anisomycin and pyrazolanthrone

Acute liver failure (ALF) is a life-threatening disease characterized by abrupt and extensive hepatic necrosis and apoptosis, resulting in high mortality. The approved drug, N-acetylcysteine (NAC), is only effective for acetaminophen (APAP)-associated ALF at the early stage. Thus, we investigate whether fluorofenidone (AKF-PD), a novel antifibrosis pyridone agent, protects against ALF in mice and explore its underlying mechanisms.. ALF mouse models were established using APAP or lipopolysaccharide/D-galactosamine (LPS/D-Gal). Anisomycin and SP600125 were used as JNK activator and inhibitor, respectively, and NAC served as a positive control. Mouse hepatic cell line AML12 and primary mouse hepatocytes were used for in vitro studies.. AKF-PD pretreatment alleviated APAP-induced ALF with decreased necrosis, apoptosis, reactive oxygen species (ROS) markers, and mitochondrial permeability transition in liver. Additionally, AKF-PD alleviated mitochondrial ROS stimulated by APAP in AML12 cells. RNA-sequencing in the liver and subsequent gene set enrichment analysis showed that AKF-PD significantly impacted MAPK and IL-17 pathway. In vitro and in vivo studies demonstrated that AKF-PD inhibited APAP-induced phosphorylation of MKK4/JNK, while SP600125 only inhibited JNK phosphorylation. The protective effect of AKF-PD was abolished by anisomycin. Similarly, AKF-PD pretreatment abolished hepatotoxicity caused by LPS/D-Gal, decreased ROS levels, and diminished inflammation. Furthermore, unlike NAC, AKF-PD, inhibited the phosphorylation of MKK4 and JNK upon pretreatment, and improved survival in cases of LPS/D-Gal-induced mortality with delayed dosing.. In summary, AKF-PD can protect against ALF caused by APAP or LPS/D-Gal, in part, via regulating MKK4/JNK pathway. AKF-PD might be a novel candidate drug for ALF.

Topics: Acetaminophen; Animals; Anisomycin; Hepatocytes; Lipopolysaccharides; Liver; Liver Failure, Acute; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Necrosis; Pyridones; Reactive Oxygen Species

Neuronal proton-gated acid-sensing ion channels (ASICs) participate in the detection of tissue acidosis, a phenomenon often encountered in painful pathologic diseases. Such conditions often involve in parallel the activation of various signaling pathways such as mitogen activated protein kinases (MAPKs) that ultimately leads to phenotype modifications of sensory neurons. Here, we identify one member of the MAPKs, c-Jun N-terminal kinase (JNK), as a new post-translational positive regulator of ASICs in rodent sensory neurons. Recombinant H

Topics: Acid Sensing Ion Channels; Amino Acid Sequence; Animals; Anisomycin; Anthracenes; Cells, Cultured; Ganglia, Spinal; HEK293 Cells; Humans; JNK Mitogen-Activated Protein Kinases; Male; Mice; Mice, Inbred C57BL; Pain; Protein Processing, Post-Translational; Protein Synthesis Inhibitors; Rats; Rats, Wistar

The study aimed to determine the effects of protease-activated receptor-2 (PAR-2) on glial scar formation after spinal cord injury (SCI) in Sprague-Dawley (SD) rats and the underlying mechanisms. Rivlin and Tator's acute extradural clip compression injury (CCI) model of severe SCI was established in this study. Animals were divided into four groups: 1) sham group (laminectomy only); 2) model group, treated with normal saline; 3) PAR-2 inhibitor group; 4) PAR-2 activator group. Enhanced GFAP and vimentin expression were the markers of glial scar formation. To determine whether JNK was involved in the effects of PAR-2 on GFAP and vimentin expression, we administered anisomycin (a JNK activator) in the presence of PAR-2 inhibitor and SP600125 (a JNK inhibitor) in the presence of PAR-2 activator. At 1, 7, 14 and 28 day after SCI, Basso, Beattie, and Bresnahan (BBB) locomotor score test was used to assess the locomotor functional recovery; immunofluorescence and western blot analysis were used to assess the expression level of GFAP, vimentin and p-JNK. Double immunofluorescence staining with GFAP and tubulin beta was used to assess the glial scar formation and the remaining neurons. Results suggested that PAR-2 is involved in glial scar formation and reduces neurons residues which can cause a further worsening in the functional outcomes after SCI via JNK signaling. Therefore, it may be effective to target PAR-2 in the treatment of SCI.

Topics: Animals; Anisomycin; Anthracenes; Cicatrix; Female; MAP Kinase Signaling System; Rats; Rats, Sprague-Dawley; Receptor, PAR-2; Spinal Cord Injuries

Ultraviolet (UV) radiation is a carcinogen that generates DNA lesions. Here, we demonstrate an unexpected role for DGCR8, an RNA binding protein that canonically functions with Drosha to mediate microRNA processing, in the repair of UV-induced DNA lesions. Treatment with UV induced phosphorylation on serine 153 (S153) of DGCR8 in both human and murine cells. S153 phosphorylation was critical for cellular resistance to UV, the removal of UV-induced DNA lesions, and the recovery of RNA synthesis after UV exposure but not for microRNA expression. The RNA-binding and Drosha-binding activities of DGCR8 were not critical for UV resistance. DGCR8 depletion was epistatic to defects in XPA, CSA, and CSB for UV sensitivity. DGCR8 physically interacted with CSB and RNA polymerase II. JNKs were involved in the UV-induced S153 phosphorylation. These findings suggest that UV-induced S153 phosphorylation mediates transcription-coupled nucleotide excision repair of UV-induced DNA lesions in a manner independent of microRNA processing.

