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1.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(5): 1463-1468, 2019 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-31607299

RESUMO

OBJECTIVE: To investigate the transcriptional regulation of transcription factor MZF-1 on acute monocytic leukemia-related gene MLAA-34. METHODS: The effect of MZF-1 on the transcriptional activity of MLAA-34 gene promoter was analyzed by luciferase reporter gene detection system and site-directed mutation technique. The EMSA and ChIP assay were used to verify whether MZF-1 directly and specifically binds to the core region of MLAA-34 promoter. The over-expression vector and interference vector of MZF-1 were constructed to transfect U937 cells, and RT-PCR and Western blot were used to detect the transcription and expression changes of MLAA-34 gene. RESULTS: The transcription factor MZF-1 had a regulatory effect on MLAA-34 gene expression, and the relative luciferase activity was decreased after MZF-1 binding point mutation (P<0.01). EMSA and ChIP experiments demonstrated that MZF-1 could directly bind to MLAA-34 promoter and play a regulatory role. In the over-expression test, the increase of MZF-1 could up-regulate the expression of MLAA-34 (P<0.05). In the interference test, the decrease of MZF-1 could down-regulate the expression of MLAA-34 (P<0.05). CONCLUSION: Transcription factor MZF-1 can bind to the transcriptional regulatory region on the promoter of MLAA-34 gene and promote the transcription of MLAA-34 gene in acute monocytic leukemia.


Assuntos
Antígenos de Neoplasias/genética , Proteínas Reguladoras de Apoptose/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Leucemia Monocítica Aguda , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Fator 1-alfa Nuclear de Hepatócito , Humanos , Regiões Promotoras Genéticas , Transcrição Genética
3.
Nat Cell Biol ; 21(10): 1179-1190, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548608

RESUMO

Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors, as is exemplified by reprogramming somatic cells to pluripotent stem cells through the expression of OCT4, KLF4, SOX2 and cMYC. How transcription factors orchestrate the complex molecular changes around their target gene loci remains incompletely understood. Here, using KLF4 as a paradigm, we provide a transcription-factor-centric view of chromatin reorganization and its association with three-dimensional enhancer rewiring and transcriptional changes during the reprogramming of mouse embryonic fibroblasts to pluripotent stem cells. Inducible depletion of KLF factors in PSCs caused a genome-wide decrease in enhancer connectivity, whereas disruption of individual KLF4 binding sites within pluripotent-stem-cell-specific enhancers was sufficient to impair enhancer-promoter contacts and reduce the expression of associated genes. Our study provides an integrative view of the complex activities of a lineage-specifying transcription factor and offers novel insights into the nature of the molecular events that follow transcription factor binding.


Assuntos
Reprogramação Celular/genética , Montagem e Desmontagem da Cromatina/genética , Elementos Facilitadores Genéticos , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Células-Tronco Pluripotentes/metabolismo
4.
Ann R Coll Surg Engl ; 101(8): 609-616, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31508984

RESUMO

INTRODUCTION: Hypothermic machine perfusion, an organ preservation modality, involves flow of chilled preservation fluid through an allograft's vasculature. This study describes a simple, reproducible, human model that allows for interrogation of flow effects during ex vivo organ perfusion. MATERIALS AND METHODS: Gonadal veins from deceased human renal allografts were subjected to either static cold storage or hypothermic machine perfusion for up to 24 hours. Caspase-3, Krüppel-like factor 2 expression and electron microscopic analysis were compared between 'flow' and 'no-flow' conditions, with living donor gonadal vein sections serving as negative controls. RESULTS: The increase in caspase-3 expression was less pronounced for hypothermic machine-perfused veins compared with static cold storage (median-fold increase 1.2 vs 2.3; P < 0.05). Transmission electron microscopy provided ultrastructural corroboration of endothelial cell apoptosis in static cold storage conditions. For static cold storage preserved veins, Krüppel-like factor 2 expression diminished in a time-dependent manner between baseline and 12 hours (P < 0.05) but was abrogated and reversed by hypothermic machine perfusion (P < 0.05). CONCLUSIONS: Our methodology is a simple, reproducible and successful model of ex vivo perfusion in the context of human organ preservation. To demonstrate the model's utility, we establish that two widely used markers of endothelial health (caspase-3 and Krüppel-like factor 2) differ between the flow and no-flow conditions of the two predominant kidney preservation modalities. These findings suggest that ex vivo perfusion may mediate the induction of a biochemically favourable endothelial niche which may contribute tohypothermic machine perfusion's association with improved renal transplantation outcomes.


