BACKGROUND The low survival rate of mesenchymal stem cells(MSCs)caused by anoikis,a form of apoptosis,limits the therapeutic efficacy of MSCs.As a proapoptotic molecule,mammalian Ste20-like kinase 1(Mst1)can increase ...BACKGROUND The low survival rate of mesenchymal stem cells(MSCs)caused by anoikis,a form of apoptosis,limits the therapeutic efficacy of MSCs.As a proapoptotic molecule,mammalian Ste20-like kinase 1(Mst1)can increase the production of reactive oxygen species(ROS),thereby promoting anoikis.Recently,we found that Mst1 inhibition could protect mouse bone marrow MSCs(mBMSCs)from H 2 O 2-induced cell apoptosis by inducing autophagy and reducing ROS production.However,the influence of Mst1 inhibition on anoikis in mBMSCs remains unclear.AIM To investigate the mechanisms by which Mst1 inhibition acts on anoikis in isolated mBMSCs.METHODS Poly-2-hydroxyethyl methacrylate-induced anoikis was used following the silencing of Mst1 expression by short hairpin RNA(shRNA)adenovirus transfection.Integrin(ITGs)were tested by flow cytometry.Autophagy and ITGα5β1 were inhibited using 3-methyladenine and small interfering RNA,respe-ctively.The alterations in anoikis were measured by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling and anoikis assays.The levels of the anoikis-related proteins ITGα5,ITGβ1,and phospho-focal adhesion kinase and the activation of caspase 3 and the autophagy-related proteins microtubules associated protein 1 light chain 3 II/I,Beclin1 and p62 were detected by Western blotting.RESULTS In isolated mBMSCs,Mst1 expression was upregulated,and Mst1 inhibition significantly reduced cell apoptosis,induced autophagy and decreased ROS levels.Mechanistically,we found that Mst1 inhibition could upregulate ITGα5 and ITGβ1 expression but not ITGα4,ITGαv,or ITGβ3 expression.Moreover,autophagy induced by upregulated ITGα5β1 expression following Mst1 inhibition played an essential role in the protective efficacy of Mst1 inhibition in averting anoikis.CONCLUSION Mst1 inhibition ameliorated autophagy formation,increased ITGα5β1 expression,and decreased the excessive production of ROS,thereby reducing cell apoptosis in isolated mBMSCs.Based on these results,Mst1 inhibition may provide a promising strategy to overcome anoikis of implanted MSCs.展开更多
Primary immune thrombocytopenia(ITP)is an autoimmune hemorrhagic disorder in which macrophages play a critical role.Mammalian sterile-20-like kinase 4(MST4),a member of the germinal-center kinase STE20 family,has been...Primary immune thrombocytopenia(ITP)is an autoimmune hemorrhagic disorder in which macrophages play a critical role.Mammalian sterile-20-like kinase 4(MST4),a member of the germinal-center kinase STE20 family,has been demonstrated to be a regulator of inflammation.Whether MST4 participates in the macrophage-dependent inflammation of ITP remains elusive.The expression and function of MST4 in macrophages of ITP patients and THP-1 cells,and of a macrophage-specific Mst4−/−(Mst4ΔM/ΔM)ITP mouse model were determined.Macrophage phagocytic assays,RNA sequencing(RNA-seq)analysis,immunofluorescence analysis,coimmunoprecipitation(co-IP),mass spectrometry(MS),bioinformatics analysis,and phosphoproteomics analysis were performed to reveal the underlying mechanisms.The expression levels of the MST4 gene were elevated in the expanded M1-like macrophages of ITP patients,and this elevated expression of MST4 was restored to basal levels in patients with remission after high-dose dexamethasone treatment.The expression of the MST4 gene was significantly elevated in THP-1-derived M1 macrophages.Silencing of MST4 decreased the expression of M1 macrophage markers and cytokines,and impaired phagocytosis,which could be increased by overexpression of MST4.In a passive ITP mouse model,macrophage-specific depletion of Mst4 reduced the numbers of M1 macrophages in the spleen and peritoneal lavage fluid,attenuated the expression of M1 cytokines,and promoted the predominance of FcγRIIb in splenic macrophages,which resulted in amelioration of thrombocytopenia.Downregulation of MST4 directly inhibited STAT1 phosphorylation,which is essential for M1 polarization of macrophages.Our study elucidates a critical role for MST4 kinase in the pathology of ITP and identifies MST4 kinase as a potential therapeutic target for refractory ITP.展开更多
