c-Jun Signaling Research Articles

Role of Dickkopf-1, an Antagonist of the Wnt/β-Catenin Signaling Pathway, in Estrogen-Induced Neuroprotection and Attenuation of Tau Phosphorylation

17beta-Estradiol (E2) has been implicated to be neuroprotective in a variety of neurodegenerative disorders, although the mechanism remains poorly understood. The current study sheds light on this issue by demonstrating that low physiological levels of E2 protects the hippocampus CA1 against global cerebral ischemia by preventing elevation of dickkopf-1 (Dkk1), an antagonist of the Wnt/beta-catenin signaling pathway, which is a principal mediator of neurodegeneration in cerebral ischemia and Alzheimer's disease. E2 inhibition of Dkk1 elevation correlated with a reduction of phospho-beta-catenin and elevation of nuclear beta-catenin levels, as well as enhancement of Wnt-3, suggesting E2 activation of the Wnt/beta-catenin signaling pathway. In agreement, the beta-catenin downstream prosurvival factor, survivin, was induced by E2 at 24 and 48 h after cerebral ischemia, an effect observed only in surviving neurons because degenerating neurons lacked survivin expression. E2 suppression of Dkk1 elevation was found to be caused by attenuation of upstream c-Jun N-terminal protein kinase (JNK)/c-Jun signaling, as E2 attenuation of JNK/c-Jun activation and a JNK inhibitor significantly blocked Dkk1 induction. Tau hyperphosphorylation has been implicated to have a prodeath role in Alzheimer's disease and cerebral ischemia, and E2 attenuates tau hyperphosphorylation. Our study demonstrates that tau hyperphosphorylation is strongly induced after global cerebral ischemia, and that E2 inhibits tau hyperphosphorylation by suppressing activation of the JNK/c-Jun/Dkk1 signaling pathway. Finally, exogenous Dkk1 replacement via intracerebroventricular administration completely reversed E2-induced neuroprotection, nuclear beta-catenin induction, and phospho-tau attenuation, further suggesting that E2 inhibition of Dkk1 is a critical mechanism underlying its neuroprotective and phospho-tau regulatory effects after cerebral ischemia.

Stopping the Primal RAGE Reaction in Myocardial Infarction

The report of Andrassy and colleagues1 in the current issue of Circulation adds to the growing body of evidence that the receptor for advanced glycation end products (RAGE) and its ligands, particularly high mobility group box-1 (HMGB1), are central mediators of ischemia/reperfusion (I/R) injury in the heart.1–4 A major cause of injury, especially in the reperfusion phase, is the influx of inflammatory cells into the stressed heart. Andrassy and colleagues show that infiltrating leukocytes express proinflammatory HMGB1 and that HMGB1 plays fundamental roles in injury responses in the I/R heart. In homeostasis, HMGB1 is largely a nuclear protein. In stress conditions, HMGB1 may be released from injured cells, particularly on necrosis. The chief receptor for HMGB1, RAGE, is expressed in multiple cell types in the I/R heart, such as inflammatory cells, and also in cardiomyocytes and vascular cells (endothelial cells and smooth muscle cells).2 Although HMGB1 may interact with distinct receptors beyond RAGE (for example, toll receptors),5 the current study of Andrassy and colleagues reveals major roles for RAGE in transducing the effects of HMGB1 in the heart, as administration of recombinant HMGB1 or antagonists of this molecule had significant proinjury effects in the wild-type mouse heart, but no additive effects were noted in mice devoid of RAGE. The biology of RAGE, however, is complex and extends beyond HMGB1 in the injured heart. Article p 3216 In addition to HMGB1, other ligands of RAGE likely play pivotal roles in the response to ischemia or I/R in the heart. For example, rapid generation of pre–advanced glycation end products (AGE) species (methylglyoxal) and AGEs in transient occlusion/reperfusion of the LAD coronary artery has been illustrated.2 In hypoxia, in both the murine heart and in isolated primary murine endothelial cells, AGE-immunoreactive species are central mediators of regulation of …

