Intrathecal administration of low‐dose nociceptin/orphanin FQ induces allodynia via c‐Jun N‐terminal kinase and monocyte chemoattractant protein‐1

Pathological chronic pain, which is frequently associated with prolonged tissue damage, inflammation, tumour invasion, and neurodegenerative diseases, gives rise to hyperalgesia and allodynia. We previously reported that intrathecal administration of nociceptin/orphanin FQ (N/OFQ), an endogenous ligand for the orphan opioid receptor‐like receptor, in the femtomole range induces touch‐evoked allodynia. N/OFQ has been implicated in multiple signalling pathways, such as inhibition of cAMP production and Ca²⁺ channels, or activation of K⁺ channels and mitogen‐activated protein kinase, although the signalling pathways of N/OFQ‐induced allodynia remain unclear. To address these issues, we developed an ex vivo mitogen‐activated protein kinase assay by using intact slices of mouse spinal cord. N/OFQ markedly increased the phosphorylation of c‐Jun N‐terminal kinase (JNK) in the superficial dorsal horn of the spinal cord. The N/OFQ‐stimulated JNK phosphorylation was significantly inhibited by pertussis toxin, the phospholipase C inhibitor U73122, and the inositol trisphosphate receptor antagonist Xestospongin C. Intrathecal administration of the JNK inhibitor SP600125 inhibited N/OFQ‐evoked allodynia. The N/OFQ‐induced increase in JNK phosphorylation was observed in astrocytes that expressed glial fibrillary acidic protein. N/OFQ also induced monocyte chemoattractant protein‐1 (MCP‐1) release via the JNK pathway, and N/OFQ‐induced JNK phosphorylation was observed in MCP‐1‐immunoreactive astrocytes. Intrathecal administration of the MCP‐1 receptor antagonist RS504393 inhibited N/OFQ‐evoked allodynia. These results suggest that, in the spinal dorsal horn, N/OFQ induces allodynia through activation of JNK via the phospholipase C–inositol trisphosphate pathway, which is coupled to pertussis toxin‐sensitive G‐protein, and following the release of MCP‐1 from astrocytes.     

Activation of NF-kB and ERK1/2 after permanent focal ischemia is abolished by simvastatin treatment

We investigated the effects of simvastatin treatment on the expression of IL-1beta and MCP-1, the activity of NF-kB, and the signaling pathways related to NF-kB activation in a rat model of permanent middle cerebral artery occlusion (pMCAO). IL-1beta and MCP-1 expression, determined using RT-PCR, was enhanced by pMCAO; this effect was inhibited by the administration of simvastatin before ischemia. Pre-treatment with simvastatin abolished the ischemia-induced activation of NF-kB observed in vehicle-treated animals. The evaluation of signal transduction pathways, including extracellular signal-regulated kinase (ERK1/2), SAPK/JNK 46/54 and p38, indicated that only ERK1/2 phosphorylation was enhanced by ischemia, and this activation was prevented by simvastatin. ERK1/2-inhibitor, U0126, reduced brain ischemia but not cytokine induction. These results provide evidence that the HMG-CoA reductase inhibitor induces its effect in the protection of ischemic brain damage with a more complex mechanism which also involve anti-inflammatory properties rather than simple inhibition of ERK1/2 signaling pathway.     