Topics: Animals; Anisomycin; Anthracenes; DNA; DNA Damage; DNA Repair; HCT116 Cells; HeLa Cells; Humans; MAP Kinase Kinase 4; Mice; MicroRNAs; Phosphorylation; Ribonuclease III; RNA Polymerase II; RNA-Binding Proteins; Ultraviolet Rays

Mitogen-activated protein kinases (MAPKs) have been demonstrated to regulate flagellar/ciliary motility of spermatozoa and miracidia of Schistosoma mansoni. However, the role of MAPKs in mediating flagella-driven motility of Leishmania donovani is unexplored. We investigated the function of MAPKs in motility regulation of L. donovani using pharmacological inhibitors and activators of various MAPKs and fast-capture videomicroscopy. Our studies have revealed that the inhibitor of p38 MAPK, PD 169316, significantly affected various motility parameters such as flagellar beat frequency, parasite swimming speed, waveform of the flagellum and resulted in reduced parasite motility. Together, our results suggest that a MAPK, similar to human p38 MAPK, is implicated in flagellar motility regulation of L. donovani.

Topics: Animals; Anisomycin; Anthracenes; Flagella; Flavonoids; Imidazoles; Leishmania donovani; MAP Kinase Signaling System; Microscopy, Video; Movement; p38 Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Protozoan Proteins

The junctional epithelium (JE) is unique with regard to its wide intercellular spaces and sparsely developed intercellular junctions. Thus, knowledge of the molecular mechanisms that regulate the formation of the intercellular junctions of the junctional epithelium may be essential to understand the pathophysiology of the JE. HOK-16B cells, a normal human gingival epithelial cell line, were used to identify the molecules involved in the regulation of the formation of intercellular E-cadherin junctions between human gingival epithelial cells. Activation of c-Jun N-terminal kinase (JNK) disrupted the intercellular junctions through the dissociation of E-cadherin. The role of JNK in the formation of these E-cadherin junctions was further confirmed by demonstrating that JNK inhibition induced the formation of intercellular E-cadherin junctions. The upstream signaling of JNK was also examined. Activation of the small GTPase RhoA disrupted the formation of E-cadherin junctions between HOK-16B cells, which was accompanied by JNK activation. Disruption of these intercellular junctions upon RhoA activation was prevented when JNK activity was inhibited. In contrast, RhoA inactivation led to HOK-16B cell aggregation and the formation of intercellular junctions, even under conditions in which the cellular junctions were naturally disrupted by growth on a strongly adhesive surface. Furthermore, the JE of mouse molars had high JNK activity associated with low E-cadherin expression, which was reversed in the other gingival epithelia, including the sulcular epithelium. Interestingly, JNK activity was increased in cells grown on a solid surface, where cells showed higher RhoA activity than those grown on soft surfaces. Together, these results indicate that the decreased formation of intercellular E-cadherin junctions within the JE may be coupled to high JNK activity, which is activated by the upregulation of RhoA on solid tooth surfaces.

Topics: Animals; Anisomycin; Anthracenes; Cadherins; Cell Adhesion; Cell Culture Techniques; Cell Line; Culture Media; Enzyme Activation; Epithelial Attachment; Epithelial Cells; Fibronectins; Gingiva; Humans; Intercellular Junctions; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; Protein Synthesis Inhibitors; rhoA GTP-Binding Protein

To investigate the involvement of stress-activated protein kinases, JNK and p38 MAPK, in the assembly of tight junctions in keratinocytes, we treated HaCaT cells with various combinations of SP600125 (an inhibitor of JNK), SB202190 (an inhibitor of p38 MAPK) and anisomycin (an activator of both JNK and p38 MAPK) and examined the localization of ZO-1, an undercoat constitutive protein of the tight junction. Short-term (8h) incubation with SP600125, SB202190 or anisomycin induced the accumulation of ZO-1 in the cell-cell contacts, with reduced ZO-1 staining in the cytoplasm, while only long-term (24h) incubation with SP600125 induced the accumulation of ZO-1. SP600125, SB202190 or SP600125 plus SB202190 treatment induced thin linear staining for ZO-1 in the cell-cell contacts. Anisomycin treatment induced thick and irregular linear staining for ZO-1, while anisomycin plus SP600125 treatment induced zipper-like staining for ZO-1. Anisomycin plus SB202190 treatment or anisomycin plus both SP600125 and SB202190 treatment for 8h failed to lead to the accumulation of ZO-1 in cell-cell contacts, but induced thin linear staining with several gaps 16 h after removal of these agents. These results suggest that the localization of ZO-1 in cell-cell contacts is differently regulated by activation and inhibition of JNK and/or p38 MAPK depending on the incubation period.

