JNK Signal Transduction Pathway Research Articles

LncRNA Riken Attenuated Sevoflurane-Induced Neuroinflammation by Regulating the MicroRNA-101a/MKP-1/JNK Pathway.

The induction of anesthesia in children makes its safety one of the most important global health problems. Neuroinflammation contributes to anesthesia-induced neurotoxicity in young individuals. However, the mechanisms underlying anesthesia-induced neurotoxicity have not been established. In this study, the level of interleukin (IL)-6 in the hippocampus of mice and N2A cells treated with sevoflurane was increased, and long noncoding RNA (LncRNA) Riken was sufficient to decrease sevoflurane-induced neurotoxicity, and the level of inflammatory cytokine IL-6. The RNA pull-down assay verified that miR-101a was bound to lncRNA Riken in N2A cells. In addition, miR-101a blocked the protective effect of lncRNA Riken on anesthesia-induced neuroinflammation. These data suggest that lncRNA Riken attenuated anesthesia-induced neuroinflammation by interacting with microRNA-101a. Finally, we also demonstrated that MAPK phosphatase 1 (MKP-1) was a downstream target of miR-101a, and lncRNA Riken can regulate the expression of MKP-1; the JNK signal transduction pathway has been implicated in sevoflurane-induced IL-6 secretion. Our findings demonstrated that lncRNA Riken alleviated the sevoflurane-induced neurotoxic effects, and the lncRNA Riken/miR-101a/MKP-1/JNK axis plays an important role in the cognitive disorder.

Paclitaxel induces apoptosis through the TAK1-JNK activation pathway.

Paclitaxel (PTX) has previously been used to treat tumours of various tissue origins, such as lung, breast, ovarian, prostate cancers and leukemia. PTX‐induced apoptosis is associated with p38 mitogen‐activated protein kinase (p38 MAPK), extracellular signal‐regulated kinase (ERK), nuclear factor‐kappa B (NF‐κB) and c‐Jun N‐terminal kinase or stress‐activated protein kinase (JNK/ SAPK) pathways. Transforming growth factor‐beta‐activated kinase 1 (TAK1) and TAK1‐binding protein 1 (TAB1) play an important role in cell apoptosis through the p38, ERK, NF‐κB and JNK signal transduction pathways. To investigate the role of TAK1 in PTX‐induced cell apoptosis, we treated HEK293 and 8305C cells with 0–20 µm PTX for 6, 12 or 24 h. To investigate whether TAK1 can cooperate with PTX for cancer treatment, we transfected cells with TAK1, TAB1 or control plasmid and treated them with PTX (3–10 µm) for 9–24 h. Apoptosis rates were analysed by flow cytometry (Annexin V/PI). Endogenous TAK1 and TAB1, caspase‐7 cleavage, poly ADP‐ribose polymerase (PARP) cleavage, Bcl‐xL level, phospho‐p44/42, phospho‐JNK and phospho‐p38 were detected by western blot. We show that in HEK293 and 8305C cells, PTX enhanced the endogenous TAK1/TAB1 level and induced cell apoptosis in a dose‐ and time‐dependent manner. Upon TAK1 overexpression in HEK293 cells treated with PTX, apoptosis rate, JNK phosphorylation and PARP cleavage increased contrary to heat‐shocked or untreated cells. CRISPR editing of the tak1 gene upon PTX treatment resulted in lower phospho‐JNK and PARP cleavage levels than in cells transfected with the control or the TAK1‐ or TAB1 + TAK1‐containing plasmids. TAK1‐K63A could not induce JNK phosphorylation or PARP cleavage. We conclude that PTX induces HEK293 and 8305C cell apoptosis through the TAK1–JNK activation pathway, potentially highlighting TAK1’s role in chemosensitivity.

Photoprotection of Cerium Oxide Nanoparticles against UVA radiation-induced Senescence of Human Skin Fibroblasts due to their Antioxidant Properties

Ultraviolet (UV) irradiation, particularly ultraviolet A (UVA), stimulates reactive oxygen species (ROS) production in the epidermis and dermis, which plays a major part in the photoageing of human skin. Several studies have demonstrated that cerium oxide nanoparticles (CeO2 NP) can exhibit an antioxidant effect and free radical scavenging activity. However, the protective role of CeO2 NP in skin photoageing and the underlying mechanisms are unclear. In this study, we investigated the effects of CeO2 NP on UVA-irradiated human skin fibroblasts (HSFs) and explored the potential signalling pathway. CeO2 NP had no apparent cytotoxicity, and could reduce the production of proinflammatory cytokines, intracellular ROS, senescence-associated β-galactosidase activity, and downregulate phosphorylation of c-Jun N-terminal kinases (JNKs) after exposure to UVA radiation. Based on our findings, CeO2 NPs have great potential against UVA radiation-induced photoageing in HSFs via regulating the JNK signal-transduction pathway to inhibit oxidative stress and DNA damage.

