Up-regulation of c-Jun NH2-terminal kinase-interacting protein 3 (JIP3) contributes to BDNF-enhanced neurotransmitter release.

Brain-derived neurotrophic factor (BDNF) has been implicated in the potent modulation of synaptic plasticity at both pre-synaptic and post-synaptic sites. However, the molecular mechanism underlying BDNF-mediated pre-synaptic modulation remains incompletely understood. Here, we report that BDNF treatment for over 4 h could significantly enhance the expression of c-Jun NH2-terminal kinase-interacting protein 3 (JIP3) in cultured hippocampal neurons. This enhancement could be blocked by the Trk inhibitor K252a or by a cAMP response element-binding protein (CREB) inhibitor. In addition, chromatin immunoprecipitation (ChIP) assays revealed that CREB could bind with the JIP3 promoter region and the BDNF treatment could increase this binding. Using dual-luciferase assays we further characterized the cAMP response element (CRE) site in the JIP3 promoter. Finally, we found that BDNF-increased JIP3 expression contributes to the BDNF-induced modulation of neurotransmitter release. Together, our studies reveal that in hippocampal neurons BDNF up-regulates JIP3 expression via CREB activation, which contributes to the enhancement of neurotransmitter release; thus, we have identified a novel mechanism that BDNF modulates pre-synaptic transmission.

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

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.

Growth-arrest-dependent expression and phosphorylation of p27kip at serine10 is mediated by the JNK pathway in C6 glioma cells.

Induction of growth arrest by differentiation is an attractive therapeutic strategy against glioma cell proliferation and tumorigenicity. The observation that the expression of the JNK3 gene is lost in many human gliomas makes the JNK pathway an interesting target for investigation. Here, the influence of the JNK pathway on the differentiation of C6 glioma cells was investigated using pharmacological inhibition and JNK3 knockdown. Growth arrest induced the expression of JNK3 on transcriptional and translational level, whereas the expression of the cell cycle inhibitor p27kip was induced on the translational level only. Transient depletion of JNK3 in growth arrested C6 cells resulted in an about 40% decrease in cell adhesion and almost completely abolished the induction in p27kip protein. In addition, overexpression of wild type JNK3 in proliferating C6 cells led to a marked inhibition of proliferation. Beside synthesis, the amount of p27kip protein is regulated by its stability, which is known to be enhanced by phosphorylation at serine10 of p27kip. Here, the JNKs were identified as kinases that are able to phosphorylate p27kip at Ser10. As a result, the stability of p27kip protein is reduced by inhibition of the JNK pathway. These results suggest that the JNK pathway influences the stability of p27kip by phosphorylation of serine10 and that JNK3 is responsible for the translational activation of p27kip protein expression in growth arrested C6 glioma cells and therefore cell cycle arrest.

Upregulation of MAPK10, TUBB2B and RASL11B may contribute to the development of neuroblastoma.

The present study aimed to investigate genes and transcription factors (TFs) that may contribute to neuroblastoma (NB) development. The GSE78061 dataset that included 25 human NB cell lines and four retinal pigment epithelial cell lines was used to analyze differentially expressed genes (DEGs) between groups. Functional enrichment analysis and protein­protein interaction (PPI) network analysis were performed for the identified DEGs. Additionally, submodule analysis and TF­target regulatory networks were conducted. The relative mRNA expression levels of mitogen­activated protein kinase 10 (MAPK10), tubulin ß 2B class IIb (TUBB2B), RAS like family 11 member B (RASL11B) and integrin subunit α 2 (ITGA2) in the NB cell line SH­SY5Y were compared with retinal pigment epithelial cell lines. A set of 386 upregulated and 542 downregulated DEGs were obtained. Upregulated DEGs were significantly associated with the 'neuron migration' and 'dopaminergic synapse signaling' pathways, whereas, downregulated DEGs were primarily involved in 'focal adhesion' such as ITGA2 and ITGA3. In the PPI networks analyzed, MAPK10, dopa decarboxylase (DDC), G protein subunit γ 2 (GNG2), paired like homeobox 2B (PHOX2B), TUBB2B, RASL11B, and ITGA2 were hub genes with high connectivity degrees. Additionally, PHOX2B was predicted to be a TF regulating TUBB2B in the regulatory network. The expressions of MAPK10, TUBB2B, RASL11B and ITGA2 were detected by reverse transcription­quantitative polymerase chain reaction in the NB cell line SH­SY5Y, and were consistent with the present bioinformatics results, suggesting that MAPK10, DDC, GNG2, PHOX2B, TUBB2B, RASL11B, ITGA2 and ITGA3 may contribute to NB development. Additionally, the present study identified a novel significant association between the increased expression levels of MAPK10, TUBB2B and RASL11B, and NB cells.

APP upregulation contributes to retinal ganglion cell degeneration via JNK3.

