目的观察不同浓度高糖刺激后大鼠肾小球系膜细胞胞内Smad泛素化调节因子-Smurf2的表达,并以蛋白酶体抑制剂MG132作为阻断剂,探讨泛素化降解在糖尿病肾病中的作用。方法将体外培养的大鼠肾系膜细胞分别设正常对照组(葡萄糖浓度5.6mmol/L)...目的观察不同浓度高糖刺激后大鼠肾小球系膜细胞胞内Smad泛素化调节因子-Smurf2的表达,并以蛋白酶体抑制剂MG132作为阻断剂,探讨泛素化降解在糖尿病肾病中的作用。方法将体外培养的大鼠肾系膜细胞分别设正常对照组(葡萄糖浓度5.6mmol/L)、20mmol/L高糖组、30mmol/L高糖组、30mmol/L高糖加MG132组等。分别用Real time Quantitative PCR法和细胞免疫荧光染色法及激光共聚焦显微镜检测各组细胞泛素连接酶Smurf2的mRNA和蛋白表达。结果①正常组细胞Smurf2的mRNA和蛋白表达较弱,高糖组Smurf2表达较正常组增强(P<0.05),呈浓度依赖性;②30mmol/L高糖组加入MG132后,Smurf2的mRNA和蛋白表达减弱。结论高糖可诱导肾系膜细胞Smurf2表达增强,MG132可阻止高糖所导致的上述变化。提示泛素-蛋白酶体途径参与了糖尿病肾病的发病。展开更多
SMURF2(smad ubiquitination regulatory factor 2)属于HECT(homologous to E6AP C terminus)家族E3泛素连接酶,有研究报道SMURF2在不同类型癌症中发挥促癌或抑癌作用.本研究探讨了SMURF2在前列腺(癌)细胞中调节STAT1(signal transducer...SMURF2(smad ubiquitination regulatory factor 2)属于HECT(homologous to E6AP C terminus)家族E3泛素连接酶,有研究报道SMURF2在不同类型癌症中发挥促癌或抑癌作用.本研究探讨了SMURF2在前列腺(癌)细胞中调节STAT1(signal transducers and activators of transcription 1)蛋白的分子机制.首先通过数据库分析SMURF2在正常组织与肿瘤组织间的表达差异,然后选取前列腺正常细胞和多种前列腺癌细胞为实验材料,通过RT-PCR,Western blotting实验检测SMURF2在前列腺(癌)细胞中的表达水平,发现相对于前列腺正常细胞,SMURF2在前列腺癌细胞中表达更高.再通过Co-IP,免疫荧光和泛素化检测实验观察SMURF2对STAT1蛋白泛素化水平的影响,发现SMURF2可以增加STAT1蛋白的泛素化水平,并进一步促进前列腺癌细胞发生EMT(epithelial mesenchymal transformation).展开更多
Transforming growth factor-β(TGF-β)and bone morphogenetic protein(BMP)play important roles in bone metabolism.Smad ubiquitination regulatory factors(Smurfs)regulate TGF-β/BMP signaling via ubiquitination,resulting ...Transforming growth factor-β(TGF-β)and bone morphogenetic protein(BMP)play important roles in bone metabolism.Smad ubiquitination regulatory factors(Smurfs)regulate TGF-β/BMP signaling via ubiquitination,resulting in degradation of signaling molecules to prevent excessive activation of TGF-β/BMP signaling.Though Smurf2 has been shown to negatively regulate TGF-β/Smad signaling,its involvement in BMP/Smad signaling in bone metabolism has not been thoroughly investigated.In the present study,we sought to evaluate the role of Smurf2 in BMP/Smad signaling in bone metabolism.Absorbable collagen sponges containing 3μg of recombinant human BMP2(rhBMP2)were implanted in the dorsal muscle pouches of wild type(WT)and Smurf2−/−mice.The rhBMP2-induced ectopic bone in Smurf2−/−mice showed greater bone mass,higher mineral apposition and bone formation rates,and greater osteoblast numbers than the ectopic bone in WT mice.In WT mice,the ectopic bone consisted of a thin discontinuous outer cortical shell and scant inner trabecular bone.In contrast,in Smurf2−/−mice,the induced bone consisted of a thick,continuous outer cortical shell and abundant inner trabecular bone.Additionally,rhBMP2-stimulated bone marrow stromal cells(BMSCs)from Smurf2−/−mice showed increased osteogenic differentiation.Smurf2 induced the ubiquitination of Smad1/5.BMP/Smad signaling was enhanced in Smurf2−/−BMSCs stimulated with rhBMP2,and the inhibition of BMP/Smad signaling suppressed osteogenic differentiation of these BMSCs.These findings demonstrate that Smurf2 negatively regulates BMP/Smad signaling,thereby identifying a new regulatory mechanism in bone metabolism.展开更多