Myostatin Promotes Interleukin-1β Expression in Rheumatoid Arthritis Synovial Fibroblasts through Inhibition of miR-21-5p.

Rheumatoid arthritis (RA) is characterized by the infiltration of a number of pro-inflammatory cytokines into synovial fluid and patients with RA often develop joint destruction and deficits in muscle mass. The growth factor myostatin is a key regulator linking muscle mass and bone structure. We sought to determine whether myostatin regulates rheumatoid synovial fibroblast activity and inflammation in RA. We found that levels of myostatin and interleukin (IL)-1β (a key pro-inflammatory cytokine in RA) in synovial fluid from RA patients were overexpressed and positively correlated. In in vitro investigations, we found that myostatin dose-dependently regulated IL-1β expression through the ERK, JNK, and AP-1 signal-transduction pathways. Computational analysis confirmed that miR-21-5p directly targets the expression of the 3′ untranslated region (3′ UTR) of IL-1β. Treatment of cells with myostatin inhibited miR-21-5p expression and miR-21-5p mimic prevented myostatin-induced enhancement of IL-1β expression, showing an inverse correlation between miR-21-5p and IL-1β expression during myostatin treatment. We also found significantly increased paw swelling in an animal model of collagen-induced arthritis (CIA), compared with controls; immunohistochemistry staining revealed substantially higher levels of myostatin and IL-1β expression in CIA tissue. Our evidence indicates that myostatin regulates IL-1β production. Thus, targeting myostatin may represent a potential therapeutic target for RA.

Complement Receptor 3 Has Negative Impact on Tumor Surveillance through Suppression of Natural Killer Cell Function.

Complement receptor 3 (CR3) is expressed abundantly on natural killer (NK) cells; however, whether it plays roles in NK cell-dependent tumor surveillance is largely unknown. Here, we show that CR3 is an important negative regulator of NK cell function, which has negative impact on tumor surveillance. Mice deficient in CR3 (CD11b−/− mice) exhibited a more activated NK phenotype and had enhanced NK-dependent tumor killing. In a B16-luc melanoma-induced lung tumor growth and metastasis model, mice deficient in CR3 had reduced tumor growth and metastases, compared with WT mice. In addition, adaptive transfer of NK cells lacking CR3 (into NK-deficient mice) mediated more efficient suppression of tumor growth and metastases, compared with the transfer of CR3 sufficient NK cells, suggesting that CR3 can impair tumor surveillance through suppression of NK cell function. In vitro analyses showed that engagement of CR3 with iC3b (classical CR3 ligand) on NK cells negatively regulated NK cell activity and effector functions (i.e. direct tumor cell killing, antibody-dependent NK-mediated tumor killing). Cell signaling analyses showed that iC3b stimulation caused activation of Src homology 2 domain-containing inositol-5-phosphatase-1 (SHIP-1) and JNK, and suppression of ERK in NK cells, supporting that iC3b mediates negative regulation of NK cell function through its effects on SHIP-1, JNK, and ERK signal transduction pathways. Thus, our findings demonstrate a previously unknown role for CR3 in dysregulation of NK-dependent tumor surveillance and suggest that the iC3b/CR3 signaling is a critical negative regulator of NK cell function and may represent a new target for preserving NK cell function in cancer patients and improving NK cell-based therapy.

Dickkopf1 destabilizes atherosclerotic plaques and promotes plaque formation by inducing apoptosis of endothelial cells through activation of ER stress

Several clinical studies reported that Dickkopf1 (DKK1) plasma levels are correlated with atherosclerosis. However, the impact of DKK1 on the formation and vulnerability of atherosclerotic plaques remains elusive. This study investigated DKK1’s effects on enlargement and destabilization of plaques by targeting endothelial cells and assessing the possible cellular mechanisms involved. The effects of DKK1 on atherogenesis and plaque stability were evaluated in ApoE−/− mice using lentivirus injections to knockdown and knock-in the DKK1 gene. The presence of DKK1 resulted in enlarged and destabilized atherosclerotic lesions and increased apoptosis, while silencing of DKK1 alleviated plaque formation and vulnerability in the whole progression of atherosclerosis. DKK1 expression was upregulated in response to ox-LDL treatment in a time- and concentration-dependent manner on human umbilical vein endothelial cell (HUVEC). The interference of DKK1 reversed ox-LDL-induced apoptosis in HUVECs. The mechanism underlying this effect was DKK1’s activation of the JNK signal transduction pathway and inhibition of canonical Wnt signaling, following by activation of the IRE1α and eif2α/CHOP pathways. In conclusion, DKK1 promotes plaque formation and vulnerability partly by inducing apoptosis in endothelial cells, which partly through inducing the JNK-endoplasmic reticulum stress pathway and inhibiting canonical Wnt signaling.

