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1.
Am J Physiol Lung Cell Mol Physiol ; 311(2): L400-11, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27343195

RESUMO

In the lung, heme oxygenase-1 (HO-1) is developmentally regulated, with its highest expression in the first days of life. In addition, neonatal mice have limited HO-1 induction in hyperoxia compared with adults. However, few reports have addressed the functional effect of microRNAs (miRNAs) in the regulation of HO-1 in vivo. The aims of the present study were to characterize changes in lung miRNA expression during postnatal development and in response to hyperoxic exposure, and to identify miRNAs that target lung HO-1 gene expression. Neonatal (<12 h old) and adult (2 mo old) mice were exposed to room air or hyperoxia (95% oxygen) for 72 h. TaqMan low-density array rodent miRNA assays were used to calculate miRNA expression changes between control and hyperoxia groups in neonatal and adult lungs. In neonates, we identified miR-196a, which binds to the 3'-untranslated region of the transcriptional repressor BTB and CNC homology 1 (Bach1) and regulates its expression, and subsequently leads to higher levels of lung HO-1 mRNA compared with levels in adults. Despite the increase at baseline, miR-196a was degraded in hyperoxia resulting in limited HO-1 induction in neonatal mice lungs. Furthermore, the developmental differences in lung HO-1 gene expression can be explained in part by the variation in miRNA-196a and its effect on Bach1. This report is the first to show developmental differences in lung miR-196a and its effect on Bach1 and HO-1 expression at baseline and in hyperoxia.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Heme Oxigenase-1/genética , Pulmão/enzimologia , Proteínas de Membrana/genética , MicroRNAs/fisiologia , Regiões 3' não Traduzidas , Animais , Animais Recém-Nascidos , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Displasia Broncopulmonar/enzimologia , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Heme Oxigenase-1/metabolismo , Pulmão/crescimento & desenvolvimento , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
4.
J Biol Chem ; 289(39): 26882-26894, 2014 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-25107906

RESUMO

With oxidative injury as well as in some solid tumors and myeloid leukemia cells, heme oxygenase-1 (HO-1), the anti-oxidant, anti-inflammatory, and anti-apoptotic microsomal stress protein, migrates to the nucleus in a truncated and enzymatically inactive form. However, the function of HO-1 in the nucleus is not completely clear. Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor and master regulator of numerous antioxidants and anti-apoptotic proteins, including HO-1, also accumulates in the nucleus with oxidative injury and in various types of cancer. Here we demonstrate that in oxidative stress, nuclear HO-1 interacts with Nrf2 and stabilizes it from glycogen synthase kinase 3ß (GSK3ß)-mediated phosphorylation coupled with ubiquitin-proteasomal degradation, thereby prolonging its accumulation in the nucleus. This regulation of Nrf2 post-induction by nuclear HO-1 is important for the preferential transcription of phase II detoxification enzymes such as NQO1 as well as glucose-6-phosphate dehydrogenase (G6PDH), a regulator of the pentose phosphate pathway. Using Nrf2 knock-out cells, we further demonstrate that nuclear HO-1-associated cytoprotection against oxidative stress depends on an HO-1/Nrf2 interaction. Although it is well known that Nrf2 induces HO-1 leading to mitigation of oxidant stress, we propose a novel mechanism by which HO-1, by modulating the activation of Nrf2, sets an adaptive reprogramming that enhances antioxidant defenses.


Assuntos
Antioxidantes/metabolismo , Núcleo Celular/metabolismo , Heme Oxigenase-1/metabolismo , Proteínas de Membrana/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Animais , Núcleo Celular/genética , Células Cultivadas , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Heme Oxigenase-1/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fosforilação/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise
5.
J Biol Chem ; 288(18): 12901-9, 2013 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-23508953

RESUMO

The scaffold protein ISCU facilitates the assembly of iron-sulfur clusters (ISCs), which are essential cofactors for many vital metabolic processes. The mTOR pathways are central to nutrient and energy-sensing networks. Here, we demonstrate that mTORC1 associates with ISCU and phosphorylates ISCU at serine 14. This phosphorylation stabilized ISCU protein. Insufficiency of ISCU triggered by mTORC1 inhibition prevented ISC assembly. Sustained ISCU protein levels enhanced by mTORC1 sensitized TSC2-null cells to iron deprivation due to constitutive ISC biogenesis-triggered iron demand, which outstrips supply. We conclude that the mTORC1 pathway serves to modulate iron metabolism and homeostasis, and we speculate that iron deprivation may be an adjunct in the treatment of cancers characterized by constitutive mTORC1 activation.