Background:Cervical cancer has the fourth highest incidence and mortality rate of all cancers in women worldwide;让seriously harms their physical and mental health.The aim of this study was to observe the roles and pr...Background:Cervical cancer has the fourth highest incidence and mortality rate of all cancers in women worldwide;让seriously harms their physical and mental health.The aim of this study was to observe the roles and preliminary mechanism of Taurine(Tau)-induced apoptosis in cervical cancer cells.Methods:Cells from the human cervical cancer cell line SiHa were transfected with the recombinant plasmid pEGFP-N1-MST1(mammalian sterile 20-like kinase 1);then,the cell proliferation activity was analyzed by the MTT assay,cell apoptosis by flow cytometry,and the related protein levels by Western blotting.Results:Tau inhibited the proliferation of SiHa cells and induced apoptosis in these cells(the apoptotic rate was 21.95%in the Tau 160 mmol/L group and 30%in the Tau 320 mmol/L group),upregulated the expression of the MST1(control,0.53;Tau 40-320 mmol/L groups,0.84-1.45)and Bax(control,0.45;Tau 40-320 mmol/L groups,0.64-1.51)proteins(P<0.01),and downregulated the expression of Bcl-2(control,1.28,Tau 40-320 mmol/L groups,0.93-0.47)(P<0.01).The overexpression of MST1 promoted the apoptosis of SiHa cells,enhanced the apoptosis-inductive effects of Tau(P<0.01),upregulated the expression of the proapoptotic proteins p73,p53,PUMA(p53 upregulated modulator of apoptosis),and caspase-3,and promoted the phosphorylation of YAP(Yes-associated protein).Conclusions:Tau inhibited the proliferation and induced the apoptosis of cervical cancer SiHa cells.The MST1 protein plays an important role in the Tau-induced apoptosis of cervical cancer cells.展开更多
基金Supported by Natural Science Foundation of Shandong Province,China,No.ZR2020MH014,No.ZR2021QH179 and No.ZR2021MH182.
文摘BACKGROUND The low survival rate of mesenchymal stem cells(MSCs)caused by anoikis,a form of apoptosis,limits the therapeutic efficacy of MSCs.As a proapoptotic molecule,mammalian Ste20-like kinase 1(Mst1)can increase the production of reactive oxygen species(ROS),thereby promoting anoikis.Recently,we found that Mst1 inhibition could protect mouse bone marrow MSCs(mBMSCs)from H 2 O 2-induced cell apoptosis by inducing autophagy and reducing ROS production.However,the influence of Mst1 inhibition on anoikis in mBMSCs remains unclear.AIM To investigate the mechanisms by which Mst1 inhibition acts on anoikis in isolated mBMSCs.METHODS Poly-2-hydroxyethyl methacrylate-induced anoikis was used following the silencing of Mst1 expression by short hairpin RNA(shRNA)adenovirus transfection.Integrin(ITGs)were tested by flow cytometry.Autophagy and ITGα5β1 were inhibited using 3-methyladenine and small interfering RNA,respe-ctively.The alterations in anoikis were measured by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling and anoikis assays.The levels of the anoikis-related proteins ITGα5,ITGβ1,and phospho-focal adhesion kinase and the activation of caspase 3 and the autophagy-related proteins microtubules associated protein 1 light chain 3 II/I,Beclin1 and p62 were detected by Western blotting.RESULTS In isolated mBMSCs,Mst1 expression was upregulated,and Mst1 inhibition significantly reduced cell apoptosis,induced autophagy and decreased ROS levels.Mechanistically,we found that Mst1 inhibition could upregulate ITGα5 and ITGβ1 expression but not ITGα4,ITGαv,or ITGβ3 expression.Moreover,autophagy induced by upregulated ITGα5β1 expression following Mst1 inhibition played an essential role in the protective efficacy of Mst1 inhibition in averting anoikis.CONCLUSION Mst1 inhibition ameliorated autophagy formation,increased ITGα5β1 expression,and decreased the excessive production of ROS,thereby reducing cell apoptosis in isolated mBMSCs.Based on these results,Mst1 inhibition may provide a promising strategy to overcome anoikis of implanted MSCs.
