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1.
Neuroscience ; 210: 21-32, 2012 May 17.
Artículo en Inglés | MEDLINE | ID: mdl-22445724

RESUMEN

Apolipoprotein (apo) E4 isoform, a major risk factor for Alzheimer disease (AD), is more susceptible to proteolysis than apoE2 and apoE3 isoforms. ApoE4 fragments have been found in AD patients' brain. In the present study, we examined the effect of full-length apoE4 and apoE4 fragments apoE4[Δ(186-299)] and apoE4[Δ(166-299)] on inflammation in human neuroblastoma SK-N-SH and human astrocytoma SW-1783 cells. Western blot and zymography analysis showed that treatment of SK-N-SH cells with apoE4[Δ(186-299)], but not full-length apoE4 or the shorter apoE4[Δ(166-299)] fragment, leads to increased extracellular levels of matrix metalloproteinase 9 (MMP9) and tissue inhibitor of metalloproteinase 1 (TIMP1). Real-time PCR showed that interleukin (IL)-1ß gene expression is also increased in SK-N-SH cells treated with apoE4[Δ(186-299)]. Treatment of SK-N-SH cells with IL-1ß leads to increased MMP9 and TIMP1 extracellular levels, suggesting that the induction of IL-1ß may be the mechanism by which apoE4[Δ(186-299)] regulates MMP9 and TIMP1 levels in these cells. In contrast to SK-N-SH cells, treatment of SW-1783 cells with apoE4[Δ(186-299)], and to a lesser extent with apoE4, leads to increased TIMP1 extracellular levels without affecting MMP9 levels. Additionally, apoE4[Δ(186-299)] leads to decreased IL-10 gene expression in SK-N-SH cells, whereas both apoE4 and apoE4[Δ(186-299)] lead to decreased TNFα gene expression without affecting IL-1ß and IL-10 gene expression in SW-1783 cells. Overall, our findings indicate that a specific apoE4 fragment (apoE4[Δ(186-299)]), with molecular mass similar that of apoE4 fragments detected in AD patients' brain, can influence the level of inflammatory molecules in brain cell lines. It is possible that these phenomena contribute to AD pathogenesis.


Asunto(s)
Apolipoproteína E4/farmacología , Encéfalo/efectos de los fármacos , Citocinas/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Enfermedad de Alzheimer/metabolismo , Apolipoproteína E4/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Western Blotting , Encéfalo/metabolismo , Línea Celular Tumoral , Citocinas/efectos de los fármacos , Humanos , Inflamación/metabolismo , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Inhibidor Tisular de Metaloproteinasa-1/efectos de los fármacos , Transfección
2.
J Intern Med ; 270(2): 136-46, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21443680

RESUMEN

OBJECTIVES: To determine whether mutations in APOA1 affect levels of high-density lipoprotein (HDL) cholesterol and to predict risk of ischaemic heart disease (IHD) and total mortality in the general population. BACKGROUND: Epidemiologically, risk of IHD is inversely related to HDL cholesterol levels. Mutations in apolipoprotein (apo) A-I, the major protein constituent of HDL, might be associated with low HDL cholesterol and predispose to IHD and early death. DESIGN: We resequenced APOA1 in 190 individuals and examined the effect of mutations on HDL cholesterol, risk of IHD, myocardial infarction (MI) and mortality in 10 440 individuals in the prospective Copenhagen City Heart Study followed for 31 years. Results were validated in an independent case-control study (n = 16 035). Additionally, we determined plasma ratios of mutant to wildtype (WT) apoA-I in human heterozygotes and functional effects of mutations in adenovirus-transfected mice. RESULTS: We identified a new mutation, A164S (1 : 500 in the general population), which predicted hazard ratios for IHD, MI and total mortality of 3.2 [95% confidence interval (CI): 1.6-6.5], 5.5 (95% CI: 2.6-11.7) and 2.5 (95% CI: 1.3-4.8), respectively, in heterozygotes compared with noncarriers. Mean reduction in survival time in heterozygotes was 10 years (P < 0.0001). Results for IHD and MI were confirmed in the case-control study. Furthermore, the ratio of mutant S164 to WT A164 apoA-I in plasma of heterozygotes was reduced. In addition, A164S heterozygotes had normal plasma lipid and lipoprotein levels, including HDL cholesterol and apoA-I, and this finding was confirmed in adenovirus-transfected mice. CONCLUSIONS: A164S is the first mutation in APOA1 to be described that predicts an increased risk of IHD, MI and total mortality without low HDL cholesterol levels.


