelial sheets to migrate, meet, and suture. Here, we show that PINCH, a protein required for integrin-dependent cell adhesion and actin–membrane anchorage, is present at the leading edge of these migrating epithelia and is required for DC. By analysis of native protein complexes, we identify RSU-1, a regulator of Ras signaling in mammalian cells, as a novel PINCH binding partner that contributes to PINCH stability. Mutation of the gene encoding RSU-1 results in wing blistering in Drosophila, demonstrating its role in integrin-dependent cell adhesion. Genetic interaction analyses reveal that both PINCH and RSU-1 antagonize JNK signaling during DC. Our results suggest that PINCH and RSU-1 contribute to the integration of JNK and integrin functions during Drosophila development. PMID:15596544

Naringenin prevents experimental liver fibrosis by blocking TGFβ-Smad3 and JNK-Smad3 pathways.

PubMed

Hernández-Aquino, Erika; Zarco, Natanael; Casas-Grajales, Sael; Ramos-Tovar, Erika; Flores-Beltrán, Rosa E; Arauz, Jonathan; Shibayama, Mineko; Favari, Liliana; Tsutsumi, Víctor; Segovia, José; Muriel, Pablo

2017-06-28

prevent NF-κB activation and the subsequent production of IL-1 and IL-10 ( P < 0.05). NAR completely prevented the increase in TGF-β, α-SMA, CTGF, Col-1, and MMP-13 proteins compared with the CCl 4 -treated group ( P < 0.05). NAR prevented Smad3 phosphorylation in the linker region by JNK since this flavonoid blocked this kinase ( P < 0.05). NAR prevents CCl 4 induced liver inflammation, necrosis and fibrosis, due to its antioxidant capacity as a free radical inhibitor and by inhibiting the NF-κB, TGF-β-Smad3 and JNK-Smad3 pathways.

Naringenin prevents experimental liver fibrosis by blocking TGFβ-Smad3 and JNK-Smad3 pathways

PubMed Central

Hernández-Aquino, Erika; Zarco, Natanael; Casas-Grajales, Sael; Ramos-Tovar, Erika; Flores-Beltrán, Rosa E; Arauz, Jonathan; Shibayama, Mineko; Favari, Liliana; Tsutsumi, Víctor; Segovia, José; Muriel, Pablo

2017-01-01

to prevent NF-κB activation and the subsequent production of IL-1 and IL-10 (P < 0.05). NAR completely prevented the increase in TGF-β, α-SMA, CTGF, Col-1, and MMP-13 proteins compared with the CCl4-treated group (P < 0.05). NAR prevented Smad3 phosphorylation in the linker region by JNK since this flavonoid blocked this kinase (P < 0.05). CONCLUSION NAR prevents CCl4 induced liver inflammation, necrosis and fibrosis, due to its antioxidant capacity as a free radical inhibitor and by inhibiting the NF-κB, TGF-β-Smad3 and JNK-Smad3 pathways. PMID:28706418

Development of Tethered Hsp90 Inhibitors Carrying Radioiodinated Probes to Specifically Discriminate and Kill Malignant Breast Tumor Cells

DTIC Science & Technology

2016-05-01

AWARD NUMBER: W81XWH-15-1-0072 TITLE: Development of Tethered Hsp90 Inhibitors Carrying Radioiodinated Probes to Specifically Discriminate and...1 May 2015 - 30 Apr 2016 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Development of Tethered Hsp90 Inhibitors Carrying Radioiodinated Probes to...inhibitor capable of carrying various radioactive iodine isotopes for early detection and ablation of metastatic breast cancers. These probes

Phosphorylation of insulin receptor substrate-1 serine 307 correlates with JNK activity in atrophic skeletal muscle

NASA Technical Reports Server (NTRS)

Hilder, Thomas L.; Tou, Janet C L.; Grindeland, Richard E.; Wade, Charles E.; Graves, Lee M.

2003-01-01

c-Jun NH(2)-terminal kinase (JNK) has been shown to negatively regulate insulin signaling through serine phosphorylation of residue 307 within the insulin receptor substrate-1 (IRS-1) in adipose and liver tissue. Using a rat hindlimb suspension model for muscle disuse atrophy, we found that JNK activity was significantly elevated in atrophic soleus muscle and that IRS-1 was phosphorylated on Ser(307) prior to the degradation of the IRS-1 protein. Moreover, we observed a corresponding reduction in Akt activity, providing biochemical evidence for the development of insulin resistance in atrophic skeletal muscle.

The c-Jun N-terminal kinase pathway is critical for cell transformation by the latent membrane protein 1 of Epstein-Barr virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kutz, Helmut; Reisbach, Gilbert; Schultheiss, Ute

The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) transforms cells activating signal transduction pathways such as NF-{kappa}B, PI3-kinase, or c-Jun N-terminal kinase (JNK). Here, we investigated the functional role of the LMP1-induced JNK pathway in cell transformation. Expression of a novel dominant-negative JNK1 allele caused a block of proliferation in LMP1-transformed Rat1 fibroblasts. The JNK-specific inhibitor SP600125 reproduced this effect in Rat1-LMP1 cells and efficiently interfered with proliferation of EBV-transformed lymphoblastoid cells (LCLs). Inhibition of the LMP1-induced JNK pathway in LCLs caused the downregulation of c-Jun and Cdc2, the essential G2/M cell cycle kinase, which was accompanied bymore » a cell cycle arrest of LCLs at G2/M phase transition. Moreover, SP600125 retarded tumor growth of LCLs in a xenograft model in SCID mice. Our data support a critical role of the LMP1-induced JNK pathway for proliferation of LMP1-transformed cells and characterize JNK as a potential target for intervention against EBV-induced malignancies.« less

Aberrant Axonal Arborization of PDF Neurons Induced by Aβ42-Mediated JNK Activation Underlies Sleep Disturbance in an Alzheimer's Model.

PubMed

Song, Qian; Feng, Ge; Huang, Zehua; Chen, Xiaoman; Chen, Zhaohuan; Ping, Yong

2017-10-01

Impaired sleep patterns are common symptoms of Alzheimer's disease (AD). Cellular mechanisms underlying sleep disturbance in AD remain largely unknown. Here, using a Drosophila Aβ42 AD model, we show that Aβ42 markedly decreases sleep in a large population, which is accompanied with postdevelopmental axonal arborization of wake-promoting pigment-dispersing factor (PDF) neurons. The arborization is mediated in part via JNK activation and can be reversed by decreasing JNK signaling activity. Axonal arborization and impaired sleep are correlated in Aβ42 and JNK kinase hemipterous mutant flies. Image reconstruction revealed that these aberrant fibers preferentially project to pars intercerebralis (PI), a fly brain region analogous to the mammalian hypothalamus. Moreover, PDF signaling in PI neurons was found to modulate sleep/wake activities, suggesting that excessive release of PDF by these aberrant fibers may lead to the impaired sleep in Aβ42 flies. Finally, inhibition of JNK activation in Aβ42 flies restores nighttime sleep loss, decreases Aβ42 accumulation, and attenuates neurodegeneration. These data provide a new mechanism by which sleep disturbance could be induced by Aβ42 burden, a key initiator of a complex pathogenic cascade in AD.

In Vitro Targeted Photodynamic Therapy with a Pyropheophorbide-a Conjugated Inhibitor of Prostate Specific Membrane Antigen

PubMed Central

Liu, Tiancheng; Wu, Lisa Y.; Choi, Joseph K.; Berkman, Clifford E.

2009-01-01

BACKROUND The lack of specific delivery of photosensitizers (PSs), represents a significant limitation of photodynamic therapy (PDT) of cancer. The biomarker prostate-specific membrane antigen (PSMA) has attracted considerable attention as a target for imaging and therapeutic applications for prostate cancer. Although recent efforts have been made to conjugate inhibitors of PSMA with imaging agents, there have been no reports on photosensitizer-conjugated PSMA inhibitors for targeted PDT of prostate cancer. The present study focuses on the use of a PSMA inhibitor-conjugate of pyropheophorbide-a (Ppa-conjugate 2) for targeted PDT to achieve apoptosis in PSMA+ LNCaP cells. METHODS Confocal laser scanning microscopy with a combination of nuclear staining and immunofluorescence methods were employed to monitor the specific imaging and PDT-mediated apoptotic effects on PSMA-positive LNCaP and PSMA-negative (PC-3) cells. RESULTS Our results demonstrated that PDT-mediated effects by Ppa-conjugate 2 were specific to LNCaP cells, but not PC-3 cells. Cell permeability was detected as early as 2 h by HOE33342/PI double-staining, becoming more intense by 4 h. Evidence for the apoptotic caspase cascade being activated was based on the appearance of PARP p85 fragment. TUNEL assay detected DNA fragmentation 16 h post-PDT, confirming apoptotic events. CONCLUSIONS Cell permeability by HOE33342/PI double-staining as well as PARP p85 fragment and TUNEL assays confirm cellular apoptosis in PSMA+ cells when treated with PS-inhibitor conjugate 2 and subsequently irradiated. It is expected that the PSMA targeting small-molecule of this conjugate can serve as a delivery vehicle for PDT and other therapeutic applications for prostate cancer. PMID:19142895

Acute rosiglitazone treatment is cardioprotective against ischemia-reperfusion injury by modulating AMPK, Akt, and JNK signaling in nondiabetic mice.

PubMed

Morrison, Alex; Yan, Xiaoyan; Tong, Chao; Li, Ji

2011-09-01

Rosiglitazone (RGZ), a peroxisome proliferator-activated receptor (PPAR)-γ agonist, has been demonstrated to possess cardioprotective properties during ischemia-reperfusion. However, this notion remains controversial as recent evidence has suggested an increased risk in cardiac events associated with long-term use of RGZ in patients with type 2 diabetes. In this study, we tested the hypothesis that acute RGZ treatment is beneficial during I/R by modulating cardioprotective signaling pathways in a nondiabetic mouse model. RGZ (1 μg/g) was injected intravenously via the tail vein 5 min before reperfusion. Myocardial infarction was significantly reduced in mice treated with RGZ compared with vehicle controls (8.7% ± 1.1% vs. 20.2% ± 2.5%, P < 0.05). Moreover, isolated hearts were subjected to 20 min of global, no-flow ischemia in an ex vivo heart perfusion system. Postischemic recovery was significantly improved with RGZ treatment administered at the onset of reperfusion compared with vehicle (P < 0.001). Immunoblot analysis data revealed that the levels of both phospho-AMP-activated protein kinase (Thr(172)) and phospho-Akt (Ser(473)) were significantly upregulated when RGZ was administered 5 min before reperfusion compared with vehicle. On the other hand, inflammatory signaling [phospho-JNK (Thr(183)/Tyr(185))] was significantly downregulated as a result of RGZ treatment compared with vehicle (P < 0.05). Intriguingly, pretreatment with the selective PPAR-γ inhibitor GW-9662 (1 μg/g iv) 10 min before reperfusion significantly attenuated these beneficial effects of RGZ on the ischemic heart. Taken together, acute treatment with RGZ can reduce ischemic injury in a nondiabetic mouse heart via modulation of AMP-activated protein kinase, Akt, and JNK signaling pathways, which is dependent on PPAR-γ activation.

The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors.

PubMed

Halestrap, A P

1975-04-01

1. Studies on the kinetics of pyruvate transport into mitochondria by an 'inhibitor-stop' technique were hampered by the decarboxylation of pyruvate by mitochondria even in the presence of rotenone. Decarboxylation was minimal at 6 degrees C. At this temperature the Km for pyruvate was 0.15 mM and Vmax. was 0.54nmol/min per mg of protein; alpha-cyano-4-hydroxycinnamate was found to be a non-competitive inhibitor, Ki 6.3 muM, and phenyl-pyruvate a competitive inhibitor, Ki 1.8 mM. 2. At 100 muM concentration, alpha-cyano-4-hydroxycinnamate rapidly and almost totally inhibited O2 uptake by rat heart mitochondria oxidizing pyruvate. Inhibition could be detected at concentrations of inhibitor as low as 1 muM although inhibition took time to develop at this concentration. Inhibition could be reversed by diluting out the inhibitor. 3. Various analogues of alpha-cyano-4-hydroxycinnamate were tested on rat liver and heart mitochondria. The important structural features appeared to be the alpha-cyanopropenoate group and the hydrophobic aromatic side chain. Alpha-Cyanocinnamate, alpha-cyano-5-phenyl-2,4-pentadienoate and compound UK 5099 [alpha-cyano-beta-(2-phenylindol-3-yl)acrylate] were all more powerful inhibitors than alpha-cyano-4-hydroxycinnamate showing 50% inhibition of pyruvate-dependent O2 consumption by rat heart mitochondria at concentrations of 200, 200 and 50 nM respectively. 4. The specificity of the carrier for its substrate was studied by both influx and efflux experiments. Oxamate, 2-oxobutyrate, phenylpyruvate, 2-oxo-4-methyl-pentanoate, chloroacetate, dichloroacetate, difluoroacetate, 2-chloropropionate, 3-chloropropionate and 2,2-dichloropropionate all exchanged with pyruvate, whereas acetate, lactate and trichloroacetate did not. 5. Pyruvate entry into the mitochondria was shown to be accompanied by the transport of a proton (or by exchange with an OH-ion). This proton flux was inhibited by alpha-cyano-4-hydroxycinnamate and allowed measurements of

Smad4 inhibits cell migration via suppression of JNK activity in human pancreatic carcinoma PANC-1 cells.

PubMed

Zhang, Xueying; Cao, Junxia; Pei, Yujun; Zhang, Jiyan; Wang, Qingyang

2016-05-01

Smad4 is a common Smad and is a key downstream regulator of the transforming growth factor-β signaling pathway, in which Smad4 often acts as a potent tumor suppressor and functions in a highly context-dependent manner, particularly in pancreatic cancer. However, little is known regarding whether Smad4 regulates other signaling pathways involved in pancreatic cancer. The present study demonstrated that Smad4 downregulates c-Jun N-terminal kinase (JNK) activity using a Smad4 loss-of-function or gain-of-function analysis. Additionally, stable overexpression of Smad4 clearly affected the migration of human pancreatic epithelioid carcinoma PANC-1 cells, but did not affect cell growth. In addition, the present study revealed that upregulation of mitogen-activated protein kinase phosphatase-1 is required for the reduction of JNK activity by Smad4, leading to a decrease in vascular endothelial growth factor expression and inhibiting cell migration. Overall, the present findings indicate that Smad4 may suppress JNK activation and inhibit the tumor characteristics of pancreatic cancer cells.

The immunoproteasome-specific inhibitor ONX 0914 reverses susceptibility to acute viral myocarditis.

PubMed

Althof, Nadine; Goetzke, Carl Christoph; Kespohl, Meike; Voss, Karolin; Heuser, Arnd; Pinkert, Sandra; Kaya, Ziya; Klingel, Karin; Beling, Antje

2018-02-01

Severe heart pathology upon virus infection is closely associated with the immunological equipment of the host. Since there is no specific treatment available, current research focuses on identifying new drug targets to positively modulate predisposing immune factors. Utilizing a murine model with high susceptibility to coxsackievirus B3-induced myocarditis, this study describes ONX 0914-an immunoproteasome-specific inhibitor-as highly protective during severe heart disease. Represented by reduced heart infiltration of monocytes/macrophages and diminished organ damage, ONX 0914 treatment reversed fulminant pathology. Virus-induced immune response features like overwhelming pro-inflammatory cytokine and chemokine production as well as a progressive loss of lymphocytes all being reminiscent of a sepsis-like disease course were prevented by ONX 0914. Although the viral burden was only minimally affected in highly susceptible mice, resulting maintenance of immune homeostasis improved the cardiac output, and saved animals from severe illness as well as high mortality. Altogether, this could make ONX 0914 a potent drug for the treatment of severe virus-mediated inflammation of the heart and might rank immunoproteasome inhibitors among drugs for preventing pathogen-induced immunopathology. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Iron overload may promote alteration of NK cells and hematopoietic stem/progenitor cells by JNK and P38 pathway in myelodysplastic syndromes.

PubMed

Hua, Yanni; Wang, Chaomeng; Jiang, Huijuan; Wang, Yihao; Liu, Chunyan; Li, Lijuan; Liu, Hui; Shao, Zonghong; Fu, Rong

2017-08-01

The objective of the study was to examine levels of intracellular iron, reactive oxygen species (ROS) and the expression of JNK and p38MAPK in NK cells and hematopoietic stem/progenitor cells (HSPCs) in MDS patients, and explore potential mechanisms by which iron overload (IOL) promotes MDS progression. Thirty-four cases of MDS and six cases of AML transformed from MDS (MDS/AML) were included. HSPCs and NK cells were isolated by magnetic absorption cell sorting. We used flow cytometry to detect the levels of ROS and intracellular JNK and P38 in NK cells and HSPCs. Total RNA and protein were extracted from NK cells and CD34 + cells to examine the expression of JNK and p38MAPK using RT-PCR and Western blotting. Intracellular iron concentration was detected. Data were analyzed by SPSS 21 statistical software. Intracellular iron concentration and ROS were increased in both NK cells and HSPCs in MDS patients with iron overload (P < 0.05). MDS patients with iron overload had higher JNK expression and lower p38 expression in NK cells, and higher p38 expression in HSPCs compared with non-iron overload group. IOL may cause alterations in NK cells and HSPCs through the JNK and p38 pathways, and play a role in the transformation to AML from MDS.

Nuclear movement regulated by non-Smad Nodal signaling via JNK is associated with Smad signaling during zebrafish endoderm specification.

PubMed

Hozumi, Shunya; Aoki, Shun; Kikuchi, Yutaka

2017-11-01

Asymmetric nuclear positioning is observed during animal development, but its regulation and significance in cell differentiation remain poorly understood. Using zebrafish blastulae, we provide evidence that nuclear movement towards the yolk syncytial layer, which comprises extraembryonic tissue, occurs in the first cells fated to differentiate into the endoderm. Nodal signaling is essential for nuclear movement, whereas nuclear envelope proteins are involved in movement through microtubule formation. Positioning of the microtubule-organizing center, which is proposed to be crucial for nuclear movement, is regulated by Nodal signaling and nuclear envelope proteins. The non-Smad JNK signaling pathway, which is downstream of Nodal signaling, regulates nuclear movement independently of the Smad pathway, and this nuclear movement is associated with Smad signal transduction toward the nucleus. Our study provides insight into the function of nuclear movement in Smad signaling toward the nucleus, and could be applied to the control of TGFβ signaling. © 2017. Published by The Company of Biologists Ltd.

Resveratrol promotes recovery of immune function of immunosuppressive mice by activating JNK/NF-κB pathway in splenic lymphocytes.

PubMed

Lai, Xin; Cao, Mei; Song, Xu; Jia, Renyong; Zou, Yuanfeng; Li, Lixia; Liang, Xiaoxia; He, Changliang; Yin, Lizi; Yue, Guizhou; Ye, Gang; Yin, Zhongqiong

2017-06-01

Resveratrol, a natural compound found in over 70 plants, is known to possess immunoregulatory effects and anti-inflammatory activity. It has been shown that resveratrol has regulatory effects on different signaling pathways in different diseases. However, few reports have evaluated the effects of resveratrol on reinforcing immunity recovery via activating nuclear factor-κB (NF-κB) pathway and Jun N-terminal kinases (JNK) pathway. The present study aimed to assess immune-enhancing activity and underlying mechanism of resveratrol in immunosuppressive mice. Previously, we reported that resveratrol could promote mouse spleen lymphocyte functions to recover the immune system effectively. In the present study, we show that resveratrol could upregulate the expressions of NF-κB, IκB kinase, JNK, and c-jun in splenic lymphocytes of immunosuppressive mice. Taken together, our results indicate that resveratrol could promote recovery of immunologic function in immunosuppressive mice by activating JNK/NF-κB pathway.