Axonal injury is a common feature of central nervous system insults. Upregulation of amyloid precursor protein (APP) is observed following central nervous system neurotrauma and is regarded as a marker of central nervous system axonal injury. However, the underlying mechanism by which APP mediates neuronal death remains to be elucidated. Here, we used mouse optic nerve axotomy (ONA) to model central nervous system axonal injury replicating aspects of retinal ganglion cell (RGC) death in optic neuropathies. APP and APP intracellular domain (AICD) were upregulated in retina after ONA and APP knockout reduced Tuj1+ RGC loss. Pathway analysis of microarray data combined with chromatin immunoprecipitation and a luciferase reporter assay demonstrated that AICD interacts with the JNK3 gene locus and regulates JNK3 expression. Moreover, JNK3 was found to be upregulated after ONA and to contribute to Tuj1+ RGC death. APP knockout reduced the ONA-induced enhanced expression of JNK3 and phosphorylated JNK (pJNK). Gamma-secretase inhibitors prevented production of AICD, reduced JNK3 and pJNK expression similarly, and protected Tuj1+ RGCs from ONA-induced cell death. Together these data indicate that ONA induces APP expression and that gamma-secretase cleavage of APP releases AICD, which upregulates JNK3 leading to RGC death. This pathway may be a novel target for neuronal protection in optic neuropathies and other forms of neurotrauma.

Recombinant Kinase Production and Fragment Screening by NMR Spectroscopy.

During the past decade fragment-based drug discovery (FBDD) has rapidly evolved and several drugs or drug candidates developed by FBDD approach are clinically in use or in clinical trials. For example, vemurafenib, a V600E mutated BRAF inhibitor, was developed by utilizing FBDD approach and approved by FDA in 2011. In FBDD, screening of fragments is the starting step for identification of hits and lead generation. Fragment screening usually relies on biophysical techniques by which the protein-bound small molecules can be detected. NMR spectroscopy has been extensively used to study the molecular interaction between the protein and the ligand, and has many advantages in fragment screening over other biophysical techniques. This chapter describes the practical aspects of fragment screening by saturation transfer difference NMR.

Inhibition of JNK3 promotes apoptosis induced by BH3 mimetic S1 in chemoresistant human ovarian cancer cells.

Previous studies have suggested that the novel BH3 mimetic S1 could induce apoptosis in diverse tumor cell lines through endoplasmic reticulum (ER) stress or mitochondrial cell death pathways. The activation of c-Jun N-terminal kinase (JNK) through inositol requiring enzyme-1 (IRE1) is closely connected to ER stress-induced apoptosis. However, the role of JNK is complex, as there are different JNK subtypes and the function of each subtype is still not entirely clear. Here we found that the mRNA expression of JNK3 was continuously high in S1-treated human ovarian cancer SKOV3/DDP cells using a human unfolded protein response (UPR) pathway PCR array. Pharmacological inhibition of JNK3 increased cell sensitivity to apoptosis induced by S1. Furthermore, inhibition of JNK3 induced accumulation of both acidic compartment and p62, and upregulated ROS production. Our results suggest that JNK3 plays a pro-survival role during ER stress through preventing the block of autophagic flux and reducing oxidative stress in SKOV3/DDP cells. Inhibition of JNK3 may be a potential method to enhance the killing effect of the Bcl-2 inhibitor S1.

Regulation of the JNK3 signaling pathway during islet isolation: JNK3 and c-fos as new markers of islet quality for transplantation.

Stress conditions generated throughout pancreatic islet processing initiate the activation of pro-inflammatory pathways and beta-cell destruction. Our goal is to identify relevant and preferably beta-specific markers to assess the activation of beta-cell stress and apoptotic mechanisms, and therefore the general quality of the islet preparation prior to transplantation. Protein expression and activation were analyzed by Western blotting and kinase assays. ATP measurements were performed by a luminescence-based assay. Oxygen consumption rate (OCR) was measured based on standard protocols using fiber optic sensors. Total RNA was used for gene expression analyzes. Our results indicate that pancreas digestion initiates a potent stress response in the islets by activating two stress kinases, c-Jun N-terminal Kinase (JNK) and p38. JNK1 protein levels remained unchanged between different islet preparations and following culture. In contrast, levels of JNK3 increased after islet culture, but varied markedly, with a subset of preparations bearing low JNK3 expression. The observed changes in JNK3 protein content strongly correlated with OCR measurements as determined by the Spearman's rank correlation coefficient rho [Formula: see text] in the matching islet samples, while inversely correlating with c-fos mRNA expression [Formula: see text]. In conclusion, pancreas digestion recruits JNK and p38 kinases that are known to participate to beta-cell apoptosis. Concomitantly, the islet isolation alters JNK3 and c-fos expression, both strongly correlating with OCR. Thus, a comparative analysis of JNK3 and c-fos expression before and after culture may provide for novel markers to assess islet quality prior to transplantation. JNK3 has the advantage over all other proposed markers to be islet-specific, and thus to provide for a marker independent of non-beta cell contamination.