Asunto(s)
Apolipoproteína A-I/genética , Lipoproteínas HDL/sangre , Mutación/genética , Isquemia Miocárdica/sangre , Isquemia Miocárdica/genética , Adulto , Anciano , Animales , Estudios de Casos y Controles , Dinamarca/epidemiología , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Isquemia Miocárdica/mortalidad , Factores de Riesgo , Análisis de Secuencia de ADN , Análisis de Supervivencia
3.
Pharmacogenomics J ; 10(3): 165-79, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19949424

RESUMEN

We have used a new ApoA-I transgenic mouse model to identify by global gene expression profiling, candidate genes that affect lipid and lipoprotein metabolism in response to fenofibrate treatment. Multilevel bioinformatical analysis and stringent selection criteria (2-fold change, 0% false discovery rate) identified 267 significantly changed genes involved in several molecular pathways. The fenofibrate-treated group did not have significantly altered levels of hepatic human APOA-I mRNA and plasma ApoA-I compared with the control group. However, the treatment increased cholesterol levels to 1.95-fold mainly due to the increase in high-density lipoprotein (HDL) cholesterol. The observed changes in HDL are associated with the upregulation of genes involved in phospholipid biosynthesis and lipid hydrolysis, as well as phospholipid transfer protein. Significant upregulation was observed in genes involved in fatty acid transport and beta-oxidation, but not in those of fatty acid and cholesterol biosynthesis, Krebs cycle and gluconeogenesis. Fenofibrate changed significantly the expression of seven transcription factors. The estrogen receptor-related gamma gene was upregulated 2.36-fold and had a significant positive correlation with genes of lipid and lipoprotein metabolism and mitochondrial functions, indicating an important role of this orphan receptor in mediating the fenofibrate-induced activation of a specific subset of its target genes.


Asunto(s)
Apolipoproteína A-I/genética , Fenofibrato/uso terapéutico , Metabolismo de los Lípidos/genética , Receptores de Estrógenos/genética , Animales , HDL-Colesterol/sangre , Femenino , Fenofibrato/farmacología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Redes y Vías Metabólicas/efectos de los fármacos , Redes y Vías Metabólicas/genética , Ratones , Ratones Transgénicos , Análisis por Matrices de Proteínas , ARN Mensajero/metabolismo , Factores de Transcripción/genética , Regulación hacia Arriba
4.
J Lipid Res ; 45(2): 287-94, 2004 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-14617740

