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1.
Cell Physiol Biochem ; 53(4): 638-647, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31556253

RESUMO

BACKGROUND/AIMS: Prolonged hyperosmotic shrinkage evokes expression of osmoprotective genes via nuclear factor NFAT5-mediated pathway and activates Na+ influx via hypertonicity-induced cation channels (HICC). In human umbilical vein endothelial cells (HUVEC) elevation of intracellular sodium concentration ([Na+]i) triggers transcription of dozens of early response genes (ERG). This study examined the role of monovalent cations in the expression of Na+i-sensitive ERGs in iso- and hyperosmotically shrunken HUVEC. METHODS: Cell volume was measured by 3D reconstruction of cell shape and as 14C-urea available space. Intracellular Na+ and K+ content was measured by flame atomic absorption spectrometry. ERG transcription was estimated by RT-PCR. RESULTS: Elevation of medium osmolality by 150 mM mannitol or cell transfer from hypo- to isosmotic medium decreased cell volume by 40-50%. Hyperosmotic medium increased [Na+]i by 2-fold whereas isosmotic shrinkage had no impact on this parameter. Hyperosmotic but not isosmotic shrinkage increased up-to 5-fold the content of EGR1, FOS, ATF3, ZFP36 and JUN mRNAs. Expression of these ERGs triggered by hyperosmotic shrinkage and Na+,K+-ATPase inhibition by 0.1 µM ouabain exhibited positive correlation (R2=0.9383, p=0.0005). Isosmotic substitution of NaCl by N-methyl-D-glucamine abolished an increment of [Na+]i and ERG expression triggered by mannitol addition. CONCLUSION: Augmented expression of ERGs in hyperosmotically shrunken HUVEC is mediated by elevation of [Na+]i.


Assuntos
Tamanho Celular , Sódio/metabolismo , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Meglumina/farmacologia , Ouabaína/farmacologia , Potássio/metabolismo , Cloreto de Sódio/farmacologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismo , Tristetraprolina/genética , Tristetraprolina/metabolismo
2.
Nat Commun ; 10(1): 4223, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31530804

RESUMO

Diseases related to impaired blood flow such as peripheral artery disease (PAD) impact nearly 10 million people in the United States alone, yet patients with clinical manifestations of PAD (e.g., claudication and limb ischemia) have limited treatment options. In ischemic tissues, stress kinases such as c-Jun N-terminal kinases (JNKs), are activated. Here, we show that inhibition of the JNK3 (Mapk10) in the neural compartment strikingly potentiates blood flow recovery from mouse hindlimb ischemia. JNK3 deficiency leads to upregulation of growth factors such as Vegfa, Pdgfb, Pgf, Hbegf and Tgfb3 in ischemic muscle by activation of the transcription factors Egr1/Creb1. JNK3 acts through Forkhead box O3 (Foxo3a) to suppress the activity of Egr1/Creb1 transcription regulators in vitro. In JNK3-deficient cells, Foxo3a is suppressed which leads to Egr1/Creb1 activation and upregulation of downstream growth factors. Collectively, these data suggest that the JNK3-Foxo3a-Egr1/Creb1 axis coordinates the vascular remodeling response in peripheral ischemia.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Membro Posterior/irrigação sanguínea , Isquemia/metabolismo , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Neurônios/metabolismo , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Membro Posterior/inervação , Membro Posterior/metabolismo , Humanos , Isquemia/genética , Isquemia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 10 Ativada por Mitógeno/genética , Músculo Esquelético/metabolismo , Fluxo Sanguíneo Regional , Transdução de Sinais
3.
Nat Commun ; 10(1): 3892, 2019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31467272

RESUMO

Life experience can leave lasting marks, such as epigenetic changes, in the brain. How life experience is translated into storable epigenetic information remains largely unknown. With unbiased data-driven approaches, we predicted that Egr1, a transcription factor important for memory formation, plays an essential role in brain epigenetic programming. We performed EGR1 ChIP-seq and validated thousands of EGR1 binding sites with methylation patterns established during postnatal brain development. More specifically, these EGR1 binding sites become hypomethylated in mature neurons but remain heavily methylated in glia. We further demonstrated that EGR1 recruits a DNA demethylase TET1 to remove the methylation marks and activate downstream genes. The frontal cortices from the knockout mice lacking Egr1 or Tet1 share strikingly similar profiles in both gene expression and DNA methylation. In summary, our study reveals EGR1 programs the brain methylome together with TET1 providing new insight into how life experience may shape the brain methylome.