MAPK信号通路在创伤性脑损伤中的作用

目的通过建立小鼠创伤性脑损伤(TBI)模型,研究丝裂原活化蛋白激酶(MAPKs)通路中的细胞外调节蛋白激酶1/2(ERK1/2)通路、JNK通路和p38通路的激活及在TBI中的作用及机制。方法建立小鼠TBI模型,通过Western blot检测ERK1/2、JNK和p38的相对磷酸化水平,确定TBI后MAPK通路的激活情况;分别加入ERK1/2通路抑制剂(PD98059,500μmol/L)、JNK通路抑制剂(SP600125,500μmol/L)和p38通路抑制剂(SB203580,500μmol/L),通过脑干湿重检测、神经功能学评分和TUNEL染色评估不同抑制剂对TBI的作用,并通过Western blot检测ERK1/2、JNK和p38的相对磷酸化水平,明确ERK1/2通路、JNK通路和p38通路之间的相互调节作用。结果 TBI可分别引起ERK1/2通路、JNK通路和p38通路的激活;抑制ERK通路和JNK通路可减轻TBI引起的脑水肿、神经功能损伤和细胞凋亡,而抑制p38通路则加重TBI引起的脑水肿、神经功能损伤和细胞凋亡;抑制JNK通路可减少ERK1/2的相对磷酸化水平,而抑制p38通路可增加ERK1/2的相对磷酸化水平。结论 TBI后,ERK1/2通路和JNK通路的激活发挥促进损伤形成的作用,而p38通路的激活则起到神经保护的作用;ERK1/2通路的激活受到JNK通路的促进和p38通路的抑制,表明MAPK通路之间存在相互调节。     

Activated glia induce neuron death via MAP kinase signaling pathways involving JNK and p38

Chronic glial activation in neurodegenerative diseases contributes to neuronal dysfunction and neuron loss through production of neuroinflammatory molecules. However, the molecular mechanisms, particularly the signal transduction pathways involved in glia-dependent neuron death, are poorly understood. As a first step to address this question, we used a neuron-glia co-culture system that allows diffusion of soluble molecules between glia and neurons to test the potential importance of mitogen-activated protein kinase (MAPK) signaling pathways in the glia-induced neuron death. Activation of glia in co-culture by lipopolysaccharide (LPS) induced apoptotic-like neuron death. The MAPKs tested (p38, JNK, ERK1/2) were activated in both glia and neurons following LPS treatment, suggesting their involvement in both glial activation and neuronal response to diffusible, glia-derived neurotoxic molecules. Inhibitors of p38 and JNK partially blocked neuron death in the LPS-treated co-culture, whereas an ERK1/2 pathway inhibitor did not protect neurons. These results show that p38 and JNK MAPKs, but not ERK1/2 MAPK, are important signal transduction pathways contributing to glia-induced neuron death.     

Implications of mitogen‐activated protein kinase signaling in glioma

Gliomas are the most common primary central nervous system tumors. Gliomas originate from astrocytes, oligodendrocytes, and neural stem cells or their precursors. According to WHO classification, gliomas are classified into four different malignant grades ranging from grade I to grade IV based on histopathological features and related molecular aberrations. The induction and maintenance of these tumors can be attributed largely to aberrant signaling networks. In this regard, the mitogen‐activated protein kinase (MAPK) network has been widely studied and is reported to be severely altered in glial tumors. Mutations in MAPK pathways most frequently affect RAS and B‐RAF in the ERK, c‐Jun N‐terminal kinase (JNK), and p38 pathways leading to malignant transformation. Also, it is linked to both inherited and sequential accumulations of mutations that control receptor tyrosine kinase (RTK)‐activated signal transduction pathways, cell cycle growth arrest pathways, and nonresponsive cell death pathways. Genetic alterations that modulate RTK signaling can also alter several downstream pathways, including RAS‐mediated MAP kinases along with JNK pathways, which ultimately regulate cell proliferation and cell death. The present review focuses on recent literature regarding important deregulations in the RTK‐activated MAPK pathway during gliomagenesis and progression. © 2015 Wiley Periodicals, Inc.     

Clozapine and the mitogen-activated protein kinase signal transduction pathway: implications for antipsychotic actions.