Assuntos
Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Proteínas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Células 3T3-L1 , Animais , Células HeLa , Homeostase/fisiologia , Humanos , Proteínas Ferro-Enxofre/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos , Fosforilação/fisiologia , Estabilidade Proteica , Proteínas/genética , Serina-Treonina Quinases TOR/genética
6.
Int J Mol Sci ; 15(6): 11111-25, 2014 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-24955789

RESUMO

Five new (4-8) and three known (1-3) dihydro-ß-agarofuran sesquiterpene polyesters were isolated from the whole plants of Parnassia wightiana. The structures of all compounds were elucidated through spectroscopic analysis including 2D-NMR and HR-MS. The absolute configuration of these compounds was established by X-ray diffraction analysis, comparison of NOESY spectra and biogenetic means. The cytotoxities of compounds 2-8 were evaluated in vitro against HL-60, SMMC-7721, A549, MCF-7 and SW480 cell lines. Compounds 5-7 exhibited the highest activities with IC50 values of 11.8-30.1 µM in most cases. The SAR revealed that the introduction of hydroxyl group was able to significantly improve the activities of the compounds for most of the cell lines.


Assuntos
Antineoplásicos Fitogênicos/química , Celastraceae/química , Extratos Vegetais/química , Sesquiterpenos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/toxicidade , Celastraceae/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Células HL-60 , Humanos , Células MCF-7 , Conformação Molecular , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/toxicidade , Relação Estrutura-Atividade
7.
J Biol Chem ; 287(9): 6230-9, 2012 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-22223647

RESUMO

The transcription factor NF-κB regulates the cellular response to inflammatory and oxidant stress. Although many studies have evaluated NF-κB activity following exposure to oxidative stress, the role of the IκB family of inhibitory proteins in modulating this activity remains unclear. Specifically, the function of IκBß in mediating the cellular response to oxidative stress has not been evaluated. We hypothesized that blocking oxidative stress-induced NF-κB signaling through IκBß would prevent apoptotic cell death. Using IκBß knock-in mice (AKBI), in which the IκBα gene is replaced with the IκBß cDNA, we show that IκBß overexpression prevented oxidative stress-induced apoptotic cell death. This was associated with retention of NF-κB subunits in the nucleus and maintenance of NF-κB activity. Furthermore, the up-regulation of pro-apoptotic genes in WT murine embryonic fibroblasts (MEFs) exposed to serum starvation was abrogated in AKBI MEFs. Inhibition of apoptosis was observed in WT MEFs overexpressing IκBß with simultaneous IκBα knockdown, whereas IκBß overexpression alone did not produce this effect. These findings represent a necessary but not sufficient role of IκBß in preventing oxidant stress-induced cell death.


Assuntos
Apoptose/fisiologia , Fibroblastos/citologia , Proteínas I-kappa B/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/fisiologia , Animais , Linhagem Celular Transformada , Meios de Cultura Livres de Soro/farmacologia , Feminino , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Técnicas de Introdução de Genes , Proteínas I-kappa B/genética , Masculino , Camundongos , Camundongos Mutantes , Gravidez , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
8.
Am J Physiol Lung Cell Mol Physiol ; 301(2): L187-96, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21571903

RESUMO

Postnatal lung development requires proliferation and differentiation of specific cell types at precise times to promote proper alveolar formation. Hyperoxic exposure can disrupt alveolarization by inhibiting cell growth; however, it is not fully understood how this is mediated. The transcription factor CCAAT/enhancer binding protein-α (C/EBPα) is highly expressed in the lung and plays a role in cell proliferation and differentiation in many tissues. After 72 h of hyperoxia, C/EBPα expression was significantly enhanced in the lungs of newborn mice. The increased C/EBPα protein was predominantly located in alveolar type II cells. Silencing of C/EBPα with a transpulmonary injection of C/EBPα small interfering RNA (siRNA) prior to hyperoxic exposure reduced expression of markers of type I cell and differentiation typically observed after hyperoxia but did not rescue the altered lung morphology at 72 h. Nevertheless, when C/EBPα hyperoxia-exposed siRNA-injected mice were allowed to recover for 2 wk in room air, lung epithelial cell proliferation was increased and lung morphology was restored compared with hyperoxia-exposed control siRNA-injected mice. These data suggest that C/EBPα is an important regulator of postnatal alveolar epithelial cell proliferation and differentiation during injury and repair.


