RESUMO
Peroxiredoxin 6 (Prdx6) is a pleiotropic oxidative stress-response protein that defends cells against reactive oxygen species (ROS)-induced damage. Curcumin, a naturally occurring agent, has diversified beneficial roles including cytoprotection. Using human lens epithelial cells (hLECs) and Prdx6-deficient cells, we show the evidence that curcumin protects cells by upregulating Prdx6 transcription via invoking specificity protein 1 (Sp1) activity against proapoptotic stimuli. Curcumin enhanced Sp1 and Prdx6 mRNA and protein expression in a concentration-dependent manner, as evidenced by western and real-time PCR analyses, and thereby negatively regulated ROS-mediated apoptosis by blunting ROS expression and lipid peroxidation. Bioinformatic analysis and DNA-protein binding assays disclosed three active Sp1 sites (-19/27, -61/69 and -82/89) in Prdx6 promoter. Co-transfection experiments with Sp1 and Prdx6 promoter-chloramphenicol acetyltransferase (CAT) constructs showed that CAT activity was dramatically increased in LECs or Sp1-deficient cells (SL2). Curcumin treatment of LECs enhanced Sp1 binding to its sites, consistent with curcumin-dependent stimulation of Prdx6 promoter with Sp1 sites and cytoprotection. Notably, disruption of Sp1 sites by point mutagenesis abolished curcumin transactivation of Prdx6. Also, curcumin failed to activate Prdx6 expression in the presence of Sp1 inhibitors, demonstrating that curcumin-mediated increased expression of Prdx6 was dependent on Sp1 activity. Collectively, the study may provide a foundation for developing transcription-based inductive therapy to reinforce endogenous antioxidant defense by using dietary supplements.
Assuntos
Curcumina/farmacologia , Células Epiteliais/efeitos dos fármacos , Peroxirredoxina VI/metabolismo , Fator de Transcrição Sp1/metabolismo , Apoptose , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Células Epiteliais/metabolismo , Humanos , Peroxidação de Lipídeos , Dados de Sequência Molecular , Mutagênese , Oligorribonucleotídeos Antissenso/farmacologia , Estresse Oxidativo , Peroxirredoxina VI/antagonistas & inibidores , Peroxirredoxina VI/genética , Regiões Promotoras Genéticas , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição Sp1/genética , Ativação Transcricional , Raios UltravioletaRESUMO
The hypothalamic peptide, nesfatin-1, derived from the precursor NEFA/nucleobindin 2 (NUCB2), was recently identified as anorexigenic signal, acting in a leptin-independent manner. Yet its participation in the regulation of other biological functions gated by body energy status remains unexplored. We show herein that NUCB2/nesfatin-1 is involved in the control of female puberty. NUCB2/nesfatin mRNA and protein were detected at the hypothalamus of pubertal female rats, with prominent signals at lateral hypothalamus (LHA), paraventricular (PVN), and supraoptic (SON) nuclei. Hypothalamic NUCB2 expression raised along pubertal transition, with detectable elevations of its mRNA levels at LHA, PVN, and SON, and threefold increase of its total protein content between late-infantile and peripubertal periods. Conditions of negative energy balance, such as 48 h fasting or sustained subnutrition, decreased hypothalamic NUCB2 mRNA and/or protein levels in pubertal females. At this age, central administration of nesfatin-1 induced modest but significant elevations of circulating gonadotropins, whose magnitude was notably augmented in conditions of food deprivation. Continuous intracerebroventricular infusion of antisense morpholino oligonucleotides (as-MONs) against NUCB2 along pubertal maturation, which markedly reduced hypothalamic NUCB2 protein content, delayed vaginal opening and decreased ovarian weights and serum luteinizing hormone (LH) levels. In contrast, in adult female rats, intracerebroventricular injection of nesfatin did not stimulate LH or follicle-stimulating hormone secretion; neither did central as-MON infusion alter preovulatory gonadotropin surges, despite suppression of hypothalamic NUCB2. In sum, our data are the first to disclose the indispensable role of NUCB2/nesfatin-1 in the central networks driving puberty onset, a function that may contribute to its functional coupling to energy homeostasis.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Hipotálamo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/farmacologia , Neuropeptídeos/farmacologia , Maturidade Sexual/efeitos dos fármacos , Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação a DNA/genética , Feminino , Hormônio Foliculoestimulante/sangue , Região Hipotalâmica Lateral/metabolismo , Injeções Intraventriculares , Hormônio Luteinizante/sangue , Proteínas do Tecido Nervoso/administração & dosagem , Proteínas do Tecido Nervoso/genética , Neuropeptídeos/administração & dosagem , Neuropeptídeos/metabolismo , Nucleobindinas , Oligorribonucleotídeos Antissenso/administração & dosagem , Oligorribonucleotídeos Antissenso/farmacologia , Núcleo Hipotalâmico Paraventricular/metabolismo , RNA Mensageiro , Ratos , Ratos Wistar , Núcleo Supraóptico/metabolismoRESUMO
BACKGROUND: Stearoyl-CoA desaturase 1 (SCD1) is a critical regulator of energy metabolism and inflammation. We have previously reported that inhibition of SCD1 in hyperlipidemic mice fed a saturated fatty acid (SFA)-enriched diet prevented development of the metabolic syndrome, yet surprisingly promoted severe atherosclerosis. In this study we tested whether dietary fish oil supplementation could prevent the accelerated atherosclerosis caused by SCD1 inhibition. METHODS AND RESULTS: LDLr(-/-), ApoB(100/100) mice were fed diets enriched in saturated fat or fish oil in conjunction with antisense oligonucleotide (ASO) treatment to inhibit SCD1. As previously reported, in SFA-fed mice, SCD1 inhibition dramatically protected against development of the metabolic syndrome, yet promoted atherosclerosis. In contrast, in mice fed fish oil, SCD1 inhibition did not result in augmented macrophage inflammatory response or severe atherosclerosis. In fact, the combined therapy of dietary fish oil and SCD1 ASO treatment effectively prevented both the metabolic syndrome and atherosclerosis. CONCLUSIONS: SCD1 ASO treatment in conjunction with dietary fish oil supplementation is an effective combination therapy to comprehensively combat the metabolic syndrome and atherosclerosis in mice.