Effect of naloxone on the induction of immediately early genes following oxygen- and glucose-deprivation in PC12 cells

Cerebral ischemia/reperfusion involves inflammatory process and naloxone is able to reduce infarct volume and has been used as a therapeutic agent for brain injury. Hypoxia induces the immediate early genes (IEGs) rapidly and transiently that may initiate a cascade of cellular responses that are necessary for survival and normal function. However, the protective effect of naloxone on ischemic/hypoxic neuronal cells was only partly studied. Thus, the effects of naloxone on oxygen- and glucose-deprivation (OGD) and OGD followed by reoxygenation (OGD/R) on the expression of IEGs were examined in PC12 cells. The result showed that lactate dehydrogenase (LDH) released in the media was reduced by naloxone. The temporal response of IEG mRNA encoding c-fos, c-jun, nur77, and zif268 was induced with different degree of intensity following hypoxia, whereas the level of GAPDH mRNA was relatively constant. However, these signals of c-fos, c-jun, and nur77 by hypoxia were reduced significantly by naloxone. Treatment with OGD also activated mitogen-activated protein kinase (MAPK) pathway. The induction of c-fos, c-jun, nur77, and zif268 by hypoxia was inhibited by naloxone (0.1 microM) and MAPK inhibitors (10 microM of U0126, D98059, SB203580). However, naloxone increased the expression of ERK1/2 by OGD concomitantly diminished the LDH release. Thus, the present studies demonstrated that OGD induced IEGs including c-fos, c-jun, nur77, and zif268 and MAPK signaling pathways were regulated differently by naloxone.

Benzo(a)pyrene-caused increased G 1–S transition requires the activation of c-Jun through p53-dependent PI-3K/Akt/ERK pathway in human embryo lung fibroblasts

Benzo(a)pyrene (B(a)P) is a potent lung carcinogen mainly derived from tobacco smoking and environmental contamination, however, the molecular mechanisms by which it accelerates the cell cycle progression and induces the abnormal cell proliferation are still far away from understood. Our current analysis of human embryo lung fibroblasts (HELF) showed that B(a)P exposure was able to promote cell cycle G 1–S phase transition. This effect was correlated with c-Jun activation because inhibition of c-Jun by its dominant negative mutant (TAM67) reversed B(a)P action on cell cycle with the down-regulation of expression of cyclin D1, pRb and E2F1. Further study found that overexpression of dominant negative mutants of, PI-3K or Akt, dramatically reduced B(a)P-induced the activation of c-Jun and extracellular signaling regulated kinase (ERK), but not c-Jun NH2 terminal kinase (JNK). Inhibition of p53 by either its inhibitor pifithrin-α or p53 siRNA markedly increased B(a)P-induced the activation of c-Jun, Akt and ERK in this context. Take together, our results indicate that c-Jun activation by p53-dependent PI-3K/Akt/ERK pathway is responsible for B(a)P-induced cell cycle alternations in human embryo lung fibroblasts.

Endoplasmic Reticulum Stress Increases the Expression of Methylenetetrahydrofolate Reductase through the IRE1 Transducer

