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1.
Mol Cell ; 62(3): 443-452, 2016 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-27151441

RESUMO

S6K1 has been implicated in a number of key metabolic responses, which contribute to obesity. Critical among these is the control of a transcriptional program required for the commitment of mesenchymal stem cells to the adipocytic lineage. However, in contrast to its role in the cytosol, the functions and targets of nuclear S6K1 are unknown. Here, we show that adipogenic stimuli trigger nuclear translocation of S6K1, leading to H2BS36 phosphorylation and recruitment of EZH2 to H3, which mediates H3K27 trimethylation. This blocks Wnt gene expression, inducing the upregulation of PPARγ and Cebpa and driving increased adipogenesis. Consistent with this finding, white adipose tissue from S6K1-deficient mice exhibits no detectable H2BS36 phosphorylation or H3K27 trimethylation, whereas both responses are highly elevated in obese humans or in mice fed a high-fat diet. These findings define an S6K1-dependent mechanism in early adipogenesis, contributing to the promotion of obesity.


Assuntos
Adipócitos/enzimologia , Adipogenia , Tecido Adiposo/enzimologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Histonas/metabolismo , Obesidade/enzimologia , Processamento de Proteína Pós-Traducional , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Tecido Adiposo/patologia , Adiposidade , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Modelos Animais de Doenças , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Epigênese Genética , Células HeLa , Histonas/genética , Humanos , Masculino , Metilação , Camundongos , Obesidade/genética , Obesidade/patologia , PPAR gama/genética , PPAR gama/metabolismo , Fosforilação , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transcrição Gênica , Transfecção , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt
2.
Biochem Biophys Res Commun ; 594: 101-108, 2022 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-35078109

RESUMO

S6K1 serves as an important signaling regulator of cell proliferation and growth in the mTOR signaling pathway. Excessive activation of the mTOR/S6K1 signaling pathway promotes abnormal cell growth and survival, thereby resulting in tumorigenesis. The roles of S6K1 in protein synthesis and metabolism are well known, but an additional role of S6K1 as a gene transcription regulator has not been much understood. Here, we demonstrated that S6K1 is dynamically distributed in the cytoplasm and nuclei of human cervical cancer cells. S6K1 nuclear localization was serum dependent and serum deprivation or rapamycin treatment inhibited S6K1 Thr389 phosphorylation and, thereby, S6K1 was retained in the cytoplasm. Furthermore, we found that endogenous S6K1 interacted with CREB in the cervical cancer cells. Additionally, S6K1 upregulated the CRE-driven promoter luciferase activity. The proto-oncogene c-JUN, which has several CREs, was attenuated in the S6K1 knockdown cervical cancer cells. The binding of CREB/S6K1 to the c-JUN promoter, altered by serum restimulation, was associated with active epigenetic markers. In HeLa cell, 891 promoter regions, to which S6K1 directly binds, were detected. Our findings suggested that active S6K1, which is dynamically translocated into the nucleus, directly binds to chromatin and could play a role in epigenetic mechanisms or transcription factor recruitment.


Assuntos
Núcleo Celular/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transporte Ativo do Núcleo Celular , Citoplasma/metabolismo , Epigênese Genética , Genoma Humano , Genômica , Células HeLa , Humanos , Fosforilação , Regiões Promotoras Genéticas , Elementos de Resposta , Transdução de Sinais , Transcrição Gênica
3.
Proc Natl Acad Sci U S A ; 116(25): 12516-12523, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31164420

RESUMO

BACE1 is the rate-limiting enzyme for amyloid-ß peptides (Aß) generation, a key event in the pathogenesis of Alzheimer's disease (AD). By an unknown mechanism, levels of BACE1 and a BACE1 mRNA-stabilizing antisense RNA (BACE1-AS) are elevated in the brains of AD patients, implicating that dysregulation of BACE1 expression plays an important role in AD pathogenesis. We found that nuclear factor erythroid-derived 2-related factor 2 (NRF2/NFE2L2) represses the expression of BACE1 and BACE1-AS through binding to antioxidant response elements (AREs) in their promoters of mouse and human. NRF2-mediated inhibition of BACE1 and BACE1-AS expression is independent of redox regulation. NRF2 activation decreases production of BACE1 and BACE1-AS transcripts and Aß production and ameliorates cognitive deficits in animal models of AD. Depletion of NRF2 increases BACE1 and BACE1-AS expression and Aß production and worsens cognitive deficits. Our findings suggest that activation of NRF2 can prevent a key early pathogenic process in AD.


Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Transtornos Cognitivos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Transtornos Cognitivos/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Isotiocianatos/farmacologia , Camundongos , Camundongos Transgênicos , Fator 2 Relacionado a NF-E2/biossíntese , Regiões Promotoras Genéticas , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Sulfóxidos , Transcrição Gênica
4.
Int J Mol Sci ; 23(24)2022 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-36555784

RESUMO

Ribosomal protein S6 kinase 1 (S6K1), a key downstream effector of the mammalian target of rapamycin (mTOR), regulates diverse functions, such as cell proliferation, cell growth, and protein synthesis. Because S6K1 was previously known to be localized in the cytoplasm, its function has been mainly studied in the cytoplasm. However, the nuclear localization and function of S6K1 have recently been elucidated and other nuclear functions are expected to exist but remain elusive. Here, we show a novel nuclear role of S6K1 in regulating the expression of the Wnt target genes. Upon activation of the Wnt signaling, S6K1 translocated from the cytosol into the nucleus and subsequently bound to ß-catenin and the cofactors of the Wnt/ß-catenin transcriptional complex, leading to the upregulation of the Wnt target genes. The depletion or repression of S6K1 downregulated the Wnt target gene expression by inhibiting the formation of the Wnt/ß-catenin transcriptional complex. The S6K1-depleted colon cancer cell lines showed lower transcription levels of the Wnt/ß-catenin target genes and a decrease in the cell proliferation and invasion compared to the control cell lines. Taken together, these results indicate that nuclear S6K1 positively regulates the expression of the Wnt target genes by inducing the reciprocal interaction of the subunits of the transcriptional complex.


Assuntos
Via de Sinalização Wnt , beta Catenina , Via de Sinalização Wnt/fisiologia , beta Catenina/genética , beta Catenina/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Núcleo Celular/metabolismo , Linhagem Celular
5.
Molecules ; 26(18)2021 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-34577136

RESUMO

Extensive epigenetic remodeling occurs during the cell fate determination of stem cells. Previously, we discovered that eudesmin regulates lineage commitment of mesenchymal stem cells through the inhibition of signaling molecules. However, the epigenetic modulations upon eudesmin treatment in genomewide level have not been analyzed. Here, we present a transcriptome profiling data showing the enrichment in PRC2 target genes by eudesmin treatment. Furthermore, gene ontology analysis showed that PRC2 target genes downregulated by eudesmin are closely related to Wnt signaling and pluripotency. We selected DKK1 as an eudesmin-dependent potential top hub gene in the Wnt signaling and pluripotency. Through the ChIP-qPCR and RT-qPCR, we found that eudesmin treatment increased the occupancy of PRC2 components, EZH2 and SUZ12, and H3K27me3 level on the promoter region of DKK1, downregulating its transcription level. According to the analysis of GEO profiles, DEGs by depletion of Oct4 showed an opposite pattern to DEGs by eudesmin treatment. Indeed, the expression of pluripotency markers, Oct4, Sox2, and Nanog, was upregulated upon eudesmin treatment. This finding demonstrates that pharmacological modulation of PRC2 dynamics by eudesmin might control Wnt signaling and maintain pluripotency of stem cells.