[Study of the effect of JNK signal transduction pathway in intense noise-induced apoptosis in cochlea of guinea pig].

PubMed

Xue, Qiuhong; Chen, Jia; Gong, Shusheng; Xie, Jing; He, Jian; Chen, Xiaolin

2009-12-01

To investigate the mechanism of intense noise-induced cochlea cells death in guinea pig, and the effect of JNK signal transduction pathway in the procedure of cochlea cells apoptosis by intense noise-induced. Thirty-two guinea pigs were randomly divided into 4 groups. The guinea pigs in the experiment groups were exposed to 4 kHz narrow band noise at 120 dB SPL for 4 h. After the noise expose for 1, 4, 14 days of the experiment guinea pigs, ABR of the guinea pigs on experiment and control groups were tested before put them to death. Four guinea pig's cochleas of every group were taken to paraffin section, and the rest was extracted the total cochlear's protein. Apoptosis was tested by terminal deoxynucleotidyl Transferase (TdT)-mediated deoxyuridine triphosphate (d-UTP) nick and labeling method (TUNEL). The phosphorylation of JNK and c-Jun were tested by immunohistochemistry and western blot methods. Tunel-Positive cells in the Corti's, SGC and SV of experiment groups, and there have significant differences compared with the control group (P<0.01) and Tunel-Positive cells are most in 1 d experiment group. The positive cells of P-JNK and P-c-Jun could be detected in guinea pig's cochleas after noise exposed, but no positive cells were found in the control. Protein levels of P-JNK and P-c-Jun were risen up and activated quickly after noise exposed, and achieved peak in 1 d, 4 d and then fallen-offs, but still maintained higher levels within 14 d. Intense noise causes cochlea cell lesion by inducing apoptosis to result in and JNK signal transduction pathway plays an important role in the procedure of apoptosis.

In vitro targeted photodynamic therapy with a pyropheophorbide--a conjugated inhibitor of prostate-specific membrane antigen.

PubMed

Liu, Tiancheng; Wu, Lisa Y; Choi, Joseph K; Berkman, Clifford E

2009-05-01

The lack of specific delivery of photosensitizers (PSs), represents a significant limitation of photodynamic therapy (PDT) of cancer. The biomarker prostate-specific membrane antigen (PSMA) has attracted considerable attention as a target for imaging and therapeutic applications for prostate cancer. Although recent efforts have been made to conjugate inhibitors of PSMA with imaging agents, there have been no reports on PS-conjugated PSMA inhibitors for targeted PDT of prostate cancer. The present study focuses on the use of a PSMA inhibitor-conjugate of pyropheophorbide-a (Ppa-conjugate 2) for targeted PDT to achieve apoptosis in PSMA+ LNCaP cells. Confocal laser scanning microscopy with a combination of nuclear staining and immunofluorescence methods were employed to monitor the specific imaging and PDT-mediated apoptotic effects on PSMA-positive LNCaP and PSMA-negative (PC-3) cells. Our results demonstrated that PDT-mediated effects by Ppa-conjugate 2 were specific to LNCaP cells, but not PC-3 cells. Cell permeability was detected as early as 2 hr by HOE33342/PI double staining, becoming more intense by 4 hr. Evidence for the apoptotic caspase cascade being activated was based on the appearance of poly-ADP-ribose polymerase (PARP) p85 fragment. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay detected DNA fragmentation 16 hr post-PDT, confirming apoptotic events. Cell permeability by HOE33342/PI double staining as well as PARP p85 fragment and TUNEL assays confirm cellular apoptosis in PSMA+ cells when treated with PS-inhibitor conjugate 2 and subsequently irradiated. It is expected that the PSMA targeting small-molecule of this conjugate can serve as a delivery vehicle for PDT and other therapeutic applications for prostate cancer. (c) 2009 Wiley-Liss, Inc.

MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Teng, E-mail: tengyu33@yahoo.com; Ji, Jiang; Guo, Yong-li

2013-11-08

Highlights: •Curcumin activates MST1 in melanoma cells. •MST1 mediates curcumin-induced apoptosis of melanoma cells. •ROS production is involved in curcumin-induced MST1 activation. •MST1 mediates curcumin-induced JNK activation in melanoma cells. •MST1 mediates curcumin-induced Foxo3a nuclear translocation and Bim expression. -- Abstract: Different groups including ours have shown that curcumin induces melanoma cell apoptosis, here we focused the role of mammalian Sterile 20-like kinase 1 (MST1) in it. We observed that curcumin activated MST1-dependent apoptosis in cultured melanoma cells. MST1 silencing by RNA interference (RNAi) suppressed curcumin-induced cell apoptosis, while MST1 over-expressing increased curcumin sensitivity. Meanwhile, curcumin induced reactive oxygen speciesmore » (ROS) production in melanoma cells, and the ROS scavenger, N-acetyl-cysteine (NAC), almost blocked MST1 activation to suggest that ROS might be required for MST1 activation by curcumin. c-Jun N-terminal protein kinase (JNK) activation by curcumin was dependent on MST1, since MST1 inhibition by RNAi or NAC largely inhibited curcumin-induced JNK activation. Further, curcumin induced Foxo3 nuclear translocation and Bim-1 (Foxo3 target gene) expression in melanoma cells, such an effect by curcumin was inhibited by MST1 RNAi. In conclusion, we suggested that MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells.« less

A Role for the p38 Mitogen-activated Protein Kinase Pathway in Myocardial Cell Growth, Sarcomeric Organization, and Cardiac-specific Gene Expression

PubMed Central

Zechner, Dietmar; Thuerauf, Donna J.; Hanford, Deanna S.; McDonough, Patrick M.; Glembotski, Christopher C.

1997-01-01

Three hallmark features of the cardiac hypertrophic growth program are increases in cell size, sarcomeric organization, and the induction of certain cardiac-specific genes. All three features of hypertrophy are induced in cultured myocardial cells by α1- adrenergic receptor agonists, such as phenylephrine (PE) and other growth factors that activate mitogen- activated protein kinases (MAPKs). In this study the MAPK family members extracellular signal–regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 were activated by transfecting cultured cardiac myocytes with constructs encoding the appropriate kinases possessing gain-of-function mutations. Transfected cells were then analyzed for changes in cell size, sarcomeric organization, and induction of the genes for the A- and B-type natriuretic peptides (NPs), as well as the α-skeletal actin (α-SkA) gene. While activation of JNK and/or ERK with MEKK1COOH or Raf-1 BXB, respectively, augmented cell size and effected relatively modest increases in NP and α-SkA promoter activities, neither upstream kinase conferred sarcomeric organization. However, transfection with MKK6 (Glu), which specifically activated p38, augmented cell size, induced NP and α-Ska promoter activities by up to 130-fold, and elicited sarcomeric organization in a manner similar to PE. Moreover, all three growth features induced by MKK6 (Glu) or PE were blocked with the p38-specific inhibitor, SB 203580. These results demonstrate novel and potentially central roles for MKK6 and p38 in the regulation of myocardial cell hypertrophy. PMID:9314533

Pathway-specific effect of caffeine on protection against UV irradiation-induced apoptosis in corneal epithelial cells.

PubMed

Wang, Ling; Lu, Luo

2007-02-01

To define the role of molecular interaction between the UV-induced JNK (c-Jun N-terminal kinase) cascade and corneal epithelial cell apoptosis and protection against apoptosis by caffeine. Rabbit and human corneal epithelial cells were cultured in DMEM/F12 medium containing 10% FBS and 5 microg/mL insulin at 37 degrees C in 5% CO(2). DNA fragmentation and ethidium bromide/acridine orange (EB/AO) nuclear staining were performed to detect cell death. Western blot, immunoprecipitation, and kinase assays were used to measure UV-induced mitogen-activated protein (MAP) kinase activity. UV irradiation-induced apoptosis through apoptosis signal-regulating kinase 1 (ASK1) and MAKK4 (SEK1) upstream from JNK was caffeine sensitive. Caffeine (1,3,7-trimethylxanthine), an agent that is one of the most popular additions to food consumed in the world and a potential enhancer of chemotherapy, effectively protected corneal epithelial cells against apoptosis by its specific effect on the JNK cascade. Theophylline (1,3-dimethylxanthine) exhibited an effect similar to that of caffeine on prevention of UV irradiation-induced apoptosis. However, alterations of either intracellular cAMP or Ca(2+) levels did not alter the effect of caffeine on the JNK signaling pathway. In addition, the blockade of PI3K-like kinases by wortmannin had no impact on the protective effect of caffeine against UV irradiation-induced apoptosis, suggesting that the protective effect of caffeine acts through a specific mechanism involving UV irradiation-induced activation of ASK1 and SEK1. In contrast, caffeine had no effects on melphalan-, hyperosmotic stress-, or IL-1beta-induced activation of the JNK signaling pathway in these cells. UV irradiation stress-induced activation of the ASK1-SEK1-JNK signaling pathway leading to apoptosis is a caffeine-sensitive process, and caffeine, as a multifunctional agent in cells, can specifically interact with the pathway to protect against apoptosis.

Prevotella intermedia stimulates tissue-type plasminogen activator and plasminogen activator inhibitor-2 expression via multiple signaling pathways in human periodontal ligament cells.

PubMed

Guan, Su-Min; He, Jian-Jun; Zhang, Ming; Shu, Lei

2011-06-01

Prevotella intermedia is an important periodontal pathogen that induces various inflammatory and immune responses. In this study, we investigated the effects of P. intermedia on the plasminogen system in human periodontal ligament (hPDL) cells and explored the signaling pathways involved. Using semi-quantitative reverse transcription (RT)-PCR and quantitative real-time RT-qPCR, we demonstrated that P. intermedia challenge increased tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor (PAI)-2 expression in a concentration- and time-dependent manner, but exerted no influence on urokinase-type plasminogen activator and PAI-1mRNA expression in hPDL cells. Prevotella intermedia stimulation also enhanced tPA protein secretion as confirmed by enzyme-linked immunosorbent assay. Western blot results revealed that P. intermedia treatment increased phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 kinase (p38). ERK, JNK and protein kinase C inhibitors significantly attenuated the P. intermedia-induced tPA and PAI-2 expression. Furthermore, p38 and phosphatidylinositol 3-kinase inhibitors markedly decreased PAI-2 expression, whereas they showed no or little inhibition on tPA expression. In contrast, inhibition of protein kinase A greatly enhanced the upregulatory effect of P. intermedia on tPA and PAI-2 expression. Our results suggest that P. intermedia may contribute to periodontal tissue destruction by upregulating tPA and PAI-2 expression in hPDL cells via multiple signaling pathways. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Wound-Induced Polyploidization: Regulation by Hippo and JNK Signaling and Conservation in Mammals

PubMed Central

Losick, Vicki P.; Jun, Albert S.; Spradling, Allan C.

2016-01-01

Tissue integrity and homeostasis often rely on the proliferation of stem cells or differentiated cells to replace lost, aged, or damaged cells. Recently, we described an alternative source of cell replacement- the expansion of resident, non-dividing diploid cells by wound-induced polyploidization (WIP). Here we show that the magnitude of WIP is proportional to the extent of cell loss using a new semi-automated assay with single cell resolution. Hippo and JNK signaling regulate WIP; unexpectedly however, JNK signaling through AP-1 limits rather than stimulates the level of Yki activation and polyploidization in the Drosophila epidermis. We found that polyploidization also quantitatively compensates for cell loss in a mammalian tissue, mouse corneal endothelium, where increased cell death occurs with age in a mouse model of Fuchs Endothelial Corneal Dystrophy (FECD). Our results suggest that WIP is an evolutionarily conserved homeostatic mechanism that maintains the size and synthetic capacity of adult tissues. PMID:26958853

Wound-Induced Polyploidization: Regulation by Hippo and JNK Signaling and Conservation in Mammals.

PubMed

Losick, Vicki P; Jun, Albert S; Spradling, Allan C

2016-01-01

Tissue integrity and homeostasis often rely on the proliferation of stem cells or differentiated cells to replace lost, aged, or damaged cells. Recently, we described an alternative source of cell replacement- the expansion of resident, non-dividing diploid cells by wound-induced polyploidization (WIP). Here we show that the magnitude of WIP is proportional to the extent of cell loss using a new semi-automated assay with single cell resolution. Hippo and JNK signaling regulate WIP; unexpectedly however, JNK signaling through AP-1 limits rather than stimulates the level of Yki activation and polyploidization in the Drosophila epidermis. We found that polyploidization also quantitatively compensates for cell loss in a mammalian tissue, mouse corneal endothelium, where increased cell death occurs with age in a mouse model of Fuchs Endothelial Corneal Dystrophy (FECD). Our results suggest that WIP is an evolutionarily conserved homeostatic mechanism that maintains the size and synthetic capacity of adult tissues.

SK-N-MC cell death occurs by distinct molecular mechanisms in response to hydrogen peroxide and superoxide anions: involvements of JAK2-STAT3, JNK, and p38 MAP kinases pathways.

PubMed

Moslehi, Maryam; Yazdanparast, Razieh

2013-07-01

Oxidative stress plays a vital role in the pathogenesis of neurodegenerative diseases. Nerve cells are incessantly exposed to environmental stresses leading to overproduction of some harmful species like reactive oxygen species (ROS). ROS including hydrogen peroxide and superoxide anion are potent inducers of various signaling pathways encompassing MAPKs and JAK-STAT pathways. In the current study, we scrutinized the effects of hydrogen peroxide and/or menadione (superoxide anion generator) on JNK/p38-MAPKs and JAK2-STAT3 pathways to elucidate the mechanism(s) by which each oxidant modulated the above-mentioned pathways leading to SK-N-MC cell death. Our results delineated that hydrogen peroxide and superoxide anion radical induced distinct responses as we showed that STAT3 and p38 were activated in response to hydrogen peroxide, but not superoxide anion radicals indicating the specificity in ROS-induced signaling pathways activations and behaviors. We also observed that menadione induced JNK-dependent p53 expression and apoptotic death in SK-N-MC cells while H2O2-induced JNK activation was p53 independent. Thus, we declare that ROS type has a key role in selective instigation of JNK/p38-MAPKs and JAK2-STAT3 pathways in SK-N-MC cells. Identifying these differential behaviors and mechanisms of hydrogen peroxide and superoxide anion functions illuminates the possible therapeutic targets in the prevention or treatment of ROS-induced neurodegenerative diseases such as Alzheimer's disease.

(Bis)urea and (Bis)thiourea Inhibitors of Lysine-Specific Demethylase 1 as Epigenetic Modulators

PubMed Central

Sharma, Shiv K.; Wu, Yu; Steinbergs, Nora; Crowley, Michael L.; Hanson, Allison S.; Casero, Robert A.; Woster, Patrick M.

2010-01-01

The recently discovered enzyme lysine-specific demethylase 1 (LSD1) plays an important role in the epigenetic control of gene expression, and aberrant gene silencing secondary to LSD1 over expression is thought to contribute to the development of cancer. We recently reported a series of (bis)guanidines and (bis)biguanides that are potent inhibitors of LSD1, and induce the re-expression of aberrantly silenced tumor suppressor genes in tumor cells in vitro. We now report a series of isosteric ureas and thioureas that are also potent inhibitors of LSD1. These compounds induce increases in methylation at the histone 3 lysine 4 (H3K4) chromatin mark, a specific target of LSD1, in Calu-6 lung carcinoma cells. In addition, these analogues increase cellular levels of secreted frizzle-related proteins (SFRP) 2 and 5, and transcription factor GATA4. These compounds represent an important new series of epigenetic modulators with the potential for use as antitumor agents. PMID:20568780

Fluoxetine a novel anti-hepatitis C virus agent via ROS-, JNK-, and PPARβ/γ-dependent pathways.

PubMed

Young, Kung-Chia; Bai, Chyi-Huey; Su, Hui-Chen; Tsai, Pei-Ju; Pu, Chien-Yu; Liao, Chao-Sheng; Lin, Yu-Min; Lai, Hsin-Wen; Chong, Lee-Won; Tsai, Yau-Sheng; Tsao, Chiung-Wen

2014-10-01

More than 20% of chronic hepatitis C (CHC) patients receiving interferon-alpha (IFN-α)-based anti-hepatitis C virus (HCV) therapy experienced significant depression, which was relieved by treatment with fluoxetine. However, whether and how fluoxetine affected directly the anti-HCV therapy remained unclear. Here, we demonstrated that fluoxetine inhibited HCV infection and blocked the production of reactive oxygen species (ROS) and lipid accumulation in Huh7.5 cells. Fluoxetine facilitated the IFN-α-mediated antiviral actions via activations of signal transducer and activator of transcription (STAT)-1 and c-Jun amino-terminal kinases (JNK). Alternatively, fluoxetine elevated peroxisome proliferator-activated receptor (PPAR) response element activity under HCV infection. The inhibitory effects of fluoxetine on HCV infection and lipid accumulation, but not production of ROS, were partially reversed by the PPAR-β, -γ, and JNK antagonists. Furthermore, fluoxetine intervention to the IFN-α-2b regimen facilitated to reduce HCV titer and alanine transaminase level for CHC patients. Therefore, fluoxetine intervention to the IFN-α-2b regimen improved the efficacy of anti-HCV treatment, which might be related to blockades of ROS generation and lipid accumulation and activation of host antiviral JNK/STAT-1 and PPARβ/γ signals. Copyright © 2014 Elsevier B.V. All rights reserved.

Balanites aegyptiaca ameliorates insulin secretion and decreases pancreatic apoptosis in diabetic rats: Role of SAPK/JNK pathway.

PubMed

Hassanin, Kamel M A; Mahmoud, Mohamed O; Hassan, Hossam M; Abdel-Razik, Abdel-Razik H; Aziz, Lourin N; Rateb, Mostafa E

2018-06-01

SAPK-JNK pathway performs a significant role in the pathogenesis of type 2 diabetes. Balanites aegyptiaca (BA) is used as an anti-diabetic agent in folk medicine however its hypoglycemic mechanism is not fully elucidated. The current study aimed to evaluate the effect of crude extract, butanol, and dichloromethane fractions from BA on the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK-JNK) pathway in experimental diabetic rats. Six groups of male Wistar rats were included: normal control, diabetic, diabetic rats treated with crude, butanol or dichloromethane fraction from BA (50 mg/kg BW) and diabetic rats treated with gliclazide as a reference drug for one month. Our results suggested a protective role of treatment of diabetic rats with BA against oxidative stress-induced SAPK-JNK pathway. Moreover, BA treatment produced a reduction in plasma glucose, HbA 1c , lactic acid, lipid profile, malondialdehyde levels and produced an increase in insulin, reduced glutathione levels, catalase and superoxide dismutase activities compared with untreated diabetic rats. Moreover, it decreased apoptosis signal-regulating kinase 1, c-Jun N-terminal kinase 1, protein 53 and increased insulin receptor substrate 1 in rat pancreas while it increased glucose transporter 4 in rat muscle. Analysis of BA extracts by LC-HRMS revealed the presence of different saponins with reported hypoglycemic effect. In conclusion, BA exerted hypoglycemic, hypolipidemic, insulinotropic and antioxidant effects. Additionally, it reduced apoptosis in pancreatic β-cells and increased glucose uptake in muscle. These results suggest that the hypoglycemic effect of BA is due to the inhibition of the SAPK-JNK pathway. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Arsenic sulfide induces apoptosis and autophagy through the activation of ROS/JNK and suppression of Akt/mTOR signaling pathways in osteosarcoma.

