Involvement Of c-Jun N-terminal Kinase Research Articles

Curcumin Analog L48H37 Induces Apoptosis in Human Oral Cancer Cells by Activating Caspase Cascades and Downregulating the Inhibitor of Apoptosis Proteins through JNK/p38 Signaling.

L48H37 is a synthetic curcumin analog that has anticancer potentials. Here, we further explored the anticancer effect of L48H37 on oral cancer cells and its mechanistic acts. Cell cycle distribution was assessed using flow cytometric analysis. Apoptosis was elucidated by staining with PI/Annexin V and activation of the caspase cascade. Cellular signaling was explored using apoptotic protein profiling, Western blotting, and specific inhibitors. Our findings showed that L48H37 significantly reduced the cell viability of SCC-9 and HSC-3 cells, resulting in sub-G1 phase accumulation and increased apoptotic cells. Apoptotic protein profiling revealed that L48H37 increased cleaved caspase-3, and downregulated cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP) in SCC-9 cells, and the downregulated cIAP1 and XIAP in both oral cancer cells were also demonstrated by Western blotting. Meanwhile, L48H37 triggered the activation of caspases and mitogen-activated protein kinases (MAPKs). The involvement of c-Jun N-terminal kinase (JNK) and p38 MAPK (p38) in the L48H37-triggered apoptotic cascade in oral cancer cells was also elucidated by specific inhibitors. Collectively, these findings indicate that L48H37 has potent anticancer activity against oral cancer cells, which may be attributed to JNK/p38-mediated caspase activation and the resulting apoptosis. This suggests a potential benefit for L48H37 for the treatment of oral cancer.

Involvement of c-Jun N-terminal kinase 2 (JNK2) in Endothelin-1 (ET-1) Mediated Neurodegeneration of Retinal Ganglion Cells.

PurposeThe goal of this study was to determine whether JNK2 played a causative role in endothelin-mediated loss of RGCs in mice.MethodsJNK2−/− and wild type (C57BL/6) mice were intravitreally injected in one eye with 1 nmole of ET-1, whereas the contralateral eye was injected with the vehicle. At two time points (two hours and 24 hours) after the intravitreal injections, mice were euthanized, and phosphorylated c-Jun was assessed in retinal sections. In a separate set of experiments, JNK2−/− and wild type mice were intravitreally injected with either 1 nmole of ET-1 or its vehicle and euthanized seven days after injection. Retinal flat mounts were stained with antibodies to the RGC marker, Brn3a, and surviving RGCs were quantified. Axonal degeneration was assessed in paraphenylenediamine stained optic nerve sections.ResultsIntravitreal ET-1 administration produced a significant increase in immunostaining for phospho c-Jun in wild type mice, which was appreciably lower in the JNK2 −/− mice. A significant (P < 0.05) 26% loss of RGCs was found in wild type mice, seven days after injection with ET-1. JNK2−/− mice showed a significant protection from RGC loss following ET-1 administration, compared to wild type mice injected with ET-1. A significant decrease in axonal counts and an increase in the collapsed axons was found in ET-1 injected wild type mice eyes.ConclusionsJNK2 appears to play a major role in ET-1 mediated loss of RGCs in mice. Neuroprotective effects in JNK2−/− mice following ET-1 administration occur mainly in the soma and not in the axons of RGCs.

Effects of Curcumin Treatment in a Diabetic Neuropathic Pain Model of Rats: Involvement of c-Jun N-Terminal Kinase Located in the Astrocytes and Neurons of the Dorsal Root Ganglion.

