Objective Vascular smooth muscle cell(VSMC)differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension,atherosclerosis...Objective Vascular smooth muscle cell(VSMC)differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension,atherosclerosis,and restenosis.MicroRNA-146a(miR-146a)has been proven to be involved in cell proliferation,migration,and tumor metabolism.However,little is known about the functional role of miR-146a in VSMC differentiation from embryonic stem cells(ESCs).This study aimed to determine the role of miR-146a in VSMC differentiation from ESCs.Methods Mouse ESCs were differentiated into VSMCs,and the cell extracts were analyzed by Western blotting and RT-qPCR.In addition,luciferase reporter assays using ESCs transfected with miR-146a/mimic and plasmids were performed.Finally,C57BL/6J female mice were injected with mimic or miR-146a-overexpressing ESCs,and immunohistochemistry,Western blotting,and RT-qPCR assays were carried out on tissue samples from these mice.Results miR-146a was significantly upregulated during VSMC differentiation,accompanied with the VSMC-specific marker genes smooth muscle-alpha-actin(SMαA),smooth muscle 22(SM22),smooth muscle myosin heavy chain(SMMHC),and h1-calponin.Furthermore,overexpression of miR-146a enhanced the differentiation process in vitro and in vivo.Concurrently,the expression of Kruppel-like factor 4(KLF4),predicted as one of the top targets of miR-146a,was sharply decreased in miR-146a-overexpressing ESCs.Importantly,inhibiting KLF4 expression enhanced the VSMC-specific gene expression induced by miR-146a overexpression in differentiating ESCs.In addition,miR-146a upregulated the mRNA expression levels and transcriptional activity of VSMC differentiation-related transcription factors,including serum response factor(SRF)and myocyte enhancer factor 2c(MEF-2c).Conclusion Our data support that miR-146a promotes ESC-VSMC differentiation through regulating KLF4 and modulating the transcription factor activity of VSMCs.展开更多
Kruppel-like factor 4（Klf4） is a zinc finger transcription factor and plays crucial roles in Xenopus embryogenesis.However, its regulation during embryogenesis is still unclear. Here, we report that Tcf711, a key do...Kruppel-like factor 4（Klf4） is a zinc finger transcription factor and plays crucial roles in Xenopus embryogenesis.However, its regulation during embryogenesis is still unclear. Here, we report that Tcf711, a key downstream transducer of the Wnt signaling pathway, could promote Klf4 transcription and stimulate Klf4 promoter activity in early Xenopus embryos. Furthermore, cycloheximide treatment showed a direct effect on Klf4 transcription facilitated by Tcf711. Moreover, the dominant negative form of Tcf711（dnTcf711）, which lacks N-terminus of the β-catenin binding motif, could still activate Klf4 transcription, suggesting that this regulation is Wnt/β-catenin independent.Taken together, our results demonstrate that Tcf711 lies upstream of Klf4 to maintain its expression level during Xenopus embryogenesis.展开更多
Viral myocarditis(VMC)is a cardiac disease associated with myocardial inflammation and injury induced by virus infection.Cardiomyocytes have recently been regarded as key players in eliciting and modulating inflammati...Viral myocarditis(VMC)is a cardiac disease associated with myocardial inflammation and injury induced by virus infection.Cardiomyocytes have recently been regarded as key players in eliciting and modulating inflammation within the myocardium.Kruppel-like factor 10(KLF10)is a crucial regulator of various pathological processes and plays different roles in a variety of diseases.However,its role in VMC induced by coxsackievirus B3(CVB3)infection remains unknown.In this study,we report that cardiac KLF10 confers enhanced protection against viral myocarditis.We found that KLF10 expression was downregulated upon CVB3 infection.KLF10 deficiency enhanced cardiac viral replication and aggravated VMC progress.Bone marrow chimera experiments indicated that KLF10 expression in nonhematopoietic cells was involved in the pathogenesis of VMC.We further identified MCP-1 as a novel target of KLF10 in cardiomyocytes,and KLF10 cooperated with histone deacetylase 1(HDAC1)to negatively regulate MCP-1 expression by binding its promoter,leading to activation of MCP-1 transcription and recruitment of Ly6C^(high) monocytes/macrophages into the myocardium.This novel mechanism of MCP-1 regulation by KLF10 might provide new insights into the pathogenesis of VMC and a potential therapeutic target for VMC.展开更多