Glioma is the tumor with the highest incidence in the brain,and it is eager to seek new and efiective treatment.The interaction of ubiquitination and deubiquitination regulates many cell activities in organisms,and pa...Glioma is the tumor with the highest incidence in the brain,and it is eager to seek new and efiective treatment.The interaction of ubiquitination and deubiquitination regulates many cell activities in organisms,and participates in tumor occurrence,development,migration,invasion and other processes.This article summarized the progress of E3 ubiquitination ligase smad ubiquitination regulatory factor 2(Smurf2)and glioma-related signaling pathways to assist clinical diagnosis and treatment of glioma.展开更多
目的:探讨miR-20a-3p基因干扰对人牙髓干细胞(hDPSCs)成牙本质分化的影响及信号通路调控机制。方法:对hDPSCs转染miR-20a-3p-mimic和miR-20a-3p-inhibitor来上调或下调其表达,然后对hDPSCs进行成牙本质分化诱导培养。通过茜素红染色观...目的:探讨miR-20a-3p基因干扰对人牙髓干细胞(hDPSCs)成牙本质分化的影响及信号通路调控机制。方法:对hDPSCs转染miR-20a-3p-mimic和miR-20a-3p-inhibitor来上调或下调其表达,然后对hDPSCs进行成牙本质分化诱导培养。通过茜素红染色观察细胞钙基质的矿化程度并检测ALP活性。通过双荧光素酶报告基因测定实验验证miR-20a-3p与mothers against decapentaplegic homolog(SMAD)特异性E3泛素蛋白连接酶1(SMURF1)的靶向调节关系。通过RT-qPCR检测hDPSCs中miR-20a-3p以及SMURF1、Runt相关转录因子2(RUNX2)、骨钙蛋白(OCN)和牙本质涎磷蛋白(DSPP)mRNA水平。结果:与Control组相比,miR-20a-3p-mimic组的茜素红相对染色强度升高了67.09%,miR-20a-3p-inhibitor组降低了46.13%(P<0.05)。与Control组相比,miR-20a-3p-mimic组的相对ALP活性升高了52.89%,miR-20a-3p-inhibitor组降低了53.32%(P<0.05)。与Control组相比,miR-20a-3p-mimic组的RUNX2、OCN和DSPP mRNA相对表达量分别升高了2.19倍、1.86倍和2.35倍,miR-20a-3p-inhibitor组分别降低了63.26%、58.84%和68.12%(P<0.05)。双荧光素酶报告基因测定显示miR-20a-3p靶向抑制SMURF1(P<0.05)。过表达SMURF1抑制了miR-20a-3p对成牙本质分化的影响(P<0.05)。结论:miR-20a-3p通过靶向抑制SMURF1及其下游基因促进hDPSCs的成牙本质分化。展开更多
文摘目的观察不同浓度高糖刺激后大鼠肾小球系膜细胞胞内Smad泛素化调节因子-Smurf2的表达,并以蛋白酶体抑制剂MG132作为阻断剂,探讨泛素化降解在糖尿病肾病中的作用。方法将体外培养的大鼠肾系膜细胞分别设正常对照组(葡萄糖浓度5.6mmol/L)、20mmol/L高糖组、30mmol/L高糖组、30mmol/L高糖加MG132组等。分别用Real time Quantitative PCR法和细胞免疫荧光染色法及激光共聚焦显微镜检测各组细胞泛素连接酶Smurf2的mRNA和蛋白表达。结果①正常组细胞Smurf2的mRNA和蛋白表达较弱,高糖组Smurf2表达较正常组增强(P<0.05),呈浓度依赖性;②30mmol/L高糖组加入MG132后,Smurf2的mRNA和蛋白表达减弱。结论高糖可诱导肾系膜细胞Smurf2表达增强,MG132可阻止高糖所导致的上述变化。提示泛素-蛋白酶体途径参与了糖尿病肾病的发病。
文摘SMURF2(smad ubiquitination regulatory factor 2)属于HECT(homologous to E6AP C terminus)家族E3泛素连接酶,有研究报道SMURF2在不同类型癌症中发挥促癌或抑癌作用.本研究探讨了SMURF2在前列腺(癌)细胞中调节STAT1(signal transducers and activators of transcription 1)蛋白的分子机制.首先通过数据库分析SMURF2在正常组织与肿瘤组织间的表达差异,然后选取前列腺正常细胞和多种前列腺癌细胞为实验材料,通过RT-PCR,Western blotting实验检测SMURF2在前列腺(癌)细胞中的表达水平,发现相对于前列腺正常细胞,SMURF2在前列腺癌细胞中表达更高.再通过Co-IP,免疫荧光和泛素化检测实验观察SMURF2对STAT1蛋白泛素化水平的影响,发现SMURF2可以增加STAT1蛋白的泛素化水平,并进一步促进前列腺癌细胞发生EMT(epithelial mesenchymal transformation).