RESUMEN

Apolipoproteins, such as apolipoprotein A-I (apoA-I), can stimulate cholesterol efflux from cells expressing the ATP binding cassette transporter A1 (ABCA1). The nature of the molecular interaction between these cholesterol acceptors and ABCA1 is controversial, and models suggesting a direct protein-protein interaction or indirect association have been proposed. To explore this issue, we performed competition binding and chemical cross-linking assays using six amphipathic plasma proteins and an 18 amino acid amphipathic helical peptide. All seven proteins stimulated lipid efflux and inhibited the cross-linking of apoA-I to ABCA1. Cross-linking of apoA-I to ABCA1 was saturable and occurred at high affinity (Kd of 7.0 +/- 1.9 nM), as was cross-linking of apoA-II. After binding to ABCA1, apoA-I rapidly dissociated (half-life of 25 min) from the complex and was released back into the medium. A mutant form of ABCA1 (W590S) that avidly binds apoA-I but fails to promote cholesterol efflux released apoA-I with similar kinetics but without transfer of cholesterol to apoA-I. Thus, a high-affinity, saturable, protein-protein interaction occurs between ABCA1 and all of its amphipathic protein ligands. Dissociation of the complex leads to the cellular release of cholesterol and the apolipoprotein. However, dissociation is not dependent on cholesterol transfer, which is a clearly separable event, distinguishable by ABCA1 mutants.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Apolipoproteínas/metabolismo , Colesterol/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Adenoviridae , Transporte Biológico/fisiología , Línea Celular Transformada , Membrana Celular/metabolismo , Humanos , Metabolismo de los Lípidos , Sustancias Macromoleculares , Mutación , Péptidos/metabolismo , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción Genética
5.
Biochemistry ; 40(45): 13670-80, 2001 Nov 13.
Artículo en Inglés | MEDLINE | ID: mdl-11695916

RESUMEN

We have used adenovirus-mediated gene transfer in apoA-I-deficient (A-I-/-) mice to probe the in vivo assembly and metabolism of HDL using apoA-I variants, focusing primarily on the role of the C-terminal 32 amino acids (helices 9-10). Lipid, lipoprotein, and apoA-I analyses showed that plasma levels of apoA-I and HDL of the mutants were 40-88% lower than that of wild type (WT) human apoA-I despite comparable levels of expression in the liver. WT apoA-I and mutant 1 (P165A, E172A) formed spherical particles with the size and density of HDL2 and HDL3. Mutant 2 (E234A, E235A, K238A, K239A) generated spherical particles with density between HDL2 and HDL3. Mutant 3 (L211V, L214V, L218V, L219V) and mutant 4 (L222K, F225K, F229K), which have substitutions of hydrophobic residues in the C-terminus, generated discoidal HDL particles indicating a defect in their conversion to mature spherical HDL. Significant amounts of mutant 4 and mutant 5 (truncated at residue 219) were found in the lipid poor fractions after ultracentrifugation of the plasma (18 and 35%, respectively, of total apoA-I). These findings suggest that hydrophobic residues in and/or between helices 9 and 10 are important for the maturation of HDL in vivo.


Asunto(s)
Apolipoproteína A-I/metabolismo , Lipoproteínas HDL/metabolismo , Hígado/metabolismo , Adenoviridae/genética , Animales , Apolipoproteína A-I/sangre , Apolipoproteína A-I/deficiencia , Apolipoproteína A-I/genética , HDL-Colesterol/sangre , Dimiristoilfosfatidilcolina/metabolismo , Eliminación de Gen , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , Lípidos/sangre , Lipoproteínas HDL/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Estructura Secundaria de Proteína , ARN Mensajero/metabolismo
7.
Biochemistry ; 40(20): 6027-35, 2001 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-11352738

RESUMEN

Apolipoprotein E (apoE) promotes receptor-mediated catabolism of apoE-containing lipoprotein remnants. Impairments in remnant clearance are associated with type III hyperlipoproteinemia and premature atherosclerosis. In humans, apoE plasma levels correlate with plasma triglyceride levels, suggesting that excess apoE may also affect plasma triglyceride levels. We have used adenovirus-mediated gene transfer in mice to map the domains of apoE required for cholesterol and triglyceride clearance, in vivo. Adenovirus expressing apoE3 and apoE4 at doses of (1-2) x 10(9) pfu increased plasma cholesterol and triglyceride levels in normal C57BL6 mice and failed to normalize the high cholesterol levels of apoE-deficient mice due to induction of hypertriglyceridemia. In contrast, an adenovirus expressing the truncated apoE 1-185 form normalized the cholesterol levels of E(-)(/)(-) mice and did not cause hypertriglyceridemia. Northern blot analysis of hepatic RNA from mice expressing the full-length and the truncated apoE forms showed comparable steady-state apoE mRNA levels of the full-length apoE forms that cause hyperlipidemia and the truncated apoE forms that do not cause hyperlipidemia. The findings suggest that the amino-terminal residues 1-185 of apoE are sufficient for the clearance of apoE-containing lipoprotein remnants by the liver, whereas domains of the carboxy-terminal one-third of apoE are required for apoE-induced hyperlipidemia.


