Our previous study showed that when glutamate receptor （GluR）6 C terminus-containing peptide conjugated with the human immunodeficiency virus Tat protein （GluR6）-9c is delivered into hippocampal neurons in a brain...Our previous study showed that when glutamate receptor （GluR）6 C terminus-containing peptide conjugated with the human immunodeficiency virus Tat protein （GluR6）-9c is delivered into hippocampal neurons in a brain ischemic model, the activation of mixed lineage kinase 3 （MLK3） and c-Jun NH2-terminal kinase （JNK） is inhibited via GluR6-postsynaptic density protein 95 （PSD95）. In the present study, we investigated whether the recombinant adenovirus （Ad） carrying GluR6c could suppress the assembly of the GluR6-PSD95-MLK3 signaling module and decrease neuronal cell death induced by kainate in hippocampal CA1 subregion. A seizure model in Sprague-Dawley rats was induced by intraperitoneal injections of kainate. The effect of Ad- Glur6-9c on the phosphorylation of INK, MLK3 and mitogen-activated ldnase kinase 7 （MKK7） was observed with western immunoblots and immunohistochemistry. Our findings revealed that overexpression of GluR6c inhibited the interaction of GluR6 with PSD95 and prevented the kainate-induced activation of INK, MLK3 and MKK7. Furthermore, kainate-mediated neuronal cell death was significantly suppressed by GluR6c. Taken together, GluR6 may play a pivotal role in neuronal cell death.展开更多
AIM： To investigate the possible mechanism by which hepatitis B virus X protein （HBx） mediates apoptosis of HepG2 cells. METHODS： HBx expression vector pcDNA3.1-X was transfected into HepG2 cells to establish an H...AIM： To investigate the possible mechanism by which hepatitis B virus X protein （HBx） mediates apoptosis of HepG2 cells. METHODS： HBx expression vector pcDNA3.1-X was transfected into HepG2 cells to establish an HBx high- expression cellular model as pcDNA3.1-X transfected group. The pcDNA3.1-X and pSilencer3.1-shHBX （HBx antagonist） were cotransfected into HepG2 cells to es- tablish an HBx low-expression model as RNAi group. Untransfected HepG2 cells and HepG2 cells transfected with negative control plasmid were used as controls. Apoptosis rate, the expression of Fas/FasL signaling pathway-related proteins and the phosphorylation lev- els of MLK3, MKK7 and JNKs, which are upstream molecules of death receptor pathways and belong to the family of mitogen-activated protein kinases （MAPKs）,were measured in each group RESULTS： Compared with HepG2 cell group and RNAi group, apoptosis rate, the expression of Fas and FasL proteins, and the activation of MLK3, MKK7 and 3NKs were increased in the pcDNA3.1-X transfected group. The activation of JNKs and expression of FasL protein were inhibited in the pcDNA3.1-X transfected group when treated with a known JNK inhibitor, SP600125. When authors treated pcDNA3.1-X transfected group with K252a, a known MLK3 inhibitor, the activation of MLK3, MKK7 and 3NKs as well as expression of FasL protein was inhibited. Furthermore, cell apoptosis rate was also significantly declined in the presence of K252a in the pcDNA3.1-X transfected group. CONCLUSION： HBx can induce HepG2 cell apoptosis via a novel active MLK3-MKK7-JNKs signaling module to upregulate FasL protein expression.展开更多
Mixed lineage kinase 3 （MLK3） is a mitogen-activated protein kinase kinase kinase that is activated by tumor necrosis factor-α （TNF-α） and specifically activates c-Jun N-terminal kinase （JNK） on TNF-a stimulat...Mixed lineage kinase 3 （MLK3） is a mitogen-activated protein kinase kinase kinase that is activated by tumor necrosis factor-α （TNF-α） and specifically activates c-Jun N-terminal kinase （JNK） on TNF-a stimulation. The mecha- nism by which TNF-α activates MLK3 is still not known. TNF receptor-associated factors （TRAFs） are adapter molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK. Here, we report that MLK3 associates with TRAF2, TRAF5 and TRAF6; however only TRAF2 can significantly induce the kinase activity of MLK3. The interaction domain of TRAF2 maps to the TRAF domain and for MLK3 to its C-terminal half （amino acids 511-847）. Endogenous TRAF2 and MLK3 associate with each other in response to TNF-α treatment in a time-dependent manner. The association between MLK3 and TRAF2 mediates MLK3 activation and competition with the TRAF2 deletion mutant that binds to MLK3 attenuates MLK3 kinase activity in a dose-dependent manner, on TNF-α treatment. Furthermore the downstream target of MLK3, JNK was activated by TNF-α in a TRAF2-dependent manner. Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-α-induced activation of MLK3 and its downstream target, JNK.展开更多