PubMed

Wang, Gangyang; Zhang, Tao; Sun, Wei; Wang, Hongsheng; Yin, Fei; Wang, Zhuoying; Zuo, Dongqing; Sun, Mengxiong; Zhou, Zifei; Lin, Binhui; Xu, Jing; Hua, Yingqi; Li, Haoqing; Cai, Zhengdong

2017-05-01

Osteosarcoma is a common primary malignant bone tumor, the cure rate of which has stagnated over the past 25-30 years. Arsenic sulfide (As 2 S 2 ), the main active ingredient of the traditional Chinese medicine realgar, has been proved to have antitumor efficacy in several tumor types including acute promyelocytic leukemia, gastric cancer and colon cancer. Here, we investigated the efficacy and mechanism of As 2 S 2 in osteosarcoma both in vitro and in vivo. In this study, we demonstrated that As 2 S 2 potently suppressed cell proliferation by inducing G2/M phase arrest in various osteosarcoma cell lines. Also, treatment with As 2 S 2 induced apoptosis and autophagy in osteosarcoma cells. The apoptosis induction was related to PARP cleavage and activation of caspase-3, -8, -9. As 2 S 2 was demonstrated to induce autophagy as evidenced by formation of autophagosome and accumulation of LC3II. Further studies showed that As 2 S 2 -induced apoptosis and autophagy could be significantly attenuated by ROS scavenger and JNK inhibitor. Moreover, we found that As 2 S 2 inhibited Akt/mTOR signaling pathway, and suppressing Akt and mTOR kinases activity can increase As 2 S 2 -induced apoptosis and autophagy. Finally, As 2 S 2 in vivo suppressed tumor growth with few side effects. In summary, our results revealed that As 2 S 2 induced G2/M phase arrest, apoptosis, and autophagy via activing ROS/JNK and blocking Akt/mTOR signaling pathway in human osteosarcoma cells. Arsenic sulfide may be a potential clinical antitumor drugs targeting osteosarcoma. Copyright © 2017. Published by Elsevier Inc.

Dual regulation of HMGB1 by combined JNK1/2-ATF2 axis with miR-200 family in nonalcoholic steatohepatitis in mice.

PubMed

Chen, Xin; Ling, Yan; Wei, Yanping; Tang, Jing; Ren, Yibing; Zhang, Baohua; Jiang, Feng; Li, Hengyu; Wang, Ruoyu; Wen, Wen; Lv, Guishuai; Wu, Mengchao; Chen, Lei; Li, Liang; Wang, Hongyang

2018-05-01

In the context of diabetes, obesity, and metabolic syndrome, the inflammatory signaling has critical roles in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), but the underlying mechanisms remain poorly delineated. Herein, early and persistently elevated, proinflammatory cytokine HMGB1 expression was detected in a high-fat diet (HFD)-induced NAFLD model in C57BL/6 mice. The expression and extracellular release of HMGB1 was rapidly and dramatically induced by saturated palmitic acid in vitro. HFD-induced inflammatory response and liver function impairment were both mitigated after the inhibition of endogenous HMGB1 by neutralizing antibody in vivo. The up-regulation of HMGB1 was thought to be modified by dual channels: in the transcriptional level, it was regulated by JNK1/JNK2-ATF2 axis; post-transcriptionally, it was regulated by the microRNA (miR)-200 family, especially miR-429. miR-429 liver conditional knockout mice (miR-429 Δhep ), fed either a normal diet or an HFD, showed severe liver inflammation and dysfunction, accompanied by greater expression of HMGB1. Intriguingly, the up-regulation and release of HMGB1 could in turn self-activate TLR4-JNK1/JNK2-ATF2 signaling, thus forming a positive feedback. Our findings reveal a novel mechanism by which HMGB1 expression was regulated by both the JNK1/2-ATF2 axis and the miR-200 family, which provides a potential new approach for the treatment of NAFLD.-Chen, X., Ling, Y., Wei, Y., Tang, J., Ren, Y., Zhang, B., Jiang, F., Li, H., Wang, R., Wen, W., Lv, G., Wu, M., Chen, L., Li, L., Wang, H. Dual regulation of HMGB1 by combined JNK1/2-ATF2 axis with miR-200 family in nonalcoholic steatohepatitis in mice.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuan, Bo; Cui, Jinquan, E-mail: jinquancuijqc@163.com; Wang, Wuliang

Several reports have indicated a role for the members of the G12 family of heterotrimeric G proteins (Gα12 and Gα13) in oncogenesis and tumor cell growth. The aims of the present study were to evaluate the role of G12 signaling in cervical cancer. We demonstrated that expression of the G12 proteins was highly upregulated in cervical cancer cells. Additionally, expression of the activated forms of Gα12/Gα13 but not expression of activated Gαq induced cell invasion through the activation of the RhoA family of G proteins, but had no effect on cell proliferation in the cervical cancer cells. Inhibition of G12more » signaling by expression of the RGS domain of the p115-Rho-specific guanine nucleotide exchange factor (p115-RGS) blocked thrombin-stimulated cell invasion, but did not inhibit cell proliferation in cervical cells, whereas the inhibition of Gαq (RGS2) had no effect. Furthermore, G12 signaling was able to activate Rho proteins, and this stimulation was inhibited by p115-RGS, and Gα12-induced invasion was blocked by an inhibitor of RhoA/B/C (C3 toxin). Pharmacological inhibition of JNK remarkably decreased G12-induced JNK activation. Both a JNK inhibitor (SP600125) and a ROCK inhibitor (Y27632) reduced G12-induced JNK and c-Jun activation, and markedly inhibited G12-induced cellular invasion. Collectively, these findings demonstrate that stimulation of G12 proteins is capable of promoting invasion through RhoA/ROCK-JNK activation. -- Highlights: •Gα12/Gα13 is upregulated in cervical cancer cell lines. •Gα12/Gα13 is not involved in cervical cancer cell proliferation. •Gα12/Gα13 promotes cervical cancer cell invasion. •The role of Rho G proteins in G12-promoted cervical cancer cell invasion. •G12 promotes cell invasion through activation of the ROCK-JNK signaling axis.« less

The dual-specificity phosphatase MKP-1 limits the cardiac hypertrophic response in vitro and in vivo.

PubMed

Bueno, O F; De Windt, L J; Lim, H W; Tymitz, K M; Witt, S A; Kimball, T R; Molkentin, J D

2001-01-19

Mitogen-activated protein kinase (MAPK) signaling pathways are important regulators of cell growth, proliferation, and stress responsiveness. A family of dual-specificity MAP kinase phosphatases (MKPs) act as critical counteracting factors that directly regulate the magnitude and duration of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) activation. Here we show that constitutive expression of MKP-1 in cultured primary cardiomyocytes using adenovirus-mediated gene transfer blocked the activation of p38, JNK1/2, and ERK1/2 and prevented agonist-induced hypertrophy. Transgenic mice expressing physiological levels of MKP-1 in the heart showed (1) no activation of p38, JNK1/2, or ERK1/2; (2) diminished developmental myocardial growth; and (3) attenuated hypertrophy in response to aortic banding and catecholamine infusion. These results provide further evidence implicating MAPK signaling factors as obligate regulators of cardiac growth and hypertrophy and demonstrate the importance of dual-specificity phosphatases as counterbalancing regulatory factors in the heart.

In silico simulations of STAT1 and STAT3 inhibitors predict SH2 domain cross-binding specificity.

PubMed

Szelag, Malgorzata; Sikorski, Krzysztof; Czerwoniec, Anna; Szatkowska, Katarzyna; Wesoly, Joanna; Bluyssen, Hans A R

2013-11-15

Signal transducers and activators of transcription (STATs) comprise a family of transcription factors that are structurally related and which participate in signaling pathways activated by cytokines, growth factors and pathogens. Activation of STAT proteins is mediated by the highly conserved Src homology 2 (SH2) domain, which interacts with phosphotyrosine motifs for specific contacts between STATs and receptors and for STAT dimerization. By generating new models for human (h)STAT1, hSTAT2 and hSTAT3 we applied comparative in silico docking to determine SH2-binding specificity of the STAT3 inhibitor stattic, and of fludarabine (STAT1 inhibitor). Thus, we provide evidence that by primarily targeting the highly conserved phosphotyrosine (pY+0) SH2 binding pocket stattic is not a specific hSTAT3 inhibitor, but is equally effective towards hSTAT1 and hSTAT2. This was confirmed in Human Micro-vascular Endothelial Cells (HMECs) in vitro, in which stattic inhibited interferon-α-induced phosphorylation of all three STATs. Likewise, fludarabine inhibits both hSTAT1 and hSTAT3 phosphorylation, but not hSTAT2, by competing with the highly conserved pY+0 and pY-X binding sites, which are less well-preserved in hSTAT2. Moreover we observed that in HMECs in vitro fludarabine inhibits cytokine and lipopolysaccharide-induced phosphorylation of hSTAT1 and hSTAT3 but does not affect hSTAT2. Finally, multiple sequence alignment of STAT-SH2 domain sequences confirmed high conservation between hSTAT1 and hSTAT3, but not hSTAT2, with respect to stattic and fludarabine binding sites. Together our data offer a molecular basis that explains STAT cross-binding specificity of stattic and fludarabine, thereby questioning the present selection strategies of SH2 domain-based competitive small inhibitors. © 2013 Elsevier B.V. All rights reserved.

The flavonoid quercetin induces apoptosis and inhibits JNK activation in intimal vascular smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perez-Vizcaino, Francisco; Bishop-Bailley, David; Lodi, Federica

Quercetin, the most abundant dietary flavonol, exerts vasodilator, anti-hypertensive, and anti-atherogenic effects and reduces the vascular remodelling associated with elevated blood pressure. Here, we have compared the effects of quercetin in intimal- and medial-type rat vascular smooth muscle cells (VSMC) in culture. After 48 h, quercetin reduced the viability of a polyclonal intimal-type cell line derived from neonatal aorta but not of a medial-type cell line derived from adult aorta. These differential effects were similar in both proliferating and quiescent VSMC. Quercetin also preferentially reduced the viability of intimal-type over medial-type VSMC in primary cultures derived from balloon-injured carotid arteries.more » The effects of quercetin on cell viability were mainly dependent upon induction of apoptosis, as demonstrated by nuclear condensation and fragmentation, and were unrelated to PPAR{gamma}, pro-oxidant effects or nitric oxide. The expression of MAPKs (ERK, p38, and JNK) and ERK phosphorylation were not different between intimal- and medial-type VSMC. p38 phosphorylation was negligible in both cell types. Medial-type showed a weak JNK phosphorylation while this was markedly increased in intimal-type cells. Quercetin reduced JNK phosphorylation but had no consistent effect on ERK phosphorylation. In conclusion, quercetin preferentially produced apoptosis in intimal-type compared to medial-type VSMC. This might play a role in the anti-atherogenic and anti-hypertensive effects of quercetin.« less

Eupafolin inhibits PGE2 production and COX2 expression in LPS-stimulated human dermal fibroblasts by blocking JNK/AP-1 and Nox2/p47{sup phox} pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsai, Ming-Horng; Lin, Zih-Chan; Liang, Chan-Jung

2014-09-01

Eupafolin, a major active component found in the methanol extracts of Phyla nodiflora, has been used to treat inflammation of skin. We examined its effects on cyclooxygenase-2 (COX-2) expression in LPS-treated human dermal fibroblasts. Lipopolysaccharide (LPS) significantly increased prostaglandin-E2 (PGE2) production associated with increased COX-2 expression in Hs68 cells. This effect was blocked by eupafolin, TLR-4 antibody, antioxidants (APO and NAC), as well as inhibitors, including U0126 (ERK1/2), SB202190 (p38), SP600125 (JNK1/2), and Tanshinone IIA (AP-1). In gene regulation level, qPCR and promoter assays revealed that COX-2 expression was attenuated by eupafolin. In addition, eupafolin also ameliorated LPS-induced p47 phoxmore » activation and decreased reactive oxygen species (ROS) generation and NADPH oxidase (Nox) activity. Moreover, pretreatment with eupafolin and APO led to reduced LPS-induced phosphorylation of ERK1/2, JNK, and p38. Further, eupafolin attenuated LPS-induced increase in AP-1 transcription factor binding activity as well as the increase in the phosphorylation of c-Jun and c-Fos. In vivo studies have shown that in dermal fibroblasts of LPS treated mice, eupafolin exerted anti-inflammation effects by decreasing COX-2 protein levels. Our results reveal a novel mechanism for anti-inflammatory and anti-oxidative effects of eupafolin that involved inhibition of LPS-induced ROS generation, suppression of MAPK phosphorylation, diminished DNA binding activity of AP-1 and attenuated COX-2 expression leading to reduced production of prostaglandin E2 (PGE2). Our results demonstrate that eupafolin may be used to treat inflammatory responses associated with dermatologic diseases. - Highlights: • LPS activates the Nox2/p47{sup phox}/JNK/AP-1 and induces COX2 expression in Hs68 cells. • Eupafolin inhibits LPS-induced COX-2 expression via Nox2/p47{sup phox} inhibition. • Eupafolin may be used in the treatment of skin diseases involving

IL-17A induces eotaxin-1/CC chemokine ligand 11 expression in human airway smooth muscle cells: role of MAPK (Erk1/2, JNK, and p38) pathways.

PubMed

Rahman, Muhammad Shahidur; Yamasaki, Akira; Yang, Jie; Shan, Lianyu; Halayko, Andrew J; Gounni, Abdelilah Soussi

2006-09-15

Recently, IL-17A has been shown to be expressed in higher levels in respiratory secretions from asthmatics and correlated with airway hyperresponsiveness. Although these studies raise the possibility that IL-17A may influence allergic disease, the mechanisms remain unknown. In this study, we investigated the molecular mechanisms involved in IL-17A-mediated CC chemokine (eotaxin-1/CCL11) production from human airway smooth muscle (ASM) cells. We found that incubation of human ASM cells with rIL-17A resulted in a significant increase of eotaxin-1/CCL11 release from ASM cells that was reduced by neutralizing anti-IL-17A mAb. Moreover, IL-17A significantly induced eotaxin-1/CCL11 release and mRNA expression, an effect that was abrogated with cycloheximide and actinomycin D treatment. Furthermore, transfection studies using a luciferase-driven reporter construct containing eotaxin-1/CCL11 proximal promoter showed that IL-17A induced eotaxin-1/CCL11 at the transcriptional level. IL-17A also enhanced significantly IL-1beta-mediated eotaxin-1/CCL11 mRNA, protein release, and promoter activity in ASM cells. Primary human ASM cells pretreated with inhibitors of MAPK p38, p42/p44 ERK, JNK, or JAK but not PI3K, showed a significant decrease in eotaxin-1/CCL11 release upon IL-17A treatment. In addition, IL-17A mediated rapid phosphorylation of MAPK (p38, JNK, and p42/44 ERK) and STAT-3 but not STAT-6 or STAT-5 in ASM cells. Taken together, our data provide the first evidence of IL-17A-induced eotaxin-1/CCL11 expression in ASM cells via MAPK (p38, p42/p44 ERK, JNK) signaling pathways. Our results raise the possibility that IL-17A may play a role in allergic asthma by inducing eotaxin-1/CCL11 production.

Substrate and inhibitor specificity of kynurenine monooxygenase from Cytophaga hutchinsonii.

PubMed

Phillips, Robert S; Anderson, Andrew D; Gentry, Harvey G; Güner, Osman F; Bowen, J Phillip

2017-04-15

Kynurenine monooxygenase (KMO) is a potential drug target for treatment of neurodegenerative disorders such as Huntington's and Alzheimer's diseases. We have evaluated substituted kynurenines as substrates or inhibitors of KMO from Cytophaga hutchinsonii. Kynurenines substituted with a halogen at the 5-position are excellent substrates, with values of k cat and k cat /K m comparable to or higher than kynurenine. However, kynurenines substituted in the 3-position are competitive inhibitors, with K I values lower than the K m for kynurenine. Bromination also enhances inhibition, and 3,5-dibromokynurenine is a potent competitive inhibitor with a K I value of 1.5μM. A pharmacophore model of KMO was developed, and predicted that 3,4-dichlorohippuric acid would be an inhibitor. The K I for this compound was found to be 34μM, thus validating the pharmacophore model. We are using these results and our model to design more potent inhibitors of KMO. Copyright © 2017 Elsevier Ltd. All rights reserved.

Rosuvastatin Attenuates CD40L-Induced Downregulation of Extracellular Matrix Production in Human Aortic Smooth Muscle Cells via TRAF6-JNK-NF-κB Pathway

PubMed Central

Wang, Xiao-Lin; Zhou, Yuan-Li; Sun, Wei; Li, Li

2016-01-01

CD40L and statins exhibit pro-inflammatory and anti-inflammatory effects, respectively. They are both pleiotropic and can regulate extracellular matrix (ECM) degeneration in an atherosclerotic plaque. Statins can decrease both the CD40 expression and the resulting inflammation. However, the effects of CD40L and stains on atherosclerotic plaque ECM production and the underlying mechanisms are not well established. Moreover, prolyl-4-hydroxylase α1 (P4Hα1) is involved in collagen synthesis but its correlations with CD40L and statins are unknown. In the present study, CD40L suppressed P4Hα1 expression in human aortic smooth muscle cells (HASMCs) in a dose- and time-dependent manner, with insignificant changes in MMP2 expression and negative enzymatic activity of MMP9. CD40L increased TRAF6 expression, JNK phosphorylation, NF-κB nuclear translocation as well as DNA binding. Furthermore, silencing TRAF6, JNK or NF-κB genes abolished CD40L-induced suppression of P4Hα1. Lower NF-κB nuclear import rates were observed when JNK or TRAF6 silenced HASMCs were stimulated with CD40L compared to HASMCs with active JNK or TRAF6. Together, these results indicate that CD40L suppresses P4Hα1 expression in HASMCs by activating the TRAF6-JNK- NF-κB pathway. We also found that rosuvastatin inhibits CD40L-induced activation of the TRAF6-JNK- NF-κB pathway, thereby significantly rescuing the CD40L stimulated P4Hα1 inhibition. The results from this study will help find potential targets for stabilizing vulnerable atherosclerotic plaques. PMID:27120457

Biarylmethoxy Nicotinamides As Novel and Specific Inhibitors of Mycobacterium tuberculosis.

PubMed

Kedari, Chaitanya Kumar; Roy Choudhury, Nilanjana; Sharma, Sreevalli; Kaur, Parvinder; Guptha, Supreeth; Panda, Manoranjan; Mukerjee, Kakoli; Ramachandran, Vasanthi; Bandodkar, Balachandra; Ramachandran, Sreekanth; Tantry, Subramanyam J

2014-05-08

A whole cell based screening effort on a focused library from corporate collection resulted in the identification of biarylmethoxy nicotinamides as novel inhibitors of M. tuberculosis (Mtu) H37Rv. The series exhibited tangible structure-activity relationships, and during hit to lead exploration, a cellular potency of 100 nM was achieved, which is an improvement of >200-fold from the starting point. The series is very specific to Mtu and noncytotoxic up to 250 μM as measured in the mammalian cell line THP-1 based cytotoxicity assay. This compound class retains its potency on several drug sensitive and single drug resistant clinical isolates, which indicate that the compounds could be acting through a novel mode of action.

Therapeutic Effect of Topical Administration of SN50, an Inhibitor of Nuclear Factor-κB, in Treatment of Corneal Alkali Burns in Mice

PubMed Central

Saika, Shizuya; Miyamoto, Takeshi; Yamanaka, Osamu; Kato, Tadashi; Ohnishi, Yoshitaka; Flanders, Kathleen C.; Ikeda, Kazuo; Nakajima, Yuji; Kao, Winston W.-Y.; Sato, Misako; Muragaki, Yasuteru; Ooshima, Akira

2005-01-01

We evaluated the therapeutic efficacy of topical administration of SN50, an inhibitor of nuclear factor-κB, in a corneal alkali burn model in mice. An alkali burn was produced with 1 N NaOH in the cornea of C57BL/6 mice under general anesthesia. SN50 (10 μg/μl) or vehicle was topically administered daily for up to 12 days. The eyes were processed for histological or immunohistochemical examination after bromodeoxyuridine labeling or for semiquantification of cytokine mRNA. Topical SN50 suppressed nuclear factor-κB activation in local cells and reduced the incidence of epithelial defects/ulceration in healing corneas. Myofibroblast generation, macrophage invasion, activity of matrix metalloproteinases, basement membrane destruction, and expression of cytokines were all decreased in treated corneas compared with controls. To elucidate the role of tumor necrosis factor (TNF)-α in epithelial cell proliferation, we performed organ culture of mouse eyes with TNF-α, SN50, or an inhibitor of c-Jun N-terminal kinase (JNK) and examined cell proliferation in healing corneal epithelium in TNF-α−/− mice treated with SN50. An acceleration of epithelial cell proliferation by SN50 treatment was found to depend on TNF-α/JNK signaling. In conclusion, topical application of SN50 is effective in treating corneal alkali burns in mice. PMID:15855640

Protection of Momordica charantia polysaccharide against intracerebral hemorrhage-induced brain injury through JNK3 signaling pathway.

