目的探讨沉默RPMI-8226 DEPTOR基因表达在非接触式共培养体系下,对破骨细胞分化因子(RANKL)蛋白表达及THP-1细胞向破骨样分化的作用,并研究其可能机制。方法构建DEPTOR sh RNA重组慢病毒载体转染RPMI-8226细胞。通过Western blot技术从...目的探讨沉默RPMI-8226 DEPTOR基因表达在非接触式共培养体系下,对破骨细胞分化因子(RANKL)蛋白表达及THP-1细胞向破骨样分化的作用,并研究其可能机制。方法构建DEPTOR sh RNA重组慢病毒载体转染RPMI-8226细胞。通过Western blot技术从蛋白水平检测RPMI-8226细胞中DEPTOR及RANKL的表达,Western blot检测自噬相关蛋白LC-3和Atg5表达。构建共培养体系,分三组:THP-1细胞单培养组、THP-1+RPMI-8226细胞组和THP-1+DEPTOR sh RNA组。应用抗酒石酸酸性磷酸酶(TRAP)染色法检测THP-1向破骨样细胞分化过程中抗酒石酸酸性磷酸酶活性改变,应用RT-PCR法检测THP-1细胞降钙素受体(CTR)和组织蛋白酶(Cathepsin)-K m RNA表达水平。结果 DEPTOR sh RNA可明显抑制RPMI-8226细胞中DEPTOR及RANKL蛋白表达(P<0.05),DEPTOR sh RNA组自噬相关蛋白LC-3和Atg5的蛋白的表达水平较阴性对照转染组及未转染组下降(P<0.05)。THP-1细胞与RPMI-8226细胞共培养条件下可诱导THP-1细胞破骨样分化,CTR和Cathepsin-K基因表达上调(P<0.05);DEPTOR sh RNA共培养条件下可抑制THP-1细胞向破骨样细胞分化,CTR和Cathepsin-K基因表达减弱,差异有统计学意义(P<0.05)。结论 DEPTOR sh RNA能明显抑制共培养体系中THP-1细胞的破骨样分化,该作用可能与DEPTOR下调RPMI-8226细胞RANKL有关,抑制自噬可阻碍破骨细胞的分化成熟。展开更多
This study explored the molecular mechanisms underlying the time-dependent autophagy and apoptosis induced by nutrient depletion in human multiple myeloma cell line RPMI8226 cells.RT-PCR and qRT-PCR were used to evalu...This study explored the molecular mechanisms underlying the time-dependent autophagy and apoptosis induced by nutrient depletion in human multiple myeloma cell line RPMI8226 cells.RT-PCR and qRT-PCR were used to evaluate the transcriptional levels of Deptor,JNK1,JNK2,JNK3,Raf-1,p53,p21 and NFκB1 at 0,6,12,18,24 and 48 h after nutrient depletion in RPMI8226 cells.We found that transcriptional levels of Deptor were increased time-dependently at 0,6,12 and 18 h,and then decreased.Its alternation was consistent with autophagy.Transcriptional levels of Raf-1,JNK1,JNK2,p53 and p21 were increased time-dependently at 0,6,12,18,24 and 48 h accompanying with the increase of apoptosis.Transcriptional levels of NFκB1 at 6,12,18,24 and 48 h were decreased as com-pared with 0 h.It was suggested that all the studied signaling molecules were involved in cellular re-sponse to nutrient depletion in RPMI8226 cells.Deptor contributed to autophagy in this process.Raf-1/JNK /p53/p21 pathway may be involved in apoptosis,and NFκB1 may play a possible role in in-hibiting apoptosis.It remained to be studied whether Deptor was involved in both autophagy and apoptosis.展开更多
The mammalian target of rapamycin,mTOR,forms various protein-protein complexes to regulate cell growth in response to the nutrient and energy status of the cell.Recently,the first crystal structure of large HEAT repea...The mammalian target of rapamycin,mTOR,forms various protein-protein complexes to regulate cell growth in response to the nutrient and energy status of the cell.Recently,the first crystal structure of large HEAT repeat protein mTOR revealed that the FAT domain interacts with the kinase domain through electrostatic effects and hydrophobic interactions.Based on the structure,the previous researches on how FAT domain regulates mTOR activity are reviewed.DEPTOR is currently known as an endogenous mTOR inhibitor,which may interact with mTOR FAT domain to suppress mTOR activity in vivo.The possible interactions of DEPTOR with the mTOR FAT domain are analyzed,too.In addition,the inhibition mechanism of DEPTOR may be similar to members of HEAT-involved RanGTP complex family,providing new mechanistic insights into mTOR kinase regulation.展开更多