Methylenetetrahydrofolate reductase (MTHFR), an enzyme in folate and homocysteine metabolism, influences many cellular processes including methionine and nucleotide synthesis, methylation reactions, and maintenance of homocysteine at nontoxic levels. Mild deficiency of MTHFR is common in many populations and modifies risk for several complex traits including vascular disease, birth defects, and cancer. We recently demonstrated that MTHFR can be up-regulated by NF-kappaB, an important mediator of cell survival that is activated by endoplasmic reticulum (ER) stress. This observation, coupled with the reports that homocysteine can induce ER stress, prompted us to examine the possible regulation of MTHFR by ER stress. We found that several well characterized stress inducers (tunicamycin, thapsigargin, and A23187) as well as homocysteine could increase Mthfr mRNA and protein in Neuro-2a cells. The induction of MTHFR was also observed after overexpression of inositol-requiring enzyme-1 (IRE1) and was inhibited by a dominant-negative mutant of IRE1. Because IRE1 triggers c-Jun signaling, we examined the possible involvement of c-Jun in up-regulation of MTHFR. Transfection of c-Jun and two activators of c-Jun (LiCl and sodium valproate) increased MTHFR expression, whereas a reported inhibitor of c-Jun (SP600125) and a dominant-negative derivative of c-Jun N-terminal kinase-1 reduced MTHFR activation. We conclude that ER stress increases MTHFR expression and that IRE1 and c-Jun mediate this activation. These findings provide a novel mechanism by which the ER can regulate homeostasis and allude to an important role for MTHFR in cell survival.

Critical roles for JNK, c‐Jun, and Fas/FasL‐Signaling in vitamin E analog‐induced apoptosis in human prostate cancer cells

Alpha-tocopherol ether-linked acetic acid (alpha-TEA), an analog of vitamin E (RRR-alpha-tocopherol), is a potent pro-apoptotic agent for human cancer cells in vivo and in vitro. alpha-TEA-induced apoptosis was investigated in LNCaP and PC-3 human prostate cancer cells. Apoptosis was measured by DAPI-staining and FACS analyses of the sub-G1 fraction. Signaling molecules involved in apoptosis were measured by Western immunoblot analyses with or without prior immunoprecipitation, FACS analyses of cell surface membrane expression, RT-PCR analyses of mRNA levels, and chromatin immunoprecipitation. Functional significance was determined using siRNAs, dominant negative mutant, chemical inhibitor, or neutralizing antibody. Alpha-TEA treatment increased Fas and Fas ligand mRNA and protein levels; as well as, levels of cell surface membrane Fas in both cell lines. Blockage of Fas signaling attenuated alpha-TEA-induced apoptosis. alpha-TEA treatment also produced prolonged, elevated levels of activated (phosphorylated) c-Jun N-terminal kinase (JNK) and its substrate c-Jun, both of which were demonstrated to be necessary for alpha-TEA-induced apoptosis. Chromatin immunoprecipitation results showed binding of c-Jun to the promoters of both Fas and FasL in alpha-TEA treated cells. Investigations of alpha-TEA-triggered apoptosis showed dual signaling from Fas with essential roles for both FADD and Daxx with FADD initiating the classical pathway mediated by caspase-8 activation and Daxx initiating an alternate pathway involving activation of JNK, c-Jun, and increased levels of Fas and FasL. Collectively, data support critical roles for JNK, c-Jun, and dual signaling from Fas/FasL via FADD and Daxx in alpha-TEA-induced apoptosis of human prostate cancer cells.

-lipoic acid promotes the growth of rat hepatic pre-neoplastic lesions in the choline-deficient model

alpha-lipoic acid (alpha-LA) is an antioxidant used in a number of conditions related to liver diseases. Herein, we investigated the effect of alpha-LA on the development of rat pre-neoplastic lesions generated by a model of hepatocarcinogenesis, which has similarities in its histopathological sequence to human hepatocellular carcinoma development with cirrhosis. Initiation of hepatocytes was achieved by treatment with a single dose of diethylnitrosamine and promotion by feeding a choline-methionine-deficient diet (CMD), with or without alpha-LA. Pre-neoplastic lesions were identified by their positivity to the placental form of glutathione S-transferase (GSTP) or to gamma glutamyl transpeptidase. alpha-LA given to rats fed a CMD for 6 weeks dramatically increased the number of GSTP-positive foci as compared with rats fed a CMD alone (96/cm(2) versus 7/cm(2)), the mean foci area (0.033 versus 0.008 mm(2)) and the percentage of GSTP-positive liver tissue (3.01 versus 0.07%). Essentially similar results were obtained after 10 weeks of treatment. Co-treatment with CMD + alpha-LA also resulted in the enhancement of fat accumulation, lipid peroxidation and hepatocyte death; increased expression of tumor necrosis factor-alpha, cytochrome 2E1 and cyclooxygenase-2, enhanced activation of c-jun N-terminal kinase and signal transducer activator of transcription 3, and chronic hepatocyte proliferation was also observed. No such effects were observed when alpha-LA was added to a choline-supplemented diet. In conclusion, administration of alpha-LA in conditions associated with hepatic damage aggravates liver injury and stimulates the development of pre-neoplastic lesions; the results also suggest caution in its use in the presence of chronic liver injury.