PubMed

Duan, Zhen-Zhen; Zhou, Xiao-Ling; Li, Yi-Hang; Zhang, Feng; Li, Feng-Ying; Su-Hua, Qi

2015-01-01

It has been well documented that Momordica charantia polysaccharide (MCP) has multiple biological effects such as immune enhancement, anti-oxidation and anti-cancer. However, the potential protective effects of MCP on stroke damage and its relative mechanisms remain unclear. Our present study demonstrated that MCP could scavenge reactive oxygen species (ROS) in intra-cerebral hemorrhage damage, significantly attenuating the neuronal death induced by thrombin in primary hippocampal neurons. Furthermore, we found that MCP prevented the activation of the c-Jun N-terminal protein kinase (JNK3), c-Jun and caspase-3, which was caused by the intra-cerebral hemorrhage injury. Taken together, our study demonstrated that MCP had a neuroprotective effect in response to intra-cerebral hemorrhage and its mechanisms involved the inhibition of JNK3 signaling pathway.

Urotensin II inhibited the proliferation of cardiac side population cells in mice during pressure overload by JNK-LRP6 signalling

PubMed Central

Chen, Zhidan; Xu, Jiahong; Ye, Yong; Li, Yang; Gong, Hui; Zhang, Guoping; Wu, Jian; jia, Jianguo; Liu, Ming; Chen, Ying; Yang, Chunjie; Tang, Yu; Zhu, Yichun; Ge, Junbo; Zou, Yunzeng

2014-01-01

Cardiac side population cells (CSPs) are promising cell resource for the regeneration in diseased heart as intrinsic cardiac stem cells. However, the relative low ratio of CSPs in the heart limited the ability of CSPs to repair heart and improve cardiac function effectively under pathophysiological condition. Which factors limiting the proliferation of CSPs in diseased heart are unclear. Here, we show that urotensin II (UII) regulates the proliferation of CSPs by c-Jun N-terminal kinase (JNK) and low density lipoprotein receptor-related protein 6 (LRP6) signalling during pressure overload. Pressure overload greatly upregulated UII level in plasma, UII receptor (UT) antagonist, urantide, promoted CSPs proliferation and improved cardiac dysfunction during chronic pressure overload. In cultured CSPs subjected to mechanical stretch (MS), UII significantly inhibited the proliferation by UT. Nanofluidic proteomic immunoassay showed that it is the JNK activation, but not the extracellular signal-regulated kinase signalling, that involved in the UII-inhibited- proliferation of CSPs during pressure overload. Further analysis in vitro indicated UII-induced-phospho-JNK regulates phosphorylation of LRP6 in cultured CSPs after MS, which is important in the inhibitory effect of UII on the CSPs during pressure overload. In conclusion, UII inhibited the proliferation of CSPs by JNK/LRP6 signalling during pressure overload. Pharmacological inhibition of UII promotes CSPs proliferation in mice, offering a possible therapeutic approach for cardiac failure induced by pressure overload. PMID:24447593

Lipopolysaccharide (LPS) of Porphyromonas gingivalis induces IL-1beta, TNF-alpha and IL-6 production by THP-1 cells in a way different from that of Escherichia coli LPS.

PubMed

Diya Zhang; Lili Chen; Shenglai Li; Zhiyuan Gu; Jie Yan

2008-04-01

Lipopolysaccharide (LPS) derived from the periodontal pathogen Porphyromonas gingivalis has been shown to differ from enterobacterial LPS in structure and function; therefore, the Toll-like receptors (TLRs) and the intracellular inflammatory signaling pathways are accordingly different. To elucidate the signal transduction pathway of P. gingivalis, LPS-induced pro-inflammatory cytokine production in the human monocytic cell line THP-1 was measured by ELISA, and the TLRs were determined by the blocking test using anti-TLRs antibodies. In addition, specific inhibitors as well as Phospho-ELISA kits were used to analyze the intracellular signaling pathways. Escherichia coli LPS was used as the control. In this study, P. gingivalis LPS showed the ability to induce cytokine production in THP-1 cells and its induction was significantly (P < 0.05) suppressed by anti-TLR2 antibody or JNK inhibitor, and the phosphorylation level of JNK was significantly increased (P < 0.05). These results indicate that TLR2-JNK is the main signaling pathway of P. gingivalis LPS-induced cytokine production, while the cytokine induction by E. coli LPS was mainly via TLR4-NF-kappaB and TLR4-p38MAPK. This suggests that P. gingivalis LPS differs from E. coli LPS in its signaling pathway in THP-1 cells, and that the TLR2-JNK pathway might play a significant role in P. gingivalis LPS-induced chronic inflammatory periodontal disease.

Arctic ground squirrel (Spermophilus parryii) hippocampal neurons tolerate prolonged oxygen– glucose deprivation and maintain baseline ERK1/2 and JNK activation despite drastic ATP loss

PubMed Central

Christian, Sherri L; Ross, Austin P; Zhao, Huiwen W; Kristenson, Heidi J; Zhan, Xinhua; Rasley, Brian T; Bickler, Philip E; Drew, Kelly L

2009-01-01

Oxygen–glucose deprivation (OGD) initiates a cascade of intracellular responses that culminates in cell death in sensitive species. Neurons from Arctic ground squirrels (AGS), a hibernating species, tolerate OGD in vitro and global ischemia in vivo independent of temperature or torpor. Regulation of energy stores and activation of mitogen-activated protein kinase (MAPK) signaling pathways can regulate neuronal survival. We used acute hippocampal slices to investigate the role of ATP stores and extracellular signal-regulated kinase (ERK)1/2 and Jun NH2-terminal kinase (JNK) MAPKs in promoting survival. Acute hippocampal slices from AGS tolerated 30 mins of OGD and showed a small but significant increase in cell death with 2 h OGD at 37°C. This tolerance is independent of hibernation state or season. Neurons from AGS survive OGD despite rapid ATP depletion by 3 mins in interbout euthermic AGS and 10 mins in hibernating AGS. Oxygen–glucose deprivation does not induce JNK activation in AGS and baseline ERK1/2 and JNK activation is maintained even after drastic depletion of ATP. Surprisingly, inhibition of ERK1/2 or JNK during OGD had no effect on survival, whereas inhibition of JNK increased cell death during normoxia. Thus, protective mechanisms promoting tolerance to OGD by AGS are downstream from ATP loss and are independent of hibernation state or season. PMID:18398417

A Tumor Cell-Selective Inhibitor of Mitogen-Activated Protein Kinase Phosphatases Sensitizes Breast Cancer Cells to Lymphokine-Activated Killer Cell Activity

PubMed Central

Kaltenmeier, Christof T.; Vollmer, Laura L.; Vernetti, Lawrence A.; Caprio, Lindsay; Davis, Keanu; Korotchenko, Vasiliy N.; Day, Billy W.; Tsang, Michael; Hulkower, Keren I.; Lotze, Michael T.

2017-01-01

Dual specificity mitogen-activated protein kinase (MAPK) phosphatases [dual specificity phosphatase/MAP kinase phosphatase (DUSP-MKP)] have been hypothesized to maintain cancer cell survival by buffering excessive MAPK signaling caused by upstream activating oncogenic products. A large and diverse body of literature suggests that genetic depletion of DUSP-MKPs can reduce tumorigenicity, suggesting that hyperactivating MAPK signaling by DUSP-MKP inhibitors could be a novel strategy to selectively affect the transformed phenotype. Through in vivo structure-activity relationship studies in transgenic zebrafish we recently identified a hyperactivator of fibroblast growth factor signaling [(E)-2-benzylidene-5-bromo-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI-215)] that is devoid of developmental toxicity and restores defective MAPK activity caused by overexpression of DUSP1 and DUSP6 in mammalian cells. Here, we hypothesized that BCI-215 could selectively affect survival of transformed cells. In MDA-MB-231 human breast cancer cells, BCI-215 inhibited cell motility, caused apoptosis but not primary necrosis, and sensitized cells to lymphokine-activated killer cell activity. Mechanistically, BCI-215 induced rapid and sustained phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) in the absence of reactive oxygen species, and its toxicity was partially rescued by inhibition of p38 but not JNK or ERK. BCI-215 also hyperactivated MKK4/SEK1, suggesting activation of stress responses. Kinase phosphorylation profiling documented BCI-215 selectively activated MAPKs and their downstream substrates, but not receptor tyrosine kinases, SRC family kinases, AKT, mTOR, or DNA damage pathways. Our findings support the hypothesis that BCI-215 causes selective cancer cell cytotoxicity in part through non-redox-mediated activation of MAPK signaling, and the findings also identify an intersection with immune cell killing that is

Upregulation of RhoB via c-Jun N-terminal kinase signaling induces apoptosis of the human gastric carcinoma NUGC-3 cells treated with NSC12618.

PubMed

Kim, Bo-Kyung; Kim, Hwan Mook; Chung, Kyung-Sook; Kim, Dong-Myung; Park, Song-Kyu; Song, Alexander; Won, Kyoung-Jae; Lee, Kiho; Oh, Yu-Kyoung; Lee, Kyeong; Song, Kyung-Bin; Simon, Julian A; Han, Gyoonhee; Won, Misun

2011-03-01

RhoB expression is reduced in most invasive tumors, with loss of RhoB expression correlating significantly with tumor stage. Here, we demonstrate that upregulation of RhoB by the potent anticancer agent NSC126188 induces apoptosis of NUGC-3 human gastric carcinoma cells. The crucial role of RhoB in NSC126188-induced apoptosis is indicated by the rescue of NUGC-3 cells from apoptosis by knockdown of RhoB. In the presence of NSC126188, c-Jun N-terminal kinase (JNK) signaling was activated, and the JNK inhibitor SP600125 reduced RhoB expression and suppressed the apoptosis of NUGC-3 cells. Knockdowns of mitogen-activated protein kinase kinase (MKK) 4/7, JNK1/2 and c-Jun downregulated RhoB expression and rescued cells from apoptotic death in the presence of NSC126188. The JNK inhibitor SP600125 suppressed transcriptional activation of RhoB in the presence of NSC126188, as indicated by a reporter assay that used luciferase under the RhoB promoter. The ability of NSC126188 to increase luciferase activity through both the p300-binding site and the inverted CCAAT sequence (iCCAAT box) suggests that JNK signaling to upregulate RhoB expression is mediated through both the p300-binding site and the iCCAAT box. However, the JNK inhibitor SP600125 did not inhibit the upregulation of RhoB by farnesyltransferase inhibitor (FTI)-277. The p300-binding site did not affect activation of the RhoB promoter by FTI-277 in NUGC-3 cells, suggesting that the transcriptional activation of RhoB by NSC126188 occurs by a different mechanism than that reported for FTIs. Our data indicate that NSC126188 increases RhoB expression via JNK-mediated signaling through a p300-binding site and iCCAAT box resulting in apoptosis of NUGC-3 cells.

Induction of apoptosis by thrombin in the cultured neurons of dorsal motor nucleus of the vagus.

PubMed

Wu, X; Zhang, W; Li, J-Y; Chai, B-X; Peng, J; Wang, H; Mulholland, M W

2011-03-01

A previous study demonstrated the presence of protease-activated receptor (PAR) 1 and 2 in the dorsal motor nucleus of vagus (DMV). The aim of this study is to characterize the effect of thrombin on the apoptosis of DMV neurons. The dorsal motor nucleus of vagus neurons were isolated from neonatal rat brainstems using micro-dissection and enzymatic digestion and cultured. Apoptosis of DMV neurons were examined in cultured neurons. Apoptotic neuron was examined by TUNEL and ELISA. Data were analyzed using anova and Student's t-test. Exposure of cultured DMV neurons to thrombin (0.1 to 10 U mL(-1)) for 24 h significantly increased apoptosis. Pretreatment of DMV neurons with hirudin attenuated the apoptotic effect of thrombin. Similar induction of apoptosis was observed for the PAR1 receptor agonist SFLLR, but not for the PAR3 agonist TFRGAP, nor for the PAR4 agonist YAPGKF. Protease-activated receptors 1 receptor antagonist Mpr(Cha) abolished the apoptotic effect of thrombin, while YPGKF, a specific antagonist for PAR4, demonstrated no effect. After administration of thrombin, phosphorylation of JNK and P38 occurred as early as 15 min, and remained elevated for up to 45 min. Pretreatment of DMV neurons with SP600125, a specific inhibitor for JNK, or SB203580, a specific inhibitor for P38, significantly inhibited apoptosis induced by thrombin. Thrombin induces apoptosis in DMV neurons through a mechanism involving the JNK and P38 signaling pathways. © 2010 Blackwell Publishing Ltd.

[The specific enzyme inhibitors for potential therapeutic use].

PubMed

Bretner, Maria

2015-01-01

Therapy for hepatitis C virus (HCV) initially consisted on administering ribavirin - having a broad spectrum of action - and pegylated interferon, and was only effective in 40-50% of patients. Appropriate was to find effective inhibitors of viral replication e.g. by inhibition of a viral enzyme, NTPase/helicase required in the process of translation and RNA replication of the HCV. We developed methods of synthesis of many compounds belonging to different groups - derivatives of nucleosides, benzotriazole, benzimidazole, tropolone and epirubicine. Some of the derivatives inhibit HCV helicase activity at low concentrations and reduces replication of the viral RNA in subgenomic replicon system. In the process of HCV replication casein kinase CK2 plays an important role. It regulates the level of phosphorylation of HCV protein NS5A, which affects the production of infectious virions of HCV. Effective and selective inhibitors of kinase CK2 could be of use in the treatment of HCV in combination with other drugs. CK2 kinase phosphorylates approximately 300 proteins that affect the growth, differentiation, proliferation or apoptosis. Elevated CK2 kinase activity has been observed in several types of cancer and other diseases, therefore, inhibitors of this enzyme are potential therapeutic importance, particularly for anti-cancer treatment. Research carried out in collaboration with prof. Shugar led to the synthesis of one of the most selective inhibitors of this enzyme which is 4,5,6,7-tetrabromo-1H-benzotriazole, used for the study of the role of kinase CK2 in a number of metabolic processes in tumor cells.

Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lasko, Loren M.; Jakob, Clarissa G.; Edalji, Rohinton P.

The dynamic and reversible acetylation of proteins, catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), is a major epigenetic regulatory mechanism of gene transcription1 and is associated with multiple diseases. Histone deacetylase inhibitors are currently approved to treat certain cancers, but progress on the development of drug-like histone actyltransferase inhibitors has lagged behind2. The histone acetyltransferase paralogues p300 and CREB-binding protein (CBP) are key transcriptional co-activators that are essential for a multitude of cellular processes, and have also been implicated in human pathological conditions (including cancer3). Current inhibitors of the p300 and CBP histone acetyltransferase domains, including natural products4,more » bi-substrate analogues5 and the widely used small molecule C6466,7, lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like catalytic inhibitor of p300 and CBP. We present a high resolution (1.95 Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 competes with acetyl coenzyme A (acetyl-CoA). A-485 selectively inhibited proliferation in lineage-specific tumour types, including several haematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen-sensitive and castration-resistant prostate cancer and inhibited tumour growth in a castration-resistant xenograft model. These results demonstrate the feasibility of using small molecule inhibitors to selectively target the catalytic activity of histone acetyltransferases, which may provide effective treatments for transcriptional activator-driven malignancies and diseases.« less

Cyclic Tensile Strain Upregulates Pro-Inflammatory Cytokine Expression Via FAK-MAPK Signaling in Chondrocytes.

PubMed

Yanoshita, Makoto; Hirose, Naoto; Okamoto, Yuki; Sumi, Chikako; Takano, Mami; Nishiyama, Sayuri; Asakawa-Tanne, Yuki; Horie, Kayo; Onishi, Azusa; Yamauchi, Yuka; Mitsuyoshi, Tomomi; Kunimatsu, Ryo; Tanimoto, Kotaro

2018-05-08

Excessive mechanical stimulation is considered an important factor in the destruction of chondrocytes. Focal adhesion kinase (FAK) is non-receptor tyrosine kinase related to a number of different signaling proteins. Little is known about the function of FAK in chondrocytes under mechanical stimulation. In the present study, we investigated the function of FAK in mechanical signal transduction and the mechanism through which cyclic tensile strain (CTS) induces expression of inflammation-related factors. Mouse ATDC5 chondrogenic cells were subjected to CTS of 0.5 Hz to 10% cell elongation with an FAK inhibitor. The expression of genes encoding COX-2, IL-1β, and TNF-α was examined using real-time RT-PCR after CTS application with FAK inhibitor. Phosphorylation of p-38, ERK, and JNK was analyzed by Western blotting. Differences in COX-2 expression following pretreatment with FAK, p-38, ERK, and JNK inhibitors were compared by Western blotting. We found that CTS increased the expression of genes encoding COX-2, IL-1β, and TNF-α and activated the phosphorylation of FAK, p-38, ERK, and JNK. Pretreatment with an FAK inhibitor for 2 h reduced the expression of genes encoding COX-2, IL-1β, and TNF-α induced by CTS-associated inflammation and decreased phosphorylation of FAK, p-38, ERK, and JNK. Pretreatment with FAK, p-38, ERK, and JNK inhibitors markedly suppressed COX-2 and IL-1β protein expression. In conclusion, FAK appears to regulate inflammation in chondrocytes under CTS via MAPK pathways.

MAPK/JNK1 activation protects cells against cadmium-induced autophagic cell death via differential regulation of catalase and heme oxygenase-1 in oral cancer cells.

PubMed

So, Keum-Young; Kim, Sang-Hun; Jung, Ki-Tae; Lee, Hyun-Young; Oh, Seon-Hee

2017-10-01

Antioxidant enzymes are related to oral diseases. We investigated the roles of heme oxygenase-1 (HO-1) and catalase in cadmium (Cd)-induced oxidative stress and the underlying molecular mechanism in oral cancer cells. Exposing YD8 cells to Cd reduced the expression levels of catalase and superoxide dismutase 1/2 and induced the expression of HO-1 as well as autophagy and apoptosis, which were reversed by N-acetyl-l-cysteine (NAC). Cd-exposed YD10B cells exhibited milder effects than YD8 cells, indicating that Cd sensitivity is associated with antioxidant enzymes and autophagy. Autophagy inhibition via pharmacologic and genetic modulations enhanced Cd-induced HO-1 expression, caspase-3 cleavage, and the production of reactive oxygen species (ROS). Ho-1 knockdown increased autophagy and apoptosis. Hemin treatment partially suppressed Cd-induced ROS production and apoptosis, but enhanced autophagy and CHOP expression, indicating that autophagy induction is associated with cellular stress. Catalase inhibition by pharmacological and genetic modulations increased Cd-induced ROS production, autophagy, and apoptosis, but suppressed HO-1, indicating that catalase is required for HO-1 induction. p38 inhibition upregulated Cd-induced phospho-JNK and catalase, but suppressed HO-1, autophagy, apoptosis. JNK suppression exhibited contrary results, enhancing the expression of phospho-p38. Co-suppression of p38 and JNK1 failed to upregulate catalase and procaspase-3, which were upregulated by JNK1 overexpression. Overall, the balance between the responses of p38 and JNK activation to Cd appears to have an important role in maintaining cellular homeostasis via the regulation of antioxidant enzymes and autophagy induction. In addition, the upregulation of catalase by JNK1 activation can play a critical role in cell protection against Cd-induced oxidative stress. Copyright © 2017 Elsevier Inc. All rights reserved.