Assuntos
Furanos , Lignanas , Transcriptoma , Diferenciação Celular , Linhagem Celular , Reposicionamento de Medicamentos , Histonas/metabolismo , Fator 3 de Transcrição de Octâmero , Complexo Repressor Polycomb 2 , Via de Sinalização Wnt
6.
Nucleic Acids Res ; 46(22): 11759-11775, 2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30335163

RESUMO

Constitutive heterochromatin undergoes a dynamic clustering and spatial reorganization during myogenic differentiation. However the detailed mechanisms and its role in cell differentiation remain largely elusive. Here, we report the identification of a muscle-specific long non-coding RNA, ChRO1, involved in constitutive heterochromatin reorganization. ChRO1 is induced during terminal differentiation of myoblasts, and is specifically localized to the chromocenters in myotubes. ChRO1 is required for efficient cell differentiation, with global impacts on gene expression. It influences DNA methylation and chromatin compaction at peri/centromeric regions. Inhibition of ChRO1 leads to defects in the spatial fusion of chromocenters, and mislocalization of H4K20 trimethylation, Suv420H2, HP1, MeCP2 and cohesin. In particular, ChRO1 specifically associates with ATRX/DAXX/H3.3 complex at chromocenters to promote H3.3 incorporation and transcriptional induction of satellite repeats, which is essential for chromocenter clustering. Thus, our results unveil a mechanism involving a lncRNA that plays a role in large-scale heterochromatin reorganization and cell differentiation.


Assuntos
Proteínas de Transporte/genética , Heterocromatina/química , Histonas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Desenvolvimento Muscular/genética , Proteínas Nucleares/genética , RNA Longo não Codificante/genética , Proteína Nuclear Ligada ao X/genética , Animais , Sistemas CRISPR-Cas , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Correpressoras , Feminino , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Células NIH 3T3 , Proteínas Nucleares/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transcrição Gênica , Proteína Nuclear Ligada ao X/metabolismo , Coesinas
7.
Int J Mol Sci ; 21(17)2020 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-32825184

RESUMO

Cisplatin is the most frequently used agent for chemotherapy against cervical cancer. However, recurrent use of cisplatin induces resistance, representing a major hurdle in the treatment of cervical cancer. Our previous study revealed that HP1γ suppresses UBE2L3, an E2 ubiquitin conjugating enzyme, thereby enhancing the stability of tumor suppressor p53 specifically in cervical cancer cells. As a follow-up study of our previous findings, here we have identified that the pharmacological substances, leptomycin B and doxorubicin, can improve the sensitivity of cervical cancer cells to cisplatin inducing HP1γ-mediated elevation of p53. Leptomycin B, which inhibits the nuclear export of HP1γ, increased cisplatin-dependent apoptosis induction by promoting the activation of p53 signaling. We also found that doxorubicin, which induces the DNA damage response, promotes HP1γ-mediated silencing of UBE2L3 and increases p53 stability. These effects resulted from the nuclear translocation and binding of HP1γ on the UBE2L3 promoter. Doxorubicin sensitized the cisplatin-resistant cervical cancer cells, enhancing their p53 levels and rate of apoptosis when administered together with cisplatin. Our findings reveal a therapeutic strategy to target a specific molecular pathway that contributes to p53 degradation for the treatment of patients with cervical cancer, particularly with cisplatin resistance.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Cisplatino/toxicidade , Resistencia a Medicamentos Antineoplásicos , Enzimas de Conjugação de Ubiquitina/metabolismo , Neoplasias do Colo do Útero/metabolismo , Apoptose/efeitos dos fármacos , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Ácidos Graxos Insaturados/farmacologia , Feminino , Células HeLa , Humanos , Proteína Supressora de Tumor p53/metabolismo , Enzimas de Conjugação de Ubiquitina/genética
8.
Molecules ; 25(14)2020 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-32650569

RESUMO

The pharmacological effects of BST204-a fermented ginseng extract-on several types of cancers have been reported. However, the effects of ginseng products or single ginsenosides against cancer stem cells are still poorly understood. In this study, we identified the anti-tumorigenic and anti-invasive activities of BST204 through the suppression of the cancer stem cell marker, CD133. The treatment of embryonic carcinoma cells with BST204 induced the expression of the tumor suppressor protein, p53, which decreased the expression of cell cycle regulatory proteins and downregulated the expression of CD133 and several stemness transcription factors. These changes resulted in both the inhibition of tumor cell proliferation and tumorigenesis. The knockdown of CD133 suggests that it has a role in tumorigenesis, but not in cancer cell proliferation or cell cycle arrest. Treatment with BST204 resulted in the reduced expression of the mesenchymal marker, N-cadherin, and the increased expression of the epithelial marker, E-cadherin, leading to the suppression of tumor cell migration and invasion. The knockdown of CD133 also exhibited an anti-invasive effect, indicating the role of CD133 in tumor invasion. The single ginsenosides Rg3 and Rh2-major components of BST204-exhibited limited effects against cancer stem cells compared to BST204, suggesting possible synergism among several ginsenoside compounds.