Curcumin (diferuloylmethane) is a major component of turmeric, which is isolated from the rhizomes of Curcuma longa L. from the family Zingiberaceae. It is used as a dietary pigment for curry and in traditional Indian medicine for its anti-inflammatory and attenuating pain effects. This study aimed to evaluate the beneficial effects of curcumin in a rat model of diabetic neuropathic pain. Additionally, we investigated the involvement of the phosphorylated form of c-Jun N-terminal kinase (pJNK) located in the neurons and astrocytes of the dorsal root ganglion (DRG). To induce diabetic neuropathic pain in rats, 50 mg/kg of streptozotocin (STZ) was intraperitoneally injected. After 4 weeks, rats were administered the vehicle, 10 mg/kg/day curcumin, or 50 mg/kg/day curcumin orally for 4 consecutive weeks. One day after the final drug administration, we performed behavioral tests to measure responses of rats to mechanical, heat, cold, and acetone-induced cold stimuli. After behavioral tests, pJNK expression in the DRG was evaluated using western blot assay and immunohistochemistry. Curcumin treatment for 4 consecutive weeks in STZ-induced diabetic neuropathic pain rats improved behavioral responses to mechanical, cold, and thermal stimuli. Increased pJNK expression in the astrocytes and neurons of the DRG in STZ-induced diabetic neuropathic pain rats was reduced by curcumin treatment for 4 consecutive weeks. We suggest that curcumin can be an option for the treatment of diabetes-related neuropathic pain, and one of the mechanisms that underlie the action of curcumin may involve pJNK expression in the astrocytes and neurons of the DRG.

Review on Potential Vitamin D Mechanism with Type 2 Diabetes Mellitus Pathophysiology in Malaysia

Evidences on vitamin D deficiency suggest there is increasing risk of diabetes. To date, some cohort, observation, cross-sectional studies on populations and randomized controlled trials in vitamin D supplements highlighting the potential of vitamin D are essentially in modifying Type 2 Diabetes Mellitus (T2DM) pathophysiology. Relevant literature sought in a various databases focus on the discovery of vitamin D studies in Malaysia, particularly in dietary, health status and disease study. However, recent data in Malaysia, the scope of the literature focuses on the deficient vitamin D mediated insulin impairment. The development of literary findings encompasses on the etiology of diabetes which highly correlates with decreased mechanism of action of vitamin D. It is important to understand diabetes etiology before explaining more about insulin resistance mechanisms which is strongly correlated with the involvement of c-Jun N-terminal kinase (JNK) pathways in insulin signalling. Furthermore, the vitamin D works synergistically with calcium homeostasis which is believed to have interaction with insulin. The purpose of this article is to illustrate the potential of vitamin D in modulating T2DM pathophysiology. Existing evidence showing the biochemical function of vitamin D is strongly involved in the pathogenesis of T2DM which requires considerable attention.

Connexin 43 enhances Bax activation via JNK activation in sunitinib-induced apoptosis in mesothelioma cells

The constituent protein of gap junctions, connexin (Cx), interacts with various proteins via its C-terminus region, including kinases, cell-adhesion proteins, and a pro-apoptotic protein, Bax. This molecular interaction may affect expression and functioning of the interacting proteins and modulate the cellular physiology. In our previous work, Cx43 was found to interact directly with Bax and in the presence of sunitinib, lead to the Bax-mediated apoptosis in mesothelioma cells. In this study, we investigated the mechanism of how Cx43 promotes Bax-mediated apoptosis using the same cell line. Treatment with sunitinib increased the expression of the active conformation of the Bax protein, which was predominantly localized at the mitochondria, only in Cx43-transfected cells. Bax oligomerization and decrease in the mitochondrial membrane potential were also observed. The involvement of c-Jun N-terminal kinase (JNK) in the interaction of Cx43 and Bax was further examined. Treatment with sunitinib increased the expression of phosphorylated (active) form of JNK only in the Cx43-transfected cells. Phosphorylated JNK and active Bax were co-localized, and the co-localization was suppressed by the knockdown of Cx43. Moreover, JNK inhibition clearly suppressed Bax activation. In conclusion, we identified a novel Cx43-JNK-Bax axis regulating the process of apoptosis for the first time.

Involvement of c-Jun N-Terminal Kinase in TNF-α-Driven Remodeling.