Asunto(s)
Apolipoproteínas E/fisiología , Hiperlipidemias/genética , Lipoproteínas/metabolismo , Fragmentos de Péptidos/fisiología , Adenoviridae/genética , Animales , Apolipoproteína E3 , Apolipoproteína E4 , Apolipoproteínas E/biosíntesis , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Transporte Biológico Activo/genética , Cromatografía Líquida de Alta Presión , Femenino , Eliminación de Gen , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Hipercolesterolemia/sangre , Hipercolesterolemia/etiología , Hipercolesterolemia/genética , Hiperlipidemias/sangre , Hiperlipidemias/etiología , Hipertrigliceridemia/sangre , Hipertrigliceridemia/etiología , Hipertrigliceridemia/genética , Lipoproteínas/sangre , Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fragmentos de Péptidos/genética , Estructura Terciaria de Proteína/genética , ARN Mensajero/metabolismo , Triglicéridos/metabolismo , Células Tumorales Cultivadas
8.
Curr Opin Lipidol ; 12(2): 181-207, 2001 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-11264990

RESUMEN

The present review summarizes recent advances in the transcriptional regulation of the human apolipoprotein genes, focusing mostly, but not exclusively, on in-vivo studies and signaling mechanisms that affect apolipoprotein gene transcription. An attempt is made to explain how interactions of transcription factors that bind to proximal promoters and distal enhancers may bring about gene transcription. The experimental approaches used and the transcriptional regulatory mechanisms that emerge from these studies may also be applicable in other gene systems that are associated with human disease. Understanding extracellular stimuli and the specific mechanisms that underlie apolipoprotein gene transcription may in the long run allow us to selectively switch on antiatherogenic genes, and switch off proatherogenic genes. This may have beneficial effects and may confer protection from atherosclerosis to humans.


Asunto(s)
Apolipoproteínas/genética , Regulación de la Expresión Génica , Transcripción Genética , Animales , Apolipoproteínas A/genética , Apolipoproteínas B/genética , Apolipoproteínas C/genética , Apolipoproteínas E/genética , Arteriosclerosis/genética , Humanos , Mutación
9.
Front Biosci ; 6: D456-504, 2001 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-11229886

