c-Jun NH2-terminal kinase（JNK）-interacting protein 3 plays an important role in brain-derived neurotrophic factor/tropomyosin-related kinase B（Trk B） anterograde axonal transport. It remains unclear whether JNK-in...c-Jun NH2-terminal kinase（JNK）-interacting protein 3 plays an important role in brain-derived neurotrophic factor/tropomyosin-related kinase B（Trk B） anterograde axonal transport. It remains unclear whether JNK-interacting protein 1 mediates similar effects, or whether JNK-interacting protein 1 affects the regulation of Trk B anterograde axonal transport. In this study, we isolated rat embryonic hippocampus and cultured hippocampal neurons in vitro. Coimmunoprecipitation results demonstrated that JNK-interacting protein 1 formed Trk B complexes in vitro and in vivo. Immunocytochemistry results showed that when JNK-interacting protein 1 was highly expressed, the distribution of Trk B gradually increased in axon terminals. However, the distribution of Trk B reduced in axon terminals after knocking out JNK-interacting protein 1. In addition, there were differences in distribution of Trk B after JNK-interacting protein 1 was knocked out compared with not. However, knockout of JNK-interacting protein 1 did not affect the distribution of Trk B in dendrites. These findings confirm that JNK-interacting protein 1 can interact with Trk B in neuronal cells, and can regulate the transport of Trk B in axons, but not in dendrites.展开更多
Background Activation of c-Jun NH2-terminal kinase （JNK） has been implicated in neuron apoptosis as well as autophagy in response to various stressors after traumatic brain injury （TBI）. However, the underlying mo...Background Activation of c-Jun NH2-terminal kinase （JNK） has been implicated in neuron apoptosis as well as autophagy in response to various stressors after traumatic brain injury （TBI）. However, the underlying molecular pathway remains unclear. Our study assessed whether JNK-mediated p53 phosphorylation might be an important mechanism for enhancing neuron autophagy in response to TBI. Methods A total of 186 male Sprague-Dawley （SD） rats （300-350 g） were used in this study. By randomized block method rats were randomly divided into four groups： sham-operated （n=46）, TBI （n=60）, TBI ＋ dimethyl sulfoxide （DMSO） （n=40）, and TBI ＋ SP600125 （n=40）. JNK was treated with SP600125, a specific JNK inhibitor. JNK, p-P53, Beclin-1, damage-regulated autophagy modulator （DRAM） and p-bcl-2 were evaluated by Western blotting analysis. The cellular localization and expression of Beclin-1 and DRAM was observed by immunofluorescence and immunohistochemistry, and the expression of Beclin-l-Bcl-2/Bcl-xL complexes was evaluated by immunoprecipitation. Multiple-group comparisons were conducted using analysis of variance （ANOVA）. P values of less than 0.05 were considered statistically significant. Results It was observed that the expression of JNK, p-P53, Beclin-1, DRAM and p-bcl-2 was increasing after TBI, and the expression of Beclin-1 and DRAM was mainly located in the cytoplasm of neurons. But these were significantly inhibited in SP600125 group compared with sham group and TBI＋SP600125 group （P 〈0.05）. The expression of Beclin-l-Bcl-2/Bcl-xL complexes was reduced after TBI. Conclusion JNK-mediated p53 phosphorylation might be an important mechanism for enhancing neuron autophagy in response to TBI.展开更多
Background： Najanalgesin, a toxin isolated from the venom ofNaja nqja atra, has been shown to exert significant analgesic effects in a neuropathic pain model in rats. However, the molecular mechanism underlying this ...Background： Najanalgesin, a toxin isolated from the venom ofNaja nqja atra, has been shown to exert significant analgesic effects in a neuropathic pain model in rats. However, the molecular mechanism underlying this protective effect ofnajanalgesin is poorly understood. The present study sought to evaluate the intracellular signaling pathways that are involved in the antinociceptive effect of najanalgesin on neuropathic pain. Methods： The antinociceptive properties of najanalgesin were tested in hind paw withdrawal thresholds in response to mechanical stimulation. We analyzed the participation of the mitogen-activated protein kinase p38, extracellular-regulated kinase （ERK）, and c-Jun N-terminal kinase （JNK） by western blot analysis. This inhibition of JNK was confirmed by immunohistochemistry. Results： The phosphorylation levels of INK （as well as its downstream molecule c-Jun）, p38, and ERK were significantly increased after injury. Najanalgesin only inhibited JNK and c-Jun phosphorylation but had no effect on either ERK or p38. This inhibition of JNK was confirmed by immunohistochemistry, which suggested that the antinociceptive effect of najanalgesin on spinal nerve ligation-induced neuropathic pain in rats is associated with JNK activation in the spinal cord. Conclusion： The antinociceptive effect of najanalgesin thnctions by inhibiting the JNK in a neuropathic pain model.展开更多
