BACKGROUND Hepatic fibrosis is a serious condition,and the development of hepatic fibrosis can lead to a series of complications.However,the pathogenesis of hepatic fibrosis remains unclear,and effective therapy optio...BACKGROUND Hepatic fibrosis is a serious condition,and the development of hepatic fibrosis can lead to a series of complications.However,the pathogenesis of hepatic fibrosis remains unclear,and effective therapy options are still lacking.Our group identified hepatitis C virus nonstructural protein 3-transactivated protein 1(NS3TP1) by suppressive subtractive hybridization and bioinformatics analysis,but its role in diseases including hepatic fibrosis remains undefined.Therefore,additional studies on the function of NS3TP1 in hepatic fibrosis are urgently needed to provide new targets for treatment.AIM To elucidate the mechanism of NS3TP1 in hepatic fibrosis and the regulatory effects of calcitriol on NS3TP1.METHODS Twenty-four male C57BL/6 mice were randomized and separated into three groups,comprising the normal,fibrosis,and calcitriol treatment groups,and liver fibrosis was modeled by carbon tetrachloride(CCl4).To evaluate the level of hepatic fibrosis in every group,serological and pathological examinations of the liver were conducted.TGF-β1 was administered to boost the in vitro cultivation of LX-2 cells.NS3TP1,α-smooth muscle actin(α-SMA),collagen I,and collagen Ⅲ in every group were examined using a Western blot and real-time quantitative polymerase chain reaction.The activity of the transforming growth factor beta 1(TGFβ1)/Smad3 and NF-κB signaling pathways in each group of cells transfected with pcDNA-NS3TP1 or siRNA-NS3TP1 was detected.The statistical analysis of the data was performed using the Student’s t test.RESULTS NS3TP1 promoted the activation,proliferation,and differentiation of hepatic stellate cells(HSCs)and enhanced hepatic fibrosis via the TGFβ1/Smad3 and NF-κB signaling pathways,as evidenced by the presence of α-SMA,collagen I,collagen Ⅲ,p-smad3,and p-p65 in LX-2 cells,which were upregulated after NS3TP1 overexpression and downregulated after NS3TP1 interference.The proliferation of HSCs was lowered after NS3TP1 interference and elevated after NS3TP1 overexpression,as shown by the luciferase assay.NS3TP1 inhibited the apoptosis of HSCs.Moreover,both Smad3 and p65 could bind to NS3TP1,and p65 increased the promoter activity of NS3TP1,while NS3TP1 increased the promoter activity of TGFβ1 receptor I,as indicated by coimmunoprecipitation and luciferase assay results.Both in vivo and in vitro,treatment with calcitriol dramatically reduced the expression of NS3TP1.Calcitriol therapy-controlled HSCs activation,proliferation,and differentiation and substantially suppressed CCl4-induced hepatic fibrosis in mice.Furthermore,calcitriol modulated the activities of the above signaling pathways via downregulation of NS3TP1.CONCLUSION Our results suggest that calcitriol may be employed as an adjuvant therapy for hepatic fibrosis and that NS3TP1 is a unique,prospective therapeutic target in hepatic fibrosis.展开更多
The biomolecular mechanisms that regulate tooth root development and odontoblast differentiation are poorly understood.We found that Atp6i deficient mice(Atp6i^(−/−))arrested tooth root formation,indicated by truncate...The biomolecular mechanisms that regulate tooth root development and odontoblast differentiation are poorly understood.We found that Atp6i deficient mice(Atp6i^(−/−))arrested tooth root formation,indicated by truncated Hertwig’s epithelial root sheath(HERS)progression.Furthermore,Atp6i deficiency significantly reduced the proliferation and differentiation of radicular odontogenic cells responsible for root formation.Atp6i^(−/−)mice had largely decreased expression of odontoblast differentiation marker gene expression profiles(Col1a1,Nfic,Dspp,and Osx)in the alveolar bone.Atp6i^(−/−)mice sample RNA-seq analysis results showed decreased expression levels of odontoblast markers.Additionally,there was a significant reduction in Smad2/3 activation,inhibiting transforming growth factor-β(TGF-β)signaling in Atp6i^(−/−)odontoblasts.Through treating pulp