AIM: To explore the role of the adaptor molecule in liver regeneration after partial hepatectomy (PH). METHODS: We used transgenic mice expressing an N-terminal truncated form of MORT1/FADD under the control of the al...AIM: To explore the role of the adaptor molecule in liver regeneration after partial hepatectomy (PH). METHODS: We used transgenic mice expressing an N-terminal truncated form of MORT1/FADD under the control of the albumin promoter. As previously shown, this transgenic protein abrogated CD95- and CD120a-mediated apoptosis in the liver. Cyclin A expression was detected using Western blotting. ELISA and RT-PCR were used to detect IL-6 and IL-6 mRNA, respectively. DNA synthesis in liver tissue was measured by BrdU staining. RESULTS: Resection of 70% of the liver was followed by a reduced early regenerative response in the transgenic group at 36 h. Accordingly, 36 h after hepatectomy, cyclin A expression was only detectable in wild-type animals. Consequently, the onset of liver mass restoration was retarded as measured by MRI volumetry and mortality was significantly higher in the transgenic group. CONCLUSION: Our data demonstrate for the first time an involvement of the death receptor molecule MORT1/ FADD in liver regeneration, beyond its well described role as part of the intracellular death signaling pathway.展开更多
RAC1 is a small-molecule G protein that regulates multiple cell cycle, cytoskeletal reorganization, cell migration, and apoptosis. FADD-dependent TRAIL can promote tumor metastasis through RAC1 and PI3K, and down-regu...RAC1 is a small-molecule G protein that regulates multiple cell cycle, cytoskeletal reorganization, cell migration, and apoptosis. FADD-dependent TRAIL can promote tumor metastasis through RAC1 and PI3K, and down-regulating RAC1 expression can reduce FasL-induced apoptosis. In addition, RIP1 bound to GTP acts as an activating protein for RAC1 and is involved in cytoskeletal reorganization. TRAF6 promotes migration and metastasis by regulating the RAS pathway in tumors. Thus, it is necessary to understand the interaction between RAC1 and TRAF6 as well as FADD and RIP1. In this study, we cultured hepatoma SK-Hep1 cells in vitro, specifically blocked the necroptosis pathway with Nec-1, and silenced FADD, RIP1 and TRAF6 gene expression using RNAi technology. At the same time, the expression of RAC1 was evaluated separately using RT-PCR and Western blot. The hepatoma SK-Hep1 cells survival rate was highest when the concentration of Nec-1 was 60 μM and the concentration of Z-vad-fmk was 20 μM. And the apoptosis rate of the transfected RAC1 siRNA cells was 3.59% compared with transfected siRNA cells 10.01% which was significantly decreased (P < 0.01). RAC1 could promote the occurrence of apoptosis in SK-Hep1 cells. RAC1 expression was suppressed in both protein and gene level in SK-Hep1 cells when the TRAF6 gene was silenced, but there was no significant change in RAC1 gene and protein expression when FADD and RIP1 genes were silenced. TRAF6 affects RAC1 expression and apoptosis in SK-Hep1 cells, while the FADD and RIP1 genes do not affect the role of RAC1. The TRAF6 gene is an important target in liver cancer cells.展开更多
Interleukin I receptor associated kinase 1 (IRAK1) is a downstream signal molecule of activated MyD88 recruitment, which can activate Fas associated death domain protein (FADD) to induce apoptosis. IRAK1 can also acti...Interleukin I receptor associated kinase 1 (IRAK1) is a downstream signal molecule of activated MyD88 recruitment, which can activate Fas associated death domain protein (FADD) to induce apoptosis. IRAK1 can also activate tumor necrosis factor-related factor 6 (TRAF6) and induce the expression of a series of downstream specific genes. IRAK1 is an essential factor in the induction of mitochondrial