Assuntos
Transplante de Rim/métodos , Rim/irrigação sanguínea , Modelos Biológicos , Soluções para Preservação de Órgãos/farmacocinética , Preservação de Órgãos/métodos , Apoptose , Biomarcadores/metabolismo , Cadáver , Caspase 3/metabolismo , Temperatura Baixa , Endotélio Vascular/metabolismo , Humanos , Rim/metabolismo , Rim/ultraestrutura , Fatores de Transcrição Kruppel-Like/metabolismo , Microscopia Eletrônica , Perfusão/métodos , Veias/metabolismo , Veias/ultraestrutura
5.
Toxicol Lett ; 314: 63-74, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31306741

RESUMO

This study aimed to verify the toxic effects of prenatal caffeine exposure (PCE) on the podocyte development in male offspring, and to explore the underlying intrauterine programming mechanisms. The pregnant rats were administered with caffeine (30 to 120 mg/kg⋅d) during gestational day (GD) 9 to 20. The male fetus on GD20 and the offspring at postnatal week (PW) 6 and PW28 were sacrificed. The results indicated that PCE caused ultrastructural abnormalities on podocyte, and inhibited the expression of podocyte marker genes such as Nephrin, Wilms tumor 1 (WT1), the histone 3 lysine 9 acetylation (H3K9ac) level in the Kruppel-like factor 4 (KLF4) promoter and its expression in the male offspring from GD20 to PW28. Meanwhile, the expression of glucocorticoid receptor (GR) and histone deacetylase 7 (HDAC7) in the fetus were increased by PCE. In vitro, corticosterone increased GR and HDAC7 whereas reduced the H3K9ac level of KLF4 and KLF4/Nephrin expression. KLF4 over-expression reversed the reduction of Nephrin expression, knockdown of HDAC7 and GR antagonist RU486 partially reversed the inhibitory effects of corticosterone on H3K9ac level and KLF4 expression. In conclusion, PCE caused podocyte developmental toxicity in male offspring, which was associated with corticosterone-induced low-functional programming of KLF4 through GR/HDAC7/H3K9ac pathway.


Assuntos
Cafeína/toxicidade , Estimulantes do Sistema Nervoso Central/toxicidade , Histonas/metabolismo , Nefropatias/induzido quimicamente , Fatores de Transcrição Kruppel-Like/metabolismo , Podócitos/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , Acetilação , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Idade Gestacional , Glucocorticoides/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Nefropatias/embriologia , Nefropatias/genética , Nefropatias/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Lisina , Masculino , Exposição Materna , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fenótipo , Podócitos/metabolismo , Podócitos/ultraestrutura , Gravidez , Regiões Promotoras Genéticas , Ratos Wistar , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais/efeitos dos fármacos
6.
Life Sci ; 233: 116641, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31295469

RESUMO

Cardiomyocyte injury caused by excessive oxidative stress underlies the pathogenesis of myocardial infarction (MI), a devastating disease leading to heart failure and death. The Krüppel-like factor 9 (KLF9) is a transcriptional factor that has recently been reported to regulate oxidative stress, however, whether it is associated with cardiomyocyte injury and MI is unknown. We found that KLF9 was upregulated in the heart from a rat MI model. In addition, KLF9 was also upregulated in cardiomyocytes exposed to ischemia in vitro, suggesting that KLF9 responds to MI-relevant stimuli. Moreover, KLF9 knockdown protected cardiomyocytes against ischemic injury. Mechanistically, KLF9 knockdown reduced reactive oxygen species (ROS) generation in ischemic cardiomyocytes through upregulating the antioxidant thioredoxin reductase 2 (Txnrd2), and more important, Txnrd2 silencing abrogated KLF9 knockdown-mediated cardioprotection in ischemic cardiomyocytes. Altogether, these results suggest that KLF9 aggravates ischemic injury in cardiomyocytes through undermining Txnrd2-mediated ROS clearance, which might offer KLF9 as a possible target in alleviating MI.