Curcumin Attenuates Inflammatory Responses of TNF-α-Stimulated Human Endothelial Cells

Curcumin, a yellow pigment of turmeric in curry, is reported to interfere with nuclear factor (NF)-kappaB. This study was designed to investigate the underlying pathway of antiinflammation of curcumin on endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with 10 ng/mL tumor necrosis factor (TNF)-alpha. Curcumin blocked the activation of NF-kappaB by TNF-alpha. Curcumin also reduced the intracellular reactive oxygen species (ROS), monocyte adhesion, phosphorylation of c-Jun N-terminal kinase (JNK), p38, and signal transducer and activator of transcription (STAT)-3 in TNF-alpha-stimulated HUVECs. The expression of intracellular cell adhesion molecule (ICAM)-1, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-8 were attenuated by curcumin at both mRNA and protein level. Curcumin, however, did not affect the expression of TNF receptor I and II in TNF-alpha-stimulated HUVECs. We suggest that curcumin could contribute to protection against the adverse vascular effect of the proinflammatory response through the modulation of p38 and STAT-3 in addition to NF-kappaB and JNK in endothelial cells.

Dexamethasone promotes osteoclastogenesis by inhibiting osteoprotegerin through multiple levels

Increased bone fragility attributed to osteopenia is a serious side effect of glucocorticoid treatment. Glucocorticoid-induced bone loss is caused primarily by hypofunction and apoptosis of osteoblasts, and secondarily by accelerated bone resorption. To explore the mechanism whereby dexamethasone (Dex) stimulates osteoclastogenesis in the coculture system, we analyzed the effect of Dex on the expression of both mouse osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL). Dex reduced OPG transcripts and OPG protein secretion by the ST2 osteoblastic cells. Since mainly the c-Jun homodimer maintains the steady-state transcription of the OPG gene, we examined the effect of Dex on c-Jun signaling in ST2 cells. Western blotting disclosed that Dex decreased the amount of phospho-c-Jun protein (p-c-Jun) and, correspondingly, the amount of the phosphorylated p46 isoform of Jun N-terminal kinase (JNK). The amount of phospho-SEK1 also decreased after Dex treatment, while the amounts of phospho-ERK and p38 remained constant. Among mitogen-activated protein (MAP) kinase inhibitors, the JNK inhibitor mimicked the inhibitory effect of Dex on OPG promoter activity. On the other hand, Dex treatment per se showed a nominal increase of RANKL gene expression. A part of Dex-mediated OPG gene suppression was achieved by the suppression of beta-catenin signaling. We speculate therefore that the bone resorptive action of Dex is mediated mainly by the inhibition of OPG by transrepressing the OPG gene through the AP-1 site, with a reduction (mediated mainly by the decrease in the p46 isoform of JNK) in the proportion of p-c-Jun in a JNK-dependent manner.