Assuntos
Encéfalo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Neurônios/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Sítios de Ligação , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce/genética , Epigenômica , Regulação da Expressão Gênica , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Neurogênese/fisiologia , Plasticidade Neuronal/fisiologia , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição , Transcriptoma
4.
J Exp Clin Cancer Res ; 38(1): 333, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370861

RESUMO

BACKGROUND: Cathepsin L (CTSL) is a cysteine protease known to have important roles in regulating cancer cellular resistance to chemotherapy. However mechanism underlying which regulates CTSL-mediated drug resistance remain largely unknown. METHODS: We used NSCLC cell lines: A549, A549/TAX (paclitaxel-resistant), A549/DDP (cisplatin-resistant), H460 and PC9 cells, to evaluate CTSL and drug resistance changes. Tumor specimens from 53 patients with NSCLC and Xenograft models was also utilized to explore the regulatory relationship of CTSL, TGF-ß, Egr-1 and CREB. RESULTS: TGF-ß and smad3 were overexpressed only in A549/TAX cells, silencing TGF-ß or smad3 in A549/TAX cells decreased the expression of CTSL and enhanced their sensitivity to paclitaxel. Smad3 binds to the Smad-binding-element(SBE) of the CTSL promoter, resulting in increased activity of the CTSL promoter and subsequent CTSL. Egr-1 and CREB were overexpressed only in A549/DDP cells, and silencing Egr-1 or CREB reduced the expression of CTSL and increased cisplatin cytotoxicity. CREB could affect the activity of the CTSL promoter by binding to it. And the potential regulatory factors of CTSL were consistent in vivo and in human lung cancer. These different regulatory mechanisms of CTSL-mediated drug resistance exist in two other NSCLC cell lines. CONCLUSION: CTSL-mediated drug resistance to paclitaxel and cisplatin may be modulated by different mechanisms. The results of our study identified different mechanisms regulating CTSL-mediated drug resistance and identified smad3 as a novel regulator of CTSL.


Assuntos
Antineoplásicos/farmacologia , Catepsina L/metabolismo , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Paclitaxel/farmacologia , Animais , Catepsina L/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Genes Reporter , Humanos , Masculino , Regiões Promotoras Genéticas , Ligação Proteica , Proteína Smad3/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
5.
BMC Cancer ; 19(1): 791, 2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399076

RESUMO

BACKGROUND: Roles of cancer stem cells and early growth response gene 1 (Egr1) in carcinogenesis have been extensively studied in lung cancer. However, the role of Egr1 in the metastasis of lung cancer remains undetermined, especially in regard to stem cell-related pathways. METHODS: Egr1, osteopontin (OPN) and Oct4 expression in human lung cancer was determined by performing immunohistochemistry. Immunoblotting, ELISA, luciferase reporter assay, chromatin immunoprecipitation assay and RT-PCR were performed to validate the regulation of Oct4-Egr1-OPN axis. Moreover, the effect of Oct4-Egr1-OPN axis on lung cancer progression was evaluated by cell migration assay and mice study. RESULTS: We detected Oct4, Egr1, and OPN expression in clinical specimens from 79 lung cancer patients, including 72 adenocarcinomas and 7 squamous cell carcinomas. High expression of Oct4, Egr1, and OPN accounted for 53, 51, and 57% of the patients, respectively. All of the three biomarkers were positively correlated in clinical human lung cancer. Patients with high expression of OPN were significantly associated with shorter disease-free survivals than those with low expression of OPN (p < 0.05). In lung cancer cells, Oct4 transactivated the Egr1 promoter and upregulated Egr1 expression. In a human lung cancer xenograft model, Oct4-overexpressing tumors expressed elevated levels of Egr1. Furthermore, overexpression of Oct4 in lung cancer cells increased the metastatic potential. CONCLUSIONS: Egr1 exerts a promoting effect on cancer metastasis in Oct4-overexpressing lung cancer. Thus, therapeutic strategies targeting the Oct4/Egr1/OPN axis may be further explored for the treatment of lung cancer, especially when lung cancer is refractory to conventional treatment due to cancer stem cells.


Assuntos
Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Osteopontina/genética , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Regiões Promotoras Genéticas
6.
Int J Mol Sci ; 20(13)2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31284541