Topics: Anisomycin; Anthracenes; Cell Communication; Cell Line; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; Keratinocytes; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Zonula Occludens-1 Protein

Podocalyxin (PODXL) has been found to increase the aggressive phenotype of a number of cancers, including astrocytoma. In addition, the progression of astrocytoma has been associated with sperm‑associated antigen 9 (SPAG9), a recently characterized oncoprotein. In the present study, the association between SPAG9 and PODXL in human astrocytoma invasion and the underlying mechanisms were investigated for the first time, to the best of our knowledge. Overexpression and knockdown of SPAG9 were performed in SW1783 (grade III astrocytoma) and U87 (grade IV astrocytoma; glioblastoma) cells, respectively. PODXL expression at both the mRNA and the protein level, as well as the PODXL gene promoter activity, were significantly increased and decreased in parallel with the overexpression and knockdown of SPAG9 in astrocytoma cells; these effects were blocked by the selective c‑Jun N‑terminal kinase (JNK) inhibitor SP600125 (5 µM) and restored by the JNK agonist anisomycin (25 ng/ml), respectively. SPAG9 overexpression significantly increased cell invasion and matrix metalloproteinase‑9 (MMP‑9) expression in SW1783 cells, and this effect was reversed by knockdown of PODXL. In U87 cells, knockdown of SPAG9 markedly decreased cell invasion and MMP‑9 expression, which was completely restored by overexpression of PODXL. In conclusion, it was demonstrated in the present study that SPAG9 upregulates PODXL expression in human astrocytoma cells at the PODXL gene promoter/transcriptional level through a JNK‑dependent mechanism and that PODXL is a critical mediator of the promoting effect of SPAG9 on astrocytoma cell invasion, possibly through upregulation of MMP‑9 expression. This study provides novel insights into the molecular mechanisms involved in astrocytoma invasion.

Topics: Adaptor Proteins, Signal Transducing; Anisomycin; Anthracenes; Astrocytoma; Cell Line, Tumor; Cell Movement; Humans; JNK Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; RNA Interference; RNA, Small Interfering; Sialoglycoproteins; Up-Regulation

Ultraviolet (UV) radiation activates cell signaling pathways in melanocytes. As a result of altered signaling pathways and UV-induced cellular damage, melanocytes can undergo oncogenesis and develop into melanomas. In this study, we investigated the effect of UV-radiation on p38 MAPK (mitogen-activated protein kinase), JNK and NFκB pathways to determine which plays a major role in stimulating TNFα secretion in human HEM (melanocytes) and MM96L (melanoma) cells. MM96L cells exhibited 3.5-fold higher p38 activity than HEM cells at 5 min following UVA + B radiation and 1.6-fold higher JNK activity at 15-30 min following UVB+A radiation, while NFκB was minimally activated in both cells. Irradiated HEM cells had the greatest fold of TNFα secretion (UVB: 109-fold, UVA + B: 103-fold & UVB+A: 130-fold) when co-exposed to IL1α. The p38 inhibitor, SB202190, inhibited TNFα release by 93% from UVB-irradiated HEM cells. In the UVB-irradiated MM96L cells, both SB202190 and sulfasalazine (NFκB inhibitor) inhibited TNFα release by 52%. Although, anisomycin was a p38 MAPK activator, it inhibited TNFα release in UV-irradiated cells. This suggests that UV-mediated TNFα release may occur via different p38 pathway intermediates compared to those stimulated by anisomycin. As such, further studies into the functional role p38 MAPK plays in regulating TNFα release in UV-irradiated melanocyte-derived cells are warranted.

Topics: Anisomycin; Anthracenes; Cell Line, Tumor; Cell Survival; Humans; Imidazoles; Interleukin-1alpha; MAP Kinase Signaling System; Melanocytes; Melanoma; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinase 9; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Processing, Post-Translational; Pyridines; Sulfasalazine; Tumor Necrosis Factor-alpha

c-Jun N-terminal kinase (JNK) activation is implicated in cardiovascular diseases and ageing, which are linked to enhanced propensity to atrial fibrillation (AF). However, the contribution of JNK to AF remains unknown. Thus, we assessed the role of JNK in remodelling of gap junction connexin43 (Cx43) and development of AF.. AF induction, optical mapping, and biochemical assays were performed in young and aged New Zealand white rabbit left atria (LA) and cultured HL-1 atrial cells. In aged rabbit LA, pacing-induced atrial arrhythmias were dramatically increased and conduction velocity (CV) was significantly slower compared with young controls. Aged rabbit LA contained 120% more activated JNK and 54% less Cx43 than young LA. Young rabbits treated with JNK activator anisomycin also exhibited increased pacing-induced atrial arrhythmias and reduced Cx43 (by 34%), similar to that found in aged LA. In HL-1 cell cultures, anisomycin treatment for 16 h led to 42% reduction in Cx43, 24% reduction in CV, and an increased incidence of irregular rapid spontaneous activities. These effects were prevented by a specific JNK inhibitor, SP600125. Moreover, a 63% reduction in Cx43 after anisomycin treatment for 24 h led to further slowed CV (by 41%) along with dramatically increased irregular rapid spontaneous activity and highly discontinuous conduction. These JNK-induced functional abnormalities were completely reversed by overexpressed exogenous wild-type Cx43, but not by inactive Cx43.. JNK activation contributes to Cx43 reductions that promote development of AF. Modulation of JNK may be a potential novel therapeutic approach to prevent and treat AF.