Assuntos
Carcinogênese , Carcinoma Embrionário , Movimento Celular/efeitos dos fármacos , Células-Tronco Neoplásicas , Extratos Vegetais/farmacologia , Antígeno AC133/biossíntese , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Embrionário/tratamento farmacológico , Carcinoma Embrionário/metabolismo , Carcinoma Embrionário/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteína Supressora de Tumor p53/biossíntese
9.
Molecules ; 25(24)2020 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-33322233

RESUMO

Obesity causes a wide range of metabolic diseases including diabetes, cardiovascular disease, and kidney disease. Thus, plenty of studies have attempted to discover naturally derived compounds displaying anti-obesity effects. In this study, we evaluated the inhibitory effects of morolic acid 3-O-caffeate (MAOC), extracted from Betula schmidtii, on adipogenesis. Treatment of 3T3-L1 cells with MAOC during adipogenesis significantly reduced lipid accumulation and decreased the expression of adiponectin, a marker of mature adipocytes. Moreover, the treatment with MAOC only during the early phase (day 0-2) sufficiently inhibited adipogenesis, comparable with the inhibitory effects observed following MAOC treatment during the whole processes of adipogenesis. In the early phase of adipogenesis, the expression level of Wnt6, which inhibits adipogenesis, increased by MAOC treatment in 3T3-L1 cells. To identify the gene regulatory mechanism, we assessed alterations in histone modifications upon MAOC treatment. Both global and local levels on the Wnt6 promoter region of histone H3 lysine 4 trimethylation, an active transcriptional histone marker, increased markedly by MAOC treatment in 3T3-L1 cells. Our findings identified an epigenetic event associated with inhibition of adipocyte generation by MAOC, suggesting its potential as an efficient therapeutic compound to cure obesity and metabolic diseases.


Assuntos
Adipogenia/efeitos dos fármacos , Adipogenia/genética , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Triterpenos/química , Triterpenos/farmacologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Camundongos , Estrutura Molecular , Proteínas Proto-Oncogênicas/genética , Proteínas Wnt/genética
10.
J Cell Physiol ; 234(4): 3800-3813, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30132867

RESUMO

Brown adipocytes are characterized by a high number of uncoupling protein 1 (UCP1)-positive mitochondrial content and increased thermogenic capacity. As UCP1-enriched cells can consume lipids by generating heat, browning of white adipocytes is now highlighted as a promising approach for the prevention of obesity and obesity-associated metabolic diseases. Upon cold exposure or ß-adrenergic stimuli, downregulation of microRNA-133 (miR-133) elevates the expression levels of PR domain containing 16 (Prdm16), which has been shown to be a brown adipose determination factor, in brown adipose tissue and subcutaneous white adipose tissues (WAT). Here, we show that treatment of reversine to white adipocytes induces browning via suppression of miR-133a. Reversine treatment promoted the expression of brown adipocyte marker genes, such as Prdm16 and UCP1, increasing the mitochondrial content, while decreasing the levels of miR-133a and white adipocyte marker genes. Ectopic expression of miR-133a mimic reversed the browning effects of the reversine treatment. Moreover, intraperitoneal administration of reversine in mice upregulated thermogenesis and resulted in resistance to high-fat diet-mediated weight gain as well as browning of subcutaneous and epididymal WAT. Taken together, we found a novel way to promote browning of white adipocytes through downregulation of miR-133a followed by activation of Prdm16, with a synthetic chemical, reversine.