Lung tissue remodeling in chronic obstructive pulmonary disease (COPD) is characterized by airway wall thickening and/or emphysema. Although the bronchial and alveolar compartments are functionally independent entities, we recently showed comparable alterations in matrix composition comprised of decreased elastin content and increased collagen and hyaluronan contents of alveolar and small airway walls. Out of several animal models tested, surfactant protein C (SPC)-TNF-α mice showed remodeling in alveolar and airway walls similar to what we observed in patients with COPD. Epithelial cells are able to undergo a phenotypic shift, gaining mesenchymal properties, a process in which c-Jun N-terminal kinase (JNK) signaling is involved. Therefore, we hypothesized that TNF-α induces JNK-dependent epithelial plasticity, which contributes to lung matrix remodeling. To this end, the ability of TNF-α to induce a phenotypic shift was assessed in A549, BEAS2B, and primary bronchial epithelial cells, and phenotypic markers were studied in SPC-TNF-α mice. Phenotypic markers of mesenchymal cells were elevated both in vitro and in vivo, as shown by the expression of vimentin, plasminogen activator inhibitor-1, collagen, and matrix metalloproteinases. Concurrently, the expression of the epithelial markers, E-cadherin and keratin 7 and 18, was attenuated. A pharmacological inhibitor of JNK attenuated this phenotypic shift in vitro, demonstrating involvement of JNK signaling in this process. Interestingly, activation of JNK signaling was also clearly present in lungs of SPC-TNF-α mice and patients with COPD. Together, these data show a role for TNF-α in the induction of a phenotypic shift in vitro, resulting in increased collagen production and the expression of elastin-degrading matrix metalloproteinases, and provide evidence for involvement of the TNF-α-JNK axis in extracellular matrix remodeling.

Involvement of cJun N-terminal kinase (JNK) signaling in dopamine transmission

Involvement of cJun N-terminal kinase (JNK) signaling in dopamine transmission

Wei Zhang, Bao-an Chen, Jun-fei Jin, You-ji He, and Yi-qi Niu, Involvement of c-Jun N-terminal kinase in reversal of multidrug resistance of human leukemia cells in hypoxia by 5-bromotetrandrine. November 2013;54(11):2506–2516. http://dx.doi.org/10.3109/10428194.2013.776681

View correction statement:Involvement of c-Jun N-terminal kinase in reversal of multidrug resistance of human leukemia cells in hypoxia by 5-bromotetrandrine

Mesenchymal stem cells promote liver regeneration and prolong survival in small-for-size liver grafts: involvement of C-Jun N-terminal kinase, cyclin D1, and NF-κB.

BackgroundThe therapeutic potential of mesenchymal stem cells (MSCs) has been highlighted recently for treatment of acute or chronic liver injury, by possibly differentiating into hepatocyte-like cells, reducing inflammation, and enhancing tissue repair. Despite recent progress, exact mechanisms of action are not clearly elucidated. In this study, we attempted to explore whether and how MSCs protected hepatocytes and stimulated allograft regeneration in small-for-size liver transplantation (SFSLT).MethodsSFSLT model was established with a 30% partial liver transplantation (30PLT) in rats. The differentiation potential and characteristics of bone marrow derived MSCs were explored in vitro. MSCs were infused transvenously immediately after graft implantation in therapy group. Expressions of apoptosis-, inflammatory-, anti-inflammatory-, and growth factor-related genes were measured by RT-PCR, activities of transcription factors AP-1 and NF-κB were analyzed by EMSA, and proliferative responses of the hepatic graft were evaluated by immunohistochemistry and western blot.ResultsMSCs were successfully induced into hepatocyte-like cells, osteoblasts and adipocytes in vitro. MSCs therapy could not only alleviate ischemia reperfusion injury and acute inflammation to promote liver regeneration, but also profoundly improve one week survival rate. It markedly up-regulated the mRNA expressions of HGF, Bcl-2, Bcl-XL, IL-6, IL-10, IP-10, and CXCR2, however, down-regulated TNF-α. Increased activities of AP-1 and NF-κB, as well as elevated expressions of p-c-Jun, cyclin D1, and proliferating cell nuclear antigen (PCNA), were also found in MSCs therapy group.ConclusionThese data suggest that MSCs therapy promotes hepatocyte proliferation and prolongs survival in SFSLT by reducing ischemia reperfusion injury and acute inflammation, and sustaining early increased expressions of c-Jun N-terminal Kinase, Cyclin D1, and NF-κB.