precursor cells with Atp6i^(−/−)or wild-type OC bone resorption-conditioned medium,we found the latter medium to promote odontoblast differentiation,as shown by increased odontoblast differentiation marker genes expression(Nfic,Dspp,Osx,and Runx2).This increased expression was significantly blocked by anti-TGF-β1 antibody neutralization,whereas odontoblast differentiation and Smad2/3 activation were significantly attenuated by Atp6i^(−/−)OC conditioned medium.Importantly,ectopic TGF-β1 partially rescued root development and root dentin deposition of Atp6i^(−/−)mice tooth germs were transplanted under mouse kidney capsules.Collectively,our novel data shows that the prevention of TGF-β1 release from the alveolar bone matrix due to OC dysfunction may lead to osteopetrosis-associated root formation via impaired radicular odontoblast differentiation.As such,this study uncovers TGF-β1/Smad2/3 as a key signaling pathway regulating odontoblast differentiation and tooth root formation and may contribute to future therapeutic approaches to tooth root regeneration.展开更多
Dioscin is a natural steroid saponin derived from several plants, showing potent anti-cancer effect against a variety of tumor cell lines. In the present study, we investigated the anti-cancer activity of dioscin agai...Dioscin is a natural steroid saponin derived from several plants, showing potent anti-cancer effect against a variety of tumor cell lines. In the present study, we investigated the anti-cancer activity of dioscin against human LNCaP cells, and evaluated the possible mechanism involved in its antineoplastic action. It was found that dioscin（1, 2 and 4 μmol/L） could significantly inhibit the viability of LNCaP cells in a time- and concentration-dependent manner. Flow cytometry revealed that the apoptosis rate was increased after treatment of LNCaP cells with dioscin for 24 h, indicating that apoptosis was an important mechanism by which dioscin inhibited cancer. Western blotting was employed to detect the expression of caspase-3, Bcl-2 and Bax in LNCaP cells. The expression of cleaved caspase-3 was significantly increased, and meanwhile procaspase-3 was markedly decreased. The expression of anti-apoptotic protein Bcl-2 was down-regulated, whereas the pro-apoptotic protein Bax was up-regulated. Moreover, the Bcl-2/Bax ratio was drastically decreased. These results suggested that dioscin possessed potential anti-tumor activity in human LNCaP cells through the apoptosis pathway, which might be associated with caspase-3 and Bcl-2 protein family.展开更多
The expression of Smad2 and Smad3 and the influence of exogenous transforming growth factorβ 1 (TGFβ 1) on them in rat bone marrow-derived mesenchymal stem cells (MSCs) cultured in vitro were investigated. The...The expression of Smad2 and Smad3 and the influence of exogenous transforming growth factorβ 1 (TGFβ 1) on them in rat bone marrow-derived mesenchymal stem cells (MSCs) cultured in vitro were investigated. The effects of different concentrations of TGFβ 1 on cell proliferation and ALP activity were detected by MTT and PNPP in MSCs respectively. The expression of Smad2 and Smad3 and the influence of exogenous TGFβ 1 on them were also examined by immunocytochemistry and Western blot assays. The exogenous TGFβ 1 induced a dose-dependent decrease in cell proliferation and a dose-dependent increase in ALP activity, which plateaued at 5 ng/ml. Smad2 and Smad3 proteins were detected only in the cytoplasm in the absence of TGFβ 1 and TGFβ 1 could stimulate the translocation of them from the cytoplasm to the nucleus. The total amount of Smad2 protein remained unchanged before and after TGFβ 1 treatment (P>0.05). The expression levels of Smad3 remained unchanged after 3 h and 6 h treatment (P>0.05), but decreased markedly after 24 h treatment (P<0.05). It was concluded that TGFβ 1 is a latent osteoinductive factor involved in osteoblastic differentiation. Both Samd2 and Smad3 mediate TGFβ 1 signaling as downstream mediators in MSCs. The biological output of TGFβ 1 triggering the osteoblastic differentiation could be entirely determined by Smad3 in MSCs.展开更多