Development of Specific Inhibitors for Breast Cancer-Associated Variants of ErbB2

DTIC Science & Technology

2015-10-01

activity measurements (Months 9-15) Specific Aim 3: Identifying inhibitors of ErbB2 mutants.* Major Task 5: Produce ErbB2 structures for drug -lead...identified the activated cancer- associated ErbB2 mutants that will be used for drug screening, and we have established enzyme assays that will be suitable...during protein expression and purification. We measured enzyme activity using two assays: (1) a continuous spectrophotometric assay. In this assay

Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans*

PubMed Central

Andrusiak, Matthew G.; Jin, Yishi

2016-01-01

Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundworm Caenorhabditis elegans was developed as a system to study genes required for development and nervous system function. The powerful genetics of C. elegans in combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components in C. elegans. PMID:26907690

Histamine acting on H1 receptor promotes inhibition of proliferation via PLC, RAC, and JNK-dependent pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Notcovich, Cintia; Laboratorio de Farmacologia de Receptores, Catedra de Quimica Medicinal, Departamento de Farmacologia, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires; Diez, Federico

2010-02-01

It is well established that histamine modulates cell proliferation through the activation of the histamine H1 receptor (H1R), a G protein-coupled receptor (GPCR) that is known to couple to phospholipase C (PLC) activation via Gq. In the present study, we aimed to determine whether H1R activation modulates Rho GTPases, well-known effectors of Gq/G{sub 11}-coupled receptors, and whether such modulation influences cell proliferation. Experiments were carried out in CHO cells stably expressing H1R (CHO-H1R). By using pull-down assays, we found that both histamine and a selective H1R agonist activated Rac and RhoA in a time- and dose-dependent manner without significant changesmore » in the activation of Cdc42. Histamine response was abolished by the H1R antagonist mepyramine, RGS2 and the PLC inhibitor U73122, suggesting that Rac and RhoA activation is mediated by H1R via Gq coupling to PLC stimulation. Histamine caused a marked activation of serum response factor activity via the H1R, as determined with a serum-responsive element (SRE) luciferase reporter, and this response was inhibited by RhoA inactivation with C3 toxin. Histamine also caused a significant activation of JNK which was inhibited by expression of the Rac-GAP {beta}2-chimaerin. On the other hand, H1R-induced ERK1/2 activation was inhibited by U73122 but not affected by C3 or {beta}2-chimaerin, suggesting that ERK1/2 activation was dependent on PLC and independent of RhoA or Rac. [{sup 3}H]-Thymidine incorporation assays showed that both histamine and the H1R agonist inhibited cell proliferation in a dose-dependent manner and that the effect was independent of RhoA but partially dependent on JNK and Rac. Our results reveal that functional coupling of the H1R to Gq-PLC leads to the activation of RhoA and Rac small GTPases and suggest distinct roles for Rho GTPases in the control of cell proliferation by histamine.« less

Chlorogenic acid alleviates autophagy and insulin resistance by suppressing JNK pathway in a rat model of nonalcoholic fatty liver disease.

PubMed

Yan, Hua; Gao, Yan-Qiong; Zhang, Ying; Wang, Huan; Liu, Gui-Sheng; Lei, Jian-Yuan

2018-06-01

Non-alcoholic fatty liver disease (NAFLD) is one of the leading causes of chronic liver diseases around the world and commonly associated with insulin resistance and hyperlipidemia. Chlorogenic acid (CG) was reported to have insulinsensitizing activity and exert hypocholesterolemic and hypoglycemic effect. However, the involvement of CG in NAFLD remains far from being addressed. In this study, a high-fat diet-induced NAFLD rat model was used to investigate the biological roles and underlying mechanism of CG in NAFLD. The results showed that high-fat diet-fed rats exhibited an increase in body weight, glucose tolerance, liver injury, insulin resistance, as well as autophagy and C-Jun N-terminal kinase (JNK) pathway. Nevertheless, all these effects were alleviated by CG treatment. Moreover, angiotensin treatment in CG group activated the JNK pathway, and promoted autophagy, insulin resistance, and liver injury. In conclusion, our findings demonstrated that CG ameliorated liver injury and insulin resistance by suppressing autophagy via inactivation of JNK pathway in a rat model of NAFLD. Therefore, CG might be a potential application for the treatment of NAFLD.

Pharmacodynamic effects of C-domain-specific ACE inhibitors on the renin-angiotensin system in myocardial infarcted rats.

PubMed

Sharp, Sarah; Poglitsch, Marko; Zilla, Peter; Davies, Neil H; Sturrock, Edward D

2015-12-01

The renin-angiotensin system (RAS) is a dynamic network that plays a critical role in blood pressure regulation and fluid and electrolyte homeostasis. Modulators of the RAS, such as angiotensin-converting enzyme (ACE) inhibitors, are widely used to treat hypertension, heart failure and myocardial infarction. The effect of ACE inhibitors (lisinopril and C-domain-selective LisW-S) on the constituent peptides of the RAS following myocardial infarction was examined in rats. Ten angiotensin peptides were analysed using a sensitive LC-MS/MS-based assay to examine both the circulating and equilibrium levels of these peptides. Administration of lisinopril or LisW-S caused a significant decrease in Ang 1-8/Ang 1-10 ratios as determined by circulating and equilibrium peptide level analysis. Furthermore, Ang 1-7 levels were elevated by both ACE inhibitors, but only lisinopril decreased the Ang 1-5/Ang 1-7 ratio. This indicates LisW-S C-domain specificity as Ang 1-5 is generated by hydrolysis of Ang 1-7 by the N-domain. Further corroboration of LisW-S C-domain specificity is that only lisinopril increased the circulating levels of the N-domain ACE substrate Ac-SDKP. LisW-S is able to effectively block ACE in vivo by C-domain-selective inhibition. The LC-MS/MS-based assay allows the evaluation of the pharmacologic impact of RAS inhibitors in different pathophysiological conditions. © The Author(s) 2015.

3,3'-Diindolylmethane is a novel mitochondrial H(+)-ATP synthase inhibitor that can induce p21(Cip1/Waf1) expression by induction of oxidative stress in human breast cancer cells.

PubMed

Gong, Yixuan; Sohn, Heesook; Xue, Ling; Firestone, Gary L; Bjeldanes, Leonard F

2006-05-01

Epidemiologic evidence suggests that high dietary intake of Brassica vegetables, such as broccoli, cabbage, and Brussels sprouts, protects against tumorigenesis in multiple organs. 3,3'-Diindolylmethane, one of the active products derived from Brassica vegetables, is a promising antitumor agent. Previous studies in our laboratory showed that 3,3'-diindolylmethane induced a G(1) cell cycle arrest in human breast cancer MCF-7 cells by a mechanism that included increased expression of p21. In the present study, the upstream events leading to p21 overexpression were further investigated. We show for the first time that 3,3'-diindolylmethane is a strong mitochondrial H(+)-ATPase inhibitor (IC(50) approximately 20 micromol/L). 3,3'-Diindolylmethane treatment induced hyperpolarization of mitochondrial inner membrane, decreased cellular ATP level, and significantly stimulated mitochondrial reactive oxygen species (ROS) production. ROS production, in turn, led to the activation of stress-activated pathways involving p38 and c-Jun NH(2)-terminal kinase. Using specific kinase inhibitors (SB203580 and SP600125), we showed the central role of p38 and c-Jun NH(2)-terminal kinase (JNK) pathways in 3,3'-diindolylmethane-induced p21 mRNA transcription. In addition, antioxidants significantly attenuated 3,3'-diindolylmethane-induced activation of p38 and JNK and induction of p21, indicating that oxidative stress is the major trigger of these events. To further support the role of ROS in 3,3'-diindolylmethane-induced p21 overexpression, we showed that 3,3'-diindolylmethane failed to induce p21 overexpression in mitochondrial respiratory chain deficient rho(0) MCF-7 cells, in which 3,3'-diindolylmethane did not stimulate ROS production. Thus, we have established the critical role of enhanced mitochondrial ROS release in 3,3'-diindolylmethane-induced p21 up-regulation in human breast cancer cells.

A Fat-Facets-Dscam1-JNK Pathway Enhances Axonal Growth in Development and after Injury

PubMed Central

Koch, Marta; Nicolas, Maya; Zschaetzsch, Marlen; de Geest, Natalie; Claeys, Annelies; Yan, Jiekun; Morgan, Matthew J.; Erfurth, Maria-Luise; Holt, Matthew; Schmucker, Dietmar; Hassan, Bassem A.

2018-01-01

Injury to the adult central nervous systems (CNS) can result in severe long-term disability because damaged CNS connections fail to regenerate after trauma. Identification of regulators that enhance the intrinsic growth capacity of severed axons is a first step to restore function. Here, we conducted a gain-of-function genetic screen in Drosophila to identify strong inducers of axonal growth after injury. We focus on a novel axis the Down Syndrome Cell Adhesion Molecule (Dscam1), the de-ubiquitinating enzyme Fat Facets (Faf)/Usp9x and the Jun N-Terminal Kinase (JNK) pathway transcription factor Kayak (Kay)/Fos. Genetic and biochemical analyses link these genes in a common signaling pathway whereby Faf stabilizes Dscam1 protein levels, by acting on the 3′-UTR of its mRNA, and Dscam1 acts upstream of the growth-promoting JNK signal. The mammalian homolog of Faf, Usp9x/FAM, shares both the regenerative and Dscam1 stabilizing activities, suggesting a conserved mechanism. PMID:29472843

WISP-2 in human gastric cancer and its potential metastatic suppressor role in gastric cancer cells mediated by JNK and PLC-γ pathways.

PubMed

Ji, Jiafu; Jia, Shuqin; Jia, Yongning; Ji, Ke; Hargest, Rachel; Jiang, Wen G

2015-09-15

It has recently been shown that WISP proteins (Wnt-inducted secreted proteins), a group of intra- and extra-cellular regulatory proteins, have been implicated in the initiation and progression of a variety of tumour types including colorectal and breast cancer. However, the role of WISP proteins in gastric cancer (GC) cells and their clinical implications have not yet been elucidated. The expression of WISP molecules in a cohort of GC patients was analysed using real-time quantitative PCR and immunohistochemistry. The expression of a panel of recognised epithelial-mesenchymal transition (EMT) markers was quantified using Q-PCR in paired tumour and normal tissues. WISP-2 knockdown (kd) sublines using ribozyme transgenes were created in the GC cell lines AGS and HGC27. Subsequently, several biological functions, including cell growth, adhesion, migration and invasion, were studied. Potential pathways for the interaction of EMT, extracellular matrix and MMP were evaluated. Overexpression of WISP-2 was detected in GC and significantly correlated with early tumour node-metastasis staging, differentiation status and positively correlated with overall survival and disease-free survival of the patients. WISP-2 expression was inversely correlated with that of Twist and Slug in paired samples. Kd of WISP-2 expression promoted the proliferation, migration and invasion of GC cells. WISP-2 suppressed GC cell metastasis through reversing EMT and suppressing the expression and activity of MMP9 and MMP2 via JNK and ERK. Cell motility analysis indicated that WISP-2 kd contributed to GC cells' motility and can be attenuated by PLC-γ and JNK small inhibitors. Increased expression of WISP-2 in GC is positively correlated with favourable clinical features and the survival of patients with GC and is a negative regulator of growth, migration and invasion in GC cells. These findings suggest that WISP-2 is a potential tumour suppressor in GC.

Ultrasound Stimulation of Different Dental Stem Cell Populations: Role of Mitogen-activated Protein Kinase Signaling.

PubMed

Gao, Qianhua; Walmsley, A Damien; Cooper, Paul R; Scheven, Ben A

2016-03-01

Mesenchymal stem cells (MSCs) from dental tissues may respond to low-intensity pulsed ultrasound (LIPUS) treatment, potentially providing a therapeutic approach to promoting dental tissue regeneration. This work aimed to compare LIPUS effects on the proliferation and MAPK signaling in MSCs from rodent dental pulp stem cells (DPSCs) compared with MSCs from periodontal ligament stem cells (PDLSCs) and bone marrow stem cells (BMSCs). Isolated MSCs were treated with 1-MHz LIPUS at an intensity of 250 or 750 mW/cm2 for 5 or 20 minutes. Cell proliferation was evaluated by 5-bromo-2-deoxyuridine (BrdU) staining after 24 hours of culture following a single LIPUS treatment. Specific ELISAs were used to determine the total and activated p38, ERK1/2, and JNK MAPK signaling proteins up to 4 hours after treatment. Selective MAPK inhibitors PD98059 (ERK1/2), SB203580 (p38), and SP600125 (JNK) were used to determine the role of activation of the particular MAPK pathways. The proliferation of all MSC types was significantly increased after LIPUS treatment. LIPUS at a 750-mW/cm2 dose induced the greatest effects on DPSCs. BMSC proliferation was stimulated in equal measures by both intensities, whereas 250 mW/cm2 LIPUS exposure exerted maximum effects on PDLSCs. ERK1/2 was activated immediately in DPSCs after treatment. Concomitantly, DPSC proliferation was specifically modulated by ERK1/2 inhibition, whereas p38 and JNK inhibition exerted no effects. In BMSCs, JNK MAPK signaling was LIPUS activated, and the increase in proliferation was blocked by specific inhibition of the JNK pathway. In PDLSCs, JNK MAPK signaling was activated immediately after LIPUS, whereas p-p38 MAPK increased significantly in these cells 4 hours after exposure. Correspondingly, JNK and p38 inhibition modulated LIPUS-stimulated PDLSC proliferation. LIPUS promoted MSC proliferation in an intensity and cell-specific dependent manner via activation of distinct MAPK pathways. Copyright © 2016 American

The gap junction inhibitor 2-aminoethoxy-diphenyl-borate protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes and c-jun N-terminal kinase activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Kuo; Williams, C. David; McGill, Mitchell R.

2013-12-15

Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the US. Although many aspects of the mechanism are known, recent publications suggest that gap junctions composed of connexin32 function as critical intercellular communication channels which transfer cytotoxic mediators into neighboring hepatocytes and aggravate liver injury. However, these studies did not consider off-target effects of reagents used in these experiments, especially the gap junction inhibitor 2-aminoethoxy-diphenyl-borate (2-APB). In order to assess the mechanisms of protection of 2-APB in vivo, male C56Bl/6 mice were treated with 400 mg/kg APAP to cause extensive liver injury. This injury was prevented whenmore » animals were co-treated with 20 mg/kg 2-APB and was attenuated when 2-APB was administered 1.5 h after APAP. However, the protection was completely lost when 2-APB was given 4–6 h after APAP. Measurement of protein adducts and c-jun-N-terminal kinase (JNK) activation indicated that 2-APB reduced both protein binding and JNK activation, which correlated with hepatoprotection. Although some of the protection was due to the solvent dimethyl sulfoxide (DMSO), in vitro experiments clearly demonstrated that 2-APB directly inhibits cytochrome P450 activities. In addition, JNK activation induced by phorone and tert-butylhydroperoxide in vivo was inhibited by 2-APB. The effects against APAP toxicity in vivo were reproduced in primary cultured hepatocytes without use of DMSO and in the absence of functional gap junctions. We conclude that the protective effect of 2-APB was caused by inhibition of metabolic activation of APAP and inhibition of the JNK signaling pathway and not by blocking connexin32-based gap junctions. - Highlights: • 2-APB protected against APAP-induced liver injury in mice in vivo and in vitro • 2-APB protected by inhibiting APAP metabolic activation and JNK signaling pathway • DMSO inhibited APAP metabolic activation as the solvent of 2

Exploring the role of MKK7 in excitotoxicity and cerebral ischemia: a novel pharmacological strategy against brain injury

PubMed Central

Vercelli, A; Biggi, S; Sclip, A; Repetto, I E; Cimini, S; Falleroni, F; Tomasi, S; Monti, R; Tonna, N; Morelli, F; Grande, V; Stravalaci, M; Biasini, E; Marin, O; Bianco, F; di Marino, D; Borsello, T

2015-01-01

Excitotoxicity following cerebral ischemia elicits a molecular cascade, which leads to neuronal death. c-Jun-N-terminal kinase (JNK) has a key role in excitotoxic cell death. We have previously shown that JNK inhibition by a specific cell-permeable peptide significantly reduces infarct size and neuronal death in an in vivo model of cerebral ischemia. However, systemic inhibition of JNK may have detrimental side effects, owing to blockade of its physiological function. Here we designed a new inhibitor peptide (growth arrest and DNA damage-inducible 45β (GADD45β-I)) targeting mitogen-activated protein kinase kinase 7 (MKK7), an upstream activator of JNK, which exclusively mediates JNK's pathological activation. GADD45β-I was engineered by optimizing the domain of the GADD45β, able to bind to MKK7, and by linking it to the TAT peptide sequence, to allow penetration of biological membranes. Our data clearly indicate that GADD45β-I significantly reduces neuronal death in excitotoxicity induced by either N-methyl-D-aspartate exposure or by oxygen–glucose deprivation in vitro. Moreover, GADD45β-I exerted neuroprotection in vivo in two models of ischemia, obtained by electrocoagulation and by thromboembolic occlusion of the middle cerebral artery (MCAo). Indeed, GADD45β-I reduced the infarct size when injected 30 min before the lesion in both models. The peptide was also effective when administrated 6 h after lesion, as demonstrated in the electrocoagulation model. The neuroprotective effect of GADD45β-I is long lasting; in fact, 1 week after MCAo the infarct volume was still reduced by 49%. Targeting MKK7 could represent a new therapeutic strategy for the treatment of ischemia and other pathologies involving MKK7/JNK activation. Moreover, this new inhibitor can be useful to further dissect the physiological and pathological role of the JNK pathway in the brain. PMID:26270349

Characterization of CAA0225, a novel inhibitor specific for cathepsin L, as a probe for autophagic proteolysis.

PubMed

Takahashi, Katsuyuki; Ueno, Takashi; Tanida, Isei; Minematsu-Ikeguchi, Naoko; Murata, Mitsuo; Kominami, Eiki

2009-03-01

We screened a series of new epoxysuccinyl peptides for the development of a lysosomal cathepsin L-specific inhibitor. Among the compounds tested, (2S,3S)-oxirane-2,3-dicarboxylic acid 2-[((S)-1-benzylcarbamoyl-2-phenyl-ethyl)-amide] 3-{[2-(4-hydroxy-phenyl)-ethyl]-amide} (compound CAA0225) was the most potent inhibitor of cathepsin L. CAA0225 inhibited rat liver cathepsin L with IC50 values of 1.9 nM, but not rat liver cathepsin B (IC50, >1000-5000 nM). To assess the contribution of cathepsin L to lysosomal proteolysis, we evaluated autophagy, which is the process of lysosomal self-degradation of cell constituents. In HeLa and Huh-7 cells cultured under nutrient-deprived conditions CAA0225 significantly inhibited degradation of long-lived proteins; however, the magnitude of inhibition was comparable to that in the presence of CA-074-OMe, which is a cathepsin B-specific inhibitor. Thus the contributions of cathepsin L and cathepsin B to autophagic protein degradation of cytoplasmic proteins are nearly equal. During autophagy, microtubule-associated protein IA/IB light chain 3-II (LC3-II) and gamma-aminobutyric acid (A) receptor-associated protein (GABARAP)-II, which are specific markers of autophagosomal membranes that engulf cytoplasmic components, also undergo degradation upon fusion of autophagosomes with lysosomes. CAA0225 effectively inhibited degradation of LC3-II and GABARAP, whereas CA-074-OMe had only a marginal effect on their levels. Therefore we conclude that cathepsin L does not play a general role in the degradation of proteins in the lumen of autophagosomes, but rather is involved specifically in the degradation of autophagosomal membrane markers.