Involvement of interleukin-17A-induced expression of heat shock protein 47 in intestinal fibrosis in Crohn's disease

ObjectiveIntestinal fibrosis is a clinically important issue in Crohn's disease (CD). Heat shock protein (HSP) 47 is a collagen-specific molecular chaperone involved in fibrotic diseases. The molecular mechanisms of HSP47...

SUMO: a (Oxidative) Stressed Protein

Redox species are produced during the physiological cellular metabolism of a normal tissue. In turn, their presence is also attributed to pathological conditions including neurodegenerative diseases. Many are the molecular changes that occur during the unbalance of the redox homeostasis. Interestingly, posttranslational protein modifications (PTMs) play a remarkable role. In fact, several target proteins are modified in their activation, localization, aggregation, and expression after the cellular stress. Among PTMs, protein SUMOylation represents a very important molecular modification pathway during "oxidative stress". It has been reported that this ubiquitin-like modification is a fine sensor for redox species. Indeed, SUMOylation pathway efficiency is affected by the exposure to oxidative species in a different manner depending on the concentration and time of application. Thus, we here report updated evidence that states the role of SUMOylation in several pathological conditions, and we also outline the key involvement of c-Jun N-terminal kinase and small ubiquitin modifier pathway cross talk.

Involvement of c-Jun N-terminal kinase in reversal of multidrug resistance of human leukemia cells in hypoxia by 5-bromotetrandrine

5-Bromotetrandrine (BrTet), a candidate multidrug resistance (MDR) modulator, is a potential compound for use in cancer therapy when combined with anticancer agents such as daunorubicin (DNR) and paclitaxel. The purposeof this study was to investigate the mechanism of reversal of P-glycoprotein (P-gp)-mediated MDR by BrTet and the involvement of the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway in both adriamycin-sensitive K562 and adriamycin-resistant K562 (KA) leukemia cells in hypoxia. The combination of BrTet and DNR decreased both phosphorylated JNK1/2 and MDR1/P-gp levels under hypoxic conditions. Furthermore, a pharmacological inhibitor of JNK, SP600125, or small interfering RNA (siRNA) oligonucleotides to both JNK1 and JNK2 reversed BrTet- or DNR-induced JNK phosphorylation and MDR1/P-gp levels. We further demonstrated that the decreased JNK phosphorylation and MDR1/P-gp levels were associated with a significant increase in intracellular accumulation of DNR, which dramatically enhanced the sensitivity of drug-resistant KA cells to DNR, and led to cellular apoptosis through activation of the caspase-3 pathway. It is concluded that using BrTet in combination with other chemotherapeutic agents and pharmacological inhibitors of JNK can abrogate the P-gp-induced MDR in adriamycin-resistant K562 cells, which has potential clinical relevance in cancer therapy for chemotherapeutic-resistant human leukemia.

Endoplasmic Reticulum Stress Plays a Pivotal Role in Cell Death Mediated by the Pan-Deacetylase Inhibitor Panobinostat in Human Hepatocellular Cancer Cells

Panobinostat, a pan-deacetylase inhibitor, represents a novel therapeutic option for cancer diseases. Besides its ability to block histone deacetylases (HDACs) by promoting histone hyperacetylation, panobinostat interferes with several cell death pathways providing a potential efficacy against tumors. We have previously demonstrated that panobinostat has a potent apoptotic activity in vitro and causes a significant growth delay of hepatocellular carcinoma (HCC) tumor xenografts in nude mice models. Here, we show that treatment with panobinostat is able to induce noncanonical apoptotic cell death in HepG2 and in Hep3B cells, involving the endoplasmic reticulum (ER) stress by up-regulation of the molecular chaperone binding immunoglobulin protein/glucose-regulated protein 78, activation of eukaryotic initiation factor 2α-activating transcription factor 4 (tax-responsive enhancer element B67) and inositol requiring 1α-X-box binding protein 1 factors, strong increase and nuclear translocation of the transcription factor C/EBP homologous protein/growth arrest and DNA damage-inducible gene 153, and involvement of c-Jun N-terminal kinase. These signaling cascades culminate into the activation of the ER-located caspase-4/12 and of executioner caspases, which finally lead to cell demise. Our results clearly show that panobinostat induces an alternative ER stress-mediated cell death pathway in HCC cells, independent of the p53 status.