BACKGROUND:Multidrug resistance(MDR)is extremely common in hepatocellular carcinoma(HCC)and is a major problem in cancer eradication by limiting the efficacy of chemotherapy.Modulation of c-Jun NH2-terminal kinase(JNK...BACKGROUND:Multidrug resistance(MDR)is extremely common in hepatocellular carcinoma(HCC)and is a major problem in cancer eradication by limiting the efficacy of chemotherapy.Modulation of c-Jun NH2-terminal kinase(JNK)activation could be a new method to reverse MDR.However,the relationship between JNK activity and MDR in HCC cells is unknown.This study aimed to explore the relationship between MDR and JNK in HCC cell lines with different degrees of MDR.METHODS:A MDR human HCC cell line,SMMC-7721/ ADM,was developed by exposing parental cells to gradually increasing concentrations of adriamycin.The MTT assay was used to determine drug sensitivity.Flow cytometry was used to analyze the cell cycle distribution and to measure the expression levels of P-glycoprotein(P-gp)and MDR-related protein(MRP)-1 in these cells.JNK1,JNK2 and JNK3 mRNA expression levels were quantified by real-time PCR.Expression and phosphorylation of JNK1,JNK2,and JNK3 were analyzed by Western blotting.RESULTS:The MDR of SMMC-7721/ADM cells resistant to 0.05 mg/L adriamycin was mainly attributed to the overexpression of P-gp but not MRP1.In addition,these cells had a significant increase in percentage in the S phase,accompanied by a decrease in percentage in the G0/G1 phase,which is likely associated with a reduced ability for cell proliferation and MDR generation.We found that JNK1,JNK2,and JNK3 activities were negatively correlated with the degree of MDR in HCC cells.CONCLUSION:This study suggests that JNK1,JNK2,and JNK3 activities are negatively correlated with the degree of MDR in HCC cells.展开更多
Aim:Paxillin is a well-known multidomain scaffold protein that is involved in the regulation of cell-matrix adhesion dynamics,a process required for the tumor cell migration and invasion.Phosphorylation of the serine ...Aim:Paxillin is a well-known multidomain scaffold protein that is involved in the regulation of cell-matrix adhesion dynamics,a process required for the tumor cell migration and invasion.Phosphorylation of the serine residue 178 requires c-Jun NH2-terminal kinase(JNK)activation,which occurs downstream of epidermal growth factor receptor(EGFR)-mediated signaling and drives cell migration.In this study,we investigated the significance of paxillin Ser178 phosphorylation in breast cancer progression.Methods:We employed the rat mammary carcinoma MTLn3 cell line with which we established stabile variants of both wild type and mutant GFP-paxillin constructs.With those,we next performed several in vitro assays including cell proliferation,migration and focal adhesion dynamics.Finally,we monitored the metastatic spread of both cell line variants in an othrotopic mouse model for breast cancer.Results:Here we show that expression of the phospho-defective mutant paxillinS178A in the metastatic mammary adenocarcinoma MTLn3 cell-line significantly decreased EGF-induced cell migration,which was correlated with impaired focal adhesion dynamics.Moreover,paxillinS178A attenuated lung metastasis formation in an orthotopic in vivo mammary gland tumor/metastasis model,demonstrating the importance of JNK-mediated paxillin phosphorylation in breast cancer progression.Expression of paxillinS178A caused a decrease in EGFR expression, ;while re-expression of EGFR in MTLn3-paxillinS178A cells fully restored EGF-driven cell motility and focal adhesion dynamics.Furthermore,re-expression of EGFR in MTLn3-paxillinS178A rescued spontaneous metastasis from breast to lung.Conclusion:Overall our data show an important role for JNK-mediated paxillin Ser178 phosphorylation in the regulation of EGFR expression and thereby,in EGF-driven cell migration and metastasis formation.展开更多
基金supported by the Henan Province Education Department Key Project of Science and Technology Research in China,No.12A350006