Arsenic trioxide mediates HAPI microglia inflammatory response and subsequent neuron apoptosis through p38/JNK MAPK/STAT3 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mao, Jiamin

Arsenic is a widely distributed toxic metalloid all over the world. Inorganic arsenic species are supposed to affect astrocytic functions and to cause neuron apoptosis in CNS. Microglias are the key cell type involved in innate immune responses in CNS, and microglia activation has been linked to inflammation and neurotoxicity. In this study, using ELISA, we showed that Arsenic trioxide up-regulated the expression and secretion of IL-1β in a dose-dependent manner and a time-dependent manner in cultured HAPI microglia cells. The secretion of IL-1β caused the apoptosis of SH-SY5Y. These pro-inflammatory responses were inhibited by the STAT3 blocker, AG490 andmore » P38/JNK MAPK blockers SB202190, SP600125. Further, Arsenic trioxide exposure could induce phosphorylation and activation of STAT3, and the translocation of STAT3 from the cytosol to the nucleus in this HAPI microglia cell line. Thus, the STAT3 signaling pathway can be activated after Arsenic trioxide treatment. However, P38/JNK MAPK blockers SB202190, SP600125 also obviously attenuated STAT3 activation and transnuclear transport induced by Arsenic trioxide. In concert with these results, we highlighted that the secretion of IL-1β and STAT3 activation induced by Arsenic trioxide can be mediated by elevation of P38/JNK MAPK in HAPI microglia cells and then induced the toxicity of neurons. - Highlights: • Arsenic trioxide exposure induced expression of IL-β in HAPI microglia. • Arsenic trioxide exposure induced activation of MAPK pathways in HAPI microglia. • Arsenic trioxide exposure induced activation of STAT3 pathways in HAPI microglia. • The expression of IL-β though P38/JNK MAPK/STAT3 pathways in HAPI microglia.« less

Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans.

PubMed

Andrusiak, Matthew G; Jin, Yishi

2016-04-08

Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundwormCaenorhabditis eleganswas developed as a system to study genes required for development and nervous system function. The powerful genetics ofC. elegansin combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components inC. elegans. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Gasdermin C is induced by ultraviolet light and contributes to MMP-1 expression via activation of ERK and JNK pathways.

PubMed

Kusumaningrum, Novi; Lee, Dong Hun; Yoon, Hyun-Sun; Kim, Yeon Kyung; Park, Chi-Hyun; Chung, Jin Ho

2018-05-01

Ultraviolet (UV) radiation plays important roles in various skin diseases including premature aging and cancer. UV has been shown to regulate the expressions of many genes including matrix metalloproteinases (MMPs). Gasdermin C (GSDMC) belongs to Gasdermin family and is known to be expressed in the epithelial cells of many tissues including the skin. However, the functions of GSDMC remain poorly understood. We aimed to investigate the role of GSDMC in UV-induced MMP-1, MMP-3, and MMP-9 expressions in human skin keratinocytes. Primary human skin keratinocytes and an immortalized human skin keratinocyte cell line (HaCaT cells) were irradiated with UV. Knockdown and overexpression of GSDMC were performed to study the effect of GSDMC. The mRNA and protein levels were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. We found that GSDMC expression is increased by UV irradiation in human skin keratinocytes. Further studies showed that GSDMC expression is increased at relatively late time points after UV irradiation and that this GSDMC induction plays important roles in the expressions of MMP-1, but not of MMP-3 and MMP-9, and the activations of ERK and JNK induced by UV. In addition, we found that overexpression of GSDMC increases the MMP-1 expression and the activities of ERK and JNK and that GSDMC-induced MMP-1 expression is suppressed by inhibition of ERK or JNK activities. Our results suggest that GSDMC is increased by UV radiation and contributes to UV-induced MMP-1 expression through the activation of ERK and JNK pathways. Copyright © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Ligand-based and structure-based approaches in identifying ideal pharmacophore against c-Jun N-terminal kinase-3.

PubMed

Kumar, B V S Suneel; Kotla, Rohith; Buddiga, Revanth; Roy, Jyoti; Singh, Sardar Shamshair; Gundla, Rambabu; Ravikumar, Muttineni; Sarma, Jagarlapudi A R P

2011-01-01

Structure and ligand based pharmacophore modeling and docking studies carried out using diversified set of c-Jun N-terminal kinase-3 (JNK3) inhibitors are presented in this paper. Ligand based pharmacophore model (LBPM) was developed for 106 inhibitors of JNK3 using a training set of 21 compounds to reveal structural and chemical features necessary for these molecules to inhibit JNK3. Hypo1 consisted of two hydrogen bond acceptors (HBA), one hydrogen bond donor (HBD), and a hydrophobic (HY) feature with a correlation coefficient (r²) of 0.950. This pharmacophore model was validated using test set containing 85 inhibitors and had a good r² of 0.846. All the molecules were docked using Glide software and interestingly, all the docked conformations showed hydrogen bond interactions with important hinge region amino acids (Gln155 and Met149)and these interactions were compared with Hypo1 features. The results of ligand based pharmacophore model (LBPM)and docking studies are validated each other. The structure based pharmacophore model (SBPM) studies have identified additional features, two hydrogen bond donors and one hydrogen bond acceptor. The combination of these methodologies is useful in designing ideal pharmacophore which provides a powerful tool for the discovery of novel and selective JNK3 inhibitors.

The contribution of c-Jun N-terminal kinase activation and subsequent Bcl-2 phosphorylation to apoptosis induction in human B-cells is dependent on the mode of action of specific stresses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muscarella, Donna E.; Bloom, Stephen E.

2008-04-01

The c-Jun N-terminal kinase (JNK) pathway can play paradoxical roles as either a pro-survival or a pro-cell death pathway depending on type of stress and cell type. The goal of the present study was to determine the role of JNK pathway signaling for regulating B-cell apoptosis in two important but contrasting situations-global proteotoxic damage, induced by arsenite and hyperthermia, versus specific microtubule inhibition, induced by the anti-cancer drug vincristine, using the EW36 B-cell line. This cell line over-expresses the Bcl-2 protein and is a useful model to identify treatments that can overcome multi-drug resistance in lymphoid cells. Exposure of EW36more » B-cells to arsenite or lethal hyperthermia resulted in activation of the JNK pathway and induction of apoptosis. However, pharmacological inhibition of the JNK pathway did not inhibit apoptosis, indicating that JNK pathway activation is not required for apoptosis induction by these treatments. In contrast, vincristine treatment of EW36 B-cells resulted in JNK activation and apoptosis that was suppressed by JNK inhibition. A critical difference between the two types of stress treatments was that only vincristine-induced JNK activation resulted in phosphorylation of Bcl-2 at threonine-56, a modification that can block its anti-apoptotic function. Importantly, Bcl-2 phosphorylation was attenuated by JNK inhibition implicating JNK as the upstream kinase. Furthermore, arsenite and hyperthermia treatments activated a p53/p21 pathway associated with apoptosis induction, whereas vincristine did not activate this pathway. These results reveal two stress-activated pathways, one JNK-dependent and another JNK-independent, either of which can bypass Bcl-2 mediated resistance, resulting in cell death.« less

Bex2 regulates cell proliferation and apoptosis in malignant glioma cells via the c-Jun NH2-terminal kinase pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Xiuping, E-mail: xpzhou@xzmc.edu.cn; Lab of Neurosurgery, Xuzhou Medical College, Xuzhou, Jiangsu; Key Laboratory of Brain Disease Biology, Affiliated Hospital of Xuzhou Medical College, Jiangsu

Highlights: Black-Right-Pointing-Pointer The expression levels of Bex2 markedly increased in glioma tissues. Black-Right-Pointing-Pointer Bex2 over-expression promoted cell proliferation, while its down-regulation inhibited cell growth. Black-Right-Pointing-Pointer Bex2 down-regulation promoted cell apoptosis via JNK/c-Jun signaling pathway. -- Abstract: The function of Bex2, a member of the Brain Expressed X-linked gene family, in glioma is controversial and its mechanism is largely unknown. We report here that Bex2 regulates cell proliferation and apoptosis in malignant glioma cells via the c-Jun NH2-terminal kinase (JNK) pathway. The expression level of Bex2 is markedly increased in glioma tissues. We observed that Bex2 over-expression promotes cell proliferation, whilemore » down-regulation of Bex2 inhibits cell growth. Furthermore, Bex2 down-regulation promotes cell apoptosis and activates the JNK pathway; these effects were abolished by administration of the JNK specific inhibitor, (SP600125). Thus, Bex2 may be an important player during the development of glioma.« less

Molecular dynamics of conformation-specific dopamine transporter-inhibitor complexes.

PubMed

Jean, Bernandie; Surratt, Christopher K; Madura, Jeffry D

2017-09-01

The recreational psychostimulant cocaine inhibits dopamine reuptake from the synapse, resulting in excessive stimulation of postsynaptic dopamine receptors in brain areas associated with reward and addiction. Cocaine binds to and stabilizes the outward- (extracellular-) facing conformation of the dopamine transporter (DAT) protein, while the low abuse potential DAT inhibitor benztropine prefers the inward- (cytoplasmic-) facing conformation. A correlation has been previously postulated between psychostimulant abuse potential and preference for the outward-facing DAT conformation. The 3β-aryltropane cocaine analogs LX10 and LX11, however, differ only in stereochemistry and share a preference for the outward-facing DAT, yet are reported to vary widely in abuse potential in an animal model. In search of the molecular basis for DAT conformation preference, complexes of cocaine, benztropine, LX10 or LX11 bound to each DAT conformation were subjected to 100ns of all-atom molecular dynamics simulation. Results were consistent with previous findings from cysteine accessibility assays used to assess an inhibitor's DAT conformation preference. The respective 2β- and 2α-substituted phenyltropanes of LX10 and LX11 interacted with hydrophobic regions of the DAT S1 binding site that were inaccessible to cocaine. Solvent accessibility measurements also revealed subtle differences in inhibitor positioning within a given DAT conformation. This work serves to advance our understanding of the conformational selectivity of DAT inhibitors and suggests that MD may be useful in antipsychostimulant therapeutic design. Copyright © 2017 Elsevier Inc. All rights reserved.

Demonstration of isoleucine 199 as a structural determinant for the selective inhibition of human monoamine oxidase B by specific reversible inhibitors.

PubMed

Hubálek, Frantisek; Binda, Claudia; Khalil, Ashraf; Li, Min; Mattevi, Andrea; Castagnoli, Neal; Edmondson, Dale E

2005-04-22

Several reversible inhibitors selective for human monoamine oxidase B (MAO B) that do not inhibit MAO A have been described in the literature. The following compounds: 8-(3-chlorostyryl)caffeine, 1,4-diphenyl-2-butene, and trans,trans-farnesol are shown to inhibit competitively human, horse, rat, and mouse MAO B with K(i) values in the low micromolar range but are without effect on either bovine or sheep MAO B or human MAO A. In contrast, the reversible competitive inhibitor isatin binds to all known MAO B and MAO A with similar affinities. Sequence alignments and the crystal structures of human MAO B in complex with 1,4-diphenyl-2-butene or with trans,trans-farnesol provide molecular insights into these specificities. These inhibitors span the substrate and entrance cavities with the side chain of Ile-199 rotated out of its normal conformation suggesting that Ile-199 is gating the substrate cavity. Ile-199 is conserved in all known MAO B sequences except bovine MAO B, which has Phe in this position (the sequence of sheep MAO B is unknown). Phe is conserved in the analogous position in MAO A sequences. The human MAO B I199F mutant protein of MAO B binds to isatin (K(i) = 3 microM) but not to the three inhibitors listed above. The crystal structure of this mutant demonstrates that the side chain of Phe-199 interferes with the binding of those compounds. This suggests that the Ile-199 "gate" is a determinant for the specificity of these MAO B inhibitors and provides a molecular basis for the development of MAO B-specific reversible inhibitors without interference with MAO A function in neurotransmitter metabolism.

Discovery of Specific Inhibitors for Intestinal E. coli   β-Glucuronidase through In Silico Virtual Screening

PubMed Central

Cheng, Ta-Chun; Cheng, Kai-Wen; Leu, Yu-Lin; Chuang, Chih-Hung; Huang, Chien-Chaio; Hsieh, Yuan-Chin; Chang, Long-Sen; Cheng, Tian-Lu

2015-01-01

Glucuronidation is a major metabolism process of detoxification for carcinogens, 4-(methylnitrosamino)-1-(3-pyridy)-1-butanone (NNK) and 1,2-dimethylhydrazine (DMH), of reactive oxygen species (ROS). However, intestinal E. coli    β-glucuronidase (eβG) has been considered pivotal to colorectal carcinogenesis. Specific inhibition of eβG may prevent reactivating the glucuronide-carcinogen and protect the intestine from ROS-mediated carcinogenesis. In order to develop specific eβG inhibitors, we found that 59 candidate compounds obtained from the initial virtual screening had high inhibition specificity against eβG but not human βG. In particular, we found that compounds 7145 and 4041 with naphthalenylidene-benzenesulfonamide (NYBS) are highly effective and selective to inhibit eβG activity. Compound 4041  (IC50 = 2.8 μM) shows a higher inhibiting ability than compound 7145  (IC50 = 31.6 μM) against eβG. Furthermore, the molecular docking analysis indicates that compound 4041 has two hydrophobic contacts to residues L361 and I363 in the bacterial loop, but 7145 has one contact to L361. Only compound 4041 can bind to key residue (E413) at active site of eβG via hydrogen-bonding interactions. These novel NYBS-based eβG specific inhibitors may provide as novel candidate compounds, which specifically inhibit eβG to reduce eβG-based carcinogenesis and intestinal injury. PMID:25839056

Discovery of specific inhibitors for intestinal E. coli  β-glucuronidase through in silico virtual screening.

PubMed

Cheng, Ta-Chun; Chuang, Kuo-Hsiang; Roffler, Steve R; Cheng, Kai-Wen; Leu, Yu-Lin; Chuang, Chih-Hung; Huang, Chien-Chaio; Kao, Chien-Han; Hsieh, Yuan-Chin; Chang, Long-Sen; Cheng, Tian-Lu; Chen, Chien-Shu

2015-01-01

Glucuronidation is a major metabolism process of detoxification for carcinogens, 4-(methylnitrosamino)-1-(3-pyridy)-1-butanone (NNK) and 1,2-dimethylhydrazine (DMH), of reactive oxygen species (ROS). However, intestinal E. coli   β-glucuronidase (eβG) has been considered pivotal to colorectal carcinogenesis. Specific inhibition of eβG may prevent reactivating the glucuronide-carcinogen and protect the intestine from ROS-mediated carcinogenesis. In order to develop specific eβG inhibitors, we found that 59 candidate compounds obtained from the initial virtual screening had high inhibition specificity against eβG but not human βG. In particular, we found that compounds 7145 and 4041 with naphthalenylidene-benzenesulfonamide (NYBS) are highly effective and selective to inhibit eβG activity. Compound 4041  (IC50 = 2.8 μM) shows a higher inhibiting ability than compound 7145  (IC50 = 31.6 μM) against eβG. Furthermore, the molecular docking analysis indicates that compound 4041 has two hydrophobic contacts to residues L361 and I363 in the bacterial loop, but 7145 has one contact to L361. Only compound 4041 can bind to key residue (E413) at active site of eβG via hydrogen-bonding interactions. These novel NYBS-based eβG specific inhibitors may provide as novel candidate compounds, which specifically inhibit eβG to reduce eβG-based carcinogenesis and intestinal injury.

Antitumor Activity and Mechanism of a Reverse Transcriptase Inhibitor, Dapivirine, in Glioblastoma.

PubMed

Liu, Weiwen; Song, Xian-Lu; Zhao, Shan-Chao; He, Minyi; Wang, Hai; Chen, Ziyang; Xiang, Wei; Yi, Guozhong; Qi, Songtao; Liu, Yawei

2018-01-01

Dapivirine is one of reverse transcriptase inhibitors (RTIs). It is the prototype of diarylpyrimidines (DAPY), formerly known as TMC120 or DAPY R147681 (IUPAC name: 4- [[4-(2, 4, 6-trimethylphenyl) amino]-2-pyrimidinyl] amino]-benzonitrile; CAS no.244767-67-7). The purpose of this study is to investigate the antitumor activity of dapivirine, one of the RTIs, on U87 glioblastoma (GBM) cells in vitro and in vivo . U87 GBM cells were cultured and treated with or without dapivirine. Cell viability was evaluated by CCK-8 (Cell Counting Kit 8, CCK-8) assay; apoptosis was analyzed by flow cytometry; cell migration was evaluated by Boyden Chamber assay; Western blotting was performed to detect proteins related to apoptosis, epithelial-to-mesenchymal transition and autophagy. PathScan intracellular signaling array kit was used to detect important and well-characterized signaling molecules. Tumor xenograft model in nude mice was used to evaluate the antitumorigenic effect in vivo . Dapivirine weakened proliferation of glioma cells and induced the apoptosis of U87 glioblastoma cells. Furthermore, dapivirine regulated autophagy and induced Akt, Bad and SAPK/JNK activations. Moreover, the inhibition of glioma cell growth by dapivirine was also observed in nude mice in vivo . In summary, in our study dapivirine exposure induces stress, resulting in JNK and PI3K/Akt pathway activation through diminished inhibition of the apoptosis and autophagy cascade in U87 GBM cells, which inhibits cell growth in vitro and in vivo .

Inhibition of c-Jun N-terminal kinase activation reverses Alzheimer disease phenotypes in APPswe/PS1dE9 mice.

PubMed

Zhou, Qiong; Wang, Man; Du, Ying; Zhang, Wei; Bai, Miao; Zhang, Zhuo; Li, Zhuyi; Miao, Jianting

2015-04-01

Growing evidence indicates that the activation of c-Jun N-terminal kinase (JNK) is implicated in the multiple major pathological features of Alzheimer disease (AD). However, whether specific inhibition of JNK activation could prevent disease progression in adult transgenic AD models at moderate stage remains unknown. Here we first investigated the potential disease-modifying therapeutic effect of systemic administration of SP600125, a small-molecule JNK-specific inhibitor, in middle-aged APPswe/PS1dE9 mice. Using behavioral, histological, and biochemical methods, outcomes of SP600125 treatment on neuropathology and cognitive deficits were studied in APPswe/PS1dE9 mice. Compared with vehicle-treated APPswe/PS1dE9 mice, chronic treatment of SP600125 for 12 weeks potently inhibited JNK activation, which resulted in a marked improvement of behavioral measures of cognitive deficits and a dramatic reduction in amyloid plaque burden, β-amyloid production, tau hyperphosphorylation, inflammatory responses, and synaptic loss in these transgenic animals. In particular, we found that SP600125 treatment strongly promoted nonamyloidogenic amyloid precursor protein (APP) processing and inhibited amyloidogenic APP processing via regulating APP-cleavage secretase expression (ie, ADAM10, BACE1, and PS1) in APPswe/PS1dE9 mice. Our findings demonstrate that chronic SP600125 treatment is powerfully effective in slowing down disease progression by markedly reducing multiple pathological features and ameliorating cognitive deficits associated with AD. This study highlights the concept that active JNK actually contributes to the development of the disease, and provides critical preclinical evidence that specific inhibition of JNK activation by SP600125 treatment may be a novel promising disease-modifying therapeutic strategy for the treatment of AD. © 2015 American Neurological Association.

Loss of MURC/Cavin-4 induces JNK and MMP-9 activity enhancement in vascular smooth muscle cells and exacerbates abdominal aortic aneurysm.