Assuntos
Aterosclerose/prevenção & controle , Gorduras Insaturadas na Dieta/farmacologia , Óleos de Peixe/farmacologia , Síndrome Metabólica/prevenção & controle , Oligorribonucleotídeos Antissenso/farmacologia , Estearoil-CoA Dessaturase/genética , Animais , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismo , Aterosclerose/tratamento farmacológico , Aterosclerose/imunologia , Terapia Combinada , Ácidos Graxos/farmacologia , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/prevenção & controle , Hiperlipidemias/tratamento farmacológico , Hiperlipidemias/prevenção & controle , Resistência à Insulina , Macrófagos/imunologia , Masculino , Síndrome Metabólica/dietoterapia , Síndrome Metabólica/imunologia , Camundongos , Camundongos Mutantes , Obesidade/tratamento farmacológico , Obesidade/prevenção & controle , Receptores de LDL/genética , Receptores de LDL/metabolismo , Estearoil-CoA Dessaturase/antagonistas & inibidores , Receptor 4 Toll-Like/imunologiaRESUMO
A growing body of evidence concerning estrogen effects cannot be explained by the classic model of hormone action, which involves the binding to estrogen receptors (ERs) alpha and ERbeta and the interaction of the steroid-receptor complex with specific DNA sequences associated with target genes. Using c-fos proto-oncogene expression as an early molecular sensor of estrogen action in ERalpha-positive MCF7 and ER-negative SKBR3 breast cancer cells, we have discovered that 17beta-estradiol (E2), and the two major phytoestrogens, genistein and quercetin, stimulate c-fos expression through ERalpha as well as through an ER-independent manner via the G protein-coupled receptor homologue GPR30. The c-fos response is repressed in GPR30-expressing SKBR3 cells transfected with an antisense oligonucleotide against GPR30 and reconstituted in GPR30-deficient MDA-MB 231 and BT-20 breast cancer cells transfected with a GPR30 expression vector. GPR30-dependent activation of ERK1/2 by E2 and phytoestrogens occurs via a Gbetagamma-associated pertussis toxin-sensitive pathway that requires both Src-related and EGF receptor tyrosine kinase activities. The ability of E2 and phytoestrogens to regulate the expression of growth-related genes such as c-fos even in the absence of ER has interesting implications for understanding breast cancer progression.
Assuntos
Neoplasias da Mama/metabolismo , Estradiol/farmacologia , Isoflavonas/farmacologia , Preparações de Plantas/farmacologia , Proteínas Proto-Oncogênicas c-fos/biossíntese , Receptores Acoplados a Proteínas G/fisiologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Receptor alfa de Estrogênio , Genisteína/farmacologia , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oligorribonucleotídeos Antissenso/farmacologia , Fosforilação , Fitoestrógenos , Plasmídeos/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fos/genética , Quercetina/farmacologia , RNA Mensageiro/biossíntese , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Ativação Transcricional/efeitos dos fármacos , Transfecção , Regulação para Cima/efeitos dos fármacosRESUMO
Two membrane-permeable and RNase-resistant antisense poly-2'-O-(2,4-dinitrophenyl)-oligoribonucleotides (poly-DNP-RNAs) have been synthesized as inhibitors of human breast cancer, with nucleotide sequences complementary to the genes of RIalpha subunit of protein kinase A (RIalpha/PKA) and erbB-2, respectively. Both compounds inhibit the proliferation of SK-Br-3 breast cancer cells in culture above the concentration of 10 microg/ml, but have no effect on nontumorigenic MCF-10A breast cells. These antisense inhibitors also block the cell colony formation in methylcellulose medium, whereas the control poly-DNP-RNA with either random or sense sequence has no effect. RT-PCR data show that the antisense inhibition decreases the concentration of the mRNA. TdT-mediated dUTP nick-end labeling (TUNEL) fluorescence assay indicates that the targeted antisense inhibition by poly-DNP-RNAs leads to apoptosis of SK-Br-3 cells but does not affect nontumorigenic MCF-10A cells. The control poly-DNP-RNAs with random or sense nucleotide sequence are completely inactive.