Comparative anti-inflammatory characterization of wild fruiting body, liquid-state fermentation, and solid-state culture of Taiwanofungus camphoratus in microglia and the mechanism of its action

Taiwanofungus camphoratus (syn. Antrodia camphorata), a medicinal mushroom in Taiwan, is reputed to provide several therapeutic benefits, but the wild fruiting body is very rare. In this study, we used Taiwanofungus camphoratus extracts from wild fruiting bodies and two types of artificial cultivation (solid-state culture and liquid-state fermentation) to examine their anti-inflammatory effects in microglia cells and their possible roles in protection against neurodegenerative diseases. First, EOC13.31 microglia was treated with various kinds of Taiwanofungus camphoratus extracts and lipopolysaccharide (LPS) and interferon-γ (IFN-γ) to evaluate the iNOS expression. Western blot and RT-PCR analysis showed that among the various kinds of extracts from wild fruiting bodies, methanol extracts were the most potent inhibitors of iNOS expression. Secondly, the potency of methanol extracts could be ranked as follows: extracts of wild fruiting body > solid-state culture > liquid-state fermentation. To clarify the mechanisms involved, methanol extracts from fruiting body were found to inhibit the phosphorylation of extracellular signal-regulated protein kinases (ERK), c-Jun NH2-terminal protein kinases (JNK) and signal transducer and activator of transcription-1 (STAT-1) induced by LPS/IFN-γ. Methanol extracts from fruiting body also inhibited NF-κB activation through the prevention of inhibitor κB (IκB) degradation. Moreover, methanol extracts from wild fruiting body inhibited both the iNOS and cyclooxygenase-2 (COX-2) expression induced by β-amyloid in microglia in a dose-dependent manner. In an animal model, we confirmed that methanol extracts from fruiting bodies were able to suppress ear edema, indicating that they have anti-inflammatory activity in vivo. These results suggest that Taiwanofungus camphoratus exhibits an anti-inflammatory activity that might contribute to the prevention of neurodegenerative diseases.

Osteoclasts: What Do They Do and How Do They Do It?

As Americans live longer, degenerative skeletal diseases, such as osteoporosis, become increasingly prevalent. Regardless of cause, osteoporosis reflects a relative enhancement of osteoclast activity. Thus, this unique bone resorptive cell is a prominent therapeutic target. A number of key observations provide insights into the mechanisms by which precursors commit to the osteoclast phenotype and how the mature cell degrades bone. The osteoclast is a member of the monocyte/macrophage family that differentiates under the aegis of two critical cytokines, namely RANK ligand and M-CSF. Tumor necrosis factor (TNF)-alpha also promotes osteoclastogenesis, particularly in states of inflammatory osteolysis such as that attending rheumatoid arthritis. Once differentiated, the osteoclast forms an intimate relationship with the bone surface via the alphavbeta3 integrin, which transmits matrix-derived, cytoskeleton-organizing, signals. These integrin-transmitted signals include activation of the associated proteins, c-src, syk, Vav3, and Rho GTPases. The organized cytoskeleton generates an isolated microenvironment between the cell's plasma membrane and the bone surface in which matrix mineral is mobilized by the acidic milieu and organic matrix is degraded by the lysosomal protease, cathepsin K. This review focuses on these and other molecules that mediate osteoclast differentiation or function and thus serve as candidate anti-osteoporosis therapeutic targets.

Kinase signaling pathways: potential therapeutic targets in Parkinson‘s disease

There is substantial preclinical evidence that the mixed-lineage kinase–JNK–c-jun signaling pathway plays a role in programmed cell death in neurons, including dopamine neurons of the substantia nigra, which degenerate in Parkinson’s disease. Therefore, this pathway is a likely target for therapeutics. However, a recent large, double-blind clinical trial, the Parkinson Research Examination of CEP-1347 Trial (PRECEPT), of a mixed-lineage kinase inhibitor, CEP1347, has failed to demonstrate efficacy in preventing the progression of the disease, and has brought this approach to a crossroads. There are many considerations related to the implementation of the trial and our limited knowledge about the neural basis of the progression of the disease, which preclude a conclusion from this negative outcome that inhibition of this kinase pathway is a failed strategy. Furthermore, growing knowledge of the molecular basis of Parkinson’s disease, derived from investigations of genetic causes, including mutations in DJ-1, PTEN-induced putative kinase 1 (PINK-1) and leucine-rich repeat kinase 2 (LRRK2), suggest that kinase signaling pathways may be central to pathogenesis. We conclude that these pathways remain important future therapeutic targets.

