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1.
Int J Biochem Cell Biol ; 78: 315-326, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27477312

RESUMO

Hepatitis C Virus (HCV) infection presents with a disturbed lipid profile and can evolve to hepatic steatosis and hepatocellular carcinoma (HCC). Hepatocyte Nuclear Factor 4 alpha (HNF4α) is the most abundant transcription factor in the liver, a key regulator of hepatic lipid metabolism and a critical determinant of Epithelial to Mesenchymal Transition and hepatic development. We have previously shown that transient inhibition of HNF4α initiates transformation of immortalized hepatocytes through a feedback loop consisting of miR-24, IL6 receptor (IL6R), STAT3, miR-124 and miR-629, suggesting a central role of HNF4α in HCC. However, the role of HNF4α in Hepatitis C Virus (HCV)-related hepatocarcinoma has not been evaluated and remains controversial. In this study, we provide strong evidence suggesting that HCV downregulates HNF4α expression at both transcriptional and translational levels. The observed decrease of HNF4α expression correlated with the downregulation of its downstream targets, HNF1α and MTP. Ectopic overexpression of HCV proteins also exhibited an inhibitory effect on HNF4α levels. The inhibition of HNF4α expression by HCV appeared to be mediated at transcriptional level as HCV proteins suppressed HNF4α gene promoter activity. HCV also up-regulated IL6R, activated STAT3 protein phosphorylation and altered the expression of acute phase genes. Furthermore, as HCV triggered the loss of HNF4α a consequent change of miR-24, miR-629 or miR-124 was observed. Our findings demonstrated that HCV-related HCC could be mediated through HNF4α-microRNA deregulation implying a possible role of HNF4α in HCV hepatocarcinogenesis. HCV inhibition of HNF4α could be sustained to promote HCC.


Assuntos
Carcinoma Hepatocelular/patologia , Hepacivirus/fisiologia , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Neoplasias Hepáticas/patologia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Fator 1-alfa Nuclear de Hepatócito/genética , Humanos , MicroRNAs/genética , Fosforilação , Regiões Promotoras Genéticas/genética , Receptores de Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Transcrição Gênica
2.
PLoS One ; 11(1): e0147117, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26784701

RESUMO

The control of hyperlipidemia plays a central role in cardiovascular disease. Previously, we have shown that camphene, a constituent of mastic gum oil, lowers cholesterol and triglycerides (TG) in the plasma of hyperlipidemic rats without affecting HMG-CoA reductase activity, suggesting that its hypocholesterolemic and hypotriglyceridemic effects are associated with a mechanism of action different than that of statins. In the present study, we examine the mechanism by which camphene exerts its hypolipidemic action. We evaluated the effect of camphene on the de novo synthesis of cholesterol and TG from [14C]-acetate in HepG2 cells, along with the statin mevinolin. Camphene inhibited the biosynthesis of cholesterol in a concentration-dependent manner, and a maximal inhibition of 39% was observed at 100 µM while mevinolin nearly abolished cholesterol biosynthesis. Moreover, treatment with camphene reduced TG by 34% and increased apolipoprotein AI expression. In contrast, mevinolin increased TG by 26% and had a modest effect on apolipoprotein AI expression. To evaluate the mode of action of camphene, we examined its effects on the expression of SREBP-1, which affects TG biosynthesis and SREBP-2, which mostly affects sterol synthesis. Interestingly, camphene increased the nuclear translocation of the mature form of SREBP-1 while mevinolin was found to increase the amount of the mature form of SREBP-2. The effect of camphene is most likely regulated through SREBP-1 by affecting MTP levels in response to a decrease in the intracellular cholesterol. We propose that camphene upregulates SREBP-1 expression and MTP inhibition is likely to be a probable mechanism whereby camphene exerts its hypolipidemic effect.