BACKGROUND: Mitogen-activated protein kinase (MAPK) signaling pathways respond to dopaminergic and serotonergic agents and mediate short- and long-term effects of intracellular signaling in neurons. Here we show that the antipsychotic agent, clozapine, selectively activates the MEK/ERK MAPK pathway, and inhibition of this pathway reverses clozapine's actions in the conditioned avoidance response (CAR) paradigm, a rodent behavioral assay of antipsychotic activity. METHODS: Phosphorylation patterns of MAPK pathway enzymes were determined by quantitative immunoblot analysis and immunohistochemistry of rat prefrontal cortex. Kinase inhibitors were used to assess the role of MAPK signaling pathways in mediating clozapine-induced suppression of CAR. RESULTS: Clozapine administration selectively increased phosphorylation of MEK1/2 but had no effect on p38 or JNK phosphorylation. Pretreatment with the 5-HT2A agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride blocked the clozapine-induced increase in MEK1/2 phosphorylation. Immunohistochemistry revealed that clozapine treatment elevated the number of cells in the prefrontal cortex positive for phosphoERK, the downstream substrate of MEK1/2. Prior administration of MEK1/2 inhibitors U0126 or Sl327, or ERK inhibitor 5-iodotubercidin, reversed suppression of CAR induced by clozapine, whereas administration of vehicle, JNK or p38 inhibitors (L-JNK-1 and SB203580, respectively) had no effect. Inhibition of kinases upstream to MEK1/2 (PI-3K, PKC, and CaMKII) by administration of LY294002, bisindolylmaleimide, or KN-62, respectively, also reversed clozapine-induced suppression of CAR. CONCLUSIONS: These data support the hypothesis that the MEK/ERK signal transduction cascade participates in clozapine's antipsychotic actions.     

MAPK/ERK激酶-ERK信号通路参与外源性bFGF对缺氧-复氧损伤后星形胶质细胞内Egr-1结合活性的调节(英文)

目的静脉注射碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)可以明显降低实验性脑缺血大鼠的脑梗死面积,但该作用的分子机制尚不清楚。本文旨在研究外源性bFGF 作用的信号转导通路。方法缺氧-复氧损伤星形胶质细胞。Western blot检测外源性bFGF作用后丝裂原活化蛋白激酶/细胞外信号调节激酶激酶(mitogen-activated protein kinase/extracellular signal-regulated kinase kinase,MEK)-细胞外信号调节激酶(extracellular signal-regulated kinase, ERK) 信号通路活化。电泳变动迁移率分析实验检测外源性bFGF 作用后核转录因子早期生长反应因子-1(early growth respons factor 1, Egr-1)的结合活性变化。结果外源性bFGF可以保护胞外信号调节激酶MEK-ERK信号通路蛋白不被氧自由基降解。MEK-ERK信号通路在外源性bFGF作用后活化。这一信号通路进一步使Egr-1结合活性升高。结论外源性bFGF可能通过激活ERK信号通路,促进内源性转录因子Egr-1的结合活性升高,进而促进内源性bFGF的表达。     

MAPK/ERK激酶-ERK信号通路参与外源性bFGF对缺氧-复氧损伤后星形胶质细胞内Egr-1结合活性的调节

目的 静脉注射碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）可以明显降低实验性脑缺血大鼠的脑梗死面积,但该作用的分子机制尚不清楚。本文旨在研究外源性bFGF 作用的信号转导通路。方法缺氧-复氧损伤星形胶质细胞。Western blot检测外源性bFGF作用后丝裂原活化蛋白激酶/细胞外信号调节激酶激酶（mitogen-activated protein kinase/extracellular signal-regulated kinase kinase,MEK）-细胞外信号调节激酶（extracellular signal-regulated kinase, ERK） 信号通路活化。电泳变动迁移率分析实验检测外源性bFGF 作用后核转录因子早期生长反应因子-1（early growth respons factor 1, Egr-1）的结合活性变化。结果外源性bFGF可以保护胞外信号调节激酶MEK-ERK信号通路蛋白不被氧自由基降解。MEK-ERK信号通路在外源性bFGF作用后活化。这一信号通路进一步使Egr-1结合活性升高。结论外源性bFGF可能通过激活ERK信号通路,促进内源性转录因子Egr-1的结合活性升高,进而促进内源性bFGF的表达。     

Herpes simplex virus type 1-induced encephalitis has an apoptotic component associated with activation of c-Jun N-terminal kinase