RESUMO

The process of arteriogenesis is severely compromised in patients with diabetes mellitus (DM). Earlier studies have reported the importance of Egr-1 in promoting collateral outward remodeling. However, the role of Egr-1 in the presence of DM in outward vessel remodeling was not studied. We hypothesized that Egr-1 expression may be compromised in DM which may lead to impaired collateral vessel growth. Here, we investigated the relevance of the transcription factor Egr-1 for the process of collateral artery growth in diabetic mice. Induction of arteriogenesis by femoral artery ligation resulted in an increased expression of Egr-1 on mRNA and protein level but was severely compromised in streptozotocin-induced diabetic mice. Diabetes mellitus mice showed a significantly reduced expression of Egr-1 endothelial downstream genes Intercellular Adhesion Molecule-1 (ICAM-1) and urokinase Plasminogen Activator (uPA), relevant for extravasation of leukocytes which promote arteriogenesis. Fluorescent-activated cell sorting analyses confirmed reduced leukocyte recruitment. Diabetes mellitus mice showed a reduced expression of the proliferation marker Ki-67 in growing collaterals whose luminal diameters were also reduced. The Splicing Factor-1 (SF-1), which is critical for smooth muscle cell proliferation and phenotype switch, was found to be elevated in collaterals of DM mice. Treatment of DM mice with insulin normalized the expression of Egr-1 and its downstream targets and restored leukocyte recruitment. SF-1 expression and the diameter of growing collaterals were normalized by insulin treatment as well. In summary, our results showed that Egr-1 signaling was impaired in DM mice; however, it can be rescued by insulin treatment.


Assuntos
Diabetes Mellitus Experimental/patologia , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Artéria Femoral/crescimento & desenvolvimento , Insulinas/farmacologia , Morfogênese/efeitos dos fármacos , Transdução de Sinais , Regulação para Cima/efeitos dos fármacos , Animais , Antígenos CD/metabolismo , Circulação Colateral/efeitos dos fármacos , Diabetes Mellitus Experimental/genética , Artéria Femoral/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Leucócitos/patologia , Masculino , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos
7.
Oral Dis ; 25(7): 1759-1768, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31357246

RESUMO

OBJECTIVES: To detect whether early growth response 1 (EGR1) in peripheral blood leucocytes (PBLs) indicates temporomandibular joint (TMJ) osteoarthritis (OA) lesions. MATERIALS AND METHODS: Egr1 mRNA expression levels in PBLs were detected in eight malocclusion patients without temporomandibular disorder (TMD) signs and 16 malocclusion patients with clinical TMD signs with (eight) or without (eight) imaging signs of TMJ OA. Twelve 6-week-old rats were randomized to a control group and a unilateral anterior crossbite (UAC) group and were sampled at 4 weeks. The Egr1 mRNA expression levels in PBLs and protein expression levels in different orofacial tissues were measured. RESULTS: Patients with TMD signs with/without TMJ OA diagnosis showed lower Egr1 mRNA expression levels in PBLs than patients without TMD signs. The lower Egr1 mRNA expression was also found in the PBLs of UAC rats, which were induced to exhibit early histo-morphological signs of TMJ OA lesions. In subchondral bone of UAC rats, EGR1 protein expression was decreased, co-localization of EGR1 with osterix or dentin matrix protein-1 was identified, and the number of EGR1 and osterix double-positive cells was reduced (all p < .05). CONCLUSION: Egr1 reduction in PBLs potentially indicates subchondral bone OA lesions at an early stage.


Assuntos
Cartilagem Articular , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Côndilo Mandibular , Osteoartrite , Transtornos da Articulação Temporomandibular/etiologia , Animais , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Proteína 1 de Resposta de Crescimento Precoce/genética , Má Oclusão/complicações , RNA Mensageiro , Distribuição Aleatória , Ratos , Articulação Temporomandibular , Transtornos da Articulação Temporomandibular/metabolismo , Tomografia Computadorizada por Raios X , Fatores de Transcrição/análise
8.
Artigo em Chinês | MEDLINE | ID: mdl-31245944

RESUMO

OBJECTIVE: To investigate the expression of EGR1 gene and the localization of EGR1 protein in bovine skeletal muscle-derived satellite cells (MDSCs), as well as to investigate the mechanism that EGR1 protein enters the nucleus. METHODS: Bovine MDSCs were cultured in differentiation medium for 1 day, 3 days and 5 days, respectively, and each group was triplicate. The expression of EGR1 gene and the localization of EGR1 protein were studied at different differentiation period in MDSCs by qRT-PC and Western blot. Moreover, the changes on the expression of endogenous EGR1 gene and EGR1 proteins were explored by CRISPRi, site-directed mutagenesis and laser confocal method. RESULTS: The results from the qRT-PCR and Western blot showed that the expressions of EGR1 gene on transcription level and translation level were significantly higher in differentiated cells than those in undifferentiated cells. The highest expression was found on the third day after the differentiation, and then began to decline. Immunofluorescence assays showed that EGR1 proteins were preferentially expressed in differentiated MDSCs, and increased along with the increase of number of myotubes. Confocal observation revealed that some EGR1 proteins were transferred into the nucleus in the differentiation of cells, however, the EGR1 proteins would not be detected in the differentiated MDSCs nuclei if a site directed mutagenesis (serine533) on EGR1 protein occurred. CONCLUSION: During the differentiation of bovine skeletal muscle satellite cells, the transcriptional level of EGR1 gene is increased, and some EGR1 proteins are transferred into the nucleus. The serine phosphorylation at position 533 of the C terminal of EGR1 protein is necessary for the nucleus transfer.


