Hypertrophic differentiation is not only the terminal process of endochondral ossification in the growth plate but is also an important pathological change in osteoarthritic cartilage.Collagen type II(COL2A1)was previ...Hypertrophic differentiation is not only the terminal process of endochondral ossification in the growth plate but is also an important pathological change in osteoarthritic cartilage.Collagen type II(COL2A1)was previously considered to be only a structural component of the cartilage matrix,but recently,it has been revealed to be an extracellular signaling molecule that can significantly suppress chondrocyte hypertrophy.However,the mechanisms by which COL2A1 regulates hypertrophic differentiation remain unclear.In our study,a Col2a1 p.Gly1170Ser mutant mouse model was constructed,and Col2a1 loss was demonstrated in homozygotes.Loss of Col2a1 was found to accelerate chondrocyte hypertrophy through the bone morphogenetic protein(BMP)-SMAD1 pathway.Upon interacting with COL2A1,integrinβ1(ITGB1),the major receptor for COL2A1,competed with BMP receptors for binding to SMAD1 and then inhibited SMAD1 activation and nuclear import.COL2A1 could also activate ITGB1-induced ERK1/2 phosphorylation and,through ERK1/2-SMAD1 interaction,it further repressed SMAD1 activation,thus inhibiting BMP-SMAD1-mediated chondrocyte hypertrophy.Moreover,COL2A1 expression was downregulated,while chondrocyte hypertrophic markers and BMP-SMAD1 signaling activity were upregulated in degenerative human articular cartilage.Our study reveals novel mechanisms for the inhibition of chondrocyte hypertrophy by COL2A1 and suggests that the degradation and decrease in COL2A1 might initiate and promote osteoarthritis progression.展开更多
目的:观察荔枝核总黄酮(total flavone from Litchi chinensis Sonn,TFL)对二甲基亚硝胺(dimethylnitrosamine,DMN)诱导的肝纤维化大鼠肝脏组Smads信号通路中关键信号传导分子Smad3、Smad4及基质金属蛋白酶抑制剂-1(tissue inhibitor of...目的:观察荔枝核总黄酮(total flavone from Litchi chinensis Sonn,TFL)对二甲基亚硝胺(dimethylnitrosamine,DMN)诱导的肝纤维化大鼠肝脏组Smads信号通路中关键信号传导分子Smad3、Smad4及基质金属蛋白酶抑制剂-1(tissue inhibitor of metalloproteinase-1TIMP-1)表达水平的变化,探讨TFL抗肝纤维化的作用机制.方法:90只S D大鼠随机平均分成正常对照组、模型组、秋水仙碱组不同浓度的TFL[200、100、50 mg/(kg·d)].用DMN腹腔注射诱导大鼠肝纤维化模型,造模同时灌胃给药.1次/d,共给6 wk,于实验第6周后处死大鼠,取血清测定谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate transaminase,AST)的含量.取留取肝脏同一部位行Masson染色观察大鼠病理改变及肝纤维化程度;免疫组织化学法检测Smad3、Smad4、TIMP-1表达量,实时荧光定量PCR检测(real-time quantitative PCR,qRT-PCR)检测Smad3、Smad4、TIMP-1m RNA表达量.结果:与模型组比较,TFL能降低血清ALT、AST含量,Masson染色病理显示TFL能显著减轻大鼠肝纤维化程度;与空白对照组比较,模型组大鼠的肝纤维化程度明显增加,肝组织Smad3、Smad4及TIMP-1的表达明显增强(P<0.05);与模型组比较,TFL各剂量组和秋水仙碱组肝组织Smad3、Smad4、TIMP-1的表达不同程度的降低(P<0.05).结论:T F L可减轻实验性大鼠肝损伤及改善肝纤维化程度,其机制与降低S m a d3、Smad4及TIMP-1的表达有密切关系,可能与改善肝功能、抑制肝细胞变性坏死,从而抑制胶原蛋白的合成和沉积减少细胞外基质有关.展开更多