Assuntos
Fatores de Transcrição Kruppel-Like/metabolismo , Traumatismo por Reperfusão Miocárdica/etiologia , Miócitos Cardíacos/patologia , Estresse Oxidativo , Tiorredoxina Redutase 2/metabolismo , Animais , Células Cultivadas , Fatores de Transcrição Kruppel-Like/genética , Masculino , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxina Redutase 2/genética , Regulação para Cima
7.
Biochimie ; 163: 152-162, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31199942

RESUMO

Extra-cellular signal regulated kinase-5 (Erk-5), a transcriptional activator and regulator of endothelial cells (ECs) homeostasis, has been implicated in shear stress-induced endothelial dysfunction (ED), however its role in oxidized low-density lipoprotein (oxLDL)- induced ED during metabolic stress is not known. Herein, regulation and function of Erk-5 in oxLDL-induced EC death, inflammation and dysfunction has been investigated. Primary Human Umbilical Vein Endothelial Cells (pHUVECs) were stimulated with oxLDL. MTT and Trypan blue exclusion assays to assess cell viability, RT-qPCR and Western blotting assays to determine expression of endothelial and inflammatory markers and ED mediators at mRNA and protein levels, respectively were performed. Monocyte adhesion assay was performed to examine monocytes adherence to oxLDL-stimulated pHUVECs. The exposure of oxLDL induced a dose- and time-dependent decrease in pHUVECs viability, which concurred with decreased Erk-5 expression. Further, oxLDL (100 µg/ml) decreased the expression of endothelial markers eNOS and vWF, and increased the expression of ICAM-1, at both mRNA and protein levels. SiRNA-mediated silencing of Erk-5 or its inhibition showed that changes in eNOS, vWF and ICAM-1 expression could be mediated through Erk-5. Furthermore, oxLDL decreased the levels of Erk-5's upstream regulator MEK5 and downstream regulators Mef2c and KLF2, which were similar to their expressions in Erk-5 silenced cells. Fisetin, a phytochemical and bioflavonoid, could reduce the effect of oxLDL in ECs by upregulating the expression of endothelial markers including Erk-5, and downregulating the expression of inflammation markers. These results suggest that Erk-5 could be a critical regulator of oxLDL-induced EC death, inflammation and dysfunction via downregulation of Erk-5/Mef2c-KLF2 signaling pathway, which can be ameliorated by a bioflavonoid, fisetin.


Assuntos
Flavonoides/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Lipoproteínas LDL/toxicidade , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Monócitos/fisiologia , Transdução de Sinais , Aterosclerose/induzido quimicamente , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/prevenção & controle , Células Cultivadas , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Inflamação/prevenção & controle , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Lipoproteínas LDL/farmacologia , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Substâncias Protetoras/farmacologia
8.
Genes Dev ; 33(15-16): 1069-1082, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31221664

RESUMO

Embryonic stem (ES) cells are regulated by a network of transcription factors that maintain the pluripotent state. Differentiation relies on down-regulation of pluripotency transcription factors disrupting this network. While investigating transcriptional regulation of the pluripotency transcription factor Kruppel-like factor 4 (Klf4), we observed that homozygous deletion of distal enhancers caused a 17-fold decrease in Klf4 transcript but surprisingly decreased protein levels by less than twofold, indicating that posttranscriptional control of KLF4 protein overrides transcriptional control. The lack of sensitivity of KLF4 to transcription is due to high protein stability (half-life >24 h). This stability is context-dependent and is disrupted during differentiation, as evidenced by a shift to a half-life of <2 h. KLF4 protein stability is maintained through interaction with other pluripotency transcription factors (NANOG, SOX2, and STAT3) that together facilitate association of KLF4 with RNA polymerase II. In addition, the KLF4 DNA-binding and transactivation domains are required for optimal KLF4 protein stability. Posttranslational modification of KLF4 destabilizes the protein as cells exit the pluripotent state, and mutations that prevent this destabilization also prevent differentiation. These data indicate that the core pluripotency transcription factors are integrated by posttranslational mechanisms to maintain the pluripotent state and identify mutations that increase KLF4 protein stability while maintaining transcription factor function.


Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Células-Tronco Embrionárias , Células HEK293 , Humanos , Camundongos , Mutação/genética , Domínios Proteicos , Estabilidade Proteica , Proteólise , RNA Polimerase II/metabolismo , Transdução de Sinais , Ubiquitinação
9.
Mol Carcinog ; 58(8): 1427-1437, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31162747