Assuntos
Colesterol/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hipolipemiantes/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Terpenos/farmacologia , Triglicerídeos/metabolismo , Animais , Monoterpenos Bicíclicos , Western Blotting , Células Hep G2 , Humanos , Proteínas de Membrana Transportadoras/genética , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína de Ligação a Elemento Regulador de Esterol 1/genética
3.
PLoS One ; 8(9): e73616, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24040001

RESUMO

Recent studies have suggested that aberrant K-ras signaling is responsible for triggering immunological responses and inflammation-driven tumorigenesis. Interleukins IL-17, IL-22, and IL-23 have been reported in various types of malignancies, but the exact mechanistic role of these molecules remains to be elucidated. Given the role of K-ras and the involvement of interleukins in colorectal tumorigenesis, research efforts are reported for the first time, showing that differentially expressed interleukin IL-17, IL-22, and IL-23 levels are associated with K-ras in a stage-specific fashion along colorectal cancer progression. Specifically, a) the effect of K-ras signaling was investigated in the overall expression of interleukins in patients with colorectal cancer and healthy controls, and b) an association was established between mutant K-ras and cytokines GM-CSF and IFN-γ. The results indicate that specific interleukins are differentially expressed in K-ras positive patients and the use of K-ras inhibitor Manumycin A decreases both interleukin levels and apoptosis in Caco-2 cells by inhibiting cell viability. Finally, inflammation-driven GM-CSF and IFN-γ levels are modulated through interleukin expression in tumor patients, with interleukin expression in the intestinal lumen and cancerous tissue mediated by aberrant K-ras signaling. Collectively, the findings a) indicate that interleukin expression is influenced by ras signaling and specific interleukins play an oncogenic promoter role in colorectal cancer, highlighting the molecular link between inflammation and tumorigenesis, and b) accentuate the interwoven molecular correlations as leads to new therapeutic approaches in the future.


Assuntos
Neoplasias Colorretais/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Interferon gama/genética , Interleucina-17/genética , Interleucina-23/genética , Interleucinas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Apoptose/genética , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Progressão da Doença , Relação Dose-Resposta a Droga , Feminino , Perfilação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Interferon gama/metabolismo , Interleucina-17/metabolismo , Interleucina-23/metabolismo , Interleucinas/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , Polienos/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Proteínas Proto-Oncogênicas p21(ras) , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Interleucina 22
4.
Cancer Lett ; 335(2): 387-96, 2013 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-23474496

RESUMO

Vanadium is known for its antitumorigenicity. Poised to investigate the impact of well-defined forms of vanadium on processes and specific biomolecules (oncogenes-proteins) involved in cancer cell physiology, a novel ternary V(V)-peroxido-betaine compound was employed in experiments targeting cell viability, apoptosis, reactive oxygen species (ROS) production, H-ras signaling, and matrix metalloproteinase-2 (MMP-2) expression in human breast cancer epithelial and lung adenocarcinoma cells. The results reveal that vanadium imparts a significant decrease in cancer cell viability, reducing H-ras and MMP-2 expression by increasing ROS-mediated apoptosis, distinctly emphasizing the nature, structure and properties of ternary ligands on vanadium anti-tumor activity and its future potential as a metallodrug.


Assuntos
Adenocarcinoma/tratamento farmacológico , Betaína/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Peróxidos/farmacologia , Vanádio/farmacologia , Adenocarcinoma de Pulmão , Apoptose/efeitos dos fármacos , Betaína/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Peróxidos/química , Proteínas Proto-Oncogênicas p21(ras)/biossíntese , Proteínas Proto-Oncogênicas p21(ras)/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Vanádio/química
5.
J Inorg Biochem ; 121: 100-7, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23376331

RESUMO

Cadmium (Cd) is a well-known metal carcinogen associated with tumor formation and carcinogenesis. It has been shown to induce cancer through various cellular mechanisms involving inhibition of DNA repair, abnormal gene expression, induction of oxidative stress, and triggering apoptosis. It is well-established that the H-ras oncogene is involved in the process of carcinogenesis with direct effects on cellular proliferation and tumorigenesis. Given the biotoxicity of cadmium and its association with carcinogenesis, the effect of that metal ion (Cd(II)) was investigated, in a concentration-dependent fashion, on cell viability, cell proliferation, caspase-3 mediated apoptosis and H-ras gene expression in human breast cancer epithelial MCF-7 cells transfected with the H-ras oncogene (wild type and G12V mutation). The findings show a significant modulation effect of cadmium on H-ras gene expression accompanied by up-regulation of caspase-3-related apoptosis in the concentration range of 100-1000 nΜ cadmium. Concurrently, there is a decrease in MCF-7 proliferation. Collectively, the results a) indicate an interplay of cadmium with H-ras(wt and G12V), with cadmium exhibiting a significant concentration-dependent effect on the modulation of H-ras expression, cell viability and proliferation, and b) project distinctly interwoven roles for both cadmium and H-ras in aberrant physiologies in cancer cells.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Cloreto de Cádmio/farmacologia , Caspase 3/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína Oncogênica p21(ras)/genética , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Mutação , Proteína Oncogênica p21(ras)/metabolismo , Transdução de Sinais/efeitos dos fármacos
6.
Eur J Nutr ; 51(2): 181-90, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21598030