Assuntos
Animais Recém-Nascidos , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proliferação de Células , Inativação Gênica , Hiperóxia/metabolismo , Pulmão/patologia , Alvéolos Pulmonares/patologia , Animais , Animais Recém-Nascidos/anatomia & histologia , Animais Recém-Nascidos/metabolismo , Biomarcadores/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diferenciação Celular , Inibidor de Quinase Dependente de Ciclina p21/antagonistas & inibidores , Células Epiteliais/classificação , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Hiperóxia/patologia , Injeções , Pulmão/irrigação sanguínea , Camundongos , Camundongos Endogâmicos C57BL , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Alvéolos Pulmonares/metabolismo , RNA Interferente Pequeno/administração & dosagem , Fatores de Tempo , Distribuição Tecidual
9.
J Biol Chem ; 284(52): 36302-36311, 2009 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-19850937

RESUMO

Zinc protoporphyrin IX (ZnPP), an endogenous heme analogue that inhibits heme oxygenase (HO) activity, represses tumor growth. It can also translocate into the nucleus and up-regulate heme oxygenase 1 (HMOX1) gene expression. Here, we demonstrate that tumor cell proliferation was inhibited by ZnPP, whereas tin protoporphyrin (SnPP), another equally potent HO-1 inhibitor, had no effect. Microarray analysis on 128 tumorigenesis related genes showed that ZnPP suppressed genes involved in cell proliferation and angiogenesis. Among these genes, CYCLIN D1 (CCND1) was specifically inhibited as were its mRNA and protein levels. Additionally, ZnPP inhibited CCND1 promoter activity through an Sp1 and Egr1 overlapping binding site (S/E). We confirmed that ZnPP modulated the S/E site, at least partially by associating with Sp1 and Egr1 proteins rather than direct binding to DNA targets. Furthermore, administration of ZnPP significantly inhibited cyclin D1 expression and progression of a B-cell leukemia/lymphoma 1 tumor in mice by preferentially targeting tumor cells. These observations show HO independent effects of ZnPP on cyclin D1 expression and tumorigenesis.


Assuntos
Ciclina D1/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/biossíntese , Protoporfirinas/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Proteína 1 de Resposta de Crescimento Precoce/antagonistas & inibidores , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Células Hep G2 , Humanos , Células K562 , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/enzimologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/enzimologia , Análise de Sequência com Séries de Oligonucleotídeos , Elementos de Resposta , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp1/metabolismo
10.
Pediatr Res ; 68(6): 484-9, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20805787

RESUMO

Inhaled NO (iNO) may be protective against hyperoxic injury in the premature lung, but the mechanism is unknown. We hypothesized that NO would prevent hyperoxia-induced nuclear factor kappa B (NF-κB) activation in neonatal pulmonary microvascular endothelial cells [human pulmonary microvascular endothelial cell (HPMEC)] and prevent the up-regulation of target genes. After hyperoxic exposure (O2 >95%), nuclear NF-κB consensus sequence binding increased and was associated with IκBα degradation. Both of these findings were prevented by exposure to NO. Furthermore, intracellular adhesion molecule (ICAM)-1 mRNA and protein levels increased in cells exposed to hyperoxia, an effect abrogated by NO. To evaluate the potentially toxic effect of NO plus hyperoxia, cell viability and proliferation were assessed. Cells exposed to NO plus hyperoxia demonstrated improved survival as measured by trypan blue exclusion when compared with cells exposed to hyperoxia alone. These differences in cell death could not be attributed to apoptosis measured by caspase-3 activity. Finally, cellular proliferation inhibited by hyperoxia was rescued by concurrent exposure to NO. These data demonstrate that NO prevents hyperoxia-induced NF-κB activation in HPMEC and results in decreased expression of adhesion molecules and decreased cellular toxicity. This may help to explain the protective effects of NO on hyperoxic injury in the developing lung vasculature.