RESUMO

The Helicobacter pylori (H. pylori) cytotoxin-associated gene A (CagA) and Krüppel-like transcription factor (KLF4) were both closely associated with the development and progression of gastric cancer (GC). However, the nature of the interactions between CagA and KLF4 in GC development has not been elucidated. Therefore, we focused on the CagA-mediated promotion of the malignant transformation of gastric epithelial cells. Herein, we first examined the expression of KLF4 in both human cancer and paracarcinoma tissues with or without H. pylori infection and found that KLF4 expression was significantly decreased in H. pylori-positive GC cells compared with the H. pylori-negative GC cells. Further functional studies revealed that the increased expression of CagA could suppress KLF4 expression and promote the malignant transformation of normal epithelial cells. Subsequently, we found that CagA could upregulate miR-155 and further restrict the expression of downstream KLF4. More importantly, the overexpression of miR-155 in GES-1 promoted epithelial-mesenchymal transition and eventually facilitated tumor growth in vivo. Overall, the identification of the CagA/miR-155/KLF4 signaling pathway provided a new insight into the development and treatment of GC.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Transformação Celular Neoplásica/patologia , Mucosa Gástrica/patologia , Fatores de Transcrição Kruppel-Like/metabolismo , Neoplasias Gástricas/patologia , Adulto , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Transformação Celular Neoplásica/genética , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Feminino , Mucosa Gástrica/citologia , Células HEK293 , Infecções por Helicobacter/patologia , Helicobacter pylori/patogenicidade , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/genética , MicroRNAs/metabolismo , Transplante de Neoplasias , Transdução de Sinais , Estômago/patologia , Neoplasias Gástricas/genética , Transplante Heterólogo
10.
Artif Cells Nanomed Biotechnol ; 47(1): 2188-2195, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31159593

RESUMO

The damage of vascular endothelial cells has become an indispensable factor in the occurrence and advancement of cardiovascular diseases. In the current study, we investigated the effect of Astragalus Polysacharin (APS) on H2O2-evoked oxidative injury in HUVECs. HUVECs cells were treated by H2O2 to induce oxidative damage. Cells viability, apoptosis and reactive oxygen species (ROS) level were detected through CCK8 assay and flow cytometry. The cell growth-related proteins and heme oxygenase-1(HO-1) and KLF2 expression were evaluated via Western blot assay. The functions of KLF2 in APS and H2O2 co-disposed HUVECs were explored after si-KLF2 transfection. MEK/ERK pathway was finally measured through Western blot. We found that H2O2 stimulation-evoked HUVECs oxidative damage meanwhile impeded HO-1 expression. APS treatment effectively suppressed H2O2-induced oxidative injury in HUVECs. KLF2 and Nrf2 expression were elevated by APS and KLF2 repression abolished the protective action of APS in H2O2-triggered cell injury. MEK/ERK pathway was activated by APS treatment. Furthermore, the MEK/ERK pathway inhibitor weakened the promoting effect of APS on the expression of KLF2. In conclusion, our study reveals that APS alleviates H2O2-triggered oxidative injury in HUVECs via elevating the expression of KLF2 via the MEK/ERK pathway.


Assuntos
Fabaceae/química , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Peróxido de Hidrogênio/farmacologia , Fatores de Transcrição Kruppel-Like/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Polissacarídeos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
11.
BMC Genomics ; 20(1): 417, 2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31126231

RESUMO

BACKGROUND: Mutations in the transcription factor, KLF1, are common within certain populations of the world. Heterozygous missense mutations in KLF1 mostly lead to benign phenotypes, but a heterozygous mutation in a DNA-binding residue (E325K in human) results in severe Congenital Dyserythropoietic Anemia type IV (CDA IV); i.e. an autosomal-dominant disorder characterized by neonatal hemolysis. RESULTS: To investigate the biochemical and genetic mechanism of CDA IV, we generated murine erythroid cell lines that harbor tamoxifen-inducible (ER™) versions of wild type and mutant KLF1 on a Klf1-/- genetic background. Nuclear translocation of wild type KLF1 results in terminal erythroid differentiation, whereas mutant KLF1 results in hemolysis without differentiation. The E to K variant binds poorly to the canonical 9 bp recognition motif (NGG-GYG-KGG) genome-wide but binds at high affinity to a corrupted motif (NGG-GRG-KGG). We confirmed altered DNA-binding specificity by quantitative in vitro binding assays of recombinant zinc-finger domains. Our results are consistent with previously reported structural data of KLF-DNA interactions. We employed 4sU-RNA-seq to show that a corrupted transcriptome is a direct consequence of aberrant DNA binding. CONCLUSIONS: Since all KLF/SP family proteins bind DNA in an identical fashion, these results are likely to be generally applicable to mutations in all family members. Importantly, they explain how certain mutations in the DNA-binding domain of transcription factors can generate neomorphic functions that result in autosomal dominant disease.