RESUMO

BACKGROUND: Flavonoids have physiological activity and a variety of pharmacological properties, including anticancer activity in vitro, but structure-anticancer activity relationships are unclear. AIM: The objectives of this work were to investigate the activity of dietary flavonol congeners against cell lines derived from human solid tumours and to examine whether the in vitro activity was associated with specific structural feature(s) of the molecules. METHODS: Antiproliferative activity of the flavonol congeners was investigated against eight different human cancer cell lines representing different types of human solid tumour, using the sulforhodamine B (SRB) assay in accordance with the instructions published by the NCI. Cell cycle perturbations caused by the congeners were monitored by flow-cytometric analysis of DNA stained with propidium iodide. RESULTS: Most of the flavonols examined had weak antiproliferative and cytotoxic activity. Of all the flavonol congeners tested peracetylated tiliroside found to be the most powerful, with significant antiproliferative and cytotoxic activity. Most flavonols induced similar cell cycle perturbations, whereas induction of apoptosis was significant only for cells treated with peracetylated tiliroside. CONCLUSIONS: These findings indicated that the -OH groups of aromatic ring B were not linked to the cytotoxic and antiproliferative activity of the tested flavonols whereas peracetylation of the glycosides resulted in moderate improvement. In contrast, acetylation of tiliroside esterified with coumaric acid at position 5 of the sugar moiety greatly improved the activity of this congener. Overall, the results of this study suggest a critical role of sugar moiety substituents in the anticancer activity of the flavonols.


Assuntos
Divisão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dieta , Flavonóis/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Ácidos Cumáricos/metabolismo , Flavonoides/farmacologia , Glicosídeos/metabolismo , Humanos , Neoplasias/patologia , Relação Estrutura-Atividade
7.
Cell ; 147(6): 1233-47, 2011 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-22153071

RESUMO

Hepatocyte nuclear factor 4α (HNF4α) is essential for liver development and hepatocyte function. Here, we show that transient inhibition of HNF4α initiates hepatocellular transformation through a microRNA-inflammatory feedback loop circuit consisting of miR-124, IL6R, STAT3, miR-24, and miR-629. Moreover, we show that, once this circuit is activated, it maintains suppression of HNF4α and sustains oncogenesis. Systemic administration of miR-124, which modulates inflammatory signaling, prevents and suppresses hepatocellular carcinogenesis by inducing tumor-specific apoptosis without toxic side effects. As we also show that this HNF4α circuit is perturbed in human hepatocellular carcinomas, our data raise the possibility that manipulation of this microRNA feedback-inflammatory loop has therapeutic potential for treating liver cancer.


Assuntos
Carcinoma Hepatocelular/metabolismo , Transformação Celular Neoplásica , Fator 4 Nuclear de Hepatócito/metabolismo , Inflamação/metabolismo , Neoplasias Hepáticas/metabolismo , MicroRNAs/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos , Receptores de Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo
8.
PLoS One ; 6(11): e20516, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22073134