Assuntos
Células Endoteliais/metabolismo , Hiperóxia/metabolismo , Pulmão/irrigação sanguínea , Microcirculação/fisiologia , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Caspase 3/metabolismo , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Células Endoteliais/citologia , Humanos , Proteínas I-kappa B/metabolismo , Recém-Nascido , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Inibidor de NF-kappaB alfa , Oxigênio/metabolismo , Transdução de Sinais/fisiologia
11.
DNA Cell Biol ; 39(5): 756-765, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32282232

RESUMO

Iron-sulfur (Fe-S) clusters are required for mitochondrial function. Fe-S cluster synthesis occurs in the mitochondria and iron uptake is required for mitochondrial biogenesis. However, Fe-S clusters inhibit the expression of the iron importer transferrin receptor 1 (TfR1), whereas lack of the Fe-S cluster stimulates TfR1 expression. Yet, it is unclear whether Fe-S cluster synthesis increases with mitochondria biogenesis and, in turn, whether this negatively modulates TfR1 expression. We manipulated peroxisome proliferator-activated receptor-gamma coactivator-1α expression to control mitochondrial biogenesis in a variety of cell types, including erythroid cells. We demonstrated that Fe-S cluster synthesis increases with mitochondria biogenesis but does not interfere with increasing TfR1 expression. In fact, TfR1 expression is stimulated through alternative means to meet iron requirement for mitochondria biogenesis. Furthermore, under enhanced mitochondria biogenesis, increased Fe-S cluster synthesis inhibits the function of iron-regulating protein (IRP)1 and hence stimulates the expression of 5'-aminolevulinate synthase 2 (ALAS2), a target of IRP1 and rate-limiting enzyme in erythroid heme biogenesis. Increased ALAS2 expression leads to enhanced heme production, hemoglobinization, and erythropoiesis. Therefore, our study also provides a mechanism to link mitochondrial biogenesis with erythropoiesis and has a potential therapeutic value in the treatment of blood disorders.


Assuntos
Ferro/metabolismo , Biogênese de Organelas , Enxofre/metabolismo , Células 3T3-L1 , 5-Aminolevulinato Sintetase/genética , Animais , Transporte Biológico/efeitos dos fármacos , Células Eritroides/citologia , Células Eritroides/metabolismo , Eritropoese/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Heme/biossíntese , Hemoglobinas/metabolismo , Humanos , Camundongos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/farmacologia
12.
Free Radic Biol Med ; 93: 177-89, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26855417

RESUMO

Diurnal oscillations in the expression of antioxidant genes imply that protection against oxidative stress is circadian-gated. We hypothesized that stabilization of the core circadian gene Rev-erbα (Nr1d1) improves cellular bioenergetics and protects against nutrient deprivation and oxidative stress. Compared to WT, mouse lung fibroblasts (MLG) stably transfected with a degradation resistant Rev-erbα (Ser(55/59) to Asp; hence referred to as SD) had 40% higher protein content, 1.5-fold higher mitochondrial area (confocal microscopy), doubled oxidative phosphorylation by high-resolution respirometry (Oroboros) and were resistant to glucose deprivation for 24h. This resulted from a 4-fold reduction in mitophagy (L3CB co-localized with MitoTracker Red) versus WT. Although PGC1α protein expression was comparable between SD and WT MLG cells, the role of mitochondrial biogenesis in explaining increased mitochondrial mass in SD cells was less clear. Embryonic fibroblasts (MEF) from C57Bl/6-SD transgenic mice, had a 9-fold induction of FoxO1 mRNA and increased mRNA of downstream antioxidant targets heme oxygenase-1 (HO-1), Mn superoxide dismutase and catalase (1.5, 2 fold and 2 fold respectively) versus WT. This allowed the SD cells to survive 1h incubation with 500 µM H2O2 as well as 24h of exposure to 95% O2 and remain attached whereas most WT cells did not. These observations establish a mechanistic link between the metabolic functions of Rev-erbα with mitochondrial homeostasis and protection against oxidative stress.