TNF-α increases lipogenesis via JNK and PI3K/Akt pathways in SZ95 human sebocytes

Tumor necrosis factor-alpha (TNF-α) is an important pathophysiologic factor involved in the development of acne. However, its role is unclear. To explore the lipogenic effect by TNF-α and possible molecular mechanisms in sebocyte. Using SZ95 human sebocytes, lipid formation by TNF-α was assessed by Oil Red O, Nile Red staining and thin layer chromatography (TLC). Expression of lipogenic genes and activation of mitogen-activated protein kinase as well as Akt were examined by real-time polymerase chain reaction and/or Western blot analysis. Activation of peroxisome proliferator-activated receptor (PPAR) was evaluated by luciferase assay using PPAR response element containing reporter plasmids. Involvement of c-Jun N-terminal kinase (JNK) and Akt in TNF-α-induced lipogenesis was investigated by molecule specific small interfering RNA and inhibitors. TNF-α treatment significantly increased formation of lipid droplets in accordance with up-regulated expression of FAS and activation of SREBP-1, but not PPARs. Suppression of phosphorylated JNK by the JNK inhibitor SP600125 greatly diminished TNF-α-induced expression of FAS and SREBP-1. TNF-α could not induce both expression of lipogenic proteins and lipid synthesis when Akt expression was attenuated with siRNA. TNF-α induces lipogenesis in SZ95 human sebocytes through the JNK and phosphoinositide-3-kinase/Akt pathways. These results will be valuable in developing therapeutic strategies for control of seborrhea and acne.

A Growth Factor Attenuates HIV-1 Tat and Morphine Induced Damage to Human Neurons: Implication in HIV/AIDS-Drug Abuse Cases

The neuropathological abnormalities of human immunodeficiency virus (HIV)-1 patients abusing illicit drugs suggest extensive interactions between the two agents, thereby leading to increased rate of progression to neurodegeneration. The role of HIV-1 transactivating protein, Tat has been elucidated in mediating neuronal damage via apoptosis, a hallmark of HIV-associated dementia (HAD), however the underlying mechanisms involved in enhanced neurodegeneration by illicit drugs remain elusive. In this study, we demonstrated that morphine enhances HIV-Tat induced toxicity in human neurons and neuroblastoma cells. Enhanced toxicity by Tat and morphine was accompanied by increased numbers of TUNEL positive apoptotic neurons, elevated caspase-3 levels and decreased ratio of anti- and pro-apoptotic proteins, Bcl2/Bax. Tat and morphine together elicited high levels of reactive oxygen species that were NADPH dependent. Significant alterations in mitochondrial membrane homeostasis were also observed with co-exposure of these agents. Extensive studies of mitogen activated protein kinase (MAPK) signaling pathways revealed the involvement of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase-1/2 (ERK1/2) pathways in enhanced toxicity of Tat and morphine. In addition to this, we found that pre-treatment of cells with platelet derived growth factor (PDGF-BB) protected neurons from HIV-Tat and morphine induced damage. PDGF-BB alleviated ROS production, maintained mitochondrial membrane potential, decreased caspase-3 activation and hence protected the cells from undergoing apoptosis. PDGF-BB mediated protection against Tat and morphine involved the phosphatidylinositol–3 kinase (PI3K) pathway, as specific inhibitor of PI3K abrogated the protection conferred by PDGF-BB. This study demonstrates the mechanism of enhanced toxicity in human neurons subjected to co-exposure of HIV protein Tat and morphine, thus implying its importance in HIV positive drug abusers, where damage to the brain is reported to be more severe than non-drug abusers. We have also showed for the first time that PDGF-BB can protect against simultaneous exposure of Tat and morphine, strengthening its role as a neuroprotective agent that could be considered for therapeutic intervention.