Assuntos
Proteína 1 de Resposta de Crescimento Precoce , Células Satélites de Músculo Esquelético , Animais , Bovinos , Diferenciação Celular , Núcleo Celular , Células Cultivadas , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Fibras Musculares Esqueléticas , Células Satélites de Músculo Esquelético/metabolismo
9.
Nat Commun ; 10(1): 2851, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253786

RESUMO

Male and female brains differ significantly in both health and disease, and yet the female brain has been understudied. Sex-hormone fluctuations make the female brain particularly dynamic and are likely to confer female-specific risks for neuropsychiatric disorders. The molecular mechanisms underlying the dynamic nature of the female brain structure and function are unknown. Here we show that neuronal chromatin organization in the female ventral hippocampus of mouse fluctuates with the oestrous cycle. We find chromatin organizational changes associated with the transcriptional activity of genes important for neuronal function and behaviour. We link these chromatin dynamics to variation in anxiety-related behaviour and brain structure. Our findings implicate an immediate-early gene product, Egr1, as part of the mechanism mediating oestrous cycle-dependent chromatin and transcriptional changes. This study reveals extreme, sex-specific dynamism of the neuronal epigenome, and establishes a foundation for the development of sex-specific treatments for disorders such as anxiety and depression.


Assuntos
Encéfalo/fisiologia , Cromatina/fisiologia , Ciclo Estral/fisiologia , Neurônios/fisiologia , Animais , Comportamento Animal , Encéfalo/citologia , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Epigenômica , Estradiol/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Progesterona/metabolismo , Ligação Proteica , RNA/genética , RNA/metabolismo
10.
Neurobiol Learn Mem ; 161: 122-134, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30965113

RESUMO

A clue to hippocampal function has been the discovery of place cells, leading to the 'spatial map' theory. Although the firing attributes of place cells are well documented, little is known about the organization of the spatial map. Unit recording studies, thus far, have reported a low coherence between neighboring cells and geometric space, leading to the prevalent view that the spatial map is not topographically organized. However, the number of simultaneously recorded units is severely limited, rendering construction of the spatial map nearly impossible. To visualize the functional organization of place cells, we used the activity-dependent immediate-early gene Zif268 in combination with behavioral, pharmacological and electrophysiological methods, in mice and rats exploring an environment. Here, we show that in animals confined to a small part of a maze, principal cells in the CA1/CA3 subfields of the dorsal hippocampus immunoreactive (IR) for Zif268 adhere to a 'cluster-type' organization. Unit recordings confirmed that the Zif268 IR clusters correspond to active place cells, while blockade of NMDAR (which alters place fields) disrupted the Zif268 IR clusters. Contrary to the prevalent view that the spatial map consists of a non-topographic neural network, our results provide evidence for a 'cluster-type' functional organization of hippocampal neurons encoding for space.


Assuntos
Região CA1 Hipocampal , Região CA3 Hipocampal , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Aprendizagem em Labirinto/fisiologia , Rede Nervosa , Células de Lugar , Percepção Espacial/fisiologia , Animais , Comportamento Animal/fisiologia , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/fisiologia , Região CA3 Hipocampal/citologia , Região CA3 Hipocampal/metabolismo , Região CA3 Hipocampal/fisiologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Rede Nervosa/citologia , Rede Nervosa/metabolismo , Rede Nervosa/fisiologia , Células de Lugar/citologia , Células de Lugar/metabolismo , Células de Lugar/fisiologia , Ratos , Ratos Long-Evans , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores
11.
Breast Cancer Res ; 21(1): 47, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30944027