PubMed

Miyagawa, Kotaro; Ogata, Takehiro; Ueyama, Tomomi; Kasahara, Takeru; Nakanishi, Naohiko; Naito, Daisuke; Taniguchi, Takuya; Hamaoka, Tetsuro; Maruyama, Naoki; Nishi, Masahiro; Kimura, Taizo; Yamada, Hiroyuki; Aoki, Hiroki; Matoba, Satoaki

2017-06-03

Abdominal aortic aneurysm (AAA) is relatively common in elderly patients with atherosclerosis. MURC (muscle-restricted coiled-coil protein)/Cavin-4 modulating the caveolae function of muscle cells is expressed in cardiomyocytes, skeletal muscle cells and smooth muscle cells. Here, we show a novel functional role of MURC/Cavin-4 in vascular smooth muscle cells (VSMCs) and AAA development. Both wild-type (WT) and MURC/Cavin-4 knockout (MURC -/- ) mice subjected to periaortic application of CaCl 2 developed AAAs. Six weeks after CaCl 2 treatment, internal and external aortic diameters were significantly increased in MURC -/- AAAs compared with WT AAAs, which were accompanied by advanced fibrosis in the tunica media of MURC -/- AAAs. The activity of JNK and matrix metalloproteinase (MMP) -2 and -9 were increased in MURC -/- AAAs compared with WT AAAs at 5 days after CaCl 2 treatment. At 6 weeks after CaCl 2 treatment, MURC -/- AAAs exhibited attenuated JNK activity compared with WT AAAs. There was no difference in the activity of MMP-2 or -9 between saline and CaCl 2 treatments. In MURC/Cavin-4-knockdown VSMCs, TNFα-induced activity of JNK and MMP-9 was enhanced compared with control VSMCs. Furthermore, WT, MURC -/- , apolipoprotein E -/- (ApoE -/- ), and MURC/Cavin-4 and ApoE double-knockout (MURC -/- ApoE -/- ) mice were subjected to angiotensin II (Ang II) infusion. In both ApoE -/- and MURC -/- ApoE -/- mice infused for 4 weeks with Ang II, AAAs were promoted. The internal aortic diameter was significantly increased in Ang II-infused MURC -/- ApoE -/- mice compared with Ang II-infused ApoE -/- mice. In MURC/Cavin-4-knockdown VSMCs, Ang II-induced activity of JNK and MMP-9 was enhanced compared with control VSMCs. Our results suggest that MURC/Cavin-4 in VSMCs modulates AAA progression at the early stage via the activation of JNK and MMP-9. MURC/Cavin-4 is a potential therapeutic target against AAA progression. Copyright © 2017 Elsevier Inc. All rights reserved.

CtBP impedes JNK- and Upd/STAT-driven cell fate misspecifications in regenerating Drosophila imaginal discs

PubMed Central

Worley, Melanie I; Alexander, Larissa A

2018-01-01

Regeneration following tissue damage often necessitates a mechanism for cellular re-programming, so that surviving cells can give rise to all cell types originally found in the damaged tissue. This process, if unchecked, can also generate cell types that are inappropriate for a given location. We conducted a screen for genes that negatively regulate the frequency of notum-to-wing transformations following genetic ablation and regeneration of the wing pouch, from which we identified mutations in the transcriptional co-repressor C-terminal Binding Protein (CtBP). When CtBP function is reduced, ablation of the pouch can activate the JNK/AP-1 and JAK/STAT pathways in the notum to destabilize cell fates. Ectopic expression of Wingless and Dilp8 precede the formation of the ectopic pouch, which is subsequently generated by recruitment of both anterior and posterior cells near the compartment boundary. Thus, CtBP stabilizes cell fates following damage by opposing the destabilizing effects of the JNK/AP-1 and JAK/STAT pathways. PMID:29372681

Inhibitor recognition specificity of MERS-CoV papain-like protease may differ from that of SARS-CoV.

PubMed

Lee, Hyun; Lei, Hao; Santarsiero, Bernard D; Gatuz, Joseph L; Cao, Shuyi; Rice, Amy J; Patel, Kavankumar; Szypulinski, Michael Z; Ojeda, Isabel; Ghosh, Arun K; Johnson, Michael E

2015-06-19

The Middle East Respiratory Syndrome coronavirus (MERS-CoV) papain-like protease (PLpro) blocking loop 2 (BL2) structure differs significantly from that of SARS-CoV PLpro, where it has been proven to play a crucial role in SARS-CoV PLpro inhibitor binding. Four SARS-CoV PLpro lead inhibitors were tested against MERS-CoV PLpro, none of which were effective against MERS-CoV PLpro. Structure and sequence alignments revealed that two residues, Y269 and Q270, responsible for inhibitor binding to SARS-CoV PLpro, were replaced by T274 and A275 in MERS-CoV PLpro, making critical binding interactions difficult to form for similar types of inhibitors. High-throughput screening (HTS) of 25 000 compounds against both PLpro enzymes identified a small fragment-like noncovalent dual inhibitor. Mode of inhibition studies by enzyme kinetics and competition surface plasmon resonance (SPR) analyses suggested that this compound acts as a competitive inhibitor with an IC50 of 6 μM against MERS-CoV PLpro, indicating that it binds to the active site, whereas it acts as an allosteric inhibitor against SARS-CoV PLpro with an IC50 of 11 μM. These results raised the possibility that inhibitor recognition specificity of MERS-CoV PLpro may differ from that of SARS-CoV PLpro. In addition, inhibitory activity of this compound was selective for SARS-CoV and MERS-CoV PLpro enzymes over two human homologues, the ubiquitin C-terminal hydrolases 1 and 3 (hUCH-L1 and hUCH-L3).

c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis

DTIC Science & Technology

2013-10-01

Treatment of Amyotrophic Lateral Sclerosis ” PRINCIPAL INVESTIGATOR: Dr. Philip LoGrasso CONTRACTING ORGANIZATION: The Scripps Research... Lateral Sclerosis ” 5a. CONTRACT NUMBER W81XWH-12-1-0431 5b. GRANT NUMBER W81XWH-12-1-0431 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Philip...Annual 3. DATES COVERED 30September2012-29September2013 4. TITLE AND SUBTITLE “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic

Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16

PubMed Central

Dixon-Clarke, Sarah E.; Shehata, Saifeldin N.; Krojer, Tobias; Sharpe, Timothy D.; vonDelft, Frank; Sakamoto, Kei

2017-01-01

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has a unique consensus substrate phosphorylation motif compared with conventional CDKs. To elucidate the structure and inhibitor-binding properties of this atypical CDK, we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multitargeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity, suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts. PMID:28057719

Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16.

PubMed

Dixon-Clarke, Sarah E; Shehata, Saifeldin N; Krojer, Tobias; Sharpe, Timothy D; von Delft, Frank; Sakamoto, Kei; Bullock, Alex N

2017-02-20

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has a unique consensus substrate phosphorylation motif compared with conventional CDKs. To elucidate the structure and inhibitor-binding properties of this atypical CDK, we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multitargeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity, suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts. © 2017 The Author(s).

Therapeutical relevance of MAP-kinase inhibitors in renal diseases: current knowledge and future clinical perspectives.

PubMed

Grande, M Teresa; López-Novoa, José M

2008-01-01

Renal failure, both acute and chronic, represents an important health problem by its social, sanitary and economic aspects. Mitogen-activated protein kinases (MAPK) are a family of mediators involved in the transduction of extracellular stimuli to intracellular responses. The best studied members of this family are extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), Jun NH(2)-terminal kinase (JNK), p38 kinase and extracellular signal regulated kinases 5 (ERK5) also known as big MAP Kinase 1 (BMK1). MAPKs plays a role in regulating renal function and all these pathways have been demonstrated to be activated in many "in vivo" and cellular models or renal failure. As MAP kinases are key regulators in the control of cell proliferation and cell death, many more or less specific inhibitors of these pathways are being developed for the treatment of tumors. The purpose of this review is to examine the data available on the role of MAPKs activation in "in vivo" models of renal failure, as well as in different renal cell types (especially in mesangial cells, podocytes, tubular epithelial cells and fibroblasts) subjected to stress or damage. We have also reviewed the effect of MAPKs inhibition on renal damage, both "in vivo" and "in vitro". Data collected allow to suggest that therapy of chronic and acute renal disease with MAPKs inhibitors is a promising therapeutic area, although much more basic and clinical studies are necessary before this kind of therapy can be used in the everyday clinic.

The Expression of Fn14 via Mechanical Stress-activated JNK Contributes to Apoptosis Induction in Osteoblasts*

PubMed Central

Matsui, Hiroyuki; Fukuno, Naoto; Kanda, Yoshiaki; Kantoh, Yusuke; Chida, Toko; Nagaura, Yuko; Suzuki, Osamu; Nishitoh, Hideki; Takeda, Kohsuke; Ichijo, Hidenori; Sawada, Yasuhiro; Sasaki, Keiichi; Kobayashi, Takayasu; Tamura, Shinri

2014-01-01

Bone mass is maintained by the balance between the activities of bone-forming osteoblasts and bone-resorbing osteoclasts. It is well known that adequate mechanical stress is essential for the maintenance of bone mass, whereas excess mechanical stress induces bone resorption. However, it has not been clarified how osteoblasts respond to different magnitudes of mechanical stress. Here we report that large-magnitude (12%) cyclic stretch induced Ca2+ influx, which activated reactive oxygen species generation in MC3T3-E1 osteoblasts. Reactive oxygen species then activated the ASK1-JNK/p38 pathways. The activated JNK led to transiently enhanced expression of FGF-inducible 14 (Fn14, a member of the TNF receptor superfamily) gene. Cells with enhanced expression of Fn14 subsequently acquired sensitivity to the ligand of Fn14, TNF-related weak inducer of apoptosis, and underwent apoptosis. On the other hand, the ASK1-p38 pathway induced expression of the monocyte chemoattractant protein 3 (MCP-3) gene, which promoted chemotaxis of preosteoclasts. In contrast, the ERK pathway was activated by small-magnitude stretching (1%) and induced expression of two osteogenic genes, collagen Ia (Col1a) and osteopontin (OPN). Moreover, activated JNK suppressed Col1a and OPN induction in large-magnitude mechanical stretch-loaded cells. The enhanced expression of Fn14 and MCP-3 by 12% stretch and the enhanced expression of Col1a and OPN by 1% stretch were also observed in mouse primary osteoblasts. These results suggest that differences in the response of osteoblasts to varying magnitudes of mechanical stress play a key role in switching the mode of bone metabolism between formation and resorption. PMID:24446436

Optimizing fluorescently tethered Hsp90 inhibitor dose for maximal specific uptake by breast tumors

NASA Astrophysics Data System (ADS)

Crouch, Brian T.; Duer, Joy; Wang, Roujia; Gallagher, Jennifer; Hall, Allison; Soo, Mary Scott; Hughes, Philip; Haystead, Timothy A. J.; Ramanujam, Nirmala

2018-03-01

Despite improvements in surgical resection, 20-40% of patients undergoing breast conserving surgery require at least one additional re-excision. Leveraging the unique surface expression of heat shock protein 90 (Hsp90), a chaperone protein involved in several key hallmarks of cancer, in breast cancer provides an exciting opportunity to identify residual disease during surgery. We developed a completely non-destructive strategy using HS-27, a fluorescently-tethered Hsp90 inhibitor, to assay surface Hsp90 expression on intact tissue specimens using a fluorescence microendoscope with a field of view of 750 μm and subcellular resolution of 4 μm. HS-27 consists of an FDA approved Hsp90 inhibitor tethered to fluorescein isothiocyanate (EX 488nm, EM 525nm). Here, we optimized ex vivo HS-27 administration in pre-clinical breast cancer models and validated our approach on 21 patients undergoing standard of care ultrasound guided core needle biopsy. HS-27 administration time was fixed at 1- minute to minimize imaging impact on clinical workflow. HS-27 and HS-217 (non-specific control) doses were modulated from 1 μM up to 100 μM to identify the dose maximizing the ratio of specific uptake (HS-27 fluorescence) to non-specific uptake (HS-217 fluorescence). The specificity ratio was maximized at 100 μM and was significantly greater than all other doses (p<0.05). We applied our optimized imaging protocol to clinical samples and demonstrated significantly greater uptake of HS-27 by tumor than non-tumor tissue (p<0.05). The ubiquitous nature of HS-27 binding to all subtypes of breast cancer makes this technology attractive for assessing tumor margins, as one agent can be used for all subtypes.

Novel synergistic mechanism for sst2 somatostatin and TNFalpha receptors to induce apoptosis: crosstalk between NF-kappaB and JNK pathways.

PubMed

Guillermet-Guibert, J; Saint-Laurent, N; Davenne, L; Rochaix, P; Cuvillier, O; Culler, M D; Pradayrol, L; Buscail, L; Susini, C; Bousquet, C

2007-02-01

Somatostatin is a multifunctional hormone that modulates cell proliferation, differentiation and apoptosis. Mechanisms for somatostatin-induced apoptosis are at present mostly unsolved. Therefore, we investigated whether somatostatin receptor subtype 2 (sst2) induces apoptosis in the nontransformed murine fibroblastic NIH3T3 cells. Somatostatin receptor subtype 2 expression induced an executioner caspase-mediated apoptosis through a tyrosine phosphatase SHP-1 (Src homology domain phosphatase-1)-dependent stimulation of nuclear factor kappa B (NF-kappaB) activity and subsequent inhibition of the mitogen-activated protein kinase JNK. Tumor necrosis factor alpha (TNFalpha) stimulated both NF-kappaB and c-Jun NH2-terminal kinase (JNK) activities, which had opposite action on cell survival. Importantly, sst2 sensitized NIH3T3 cells to TNFalpha-induced apoptosis by (1) upregulating TNFalpha receptor protein expression, and sensitizing to TNFalpha-induced caspase-8 activation; (2) enhancing TNFalpha-mediated activation of NF-kappaB, resulting in JNK inhibition and subsequent executioner caspase activation and cell death. We have here unraveled a novel signaling mechanism for a G protein-coupled receptor, which directly triggers apoptosis and crosstalks with a death receptor to enhance death ligand-induced apoptosis.

Opposing effects of cancer-type-specific SPOP mutants on BET protein degradation and sensitivity to BET inhibitors.

PubMed

Janouskova, Hana; El Tekle, Geniver; Bellini, Elisa; Udeshi, Namrata D; Rinaldi, Anna; Ulbricht, Anna; Bernasocchi, Tiziano; Civenni, Gianluca; Losa, Marco; Svinkina, Tanya; Bielski, Craig M; Kryukov, Gregory V; Cascione, Luciano; Napoli, Sara; Enchev, Radoslav I; Mutch, David G; Carney, Michael E; Berchuck, Andrew; Winterhoff, Boris J N; Broaddus, Russell R; Schraml, Peter; Moch, Holger; Bertoni, Francesco; Catapano, Carlo V; Peter, Matthias; Carr, Steven A; Garraway, Levi A; Wild, Peter J; Theurillat, Jean-Philippe P

2017-09-01

It is generally assumed that recurrent mutations within a given cancer driver gene elicit similar drug responses. Cancer genome studies have identified recurrent but divergent missense mutations affecting the substrate-recognition domain of the ubiquitin ligase adaptor SPOP in endometrial and prostate cancers. The therapeutic implications of these mutations remain incompletely understood. Here we analyzed changes in the ubiquitin landscape induced by endometrial cancer-associated SPOP mutations and identified BRD2, BRD3 and BRD4 proteins (BETs) as SPOP-CUL3 substrates that are preferentially degraded by endometrial cancer-associated SPOP mutants. The resulting reduction of BET protein levels sensitized cancer cells to BET inhibitors. Conversely, prostate cancer-specific SPOP mutations resulted in impaired degradation of BETs, promoting their resistance to pharmacologic inhibition. These results uncover an oncogenomics paradox, whereby mutations mapping to the same domain evoke opposing drug susceptibilities. Specifically, we provide a molecular rationale for the use of BET inhibitors to treat patients with endometrial but not prostate cancer who harbor SPOP mutations.

Wheat Subtilisin/Chymotrypsin Inhibitor (WSCI) as a scaffold for novel serine protease inhibitors with a given specificity.

PubMed

Tedeschi, Francesca; Di Maro, Antimo; Facchiano, Angelo; Costantini, Susan; Chambery, Angela; Bruni, Natalia; Capuzzi, Valeria; Ficca, Anna Grazia; Poerio, Elia

2012-10-30

WSCI (Wheat Subtilisin/Chymotrypsin Inhibitor) is a small protein belonging to the Potato inhibitor I family exhibiting a high content of essential amino acid. In addition to bacterial subtilisins and mammalian chymotrypsins, WSCI inhibits chymotrypsin-like activities isolated from digestive traits of a number of insect larvae. In vivo, as suggested for many plant proteinase inhibitors, WSCI seems to play a role of natural defence against attacks of pests and pathogens. The functional region of WSCI, containing the inhibitor reactive site (Met48-Glu49), corresponds to an extended flexible loop (Val42-Asp53) whose architecture is somehow stabilized by a number of secondary interactions established with a small β-sheet located underneath. The aim of this study was to employ a WSCI molecule as a stable scaffold to obtain recombinant inhibitors with new acquired anti-proteinase activity or, alternatively, inactive WSCI variants. A gene sequence coding for the native WSCI, along with genes coding for muteins with different specficities, could be exploited to obtain transformed non-food use plants with improved insect resistance. On the other hand, the genetic transformation of cereal plants over-expressing inactive WSCI muteins could represent a possible strategy to improve the nutritional quality of cereal-based foods, without risk of interference with human or animal digestive enzymes. Here, we described the characterization of four muteins containing single/multiple amino acid substitutions at the WSCI reactive site and/or at its proximity. Modalities of interaction of these muteins with proteinases (subtilisin, trypsin and chymotrypsin) were investigated by time course hydrolysis and molecular simulations studies.

Curcumin attenuates BPA-induced insulin resistance in HepG2 cells through suppression of JNK/p38 pathways.

PubMed

Geng, Shanshan; Wang, Shijia; Zhu, Weiwei; Xie, Chunfeng; Li, Xiaoting; Wu, Jieshu; Zhu, Jianyun; Jiang, Ye; Yang, Xue; Li, Yuan; Chen, Yue; Wang, Xiaoqian; Meng, Yu; Zhu, Mingming; Wu, Rui; Huang, Cong; Zhong, Caiyun

2017-04-15

Bisphenol A (BPA) is an artificial environmental endocrine disrupting chemicals. Accumulating evidence indicates that exposure to BPA contributes to insulin resistance through diverse mechanism including inflammation and oxidative stress. Previous studies have suggested curcumin as a safe phytochemical which can improve obesity-related insulin resistance, inflammation and oxidative stress. The present study aimed to investigate the ability of curcumin to prevent BPA-induced insulin resistance in vitro and the underlying mechanism. Following the establishmet of in vitro insulin resistance via BPA treatment in human liver HepG2 cells, the protective effects of curcumin were determiend. We showed that treatment of HepG2 cells with 100nM BPA for 5days induced significantly decreased glucose consumption, impaired insulin signaling, elevation of pro-inflammatory cytokines and oxidative stress, and activation of signaling pathways; inhibition of JNK and p38 pathways, but not ERK nor NF-κB pathways, improved glucose consumption and insulin signaling in BPA-treated HepG2 cells. Moreover, we revealed that curcumin effectively attenuated the spectrum of effects of BPA-triggered insulin resistance, whereas pretreatment with JNK and p38 agonist anisomycin could significantly compensate the effects caused by curcumin. These data illustrated the role of JNK/p38 activation in BPA-induced insulin resistance and suggested curcumin as a promising candidate for the intervention of BPA-induced insulin resistance. Copyright © 2017 Elsevier B.V. All rights reserved.