Apoptosis induced by genipin in human leukemia K562 cells: involvement of c-Jun N-terminal kinase in G2/M arrest

To investigate the effect of genipin on apoptosis in human leukemia K562 cells in vitro and elucidate the underlying mechanisms. The effect of genipin on K562 cell viability was measured using trypan blue dye exclusion and cell counting. Morphological changes were detected using phase-contrast microscopy. Apoptosis was analyzed using DNA ladder, propidium iodide (PI)-labeled flow cytometry (FCM) and Hoechst 33258 staining. The influence of genipin on cell cycle distribution was determined using PI staining. Caspase 3 activity was analyzed to detect apoptosis at different time points. Protein levels of phospho-c-Jun, phosphor-c-Jun N-terminal kinase (p-JNK), phosphor-p38, Fas-L, p63, and Bax and the release of cytochrome c were detected using Western blot analysis. Genipin reduced the viability of K562 cells with an IC(50) value of approximately 250 μmol/L. Genipin 200-400 μmol/L induced formation of typical apoptotic bodies and DNA fragmentation. Additionally, genipin 400 μmol/L significantly increased the caspase 3 activity from 8-24 h and arrested the cells in the G₂/M phase. After stimulation with genipin 500 μmol/L, the levels of p-JNK, p-c-Jun, Fas-L, Bax, and cytochrome c were remarkably upregulated, but there were no obvious changes of p-p38. Genipin 200-500 μmol/L significantly upregulated the Fas-L expression and downregulated p63 expression. Dicoumarol 100 μmol/L, a JNK1/2 inhibitor, markedly suppressed the formation of apoptotic bodies and JNK activation induced by genipin 400 μmol/L. These results suggest that genipin inhibits the proliferation of K562 cells and induces apoptosis through the activation of JNK and induction of the Fas ligand.

398 Glycan gene expression signatures distinguish normal and malignant breast tissue; possible role in diagnosis and progression

Panobinostat, a pan-deacetylase inhibitor, represents a novel therapeutic option for cancer diseases. Besides its ability to block histone deacetylases (HDACs) by promoting histone hyperacetylation, panobinostat interferes with several cell death pathways providing a potential efficacy against tumors. We have previously demonstrated that panobinostat has a potent apoptotic activity in vitro and causes a significant growth delay of hepatocellular carcinoma (HCC) tumor xenografts in nude mice models. Here, we show that treatment with panobinostat is able to induce noncanonical apoptotic cell death in HepG2 and in Hep3B cells, involving the endoplasmic reticulum (ER) stress by up-regulation of the molecular chaperone binding immunoglobulin protein/glucose-regulated protein 78, activation of eukaryotic initiation factor 2α-activating transcription factor 4 (tax-responsive enhancer element B67) and inositol requiring 1α-X-box binding protein 1 factors, strong increase and nuclear translocation of the transcription factor C/EBP homologous protein/growth arrest and DNA damage-inducible gene 153, and involvement of c-Jun N-terminal kinase. These signaling cascades culminate into the activation of the ER-located caspase-4/12 and of executioner caspases, which finally lead to cell demise. Our results clearly show that panobinostat induces an alternative ER stress-mediated cell death pathway in HCC cells, independent of the p53 status.

Anti-inflammatory intestinal activity of Abarema cochliacarpos (Gomes) Barneby & Grimes in TNBS colitis model

Aim of the study To assess the anti-inflammatory effect of butanolic fraction of methanolic extract from bark of Abarema cochliacarpos in acute ulcerative colitis model induced by intracolonic administration of trinitrobenzene sulfonic acid (TNBS) in Wistar rats. Materials and methods Abarema cochliacarpos (100 and 150 mg/kg/day) was administered by gavage 48, 24 and 1 h prior to the induction of colitis with 10 mg/kg of TNBS and, 24 h later. Results Phytochemical studies by mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) revealed that catechins were a major component into condensate class of tannins. Treatment with Abarema cochliacarpos decreased significantly macroscopic damage as compared with TNBS (p < 0.05). Histological analysis showed that both doses of the extract improved the microscopic structure and preserved some areas of the colonic mucosa structure. In addition, myeloperoxidase activity (MPO), as a marker of neutrophil infiltration, was decreased in a dose-dependent way ( p < 0.01 and p < 0.001 respectively), TNF-α level was also diminished with the highest dose of the extract ( p < 0.001) and, IL-10 level obtained no significant results. In order to elucidate some of the mechanisms, expression of inducible inflammatory enzymes, such as cyclooxygenase (COX)-2 and nitric oxide synthase (iNOS), were studied showing a significant reduction. Finally, the involvement of c-Jun N-terminal kinase (JNK) signalling demonstrated a reduction in the JNK activation with the highest dose ( p < 0.05 vs TNBS). Conclusions We have shown for the first time that the extracts obtained from Abarema cochliacarpos bark possess active substances, which exert marked protective effects in acute experimental colitis, confirming and justifying, at least in part, the popular use of this plant to treat gastrointestinal diseases.