Assuntos
Antioxidantes/metabolismo , Mitocôndrias/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Estresse Oxidativo/genética , Animais , Catalase/biossíntese , Metabolismo Energético/genética , Fibroblastos/metabolismo , Heme Oxigenase-1/biossíntese , Peróxido de Hidrogênio/metabolismo , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/biossíntese , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Superóxido Dismutase/biossíntese
13.
Oncogene ; 22(2): 198-210, 2003 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-12527889

RESUMO

Transforming growth factor-beta1 (TGF-beta) is a growth factor that has multiple functions including potent inhibition of cell growth. TGF-beta signals by binding to its cell surface serine/threonine kinase receptors, which in turn phosphorylate downstream signal transducers, Smad2 and Smad3. Phosphorylated Smad2 and Smad3, together with Smad4, enter the nucleus and associate with various transcription factors. This complex of transcription factors regulates transcription of a diverse group of genes, leading to growth arrest at G1 phase. Through a functional expression cloning approach, a gag-retinoid X receptor beta (gag-RXRbeta) fusion protein was found to antagonize TGF-beta-induced growth inhibition of mink lung epithelial cells and the fusion between gag and RXRbeta is essential for resistance to the growth inhibition. Like gag-RXRbeta, the oncogenic PLZF-RARalpha fusion protein also antagonizes TGF-beta-induced growth inhibition, and the fusion between PLZF and RARalpha is essential for resistance to TGF-beta. Moreover, TGF-beta and retinoic acid (RA) cooperatively induce growth inhibition as well as transcription of the p15(ink4b) gene, while PLZF-RARalpha represses TGF-beta-induced expression of the p15(ink4b) gene. Together, these results suggest that the TGF-beta and RA pathways cooperate to inhibit cell growth and that PLZF-RARalpha -mediated resistance to TGF-beta may facilitate the development of the PLZF-RARalpha-induced leukemia.


Assuntos
Pulmão/citologia , Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Supressoras de Tumor , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Células Cultivadas , Clonagem Molecular , Inibidor de Quinase Dependente de Ciclina p15 , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Humanos , Vison , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Receptores do Ácido Retinoico/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides , Proteína Smad2 , Proteína Smad3 , Transativadores/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Fator de Crescimento Transformador beta/farmacologia , Tretinoína/metabolismo , Tretinoína/farmacologia
14.
Endocrinology ; 145(7): 3443-50, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15044367

RESUMO

Multiple endocrine neoplasia type I (MEN1) is an inherited tumor syndrome characterized by development of tumors in multiple endocrine organs. The gene mutated in MEN1 patients, Men1, encodes a nuclear protein, menin. Menin interacts with several transcription factors and inhibits their activities. However, it is unclear whether menin is essential for the repression of the expression of endogenous genes. Here, using menin-null cells, we show that menin is essential for repression of the endogenous IGFBP-2, a gene that can regulate cell proliferation. Additionally, complementation of menin-null cells with wild-type menin, but not with a MEN1 disease-related point mutant, restores the function of menin in repressing IGFBP-2. Consistent with this, the promoter of IGFBP-2 is repressed by wild-type menin, but not by a MEN1-related point mutant. Menin also alters the structure of the chromatin surrounding the promoter of the IGFBP-2 gene, as demonstrated by the deoxyribonuclease I hypersensitivity assay. Furthermore, nuclear localization signals in menin are crucial for repressing the expression of IGFBP-2. Together, these results suggest that menin regulates the expression of the endogenous IGFBP-2 gene at least in part through the promoter of IGFBP-2.


Assuntos
Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Neoplasia Endócrina Múltipla Tipo 1/fisiopatologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Animais , Divisão Celular , Linhagem Celular , Fibroblastos/citologia , Regulação Neoplásica da Expressão Gênica , Teste de Complementação Genética , Humanos , Camundongos , Camundongos Mutantes , Mutagênese , Regiões Promotoras Genéticas/fisiologia , Regulação para Cima
15.
Antioxid Redox Signal ; 21(1): 17-32, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24252172