Topics: Aging; Animals; Anisomycin; Anthracenes; Arrhythmias, Cardiac; Atrial Fibrillation; Cell Communication; Cells, Cultured; Connexin 43; Disease Models, Animal; Enzyme Inhibitors; Heart Atria; Heart Conduction System; In Vitro Techniques; JNK Mitogen-Activated Protein Kinases; Male; Rabbits

JDP2 (c-Jun dimerization protein 2) is a member of the basic leucine zipper family of transcription factors that is ubiquitously expressed in all examined cell types. JDP2 is phosphorylated on Thr148 by JNK (c-Jun N-terminal kinase) and p38 kinase, although the functional role of its phosphorylation is unknown. In the present paper we show that the JDP2 protein level is dramatically reduced in response to serum stimulation, anisomycin treatment, ultraviolet light irradiation and cycloheximide treatment, all of which activate the JNK pathway. In addition, endogenous and overexpressed JDP2 are phosphorylated in response to these stimuli. Replacement of Thr148 with an alanine residue stabilizes ectopically expressed JDP2 in the presence of the stimuli; conversely, substitution with glutamic acid destabilizes it. Serum-induced phosphorylation and degradation of JDP2 are specific to JNK activation since a JNK inhibitor (SP600125) abolishes these effects, whereas p38 and MEK inhibitors (SB203580 and UO126) have no effect. In the presence of cycloheximide, JDP2 is rapidly phosphorylated and degraded due to the combined effects of protein synthesis inhibition and activation of JNK. Pre-treatment of cells with SP600125 prior to cycloheximide treatment significantly prolongs the half-life of JDP2 that is found mainly in the unphosphorylated form. Lastly, the proteasome inhibitor (MG132) rescues JDP2 degradation following cycloheximide treatment and increases the expression of the JDP2 phospho-mimetic T148E mutant. Collectively, these results suggest that phosphorylation of JDP2 on thr148 by JNK targets it to the proteasome for degradation.

Topics: Animals; Anisomycin; Anthracenes; HEK293 Cells; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; NIH 3T3 Cells; Phosphorylation; Proteasome Endopeptidase Complex; Repressor Proteins; Serum; Threonine

Sulforaphane (SFN), mainly derived from cruciferous vegetables, has received much attention for its cancer chemopreventive property. Though there have been a few epidemiological studies supporting its beneficial effect on cardiovascular diseases, much experimental evidence are still required to understand its mechanism. In this study, human vascular endothelial cell, a barrier of blood, was used as an in vitro model to investigate the protective effect of sulforaphane on inflammatory damage induced by lipopolysaccharide (LPS). The results showed that sulforaphane inhibited the expression of COX-2 and iNOS stimulated by lipopolysaccharide in a dose- and time-dependent manner. Moreover, sulforaphane suppressed the phosphorylation of ERK1/2, JNK, and p38 activated by lipopolysaccharide. Pretreatment with SB202190, the specific inhibitor of p38, abolished the expression of COX-2 induced by LPS. Likewise, SP600125, inhibitor of JNK, abrogated iNOS expression stimulated by LPS. Moreover, pretreatment with anisomycin (AM), an activator of p38 and JNK, instead of LPS, the expression of COX-2 and iNOS is still inhibited by sulforaphane. Interestingly, SFN significantly induced HO-1 and TR expression down-regulated by LPS. Taken together, these data indicated that sulforaphane exhibited the protective role against the inflammatory injury in vascular endothelia cells, through inactivating p38 MAPK and JNK, as well as inducing phase 2 enzymes.

Topics: Anisomycin; Anthracenes; Anticarcinogenic Agents; Blotting, Western; Cell Line; Cyclooxygenase 2; Drug Antagonism; Endothelium, Vascular; Enzyme Inhibitors; Heme Oxygenase-1; Humans; Imidazoles; Isothiocyanates; Lipopolysaccharides; Membrane Proteins; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Nitric Oxide Synthase Type II; Phosphorylation; Pyridines; Sulfoxides; Thiocyanates; Vasculitis

TNF-alpha and IGF-I exert opposing effects on mammary epithelial cell (MEC) growth and survival. However, both increase IGF binding protein-3 (IGFBP-3) expression, a multifunctional protein that plays both IGF-dependent as well as independent roles in these processes. We have reported that IGF-I utilizes the PI3-K and MAPK pathways to induce IGFBP-3 expression in bovine MEC. Here we show that TNF-alpha requires the SAPK pathway p38, but not JNK, to induce IGFBP-3 expression. Contrary to reports in cancer cell lines, TNF-alpha retained its ability to decrease DNA synthesis in cells transfected with IGFBP-3 siRNA. It also retained its ability to inhibit IGF-I-stimulated DNA synthesis in these cells. In contrast, the ability of IGF-I to increase DNA synthesis was attenuated with IGFBP-3 knockdown. IGFBP-3 knockdown also decreased basal DNA synthesis, indicating that a certain level of IGFBP-3 may be required for cell proliferation. While TNF-alpha alone failed to induce apoptosis, it increased cell death when added with the JNK agonist anisomycin (ANS). TNF-alpha and ANS were unable to induce apoptosis when either IGFBP-3 or JNK-2 was knocked-down, suggesting that both JNK and IGFBP-3 may interact with a downstream molecule central to apoptosis. There are reports that IGFBP-3 promotes either cell proliferation or apoptosis in different cell systems. However, this is the first report that endogenous IGFBP-3 is required for the action of both stimulatory and inhibitory factors within the same cell line. Therefore, the actions of IGFBP-3 are not pre-determined, but instead governed by cellular context such as JNK activation.