基金the JSPS Grant-in-Aid(C)grant number 17K11005the JSPS bilateral Joint Research Project grant number 1007397 to T.K.,MEXT/JSPS grant number JP19K12218 to T.S.,MEXT/JSPS grant number JP15H05952(“Resonance Bio”)to T.S.and T.I.,and MEXT/JSPS KAKENHI grant number JP16H06280(“Advanced Bioimaging Support”)。
文摘Transforming growth factor-β(TGF-β)and bone morphogenetic protein(BMP)play important roles in bone metabolism.Smad ubiquitination regulatory factors(Smurfs)regulate TGF-β/BMP signaling via ubiquitination,resulting in degradation of signaling molecules to prevent excessive activation of TGF-β/BMP signaling.Though Smurf2 has been shown to negatively regulate TGF-β/Smad signaling,its involvement in BMP/Smad signaling in bone metabolism has not been thoroughly investigated.In the present study,we sought to evaluate the role of Smurf2 in BMP/Smad signaling in bone metabolism.Absorbable collagen sponges containing 3μg of recombinant human BMP2(rhBMP2)were implanted in the dorsal muscle pouches of wild type(WT)and Smurf2−/−mice.The rhBMP2-induced ectopic bone in Smurf2−/−mice showed greater bone mass,higher mineral apposition and bone formation rates,and greater osteoblast numbers than the ectopic bone in WT mice.In WT mice,the ectopic bone consisted of a thin discontinuous outer cortical shell and scant inner trabecular bone.In contrast,in Smurf2−/−mice,the induced bone consisted of a thick,continuous outer cortical shell and abundant inner trabecular bone.Additionally,rhBMP2-stimulated bone marrow stromal cells(BMSCs)from Smurf2−/−mice showed increased osteogenic differentiation.Smurf2 induced the ubiquitination of Smad1/5.BMP/Smad signaling was enhanced in Smurf2−/−BMSCs stimulated with rhBMP2,and the inhibition of BMP/Smad signaling suppressed osteogenic differentiation of these BMSCs.These findings demonstrate that Smurf2 negatively regulates BMP/Smad signaling,thereby identifying a new regulatory mechanism in bone metabolism.
文摘Glioma is the tumor with the highest incidence in the brain,and it is eager to seek new and efiective treatment.The interaction of ubiquitination and deubiquitination regulates many cell activities in organisms,and participates in tumor occurrence,development,migration,invasion and other processes.This article summarized the progress of E3 ubiquitination ligase smad ubiquitination regulatory factor 2(Smurf2)and glioma-related signaling pathways to assist clinical diagnosis and treatment of glioma.
文摘目的:探讨miR-20a-3p基因干扰对人牙髓干细胞(hDPSCs)成牙本质分化的影响及信号通路调控机制。方法:对hDPSCs转染miR-20a-3p-mimic和miR-20a-3p-inhibitor来上调或下调其表达,然后对hDPSCs进行成牙本质分化诱导培养。通过茜素红染色观察细胞钙基质的矿化程度并检测ALP活性。通过双荧光素酶报告基因测定实验验证miR-20a-3p与mothers against decapentaplegic homolog(SMAD)特异性E3泛素蛋白连接酶1(SMURF1)的靶向调节关系。通过RT-qPCR检测hDPSCs中miR-20a-3p以及SMURF1、Runt相关转录因子2(RUNX2)、骨钙蛋白(OCN)和牙本质涎磷蛋白(DSPP)mRNA水平。结果:与Control组相比,miR-20a-3p-mimic组的茜素红相对染色强度升高了67.09%,miR-20a-3p-inhibitor组降低了46.13%(P<0.05)。与Control组相比,miR-20a-3p-mimic组的相对ALP活性升高了52.89%,miR-20a-3p-inhibitor组降低了53.32%(P<0.05)。与Control组相比,miR-20a-3p-mimic组的RUNX2、OCN和DSPP mRNA相对表达量分别升高了2.19倍、1.86倍和2.35倍,miR-20a-3p-inhibitor组分别降低了63.26%、58.84%和68.12%(P<0.05)。双荧光素酶报告基因测定显示miR-20a-3p靶向抑制SMURF1(P<0.05)。过表达SMURF1抑制了miR-20a-3p对成牙本质分化的影响(P<0.05)。结论:miR-20a-3p通过靶向抑制SMURF1及其下游基因促进hDPSCs的成牙本质分化。