RESUMO

BACKGROUND: Central to the pathology of coronary heart disease is the accumulation of lipids, cholesterol and triglycerides, within the intima of arterial blood vessels. The search for drugs to treat dislipidemia, remains a major pharmaceutical focus. In this study, we evaluated the hypolipidemic properties of the essential oil from Chios mastic gum (MGO). METHODOLOGY/PRINCIPAL FINDINGS: The hypolipidemic effect of MGO was investigated in naïve as well as in rats susceptible to detergent-induced hyperlipidemia. Serum cholesterol and triglycerides were determined using commercial kits. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase activity was measured in HepG2 cell extracts using a radioactive assay; cellular cholesterol and cholesterol esters were assessed using gas chromatography. MGO administration into naïve rats resulted in a dose-dependent reduction in the constitutive synthesis of serum cholesterol and triglycerides. In hyperlipidemic rats, MGO treatment had also a strong hypolipidemic effect. By testing various components of MGO, we show for the first time that the hypolipidemic action is associated with camphene. Administration of camphene at a dose of 30 µg/gr of body weight in hyperlipidemic rats resulted in a 54.5% reduction of total cholesterol (p<0.001), 54% of Low Density Lipoprotein (LDL)-cholesterol (p<0.001) and 34.5% of triglycerides (p<0.001). Treatment of HepG2 cells with camphene led to a decrease in cellular cholesterol content to the same extend as mevinolin, a known HMG-CoA reductase inhibitor. The hypolipidemic action of camphene is independent of HMG-CoA reductase activity, suggesting that its hypocholesterolemic and hypotriglyceridemic effects are associated with a mechanism of action different than that of statins. CONCLUSIONS: Given the critical role that the control of hyperlipidemia plays in cardiovascular disease, the results of our study provide insights into the use of camphene as an alternative lipid lowering agent and merits further evaluation.


Assuntos
Acil Coenzima A/metabolismo , Colesterol/sangue , Hipolipemiantes/uso terapêutico , Terpenos/uso terapêutico , Triglicerídeos/sangue , Animais , Monoterpenos Bicíclicos , Linhagem Celular , Cromatografia Gasosa , Relação Dose-Resposta a Droga , Feminino , Humanos , Ratos , Ratos Endogâmicos F344
9.
FEBS Lett ; 585(1): 78-84, 2011 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-21130767

RESUMO

A significant amount of nuclear p53 is found associated with the nuclear matrix in cells that were exposed to genotoxic stress. In this study we identified Scaffold attachment factor B1 (SAFB1), a nuclear matrix-associated protein that binds the scaffold or matrix attachment regions (S/MARs) of genomic DNA, as a novel p53-interacting protein. SAFB1 was able to associate with p53 through its C-terminal domain, while significant co-localization of the two proteins was observed in cells treated with 5-fluorouracil or mithramycin. Binding of p53 to SAFB1 had a significant functional outcome, since SAFB1 was shown to suppress p53-mediated reporter gene expression. These data suggest that nuclear matrix-associated proteins may play a critical role in regulating p53 localization and activity.


Assuntos
Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Receptores de Estrogênio/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Antineoplásicos/farmacologia , Núcleo Celular/metabolismo , Fluoruracila/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Immunoblotting , Células K562 , Proteínas de Ligação à Região de Interação com a Matriz/genética , Microscopia de Fluorescência , Proteínas Associadas à Matriz Nuclear/genética , Plicamicina/farmacologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Receptores de Estrogênio/genética , Transfecção , Proteína Supressora de Tumor p53/genética , Técnicas do Sistema de Duplo-Híbrido
10.
Mol Cell Biochem ; 340(1-2): 291-300, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20232113

RESUMO

Besides the well-characterized genomic action of thyroid hormone (TH), mediated by thyroid hormone receptors (TRs), accumulating data support the so-called non-genomic action of TH, which is often related to activation of signalling pathways. In this study, we sought to determine whether TH activates intracellular signalling pathways in the adult cardiac myocytes and whether such activation modulates cell growth and the expression of target proteins important in cardiac function. We demonstrate that TH promotes a rapid increase in the phosphorylation of several kinases, ERK1/2, PKCdelta, p38-MAPK and Akt. This activation is inhibited by triiodothyroacetic acid (triac), which is a TH analogue known to displace the hormone from membrane bound receptors, indicating that this TH effect is mediated through a cell membrane-initiated mechanism. Furthermore, using specific inhibitors of the TH-activated kinases, we show that the long-term effects of TH on the expression of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), alpha- and beta-myosin heavy chain (MHC) and cell growth are reverted, implying that what is initiated as a non-genomic action of the hormone interfaces with genomic effects. These data provide further insights into the underlying mechanisms of TH action in the heart with potentially important implications in the management of cardiac pathology.