Although titanate nanofibers(TiNFs)and titanate nanotubes(TiNTs)have been proposed as relatively biocompatible nanomaterials(NMs),there is currently lacking of systemic studies which investigated the toxicity of TiNFs...Although titanate nanofibers(TiNFs)and titanate nanotubes(TiNTs)have been proposed as relatively biocompatible nanomaterials(NMs),there is currently lacking of systemic studies which investigated the toxicity of TiNFs and TiNTs to endothelium.In this study,we developed endothelial monolayer model by using cell culture inserts,and systemically investigated the toxicity of TiNFs and TiNTs by RNA-seq,with a focus on Kruppel-like factor(KLF)-mediated effects,since KLF are transcription factors(TF)involved in the regulation of vascular biology.It was shown that NMs did not significantly induce cytotoxicity despite substantial internalization.However,the expression of many KLF was altered,and Western blot further confirmed that NMs down-regulated KLF2 proteins.Ingenuity pathway analysis(IPA)revealed that NMs altered the expression of KLF2-targed genes,typically the genes involved in inflammatory responses.KLF2-related Gene Ontology(GO)terms and Kyoto Encyclopedia of Gene and Genomes(KEGG)pathways were also altered,and it should be noticed that NMs altered GO terms and KEGG pathways related with endothelial NO synthase(eNOS).This study further verified that NMs decreased intracellular NO and eNOS proteins.All the observed effects were more obvious for TiNFs compared with TiNTs.Combined,this study showed that TiNFs or TiNTs we re non-cytotoxic to endothelial monolayer model,but TiNFs and more modestly TiNTs decreased KLF2 leading to decreased eNOS proteins and NO production.Our data may provide novel understanding about the toxicity of TiNFs as well as other Ti-based NMs to endothelium.展开更多
Objective Hepatic stellate cells(HSCs)play a crucial role in liver fibrosis.Early-stage liver fibrosis is reversible and intimately associated with the state of HSCs.Kruppel-like factor 4(KLF4)plays a pivotal role in ...Objective Hepatic stellate cells(HSCs)play a crucial role in liver fibrosis.Early-stage liver fibrosis is reversible and intimately associated with the state of HSCs.Kruppel-like factor 4(KLF4)plays a pivotal role in a wide array of physiological and pathological processes.This study aimed to investigate the effect of KLF4 on the proliferation,apoptosis and phenotype of quiescent HSCs Methods We designed a KLF4 lentiviral vector and a KLF4 siRNA lentiviral vector,to upregulate and silence KLF4 expression in human HSC LX-2 cells via transfection.Cell proliferation was assessed using the CCK-8 assay.Flow cytometry was used to detect the cell cycle distribution and apoptosis rate.Western blotting was used to determine the levels of some quiescence and activation markers of HSCs Results Overexpression of KLF4 significantly increased the levels of E-cadherin and ZO-1,which are quiescent HSC markers,while significantly decreased the levels of N-cadherin and a-SMA,known activated HSC markers.In contrast,cell proliferation and apoptosis rates were elevated in LX-2 cells in which KLF4 expression was silenced Conclusion KLF4 inhibits the proliferation and activation of human LX-2 HSCs.It might be a key regulatory protein in the maintenance of HSC quiescence and may serve as a target for the inhibition of hepatic fibrosis.展开更多
基金funded by the National Natural Science Foundation of China(No.82070376 and No.81873491)the Natural Science Foundation of Zhejiang Province(No.LY21H020005)+1 种基金the Zhejiang Medical Science and Technology Project(No.2019KY376 and No.2018KY071)a Ningbo Science and Technology Project(No.202002N3173).