The Effect of Minimally Invasive Hematoma Aspiration on the JNK Signal Transduction Pathway after Experimental Intracerebral Hemorrhage in Rats.

Objective: To explore the effect of minimally invasive hematoma aspiration (MIHA) on the c-Jun NH2-terminal kinase (JNK) signal transduction pathway after intracerebral hemorrhage (ICH). Methods: In this experiment, 300 adult male Wistar rats were randomly and averagely divided into sham-operated group, ICH group and MIHA group. In each group, 60 rats were used in the detection of indexes in this experiment, while the other 40 rats were used to replace rats which reached the exclusion criteria (accidental death or operation failure). In ICH group and MIHA group, ICH was induced by injection of 70 µL of autologous arterial blood into rat brain, while only the rats in MIHA group were treated by MIHA 6 h after ICH. Rats in sham-operated group were injected nothing into brains, and they were not treated either, like rats in ICH group. In each group, six rats were randomly selected to observe their Bederson’s scales persistently (6, 24, 48, 72, 96, 120 h after ICH). According to the time they were sacrificed, the remaining rats in each group were divided into 3 subgroups (24, 72, 120 h). The change of brain water content (BWC) was measured by the wet weight to dry weight ratio method. The morphology of neurons in cortex was observed by the hematoxylin–eosin (HE) staining. The expressions of phospho-c-Jun NH2-terminal kinase (pJNK) and JNK in peri-hematomal brain tissue were determined by the immunohistochemistry (IHC) and Western blotting (WB). Results: At all time points, compared with the ICH groups, the expression of pJNK decreased obviously in MIHA groups (p < 0.05), while their Bederson’s scales and BWC declined, and neuron injury in the cortex was relieved. The expression level of JNK was not altered at different groups. The data obtained by IHC and WB indicated a high-level of consistency, which provided a certain dependability of the test results. Conclusion: The JNK signal transduction pathway could be activated after intracerebral hemorrhage, with the expressions of pJNK increasing. MIHA could relieve the histo-pathological damage of nerve cells, reducing brain edema and neurological deficits, and these neuroprotective effects might be associated with suppression of JNK signal transduction pathway.

Accessible Method for the Development of Novel Sterol Analogues with Dipeptide-like Side Chains That Act as Neuroinflammation Inhibitors.

A number of novel sterol derivatives with dipeptide-like side chains were synthesized using an Ugi four-component condensation method and assayed to test their anti-inflammatory effects in activated microglial cells. Compound 18b ((3S,10R,13S)-N-((R)-1-(tert-butylamino)-1-oxo-3-phenylpropan-2-yl)-3-hydroxy-N,10,13-trimethyl-2,3,4,7,8,9,10,11,12,13,14,15-dodecahydro-1H-cyclopenta[a]phenanthrene-17-carboxamide) was identified as the most potent anti-inflammatory agent in the series of compounds analyzed. Compound 18b markedly inhibited the expression of proinflammatory factors, including inducible nitric oxide synthase, interleukin (IL)-6, IL-1β, tumor necrosis factor-α, and cyclooxygenase-2 in lipopolysaccharide-stimulated microglial cells. Further studies showed that compound 18b significantly suppressed the transcriptional activity of AP-1 and NF-κB in activated microglial cells, which was likely mediated by the inhibition of the p38 MAPK and JNK signal transduction pathways. In addition, compound 18b displayed neuroprotective effects in a microglial-conditioned medium/neuron coculture and an experimental focal ischemic mouse model.

Abstract 4668: Targeted, massively parallel sequencing identifies novel genetic subsets of cutaneous melanoma