Herpes simplex virus type 1 (HSV-1) triggered apoptosis in hippocampal cultures, as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and immunohistochemistry with antibody specific for the large fragment of activated caspase 3. The levels of phosphorylated (activated) c-Jun N-terminal kinase (JNK) were also increased in HSV-1-infected hippocampal cultures as were the levels of activated c-Jun, its target. JNK activation was involved in HSV-1-induced apoptosis as evidenced by apoptosis inhibition with the JNK inhibitor SP600125. HSV-2 activated the mitogen-activated protein kinase/extracellular regulated protein kinase (MEK/ERK) survival pathway and did not trigger apoptosis in hippocampal cultures. The MEK specific inhibitor U0126 inhibited ERK activation and caused a significant increase in the percent TUNEL(+) cells in HSV-2-infected cultures, indicating that the failure of HSV-2 to trigger apoptosis is due to its ability to activate the MEK/ERK survival pathway. JNK was also activated in brain tissues from patients with HSV-associated acute focal encephalitis (HSE) that were positive for HSV-1 antigen. JNK activation correlated with apoptosis, as determined by immunohistochemistry with antibody to activated caspase 3 or cleaved poly (ADP-ribose) polymerase (PARP). The data suggest that HSE has an apoptotic component that may contribute to disease pathogenesis.     

Differential MAP kinases activation during semaphorin3A-induced repulsion or apoptosis of neural progenitor cells

Semaphorins are multifunctional factors implicated in various developmental processes. Little is known about the intracellular pathways ensuring appropriate signal transduction that encode the diverse functions observed. In this study, we investigated whether mitogen-activated protein kinases (MAPK), which are key elements of signal transduction in eukaryotic cells, were activated during semaphorin 3A (Sema3A)-induced repulsion or apoptosis of neural progenitor cells. We found that selective recruitment of the ERK1/2 pathway occurred during Sema3A-induced neural progenitor cell repulsion, whereas p38 MAPK activation was necessary for induction of apoptosis. Moreover, we provide evidence for the involvement of vascular endothelial growth factor receptor 1 (VEGFR1) in the activation of ERK1/2. Additional experiments performed with native cerebellar progenitors confirmed such a selective recruitment of MAPK during Sema3A-dependent migration or apoptosis. Altogether, our results suggest a model to explain how a single factor can exert different functions for a given cell type by the selective recruitment of intracellular pathways.     

Aquaporin‐4 in manganese‐treated cultured astrocytes

Manganese in excess is neurotoxic and causes CNS injury resembling that of Parkinson's disease. In brain, astrocytes predominantly take up and accumulate manganese and are thus vulnerable to its toxicity. Manganese was shown to induce cell swelling in cultured astrocytes, and oxidative/nitrosative stress (ONS) mediates such swelling. As aquaporin‐4 (AQP4) is important in the mechanism of astrocyte swelling, we examined the effect of manganese on AQP4 protein levels in cultured astrocytes. Treatment of cultures with manganese increased AQP4 protein in the plasma membrane (PM), whereas total cellular AQP4 protein and mRNA levels were unchanged, suggesting that increased AQP4 levels is due to its increased stability and/or increased trafficking to the PM and not to its neosynthesis. AQP4 gene silencing by small interfering ribonucleic acid resulted in a marked reduction in astrocyte swelling by manganese. Antioxidants, as well as an inhibitor of nitric oxide synthase, diminished the increase in AQP4 protein expression, suggesting a role of ONS in the mechanism of AQP4 increase. As ONS is known to activate mitogen‐activated protein kinases (MAPKs) and MAPK activation has been implicated in astrocyte swelling, we examined the effect of manganese on the activation of MAPKs and found an increased phosphorylation of extracellular signal‐regulated kinase (ERK)1/2, C‐Jun amino‐terminal kinase (JNK)1/2/3, and p38‐MAPK. Inhibitors of ERK1/2 and p38‐MAPK (but not of JNK) blocked (40–60%) the manganese‐induced increase in AQP4 protein content and astrocyte swelling, suggesting the involvement of these kinases in the increased AQP4 content. Inhibition of oxidative stress or MAPKs may represent potential strategies for counteracting AQP4‐related neurological complications associated with manganese toxicity. © 2010 Wiley‐Liss, Inc.     