Topics: Animals; Anisomycin; Anthracenes; Apoptosis; Cattle; Cell Line; Cell Proliferation; DNA Replication; Enzyme Activation; Epithelial Cells; Female; Imidazoles; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor I; JNK Mitogen-Activated Protein Kinases; Mammary Glands, Animal; p38 Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Pyridines; RNA Interference; RNA, Messenger; RNA, Small Interfering; Time Factors; Tumor Necrosis Factor-alpha; Up-Regulation

Because macrophages play a major role in atherosclerotic plaque destabilization, selective removal of macrophages represents a promising approach to stabilize plaques. We showed recently that the protein synthesis inhibitor cycloheximide, in contrast to puromycin, selectively depleted macrophages in rabbit atherosclerotic plaques without affecting smooth muscle cells (SMCs). The mechanism of action of these two translation inhibitors is dissimilar and could account for the differential effects on SMC viability. It is not known whether selective depletion of macrophages is confined to cycloheximide or whether it can also be achieved with translation inhibitors that have a similar mechanism of action. Therefore, in the present study, we investigated the effect of anisomycin, a translation inhibitor with a mechanism of action similar to cycloheximide, on macrophage and SMC viability. In vitro, anisomycin induced apoptosis of macrophages in a concentration-dependent manner, whereas SMCs were only affected at higher concentrations. In vivo, anisomycin selectively decreased the macrophage content of rabbit atherosclerotic plaques through apoptosis. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole] prevented anisomycin-induced macrophage death, without affecting SMC viability. SB202190 decreased anisomycin-induced p38 MAPK phosphorylation, did not alter c-Jun NH(2)-terminal kinase (JNK) phosphorylation, and increased extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. The latter effect was abolished by the mitogen-activated protein kinase kinase 1/2 inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene ethanolate], although the prevention of anisomycin-induced macrophage death by SB202190 remained unchanged. The JNK phosphorylation inhibitor SP600125 did not affect anisomycin-induced macrophage or SMC death. In conclusion, anisomycin selectively decreased the macrophage content in rabbit atherosclerotic plaques, indicating that this effect is not confined to cycloheximide. p38 MAPK, but not ERK1/2 or JNK, plays a major role in anisomycin-induced macrophage death.

Topics: Animals; Anisomycin; Anthracenes; Aorta; Apoptosis; Butadienes; Carotid Arteries; Carotid Stenosis; Cell Line, Tumor; Cells, Cultured; Extracellular Signal-Regulated MAP Kinases; Imidazoles; Macrophages; Macrophages, Alveolar; Mice; Mitogen-Activated Protein Kinases; Myocytes, Smooth Muscle; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase Inhibitors; Pyridines; Rabbits; Tunica Intima; Tunica Media

Norbinaltorphimine (NorBNI), guanidinonaltrindole, and atrans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl) piperidine (JDTic) are selective kappa opioid receptor (KOR) antagonists having very long durations of action in vivo despite binding non-covalently in vitro and having only moderately high affinities. Consistent with this, we found that antagonist treatment significantly reduced the subsequent analgesic response of mice to the KOR agonist U50,488 in the tail-withdrawal assay for 14-21 days. Receptor protection assays were designed to distinguish between possible explanations for this anomalous effect, and we found that mice pretreated with the readily reversible opioid antagonists naloxone or buprenorphine before norBNI responded strongly in the tail-flick analgesia assay to a subsequent challenge with U50,488 1 week later. Protection by a rapidly cleared reagent indicates that norBNI did not persist at the site of action. In vitro binding of [(3)H]U69,593 to KOR showed that K(d) and Bmax values were not significantly affected by prior in vivo norBNI exposure, indicating that the agonist binding site was intact. Consistent with the concept that the long-lasting effects might be caused by a functional disruption of KOR signaling, both norBNI and JDTic were found to stimulate c-Jun N-terminal kinase (JNK) phosphorylation in HEK293 cells expressing KOR-GFP but not in untransfected cells. Similarly, norBNI increased phospho-JNK in both the striatum and spinal cord in wild type mice but not in KOR knock-out mice. Pretreatment of mice with the JNK inhibitor SP600125 before norBNI attenuated the long acting antagonism. Together, these results suggest that the long duration KOR antagonists disrupt KOR signaling by activating JNK.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Animals; Anthracenes; Enzyme Activation; Enzyme Inhibitors; Humans; JNK Mitogen-Activated Protein Kinases; Kinetics; Mice; Mice, Knockout; Models, Biological; Phosphorylation; Receptors, Opioid, kappa; Signal Transduction

Wnt factors are a large family of signaling molecules that play important roles in the regulation of cell fate specification, tissue polarity and cell movement. In the nervous system, Wnts also regulates the formation of neuronal connection acting as retrograde signals that regulate the remodeling of the axons prior to the assembly of the presynaptic apparatus. The scaffold protein Dishevelled (Dvl) mimics the effect of Wnt on the neuronal cytoskeleton by increasing the number of stable microtubule along the axon shaft and inducing the formation of looped microtubules (MT) at enlarged growth cones. A divergent Wnt-Dvl canonical pathway which bifurcates downstream of Gsk3beta regulates MT dynamics.. Here we show that the Wnt pathway also activates c-Jun N-terminal kinase (JNK) to regulate MT stabilization. Although in the Wnt planar cell polarity (PCP) pathway, JNK lays downstream of Rho GTPases, these GTPases are not required for Wnt-mediated MTs stability. Epistatic analyses and pharmacological studies suggest that the Wnt-Dvl signalling regulates the dynamic of the cytoskeleton through two different pathways that lead to inhibition of Gsk3beta and activation of JNK in the same cell.. We demonstrate a novel role for JNK in Wnt-mediated MT stability. Wnt-Dvl pathway increases MT stability through a transcription independent mechanism that requires the concomitant inhibition of Gsk3beta and activation of JNK. These studies demonstrate that Wnts can simultaneously activate different signalling pathways to modulate cytoskeleton dynamics.