Large Hepatitis Delta Antigen Modulates Transforming Growth Factor-β Signaling Cascades: Implication of Hepatitis Delta Virus–Induced Liver Fibrosis

Transforming growth factor-beta (TGF-beta) has been implicated in the pathogenesis of liver disease. TGF-beta is involved in liver regeneration and in the fibrotic and cirrhotic transformation with hepatitis viral infection. Hepatitis delta virus (HDV) infection causes fulminant hepatitis and liver cirrhosis. To elucidate the molecular mechanism of HDV pathogenesis, we examined the effects of HDV-encoded-only protein, the small hepatitis delta antigen (SHDAg), and the large hepatitis delta antigen (LHDAg), on TGF-beta- and c-Jun-induced signaling cascades. The effects of either SHDAg or LHDAg on TGF-beta- and c-Jun-induced signaling cascades in Huh7 and Cos7 cells were investigated by luciferase reporter gene assay, immunoprecipitation assay, electrophoretic mobility shift assay, Western blot analysis, and confocal microscopy analysis. The LHDAg, but not the SHDAg, potentiated TGF-beta- and c-Jun-induced signal activation, and the isoprenylation of LHDAg played a major role in signaling cascades. LHDAg synergistically activated hepatitis B virus X protein-mediated TGF-beta and AP-1 signaling cascades. In addition, LHDAg enhanced the protein expression level of TGF-beta-induced plasminogen activator inhibitor-1. LHDAg may induce liver fibrosis through the regulation of TGF-beta-induced signal transductions. This regulation of TGF-beta-mediated signaling is accomplished by the isoprenylation of LHDAg, which is a novel mechanism involved in HDV pathogenesis.

The AP-1/CJUN signaling cascade is involved in muscle differentiation: Implications in muscle wasting during cancer cachexia

The aim of the present study was to investigate a possible role of the AP-1 signaling cascade in the process of wasting associated with cancer cachexia at the level of skeletal muscle. The injection of virus containing the TAM67 protein (a blocker of the AP-1 protein) to the gastrocnemius muscle of tumour-bearing rats resulted in a significant recovery of the muscle mass (which is dramatically reduced as a result of tumour burden), therefore suggesting that AP-1 is certainly involved in the signaling associated with muscle protein accretion. In conclusion, the gene therapy approach presented here clearly suggests an important role for AP-1 in muscle signaling during catabolic states.

Jnk1 but not jnk2 promotes the development of steatohepatitis in mice†

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis and varying degrees of necroinflammation. Although chronic oxidative stress, inflammatory cytokines, and insulin resistance have been implicated in the pathogenesis of NAFLD, the mechanisms that underlie the initiation and progression of this disease remain unknown. c-Jun N-terminal kinase (JNK) is activated by oxidants and cytokines and regulates hepatocellular injury and insulin resistance, suggesting that this kinase may mediate the development of steatohepatitis. The presence and function of JNK activation were therefore examined in the murine methionine- and choline-deficient (MCD) diet model of steatohepatitis. Activation of hepatic JNK, c-Jun, and AP-1 signaling occurred in parallel with the development of steatohepatitis in MCD diet-fed mice. Investigations in jnk1 and jnk2 knockout mice demonstrated that jnk1, but not jnk2, was critical for MCD diet-induced JNK activation. JNK promoted the development of steatohepatitis as MCD diet-fed jnk1 null mice had significantly reduced levels of hepatic triglyceride accumulation, inflammation, lipid peroxidation, liver injury, and apoptosis compared with wild-type and jnk2 -/- mice. Ablation of jnk1 led to an increase in serum adiponectin but had no effect on serum levels of tumor necrosis factor-alpha. In conclusion, JNK1 is responsible for JNK activation that promotes the development of steatohepatitis in the MCD diet model. These findings also provide additional support for the critical mechanistic involvement of JNK1 overactivation in conditions associated with insulin resistance and the metabolic syndrome.