Assuntos
Anemia Diseritropoética Congênita/genética , DNA/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Mutação Puntual , Animais , Linhagem Celular , DNA/química , Regulação da Expressão Gênica , Camundongos , Motivos de Nucleotídeos , Ligação Proteica , Transcrição Genética
12.
Int J Mol Sci ; 20(10)2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31100930

RESUMO

The juvenile hormone (JH) signalling and ecdysone signalling pathways are crucial endocrine signalling pathways that orchestrate the metamorphosis of insects. The metamorphic process, the morphological change from the immature to adult forms, is orchestrated by the dramatic reduction of JH and downstream transcription factors. The Krüppel-homologue 1 (Kr-h1), a downstream transcription factor of the JH signalling pathway, represses E93 expression with an anti-metamorphic effect. However, the biochemical interaction between Kr-h1 and E93 and how the interaction regulates ovary development, a sensitive readout for endocrine regulation, remain unknown. In brown planthopper, Nilaparvata lugens, we found that the downregulation of Kr-h1 partially recovered the deteriorating effect of E93 knock-down on metamorphosis. Dual knock down of E93 and Kr-h1 increased ovary development and the number of eggs laid when compared to the effects of the knock down of E93 alone, indicating that the knock down of Kr-h1 partially recovered the deteriorating effect of the E93 knock-down on ovary development. In summary, our results indicated that E93 and Kr-h1 have antagonistic effects on regulating metamorphosis and ovary development. We tested the biochemical interaction between these two proteins and found that these molecules interact directly. Kr-h1 V and E93 II undergo strong and specific interactions, indicating that the potential interacting domain may be located in these two regions. We inferred that the nuclear receptor interaction motif (NR-box) and helix-turn-helix DNA binding motifs of the pipsqueak family (RHF1) are candidate domains responsible for the protein-protein interaction between E93 and Kr-h1. Moreover, the HA-tagged E93 and FLAG-tagged Kr-h1 were co-localized in the nucleus, and the expression of E93 was increased when Kr-h1 was downregulated, supporting that these two proteins may interact antagonistically. JH and ecdysone signalling are critical for the control of ovary development and pest populations. Our result is important for understanding the interactions between E93 and related proteins, which makes it possible to identify potential targets and develop new pesticides for pest management.


Assuntos
Hemípteros/metabolismo , Proteínas de Insetos/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Metamorfose Biológica/fisiologia , Ovário/metabolismo , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição/metabolismo , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ecdisona , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Proteínas de Insetos/genética , Hormônios Juvenis/metabolismo , Metamorfose Biológica/genética , Ovário/crescimento & desenvolvimento , Transdução de Sinais , Fatores de Transcrição/genética
13.
Phytomedicine ; 58: 152754, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31009837

RESUMO

BACKGROUND: Salvianolic acid B (Sal B), a water-soluble compound extracted from Salvia miltiorrhiza that has been widely used to treat cardiovascular diseases for hundreds of years in China, exerts cardiovascular protection by multiple mechanisms. miR-146a is involved in vascular smooth muscle cell (VSMC) phenotypic modulation and proliferation. However, it has yet to be investigated whether the cardiovascular protective effect of Sal B is mediated by miR-146a. PURPOSE: To determine the relationship among the cardiovascular protective effect of Sal B, miR-146a expression, and VSMC proliferation. METHODS: MTS assay and cell counting were performed to evaluate the effect of Ang II, Sal B and miR-146a on VSMC proliferation. The neointima hyperplasia was assessed by hematoxylin/eosin staining. qRT-PCR was used to detect the expression of miR-146a, KLF5, cyclin D1 and PCNA. Western blot analysis was used to detect the expressions of KLF5, cyclin D1 and PCNA after miR-20b-5p was knocked down or overexpressed in VSMC. RESULTS: Sal B suppressed intimal hyperplasia induced by carotid artery ligation and decreased Ang II-induced VSMC proliferation by down-regulating the positive cell-cycle regulators KLF5 and cyclin D1. Further experiments showed that VSMC proliferation and upregulation of KLF5 and cyclin D1 induced by Ang II were accompanied by elevated miR-146a level. Furthermore, overexpression of miR-146a promoted and knockdown of miR-146a reduced Ang II-induced VSMC proliferation and ameliorated intimal hyperplasia induced by carotid artery ligation. Sal B inhibited Ang II-induced VSMC proliferation by suppressing miR-146a expression. CONCLUSION: Sal B inhibited Ang II-induced VSMC proliferation in vitro and intimal hyperplasia in vivo by downregulating miR-146a expression.