Abstract Despite the prevalence of recurrent, high activating BRAF V600 mutations in 45% of tumors, cutaneous melanoma (CM) is a heterogeneous malignancy resulting from aberrant signaling in multiple pathways. It has been traditionally characterized by activation of the MAPK and PI3K signaling pathways, as well as cell cycle disruption. In recent years, whole-genome and exome sequencing studies have identified several new genes associated with melanomagenesis. However, a comprehensive understanding of concurrent, and mutually exclusive, mutations in tumors is currently lacking. Using a custom targeted capture of 108 genes previously implicated in melanoma pathogenesis, massively parallel sequencing was performed on 94 human melanoma cell lines, 67 patient-derived xenografts (PDX), and 5 cell lines made from PDX, all untreated. Samples were then clustered into groups based on deleterious mutations. 83% of samples had deleterious mutations in the MAPK signaling pathway, including 92 high activity BRAF (55%), 35 RAS codon 61 (21%), 7 with multiple mutations (e.g. low activity BRAF/RAS codons 12/13) (4%) and 10 NF1 (6%) mutated samples. Likely deleterious NF1 mutations were found in several BRAF or NRAS-mutated samples. PI3K pathway mutations were found in 10% of samples, predominantly associated with BRAF mutations. TP53 mutations were found in 24% of samples and were associated with all MAPK signaling mutations. Mutations in chromatin remodeling genes (ARID1A/1B, ARID2, TRRAP, and BAP1) were mutually exclusive with each other and primarily found in tumors with high activity BRAF or NRAS mutations. The majority of BRAF or RAS-mutated samples with a mutation in a chromatin remodeling gene lacked mutations in cell cycle, TP53, and PI3K signaling genes; however, 100% of deleterious, or likely deleterious, NF1-mutated samples with a chromatin remodeling gene mutation harbored additional mutations in cell cycle, TP53, and/or PI3K signaling genes. Of particular interest, five of the 10 NF1-mutated samples (50%) lacked BRAF, RAS, and MEK1/2 mutations but harbored likely deleterious mutations in MAP3K5 or MAP3K9, suggesting the potential involvement of the JNK signal transduction pathway in this particular cohort. Only 4% of samples did not have a deleterious mutation in any of the genes on the panel. These data reveal novel insights into the genetics of melanomas lacking a canonical BRAF V600 mutation. Functional assays are needed to confirm the biological relevance of likely deleterious mutations, which will further facilitate a more thorough classification of CM subsets. Citation Format: Bradley Garman, Clemens Krepler, Katrin Sproesser, Patrica Brafford, Melissa Wilson, Bradley Wubbenhorst, Ravi Amaravadi, Joseph Bennett, Marilda Beqiri, Michael Davies, David Elder, Keith Flaherty, Dennie Frederick, Tara C. Gangadhar, Michael Guarino, David Hoon, Giorgos Karakousis, Nandita Mitra, Nicholas J. Petrelli, Lynn Schuchter, Batool Shannan, Jennifer Wargo, Min Xiao, Wei Xu, Xaiowei Xu, Meenhard Herlyn, Katherine Nathanson. Targeted, massively parallel sequencing identifies novel genetic subsets of cutaneous melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4668. doi:10.1158/1538-7445.AM2015-4668

Interleukin-1β regulates the expression of glucocorticoid receptor isoforms in nasal polyps in vitro via p38 MAPK and JNK signal transduction pathways.

BackgroundTo explore the upstream signal transduction mechanisms responsible for the imbalanced expression of glucocorticoid receptor (GR) isoforms in chronic rhinosinusitis (CRS) mucosa.MethodsAn in vitro model of Glucocorticoid resistance was established by inducing nasal polyp tissue with IL-1β. Changes in the protein and mRNA expression of GRα, GRβ and the key enzymes in the p38 MAPK and JNK signal pathways were measured, respectively, before and after being induced with different doses of IL-1β and specific inhibitors of p38 MAPK, JNK, ERK, PI3K and PKC. The Glucocorticoid sensitivity was measured using in vitro Glucocorticoid binding assay. Analysis of variance (ANOVA) was used to analyze the data.ResultsThe mRNA and protein expression levels of GRα, GRβ and key enzymes of the p38 MAPK and JNK pathways increased both in time- and concentration-dependent manners in IL-1β-induced nasal polyp tissue. The expression of GRβ increased more significantly than that of GRα, and the GRα/GRβ ratio decreased in time- and concentration-dependent manners. Statistically significant differences were found in the GRα/GRβ ratio and the mRNA expression of phospho-p38 MAPK and phospho-JNK between the IL-1β-induced groups and the control groups (P < 0.05). Either a specific inhibitor of the p38 MAPK pathway or a specific inhibitor of the JNK pathway increased the GRα/GRβ ratio and the Glucocorticoid affinity. None of the specific inhibitors of ERK, PI3K or PKC had any influence on the expression of GR isoforms.ConclusionOur results demonstrated that the imbalanced expression of GR isoforms in nasal polyp tissue induced by IL-1β in vitro is mediated through the p38 MAPK and JNK signal pathways.

Spinal sample showing p-JNK and P38 associated with the pain signaling transduction of glial cell in neuropathic pain.

To investigate the signaling pathways after astrocytes were activated in neuropathic pain. Thirty-six Sprague Dawley (s.d.) rats were randomly divided into two groups (each group with 18 s.d. rats) including chronic constriction injury (CCI) of the sciatic nerve model group and sham operation group. Operation was perform ed on the right leg in all rats. The lumbar spin al cord (L4 and L5) was taken to make paraffin slices on the 1st day before operation and the 1st, 3rd, 7th, 14th and 28th day after operation in each group. Paraffin slices were labeled with p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) by immunofluorescence staining, and then were co-labeled with hexaribonucleotide binding protein-3 (NeuN), glial fibrillary acidic protein (GFAP) and anti-integrin αM (CD11b) antibody (OX-42) to explore the associations of p38MAPK and JNK with nerve cells or glial cell. Compared with sham group, the pain threshold was significantly decreased, and astrocyte-activated markers, GFAP and vimentin were significantly increased in CCI group. The mean fluorescence intensities of p38MAPK and JNK were increased in the right spinal dorsal horn of CCI group. The coexpression of JNK and GFAP was found in astrocytes of the spinal dorsal horn in CCI group. JNK signal transduction pathway is involved in the pain signaling transduction of astrocytes.