Semaphorin 3A and neurotrophins: a balance between apoptosis and survival signaling in embryonic DRG neurons

Large numbers of neurons are eliminated by apoptosis during nervous system development. For instance, in the mouse dorsal root ganglion (DRG), the highest incidence of cell death occurs between embryonic days 12 and 14 (E12-E14). While the cause of cell death and its biological significance in the nervous system is not entirely understood, it is generally believed that limiting quantities of neurotrophins are responsible for neuronal death. Between E12 and E14, developing DRG neurons pass through tissues expressing high levels of axonal guidance molecules such as Semaphorin 3A (Sema3A) while navigating to their targets. Here, we demonstrate that Sema3A acts as a death-inducing molecule in neurotrophin-3 (NT-3)-, brain-derived neurotrophic factor (BDNF)- and nerve growth factor (NGF)-dependent E12 and E13 cultured DRG neurons. We show that Sema3A most probably induces cell death through activation of the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway, and that this cell death is blocked by a moderate increase in NGF concentration. Interestingly, increasing concentrations of other neurotrophic factors, such as NT-3 or BDNF, do not elicit similar effects. Our data suggest that the number of DRG neurons is determined by a fine balance between neurotrophins and Semaphorin 3A, and not only by neurotrophin levels.

Knock‐down of POSH expression is neuroprotective through down‐regulating activation of the MLK3–MKK4–JNK pathway following cerebral ischaemia in the rat hippocampal CA1 subfield

We investigated the expression and subcellular localization of the multidomain protein POSH (plenty of SH3s) by immunohistochemistry and western blot analysis, as well as its role in the selective activation of mixed-lineage kinases (MLKs) 3, MAP kinase kinase (MKK) 4, c-Jun N-terminal kinases (JNKs) and the c-Jun signalling cascade in the rat hippocampal CA1 region following cerebral ischaemia. Our results indicated that the cytosol immunoreactivity of POSH was strong in the CA1-CA3 pyramidal cell but weak in the DG granule cell of the rat hippocampus both in sham control and after reperfusion. Co-immunoprecipitation experiments showed that the interactions of MLK3, MKK4 and phospho-JNKs with POSH were persistently enhanced during the early (30 min) and the later reperfusion period (from 1 to 3 days) compared with sham controls. Consistently, MLK3-MKK4-JNK activation was rapidly increased with peaks both at 30 min and 3 days of reperfusion. Intracerebroventricular infusion of POSH antisense oligodeoxynucleotides (AS-ODNs) not only significantly reduced the protein level of POSH, markedly decreased its interactions with MLK3, MKK4 and phospho-JNKs, but also attenuated the activation of the JNK signalling pathway. In addition, infusion of POSH AS-ODNs significantly increased the neuronal density in the CA1 region at 5 days of reperfusion. Our results suggest that POSH might serve as a scaffold mediating JNK signalling activation in the hippocampal CA1 region following cerebral ischaemia, and POSH AS-ODNs exerts its protective effects on ischaemic injury through a mechanism of inhibition of the MLK3-MKK4-JNK signalling pathway, involving c-Jun and caspase 3 activation.

Novel curcumin- and emodin-related compounds identified by in silico 2D/3D conformer screening induce apoptosis in tumor cells