RESUMO

BACKGROUND: Our previous genome-wide association study using the MA.27 aromatase inhibitors adjuvant trial identified SNPs in the long noncoding RNA MIR2052HG associated with breast cancer-free interval. MIR2052HG maintained ERα both by promoting AKT/FOXO3-mediated ESR1 transcription and by limiting ubiquitin-mediated ERα degradation. Our goal was to further elucidate MIR2052HG's mechanism of action. METHODS: RNA-binding protein immunoprecipitation assays were performed to demonstrate that the transcription factor, early growth response protein 1 (EGR1), worked together with MIR2052HG to regulate that lemur tyrosine kinase-3 (LMTK3) transcription in MCF7/AC1 and CAMA-1 cells. The location of EGR1 on the LMTK3 gene locus was mapped using chromatin immunoprecipitation assays. The co-localization of MIR2052HG RNA and the LMTK3 gene locus was determined using RNA-DNA dual fluorescent in situ hybridization. Single-nucleotide polymorphisms (SNP) effects were evaluated using a panel of human lymphoblastoid cell lines. RESULTS: MIR2052HG depletion in breast cancer cells results in a decrease in LMTK3 expression and cell growth. Mechanistically, MIR2052HG interacts with EGR1 and facilitates its recruitment to the LMTK3 promoter. LMTK3 sustains ERα levels by reducing protein kinase C (PKC) activity, resulting in increased ESR1 transcription mediated through AKT/FOXO3 and reduced ERα degradation mediated by the PKC/MEK/ERK/RSK1 pathway. MIR2052HG regulated LMTK3 in a SNP- and aromatase inhibitor-dependent fashion: the variant SNP increased EGR1 binding to LMTK3 promoter in response to androstenedione, relative to wild-type genotype, a pattern that can be reversed by aromatase inhibitor treatment. Finally, LMTK3 overexpression abolished the effect of MIR2052HG on PKC activity and ERα levels. CONCLUSIONS: Our findings support a model in which the MIR2052HG regulates LMTK3 via EGR1, and LMTK3 regulates ERα stability via the PKC/MEK/ERK/RSK1 axis. These results reveal a direct role of MIR2052HG in LMTK3 regulation and raise the possibilities of targeting MIR2052HG or LMTK3 in ERα-positive breast cancer.


Assuntos
Inibidores da Aromatase/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Proteína 1 de Resposta de Crescimento Precoce/genética , Receptor alfa de Estrogênio/genética , Proteínas de Membrana/genética , Proteínas Serina-Treonina Quinases/genética , RNA Longo não Codificante/genética , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Proteínas de Membrana/metabolismo , Modelos Biológicos , Polimorfismo de Nucleotídeo Único , Estabilidade Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Transcrição Genética
12.
Med Sci Monit ; 25: 2293-3004, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31013265

RESUMO

BACKGROUND The occurrence of nonalcoholic fatty liver disease (NAFLD) is closely related to type 2 diabetes, especially in patients with insulin resistance. The purpose of this research was to elucidate the major genes and transcriptional regulation of insulin resistance in the progression of NAFLD. MATERIAL AND METHODS We downloaded the gene expression matrix of GSE89632 from Gene Expression Omnibus. Then the principal component analysis was used to identify whether the samples were clustered. Differentially expressed genes were identified by limma R package. Enrichment analysis and protein­protein interaction network was used to find potential function and screening hub genes. We further used ChIP-seq data from ENCODE to predict the transcriptional regulation of hub genes. Finally, we verified the functions of hub genes with clinical information. RESULTS These hub genes were significantly enriched in "response to insulin", "response to glucose", and "fat cell differentiation". ChIP-seq data showed that EGR1 (early growth response gene-1) may play an important role in the transcriptional regulation of SOCS1 (suppressor of cytokine signaling 1), SOCS3 (suppressor of cytokine signaling 3), and Fos gene family in the liver, as the low expression of EGR1 in patients with insulin resistance may promote the occurrence and development of NAFLD. Similarly, correlation analysis showed that EGR1 was positively correlated with the expression of SOCS1, SOCS3, and the genes of Fos gene family, and EGR1 was negatively correlated with the degree of steatosis. CONCLUSIONS Newly identified hub genes and their transcriptional regulation may promote understanding of the molecular mechanisms underlying insulin resistance related to the progression of NAFLD and provide a new therapy target and biomarkers.


Assuntos
Proteína 1 de Resposta de Crescimento Precoce/genética , Resistência à Insulina/genética , Hepatopatia Gordurosa não Alcoólica/genética , Adulto , Biomarcadores/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Progressão da Doença , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Insulina/metabolismo , Fígado/metabolismo , Masculino , Hepatopatia Gordurosa não Alcoólica/metabolismo , Análise de Componente Principal , Mapas de Interação de Proteínas , Proteína 1 Supressora da Sinalização de Citocina/genética , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo
13.
Nat Commun ; 10(1): 1935, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31028249

RESUMO

Despite their location at the cell surface, several receptor tyrosine kinases (RTK) are also found in the nucleus, as either intracellular domains or full length proteins. However, their potential nuclear functions remain poorly understood. Here we find that a fraction of full length Colony Stimulating Factor-1 Receptor (CSF-1R), an RTK involved in monocyte/macrophage generation, migrates to the nucleus upon CSF-1 stimulation in human primary monocytes. Chromatin-immunoprecipitation identifies the preferential recruitment of CSF-1R to intergenic regions, where it co-localizes with H3K4me1 and interacts with the transcription factor EGR1. When monocytes are differentiated into macrophages with CSF-1, CSF-1R is redirected to transcription starting sites, colocalizes with H3K4me3, and interacts with ELK and YY1 transcription factors. CSF-1R expression and chromatin recruitment is modulated by small molecule CSF-1R inhibitors and altered in monocytes from chronic myelomonocytic leukemia patients. Unraveling this dynamic non-canonical CSF-1R function suggests new avenues to explore the poorly understood functions of this receptor and its ligands.