Gallic Acid Induces a Reactive Oxygen Species-Provoked c-Jun NH2-Terminal Kinase-Dependent Apoptosis in Lung Fibroblasts

PubMed Central

Chen, Chiu-Yuan; Chen, Kun-Chieh; Yang, Tsung-Ying; Liu, Hsiang-Chun; Hsu, Shih-Lan

2013-01-01

Idiopathic pulmonary fibrosis is a chronic lung disorder characterized by fibroblasts proliferation and extracellular matrix accumulation. Induction of fibroblast apoptosis therefore plays a crucial role in the resolution of this disease. Gallic acid (3,4,5-trihydroxybenzoic acid), a common botanic phenolic compound, has been reported to induce apoptosis in tumor cell lines and renal fibroblasts. The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in lung fibroblasts apoptosis induced by gallic acid. We found that treatment with gallic acid resulted in activation of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB, Akt), but not p38MAPK, in mouse lung fibroblasts. Inhibition of JNK using pharmacologic inhibitor (SP600125) and genetic knockdown (JNK specific siRNA) significantly inhibited p53 accumulation, reduced PUMA and Fas expression, and abolished apoptosis induced by gallic acid. Moreover, treatment with antioxidants (vitamin C, N-acetyl cysteine, and catalase) effectively diminished gallic acid-induced hydrogen peroxide production, JNK and p53 activation, and cell death. These observations imply that gallic acid-mediated hydrogen peroxide formation acts as an initiator of JNK signaling pathways, leading to p53 activation and apoptosis in mouse lung fibroblasts. PMID:23533505

Reactive oxygen species activate differentiation gene transcription of acute myeloid leukemia cells via the JNK/c-JUN signaling pathway.

PubMed

Lam, Chung Fan; Yeung, Hoi Ting; Lam, Yuk Man; Ng, Ray Kit

2018-05-01

Reactive oxygen species (ROS) and altered cellular redox status are associated with many malignancies. Acute myeloid leukemia (AML) cells are maintained at immature state by differentiation blockade, which involves deregulation of transcription factors in myeloid differentiation. AML cells can be induced to differentiate by phorbol-12-myristate-13-acetate (PMA), which possesses pro-oxidative activity. However, the signaling events mediated by ROS in the activation of transcriptional program during AML differentiation has not been fully elucidated. Here, we investigated AML cell differentiation by treatment with PMA and ROS scavenger N-acetyl-l-cysteine (NAC). We observed elevation of intracellular ROS level in the PMA-treated AML cells, which correlated with differentiated cell morphology and increased CD11b + mature cell population. The effect of PMA can be abolished by NAC co-treatment, supporting the involvement of ROS in the process. Moreover, we demonstrated that short ROS elevation mediated cell cycle arrest, but failed to activate myeloid gene transcription; whereas prolonged ROS elevation activated JNK/c-JUN signaling pathway. Inhibition of JNK suppressed the expression of key myeloid transcriptional regulators c-JUN, SPI-1 and MAFB, and prevented AML cells from undergoing terminal differentiation. These findings provide new insights into the crucial role of JNK/c-Jun signaling pathway in the activation of transcriptional program during ROS-mediated AML differentiation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Treatment with proteasome inhibitor bortezomib enhances antigen-specific CD8+ T cell-mediated antitumor immunity induced by DNA vaccination

PubMed Central

Tseng, Chih Wen; Monie, Archana; Wu, Chao-Yi; Huang, Bruce; Wang, Mei-Cheng; Hung, Chien-Fu; Wu, T.-C.

2008-01-01

There is an urgent need to develop new innovative therapies for the control of cancer. Antigen-specific immunotherapy and the employment of proteasome inhibitors have emerged as two potentially plausible approaches for the control of cancer. In the current study, we explored the combination of the DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 (HPV-16) E7 antigen (CRT/E7) with the proteasome inhibitor; bortezomib for their ability to generate E7-specific immune responses and antitumor effects in vaccinated mice. We found that the combination of treatment with bortezomib and CRT/E7(detox) DNA generated more potent E7-specific CD8+ T cell immune responses and better therapeutic effects against TC-1 tumors in tumor bearing mice compared to monotherapy. Furthermore, we found that treatment with bortezomib led to increased apoptosis of TC-1 tumor cells and could render the TC-1 tumor cells more susceptible to lysis by E7-specific CTLs. Our data has significant implications for future clinical translation. PMID:18542898

Drug insight: If inhibitors as specific heart-rate-reducing agents.

PubMed

Borer, Jeffrey S

2004-12-01

Heart rate is determined primarily by spontaneously repeating net inward current carried by sodium ions and potassium ions through hyperpolarization-activated cyclic-nucleotide-gated channels. Within the heart, these channels are found most abundantly in sinoatrial cardiomyocytes. The channels open in response to membrane hyperpolarization, modulated by local cAMP concentrations. They permit activation of the I(f) current, which can be blocked specifically by molecules characterized by linked benzazepinone and benzocyclobutane rings, and which are devoid of effects on cardiac conduction, inotropy or peripheral vascular tone. The resulting heart-rate reduction has been effective in angina prevention in clinical trials involving 4,000 patients, using the prototype I(f) inhibitor, ivabradine. No serious adverse events have been attributed to the treatment; the most prominent side-effect is dose-related, always reversible and often transient visual symptoms that seldom result in voluntary drug discontinuation.

Anti-leukaemic activity of the TYK2 selective inhibitor NDI-031301 in T-cell acute lymphoblastic leukaemia.

PubMed

Akahane, Koshi; Li, Zhaodong; Etchin, Julia; Berezovskaya, Alla; Gjini, Evisa; Masse, Craig E; Miao, Wenyan; Rocnik, Jennifer; Kapeller, Rosana; Greenwood, Jeremy R; Tiv, Hong; Sanda, Takaomi; Weinstock, David M; Look, A Thomas

2017-04-01

Activation of tyrosine kinase 2 (TYK2) contributes to the aberrant survival of T-cell acute lymphoblastic leukaemia (T-ALL) cells. Here we demonstrate the anti-leukaemic activity of a novel TYK2 inhibitor, NDI-031301. NDI-031301 is a potent and selective inhibitor of TYK2 that induced robust growth inhibition of human T-ALL cell lines. NDI-031301 treatment of human T-ALL cell lines resulted in induction of apoptosis that was not observed with the JAK inhibitors tofacitinib and baricitinib. Further investigation revealed that NDI-031301 treatment uniquely leads to activation of three mitogen-activated protein kinases (MAPKs), resulting in phosphorylation of ERK, SAPK/JNK and p38 MAPK coincident with PARP cleavage. Activation of p38 MAPK occurred within 1 h of NDI-031301 treatment and was responsible for NDI-031301-induced T-ALL cell death, as pharmacological inhibition of p38 MAPK partially rescued apoptosis induced by TYK2 inhibitor. Finally, daily oral administration of NDI-031301 at 100 mg/kg bid to immunodeficient mice engrafted with KOPT-K1 T-ALL cells was well tolerated, and led to decreased tumour burden and a significant survival benefit. These results support selective inhibition of TYK2 as a promising potential therapeutic strategy for T-ALL. © 2017 John Wiley & Sons Ltd.

TIPE attenuates the apoptotic effect of radiation and cisplatin and promotes tumor growth via JNK and p38 activation in Raw264.7 and EL4 cells.

PubMed

Liu, Yao; Ni, Xiao Yan; Chen, Rui Ling; Li, Juan; Gao, Feng Guang

2018-06-01

Tumor necrosis factor α‑induced protein 8 (TIPE) is highly expressed in many types of malignancies. Apoptosis is the process of programmed cell death which maintains the balance of cell survival and death. TIPE is involved in the carcinogenesis of many tumor types, yet the exact role of TIPE in defective apoptosis‑associated carcinogenesis remains uncertain. In the present study, TIPE‑overexpressing Raw264.7 and EL4 cells and vector control cells were treated with 4 mJ/cm2 ultraviolet radiation or 2 µg/ml cisplatin. Following ultraviolet irradiation, TIPE overexpression decreased the percentage of apoptotic cells as detected by flow cytometric and reversed the cisplatin‑mediated decrease in mitochondrial membrane potential by JC‑1 assay. Western blot analyses also revealed that TIPE overexpression inhibited cisplatin‑induced activation of caspase‑3 and ‑9 and PARP. Secondly, TIPE overexpression increased the levels of phosphorylated JNK, MEK and p38. Moreover, inhibition of JNK and p38, but not MEK, efficiently abolished the cell pro‑survival effect of TIPE. Most importantly, an in vivo tumor implantation model revealed that TIPE overexpression augmented the volume and weight of the implanted tumors, indicating that TIPE facilitated tumor formation. We found that TIPE exhibited an anti‑apoptotic effect via JNK and p38 activation, which ultimately promoted tumor. Hence, the present study revealed that activation of JNK and p38 kinases contribute to the TIPE‑mediated anti‑apoptotic effect, indicating that JNK and p38 may be potential therapeutic molecules for TIPE overexpression‑associated diseases.

3-MCPD 1-palmitate induced renal tubular cell apoptosis in vivo via JNK/p53 pathway

USDA-ARS?s Scientific Manuscript database

Fatty acid esters of 3-chloro-1, 2-propanediol (3-MCPD esters) are a group of processing-induced food contaminants with nephrotoxicity, but the molecular mechanism(s) remains unclear. This study investigated whether and how the JNK/p53 pathway may play a role in the nephrotoxic effect of 3-MCPD este...

Aminopyrazinamides: novel and specific GyrB inhibitors that kill replicating and nonreplicating Mycobacterium tuberculosis.

PubMed

Shirude, Pravin S; Madhavapeddi, Prashanti; Tucker, Julie A; Murugan, Kannan; Patil, Vikas; Basavarajappa, Halesha; Raichurkar, Anandkumar V; Humnabadkar, Vaishali; Hussein, Syeed; Sharma, Sreevalli; Ramya, V K; Narayan, Chandan B; Balganesh, Tanjore S; Sambandamurthy, Vasan K

2013-03-15

Aminopyrazinamides originated from a high throughput screen targeting the Mycobacterium smegmatis (Msm) GyrB ATPase. This series displays chemical tractability, robust structure-activity relationship, and potent antitubercular activity. The crystal structure of Msm GyrB in complex with one of the aminopyrazinamides revealed promising attributes of specificity against other broad spectrum pathogens and selectivity against eukaryotic kinases due to novel interactions at hydrophobic pocket, unlike other known GyrB inhibitors. The aminopyrazinamides display excellent mycobacterial kill under in vitro, intracellular, and hypoxic conditions.

Structural Basis for Specific Inhibition of tRNA Synthetase by an ATP Competitive Inhibitor.

PubMed

Fang, Pengfei; Han, Hongyan; Wang, Jing; Chen, Kaige; Chen, Xin; Guo, Min

2015-06-18

Pharmaceutical inhibitors of aminoacyl-tRNA synthetases demand high species and family specificity. The antimalarial ATP-mimetic cladosporin selectively inhibits Plasmodium falciparum LysRS (PfLysRS). How the binding to a universal ATP site achieves the specificity is unknown. Here we report three crystal structures of cladosporin with human LysRS, PfLysRS, and a Pf-like human LysRS mutant. In all three structures, cladosporin occupies the class defining ATP-binding pocket, replacing the adenosine portion of ATP. Three residues holding the methyltetrahydropyran moiety of cladosporin are critical for the specificity of cladosporin against LysRS over other class II tRNA synthetase families. The species-exclusive inhibition of PfLysRS is linked to a structural divergence beyond the active site that mounts a lysine-specific stabilizing response to binding cladosporin. These analyses reveal that inherent divergence of tRNA synthetase structural assembly may allow for highly specific inhibition even through the otherwise universal substrate binding pocket and highlight the potential for structure-driven drug development. Copyright © 2015 Elsevier Ltd. All rights reserved.

Analysis of binding properties and specificity through identification of the interface forming residues (IFR) for serine proteases in silico docked to different inhibitors.

PubMed

Ribeiro, Cristina; Togawa, Roberto C; Neshich, Izabella A P; Mazoni, Ivan; Mancini, Adauto L; Minardi, Raquel C de Melo; da Silveira, Carlos H; Jardine, José G; Santoro, Marcelo M; Neshich, Goran

2010-10-20

Enzymes belonging to the same super family of proteins in general operate on variety of substrates and are inhibited by wide selection of inhibitors. In this work our main objective was to expand the scope of studies that consider only the catalytic and binding pocket amino acids while analyzing enzyme specificity and instead, include a wider category which we have named the Interface Forming Residues (IFR). We were motivated to identify those amino acids with decreased accessibility to solvent after docking of different types of inhibitors to sub classes of serine proteases and then create a table (matrix) of all amino acid positions at the interface as well as their respective occupancies. Our goal is to establish a platform for analysis of the relationship between IFR characteristics and binding properties/specificity for bi-molecular complexes. We propose a novel method for describing binding properties and delineating serine proteases specificity by compiling an exhaustive table of interface forming residues (IFR) for serine proteases and their inhibitors. Currently, the Protein Data Bank (PDB) does not contain all the data that our analysis would require. Therefore, an in silico approach was designed for building corresponding complexes. The IFRs are obtained by "rigid body docking" among 70 structurally aligned, sequence wise non-redundant, serine protease structures with 3 inhibitors: bovine pancreatic trypsin inhibitor (BPTI), ecotine and ovomucoid third domain inhibitor. The table (matrix) of all amino acid positions at the interface and their respective occupancy is created. We also developed a new computational protocol for predicting IFRs for those complexes which were not deciphered experimentally so far, achieving accuracy of at least 0.97. The serine proteases interfaces prefer polar (including glycine) residues (with some exceptions). Charged residues were found to be uniquely prevalent at the interfaces between the "miscellaneous-virus" subfamily

Analysis of binding properties and specificity through identification of the interface forming residues (IFR) for serine proteases in silico docked to different inhibitors

PubMed Central

2010-01-01

Background Enzymes belonging to the same super family of proteins in general operate on variety of substrates and are inhibited by wide selection of inhibitors. In this work our main objective was to expand the scope of studies that consider only the catalytic and binding pocket amino acids while analyzing enzyme specificity and instead, include a wider category which we have named the Interface Forming Residues (IFR). We were motivated to identify those amino acids with decreased accessibility to solvent after docking of different types of inhibitors to sub classes of serine proteases and then create a table (matrix) of all amino acid positions at the interface as well as their respective occupancies. Our goal is to establish a platform for analysis of the relationship between IFR characteristics and binding properties/specificity for bi-molecular complexes. Results We propose a novel method for describing binding properties and delineating serine proteases specificity by compiling an exhaustive table of interface forming residues (IFR) for serine proteases and their inhibitors. Currently, the Protein Data Bank (PDB) does not contain all the data that our analysis would require. Therefore, an in silico approach was designed for building corresponding complexes The IFRs are obtained by "rigid body docking" among 70 structurally aligned, sequence wise non-redundant, serine protease structures with 3 inhibitors: bovine pancreatic trypsin inhibitor (BPTI), ecotine and ovomucoid third domain inhibitor. The table (matrix) of all amino acid positions at the interface and their respective occupancy is created. We also developed a new computational protocol for predicting IFRs for those complexes which were not deciphered experimentally so far, achieving accuracy of at least 0.97. Conclusions The serine proteases interfaces prefer polar (including glycine) residues (with some exceptions). Charged residues were found to be uniquely prevalent at the interfaces between the

Chemical probing of the human sirtuin 5 active site reveals its substrate acyl specificity and peptide-based inhibitors.

PubMed

Roessler, Claudia; Nowak, Theresa; Pannek, Martin; Gertz, Melanie; Nguyen, Giang T T; Scharfe, Michael; Born, Ilona; Sippl, Wolfgang; Steegborn, Clemens; Schutkowski, Mike

2014-09-26

Sirtuins are NAD(+)-dependent deacetylases acting as sensors in metabolic pathways and stress response. In mammals there are seven isoforms. The mitochondrial sirtuin 5 is a weak deacetylase but a very efficient demalonylase and desuccinylase; however, its substrate acyl specificity has not been systematically analyzed. Herein, we investigated a carbamoyl phosphate synthetase 1 derived peptide substrate and modified the lysine side chain systematically to determine the acyl specificity of Sirt5. From that point we designed six potent peptide-based inhibitors that interact with the NAD(+) binding pocket. To characterize the interaction details causing the different substrate and inhibition properties we report several X-ray crystal structures of Sirt5 complexed with these peptides. Our results reveal the Sirt5 acyl selectivity and its molecular basis and enable the design of inhibitors for Sirt5. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

4-Hydroxybenzoic acid derivatives as HDAC6-specific inhibitors modulating microtubular structure and HSP90α chaperone activity against prostate cancer.

PubMed

Seidel, Carole; Schnekenburger, Michael; Mazumder, Aloran; Teiten, Marie-Hélène; Kirsch, Gilbert; Dicato, Mario; Diederich, Marc

2016-01-01

Histone deacetylase (HDAC)6 is a unique isoenzyme targeting specific substrates including α-tubulin and heat shock protein (HSP)90. HDAC6 is involved in protein trafficking and degradation, cell shape and migration. Deregulation of HDAC6 activity is associated with a variety of diseases including cancer leading to a growing interest for developing HDAC6 inhibitors. Here, we identified two new structurally related 4-hydroxybenzoic acids as selective HDAC6 inhibitors reducing proliferation, colony and spheroid formation as well as viability of prostate cancer cells. Both compounds strongly enhanced α-tubulin acetylation leading to remodeling of microtubular organization. Furthermore, 4-hydroxybenzoic acids decreased HSP90α regulation of the human androgen receptor in prostate cancer cells by increasing HSP90α acetylation levels. Collectively, our data support the potential of 4-hydroxybenzoic acid derivatives as HDAC6-specific inhibitors with anti-cancer properties. Copyright © 2015 Elsevier Inc. All rights reserved.

Differential regulation of CD44 expression by lipopolysaccharide (LPS) and TNF-alpha in human monocytic cells: distinct involvement of c-Jun N-terminal kinase in LPS-induced CD44 expression.

PubMed

Gee, Katrina; Lim, Wilfred; Ma, Wei; Nandan, Devki; Diaz-Mitoma, Francisco; Kozlowski, Maya; Kumar, Ashok

2002-11-15

Alterations in the regulation of CD44 expression play a critical role in modulating cell adhesion, migration, and inflammation. LPS, a bacterial cell wall component, regulates CD44 expression and may modulate CD44-mediated biological effects in monocytic cells during inflammation and immune responses. In this study, we show that in normal human monocytes, LPS and LPS-induced cytokines IL-10 and TNF-alpha enhance CD44 expression. To delineate the mechanism underlying LPS-induced CD44 expression, we investigated the role of the mitogen-activated protein kinases (MAPKs), p38, p42/44 extracellular signal-regulated kinase, and c-Jun N-terminal kinase (JNK) by using their specific inhibitors. We demonstrate the involvement, at least in part, of p38 MAPK in TNF-alpha-induced CD44 expression in both monocytes and promonocytic THP-1 cells. However, neither p38 nor p42/44 MAPKs were involved in IL-10-induced CD44 expression in monocytes. To further dissect the TNF-alpha and LPS-induced signaling pathways regulating CD44 expression independent of IL-10-mediated effects, we used IL-10 refractory THP-1 cells as a model system. Herein, we show that CD44 expression induced by the LPS-mediated pathway predominantly involved JNK activation. This conclusion was based on results derived by transfection of THP-1 cells with a dominant-negative mutant of stress-activated protein/extracellular signal-regulated kinase kinase 1, and by exposure of cells to JNK inhibitors dexamethasone and SP600125. All these treatments prevented CD44 induction in LPS-stimulated, but not in TNF-alpha-stimulated, THP-1 cells. Furthermore, we show that CD44 induction may involve JNK-dependent early growth response gene activation in LPS-stimulated monocytic cells. Taken together, these results suggest a predominant role of JNK in LPS-induced CD44 expression in monocytic cells.