基金supported by grants from the National Natural Science Foundation of China(82370130,81870098,82300146)the Program of the Shanghai Academic/Technology Researcher Leader(20XD1401000)+2 种基金the Shanghai Engineering Research Center of Tumor Multi-Target Gene Diagnosis(20DZ2254300)the Key Subject Construction Program of the Shanghai Health Administrative Authority(ZK2019B30)the Science and Technology Commission of the Shanghai Municipality(21ZR1459000).
文摘Primary immune thrombocytopenia(ITP)is an autoimmune hemorrhagic disorder in which macrophages play a critical role.Mammalian sterile-20-like kinase 4(MST4),a member of the germinal-center kinase STE20 family,has been demonstrated to be a regulator of inflammation.Whether MST4 participates in the macrophage-dependent inflammation of ITP remains elusive.The expression and function of MST4 in macrophages of ITP patients and THP-1 cells,and of a macrophage-specific Mst4−/−(Mst4ΔM/ΔM)ITP mouse model were determined.Macrophage phagocytic assays,RNA sequencing(RNA-seq)analysis,immunofluorescence analysis,coimmunoprecipitation(co-IP),mass spectrometry(MS),bioinformatics analysis,and phosphoproteomics analysis were performed to reveal the underlying mechanisms.The expression levels of the MST4 gene were elevated in the expanded M1-like macrophages of ITP patients,and this elevated expression of MST4 was restored to basal levels in patients with remission after high-dose dexamethasone treatment.The expression of the MST4 gene was significantly elevated in THP-1-derived M1 macrophages.Silencing of MST4 decreased the expression of M1 macrophage markers and cytokines,and impaired phagocytosis,which could be increased by overexpression of MST4.In a passive ITP mouse model,macrophage-specific depletion of Mst4 reduced the numbers of M1 macrophages in the spleen and peritoneal lavage fluid,attenuated the expression of M1 cytokines,and promoted the predominance of FcγRIIb in splenic macrophages,which resulted in amelioration of thrombocytopenia.Downregulation of MST4 directly inhibited STAT1 phosphorylation,which is essential for M1 polarization of macrophages.Our study elucidates a critical role for MST4 kinase in the pathology of ITP and identifies MST4 kinase as a potential therapeutic target for refractory ITP.
基金the National Natural Science Foundation(No.81360032)Jiangxi Provincial Natural Science Foundation(No.20171BAB215059).
文摘Background:Cervical cancer has the fourth highest incidence and mortality rate of all cancers in women worldwide;让seriously harms their physical and mental health.The aim of this study was to observe the roles and preliminary mechanism of Taurine(Tau)-induced apoptosis in cervical cancer cells.Methods:Cells from the human cervical cancer cell line SiHa were transfected with the recombinant plasmid pEGFP-N1-MST1(mammalian sterile 20-like kinase 1);then,the cell proliferation activity was analyzed by the MTT assay,cell apoptosis by flow cytometry,and the related protein levels by Western blotting.Results:Tau inhibited the proliferation of SiHa cells and induced apoptosis in these cells(the apoptotic rate was 21.95%in the Tau 160 mmol/L group and 30%in the Tau 320 mmol/L group),upregulated the expression of the MST1(control,0.53;Tau 40-320 mmol/L groups,0.84-1.45)and Bax(control,0.45;Tau 40-320 mmol/L groups,0.64-1.51)proteins(P<0.01),and downregulated the expression of Bcl-2(control,1.28,Tau 40-320 mmol/L groups,0.93-0.47)(P<0.01).The overexpression of MST1 promoted the apoptosis of SiHa cells,enhanced the apoptosis-inductive effects of Tau(P<0.01),upregulated the expression of the proapoptotic proteins p73,p53,PUMA(p53 upregulated modulator of apoptosis),and caspase-3,and promoted the phosphorylation of YAP(Yes-associated protein).Conclusions:Tau inhibited the proliferation and induced the apoptosis of cervical cancer SiHa cells.The MST1 protein plays an important role in the Tau-induced apoptosis of cervical cancer cells.