Assuntos
Adipócitos Brancos/efeitos dos fármacos , Tecido Adiposo Marrom/efeitos dos fármacos , Fármacos Antiobesidade/farmacologia , MicroRNAs/metabolismo , Morfolinas/farmacologia , Obesidade/prevenção & controle , Purinas/farmacologia , Aumento de Peso/efeitos dos fármacos , Células 3T3-L1 , Adipócitos Brancos/metabolismo , Adipócitos Brancos/patologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Marrom/patologia , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Regulação para Baixo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/patologia , Fenótipo , Transdução de Sinais , Termogênese/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo
11.
BMC Cancer ; 19(1): 773, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387554

RESUMO

BACKGROUND: The mTOR/S6K1 signaling pathway is often activated in cervical cancer, and thus considered a molecular target for cervical cancer therapies. Inhibiting mTOR is cytotoxic to cervical cancer cells and creates a synergistic anti-tumor effect with conventional chemotherapy agents. In this study, we identified a novel S6K1 inhibitor, rosmarinic acid methyl ester (RAME) for the use of therapeutic agent against cervical cancer. METHODS: Combined structure- and ligand-based virtual screening was employed to identify novel S6K1 inhibitors among the in house natural product library. In vitro kinase assay and immunoblot assay was used to examine the effects of RAME on S6K1 signaling pathway. Lipidation of LC3 and mRNA levels of ATG genes were observed to investigate RAME-mediated autophagy. PARP cleavage, mRNA levels of apoptotic genes, and cell survival was measured to examine RAME-mediated apoptosis. RESULTS: RAME was identified as a novel S6K1 inhibitor through the virtual screening. RAME, not rosmarinic acid, effectively reduced mTOR-mediated S6K1 activation and the kinase activity of S6K1 by blocking the interaction between S6K1 and mTOR. Treatment of cervical cancer cells with RAME promoted autophagy and apoptosis, decreasing cell survival rate. Furthermore, we observed that combination treatment with RAME and cisplatin greatly enhanced the anti-tumor effect in cisplatin-resistant cervical cancer cells, which was likely due to mTOR/S6K1 inhibition-mediated autophagy and apoptosis. CONCLUSIONS: Our findings suggest that inhibition of S6K1 by RAME can induce autophagy and apoptosis in cervical cancer cells, and provide a potential option for cervical cancer treatment, particularly when combined with cisplatin.


Assuntos
Antineoplásicos/farmacologia , Cinamatos/farmacologia , Depsídeos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cinamatos/química , Cisplatino/farmacologia , Depsídeos/química , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Técnicas de Silenciamento de Genes , Humanos , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/química , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Neoplasias do Colo do Útero
12.
J Neurosci ; 37(20): 5099-5110, 2017 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-28432138

RESUMO

Excessive mitochondrial fission is a prominent early event and contributes to mitochondrial dysfunction, synaptic failure, and neuronal cell death in the progression of Alzheimer's disease (AD). However, it remains to be determined whether inhibition of excessive mitochondrial fission is beneficial in mammal models of AD. To determine whether dynamin-related protein 1 (Drp1), a key regulator of mitochondrial fragmentation, can be a disease-modifying therapeutic target for AD, we examined the effects of Drp1 inhibitor on mitochondrial and synaptic dysfunctions induced by oligomeric amyloid-ß (Aß) in neurons and neuropathology and cognitive functions in Aß precursor protein/presenilin 1 double-transgenic AD mice. Inhibition of Drp1 alleviates mitochondrial fragmentation, loss of mitochondrial membrane potential, reactive oxygen species production, ATP reduction, and synaptic depression in Aß-treated neurons. Furthermore, Drp1 inhibition significantly improves learning and memory and prevents mitochondrial fragmentation, lipid peroxidation, BACE1 expression, and Aß deposition in the brain in the AD model. These results provide evidence that Drp1 plays an important role in Aß-mediated and AD-related neuropathology and in cognitive decline in an AD animal model. Therefore, inhibiting excessive Drp1-mediated mitochondrial fission may be an efficient therapeutic avenue for AD.SIGNIFICANCE STATEMENT Mitochondrial fission relies on the evolutionary conserved dynamin-related protein 1 (Drp1). Drp1 activity and mitochondria fragmentation are significantly elevated in the brains of sporadic Alzheimer's disease (AD) cases. In the present study, we first demonstrated that the inhibition of Drp1 restored amyloid-ß (Aß)-mediated mitochondrial dysfunctions and synaptic depression in neurons and significantly reduced lipid peroxidation, BACE1 expression, and Aß deposition in the brain of AD mice. As a result, memory deficits in AD mice were rescued by Drp1 inhibition. These results suggest that neuropathology and combined cognitive decline can be attributed to hyperactivation of Drp1 in the pathogenesis of AD. Therefore, inhibitors of excessive mitochondrial fission, such as Drp1 inhibitors, may be a new strategy for AD.