Assuntos
Crescimento Celular , Miócitos Cardíacos/metabolismo , Transdução de Sinais , Tri-Iodotironina/metabolismo , Fatores Etários , Animais , Crescimento Celular/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática , Regulação da Expressão Gênica , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Fosforilação , Proteína Quinase C-delta/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Tri-Iodotironina/análogos & derivados , Tri-Iodotironina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
11.
FEBS J ; 273(9): 1948-58, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16640558

RESUMO

Hepatocyte nuclear factor-4 (HNF-4alpha), a member of the nuclear receptor superfamily, binds DNA exclusively as a homodimer. Dimerization controls important aspects of receptor function, such as DNA binding, protein stability, ligand binding and interaction with coactivators. Crystallographic data of the HNF-4alpha ligand-binding domain (LBD) demonstrated that the homodimer interface is composed of residues in helices 7, 9 and 10 with intermolecular salt bridges, hydrogen bonds and hydrophobic interactions contributing to the stability of the interface. To investigate the importance of the proposed ionic interactions for HNF-4alpha dimerization, interactions critical for formation of the LBD homodimer interface were disrupted by introducing point mutations in residues D261N (H7), E269Q (H7), Q307L (H9), D312N (H9) and Q336L (H10). Mutants were analysed for transactivation, coactivator interaction, DNA binding and dimerization. EMSA analysis showed that the mutants are able to bind DNA as dimers and coimmunoprecipitation assays confirmed dimerization in solution. Furthermore, the mutations do not compromise HNF-4alpha activity and are responsive to PPAR-gamma coactivator-1 (PGC-1). Finally, residue R324, located in the H9/H10 loop, which was suspected to be involved in dimer stabilization via an ionic interaction with residue E276, was studied. In contrast to the conservative substitution R324H the mutation R324L abolishes HNF-4alpha transcriptional activity and coactivator recruitment, revealing that the nature of substitution may play an important role in HNF-4alpha function.


Assuntos
Fator 4 Nuclear de Hepatócito/química , Fator 4 Nuclear de Hepatócito/genética , Mutação , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Células COS , Chlorocebus aethiops , DNA/metabolismo , Dimerização , Fator 4 Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/fisiologia , Humanos , Dados de Sequência Molecular , Ligação Proteica/genética , Ratos
12.
J Biol Chem ; 280(23): 21810-9, 2005 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-15826954

RESUMO

Hepatocyte nuclear factor-4 (HNF-4) is a transcription factor of the nuclear hormone receptor superfamily that is constitutively active without the addition of exogenous ligand. Crystallographic analysis of the HNF-4alpha and HNF-4gamma ligand binding domains (LBDs) demonstrated the presence of endogenous ligands that may act as structural cofactors for HNF-4. It was also proposed by crystallographic studies that a combination of ligand and coactivator might be required to lock the receptor in its active state. We previously showed that mutations in amino acid residues Ser-181 and Met-182 in H3, Leu-219 and Leu-220 and Arg-226 in H5, Ileu-338 in H10, and Ileu-346 in H11, which line the LBD pocket in HNF-4alpha and come in contact with the ligand, impair its transactivation potential. In the present study, physical and functional interaction assays were utilized with two different coactivators, PGC-1 and SRC-3, to address the role of coactivators in HNF-4 function. We show that the integrity of the hinge (D) domain of HNF-4alpha and the activation function (AF)-2 activation domain region are critical for coactivation. Surprisingly, a different mode of coactivation is observed among the LBD point mutants that lack transcriptional activity. In particular, coactivation is maintained in mutants Ser-181, Arg-226, and Ile-346 but is abolished in mutants Met-182, Leu-219, and Ile-338. Physical interactions confirm this pattern of activation, implying that distinct amino acid residues may be involved in coactivator and ligand interactions, although some residues may be critical for both functions. Our results provide evidence and expand predictions based on the crystallographic data as to the role of coactivators in HNF-4alpha constitutive transcriptional activity.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Fosfoproteínas/fisiologia , Fatores de Transcrição/fisiologia , Aminoácidos/química , Animais , Arginina/química , Biotina/química , Células COS , Linhagem Celular , Cloranfenicol O-Acetiltransferase/metabolismo , Proteínas de Ligação a DNA/química , Detergentes/farmacologia , Dimerização , Deleção de Genes , Vetores Genéticos , Glutationa Transferase/metabolismo , Fator 4 Nuclear de Hepatócito , Humanos , Isoleucina/química , Leucina/química , Ligantes , Metionina/química , Modelos Biológicos , Mutação , Fosfoproteínas/química , Plasmídeos/metabolismo , Mutação Puntual , Ligação Proteica , Estrutura Terciária de Proteína , Serina/química , Fatores de Transcrição/química , Transcrição Gênica , Transfecção
13.
J Mol Cell Cardiol ; 37(5): 1001-11, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15522277