Neuronal injury induces microglial production of macrophage inflammatory protein-1α in rat corticostriatal slice cultures

Chemokines are potent chemoattractants for immune and hematopoietic cells. In the central nervous system, chemokines play an important role in inflammatory responses through activation of infiltrating leukocytes and/or resident glial cells. We previously demonstrated that N‐methyl‐D‐aspartate (NMDA)‐evoked neuronal injury induced astrocytic production of monocyte chemoattractant protein‐1 (MCP‐1, CCL2) via sustained activation of extracellular signal‐regulated kinase (ERK) in rat organotypic slice cultures. In the present study, we examined mRNA expression and protein production of macrophage inflammatory protein‐1α (MIP‐1α, CCL3) induced by NMDA‐evoked neuronal injury in the slice cultures. MIP‐1α mRNA expression was transiently increased by NMDA treatment in a concentration‐dependent manner. Double‐fluorescence immunohistochemistry revealed that MIP‐1α was produced predominantly in microglia. Depletion of microglial cells from the slice cultures by pretreatment with liposome‐encapsulated clodronate abrogated the increase in MIP‐1α mRNA expression after NMDA treatment. NMDA‐induced MIP‐1α mRNA expression was partially but significantly inhibited by the c‐Jun N‐terminal kinase inhibitor SP600125; conversely, the p38 mitogen‐activated protein (MAP) kinase inhibitor SB203580 enhanced it. U0126, a MAP kinase/ERK kinase inhibitor, did not affect mRNA expression. These results, combined with our previous findings, demonstrate that NMDA‐evoked neuronal injury differentially induces MIP‐1α and MCP‐1 production in microglia and astrocytes, respectively, through activation of different intracellular signaling pathways. © 2012 Wiley Periodicals, Inc.     

Lead-induced cell signaling cascades in GT1-7 cells

The effects of lead on the signal transduction pathways that may be involved in the release of gonadotropin-releasing hormone (GnRH) from neurons in the hypothalamus have not been well defined. Using the GT1-7 cell line, an in vitro model for GnRH-secreting neurons, we examined signal transduction pathways directly affected by lead. We found that lead-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2), as well as p90RSK and cAMP response element-binding protein (CREB), but did not induce IkappaB degradation. MEK1/2 inhibitor (PD98059) suppressed lead-induced ERK and p90RSK activation. Neither PKC inhibitors (Go6983, Go6976) nor CaMKII inhibitor (KN-62) had a pronounced effect on lead-induced ERK1 and ERK2 phosphorylation. However, MEK1/2 inhibitor, CaMKII inhibitor, and PKC inhibitor significantly suppressed lead-induced CREB phosphorylation. These results indicate that lead-activated PKC, CaMKII and MEK/ERK/p90RSK pathways simultaneously, all of which contributed to CREB phosphorylation. Our results also indicate that lead-induced p90RSK and CREB activation does not alter expression of early response genes like c-fos. We conclude that lead activates PKC, CaMKII or MEK-ERK-p90RSK pathways in GT1-7 cells, leading to CREB phosphorylation and modulation of gene expression.     

MMP‐9 controls Schwann cell proliferation and phenotypic remodeling via IGF‐1 and ErbB receptor‐mediated activation of MEK/ERK pathway

Phenotypic remodeling of Schwann cells is required to ensure successful regeneration of damaged peripheral axons. After nerve damage, Schwann cells produce an over 100‐fold increase in metalloproteinase‐9 (MMP‐9), and therapy with an MMP inhibitor increases the number of resident (but not infiltrating) cells in injured nerve. Here, we demonstrate that MMP‐9 regulates proliferation and trophic signaling of Schwann cells. Using in vivo BrdU incorporation studies of axotomized sciatic nerves of MMP‐9?/? mice, we found increased Schwann cell mitosis in regenerating (proximal) stump relative to wild‐type mice. Treatment of cultured primary Schwann cells with recombinant MMP‐9 suppressed their growth, mitogenic activity, and produced a dose‐dependent, biphasic, and selective activation of ERK1/2, but not JNK and p38 MAPK. MMP‐9 induced ERK1/2 signaling in both undifferentiated and differentiated (using dbcAMP) Schwann cells. Using inhibitors to MEK and trophic tyrosine kinase receptors, we established that MMP‐9 regulates Ras/Raf/MEK—ERK pathways through IGF‐1, ErbB, and PDGF receptors. We also report on the early changes of MMP‐9 mRNA expression (within 24 h) after axotomy. These studies establish that MMP‐9 controls critical trophic signal transduction pathways and phenotypic remodeling of Schwann cells. © 2009 Wiley‐Liss, Inc.     