As a prototype of the TGF-β superfamily cytokines, TGF-β is well known for its diverse roles in embryogenesis and adult tis- sue homeostasis. TGF-β evokes cellular responses by signaling mainly through cell membran...As a prototype of the TGF-β superfamily cytokines, TGF-β is well known for its diverse roles in embryogenesis and adult tis- sue homeostasis. TGF-β evokes cellular responses by signaling mainly through cell membrane receptors and transcription fac- tor R-Smads and Co-Smad （Smad4）, while an inhibitory Smad, Smad7, acts as a critical negative regulator of TGF-β signaling. Smad7 antagonizes TGF-β signaling by regulating the stability or activity of the receptors or blocking the DNA binding of the functional R-Smad-Smad4 complex in the nucleus. However, the function of Smad7 in the nucleus is not fully understood. Yin Yang 1 （YY1） is a ubiquitously expressed transcription factor with multiple functions. It has been reported that YY1 can inhib- it Smad-dependent transcriptional responses and TGF-β/BMP-induced cell differentiation independently of its DNA binding ability. In this study, we found that Smad7 interacts with YY1 and the interaction is attenuated by TGF-β signaling. Reporter assays and target gene expression analyses revealed that Smad7 and YY1 act in concert to inhibit TGF-β-induced transcription in the nucleus. Furthermore, Smad7 could enhance the interaction of YY1 with the histone deacetylase HDAC1. Consistently, YY 1 and HDAC 1 augmented the transcription repression activity of Smad7 in Gal4-1uciferase reporter analysis. Therefore, our findings define a novel mechanism of Smad7 and YY1 to antagonize TGF-β signaling.展开更多
目的:探讨TGF-β/Smad4基因在结直肠癌肿瘤细胞高低转移细胞株中的表达及其作用。方法:免疫细胞化学方法检测Smad4在SW-480和SW-620细胞系中的定位,免疫印迹法检测Smad4蛋白在SW-480和SW-620细胞系中表达的差异,并构建Smad4真核表达载体...目的:探讨TGF-β/Smad4基因在结直肠癌肿瘤细胞高低转移细胞株中的表达及其作用。方法:免疫细胞化学方法检测Smad4在SW-480和SW-620细胞系中的定位,免疫印迹法检测Smad4蛋白在SW-480和SW-620细胞系中表达的差异,并构建Smad4真核表达载体,脂质体法瞬时转染Smad4基因,转染后24h应用蛋白质印迹法检测Smad4、PAI-1、VEGF和E-cad-herin的表达。结果:免疫细胞化学方法分析显示,Smad4基因在细胞核(质)表达,且Smad4在SW-480细胞中表达的平均光强度为2.356±0.431,而在SW-620细胞中表达的平均光强度为1.023±0.502,两者比较差异有统计学意义,P<0.05。蛋白质印迹法检测Smad4基因在SW-480细胞中表达高于SW-620细胞(0.675±0.356 vs0.224±0.243),瞬时转染Smad4基因24h后,转染前后PAI-1表达显著下调(4.355±0.674 vs2.110±0.524),E-cadherin表达明显上调(0.201±0.254 vs 0.899±0.325),而转染前后VEGF表达(0.867±0.359 vs 0.745±0.479)差异无统计学意义。结论:TGF-β/Smad4基因在SW-480中高表达,在SW-620中低表达,表明Smad4基因与肿瘤转移有关。TGF-β/Smad4基因通过调节PAI-1和E-cadherin的表达而抑制肿瘤转移。展开更多
AIM: To investigate the role of genes and kinetics of specific transcription factors in liver regeneration, and to analyze the gene expression and the activity of some molecules crucially involved in hepatic regenerat...AIM: To investigate the role of genes and kinetics of specific transcription factors in liver regeneration, and to analyze the gene expression and the activity of some molecules crucially involved in hepatic regeneration. METHODS: USING gel-shift assay and RT-PCR, transcription factors, such as NF-κB, STAT-3, SMAD3 and AP-1, and gene expression of inducible nitric oxide synthase (iNOS), hepatocyte growth factor (HGF) and c-met were analyzed in an animal model of chemically induced hepatectomy. RESULTS: Gene expression of HGF and its receptor c-met peaked at 3 h and 24 h after acute CCl4 intoxi- cation. iNOS expression was only observed from 6 to 48 h. Transcriptional factor NF-κB had an early activation at 30 min after acute liver damage. STAT-3 peaked 3 h post- intoxication, while AP-1 displayed a peak of activation at 48 h. SMAD3 showed a high activity at all analyzed times. CONCLUSION: TNF-α and IL-6 play a central role in hepatic regeneration. These two molecules are responsible for triggering the cascade of events and switch-on of genes involved in cell proliferation, such as growth factors, kinases and cyclins which are direct participants of cell proliferation.展开更多