基金supported by the National Natural Science Foundation of China,No.30800309,81372172the Educational Science Foundation of Jiangsu Province,China,No.10KJB350005+2 种基金the Xuzhou Science Foundation in China,No.XZZD1153the President Special Grant of Xuzhou Medical College in China,No.09KJZ20a grant from the Zhenxing Project Foundation of XZMC
文摘Our previous study showed that when glutamate receptor （GluR）6 C terminus-containing peptide conjugated with the human immunodeficiency virus Tat protein （GluR6）-9c is delivered into hippocampal neurons in a brain ischemic model, the activation of mixed lineage kinase 3 （MLK3） and c-Jun NH2-terminal kinase （JNK） is inhibited via GluR6-postsynaptic density protein 95 （PSD95）. In the present study, we investigated whether the recombinant adenovirus （Ad） carrying GluR6c could suppress the assembly of the GluR6-PSD95-MLK3 signaling module and decrease neuronal cell death induced by kainate in hippocampal CA1 subregion. A seizure model in Sprague-Dawley rats was induced by intraperitoneal injections of kainate. The effect of Ad- Glur6-9c on the phosphorylation of INK, MLK3 and mitogen-activated ldnase kinase 7 （MKK7） was observed with western immunoblots and immunohistochemistry. Our findings revealed that overexpression of GluR6c inhibited the interaction of GluR6 with PSD95 and prevented the kainate-induced activation of INK, MLK3 and MKK7. Furthermore, kainate-mediated neuronal cell death was significantly suppressed by GluR6c. Taken together, GluR6 may play a pivotal role in neuronal cell death.
基金Supported by Natural Science Foundation of Jiangsu Province,No.10KJD310002The Graduate Innovation Program in Science and Technology of Xuzhou Medical College,No.XYCX201005
文摘AIM： To investigate the possible mechanism by which hepatitis B virus X protein （HBx） mediates apoptosis of HepG2 cells. METHODS： HBx expression vector pcDNA3.1-X was transfected into HepG2 cells to establish an HBx high- expression cellular model as pcDNA3.1-X transfected group. The pcDNA3.1-X and pSilencer3.1-shHBX （HBx antagonist） were cotransfected into HepG2 cells to es- tablish an HBx low-expression model as RNAi group. Untransfected HepG2 cells and HepG2 cells transfected with negative control plasmid were used as controls. Apoptosis rate, the expression of Fas/FasL signaling pathway-related proteins and the phosphorylation lev- els of MLK3, MKK7 and JNKs, which are upstream molecules of death receptor pathways and belong to the family of mitogen-activated protein kinases （MAPKs）,were measured in each group RESULTS： Compared with HepG2 cell group and RNAi group, apoptosis rate, the expression of Fas and FasL proteins, and the activation of MLK3, MKK7 and 3NKs were increased in the pcDNA3.1-X transfected group. The activation of JNKs and expression of FasL protein were inhibited in the pcDNA3.1-X transfected group when treated with a known JNK inhibitor, SP600125. When authors treated pcDNA3.1-X transfected group with K252a, a known MLK3 inhibitor, the activation of MLK3, MKK7 and 3NKs as well as expression of FasL protein was inhibited. Furthermore, cell apoptosis rate was also significantly declined in the presence of K252a in the pcDNA3.1-X transfected group. CONCLUSION： HBx can induce HepG2 cell apoptosis via a novel active MLK3-MKK7-JNKs signaling module to upregulate FasL protein expression.
文摘Mixed lineage kinase 3 （MLK3） is a mitogen-activated protein kinase kinase kinase that is activated by tumor necrosis factor-α （TNF-α） and specifically activates c-Jun N-terminal kinase （JNK） on TNF-a stimulation. The mecha- nism by which TNF-α activates MLK3 is still not known. TNF receptor-associated factors （TRAFs） are adapter molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK. Here, we report that MLK3 associates with TRAF2, TRAF5 and TRAF6; however only TRAF2 can significantly induce the kinase activity of MLK3. The interaction domain of TRAF2 maps to the TRAF domain and for MLK3 to its C-terminal half （amino acids 511-847）. Endogenous TRAF2 and MLK3 associate with each other in response to TNF-α treatment in a time-dependent manner. The association between MLK3 and TRAF2 mediates MLK3 activation and competition with the TRAF2 deletion mutant that binds to MLK3 attenuates MLK3 kinase activity in a dose-dependent manner, on TNF-α treatment. Furthermore the downstream target of MLK3, JNK was activated by TNF-α in a TRAF2-dependent manner. Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-α-induced activation of MLK3 and its downstream target, JNK.