Assuntos
Benzofuranos/farmacologia , Artérias Carótidas/patologia , MicroRNAs/genética , Músculo Liso Vascular/efeitos dos fármacos , Túnica Íntima/patologia , Angiotensina II/farmacologia , Animais , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/cirurgia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperplasia/tratamento farmacológico , Hiperplasia/genética , Hiperplasia/patologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/efeitos dos fármacos , Neointima/tratamento farmacológico , Neointima/genética , Neointima/patologia , Túnica Íntima/efeitos dos fármacos
14.
Biomed Res ; 40(2): 67-78, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30982802

RESUMO

T1R1 and T1R3 are receptors expressed in taste buds that detect L-amino acids. These receptors are also expressed throughout diverse organ systems, such as the digestive system and muscle tissue, and are thought to function as amino acid sensors. The mechanism of transcriptional regulation of the mouse T1R1 gene (Tas1r1) has not been determined; therefore, in this study, we examined the function of Tas1r1 promoter in the mouse myoblast cell line, C2C12. Luciferase reporter assays showed that a 148-bp region upstream of the ATG start codon of Tas1r1 had a promoter activity. The GT box in the Tas1r1 promoter was conserved in the dog, human, mouse, and pig. Site-directed mutagenesis of this GT box significantly reduced the promoter activation. The GT box in promoters is a recurring motif for Sp/KLF family members. RNAi-mediated depletion of Sp4 and Klf5 decreased Tas1r1 expression, while overexpression of Klf5, but not Sp4, significantly increased Tas1r1 expression. The ENCODE data of chromatin immunoprecipitation and sequencing (ChIP-seq) showed that Klf5 bound to the GT box during the myogenic differentiation. Furthermore, the Klf5 knockout cell lines led to a considerable decrease in the levels of Tas1r1 expression. Collectively, these results showed that Klf5 binds to the GT box in the Tas1r1 promoter and regulates Tas1r1 expression in C2C12 cells.


Assuntos
Fatores de Transcrição Kruppel-Like/genética , Mioblastos/metabolismo , Regiões Promotoras Genéticas , Receptores Acoplados a Proteínas-G/genética , Fator de Transcrição Sp4/genética , Sítio de Iniciação de Transcrição , Animais , Sequência de Bases , Sítios de Ligação , Diferenciação Celular , Linhagem Celular , Sequência Conservada , Cães , Regulação da Expressão Gênica , Genes Reporter , Humanos , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/metabolismo , Luciferases/genética , Luciferases/metabolismo , Camundongos , Desenvolvimento Muscular/genética , Mioblastos/citologia , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Fator de Transcrição Sp4/antagonistas & inibidores , Fator de Transcrição Sp4/metabolismo , Suínos
15.
Chem Biol Interact ; 305: 105-111, 2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-30928399

RESUMO

Kruppel-like factor 2 (KLF2) regulates endothelial functions by modulating endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) pathway. Tetrahydrobiopterin (BH4) and S-glutathionylation of eNOS play essential roles in eNOS uncoupling and activation. However, the influence of KLF2 on eNOS uncoupling and the mechanism of eNOS activation still remain unknown. A hypoxia and reoxygenation (H/R) model of human umbilical vein endothelial cells (HUVECs) was utilized in this study. Cell viability and the eNOS uncoupling-related oxidative stress index were measured. The Nrf2 inhibitor ML385 and HO-1 siRNA were used to elucidate the mechanism of activation. The results show that overexpression of KLF2 increased the cell viability, reduced the lactate dehydrogenase leakage rate, downregulated the generation of O2•- and ONOO-, and increased NO levels and eNOS activity. Overexpression of KLF2 also increased the BH4/BH2 ratio and the GSH/GSSG ratio, thus significantly improving eNOS uncoupling in the H/R model. KLF2 has no regulatory effect on the upstream-associated proteins in eNOS activation. However, when combined with the Nrf2 inhibitor or HO-1 siRNA, the regulatory effect of KLF2 on eNOS uncoupling was strongly reduced. These results suggest that KLF2 could improve eNOS uncoupling via Nrf2/HO-1 in H/R-induced endothelial injury.