division and necroptosis. In the current study, RNAi technique was used to silence IRAK1, and the apoptosis and necroptosis rate of SK-Hep1 cells were detected by flow cytometry. The apoptosis and the necroptosis pathway of hepatoma SK-Hep1 cells were blocked separately, and the expressions of FADD, RIP1 and TRAF6 genes were silenced separately. The results showed when the expression of IRAK1 was down-regulated, the apoptosis and necroptosis rate of SK-Hep1 cells were significantly increased. With silenced FADD, RIP1 and TRAF6, respectively, the expression of IRAK1 protein had no significant change. However, the expression of IRAK1 mRNA decreased significantly (p < 0.01) after the silencing of RIP1 and TRAF6 genes, while the IRAK1 mRNA did not change significantly after the silencing of FADD genes;when z-VAD-FMK was interfered, the expression of IRAK1 mRNA decreased significantly after the silencing of TRAF6 genes, while the IRAK1 mRNA did not change significantly after the silencing of FADD and RIP1genes. The study shows that RAK1 gene inhibits apoptosis and necroptosis in SK-Hep1 cells. TRAF6 gene affected the role of IRAK1 in apoptosis and necroptosis, RIP1 gene affected the role of IRAK1 in apoptosis, while FADD gene did not affect the role of IRAK1 in apoptosis and necroptosis.展开更多
FADD(Fas-associated death domain protein)存在于Fas/FasL系统中,是信号传导通路的一个信号连接蛋白,FADD通过传递凋亡信号,从而介导细胞凋亡。为进一步验证FADD基因抑制细胞增殖和促进细胞凋亡的作用,从牛卵巢组织中扩增FADD基因,将F...FADD(Fas-associated death domain protein)存在于Fas/FasL系统中,是信号传导通路的一个信号连接蛋白,FADD通过传递凋亡信号,从而介导细胞凋亡。为进一步验证FADD基因抑制细胞增殖和促进细胞凋亡的作用,从牛卵巢组织中扩增FADD基因,将FADD基因连接到带有绿色荧光蛋白报告基因的真核表达载体pAcGFP-N1中,构建过表达FADD基因载体,并构建FADD基因的RNAi载体;用脂质体介导法将FADD基因RNAi载体、真核表达载体转染到牛胎儿成纤维细胞中,观察有无荧光的表达,并使用Real-Time qPCR和Western blot方法检测FADD基因mRNA、蛋白水平的表达情况。结果表明:成功构建出FADD基因RNAi载体和高表达载体,重组质粒转染牛胎儿成纤维细胞24 h后在荧光显微镜下可观察到绿色荧光,转染效率可达50%。展开更多
非酒精性脂肪性肝炎(nonalcoholic steatohepatitis,NASH)逐渐成为发病率最高的慢性肝病类型。NASH发病常常伴随全身代谢综合征,疾病进展具有发生肝硬化甚至肝癌的高风险。然而,目前临床上尚无一种获批的针对NASH的有效治疗药物。我们...非酒精性脂肪性肝炎(nonalcoholic steatohepatitis,NASH)逐渐成为发病率最高的慢性肝病类型。NASH发病常常伴随全身代谢综合征,疾病进展具有发生肝硬化甚至肝癌的高风险。然而,目前临床上尚无一种获批的针对NASH的有效治疗药物。我们的最新研究结果发现,天然免疫重要分子CFLAR[CASP8 and FADD(Fas-associating protein with death domain)-like apoptosis regulator]直在NASH疾病进程中的关键负调控作用。深入的分子机制探索证实,CFLAR接靶向激酶MAP3K5[mitogen-activated protein kinase kinase kinase 5,也称为ASK1(apoptosis signal-regulating kinase 1)]并阻断其N-端二聚化,从而抑制ASK1和激酶MAPK8[mitogen-activated protein kinase 8,也称为JNK1(c-Jun N-terminal kinase 1)]的信号通路。此外,我们鉴定出源于CFLAR的一个小肽片段(S1)可以有效发挥CFLAR对ASK1的抑制作用。应用CFLAR(S1)治疗可有效改善并逆转小鼠和猴子中的NASH及并发的代谢综合征。综上所述,我们发现,CFLAR是控制NASH疾病进展的关键抑制子。CFLAR(S1)特异性抑制ASK 1激活的作用机制,为开发或筛选NASH的靶向治疗药物提供了可行的新方案。展开更多
基金Supported by the Intramural grant (MAIFOR) to M.S.
文摘AIM: To explore the role of the adaptor molecule in liver regeneration after partial hepatectomy (PH). METHODS: We used transgenic mice expressing an N-terminal truncated form of MORT1/FADD under the control of the albumin promoter. As previously shown, this transgenic protein abrogated CD95- and CD120a-mediated apoptosis in the liver. Cyclin A expression was detected using Western blotting. ELISA and RT-PCR were used to detect IL-6 and IL-6 mRNA, respectively. DNA synthesis in liver tissue was measured by BrdU staining. RESULTS: Resection of 70% of the liver was followed by a reduced early regenerative response in the transgenic group at 36 h. Accordingly, 36 h after hepatectomy, cyclin A expression was only detectable in wild-type animals. Consequently, the onset of liver mass restoration was retarded as measured by MRI volumetry and mortality was significantly higher in the transgenic group. CONCLUSION: Our data demonstrate for the first time an involvement of the death receptor molecule MORT1/ FADD in liver regeneration, beyond its well described role as part of the intracellular death signaling pathway.