Assuntos
Regulação da Expressão Gênica , Leucemia Mielomonocítica Crônica/genética , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Sistemas CRISPR-Cas , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cromatina/química , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Corantes Fluorescentes/química , Edição de Genes , Células HEK293 , Histonas/genética , Histonas/metabolismo , Humanos , Leucemia Mielomonocítica Crônica/metabolismo , Leucemia Mielomonocítica Crônica/patologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Maleimidas/química , Cultura Primária de Células , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Transdução de Sinais , Células THP-1 , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/metabolismo
14.
Cells ; 8(3)2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30866413

RESUMO

Chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) is an orphan receptor and member of the nuclear receptor superfamily. Among a series of methylene substituted diindolylmethanes (C-DIMs) containing substituted phenyl and heteroaromatic groups, we identified 1,1-bis(3'-indolyl)-1-(4-pyridyl)-methane (DIM-C-Pyr-4) as an activator of COUP-TFI. Structure activity studies with structurally diverse heteroaromatic C-DIMs showed that the pyridyl substituted compound was active and the 4-pyridyl substituent was more potent than the 2- or 3-pyridyl analogs in transactivation assays in breast cancer cells. The DIM-C-Pyr-4 activated chimeric GAL4-COUP-TFI constructs containing full length, C- or N-terminal deletions, and transactivation was inhibited by phosphatidylinositol-3-kinase and protein kinase A inhibitors. However, DIM-C-Pyr-4 also induced transactivation and interactions of COUP-TFI and steroid receptor coactivators-1 and -2 in mammalian two-hybrid assays, and ligand-induced interactions of the C-terminal region of COUP-TFI were not affected by kinase inhibitors. We also showed that DIM-C-Pyr-4 activated COUP-TFI-dependent early growth response 1 (Egr-1) expression and this response primarily involved COUP-TFI interactions with Sp3 and to a lesser extent Sp1 bound to the proximal region of the Egr-1 promoter. Modeling studies showed interactions of DIM-C-Pyr-4 within the ligand binding domain of COUP-TFI. This report is the first to identify a COUP-TFI agonist and demonstrate activation of COUP-TFI-dependent Egr-1 expression.


Assuntos
Fator I de Transcrição COUP/metabolismo , Indóis/farmacologia , Animais , Fator I de Transcrição COUP/química , Linhagem Celular Tumoral , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Humanos , Ligantes , Camundongos , Modelos Moleculares , Correpressor 2 de Receptor Nuclear/metabolismo , Fatores de Transcrição Sp/metabolismo
15.
J Biol Chem ; 294(15): 5784-5789, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30846562

RESUMO

In mammals, leptin production in adipocytes is up-regulated by feeding and insulin. Although this regulatory connection is central to all physiological effects of leptin, its molecular mechanism remains unknown. Here, we show that the transcription factor early growth response 1, Egr1, is rapidly but transiently induced by insulin in adipose cells both in vitro and in vivo, and its induction is followed by an increase in leptin transcription. ChIP and luciferase assays demonstrate that Egr1 directly binds to and activates the leptin promoter. Interestingly, the lipid droplet protein FSP27 may work as a co-factor for Egr1 in regulating leptin expression. By using siRNA-mediated knockout of Egr1 along with its overexpression in adipocytes, we demonstrate that Egr1 is both necessary and sufficient for the stimulatory effect of insulin on leptin transcription.


Assuntos
Adipócitos/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Insulina/metabolismo , Leptina/biossíntese , Elementos de Resposta , Transcrição Genética , Células 3T3-L1 , Adipócitos/citologia , Animais , Proteína 1 de Resposta de Crescimento Precoce/genética , Regulação da Expressão Gênica , Insulina/genética , Leptina/genética , Masculino , Camundongos , Proteínas/genética , Proteínas/metabolismo
16.
Life Sci ; 223: 47-53, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30858120