文摘Objective Vascular smooth muscle cell(VSMC)differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension,atherosclerosis,and restenosis.MicroRNA-146a(miR-146a)has been proven to be involved in cell proliferation,migration,and tumor metabolism.However,little is known about the functional role of miR-146a in VSMC differentiation from embryonic stem cells(ESCs).This study aimed to determine the role of miR-146a in VSMC differentiation from ESCs.Methods Mouse ESCs were differentiated into VSMCs,and the cell extracts were analyzed by Western blotting and RT-qPCR.In addition,luciferase reporter assays using ESCs transfected with miR-146a/mimic and plasmids were performed.Finally,C57BL/6J female mice were injected with mimic or miR-146a-overexpressing ESCs,and immunohistochemistry,Western blotting,and RT-qPCR assays were carried out on tissue samples from these mice.Results miR-146a was significantly upregulated during VSMC differentiation,accompanied with the VSMC-specific marker genes smooth muscle-alpha-actin(SMαA),smooth muscle 22(SM22),smooth muscle myosin heavy chain(SMMHC),and h1-calponin.Furthermore,overexpression of miR-146a enhanced the differentiation process in vitro and in vivo.Concurrently,the expression of Kruppel-like factor 4(KLF4),predicted as one of the top targets of miR-146a,was sharply decreased in miR-146a-overexpressing ESCs.Importantly,inhibiting KLF4 expression enhanced the VSMC-specific gene expression induced by miR-146a overexpression in differentiating ESCs.In addition,miR-146a upregulated the mRNA expression levels and transcriptional activity of VSMC differentiation-related transcription factors,including serum response factor(SRF)and myocyte enhancer factor 2c(MEF-2c).Conclusion Our data support that miR-146a promotes ESC-VSMC differentiation through regulating KLF4 and modulating the transcription factor activity of VSMCs.
基金supported by the Start-up Funding of Henan University of Science and Technology(13480027) to Q. C.the Key Science Foundation of Nanjing Medical University(2015NJMUZD002)+2 种基金the Natural Science Foundation of Higher Education Institutions of Jiangsu Province(16KJB-180020)Natural Science Foundation of Jiangsu Province (BK20171053)National Natural Science Funds of China (81702747) to C.L
文摘Kruppel-like factor 4（Klf4） is a zinc finger transcription factor and plays crucial roles in Xenopus embryogenesis.However, its regulation during embryogenesis is still unclear. Here, we report that Tcf711, a key downstream transducer of the Wnt signaling pathway, could promote Klf4 transcription and stimulate Klf4 promoter activity in early Xenopus embryos. Furthermore, cycloheximide treatment showed a direct effect on Klf4 transcription facilitated by Tcf711. Moreover, the dominant negative form of Tcf711（dnTcf711）, which lacks N-terminus of the β-catenin binding motif, could still activate Klf4 transcription, suggesting that this regulation is Wnt/β-catenin independent.Taken together, our results demonstrate that Tcf711 lies upstream of Klf4 to maintain its expression level during Xenopus embryogenesis.
基金This work was supported by the Chinese National Natural Science Foundation(31400769,31870903,31870868,and 31670930)Jiangsu Province Natural Science Foundation(BK20140371)+1 种基金Jiangsu Postdoctoral Science Foundation(1402176C)Priority Academic Program Development of Jiangsu Higher Education Institutions.