文摘RAC1 is a small-molecule G protein that regulates multiple cell cycle, cytoskeletal reorganization, cell migration, and apoptosis. FADD-dependent TRAIL can promote tumor metastasis through RAC1 and PI3K, and down-regulating RAC1 expression can reduce FasL-induced apoptosis. In addition, RIP1 bound to GTP acts as an activating protein for RAC1 and is involved in cytoskeletal reorganization. TRAF6 promotes migration and metastasis by regulating the RAS pathway in tumors. Thus, it is necessary to understand the interaction between RAC1 and TRAF6 as well as FADD and RIP1. In this study, we cultured hepatoma SK-Hep1 cells in vitro, specifically blocked the necroptosis pathway with Nec-1, and silenced FADD, RIP1 and TRAF6 gene expression using RNAi technology. At the same time, the expression of RAC1 was evaluated separately using RT-PCR and Western blot. The hepatoma SK-Hep1 cells survival rate was highest when the concentration of Nec-1 was 60 μM and the concentration of Z-vad-fmk was 20 μM. And the apoptosis rate of the transfected RAC1 siRNA cells was 3.59% compared with transfected siRNA cells 10.01% which was significantly decreased (P < 0.01). RAC1 could promote the occurrence of apoptosis in SK-Hep1 cells. RAC1 expression was suppressed in both protein and gene level in SK-Hep1 cells when the TRAF6 gene was silenced, but there was no significant change in RAC1 gene and protein expression when FADD and RIP1 genes were silenced. TRAF6 affects RAC1 expression and apoptosis in SK-Hep1 cells, while the FADD and RIP1 genes do not affect the role of RAC1. The TRAF6 gene is an important target in liver cancer cells.
文摘Interleukin I receptor associated kinase 1 (IRAK1) is a downstream signal molecule of activated MyD88 recruitment, which can activate Fas associated death domain protein (FADD) to induce apoptosis. IRAK1 can also activate tumor necrosis factor-related factor 6 (TRAF6) and induce the expression of a series of downstream specific genes. IRAK1 is an essential factor in the induction of mitochondrial division and necroptosis. In the current study, RNAi technique was used to silence IRAK1, and the apoptosis and necroptosis rate of SK-Hep1 cells were detected by flow cytometry. The apoptosis and the necroptosis pathway of hepatoma SK-Hep1 cells were blocked separately, and the expressions of FADD, RIP1 and TRAF6 genes were silenced separately. The results showed when the expression of IRAK1 was down-regulated, the apoptosis and necroptosis rate of SK-Hep1 cells were significantly increased. With silenced FADD, RIP1 and TRAF6, respectively, the expression of IRAK1 protein had no significant change. However, the expression of IRAK1 mRNA decreased significantly (p < 0.01) after the silencing of RIP1 and TRAF6 genes, while the IRAK1 mRNA did not change significantly after the silencing of FADD genes;when z-VAD-FMK was interfered, the expression of IRAK1 mRNA decreased significantly after the silencing of TRAF6 genes, while the IRAK1 mRNA did not change significantly after the silencing of FADD and RIP1genes. The study shows that RAK1 gene inhibits apoptosis and necroptosis in SK-Hep1 cells. TRAF6 gene affected the role of IRAK1 in apoptosis and necroptosis, RIP1 gene affected the role of IRAK1 in apoptosis, while FADD gene did not affect the role of IRAK1 in apoptosis and necroptosis.
文摘非酒精性脂肪性肝炎(nonalcoholic steatohepatitis,NASH)逐渐成为发病率最高的慢性肝病类型。NASH发病常常伴随全身代谢综合征,疾病进展具有发生肝硬化甚至肝癌的高风险。然而,目前临床上尚无一种获批的针对NASH的有效治疗药物。我们的最新研究结果发现,天然免疫重要分子CFLAR[CASP8 and FADD(Fas-associating protein with death domain)-like apoptosis regulator]直在NASH疾病进程中的关键负调控作用。深入的分子机制探索证实,CFLAR接靶向激酶MAP3K5[mitogen-activated protein kinase kinase kinase 5,也称为ASK1(apoptosis signal-regulating kinase 1)]并阻断其N-端二聚化,从而抑制ASK1和激酶MAPK8[mitogen-activated protein kinase 8,也称为JNK1(c-Jun N-terminal kinase 1)]的信号通路。此外,我们鉴定出源于CFLAR的一个小肽片段(S1)可以有效发挥CFLAR对ASK1的抑制作用。应用CFLAR(S1)治疗可有效改善并逆转小鼠和猴子中的NASH及并发的代谢综合征。综上所述,我们发现,CFLAR是控制NASH疾病进展的关键抑制子。CFLAR(S1)特异性抑制ASK 1激活的作用机制,为开发或筛选NASH的靶向治疗药物提供了可行的新方案。