Assuntos
Hipóxia Celular , Heme Oxigenase-1/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Oxigênio/química , Sobrevivência Celular/efeitos dos fármacos , Glutationa/metabolismo , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Oxigênio/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo
16.
PLoS Genet ; 15(4): e1008058, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30933982

RESUMO

In the skin and gill epidermis of fish, ionocytes develop alongside keratinocytes and maintain body fluid ionic homeostasis that is essential for adaptation to environmental fluctuations. It is known that ionocyte progenitors in zebrafish embryos are specified from p63+ epidermal stem cells through a patterning process involving DeltaC (Dlc)-Notch-mediated lateral inhibition, which selects scattered dlc+ cells into the ionocyte progenitor fate. However, mechanisms by which the ionocyte progenitor population is modulated remain unclear. Krüppel-like factor 4 (Klf4) transcription factor was previously implicated in the terminal differentiation of mammalian skin epidermis and is known for its bifunctional regulation of cell proliferation in a tissue context-dependent manner. Here, we report novel roles for zebrafish Klf4 in the ventral ectoderm during embryonic skin development. We found that Klf4 was expressed in p63+ epidermal stem cells of the ventral ectoderm from 90% epiboly onward. Knockdown or knockout of klf4 expression reduced the proliferation rate of p63+ stem cells, resulting in decreased numbers of p63+ stem cells, dlc-p63+ keratinocyte progenitors and dlc+ p63+ ionocyte progenitor cells. These reductions subsequently led to diminished keratinocyte and ionocyte densities and resulted from upregulation of the well-known cell cycle regulators, p53 and cdkn1a/p21. Moreover, mutation analyses of the KLF motif in the dlc promoter, combined with VP16-klf4 or engrailed-klf4 mRNA overexpression analyses, showed that Klf4 can bind the dlc promoter and modulate lateral inhibition by directly repressing dlc expression. This idea was further supported by observing the lateral inhibition outcomes in klf4-overexpressing or knockdown embryos. Overall, our experiments delineate novel roles for zebrafish Klf4 in regulating the ionocyte progenitor population throughout early stem cell stage to initiation of terminal differentiation, which is dependent on Dlc-Notch-mediated lateral inhibition.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Epidérmicas/citologia , Células Epidérmicas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Padronização Corporal , Diferenciação Celular , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ectoderma/citologia , Ectoderma/embriologia , Ectoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Brânquias/citologia , Brânquias/embriologia , Brânquias/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transporte de Íons , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas , Receptores Notch/genética , Receptores Notch/metabolismo , Transativadores/genética , Transativadores/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
17.
Nat Commun ; 10(1): 1791, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996251

RESUMO

Apoptotic death of cells damaged by genotoxic stress requires regulatory input from surrounding tissues. The C. elegans scaffold protein KRI-1, ortholog of mammalian KRIT1/CCM1, permits DNA damage-induced apoptosis of cells in the germline by an unknown cell non-autonomous mechanism. We reveal that KRI-1 exists in a complex with CCM-2 in the intestine to negatively regulate the ERK-5/MAPK pathway. This allows the KLF-3 transcription factor to facilitate expression of the SLC39 zinc transporter gene zipt-2.3, which functions to sequester zinc in the intestine. Ablation of KRI-1 results in reduced zinc sequestration in the intestine, inhibition of IR-induced MPK-1/ERK1 activation, and apoptosis in the germline. Zinc localization is also perturbed in the vasculature of krit1-/- zebrafish, and SLC39 zinc transporters are mis-expressed in Cerebral Cavernous Malformations (CCM) patient tissues. This study provides new insights into the regulation of apoptosis by cross-tissue communication, and suggests a link between zinc localization and CCM disease.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Zinco/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/efeitos da radiação , Proteínas Reguladoras de Apoptose/genética , Encéfalo/patologia , Encéfalo/cirurgia , Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/efeitos da radiação , Proteínas de Caenorhabditis elegans/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/cirurgia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína KRIT1/genética , Proteína KRIT1/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutagênese , Mutação , Fosforilação/fisiologia , Alinhamento de Sequência , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
18.
Int Immunopharmacol ; 70: 486-497, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30870679

RESUMO

Macrophages play a pivotal role in destabilizing atherosclerotic plaque. The diverse phenotypes and complex autophagy in macrophage are observed in atherosclerotic lesions. Tanshinone IIA (TNA) is known as the major component extracted from the root of Chinese herb Salvia miltiorrhiza, used for treatment of cardiovascular diseases. However, the therapeutic mechanism of TNA is not clear yet. In this study, we identified inflammation-related gene expression by microarray in atherosclerotic plaques in ApoE knockout mice fed with high fat diet and found miR-375 was one of the significantly high expressed microRNAs compared with wild type mice and TNA treated mice. Then we compared the levels of proteins related to the signal pathway of autophagy, and the phenotype of macrophages in atherosclerotic plaques ex vivo. We predicted KLF4 might be the key target of miR-375 that mediated the crosstalk between autophagy and polarization by TNA. Furthermore, we detected the expression of signal pathway in ox-LDL induced macrophages after treatment with TNA in vitro to verify this predict. The results suggest TNA could activate KLF4 and enhance autophagy as well as M2 polarization of macrophages by inhibiting miR-375 to Attenuate Atherosclerosis.