文摘c-Jun NH2-terminal kinase（JNK）-interacting protein 3 plays an important role in brain-derived neurotrophic factor/tropomyosin-related kinase B（Trk B） anterograde axonal transport. It remains unclear whether JNK-interacting protein 1 mediates similar effects, or whether JNK-interacting protein 1 affects the regulation of Trk B anterograde axonal transport. In this study, we isolated rat embryonic hippocampus and cultured hippocampal neurons in vitro. Coimmunoprecipitation results demonstrated that JNK-interacting protein 1 formed Trk B complexes in vitro and in vivo. Immunocytochemistry results showed that when JNK-interacting protein 1 was highly expressed, the distribution of Trk B gradually increased in axon terminals. However, the distribution of Trk B reduced in axon terminals after knocking out JNK-interacting protein 1. In addition, there were differences in distribution of Trk B after JNK-interacting protein 1 was knocked out compared with not. However, knockout of JNK-interacting protein 1 did not affect the distribution of Trk B in dendrites. These findings confirm that JNK-interacting protein 1 can interact with Trk B in neuronal cells, and can regulate the transport of Trk B in axons, but not in dendrites.
基金This work was supported by a grant from Hebei Province Natural Science Foundation （No. C2009001247）. The authors declare that there are no conflicts of interest.Acknowledgments： We thank Dr. CUI Jian-zhong and Dr. GAO Jun-ling for their help and generous gift of antibodies for Western blotting.
文摘Background Activation of c-Jun NH2-terminal kinase （JNK） has been implicated in neuron apoptosis as well as autophagy in response to various stressors after traumatic brain injury （TBI）. However, the underlying molecular pathway remains unclear. Our study assessed whether JNK-mediated p53 phosphorylation might be an important mechanism for enhancing neuron autophagy in response to TBI. Methods A total of 186 male Sprague-Dawley （SD） rats （300-350 g） were used in this study. By randomized block method rats were randomly divided into four groups： sham-operated （n=46）, TBI （n=60）, TBI ＋ dimethyl sulfoxide （DMSO） （n=40）, and TBI ＋ SP600125 （n=40）. JNK was treated with SP600125, a specific JNK inhibitor. JNK, p-P53, Beclin-1, damage-regulated autophagy modulator （DRAM） and p-bcl-2 were evaluated by Western blotting analysis. The cellular localization and expression of Beclin-1 and DRAM was observed by immunofluorescence and immunohistochemistry, and the expression of Beclin-l-Bcl-2/Bcl-xL complexes was evaluated by immunoprecipitation. Multiple-group comparisons were conducted using analysis of variance （ANOVA）. P values of less than 0.05 were considered statistically significant. Results It was observed that the expression of JNK, p-P53, Beclin-1, DRAM and p-bcl-2 was increasing after TBI, and the expression of Beclin-1 and DRAM was mainly located in the cytoplasm of neurons. But these were significantly inhibited in SP600125 group compared with sham group and TBI＋SP600125 group （P 〈0.05）. The expression of Beclin-l-Bcl-2/Bcl-xL complexes was reduced after TBI. Conclusion JNK-mediated p53 phosphorylation might be an important mechanism for enhancing neuron autophagy in response to TBI.
基金This study was supported by the grants from the National Natural Science Foundation of China，the Shandong Province National Natural Science Foundation，the project from Weifang Science and Technology Bureau，the Weifang Medical Technology Innovation Project
文摘Background： Najanalgesin, a toxin isolated from the venom ofNaja nqja atra, has been shown to exert significant analgesic effects in a neuropathic pain model in rats. However, the molecular mechanism underlying this protective effect ofnajanalgesin is poorly understood. The present study sought to evaluate the intracellular signaling pathways that are involved in the antinociceptive effect of najanalgesin on neuropathic pain. Methods： The antinociceptive properties of najanalgesin were tested in hind paw withdrawal thresholds in response to mechanical stimulation. We analyzed the participation of the mitogen-activated protein kinase p38, extracellular-regulated kinase （ERK）, and c-Jun N-terminal kinase （JNK） by western blot analysis. This inhibition of JNK was confirmed by immunohistochemistry. Results： The phosphorylation levels of INK （as well as its downstream molecule c-Jun）, p38, and ERK were significantly increased after injury. Najanalgesin only inhibited JNK and c-Jun phosphorylation but had no effect on either ERK or p38. This inhibition of JNK was confirmed by immunohistochemistry, which suggested that the antinociceptive effect of najanalgesin on spinal nerve ligation-induced neuropathic pain in rats is associated with JNK activation in the spinal cord. Conclusion： The antinociceptive effect of najanalgesin thnctions by inhibiting the JNK in a neuropathic pain model.