The LIM domain-containing proteins Pinch1/2 regulate integrin activation and cell–extracellular matrix interaction and adhesion.Here,we report that deleting Pinch1 in limb mesenchymal stem cells(MSCs)and Pinch2 globa...The LIM domain-containing proteins Pinch1/2 regulate integrin activation and cell–extracellular matrix interaction and adhesion.Here,we report that deleting Pinch1 in limb mesenchymal stem cells(MSCs)and Pinch2 globally(double knockout;dKO)in mice causes severe chondrodysplasia,while single mutant mice do not display marked defects.Pinch deletion decreases chondrocyte proliferation,accelerates cell differentiation and disrupts column formation.Pinch loss drastically reduces Smad2/3 protein expression in proliferative zone(PZ)chondrocytes and increases Runx2 and Col10a1 expression in both PZ and hypertrophic zone(HZ)chondrocytes.Pinch loss increases sclerostin and Rankl expression in HZ chondrocytes,reduces bone formation,and increases bone resorption,leading to low bone mass.In vitro studies revealed that Pinch1 and Smad2/3 colocalize in the nuclei of chondrocytes.Through its C-terminal region,Pinch1 interacts with Smad2/3 proteins.Pinch loss increases Smad2/3 ubiquitination and degradation in primary bone marrow stromal cells(BMSCs).Pinch loss reduces TGF-β-induced Smad2/3 phosphorylation and nuclear localization in primary BMSCs.Interestingly,compared to those from single mutant mice,BMSCs from dKO mice express dramatically lower protein levels ofβ-catenin and Yap1/Taz and display reduced osteogenic but increased adipogenic differentiation capacity.Finally,ablating Pinch1 in chondrocytes and Pinch2 globally causes severe osteopenia with subtle limb shortening.Collectively,our findings demonstrate critical roles for Pinch1/2 and a functional redundancy of both factors in the control of chondrogenesis and bone mass through distinct mechanisms.展开更多
Objective： Impaired signal transduction is associated with tumorigenesis and progression of various kinds of human cancers. Transforming growth factor （TGF）-beta/Smad and ras-mitogen activated protein kinase （MAPK...Objective： Impaired signal transduction is associated with tumorigenesis and progression of various kinds of human cancers. Transforming growth factor （TGF）-beta/Smad and ras-mitogen activated protein kinase （MAPK） are two major signal transduction pathways for adjusting cell proliferation and differentiation. Little is known about TGF-beta/Smad4 in non-small cell lung cancer （NSCLC）. Hereby, we investigated the expression of Smad4 in NSCLC, its correlation with MAPK proteins （including p38, ERK1 and JNK1 proteins） and their clinical significance in NSCLC. Methods： The expressions of Smad4, p38, ERK1 and JNK1 were detected at protein level with Western blotting and immunohistochemistry, at transcription level with RT-PCR. Statistical analysis was performed for the comparisons of expressions of Smad4, p38, ERK1 and JNK1, and their correlation with various clinicopathological parameters and the prognosis of NSCLC. Results： The levels of protein and mRNA expression of Smad4 in lung cancer tissues were significantly lower than in normal tissues （P〈0.05）. All these four proteins were associated with TNM staging. There was a strongly negative correlation between p38 and Smad4. Expressions of Smad4, p38 and JNK1, as well as tumor differentiation and staging were significantly correlated with the prognosis of NSCLC by univariate analysis. By multivariate analysis, only Smad4, p38, tumor differentiation and staging were correlated with the prognosis. Taken together, the negative expression of p38 and positive expression of Smad4 were associated with a better prognosis of NSCLC. Conclusion： Smad4 could be of vital importance for the initiation and development of NSCLC. The expression of Smad4 might be inhibited by p38, supporting a cross-talk between main proteins of TGF-beta/Smad and ras-MAPK signal transduction pathways. Smad4 and p38 could be possible prognostic factors for NSCLC.展开更多
基金the National Key Research and Development Program of China,No.2017YFC0908104National Science and Technology Projects,No.2017ZX10203201,No.2017ZX10201201,and No.2017ZX10202202.