RESUMO

Psoriasis is an inflammatory skin disease with preference for the skin and joints that occurs due to hyper-proliferation and abnormal apoptosis of keratinocytes. DUSP1 expression in dermal mesenchymal stem cells (MSCs) is obviously lower in psoriasis patients than that in healthy individuals. The present study aimed to explore the roles of DUSP1 in the proliferation and apoptosis of HaCaT cells treated with a cocktail of M5. We showed that DUSP1 was markedly reduced in psoriasis patients and M5-treated HaCaT cells compared with the control subjects. MTT and BrdU assays revealed that overexpression of DUSP1 significantly suppressed the proliferation of HaCaT cells. Furthermore, DUSP1 decreased M5-induced the upregulation of cyclin D1 and Rb. In addition, we demonstrated that forced overexpression of DUSP1 caused an augment of cell apoptosis rate, c-caspase 3 protein level and the Bax/Bcl-2 ratio. Finally, we determined that enhancing DUSP1 expression resulted in the reduction of p-ERK, p-Elk-1 and Egr-1 protein levels using western blot, and the Chromatin immunoprecipitation (ChIP) assay displayed that p-Elk-1 binds to the promoter of Egr-1 in HaCaT cells. The roles of DUSP1 in cell proliferation and apoptosis were abolished by overexpression of Egr-1. In summary, gain function of DUSP1 regulates proliferation and apoptosis of HaCaT cells through the ERK/Elk-1/Egr-1 signaling pathway.


Assuntos
Apoptose/fisiologia , Proliferação de Células/fisiologia , Fosfatase 1 de Especificidade Dupla/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Queratinócitos/patologia , Sistema de Sinalização das MAP Quinases/fisiologia , Psoríase/patologia , Proteínas Elk-1 do Domínio ets/metabolismo , Apoptose/efeitos dos fármacos , Estudos de Casos e Controles , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Meios de Cultura , Citocinas/farmacologia , Fosfatase 1 de Especificidade Dupla/genética , Proteína 1 de Resposta de Crescimento Precoce/genética , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Psoríase/metabolismo , Pele/metabolismo , Pele/patologia
17.
J Biol Chem ; 294(17): 6871-6887, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-30824540

RESUMO

EPHB6 is a member of the erythropoietin-producing hepatocellular kinase (EPH) family and a receptor tyrosine kinase with a dead kinase domain. It is involved in blood pressure regulation and adrenal gland catecholamine (CAT) secretion, but several facets of EPHB6-mediated CAT regulation are unclear. In this study, using biochemical, quantitative RT-PCR, immunoblotting, and gene microarray assays, we found that EPHB6 up-regulates CAT biosynthesis in adrenal gland chromaffin cells (AGCCs). We observed that epinephrine content is reduced in the AGCCs from male Ephb6-KO mice, caused by decreased expression of tyrosine hydroxylase, the rate-limiting enzyme in CAT biosynthesis. We demonstrate that the signaling pathway from EPHB6 to tyrosine hydroxylase expression in AGCCs involves Rac family small GTPase 1 (RAC1), MAP kinase kinase 7 (MKK7), c-Jun N-terminal kinase (JNK), proto-oncogene c-Jun, activator protein 1 (AP1), and early growth response 1 (EGR1). On the other hand, signaling via extracellular signal-regulated kinase (ERK1/2), p38 mitogen-activated protein kinase, and ELK1, ETS transcription factor (ELK1) was not affected by EPHB6 deletion. We further report that EPHB6's effect on AGCCs was via reverse signaling through ephrin B1 and that EPHB6 acted in concert with the nongenomic effect of testosterone to control CAT biosynthesis. Our findings elucidate the mechanisms by which EPHB6 modulates CAT biosynthesis and identify potential therapeutic targets for diseases, such as hypertension, caused by dysfunctional CAT biosynthesis.


Assuntos
Glândulas Suprarrenais/enzimologia , Células Cromafins/enzimologia , Epinefrina/biossíntese , Receptor EphB6/fisiologia , Transcrição Genética/fisiologia , Tirosina 3-Mono-Oxigenase/genética , Regulação para Cima/fisiologia , Glândulas Suprarrenais/citologia , Animais , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Elementos Facilitadores Genéticos , Epinefrina/metabolismo , Feminino , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor EphB6/genética , Transdução de Sinais , Testosterona/fisiologia , Tirosina 3-Mono-Oxigenase/metabolismo
18.
Int J Dev Neurosci ; 74: 18-26, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30822517

RESUMO

The decline in neurogenesis is a very critical problem in Alzheimer disease. Different biological activities have been reported for medicinal application of quercetin. Herein, we investigated the neurogenesis potential of quercetin in a rat model of Alzheimer's disease induced by amyloid-beta injection. Rats were randomly divided into Control, Alzheimer + Saline and Alzheimer + Quercetin groups. Following the administration of Amyloid-beta, rats in the Alzheimer + Quercetin group received 40 mg/kg/day quercetin orally for one month. Our data demonstrated amyloid-ß injection could impair learning and memory processing in rats indicated by passive avoidance test evaluation. We noted that one-month quercetin treatment alleviated the detrimental effects of amyloid-ß on spatial learning and memory parameters using Morris water maze analysis. Quercetin was found to increase the number of proliferating neural stem/progenitor cells. Notably, quercetin increased the number of DCX-expressing cells, indicating the active dynamic growth of neural progenitor cells in the dentate gyrus of the hippocampus. We further observed that the quercetin improved the number of BrdU/NeuN positive cells contributed to enhanced adult neurogenesis. Based on our results, quercetin had the potential to promote the expression of BDNF, NGF, CREB, and EGR-1 genes involved in regulating neurogenesis. These data suggest that quercetin can play a valuable role in alleviating Alzheimer's disease symptoms by enhancing adult neurogenesis mechanism.