Hypoxic conditioned medium from rat cerebral cortical cells enhances the proliferation and differentiation of neural stem cells mainly through PI3-K/Akt pathways.

PurposeTo investigate the effects of hypoxic conditioned media from rat cerebral cortical cells on the proliferation and differentiation of neural stem cells (NSCs) in vitro, and to study the roles of PI3-K/Akt and JNK signal transduction pathways in these processes.MethodsCerebral cortical cells from neonatal Sprague–Dawley rat were cultured under hypoxic and normoxic conditions; the supernatant was collected and named ‘hypoxic conditioned medium’ (HCM) and ‘normoxic conditioned medium’ (NCM), respectively. We detected the protein levels (by ELISA) of VEGF and BDNF in the conditioned media and mRNA levels (by RT-PCR) in cerebral cortical cells. The proliferation (number and size of neurospheres) and differentiation (proportion of neurons and astrocytes over total cells) of NSCs was assessed. LY294002 and SP600125, inhibitors of PI3-K/Akt and JNK, respectively, were applied, and the phosphorylation levels of PI3-K, Akt and JNK were measured by western blot.ResultsThe protein levels and mRNA expressions of VEGF and BDNF in 4% HCM and 1% HCM were both higher than that of those in NCM. The efficiency and speed of NSCs proliferation was enhanced in 4% HCM compared with 1% HCM. The highest percentage of neurons and lowest percentage of astrocytes was found in 4% HCM. However, the enhancement of NSCs proliferation and differentiation into neurons accelerated by 4% HCM was inhibited by LY294002 and SP600125, with LY294002 having a stronger inhibitory effect. The increased phosphorylation levels of PI3-K, Akt and JNK in 4% HCM were blocked by LY294002 and SP600125.Conclusions4%HCM could promote NSCs proliferation and differentiation into high percentage of neurons, these processes may be mainly through PI3-K/Akt pathways.

Abstract 2328: COX-2 induce mediated multidrug resistance by activating JNK signal transduction pathway in colorectal cancer

Abstract Chemotherapy is an important therapeutic option for most cancer patients; however, drug resistance causes the failure of chemotherapy and becomes a major obstacle for successful chemotherapy. The overexpression of P-gp has been associated with the resistance to a wide range of anticancer drugs. In the present study, drug-sensitive and multidrug resistant MDR colon carcinoma cell lines were used to examine the role of JNK signaling pathway in P-gp-mediated MDR associated with COX-2. We have found that sp600125, a JNK inhibitor or NS-398, a COX-2 inhibitor, reduced the degree of MDR in MDR cells. This phenomenon was accompanied by a significant decrease in gene level of ABCB1 and protein level of P-gp in MDR cells. In contrast, addition of a JNK inhibitor had no significant effect on the expression of COX-2. Interestingly, inhibition of COX-2 activity led to a decreased level of p-c-Jun at Ser63/73 due to JNK activation. In addition, there is an overexpression of p-c-Jun in MDR cells transfected with recombinant vector pGL3-Basic-COX-2 promoter. Moreover, the intracellular vincristine accumulation significantly increased with treatment of sp600125, or NS-398. In conclusion, our study has provided the first direct evidence that COX-2 induced up-regulation of P-gp in MDR cells, which was associated with an enhanced activity of the c-Jun phosphorylation at Ser63/73. Our findings may facilitate the development of novel antitumor drugs that are more effective against both chemosensitive and MDR cells. Note: This abstract was not presented at the meeting. Citation Format: Qi Li, Hua Sui. COX-2 induce mediated multidrug resistance by activating JNK signal transduction pathway in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2328. doi:10.1158/1538-7445.AM2014-2328

Role of JNK Signal-Transduction Pathway in the Differentiation of Bone Marrow Mesenchymal Stem Cells into Hepatocytes In Vitro.