文摘BACKGROUND Hepatic fibrosis is a serious condition,and the development of hepatic fibrosis can lead to a series of complications.However,the pathogenesis of hepatic fibrosis remains unclear,and effective therapy options are still lacking.Our group identified hepatitis C virus nonstructural protein 3-transactivated protein 1(NS3TP1) by suppressive subtractive hybridization and bioinformatics analysis,but its role in diseases including hepatic fibrosis remains undefined.Therefore,additional studies on the function of NS3TP1 in hepatic fibrosis are urgently needed to provide new targets for treatment.AIM To elucidate the mechanism of NS3TP1 in hepatic fibrosis and the regulatory effects of calcitriol on NS3TP1.METHODS Twenty-four male C57BL/6 mice were randomized and separated into three groups,comprising the normal,fibrosis,and calcitriol treatment groups,and liver fibrosis was modeled by carbon tetrachloride(CCl4).To evaluate the level of hepatic fibrosis in every group,serological and pathological examinations of the liver were conducted.TGF-β1 was administered to boost the in vitro cultivation of LX-2 cells.NS3TP1,α-smooth muscle actin(α-SMA),collagen I,and collagen Ⅲ in every group were examined using a Western blot and real-time quantitative polymerase chain reaction.The activity of the transforming growth factor beta 1(TGFβ1)/Smad3 and NF-κB signaling pathways in each group of cells transfected with pcDNA-NS3TP1 or siRNA-NS3TP1 was detected.The statistical analysis of the data was performed using the Student’s t test.RESULTS NS3TP1 promoted the activation,proliferation,and differentiation of hepatic stellate cells(HSCs)and enhanced hepatic fibrosis via the TGFβ1/Smad3 and NF-κB signaling pathways,as evidenced by the presence of α-SMA,collagen I,collagen Ⅲ,p-smad3,and p-p65 in LX-2 cells,which were upregulated after NS3TP1 overexpression and downregulated after NS3TP1 interference.The proliferation of HSCs was lowered after NS3TP1 interference and elevated after NS3TP1 overexpression,as shown by the luciferase assay.NS3TP1 inhibited the apoptosis of HSCs.Moreover,both Smad3 and p65 could bind to NS3TP1,and p65 increased the promoter activity of NS3TP1,while NS3TP1 increased the promoter activity of TGFβ1 receptor I,as indicated by coimmunoprecipitation and luciferase assay results.Both in vivo and in vitro,treatment with calcitriol dramatically reduced the expression of NS3TP1.Calcitriol therapy-controlled HSCs activation,proliferation,and differentiation and substantially suppressed CCl4-induced hepatic fibrosis in mice.Furthermore,calcitriol modulated the activities of the above signaling pathways via downregulation of NS3TP1.CONCLUSION Our results suggest that calcitriol may be employed as an adjuvant therapy for hepatic fibrosis and that NS3TP1 is a unique,prospective therapeutic target in hepatic fibrosis.
基金supported by the National Institutes of Health[DE023813 and DE028264 to Y.P.L.,and AG056438 and AR070135 to W.C.]the UAB National Institutes of Health National Institute of Dental and Craniofacial Research[Dental Academic Research Training Grant(DART)5T90DE022736 to J.W.].