Topics: Adaptor Proteins, Signal Transducing; Animals; Animals, Newborn; Anisomycin; Anthracenes; Bucladesine; Cell Differentiation; Cell Line, Tumor; Cells, Cultured; Cerebellum; Dishevelled Proteins; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; JNK Mitogen-Activated Protein Kinases; Mice; Microtubules; Neuroblastoma; Neurons; Nocodazole; Phosphoproteins; Time Factors; Transfection; Wnt Proteins

Upon binding to the glycolipid receptor globotriaosylceramide, Shiga toxins (Stxs) undergo retrograde transport to reach ribosomes, cleave 28S rRNA, and inhibit protein synthesis. Stxs induce the ribotoxic stress response and cytokine and chemokine expression in some cell types. Signaling mechanisms necessary for cytokine expression in the face of toxin-mediated protein synthesis inhibition are not well characterized. Stxs may regulate cytokine expression via multiple mechanisms involving increased gene transcription, mRNA transcript stabilization, and/or increased translation initiation efficiency. We show that treatment of differentiated THP-1 cells with purified Stx1 resulted in prolonged activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) cascades, and lipopolysaccharides (LPS) rapidly triggered transient activation of JNK and p38 and prolonged activation of extracellular signal-regulated kinase cascades. Simultaneous treatment with Stx1 + LPS mediated prolonged p38 MAPK activation. Stx1 increased eukaryotic translation initiation factor 4E (eIF4E) activation by 4.3-fold within 4-6 h, and LPS or Stx1 + LPS treatment increased eIF4E activation by 7.8- and 11-fold, respectively, within 1 h. eIF4E activation required Stx1 enzymatic activity and was mediated by anisomycin, another ribotoxic stress inducer. A combination of MAPK inhibitors or a MAPK-interacting kinase 1 (Mnk1)-specific inhibitor blocked eIF4E activation by all stimulants. Mnk1 inhibition blocked the transient increase in total protein synthesis detected in Stx1-treated cells but failed to block long-term protein synthesis inhibition. The MAPK inhibitors or Mnk1 inhibitor blocked soluble interleukin (IL)-1beta and IL-8 production or release by 73-96%. These data suggest that Stxs may regulate cytokine expression in part through activation of MAPK cascades, activation of Mnk1, and phosphorylation of eIF4E.

Topics: Aniline Compounds; Anisomycin; Anthracenes; Cell Line, Tumor; Cytokines; Dose-Response Relationship, Drug; Eukaryotic Initiation Factor-4E; Flavonoids; Humans; Imidazoles; Lipopolysaccharides; Macrophages; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Phosphorylation; Purines; Pyridines; RNA, Messenger; Shiga Toxin 1; Time Factors

Abnormal phosphorylation of amyloid-beta precursor protein (APP) is a pathologic feature of Alzheimer's disease. To begin to understand the mechanism of APP phosphorylation, we studied this process in differentiating neurons under normal physiological conditions. We found that c-Jun NH2-terminal kinase (JNK), not cyclin-dependent kinase 5, is required for APP phosphorylation, leading to localized accumulation of phosphorylated APP (pAPP) in neurites. We show that JNK-interacting protein-3 (JIP-3), a JNK scaffolding protein that does not bind APP, selectively increases APP phosphorylation, accumulation of pAPP into processes, and stimulates process extension in both neurons and COS-1 cells. Downregulation of JIP-3 by small interfering RNA impairs neurite extension and reduces the amount of localized pAPP. Finally, whereas stress-activated JNK generates pAPP only in the cell body, concomitant expression of JIP-3 restores pAPP accumulation into neurites. Thus, APP phosphorylation, transport of the generated pAPP into neurites, and neurite extension are interdependent processes regulated by JIP-3/JNK, in a pathway distinct from stress-activated JNK signaling.

Topics: Adaptor Proteins, Signal Transducing; Amyloid beta-Protein Precursor; Animals; Anisomycin; Anthracenes; Blotting, Western; Cell Line; Chlorocebus aethiops; Enzyme Inhibitors; Gene Expression Regulation; Growth Inhibitors; Humans; Immunohistochemistry; Immunoprecipitation; Mice; Mitogen-Activated Protein Kinase 10; Models, Biological; Neurites; Neurons; Phosphorylation; Protein Binding; Purines; RNA, Small Interfering; Roscovitine; Transfection