Assuntos
Aterosclerose/tratamento farmacológico , Diterpenos de Abietano/uso terapêutico , Medicamentos de Ervas Chinesas/uso terapêutico , Fatores de Transcrição Kruppel-Like/metabolismo , Macrófagos/fisiologia , MicroRNAs/genética , Animais , Apolipoproteínas E/genética , Autofagia , Diferenciação Celular , Células Cultivadas , Dieta Hiperlipídica , Modelos Animais de Doenças , Humanos , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor Cross-Talk , Salvia miltiorrhiza/imunologia , Transdução de Sinais
19.
Transl Res ; 208: 105-118, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30904443

RESUMO

Prader-Willi syndrome (PWS) is a complex and multisystem neurobehavioral disorder. The molecular mechanism of PWS is deficiency of paternally expressed gene gene or genes from the chromosome 15q11-q13. Due to imprinted gene regulation, the same genes in the maternal chromosome 15q11-q13 are structurally intact but transcriptionally repressed by an epigenetic mechanism. The unique molecular defect underlying PWS renders an exciting opportunity to explore epigenetic-based therapy to reactivate the expression of repressed PWS genes from the maternal chromosome. Inactivation of H3K9m3 methyltransferase SETDB1 and zinc finger protein ZNF274 results in reactivation of SNRPN and SNORD116 cluster from the maternal chromosomes in PWS patient iPSCs and iPSC-derived neurons, respectively. High content screening of small molecule libraries using cells derived from transgenic mice carrying the SNRPN-EGFP fusion protein has discovered that inhibitors of EHMT2/G9a, a histone 3 lysine 9 methyltransferase, are capable of reactivating expression of paternally expressed SNRPN and SNORD116 from the maternal chromosome, both in cultured PWS patient-derived fibroblasts and in a PWS mouse model. Treatment with an EMHT2/G9a inhibitor also rescues perinatal lethality and failure to thrive phenotypes in a PWS mouse model. These findings present the first evidence to support a proof-of-principle for epigenetic-based therapy for the PWS in humans.


Assuntos
Epigênese Genética , Síndrome de Prader-Willi/genética , Animais , Cromossomos Humanos Par 15 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Transgênicos , Metiltransferases de Proteína/metabolismo , RNA Nucleolar Pequeno/genética
20.
Oxid Med Cell Longev ; 2019: 9425183, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30881601

RESUMO

The Yiqi-Huoxue granule (YQHX) is a traditional Chinese medication widely used in the therapy of the traditional Chinese medicine diagnosis "Qi deficiency" or "blood stasis" in China. Both these symptoms are related to inflammation, but the mechanisms of YQHX against inflammation are largely unknown. Thus, our present study investigated the effects of YQHX on regulating inflammatory responses induced by lipopolysaccharides (LPS) in HUVECs. Our data found that YQHX remarkably inhibits the production of prothrombotic factors, plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF), while it upregulates the protein expression of Kruppel-like factor 2 (KLF2). The increase in PAI-1 and TF was significantly attenuated through a transgenic knockdown in KLF2 with a Lenti-shKLF2 vector. YQHX also decreases the phosphorylation of nuclear factor-κB (NF-κB) p65 and IκB following LPS stimulation, and it effectively suppresses PAI-1 and TF via a NF-κB-dependent mechanism. Taken together, our results suggest that YQHX provides a notable antithrombotic activity via regulating the KLF2 expression and NF-κB signaling pathway in HUVECs. The KLF2 and NF-κB may be potential therapeutic targets for interventions of inflammation associated with atherosclerosis.


Assuntos
Medicamentos de Ervas Chinesas/uso terapêutico , Fatores de Transcrição Kruppel-Like/metabolismo , NF-kappa B/metabolismo , Regulação para Baixo , Medicamentos de Ervas Chinesas/farmacologia , Humanos , Lipopolissacarídeos , Transdução de Sinais , Transfecção
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