基金supported by the National Natural Science Foundation of China (No.81371907,No.81572134,and No.81802217)the China Postdoctoral Science Foundation (No.2017M622873)+2 种基金the Natural Science Foundation of Guangdong Province,China (No.2018A0303130260,No.2016A030313284,and No.2017A030311008)the Guangzhou Science and Technology Plan (No.201804010057)the Fundamental Research Funds for the Central Universities (No.17ykpy06)
文摘Hypertrophic differentiation is not only the terminal process of endochondral ossification in the growth plate but is also an important pathological change in osteoarthritic cartilage.Collagen type II(COL2A1)was previously considered to be only a structural component of the cartilage matrix,but recently,it has been revealed to be an extracellular signaling molecule that can significantly suppress chondrocyte hypertrophy.However,the mechanisms by which COL2A1 regulates hypertrophic differentiation remain unclear.In our study,a Col2a1 p.Gly1170Ser mutant mouse model was constructed,and Col2a1 loss was demonstrated in homozygotes.Loss of Col2a1 was found to accelerate chondrocyte hypertrophy through the bone morphogenetic protein(BMP)-SMAD1 pathway.Upon interacting with COL2A1,integrinβ1(ITGB1),the major receptor for COL2A1,competed with BMP receptors for binding to SMAD1 and then inhibited SMAD1 activation and nuclear import.COL2A1 could also activate ITGB1-induced ERK1/2 phosphorylation and,through ERK1/2-SMAD1 interaction,it further repressed SMAD1 activation,thus inhibiting BMP-SMAD1-mediated chondrocyte hypertrophy.Moreover,COL2A1 expression was downregulated,while chondrocyte hypertrophic markers and BMP-SMAD1 signaling activity were upregulated in degenerative human articular cartilage.Our study reveals novel mechanisms for the inhibition of chondrocyte hypertrophy by COL2A1 and suggests that the degradation and decrease in COL2A1 might initiate and promote osteoarthritis progression.
文摘目的:观察荔枝核总黄酮(total flavone from Litchi chinensis Sonn,TFL)对二甲基亚硝胺(dimethylnitrosamine,DMN)诱导的肝纤维化大鼠肝脏组Smads信号通路中关键信号传导分子Smad3、Smad4及基质金属蛋白酶抑制剂-1(tissue inhibitor of metalloproteinase-1TIMP-1)表达水平的变化,探讨TFL抗肝纤维化的作用机制.方法:90只S D大鼠随机平均分成正常对照组、模型组、秋水仙碱组不同浓度的TFL[200、100、50 mg/(kg·d)].用DMN腹腔注射诱导大鼠肝纤维化模型,造模同时灌胃给药.1次/d,共给6 wk,于实验第6周后处死大鼠,取血清测定谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate transaminase,AST)的含量.取留取肝脏同一部位行Masson染色观察大鼠病理改变及肝纤维化程度;免疫组织化学法检测Smad3、Smad4、TIMP-1表达量,实时荧光定量PCR检测(real-time quantitative PCR,qRT-PCR)检测Smad3、Smad4、TIMP-1m RNA表达量.结果:与模型组比较,TFL能降低血清ALT、AST含量,Masson染色病理显示TFL能显著减轻大鼠肝纤维化程度;与空白对照组比较,模型组大鼠的肝纤维化程度明显增加,肝组织Smad3、Smad4及TIMP-1的表达明显增强(P<0.05);与模型组比较,TFL各剂量组和秋水仙碱组肝组织Smad3、Smad4、TIMP-1的表达不同程度的降低(P<0.05).结论:T F L可减轻实验性大鼠肝损伤及改善肝纤维化程度,其机制与降低S m a d3、Smad4及TIMP-1的表达有密切关系,可能与改善肝功能、抑制肝细胞变性坏死,从而抑制胶原蛋白的合成和沉积减少细胞外基质有关.