Genes encoding numerous proto-oncogenes and cytokines, as well as a number of G-protein coupled receptors, are regulated post-transcriptionally at the level of mRNA stability. A common feature of all of these genes is the presence of A + U-rich elements (AREs) within their 3' untranslated regions. We, and others, have demonstrated previously that mRNAs encoding beta-adrenergic receptors (beta-ARs) are destabilized by agonist stimulation of the beta-AR/Galphas/adenylylcyclase pathway. However, in addition to PK-A, beta-ARs can also activate or inhibit mitogen activated kinase (MAPK) cascades, in a cell-type dependent basis. Recent evidence points to an important role for MAPKs in regulating the turnover of cytokine mRNAs, such as TNFalpha. We hypothesized that activation of MAPK's may also regulate beta-AR mRNA stability. The studies conducted herein demonstrate that generalized stimulation of MAPKs (JNK, p38) with anisomycin resulted in marked stabilization of beta-AR mRNA. Reciprocally, selective inhibition of JNK with SP600125 significantly decreased beta-AR mRNA half-life. Similarly, inhibition of the MEK/ERK pathway with either PD98059 or U0126 decreased beta-AR mRNA stability substantially. However, inhibition of p38 MAPK with SB203580 produced destabilization of beta-AR mRNA only at higher, non pharmacologically selective concentrations. In contrast to their effects on several other ARE containing mRNAs, inhibition of tyrosine kinases by genistein or PI3K by wortmannin, had no detectable effect on beta-AR mRNA stability. In summary, these results demonstrate for the first time that modulation of MAPK pathways can bi-directionally influence beta-AR mRNA stability.

Topics: 3' Untranslated Regions; Animals; Anisomycin; Anthracenes; Butadienes; Cell Line; Cricetinae; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Heterogeneous-Nuclear Ribonucleoproteins; Imidazoles; Mitogen-Activated Protein Kinase Kinases; Nitriles; Nucleic Acid Synthesis Inhibitors; Phosphatidylinositol 3-Kinases; Pyridines; Receptors, Adrenergic, beta; RNA Stability; Signal Transduction

cAMP significantly inhibits IL-1beta+IFNgamma-induced iNOS gene expression in hepatocytes, but the signaling pathways responsible for the effect are not known. PKA inhibitors, H89, PKI, and KT5720, had no effect on the recovery of the inhibitory effects of cAMP on cytokine-induced hepatocyte iNOS expression and activity. The JNK inhibitor, SP 600125, effectively reversed the inhibitory effects of cAMP on iNOS expression and significantly increased iNOS promoter activity. A cAMP analogue, dbcAMP, significantly induced JNK signaling and increased AP-1 binding activity in hepatocytes. The JNK activator, anisomycin, inhibited iNOS expression and transcription in hepatocytes as well as AP-1 binding activity; and SP600125 reversed this effect of anisomycin. Overexpression of c-Jun in hepatocytes inhibited IL-1beta+IFNgamma-induced nitrite accumulation and iNOS promoter activity while dominant negative c-Jun partially reversed the inhibitory effects of cAMP on nitrite accumulation. We conclude that JNK signaling plays an important role in the inhibitory effects of cAMP on IL-1beta+IFNgamma-induced iNOS gene expression in cultured hepatocytes.

Topics: Animals; Anisomycin; Anthracenes; Blotting, Western; Cell Nucleus; Cyclic AMP; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hepatocytes; Humans; Interferon-gamma; Interleukin-1; JNK Mitogen-Activated Protein Kinases; Luciferases; Male; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nucleic Acid Synthesis Inhibitors; Plasmids; Rats; Rats, Sprague-Dawley; Signal Transduction; Time Factors; Transfection

Numerous enzymes hyperphosphorylate Tau in vivo, leading to the formation of neurofibrillary tangles (NFTs) in the neurons of Alzheimer's disease (AD). Compared with age-matched normal controls, we demonstrated here that the protein levels of WW domain-containing oxidoreductase WOX1 (also known as WWOX or FOR), its Tyr33-phosphorylated form, and WOX2 were significantly down-regulated in the neurons of AD hippocampi. Remarkably knock-down of WOX1 expression by small interfering RNA in neuroblastoma SK-N-SH cells spontaneously induced Tau phosphorylation at Thr212/Thr231 and Ser515/Ser516, enhanced phosphorylation of glycogen synthase kinase 3beta (GSK-3beta) and ERK, and enhanced NFT formation. Also an increased binding of phospho-GSK-3beta with phospho-Tau was observed in these WOX1 knock-down cells. In comparison, increased phosphorylation of Tau, GSK-3beta, and ERK, as well as NFT formation, was observed in the AD hippocampi. Activation of JNK1 by anisomycin further increased Tau phosphorylation, and SP600125 (a JNK inhibitor) and PD-98059 (an MEK1/2 inhibitor) blocked Tau phosphorylation and NFT formation in these WOX1 knock-down cells. Ectopic or endogenous WOX1 colocalized with Tau, JNK1, and GSK-3beta in neurons and cultured cells. 17Beta-estradiol, a neuronal protective hormone, increased the binding of WOX1 and GSK-3beta with Tau. Mapping analysis showed that WOX1 bound Tau via its COOH-terminal short-chain alcohol dehydrogenase/reductase domain. Together WOX1 binds Tau via its short-chain alcohol dehydrogenase/reductase domain and is likely to play a critical role in regulating Tau hyperphosphorylation and NFT formation in vivo.