Assuntos
Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Transtornos Cognitivos/fisiopatologia , Dinaminas/metabolismo , Depressão Sináptica de Longo Prazo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Doença de Alzheimer/complicações , Animais , Encéfalo/fisiopatologia , Transtornos Cognitivos/complicações , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibição Neural
13.
J Cell Biochem ; 119(8): 6674-6683, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29665055

RESUMO

The failure of insulin production by pancreatic ß cells is a common hallmark of type 1 diabetes mellitus (T1DM). Because administration of exogenous insulin is associated with diabetes-derived complications, endogenous α to ß cell transition can be an attractive alternative. Although decreased ß cell size and hypoinsulinaemia have been observed in S6K1-deficient mice, the molecular mechanism underlying the involvement of S6K1 in the transcriptional regulation of insulin remains elusive. Here, we show that the hypoinsulinaemic phenotype of S6K1-deficient mice stems from the dysregulated transcription of a set of genes required for insulin and glucagon production. First, we observed that increased expression of α cell marker genes and decreased expression of ß cell marker genes in pancreas tissues from S6K1-deficient mice. Furthermore, S6K1 was highly activated in murine ß cell line, ßTC6, compared to murine α cell line αTC1. In both α and ß cells, active S6K1 promoted the transcription of ß cell marker genes, including insulin, whereas S6K1 inhibition increased the transcription of α cell marker genes. Moreover, S6K1 mediated pancreatic gene regulation by modifying two histone marks (activating H3K4me3 and repressing H3K27me3) on gene promoters. These results suggest that S6K1 drives the α to ß transition through the epigenetic regulation of cell-specific genes, including insulin and glucagon. This novel role of S6K1 in islet cells provides basic clues to establish therapeutic strategies against T1DM.


Assuntos
Antígenos de Diferenciação/biossíntese , Epigênese Genética , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transcrição Gênica , Animais , Antígenos de Diferenciação/genética , Células Secretoras de Glucagon/citologia , Células Secretoras de Insulina/citologia , Camundongos , Camundongos Mutantes , Proteínas Quinases S6 Ribossômicas 90-kDa/genética
14.
Biochem Biophys Res Commun ; 505(4): 1148-1153, 2018 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-30316515

RESUMO

Eudesmin has been reported to possess diverse therapeutic effects, including anti-tumor, anti-inflammatory, and anti-bacterial activities. However, its molecular action has not been implicated in metabolic disease. In this study, we show that treatment of mesenchymal stem cells (MSCs) with eudesmin disturbs adipogenesis via suppression of S6K1 signaling pathway. Eudesmin treatment inhibited activation and nuclear translocation of S6K1. Consequently, S6K1-mediated phosphorylation of H2B at serine 36 (H2BS36p) was reduced upon eudesmin treatment, further inducing the expression of Wnt6, Wnt10a, and Wnt10b, which disturbed adipogenic differentiation. Moreover, eudesmin promoted myogenic and osteogenic gene expression in MSCs. Taken together, we found a novel small molecule, eudesmin, to block adipogenesis through down-regulation of S6K1-H2BS36p axis, followed by regulation of cell fate determination genes. This study suggests a promising therapeutic approach with eudesmin to cure obesity and metabolic diseases.