RESUMO

AIMS: The response to oxidative stress and inflammation varies with diurnal rhythms. Nevertheless, it is not known whether circadian genes are regulated by these stimuli. We evaluated whether Rev-erbα, a key circadian gene, was regulated by oxidative stress and/or inflammation in vitro and in a mouse model. RESULTS: A unique sequence consisting of overlapping AP-1 and nuclear factor kappa B (NFκB) consensus sequences was identified on the mouse Rev-erbα promoter. This sequence mediates Rev-erbα promoter activity and transcription in response to oxidative stress and inflammation. This region serves as an NrF2 platform both to receive oxidative stress signals and to activate Rev-erbα, as well as an NFκB-binding site to repress Rev-erbα with inflammatory stimuli. The amplitude of the rhythmicity of Rev-erbα was altered by pre-exposure to hyperoxia or disruption of NFκB in a cell culture model of circadian simulation. Oxidative stress overcame the inhibitory effect of NFκB binding on Rev-erbα transcription. This was confirmed in neonatal mice exposed to hyperoxia, where hyperoxia-induced lung Rev-erbα transcription was further increased with NFκB disruption. Interestingly, this effect was not observed in similarly exposed adult mice. INNOVATION: These data provide novel mechanistic insights into how key circadian genes are regulated by oxidative stress and inflammation in the neonatal lung. CONCLUSION: Rev-erbα transcription and circadian oscillation are susceptible to oxidative stress and inflammation in the neonate. Due to Rev-erbα's role in cellular metabolism, this could contribute to lung cellular function and injury from inflammation and oxidative stress.


Assuntos
Ritmo Circadiano/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Sítios de Ligação , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ensaio Cometa , Dano ao DNA/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , RNA Mensageiro , Transdução de Sinais/efeitos dos fármacos
16.
PLoS One ; 9(3): e90936, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24599172

RESUMO

Premature infants exposed to hyperoxia suffer acute and long-term pulmonary consequences. Nevertheless, neonates survive hyperoxia better than adults. The factors contributing to neonatal hyperoxic tolerance are not fully elucidated. In contrast to adults, heme oxygenase (HO)-1, an endoplasmic reticulum (ER)-anchored protein, is abundant in the neonatal lung but is not inducible in response to hyperoxia. The latter may be important, because very high levels of HO-1 overexpression are associated with significant oxygen cytotoxicity in vitro. Also, in contrast to adults, HO-1 localizes to the nucleus in neonatal mice exposed to hyperoxia. To understand the mechanisms by which HO-1 expression levels and subcellular localization contribute to hyperoxic tolerance in neonates, lung-specific transgenic mice expressing high or low levels of full-length HO-1 (cytoplasmic, HO-1-FL(H) or HO-1-FL(L)) or C-terminally truncated HO-1 (nuclear, Nuc-HO-1-TR) were generated. In HO-1-FL(L), the lungs had a normal alveolar appearance and lesser oxidative damage after hyperoxic exposure. In contrast, in HO-1-FL(H), alveolar wall thickness with type II cell hyperproliferation was observed as well worsened pulmonary function and evidence of abnormal lung cell hyperproliferation in recovery from hyperoxia. In Nuc-HO-1-TR, the lungs had increased DNA oxidative damage, increased poly (ADP-ribose) polymerase (PARP) protein expression, and reduced poly (ADP-ribose) (PAR) hydrolysis as well as reduced pulmonary function in recovery from hyperoxia. These data indicate that low cytoplasmic HO-1 levels protect against hyperoxia-induced lung injury by attenuating oxidative stress, whereas high cytoplasmic HO-1 levels worsen lung injury by increasing proliferation and decreasing apoptosis of alveolar type II cells. Enhanced lung nuclear HO-1 levels impaired recovery from hyperoxic lung injury by disabling PAR-dependent regulation of DNA repair. Lastly both high cytoplasmic and nuclear expression of HO-1 predisposed to long-term abnormal lung cellular proliferation. To maximize HO-1 cytoprotective effects, therapeutic strategies must account for the specific effects of its subcellular localization and expression levels.