文摘The biomolecular mechanisms that regulate tooth root development and odontoblast differentiation are poorly understood.We found that Atp6i deficient mice(Atp6i^(−/−))arrested tooth root formation,indicated by truncated Hertwig’s epithelial root sheath(HERS)progression.Furthermore,Atp6i deficiency significantly reduced the proliferation and differentiation of radicular odontogenic cells responsible for root formation.Atp6i^(−/−)mice had largely decreased expression of odontoblast differentiation marker gene expression profiles(Col1a1,Nfic,Dspp,and Osx)in the alveolar bone.Atp6i^(−/−)mice sample RNA-seq analysis results showed decreased expression levels of odontoblast markers.Additionally,there was a significant reduction in Smad2/3 activation,inhibiting transforming growth factor-β(TGF-β)signaling in Atp6i^(−/−)odontoblasts.Through treating pulp precursor cells with Atp6i^(−/−)or wild-type OC bone resorption-conditioned medium,we found the latter medium to promote odontoblast differentiation,as shown by increased odontoblast differentiation marker genes expression(Nfic,Dspp,Osx,and Runx2).This increased expression was significantly blocked by anti-TGF-β1 antibody neutralization,whereas odontoblast differentiation and Smad2/3 activation were significantly attenuated by Atp6i^(−/−)OC conditioned medium.Importantly,ectopic TGF-β1 partially rescued root development and root dentin deposition of Atp6i^(−/−)mice tooth germs were transplanted under mouse kidney capsules.Collectively,our novel data shows that the prevention of TGF-β1 release from the alveolar bone matrix due to OC dysfunction may lead to osteopetrosis-associated root formation via impaired radicular odontoblast differentiation.As such,this study uncovers TGF-β1/Smad2/3 as a key signaling pathway regulating odontoblast differentiation and tooth root formation and may contribute to future therapeutic approaches to tooth root regeneration.
基金supported by the National Natural Science Foundation of China(No.81173065)
文摘Dioscin is a natural steroid saponin derived from several plants, showing potent anti-cancer effect against a variety of tumor cell lines. In the present study, we investigated the anti-cancer activity of dioscin against human LNCaP cells, and evaluated the possible mechanism involved in its antineoplastic action. It was found that dioscin（1, 2 and 4 μmol/L） could significantly inhibit the viability of LNCaP cells in a time- and concentration-dependent manner. Flow cytometry revealed that the apoptosis rate was increased after treatment of LNCaP cells with dioscin for 24 h, indicating that apoptosis was an important mechanism by which dioscin inhibited cancer. Western blotting was employed to detect the expression of caspase-3, Bcl-2 and Bax in LNCaP cells. The expression of cleaved caspase-3 was significantly increased, and meanwhile procaspase-3 was markedly decreased. The expression of anti-apoptotic protein Bcl-2 was down-regulated, whereas the pro-apoptotic protein Bax was up-regulated. Moreover, the Bcl-2/Bax ratio was drastically decreased. These results suggested that dioscin possessed potential anti-tumor activity in human LNCaP cells through the apoptosis pathway, which might be associated with caspase-3 and Bcl-2 protein family.
基金This project was supported by a grant from the NationalNatural Science Foundation of China (No. 30170270).
文摘The expression of Smad2 and Smad3 and the influence of exogenous transforming growth factorβ 1 (TGFβ 1) on them in rat bone marrow-derived mesenchymal stem cells (MSCs) cultured in vitro were investigated. The effects of different concentrations of TGFβ 1 on cell proliferation and ALP activity were detected by MTT and PNPP in MSCs respectively. The expression of Smad2 and Smad3 and the influence of exogenous TGFβ 1 on them were also examined by immunocytochemistry and Western blot assays. The exogenous TGFβ 1 induced a dose-dependent decrease in cell proliferation and a dose-dependent increase in ALP activity, which plateaued at 5 ng/ml. Smad2 and Smad3 proteins were detected only in the cytoplasm in the absence of TGFβ 1 and TGFβ 1 could stimulate the translocation of them from the cytoplasm to the nucleus. The total amount of Smad2 protein remained unchanged before and after TGFβ 1 treatment (P>0.05). The expression levels of Smad3 remained unchanged after 3 h and 6 h treatment (P>0.05), but decreased markedly after 24 h treatment (P<0.05). It was concluded that TGFβ 1 is a latent osteoinductive factor involved in osteoblastic differentiation. Both Samd2 and Smad3 mediate TGFβ 1 signaling as downstream mediators in MSCs. The biological output of TGFβ 1 triggering the osteoblastic differentiation could be entirely determined by Smad3 in MSCs.