Topics: Alzheimer Disease; Animals; Anisomycin; Anthracenes; Brain; Cell Line; Cell Line, Tumor; COS Cells; Down-Regulation; Enzyme Inhibitors; Estradiol; Flavonoids; Genetic Vectors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hippocampus; Humans; Immunohistochemistry; In Vitro Techniques; Mice; Microscopy, Fluorescence; Mitogen-Activated Protein Kinases; Neurons; Oxidoreductases; Phosphorylation; Precipitin Tests; Protein Binding; Protein Structure, Tertiary; Retroviridae; RNA, Small Interfering; Serine; tau Proteins; Temperature; Threonine; Time Factors; Tumor Suppressor Proteins; Two-Hybrid System Techniques; Tyrosine; WW Domain-Containing Oxidoreductase

To further evaluate the significance of p38 MAPK as trigger or mediator in ischaemic preconditioning, anisomycin and SB 203580 were used to manipulate its activation status. Special attention was given to the concentration of the drugs and protocols used. The isolated perfused rat heart, subjected to either 25 min global ischaemia or 35 min regional ischaemia, was used as experimental model. This was preceded by anisomycin (2 or 5 muM: 3 x 5 min; 5 muM: 5 min or 10 min; 5 muM: 10 min + 10 min washout or 20 muM: 20 min) or SB 203580 (2 muM: 3 x 5 min; before and during 3 x 5 min or 1 x 5 min ischaemic preconditioning; 10 min). Endpoints were functional recovery during reperfusion and infarct size.Anisomycin, regardless of the protocol, reduced infarct size, but did not improve functional recovery. In a number of experiments activation of JNK by anisomycin was blocked by SP 600125 (10 muM). SP 600125 had no effect on the anisomycin-induced reduction in infarct size. SB 203580 when administered for 10 min before sustained ischaemia, improved functional recovery and reduced infarct size. SB 203580 could not abolish the beneficial effects of a multi-cycle preconditioning protocol, but it significantly reduced the outcome of 1 x 5 min preconditioning. In all hearts improved functional recovery and reduction in infarct size were associated with attenuation of p38 MAPK activation during sustained ischaemia-reperfusion. The results indicate that activation of p38 MAPK acts as a trigger of preconditioning, while attenuation of its activation is a prerequisite for improved recovery and a reduction in infarct size.

Topics: Animals; Anisomycin; Anthracenes; Cardiac Output; Coronary Circulation; Disease Models, Animal; Enzyme Activation; Heart Rate; Imidazoles; Ischemic Preconditioning, Myocardial; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Myocardial Infarction; Myocardial Ischemia; p38 Mitogen-Activated Protein Kinases; Pyridines; Rats; Rats, Wistar

Prolonged activation of the c-Jun N-terminal kinase (JNK) has been suggested as a signal for apoptosis in response to a wide variety of stimuli. Using three cytocidal RNA or protein synthesis inhibitors (actinomycin D, anisomycin, and emetine), the potential role of JNK in activation of the mitochondrial apoptotic cascade was investigated in A549-S cells. Protein synthesis inhibition per se was not the cause of cell death as cycloheximide induced only growth arrest. All the cytocidal inhibitors induced cytochrome c release and caspases 9 activation within hours, but only anisomycin caused persistent JNK activation. Although, the JNK inhibitor, SP600125, inhibited JNK-dependent anisomycin-induced c-Jun phosphorylation, it was ineffective in preventing anisomycin-induced caspase activation and cell death. Thus, all three lethal macromolecule synthesis inhibitors can activate the mitochondrial apoptotic machinery independent of JNK activation, demonstrating that the mitochondrial apoptotic pathway can be activated independently of the JNK pathway in the absence of protein synthesis.

Topics: Anisomycin; Anthracenes; Apoptosis; Caspase 9; Caspases; Cytochrome c Group; Dactinomycin; Dose-Response Relationship, Drug; Emetine; Enzyme Activation; Enzyme Inhibitors; Humans; JNK Mitogen-Activated Protein Kinases; Mitochondria; Mitogen-Activated Protein Kinases; Nucleic Acid Synthesis Inhibitors; Protein Biosynthesis; Protein Synthesis Inhibitors; Proteins; Time Factors

Loss of gap junctions and impaired intercellular communication are characteristic features of pathological remodeling in heart failure as a result of stress or injury, yet the underlying regulatory mechanism has not been identified. Here, we report that in cultured myocytes, rapid loss of the gap junction protein connexin43 (Cx43) occurs in conjunction with the activation of c-Jun N-terminal kinase (JNK), a stress-activated protein kinase, on stress stimulation. To investigate the specific role of JNK activation in the regulation of connexin in cardiomyocytes, an activated mutant of mitogen-activated protein kinase kinase 7 (mutant D), a JNK-specific upstream activator, was expressed in myocytes by adenovirus-mediated gene transfer. JNK activation in infected cardiomyocytes resulted in significant reduction of Cx43 expression at both mRNA and protein levels and impaired cell-cell communication. To evaluate the role of JNK in the regulation of Cx43 expression and gap junction structure in vivo, a Cre-LoxP-mediated gene-switch system was used to establish a transgenic animal model with targeted activation of JNK in ventricular myocardium. The transgenic hearts exhibited significant downregulation of Cx43 expression and loss of gap junctions in myocardium that may contribute to the cardiac dysfunction and premature death phenotype. Our report represents the first evidence, both in vitro and in vivo, implicating JNK as an important mediator of stress-induced Cx43 downregulation and impaired intercellular communication in the failing heart.

Topics: Animals; Animals, Genetically Modified; Anisomycin; Anthracenes; Cell Communication; Cell Death; Cells, Cultured; Connexin 43; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Gap Junctions; Gene Targeting; Heart Failure; Heart Ventricles; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases; Myocardium; Protein Synthesis Inhibitors; Rats; Rats, Sprague-Dawley; RNA, Messenger