PURPOSE To explore the effect of JNK signal-transduction pathway in the differentiation of bone marrow mesenchymal stem cells ( BMMSCs) into hepatocytes in the presence of conditioned medium. MATERIAL AND METHODS The injured liver from the mice fed with hepatotoxic reagents carbon tetrachloride ( CCl4 ) was cultured. And the conditioned culture medium was obtained from the supernatant. BMMSCs were isolated, expanded and purified by density gradient centrifugation combined with adherent culture. BMMSCs(3 generation) were divided into three groups: control group, normal inductiongroup and inhibitor group. Control group was cultured in DMEM supplemented with 15% FBS, normal induction group was cultured in the conditioned medium, and inhibitor group was cultured in the conditioned medium which was added into SP600125(JNK signal-transduction pathway inhibitor) till the final concentration of 15μmol/L. The morphology of the BMMSCs of different groups will be observed. The expression of AFP, ALB and urea will be detected. Results After 21 days, BMMSCs were observed under phase contrastmicroscope, and we can find that cell morphology of normal induction group changed from spindle-shaped, fiber-like cells to polygonal or round, clear nucleus, and dual-core or multi-core emerged. But inhibitor group, the proportion of cell morphological changes decreased significantly. After adding SP600125(JNK signal-transduction pathway inhibitor), the expression of AFP on the 7th inducing day and the expression of ALB and urea on the 14th and 21st inducing day diminished considerably compared with normal induction group( P < 0.05). Conclusion Activation of JNK may play an important role in the differentiation of BMMSCs into hepatocytes.

Syk/JNK/AP-1 Signaling Pathway Mediates Interleukin-6-Promoted Cell Migration in Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) typically migrates and metastasizes. Interleukin-6 (IL-6) is a multifunctional cytokine associated with disease status and cancer outcomes. The effect of IL-6 on human OSCC cells, however, is unknown. Here, we showed that IL-6 increased cell migration and Intercellular adhesion molecule-1 (ICAM-1) expression in OSCC cells. Pretreatment of OSCC cells with IL-6R monoclonal antibody (mAb) significantly abolished IL-6-induced cell migration and ICAM-1 expression. By contrast, IL-6-mediated cell motility and ICAM-1 upregulation were attenuated by the Syk and c-Jun N-terminal kinase (JNK) inhibitors. Stimulation of OSCC cells with IL-6 promoted Syk and JNK phosphorylation. Furthermore, IL-6 enhanced AP-1 activity, and the IL-6R mAb, Syk inhibitor, or JNK inhibitor all reduced IL-6-mediated c-Jun phosphorylation, c-Jun binding to the ICAM-1 promoter, and c-Jun translocation into the nucleus. Our results indicate that IL-6 enhances the migration of OSCC cells by increasing ICAM-1 expression through the IL-6R receptor and the Syk, JNK, and AP-1 signal transduction pathways.

Higher TRAF6 and Fbxo 3 Expression Is With Progressive Multiple Myeloma and Blockage Of TRAF6 Inhibits Tumor Growth and Bone Resorption

Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been implicated in regulating the NF-kB and JNK signal transduction pathways; and, thus, is likely to promote tumor cell proliferation and osteoclast formation. F-box proteins such as the E3 ligase component Fbxo3 and another F-box family member Fbxl 2, regulate TRAF protein signaling.First, we investigated TRAF6 expression in bone marrow mononuclear cells (BMMCs) from multiple myeloma (MM) patients with progressive disease or in remission and healthy subjects. The results showed higher TRAF6 protein expression among MM patients with progressive disease than among those in remission or healthy subjects. Notably, changes in TRAF6 protein expression in MM BMMCs were found to correlate with response of individual patients to treatment with the proteasome inhibitors bortezomib or carfilzomib. We have previously reported inhibition of MM cell proliferation and increase of apoptosis through regulation of the NF-kB and JNK pathways using a silencing TRAF6 C-domain mRNA construct. In this study, we cloned a 167 amino acid (in residues 333 to 508) portion of the TRAF6 dominant negative (TRAF6dn) and synthesized decoy peptides of the TRAF6 interaction domain with CD40 and the TRAF6-RANK binding domain. Decreases in cell proliferation and increase in cell apoptosis in MM BMMCs treated with TRAF6dn occurred in a concentrationdependent fashion. We also found TRAF6dn markedly inhibited osteoclast cell formation of monocytes induced by RANKL and mCSF in a dose-dependent manner. Next, we examined the effect of TRAF6dn on NF-kB and JNK by determining phosphorylation of JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. MM BMMCs exposed to the TRAF6dn fragment or TRAF6 decoy peptide showed reduced phosphoNF-kB protein and phosphorylation of JNKK. These studies suggest that the TRAF6dn or combined TRAF6 decoy and CD40 decoy peptide may be excellent agents to block both MM cell growth and osteoclast formation in MM.We further investigated the protein expression of Fbxo 3, Fbxl 2 and TRAF6 in fresh BMMCs from MM patients with progressive disease or in remission. Results of Western blot analysis showed protein expression of Fbxo 3 and TRAF6 was increased and Fbxl 2 was decreased among patients with progressive disease compared to patients in remission.Thus, these results may offer a new mechanism through which MM tumor cells are regulated and provide a new therapeutic approach to treat MM and reduce the clinical consequences resulting from enhanced bone loss that commonly occur in these patients. Disclosures:No relevant conflicts of interest to declare.