文摘Viral myocarditis(VMC)is a cardiac disease associated with myocardial inflammation and injury induced by virus infection.Cardiomyocytes have recently been regarded as key players in eliciting and modulating inflammation within the myocardium.Kruppel-like factor 10(KLF10)is a crucial regulator of various pathological processes and plays different roles in a variety of diseases.However,its role in VMC induced by coxsackievirus B3(CVB3)infection remains unknown.In this study,we report that cardiac KLF10 confers enhanced protection against viral myocarditis.We found that KLF10 expression was downregulated upon CVB3 infection.KLF10 deficiency enhanced cardiac viral replication and aggravated VMC progress.Bone marrow chimera experiments indicated that KLF10 expression in nonhematopoietic cells was involved in the pathogenesis of VMC.We further identified MCP-1 as a novel target of KLF10 in cardiomyocytes,and KLF10 cooperated with histone deacetylase 1(HDAC1)to negatively regulate MCP-1 expression by binding its promoter,leading to activation of MCP-1 transcription and recruitment of Ly6C^(high) monocytes/macrophages into the myocardium.This novel mechanism of MCP-1 regulation by KLF10 might provide new insights into the pathogenesis of VMC and a potential therapeutic target for VMC.
基金financially supported by Hunan Innovative Province Construction Special Major Landmark Innovation Demonstration Project(No.2019XK2303)Xiangtan Science and Technology Project(No.ZD-ZD20191007)。
文摘Although titanate nanofibers(TiNFs)and titanate nanotubes(TiNTs)have been proposed as relatively biocompatible nanomaterials(NMs),there is currently lacking of systemic studies which investigated the toxicity of TiNFs and TiNTs to endothelium.In this study,we developed endothelial monolayer model by using cell culture inserts,and systemically investigated the toxicity of TiNFs and TiNTs by RNA-seq,with a focus on Kruppel-like factor(KLF)-mediated effects,since KLF are transcription factors(TF)involved in the regulation of vascular biology.It was shown that NMs did not significantly induce cytotoxicity despite substantial internalization.However,the expression of many KLF was altered,and Western blot further confirmed that NMs down-regulated KLF2 proteins.Ingenuity pathway analysis(IPA)revealed that NMs altered the expression of KLF2-targed genes,typically the genes involved in inflammatory responses.KLF2-related Gene Ontology(GO)terms and Kyoto Encyclopedia of Gene and Genomes(KEGG)pathways were also altered,and it should be noticed that NMs altered GO terms and KEGG pathways related with endothelial NO synthase(eNOS).This study further verified that NMs decreased intracellular NO and eNOS proteins.All the observed effects were more obvious for TiNFs compared with TiNTs.Combined,this study showed that TiNFs or TiNTs we re non-cytotoxic to endothelial monolayer model,but TiNFs and more modestly TiNTs decreased KLF2 leading to decreased eNOS proteins and NO production.Our data may provide novel understanding about the toxicity of TiNFs as well as other Ti-based NMs to endothelium.
基金supported by the National Natural Science Foundation of China(No.81071541).
文摘Objective Hepatic stellate cells(HSCs)play a crucial role in liver fibrosis.Early-stage liver fibrosis is reversible and intimately associated with the state of HSCs.Kruppel-like factor 4(KLF4)plays a pivotal role in a wide array of physiological and pathological processes.This study aimed to investigate the effect of KLF4 on the proliferation,apoptosis and phenotype of quiescent HSCs Methods We designed a KLF4 lentiviral vector and a KLF4 siRNA lentiviral vector,to upregulate and silence KLF4 expression in human HSC LX-2 cells via transfection.Cell proliferation was assessed using the CCK-8 assay.Flow cytometry was used to detect the cell cycle distribution and apoptosis rate.Western blotting was used to determine the levels of some quiescence and activation markers of HSCs Results Overexpression of KLF4 significantly increased the levels of E-cadherin and ZO-1,which are quiescent HSC markers,while significantly decreased the levels of N-cadherin and a-SMA,known activated HSC markers.In contrast,cell proliferation and apoptosis rates were elevated in LX-2 cells in which KLF4 expression was silenced Conclusion KLF4 inhibits the proliferation and activation of human LX-2 HSCs.It might be a key regulatory protein in the maintenance of HSC quiescence and may serve as a target for the inhibition of hepatic fibrosis.