Regulation of prostaglandin E2 synthase expression in activated primary rat microglia: Evidence for uncoupled regulation of mPGES‐1 and COX‐2

Prostaglandin E2 (PGE2) is among the most important mediators involved in neuroinflammatory processes. The final step of its synthesis is regulated by enzymes termed prostaglandin E2 synthases (PGES). Three PGES are known, cytosolic (c)PGES, membrane-associated (m)PGES-1 and mPGES-2. The expression of mPGES-1 is induced by inflammatory stimuli such as lipopolysaccharide (LPS), interleukin (IL)-1beta, and tumor necrosis factor (TNF)-alpha. Although some roles of mPGES-1 have already been suggested, its function in the CNS and the signaling pathways involved in its upregulation are poorly understood. In this study, we examined the regulation of mPGES-1 in primary rat microglia and the signaling pathways involved in its expression. Whereas the expression of cPGES and mPGES-2 was not stimulated by LPS, low doses of LPS (0.1-1 ng/mL) sufficiently stimulated mPGES-1 mRNA expression. A corresponding protein synthesis, however, was obtained only with higher doses (10-100 ng/mL). The LPS-induced increase of mPGES-1 was inhibited by different signaling pathway inhibitors, such as SP600125, LY294002, GF109203X, and SC-514, suggesting the involvement of c-Jun N-terminal kinase (JNK), phosphatidylinositol 3-kinase (PI-3K)/Akt, protein kinase C (PKC) pathways, and the nuclear factor (NF)-kappaB, respectively. In contrast to other reports, LPS-induced mPGES-1 synthesis was not invariably coupled to the synthesis of COX-2, since inhibition of PI-3K with LY294002 decreased mPGES-1 but increased COX-2 levels. This detailed view of the intracellular signaling pathways involved in mPGES-1 expression in activated microglia opens a new avenue in the search for novel potential therapeutic targets to reduce neuroinflammation, and demonstrates that mPGES-1 expression is not strictly coupled to the expression of COX-2.

Neuron-specific phosphorylation of c-Jun N-terminal kinase increased in the brain of hypoxic preconditioned mice

Accumulated studies have suggested that mitogen-activated protein kinase (MAPK) play a pivotal role in the development of cerebral hypoxic preconditioning (HPC). By using our "auto-hypoxia"-induced HPC mouse model, we have reported increased phosphorylation level of p38 MAPK, and decreased phosphorylation and protein expression levels of extracellular signal regulated kinases 1/2 (ERK1/2) in the brain of HPC mice. In the current study, we investigated the involvement of c-Jun N-terminal kinase (JNK) in the brain of HPC mice. By using Western blot analysis, we found that the phosphorylation levels of JNK at Thr183 and Tyr185 sites (phospho-Thr183/Tyr185 JNK), but not its protein expression, increased significantly (p<0.05, n=6 for each group) both in the hippocampus and frontal cortex of early (H1-H4) and delayed (H5 and H6) HPC mice than that of the normoxic group (H0, n=6). Similarly, enhanced phospho-Thr183/Tyr185 JNK was also observed by immunostaining in the hippocampus and frontal cortex of mice following series of hypoxic exposures (H3 and H6). In addition, we found that phospho-Thr183/Tyr185 JNK predominantly co-localized with a neuron-specific protein, neurogranin, in both the hippocampus and frontal cortex of HPC mice (H3) by using double-labeled immunofluorescence. These results suggest that the increased neuron-specific phosphorylation of JNK at Thr183/Tyr185, not protein expression, might be involved in the development of cerebral HPC of mice.