文摘目的探讨沉默RPMI-8226 DEPTOR基因表达在非接触式共培养体系下,对破骨细胞分化因子(RANKL)蛋白表达及THP-1细胞向破骨样分化的作用,并研究其可能机制。方法构建DEPTOR sh RNA重组慢病毒载体转染RPMI-8226细胞。通过Western blot技术从蛋白水平检测RPMI-8226细胞中DEPTOR及RANKL的表达,Western blot检测自噬相关蛋白LC-3和Atg5表达。构建共培养体系,分三组:THP-1细胞单培养组、THP-1+RPMI-8226细胞组和THP-1+DEPTOR sh RNA组。应用抗酒石酸酸性磷酸酶(TRAP)染色法检测THP-1向破骨样细胞分化过程中抗酒石酸酸性磷酸酶活性改变,应用RT-PCR法检测THP-1细胞降钙素受体(CTR)和组织蛋白酶(Cathepsin)-K m RNA表达水平。结果 DEPTOR sh RNA可明显抑制RPMI-8226细胞中DEPTOR及RANKL蛋白表达(P<0.05),DEPTOR sh RNA组自噬相关蛋白LC-3和Atg5的蛋白的表达水平较阴性对照转染组及未转染组下降(P<0.05)。THP-1细胞与RPMI-8226细胞共培养条件下可诱导THP-1细胞破骨样分化,CTR和Cathepsin-K基因表达上调(P<0.05);DEPTOR sh RNA共培养条件下可抑制THP-1细胞向破骨样细胞分化,CTR和Cathepsin-K基因表达减弱,差异有统计学意义(P<0.05)。结论 DEPTOR sh RNA能明显抑制共培养体系中THP-1细胞的破骨样分化,该作用可能与DEPTOR下调RPMI-8226细胞RANKL有关,抑制自噬可阻碍破骨细胞的分化成熟。
基金supported by a grant from National Natural Science Foundation of China (No. 30871036)
文摘This study explored the molecular mechanisms underlying the time-dependent autophagy and apoptosis induced by nutrient depletion in human multiple myeloma cell line RPMI8226 cells.RT-PCR and qRT-PCR were used to evaluate the transcriptional levels of Deptor,JNK1,JNK2,JNK3,Raf-1,p53,p21 and NFκB1 at 0,6,12,18,24 and 48 h after nutrient depletion in RPMI8226 cells.We found that transcriptional levels of Deptor were increased time-dependently at 0,6,12 and 18 h,and then decreased.Its alternation was consistent with autophagy.Transcriptional levels of Raf-1,JNK1,JNK2,p53 and p21 were increased time-dependently at 0,6,12,18,24 and 48 h accompanying with the increase of apoptosis.Transcriptional levels of NFκB1 at 6,12,18,24 and 48 h were decreased as com-pared with 0 h.It was suggested that all the studied signaling molecules were involved in cellular re-sponse to nutrient depletion in RPMI8226 cells.Deptor contributed to autophagy in this process.Raf-1/JNK /p53/p21 pathway may be involved in apoptosis,and NFκB1 may play a possible role in in-hibiting apoptosis.It remained to be studied whether Deptor was involved in both autophagy and apoptosis.
基金supported by the National Natural Science Foundation of China(21133003,21273023)Specialized Research Fund for the Doctoral Program of Higher Education(20110003120013)
文摘The mammalian target of rapamycin,mTOR,forms various protein-protein complexes to regulate cell growth in response to the nutrient and energy status of the cell.Recently,the first crystal structure of large HEAT repeat protein mTOR revealed that the FAT domain interacts with the kinase domain through electrostatic effects and hydrophobic interactions.Based on the structure,the previous researches on how FAT domain regulates mTOR activity are reviewed.DEPTOR is currently known as an endogenous mTOR inhibitor,which may interact with mTOR FAT domain to suppress mTOR activity in vivo.The possible interactions of DEPTOR with the mTOR FAT domain are analyzed,too.In addition,the inhibition mechanism of DEPTOR may be similar to members of HEAT-involved RanGTP complex family,providing new mechanistic insights into mTOR kinase regulation.