Assuntos
Antioxidantes/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Giro Denteado/patologia , Células-Tronco Neurais/fisiologia , Neurogênese/efeitos dos fármacos , Quercetina/uso terapêutico , Doença de Alzheimer/induzido quimicamente , Doença de Alzheimer/complicações , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/toxicidade , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína de Ligação a CREB/metabolismo , Giro Denteado/efeitos dos fármacos , Modelos Animais de Doenças , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Neuropeptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Ratos , Ratos Wistar
19.
Mol Med Rep ; 19(4): 3305-3313, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30816527

RESUMO

Progressive renal fibrosis is a common complication of chronic kidney disease that results in end­stage renal disorder. It is well established that several microRNAs (miRs) function as critical regulators implicated in fibrotic diseases. However, the role of miR­181 in the development and progression of renal fibrosis remains unclear, and the precise mechanism has not yet been fully defined. The present study identified the functional implications of miR­181 expression during renal fibrosis. miR­181 exhibited significantly reduced expression in the serum of renal fibrosis patients and in the kidneys of mice with unilateral ureteral obstruction (UUO). In addition, miR­181 downregulated the expression of human α­smooth muscle actin (α­SMA) in response to angiotensin II stimulation. Transfection with miR­181 mimics significantly suppressed the expression levels of α­SMA, connective tissue growth factor, collagen type I α1 (COL1A1) and collagen type III α1 (COL3A1) in NRK49F cells. Notably, early growth response factor­1 (Egr1) was identified as a direct target gene of miR­181. Furthermore, in vivo experiments revealed that treatment with miR­181 agonist strongly rescued kidney impairment induced by UUO, as supported by Masson's trichrome staining of kidney tissues and reverse transcription­quantitative polymerase chain reaction analysis of COL1A1 and COL3A1 mRNA levels. Therefore, miR­181 may be regarded as an important mediator in the control of profibrotic markers during renal fibrosis via binding to Egr1, and may be a promising new target in the diagnosis and therapy of renal fibrosis.


Assuntos
Proteína 1 de Resposta de Crescimento Precoce/genética , Regulação da Expressão Gênica , Nefropatias/genética , MicroRNAs/genética , Interferência de RNA , Actinas/genética , Actinas/metabolismo , Adulto , Idoso , Animais , Biomarcadores , Linhagem Celular , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Feminino , Fibrose , Humanos , Imuno-Histoquímica , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Pessoa de Meia-Idade , Ratos
20.
J Exp Clin Cancer Res ; 38(1): 61, 2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30732622

RESUMO

BACKGROUND: Ionizing radiation (IR) is one of the major clinical therapies of cancer, although it increases the epithelial-mesenchymal transition (EMT) of non-small cell lung cancer (NSCLC), unexpectedly. The cellular and molecular mechanisms underlying this role are not completely understood. METHODS: We used NSCLC cell lines as well as tumor specimens from 78 patients with NSCLC to evaluate p53, Cathepsin L (CTSL) and EMT phenotypic changes. Xenograft models was also utilized to examine the roles of mutant p53 (mut-p53) and CTSL in regulating IR-induced EMT of NSCLC. RESULTS: Expression of CTSL was markedly increased in human NSCLC tissues with mutant p53 (mut-p53), and p53 mutation positively correlated with metastasis of NSCLC patients. In human non-small cell lung cancer cell line, H1299 cells transfected with various p53 lentivirus vectors, mut-p53 could promote the invasion and motility of cells under IR, mainly through the EMT. This EMT process was induced by elevating intranuclear CTSL which was regulated by mut-p53 depending on Early growth response protein-1 (Egr-1) activation. In the subcutaneous tumor xenograft model, IR promoted the EMT of the cancer cells in the presence of mut-p53, owing to increase expression and nuclear transition of its downstream protein CTSL. CONCLUSION: Taken together, these data reveal the role of the mut-p53/Egr-1/CTSL axis in regulating the signaling pathway responsible for IR-induced EMT.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Catepsina L/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias Pulmonares/genética , Proteína Supressora de Tumor p53/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Pessoa de Meia-Idade , Radiação Ionizante , Transfecção
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