BackgroundInhibition of the COP9 signalosome (CSN) associated kinases CK2 and PKD by curcumin causes stabilization of the tumor suppressor p53. It has been shown that curcumin induces tumor cell death and apoptosis. Curcumin and emodin block the CSN-directed c-Jun signaling pathway, which results in diminished c-Jun steady state levels in HeLa cells. The aim of this work was to search for new CSN kinase inhibitors analogue to curcumin and emodin by means of an in silico screening method.MethodsHere we present a novel method to identify efficient inhibitors of CSN-associated kinases. Using curcumin and emodin as lead structures an in silico screening with our in-house database containing more than 106 structures was carried out. Thirty-five compounds were identified and further evaluated by the Lipinski's rule-of-five. Two groups of compounds can be clearly discriminated according to their structures: the curcumin-group and the emodin-group. The compounds were evaluated in in vitro kinase assays and in cell culture experiments.ResultsThe data revealed 3 compounds of the curcumin-group (e.g. piceatannol) and 4 of the emodin-group (e.g. anthrachinone) as potent inhibitors of CSN-associated kinases. Identified agents increased p53 levels and induced apoptosis in tumor cells as determined by annexin V-FITC binding, DNA fragmentation and caspase activity assays.ConclusionOur data demonstrate that the new in silico screening method is highly efficient for identifying potential anti-tumor drugs.

Phosphorylation of c-Jun in Avian and Mammalian Motoneurons In Vivo during Programmed Cell Death: An Early Reversible Event in the Apoptotic Cascade

c-Jun is a transcription factor that is involved in various cellular events, including apoptotic cell death. For example, phosphorylation of c-Jun is one of the earliest biochemical changes detected in dying sympathetic neurons after NGF deprivation in vitro. However, currently, it is not known whether a similar molecular event is involved in the developmental programmed cell death (PCD) of neurons in vivo. We observed that only a subpopulation of motoneurons (MNs) exhibit c-Jun phosphorylation during the PCD period in chick [embryonic day 5 (E5)-E12] and mouse (E13-E18) embryos. Experimental perturbation of MN survival-promoting signals by limb bud removal (reduced signals) or by activity blockade (increased signals) in the chick embryo demonstrated that the presence of those signals is negatively correlated with the number of c-Jun-phosphorylated MNs. This suggests that insufficient survival signals (e.g., neurotrophic factors) may induce c-Jun phosphorylation of MNs in vivo. Consistent with the idea that c-Jun phosphorylation is a reversible event during normal PCD of MNs, we found that c-Jun phosphorylation was transiently observed in a subpopulation of mouse MNs rescued from PCD by deletion of the proapoptotic gene Bax. Inhibition of c-Jun signaling significantly reduced MN death in chick embryo, indicating that activation of c-Jun signaling is necessary for the PCD of MNs. Together, c-Jun phosphorylation appears to be required for the initiation of an early and reversible event in the intracellular PCD cascade in vivo after loss of survival-promoting signals such as neurotrophic factors.

Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation

Receptor activator of NF-κB ligand (RANKL) plays an essential role in osteoclast formation and bone resorption. Although genetic and biochemical studies indicate that RANKL regulates osteoclast differentiation by activating receptor activator of NF-κB and associated signaling molecules, the molecular mechanisms of RANKL-regulated osteoclast differentiation have not yet been fully established. We investigated the role of the transcription factor c-Jun, which is activated by RANKL, in osteoclastogenesis using transgenic mice expressing dominant-negative c-Jun specifically in the osteoclast lineage. We found that the transgenic mice manifested severe osteopetrosis due to impaired osteoclastogenesis. Blockade of c-Jun signaling also markedly inhibited soluble RANKL-induced osteoclast differentiation in vitro. Overexpression of nuclear factor of activated T cells 1 (NFAT1) (NFATc2/NFATp) or NFAT2 (NFATc1/NFATc) promoted differentiation of osteoclast precursor cells into tartrate-resistant acid phosphatase–positive (TRAP–positive) multinucleated osteoclast-like cells even in the absence of RANKL. Overexpression of NFAT1 also markedly transactivated the TRAP gene promoter. These osteoclastogenic activities of NFAT were abrogated by overexpression of dominant-negative c-Jun. Importantly, osteoclast differentiation and induction of NFAT2 expression by NFAT1 overexpression or soluble RANKL treatment were profoundly diminished in spleen cells of the transgenic mice. Collectively, these results indicate that c-Jun signaling in cooperation with NFAT is crucial for RANKL-regulated osteoclast differentiation.