RESUMO

cAMP responsive element binding protein (CREB) is a stimulus induced transcription factor with possible relevance for the pathophysiology of the heart. In the present study, we provide evidence that the hypertrophic agonist, phenylephrine (PE), promotes phosphorylation of CREB in adult rat cardiac myocytes through alpha(1)- and beta-adrenergic receptors. PE-induced phosphorylation of CREB was partially inhibited by Ro318220 and H89, which were shown to be potent inhibitors of mitogen- and stress-activated protein kinase-1 (MSK1) activation, implicating the involvement of this kinase in the response. Similar results were obtained when cardiac myocytes were treated with the inhibitors of ERK1/2 and p38 MAPK pathways. In addition, inhibition of protein kinase A by RpcAMP reduced phosphorylation of CREB, suggesting that this pathway is also involved. Furthermore, PE stimulation was accompanied by an increase in CRE-binding activity, which was reduced by drugs that prevented phosphorylation of CREB. An enhanced CBP/phospho-CREB complex formation was also observed, suggesting recruitment of CBP to phosphorylated CREB. These results suggest that PE stimulates phosphorylation and DNA binding activity of CREB in adult rat ventricular myocytes through multiple signaling pathways involving ERK1/2, p38 MAPK, MSK1 and PKA. The same pathways seem to regulate atrial natriuretic peptide (ANF) mRNA expression, a highly conserved marker gene of cardiac hypertrophy, suggesting that the PE-stimulated activation of CREB is likely to play an important role in the hypertrophic response.


Assuntos
Agonistas Adrenérgicos/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Miócitos Cardíacos/enzimologia , Fenilefrina/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/fisiologia , Agonistas de Receptores Adrenérgicos alfa 1 , Animais , Fator Natriurético Atrial/genética , Proteína de Ligação a CREB , Cardiomegalia/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Regulação da Expressão Gênica/efeitos dos fármacos , Imunoprecipitação , Indóis/farmacologia , Isoquinolinas/farmacologia , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ratos , Receptores Adrenérgicos alfa 1/fisiologia , Receptores Adrenérgicos beta/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sulfonamidas/farmacologia , Transativadores/imunologia , Transativadores/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia
14.
J Biol Chem ; 279(29): 30680-8, 2004 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-15123688

RESUMO

Hepatocyte nuclear factor-4alpha (HNF-4alpha), a member of the nuclear receptor superfamily, is a crucial regulator of a large number of genes involved in glucose, cholesterol, and fatty acid metabolism. Unlike other members of the superfamily, HNF-4alpha activates transcription in the absence of exogenously added ligand. Recently published crystallographic data show that fatty acids are endogenous ligands for HNF-4. Transcriptional analysis of point mutations of the residues that are located in helices H3, H5, H10, and H11, which have been shown to come in contact with the ligand, resulted in a dramatic decrease in activity, without affecting DNA binding and dimerization. Our results show the importance of residues Ser-181, Met-182 in H3, Leu-219, Leu-220 and Arg-226 in H5, Ile-338 in H10, and Ile-346 in H11 that line the ligand-binding domain pocket in HNF-4alpha and impair its transactivation potential. Structural modeling reveals that the mutations do not cause any large scale structural alterations, and the observed loss in transactivation can be attributed to local changes, demonstrating that these residues play a significant role in maintaining the structural integrity of the HNF-4alpha ligand binding pocket.