基金the National Key Research and Development Program of China Grant(2019YFA0906004,2019YFA0906001)the National Natural Science Foundation of China(81991513,82022047,81630066,81870532,and 81972100)+1 种基金the Guangdong Provincial Science and Technology Innovation Council(2017B030301018)Science and Technology Innovation Commission of Shenzhen Municipal Government(JCYJ20180302174117738,JCYJ20180302174246105,KQJSCX20180319114434843,and JSGG20180503182321166).
文摘The LIM domain-containing proteins Pinch1/2 regulate integrin activation and cell–extracellular matrix interaction and adhesion.Here,we report that deleting Pinch1 in limb mesenchymal stem cells(MSCs)and Pinch2 globally(double knockout;dKO)in mice causes severe chondrodysplasia,while single mutant mice do not display marked defects.Pinch deletion decreases chondrocyte proliferation,accelerates cell differentiation and disrupts column formation.Pinch loss drastically reduces Smad2/3 protein expression in proliferative zone(PZ)chondrocytes and increases Runx2 and Col10a1 expression in both PZ and hypertrophic zone(HZ)chondrocytes.Pinch loss increases sclerostin and Rankl expression in HZ chondrocytes,reduces bone formation,and increases bone resorption,leading to low bone mass.In vitro studies revealed that Pinch1 and Smad2/3 colocalize in the nuclei of chondrocytes.Through its C-terminal region,Pinch1 interacts with Smad2/3 proteins.Pinch loss increases Smad2/3 ubiquitination and degradation in primary bone marrow stromal cells(BMSCs).Pinch loss reduces TGF-β-induced Smad2/3 phosphorylation and nuclear localization in primary BMSCs.Interestingly,compared to those from single mutant mice,BMSCs from dKO mice express dramatically lower protein levels ofβ-catenin and Yap1/Taz and display reduced osteogenic but increased adipogenic differentiation capacity.Finally,ablating Pinch1 in chondrocytes and Pinch2 globally causes severe osteopenia with subtle limb shortening.Collectively,our findings demonstrate critical roles for Pinch1/2 and a functional redundancy of both factors in the control of chondrogenesis and bone mass through distinct mechanisms.
基金This work was supported by the National Natural Science Foundation of China(No.30100220)
文摘Objective： Impaired signal transduction is associated with tumorigenesis and progression of various kinds of human cancers. Transforming growth factor （TGF）-beta/Smad and ras-mitogen activated protein kinase （MAPK） are two major signal transduction pathways for adjusting cell proliferation and differentiation. Little is known about TGF-beta/Smad4 in non-small cell lung cancer （NSCLC）. Hereby, we investigated the expression of Smad4 in NSCLC, its correlation with MAPK proteins （including p38, ERK1 and JNK1 proteins） and their clinical significance in NSCLC. Methods： The expressions of Smad4, p38, ERK1 and JNK1 were detected at protein level with Western blotting and immunohistochemistry, at transcription level with RT-PCR. Statistical analysis was performed for the comparisons of expressions of Smad4, p38, ERK1 and JNK1, and their correlation with various clinicopathological parameters and the prognosis of NSCLC. Results： The levels of protein and mRNA expression of Smad4 in lung cancer tissues were significantly lower than in normal tissues （P〈0.05）. All these four proteins were associated with TNM staging. There was a strongly negative correlation between p38 and Smad4. Expressions of Smad4, p38 and JNK1, as well as tumor differentiation and staging were significantly correlated with the prognosis of NSCLC by univariate analysis. By multivariate analysis, only Smad4, p38, tumor differentiation and staging were correlated with the prognosis. Taken together, the negative expression of p38 and positive expression of Smad4 were associated with a better prognosis of NSCLC. Conclusion： Smad4 could be of vital importance for the initiation and development of NSCLC. The expression of Smad4 might be inhibited by p38, supporting a cross-talk between main proteins of TGF-beta/Smad and ras-MAPK signal transduction pathways. Smad4 and p38 could be possible prognostic factors for NSCLC.