Selective modulation of microglial signal transduction by PACAP

We have investigated the possible effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on signal transduction pathways associated with inflammatory activation of BV-2 mouse microglia cells. Pretreatment of the cells with PACAP resulted in a significant decrease in LPS- or IFNgamma-induced NO production as well as iNOS and IL-1beta mRNA levels. The inhibitory effect of PACAP appeared to be mediated through an increase in intracellular cAMP. PACAP inhibition of LPS-induced NO production was accompanied by inhibition of p38 MAPK activation, but not ERK, JNK, or NF-kappaB. IFNgamma-induced STAT-1 activation or IRF-1 induction was not significantly influenced by PACAP. Therefore, PACAP appears to suppress inflammatory activation of BV-2 microglia via specific inhibition of LPS-induced p38 MAPK pathway.     

Activation of mitogen-activated protein kinases after permanent cerebral artery occlusion in mouse brain.

The purpose of this study was to examine the activation, topographic distribution, and cellular location of three mitogen-activated protein kinases (MAPKs) after permanent middle cerebral artery occlusion (MCAO) in mice. Phosphorylated MAPKs expression in the ischemic region was quantified using Western blot analysis and localized immunohistochemically using the diaminobenzide staining and double-labeled immunostaining. Extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), p38 mitogen-activated protein (p38), and c-Jun NH2-terminal kinase or stress-activated protein kinase (SAPK/JNK) were initially activated at 30 minutes, 10 minutes, and 5 minutes, respectively, after focal cerebral ischemia. Peak expression represented a 2.7-fold, 3.7-fold, and 4.8-fold increase in each of these MAPKs, respectively. The immunohistochemical expressions of ERK1, ERK2, p38, and SAPK/JNK protein paralleled the Western blot analysis results. Double-labeled immunofluorescent staining demonstrated that the neurons and astrocytes expressed ERK1, ERK2, p38, and SAPK/JNK during the early time points after MCAO. The current results demonstrate that brain damage after ischemia rapidly triggers time-dependent ERK1, ERK2, p38, and SAPK/ JNK phosphorylation, and reveals that neurons and astrocytes are involved in the activation of the MAPK pathway. This very early expression of MAPKs suggests that MAPKs may be closely involved in signal transduction during cerebral ischemia.     

The Inhibition of ERK Activation Mediates the Protection of Necrostatin-1 on Glutamate Toxicity in HT-22 Cells

Receptor-interacting protein 1 (RIP1), a molecular switch protein from apoptosis to necroptosis, is regarded to play an essential role in necroptotic cell death. Although the increased RIP1 activity induced by tumor necrosis factor α activates mitogen-activated protein kinases (MAPKs) including ERK and leads to apoptotic or necrotic cell death, it is unclear what is the role of ERK during the process of necroptosis. In this study, our data demonstrated that ERK inhibitors U0126 and PD98059 blocked glutamate-induced necroptosis in HT-22 cells, indicating the critical role of ERK activation in necroptosis. Further, we found glutamate treatment increased phosphorylated ERK1/2 level, but the specific necroptosis inhibitor Necrostatin-1 (Nec-1) significantly inhibited the phosphorylation of ERK1 (P44) at 5, 10, and 15 min after glutamate treatment; the phosphorylation of ERK2 (P42) level was also markedly reduced by Nec-1 at 10 min after glutamate treatment. The phosphorylation of JNK and P38, two other MAPK members, were slightly increased after glutamate treatment, but Nec-1 had no inhibitory effect on JNK and P38 activation. Our finding suggested that ERK activation may play an important role in necroptotic cell death and the inhibition of ERK activation mediated the protection of Nec-1 on glutamate-induced necroptosis. Since ERK is considered as a downstream of RIP1, the RIP1/ERK signal pathway may provide new therapeutic avenues for the treatment of ischemia–reperfusion damage and neurodegenerative diseases-containing necroptotic cell death.