基金supported by grants from the Medical Innovation Fundation of Fujian Province(No.2007-CXB-7)the Natural Science Foundation of Fujian Province(No.2009D010)
文摘BACKGROUND:Multidrug resistance(MDR)is extremely common in hepatocellular carcinoma(HCC)and is a major problem in cancer eradication by limiting the efficacy of chemotherapy.Modulation of c-Jun NH2-terminal kinase(JNK)activation could be a new method to reverse MDR.However,the relationship between JNK activity and MDR in HCC cells is unknown.This study aimed to explore the relationship between MDR and JNK in HCC cell lines with different degrees of MDR.METHODS:A MDR human HCC cell line,SMMC-7721/ ADM,was developed by exposing parental cells to gradually increasing concentrations of adriamycin.The MTT assay was used to determine drug sensitivity.Flow cytometry was used to analyze the cell cycle distribution and to measure the expression levels of P-glycoprotein(P-gp)and MDR-related protein(MRP)-1 in these cells.JNK1,JNK2 and JNK3 mRNA expression levels were quantified by real-time PCR.Expression and phosphorylation of JNK1,JNK2,and JNK3 were analyzed by Western blotting.RESULTS:The MDR of SMMC-7721/ADM cells resistant to 0.05 mg/L adriamycin was mainly attributed to the overexpression of P-gp but not MRP1.In addition,these cells had a significant increase in percentage in the S phase,accompanied by a decrease in percentage in the G0/G1 phase,which is likely associated with a reduced ability for cell proliferation and MDR generation.We found that JNK1,JNK2,and JNK3 activities were negatively correlated with the degree of MDR in HCC cells.CONCLUSION:This study suggests that JNK1,JNK2,and JNK3 activities are negatively correlated with the degree of MDR in HCC cells.
基金the EU FP7 Metafight project(HEALTH-F2-2007-201862)Dutch Cancer Society(KWF-UL2007-3860)NWO grant(911-02-022).
文摘Aim:Paxillin is a well-known multidomain scaffold protein that is involved in the regulation of cell-matrix adhesion dynamics,a process required for the tumor cell migration and invasion.Phosphorylation of the serine residue 178 requires c-Jun NH2-terminal kinase(JNK)activation,which occurs downstream of epidermal growth factor receptor(EGFR)-mediated signaling and drives cell migration.In this study,we investigated the significance of paxillin Ser178 phosphorylation in breast cancer progression.Methods:We employed the rat mammary carcinoma MTLn3 cell line with which we established stabile variants of both wild type and mutant GFP-paxillin constructs.With those,we next performed several in vitro assays including cell proliferation,migration and focal adhesion dynamics.Finally,we monitored the metastatic spread of both cell line variants in an othrotopic mouse model for breast cancer.Results:Here we show that expression of the phospho-defective mutant paxillinS178A in the metastatic mammary adenocarcinoma MTLn3 cell-line significantly decreased EGF-induced cell migration,which was correlated with impaired focal adhesion dynamics.Moreover,paxillinS178A attenuated lung metastasis formation in an orthotopic in vivo mammary gland tumor/metastasis model,demonstrating the importance of JNK-mediated paxillin phosphorylation in breast cancer progression.Expression of paxillinS178A caused a decrease in EGFR expression, ;while re-expression of EGFR in MTLn3-paxillinS178A cells fully restored EGF-driven cell motility and focal adhesion dynamics.Furthermore,re-expression of EGFR in MTLn3-paxillinS178A rescued spontaneous metastasis from breast to lung.Conclusion:Overall our data show an important role for JNK-mediated paxillin Ser178 phosphorylation in the regulation of EGFR expression and thereby,in EGF-driven cell migration and metastasis formation.