Remifentanil preconditioning alleviating brain damage of cerebral ischemia reperfusion rats by regulating the JNK signal pathway and TNF-α/TNFR1 signal pathway

Tumor necrosis factor (TNF) and the TNF receptor (TNFR) superfamily play very important roles for cell death as well as normal immune regulation. Previous studies have strongly suggested that c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. The purpose of this investigation was to examine the protective effect of remifentanil preconditioning in cerebral ischemia/reperfusion injury (CIR) and its possible molecular mechanism. Results showed that Remifentanil pretreatment significantly decreased the CD4(+) and increased the CD8(+) in cerebral tissues. Additionally, CD4(+)/CD8(+) in CIR+Remifentanil group was markedly lower than that in CIR group. TNF-α and TNFR1 in CIR+Remifentanil group rats was found to be significant lower than that in CIR group rats. The expression levels of Cyt-c, caspase-3, caspase-9 and pJNK proteins in brain of CIR+Remifentanil group rats were found to significantly decreased compared to CIR group rats. In addition, decreased ROS level indirectly inhibit JNK activation and cell death in CIR rat receiving Remifentanil preconditioning. From current experiment results, at least two signal pathways involve into the process of Remifentanil preconditioning inhibiting cerebral damage induced by ischemia reperfusion. The inhibitory effects of Remifentanil preconditioning on the brain damage are achieved probably through blocking the activation of TNF-α/TNFR1, JNK signal transduction pathways, which implies that Remifentanil preconditioning may be a potential and effective way for prevention of the ischemic/reperfusion injury through the suppression extrinsic apoptotic signal pathway induced by TNF-α/TNFR1, JNK signal pathways. Taken together, this study indicated that regulation of the TNF-α/TNFR1 and JNK signal pathways may provide a new therapy for cerebral damage induced by ischemia and reperfusion.

Expression of aquaporin 3 and aquaporin 9 is regulated by oleic acid through the PI3K/Akt and p38 MAPK signaling pathways

To study the effect of oleic acid (OA) on expression of aquaglyceroporin genes, AQP3 and AQP9, in hepatocyte steatosis and to investigate the underlying molecular mechanisms using an in vitro system. HepG2 cells were treated with OA at different concentration to establish in vitro models of nonalcoholic hepatocyte steatosis. The corresponding extents of hepatic steatosis modeling were assessed by oil red O staining and optical density (OD) measurements of the intracellular fat content. The model lines were then treated with inhibitors of the PI3K/Akt and p38 MAPK signaling pathway factors and effects on AQP3/9 expression was measured by real time RT-PCR and western blotting. The fat concentration, indicative of hepatic steatosis, increased in conjunction with increased concentrations of OA (0 less than 250 less than 500 mumol/L). OA exposure also down-regulated AQP3 mRNA and up-regulated AQP9 mRNA levels in a concentration-dependent manner. The most robust changes in expression occurred in response to the 500 mumol/L concentration of OA for both AQP3 (0.47+/-0.18; t = 4.5450, P less than 0.05) and AQP9 (1.57+/-0.21; t = 3.0306, P less than 0.05). Treatment with OA + PI3K pathway inhibitor (LY294004) significantly decreased AQP9 mRNA expression (4.55+/-0.62) as compared to the control group (1.00+/-0.10; t = 9.7909, P less than 0.01), that 500 mumol/L OA group (2.43+/-0.53; t = 4.5018, P less than 0.05), and the LY294002 group (1.90+/-0.16; t = 7.1683, P less than 0.01). Treatment with p38 MAPK pathway inhibitor (SB230580) significantly increased the OA-suppressed level of AQP3 mRNA to the level detected in the control group (1.27+/-0.11; t = 5.7455, P less than 0.01) and decreased the OA-stimulated AQP9 mRNA (0.38+/-0.09; t = 6.5727, P less than 0.01). No significant changes in mRNA expression of AQP3/9 were observed with inhibition of the ERK1/2 and JNK signal transduction pathways. The OA-induced changes in protein expression levels of AQR3 and AQP9 followed a similar trend of the genes. Finally, OA suppressed the level of phosphorylated Akt (from 0.21+/-0.02 to 0.13+/-0.03; t = 3.8431, P less than 0.05) but elevated the level of phosphorylated p38 (from 0.58+/-0.06 to 1.02+/-0.10; t = 12.5289, P less than 0.01). Again, OA treatment produced no significant affect on ERK1/2 and JNK phosphorylation. OA down-regulates AQP3 expression by stimulating the p38 MAPK signaling pathway, and up-regulates the AQP9 by blocking the PI3K/Akt pathway and activating the p38 MAPK signaling pathway.