基金supported by the National Natural Science Foundation of China(91019003)National Basic Research Program of China(2011CB943803 and 2013CB933701)
文摘As a prototype of the TGF-β superfamily cytokines, TGF-β is well known for its diverse roles in embryogenesis and adult tis- sue homeostasis. TGF-β evokes cellular responses by signaling mainly through cell membrane receptors and transcription fac- tor R-Smads and Co-Smad （Smad4）, while an inhibitory Smad, Smad7, acts as a critical negative regulator of TGF-β signaling. Smad7 antagonizes TGF-β signaling by regulating the stability or activity of the receptors or blocking the DNA binding of the functional R-Smad-Smad4 complex in the nucleus. However, the function of Smad7 in the nucleus is not fully understood. Yin Yang 1 （YY1） is a ubiquitously expressed transcription factor with multiple functions. It has been reported that YY1 can inhib- it Smad-dependent transcriptional responses and TGF-β/BMP-induced cell differentiation independently of its DNA binding ability. In this study, we found that Smad7 interacts with YY1 and the interaction is attenuated by TGF-β signaling. Reporter assays and target gene expression analyses revealed that Smad7 and YY1 act in concert to inhibit TGF-β-induced transcription in the nucleus. Furthermore, Smad7 could enhance the interaction of YY1 with the histone deacetylase HDAC1. Consistently, YY 1 and HDAC 1 augmented the transcription repression activity of Smad7 in Gal4-1uciferase reporter analysis. Therefore, our findings define a novel mechanism of Smad7 and YY1 to antagonize TGF-β signaling.
文摘目的:探讨TGF-β/Smad4基因在结直肠癌肿瘤细胞高低转移细胞株中的表达及其作用。方法:免疫细胞化学方法检测Smad4在SW-480和SW-620细胞系中的定位,免疫印迹法检测Smad4蛋白在SW-480和SW-620细胞系中表达的差异,并构建Smad4真核表达载体,脂质体法瞬时转染Smad4基因,转染后24h应用蛋白质印迹法检测Smad4、PAI-1、VEGF和E-cad-herin的表达。结果:免疫细胞化学方法分析显示,Smad4基因在细胞核(质)表达,且Smad4在SW-480细胞中表达的平均光强度为2.356±0.431,而在SW-620细胞中表达的平均光强度为1.023±0.502,两者比较差异有统计学意义,P<0.05。蛋白质印迹法检测Smad4基因在SW-480细胞中表达高于SW-620细胞(0.675±0.356 vs0.224±0.243),瞬时转染Smad4基因24h后,转染前后PAI-1表达显著下调(4.355±0.674 vs2.110±0.524),E-cadherin表达明显上调(0.201±0.254 vs 0.899±0.325),而转染前后VEGF表达(0.867±0.359 vs 0.745±0.479)差异无统计学意义。结论:TGF-β/Smad4基因在SW-480中高表达,在SW-620中低表达,表明Smad4基因与肿瘤转移有关。TGF-β/Smad4基因通过调节PAI-1和E-cadherin的表达而抑制肿瘤转移。
基金Supported by CONACyT grant # 28827-M to Adriana Salazar-Montes and, in part, by COECyTJal grant # 08-2004 to Juan Armendariz-Borunda
文摘AIM: To investigate the role of genes and kinetics of specific transcription factors in liver regeneration, and to analyze the gene expression and the activity of some molecules crucially involved in hepatic regeneration. METHODS: USING gel-shift assay and RT-PCR, transcription factors, such as NF-κB, STAT-3, SMAD3 and AP-1, and gene expression of inducible nitric oxide synthase (iNOS), hepatocyte growth factor (HGF) and c-met were analyzed in an animal model of chemically induced hepatectomy. RESULTS: Gene expression of HGF and its receptor c-met peaked at 3 h and 24 h after acute CCl4 intoxi- cation. iNOS expression was only observed from 6 to 48 h. Transcriptional factor NF-κB had an early activation at 30 min after acute liver damage. STAT-3 peaked 3 h post- intoxication, while AP-1 displayed a peak of activation at 48 h. SMAD3 showed a high activity at all analyzed times. CONCLUSION: TNF-α and IL-6 play a central role in hepatic regeneration. These two molecules are responsible for triggering the cascade of events and switch-on of genes involved in cell proliferation, such as growth factors, kinases and cyclins which are direct participants of cell proliferation.