Assuntos
Adipogenia/efeitos dos fármacos , Furanos/farmacologia , Lignanas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Células Musculares/citologia , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/genética
15.
Biologicals ; 45: 1-8, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27876270

RESUMO

Coagulation factors (II, VII, IX, X, and particularly XIa) remaining in high concentrations in intravenous immunoglobulin (IVIG) preparations can form thrombi, causing thromboembolic events, and in serious cases, result in death. Therefore, manufacturers of biological products must investigate the ability of their production processes to remove procoagulant activities. Previously, we were able to remove coagulation factors II, VII, IX, and X from our IVIG preparation through ethanol precipitation, but factor XIa, which plays an important role in thrombosis, remained in the intermediate products. Here, we used a chromatographic process using a new resin that binds with high capacity to IgG and removes procoagulant activities. The procoagulant activities were reduced to low levels as determined by the thrombin generation assay: <1.56 mIU/mL, chromogenic FXIa assay: <0.16 mIU/mL, non-activated partial thromboplastin time (NaPTT): >250 s, FXI/FXIa ELISA: <0.31 ng/mL. Even after spiking with FXIa at a concentration 32.5 times higher than the concentration in normal specimens, the procoagulant activities were below the detection limit (<0.31 ng/mL). These results demonstrate the ability of our manufacturing process to remove procoagulant activities to below the detection limit (except by NaPTT), suggesting a reduced risk of thromboembolic events that maybe potentially caused by our IVIG preparation.


Assuntos
Fatores de Coagulação Sanguínea/química , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Imunoglobulinas Intravenosas/química , Imunoglobulinas Intravenosas/isolamento & purificação , Humanos , Tromboembolia/induzido quimicamente , Tromboembolia/prevenção & controle
17.
Nucleic Acids Res ; 41(10): 5199-209, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23563152

RESUMO

The mammalian genome encodes multiple variants of histone H3 including H3.1/H3.2 and H3.3. In contrast to H3.1/H3.2, H3.3 is enriched in the actively transcribed euchromatin and the telomeric heterochromatins. However, the mechanism for H3.3 to incorporate into the different domains of chromatin is not known. Here, taking the advantage of well-defined transcription analysis system of yeast, we attempted to understand the molecular mechanism of selective deposition of human H3.3 into actively transcribed genes. We show that there are systemic H3 substrate-selection mechanisms operating even in yeasts, which encode a single type of H3. Yeast HIR complex mediated H3-specific recognition specificity for deposition of H3.3 in the transcribed genes. A critical component of this process was the H3 A-IG code composed of amino acids 87, 89 and 90. The preference toward H3.3 was completely lost when HIR subunits were absent and partially suppressed by human HIRA. Asf1 allows the influx of H3, regardless of H3 type. We propose that H3.3 is introduced into the active euchromatin by targeting the recycling pathway that is mediated by HIRA (or HIR), and this H3-selection mechanism is highly conserved through the evolution. These results also uncover an unexpected role of RI chaperones in evolution of variant H3s.


Assuntos
Evolução Biológica , Chaperonas de Histonas/fisiologia , Histonas/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Sequência Conservada , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiologia , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/química , Humanos , Mutação , Estrutura Terciária de Proteína , Fatores de Transcrição/genética , Leveduras/genética , Leveduras/metabolismo
18.
Biochem Biophys Res Commun ; 451(2): 308-13, 2014 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-25088994

RESUMO

Menin is a gene product of multiple endocrine neoplasia type1 (Men1), an inherited familial cancer syndrome characterized by tumors of endocrine tissues. To gain insight about how menin performs an endocrine cell-specific tumor suppressor function, we investigated the possibility that menin was integrated in a cancer-associated inflammatory pathway in a cell type-specific manner. Here, we showed that the expression of IL-6, a proinflammatory cytokine, was specifically elevated in mouse islet tumor cells upon depletion of menin and Men(-/-) MEF cells, but not in hepatocellular carcinoma cells. Histone H3 lysine (K) 9 methylation, but not H3 K27 or K4 methylation, was involved in menin-dependent IL-6 regulation. Menin occupied the IL-6 promoter and recruited SUV39H1 to induce H3 K9 methylation. Our findings provide a molecular insight that menin-dependent induction of H3 K9 methylation in the cancer-associated interleukin gene might be linked to preventing endocrine-specific tumorigenesis.