Assuntos
Proteínas de Ligação a DNA/química , Fosfoproteínas/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Western Blotting , Células COS , Linhagem Celular , Cloranfenicol O-Acetiltransferase/metabolismo , Cristalografia por Raios X , DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Fator 4 Nuclear de Hepatócito , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Plasmídeos/metabolismo , Mutação Puntual , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Transfecção
15.
Mol Biol Cell ; 14(3): 1279-94, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12631740

RESUMO

We have shown previously that the transforming growth factor-beta (TGFbeta)-regulated Sma-Mad (Smad) protein 3 and Smad4 proteins transactivate the apolipoprotein C-III promoter in hepatic cells via a hormone response element that binds the nuclear receptor hepatocyte nuclear factor 4 (HNF-4). In the present study, we show that Smad3 and Smad4 but not Smad2 physically interact with HNF-4 via their Mad homology 1 domains both in vitro and in vivo. The synergistic transactivation of target promoters by Smads and HNF-4 was shown to depend on the specific promoter context and did not require an intact beta-hairpin/DNA binding domain of the Smads. Using glutathione S-transferase interaction assays, we established that two regions of HNF-4, the N-terminal activation function 1 (AF-1) domain (aa 1-24) and the C-terminal F domain (aa 388-455) can mediate physical Smad3/HNF-4 interactions in vitro. In vivo, Smad3 and Smad4 proteins enhanced the transactivation function of various GAL4-HNF-4 fusion proteins via the AF-1 and the adjacent DNA binding domain, whereas a single tyrosine to alanine substitution in AF-1 abolished coactivation by Smads. The findings suggest that the transcriptional cross talk between the TGFbeta-regulated Smads and HNF-4 is mediated by specific functional domains in the two types of transcription factors. Furthermore, the specificity of this interaction for certain target promoters may play an important role in various hepatocyte functions, which are regulated by TGFbeta and the Smads.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Apolipoproteína C-III , Apolipoproteínas C/genética , Apolipoproteínas C/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Fator I de Transcrição COUP , Linhagem Celular , Regulação da Expressão Gênica , Fator 4 Nuclear de Hepatócito , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Proteína Smad3 , Proteína Smad4 , Fatores de Transcrição/genética , Ativação Transcricional
16.
Biochemistry ; 41(4): 1217-28, 2002 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-11802721

RESUMO

We have investigated the mechanism of functional cooperativity between specificity protein 1 (Sp1) and hepatocyte nuclear factor-4 (HNF-4) on the human apolipoprotein CIII (apoCIII) promoter. Cotransfections in Drosophila SL2 cells that lack endogenous Sp1 or Sp1-related activities showed that HNF-4 and Sp1 synergistically transactivate the -890/+24 apoCIII promoter up to 150-fold. Synergistic transactivation required the HNF-4 binding site of the apoCIII enhancer. Deletion of part of the Ser/Thr-rich and Gln-rich domain or the C-terminal domain of Sp1 decreased, and deletion of residues 501-610 of Sp1 increased, the functional cooperativity between Sp1 and HNF-4. Physical interactions between the two factors were demonstrated by glutathione S-transferase pull-down and co-immunoprecipitation assays. The amino terminal domain of both factors and the carboxy terminal domain of Sp1 contribute to these interactions. Antagonism between HNF-4 and Sp1 was demonstrated on homopolymeric promoters containing multiple binding sites for either factor, suggesting that the synergism between the two factors occurs only when both factors are bound simultaneously to the DNA. The observed physical interactions between Sp1 and HNF-4 in the context of the apoCIII promoter may explain in part their in vitro and in vivo synergism in the transcriptional activation of the apolipoprotein A-I/apoCIII/apolipoprotein A-IV gene cluster.


Assuntos
Apolipoproteínas C/genética , Proteínas de Ligação a DNA , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Apolipoproteína C-III , Apolipoproteínas C/química , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Células Cultivadas , Primers do DNA , Regulação para Baixo , Fator 4 Nuclear de Hepatócito , Humanos , Ligação Proteica , Deleção de Sequência
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