Effects of isoflurane postconditioning on cerebral ischemia-reperfusion injury in rats

Objective To evaluate the effects of isoflurane postconditioning on cerebral ischemia-reperfusion (I/R) injury in rats.Methods Thirty-two male Sprague-Dawley rats,weighing 280-320 g,were randomly divided into 4 groups (n=8 each):group sham operation (group S),group I/R,group isoflurane preconditioning (group Ⅰ-pre),and group isoflurane postconditioning (group Ⅰ-post).Global cerebral I/R was induced by 4-vessel occlusion method described by Pulsinelli.1.5％ isoflurane was inhaled for 2h before ischemia in group Ⅰ-pre.1.5％ isoflurane was inhaled for 30 min immediately after onset of reperfusion in group Ⅰ-post.Neurological function was assessed and scored at 24h of reperfusion.The rats were sacrificed at 72h of reperfusion and hippocampi were isolated for determination of neuronal apoptosis (by TUNEL),caspase-3 expression (by immuno-histochemistry),and phosphorylated c-Jun N-terminal kinase (p-JNK) protein expression (by using Western blot) in hippocampal tissues.Apoptotic rate was calculated.Results Compared with S group,the number of grid cross was decreased,twist time was prolonged,hanging time was shortened,apoptotic rate was increased,and the expression of caspase-3 and p-JNK protein was up-regulated in I/R,I-pre and Ⅰ-post groups (P ＜ 0.05).Compared in I/R group,the number of grid cross was increased,twist time was shortened,hanging time was prolonged,apoptotic rate was decreased,and the expression of caspase-3 and p-JNK protein was down-regulated in Ⅰ-pre and Ⅰ-post groups (P ＜ 0.05).Compared with I/R group,the number of grid cross was decreased,hanging time was shortened,apoptotic rate was increased,and the expression of caspase-3 was up-regulated (P ＜ 0.05),and no significant changes in the expression of p-JNK protein were found in Ⅰ-post group (P ＞ 0.05).Conclusion Isoflurane postconditioning can reduce cerebral I/R injury,the efficacy is weaker than that of preconditioning and the mechanism is related to activation of JNK signal transduction pathway and inhibition of neuronal apoptosis in hippocampus of rats. Key words: Isoflurane; Ischemic preconditioning; Brain; Reperfusion injury; Postconditioning

Regulating effect of N-acetyl-seryl-aspartyl-lysyl-proline on activation of c-jun N-terminal kinase pathway in rats with silicosis

To investigate the regulatory effect of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) on the activation of c-jun N-terminal kinase (JNK) signal transduction pathway and its role in silicotic fibrosis. A rat model of silicosis was developed by intratracheal instillation. Sixty rats were randomly divided into 4-week control group (n = 10), 8-week control group (n = 10), 4-week silicosis model group (n = 10), 8-week silicosis model group (n = 10), AcSDKP treatment group (n = 10), and AcSDKP prevention group (n = 10). The content of hydroxyproline in lung tissue was measured using a p-dimethylaminoben-zaldehyde reagent; the expression levels of transforming growth factor (TGF)-beta 1 (TGF-β1), phospho-JNK, JNK, and c-jun in lung tissue were measured by Western blot. The lung fibroblasts from neonatal rats were cultured, and the 4th generation of cells were used in the experiment; these cells were divided into control group, TGF-β1 stimulation group, SP600125 intervention group, and AcSDKP intervention group. The distributions of phospho-JNK and c-jun in lung fibroblasts were observed by immunocytochemistry; the expression levels of type I collagen and type III collagen in lung fibroblasts were measured by Western blot. The expression levels of TGF-β1, phospho-JNK, and c-jun and the content of hydroxyproline in the AcSDKP treatment group were 70.60%, 78.03%, 79.85%, and 71.28%, respectively, of those in the 4-week silicosis model group (P < 0.05) and 77.99%, 66.73%, 69.94%, and 64.82%, respectively, of those in the 8-week silicosis model group (P < 0.05); the expression levels of TGF-β1, phospho-JNK, and c-jun and the content of hydroxyproline in the AcSDKP prevention group were 84.56%, 61.18%, 64.73%, and 74.96%, respectively, of those in the 8-week silicosis model group (P < 0.05). The expression levels of phospho-JNK and c-jun in the AcSDKP intervention group were 54.59% and 55.56%, respectively, of those in the TGF-β1 stimulation group; the expression levels of type I collagen and type III collagen in the AcSDKP intervention group were 79.9% and 84.4%, respectively, of those in the TGF-β1 stimulation group (P < 0.05). AcSDKP exerts anti-silicotic fibrosis effect probably by inhibiting the activation of JNK signal transduction pathway mediated by TGF-β1 and the deposition of interstitial collagen.