Assuntos
Insulinoma/genética , Insulinoma/metabolismo , Interleucina-6/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HeLa , Células Hep G2 , Histamina N-Metiltransferase/metabolismo , Humanos , Interleucina-6/metabolismo , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética
19.
Biochem Biophys Res Commun ; 445(1): 255-62, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24513286

RESUMO

Reversine has been shown to induce dedifferentiation of C2C12 murine myoblasts into multipotent progenitor cells. However, little is known about the key regulators mediating the dedifferentiation induced by reversine. Here, we show that large scale miRNA gene expression profiling of reversine-treated C2C12 myoblasts identifies a down-regulated miRNA, miR-133a, involved in dedifferentiation of myoblasts. Reversine treatment results in up- and down-regulated miRNA profiles. Among miRNAs affected by reversine, the level of muscle-specific miR-133a, which has been shown to be up-regulated during muscle development and to suppress differentiation into other lineages, is markedly reduced by treatment of C2C12 myoblasts with reversine. In parallel, reversine decreases the expression and recruitment of myogenic factor, SRF, to the enhancer regions of miR-133a. Sequentially, down-regulation of miR-133a by reversine is accompanied by a decrease in active histone modifications including trimethylation of histone H3K4 and H3K36, phosphorylation of H3S10, and acetylation of H3K14 on the miR-133a promoter, leading to dissociation of RNA polymerase II from the promoter. Furthermore, inhibition of miR-133a by transfection of C2C12 myoblasts with miR-133a inhibitor increases the expression of osteogenic lineage marker, Ogn, and adipotenic lineage marker, ApoE, similar to that in response to reversine. In contrast, the co-overexpression of miR-133a mimic reversed the effect of reversine on C2C12 myoblast dedifferentiation. Taken together, the results indicate that reversine induces a multipotency of C2C12 myoblasts by suppression of miR-133a expression through depletion of active histone modifications, and suggest that miR-133a is a potential miRNA regulating the reversine-induced dedifferentiation. Collectively, our findings provide a mechanistic rationale for the application of reversine to dedifferentiation of somatic cells.


Assuntos
Epigênese Genética/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , MicroRNAs/genética , Morfolinas/farmacologia , Células-Tronco Multipotentes/efeitos dos fármacos , Purinas/farmacologia , Acetilação/efeitos dos fármacos , Animais , Western Blotting , Desdiferenciação Celular/efeitos dos fármacos , Desdiferenciação Celular/genética , Linhagem Celular , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Perfilação da Expressão Gênica , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Metilação/efeitos dos fármacos , Camundongos , Células-Tronco Multipotentes/metabolismo , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Resposta Sérica/genética , Fator de Resposta Sérica/metabolismo
20.
Proc Natl Acad Sci U S A ; 108(1): 85-90, 2011 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-21173268

RESUMO

In mammals, the canonical histone H3 and the variant H3.3 are assembled into chromatin through replication-coupled and replication-independent (RI) histone deposition pathways, respectively, to play distinct roles in chromatin function. H3.3 is largely associated with transcriptionally active regions via the activity of RI histone chaperone, HIRA. However, the precise role of the RI pathway and HIRA in active transcription and the mechanisms by which H3.3 affects gene activity are not known. In this study, we show that HIRA is an essential factor for muscle development by establishing MyoD activation in myotubes. HIRA and Asf1a, but not CHD1 or Asf1b, mediate H3.3 incorporation in the promoter and the critical upstream regulatory regions of the MyoD gene. HIRA and H3.3 are required for epigenetic transition into the more permissive chromatin structure for polymerase II recruitment to the promoter, regardless of transcription-associated covalent modification of histones. Our results suggest distinct epigenetic management of the master regulator with RI pathway components for cellular differentiation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Desenvolvimento Muscular/fisiologia , Proteína MyoD/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/fisiologia , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Primers do DNA/genética , Imunofluorescência , Immunoblotting , Imunoprecipitação , Camundongos , Análise em Microsséries , Interferência de RNA , RNA Nuclear Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional/genética , Transfecção
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