Assuntos
Citoproteção , Heme Oxigenase-1/metabolismo , Lesão Pulmonar/enzimologia , Lesão Pulmonar/patologia , Animais , Animais Recém-Nascidos , Apoptose , Carcinogênese/patologia , Proliferação de Células , DNA/metabolismo , Dano ao DNA , Modelos Animais de Doenças , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Humanos , Hidrólise , Hiperóxia/enzimologia , Hiperóxia/patologia , Hiperóxia/fisiopatologia , Pulmão/enzimologia , Pulmão/patologia , Pulmão/fisiopatologia , Lesão Pulmonar/fisiopatologia , Imageamento por Ressonância Magnética , Camundongos , Camundongos Transgênicos , Oxirredução , Estresse Oxidativo , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Alvéolos Pulmonares/enzimologia , Alvéolos Pulmonares/patologia , Alvéolos Pulmonares/fisiopatologia , Testes de Função Respiratória , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/enzimologia
17.
Redox Biol ; 1: 234-43, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24024157

RESUMO

In the newborn, alveolarization continues postnatally and can be disrupted by hyperoxia, leading to long-lasting consequences on lung function. We wanted to better understand the role of heme oxygenase (HO)-1, the inducible form of the rate-limiting enzyme in heme degradation, in neonatal hyperoxic lung injury and repair. Although it was not observed after 3 days of hyperoxia alone, when exposed to hyperoxia and allowed to recover in air (O2/air recovered), neonatal HO-1 knockout (KO) mice had enlarged alveolar spaces and increased lung apoptosis as well as decreased lung protein translation and dysregulated gene expression in the recovery phase of the injury. Associated with these changes, KO had sustained low levels of active ß-catenin and lesser lung nuclear heterogeneous nuclear ribonucleoprotein K (hnRNPK) protein levels, whereas lung nuclear hnRNPK was increased in transgenic mice over-expressing nuclear HO-1. Disruption of HO-1 may enhance hnRNPK-mediated inhibition of protein translation and subsequently impair the ß-catenin/hnRNPK regulated gene expression required for coordinated lung repair and regeneration.


Assuntos
Heme Oxigenase-1/genética , Hiperóxia/metabolismo , Lesão Pulmonar/metabolismo , Pulmão/patologia , Proteínas de Membrana/genética , Ribonucleoproteínas/metabolismo , beta Catenina/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Heme Oxigenase-1/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo K , Hiperóxia/genética , Lesão Pulmonar/genética , Lesão Pulmonar/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
18.
Ther Apher Dial ; 17(1): 78-83, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23379498

RESUMO

To evaluate the effects of pulse high-volume hemofiltration (PHVHF) on severe acute pancreatitis (SAP) with multiple organ dysfunction syndrome (MODS). Thirty patients were divided into two groups: PHVHF group and continuous venovenous hemofiltration (CVVH) group. They were evaluated in terms of clinical symptoms, acute physiology and chronic health evaluation (APACHE) II score, sequential organ failure assessment (SOFA) score, simplified acute physiology (SAPS) II score and biochemical changes. The levels of IL-6, IL-10 and TNF-α in plasma were assessed by ELISA before and after treatment. The doses of dopamine used in shock patients were also analyzed. In the two groups, symptoms were markedly improved after treatment. Body temperature (BT), breath rate (BR), heart rate (HR), APACHE II score, SOFA score, SAPS II score, serum amylase, white blood cell count and C-reactive protein were decreased after hemofiltration (P < 0.05). The PHVHF group was superior to the CVVH group, especially in APACHE II score, CRP (P < 0.01), HR, temperature, SOFA score and SAPS II score (P < 0.05). The doses of dopamine for shock patients were also decreased in the two groups (P < 0.05), with more reduction in the PHVHF group than the CVVH group (P < 0.05). The levels of IL-6, IL-10 and TNF-α decreased (P < 0.05) in the PHVHF group more significantly than the CVVH group (P < 0.01). PHVHF appears to be superior to CVVH in the treatment of SAP with MODS.


Assuntos
Hemofiltração/métodos , Insuficiência de Múltiplos Órgãos/terapia , Pancreatite/terapia , Choque/terapia , APACHE , Doença Aguda , Adulto , Dopamina/administração & dosagem , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Interleucina-10/sangue , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Insuficiência de Múltiplos Órgãos/fisiopatologia , Pancreatite/fisiopatologia , Índice de Gravidade de Doença , Choque/tratamento farmacológico , Fator de Necrose Tumoral alfa/sangue
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