RESUMEN

This review provides experiments and putative mechanisms which underlie the transcription of the human apolipoprotein genes in vitro and in vivo. Summarized below are the key findings for individual genes and gene clusters. ApoA-II. 1- The -911/+29 promoter is sufficient to direct expression of a reporter gene exclusively in the liver and thus represents a liver-specific promoter. 2- Important factors for the activity of this promoter are hormone nuclear receptors and the ubiquitous factor USF. 3. SREBP-1 and SREBP-2 bind to five and four sites respectively and transactivate the apoA-II promoter. Their role in the in vivo transcription of the apoA-II gene has not been established. ApoB. 1. Regulatory sequence extending 5 Kb upstream and 1.5 Kb downstream of the apoB promoter are sufficient to direct hepatic expression of the apoB gene. The intestinal expression of the apoB gene requires in addition a 315 bp intestinal enhancer located 56 Kb upstream of the apoB gene. 2. Important factors for apoB gene transcription appear to be C/EBP, HNF-3, HNF-4 and other nuclear receptors which bind both on the proximal promoter and the intestinal enhancer. ApoE/ApoCI/ApoCIV/ApoCII Cluster. 1. The expression of the genes of the apoE/apoCI/apoCII/apoE cluster are controlled by two homologous hepatic control regions designated HCR-1 and HCR-2 of approximately 600 bp located 15 and 27 Kb 3? of the apoE gene. Either region is sufficient to direct gene expression in vivo, although HCR-1 appears to have a dominant effect on apoE and apoCI and HCR-2 has a dominant effect on apoCIV and apoCII gene expression. 2. Two other homologous regulatory regions designated ME-1 and ME-2 located 3.3 and 15.9 Kb downstream of the apoE gene can direct independently the expression of the apoE gene in macrophages and adipocytes. 3. Important factors for apoE gene regulation appear to be SP1 on the proximal promoter, and possibly HNF-3, C/EBP and hormone nuclear receptors on the enhancers. 4. Important factors for apoCII gene transcription appear to be HNF-4 and RXR-alpha/T3R-beta which binds to a thyroid response element of the proximal promoter. ApoA-I/ApoCIII/ApoA-IV Gene Cluster. 1. The transcription of the apoA-I/apoCIII/apoA-IV gene cluster is controlled by a common enhancer located 590 to 790 nucleotides upstream of the apoCIII gene. 2. Important factors for the activity of the enhancer are SP1, HNF-4 and possibly other nuclear receptors. Important factors for the activity of the proximal promoters are HNF-4, and possibly other nuclear receptors. 3. The HNF-4 binding site of the apoCIII enhancer is required for the intestinal expression of apoA-I and apoCIII gene and enhances synergistically the hepatic transcription of the two genes and possibly of apoA-IV in vivo. The three SP1 sites of the enhancer are also required for the intestinal expression of apoA-I and apoCIII genes in vivo and for the enhancement of the hepatic transcription. 4. Pro-inflammatory cytokines such as TNF-alpha and IL-1 repress, and TGF-beta stimulates the apoCIII promoter activity. The TGF-beta pathway activates SMAD3/4 proteins which interact with HNF-4 bound to the apoCIII promoter and enhancer and increase its activity. 5. It appears that other factors activated by different signaling pathways (NF-kappa-B, Jun and others) interact with HNF-4 bound to the enhancer and thus repress the activity of apoCIII promoter. Understanding the transcriptional regulatory mechanism of the apolipoprotein genes may allow, in the long run, selective increase of anti-atherogenic lipoproteins and thus reduce the risk of cardiovascular disease.


Asunto(s)
Apolipoproteínas/genética , Regulación de la Expresión Génica , Humanos , Factores de Transcripción/fisiología , Transcripción Genética
10.
J Biol Chem ; 276(23): 19778-86, 2001 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-11279066

RESUMEN

Apolipoprotein (apo) E has been implicated in cholesterol and triglyceride homeostasis in humans. At physiological concentration apoE promotes efficient clearance of apoE-containing lipoprotein remnants. However, high apoE plasma levels correlate with high plasma triglyceride levels. We have used adenovirus-mediated gene transfer in apoE-deficient mice (E(-)/-) to define the domains of apoE required for cholesterol and triglyceride homeostasis in vivo. A dose of 2 x 10(9) plaque-forming units of apoE4-expressing adenovirus reduced slightly the cholesterol levels of E(-)/- mice and resulted in severe hypertriglyceridemia, due to accumulation of cholesterol and triglyceride-rich very low density lipoprotein particles in plasma. In contrast, the truncated form apoE4-202 resulted in a 90% reduction in the plasma cholesterol levels but did not alter plasma triglyceride levels in the E(-)/- mice. ApoE secretion by cell cultures, as well as the steady-state hepatic mRNA levels in individual mice expressing apoE4 or apoE4-202, were similar. In contrast, very low density lipoprotein-triglyceride secretion in mice expressing apoE4, but not apoE4-202, was increased 10-fold, as compared with mice infected with a control adenovirus. The findings suggest that the amino-terminal 1-202 region of apoE4 contains the domains required for the in vivo clearance of lipoprotein remnants. Furthermore, the carboxyl-terminal 203-299 residues of apoE promote hepatic very low density lipoprotein-triglyceride secretion and contribute to apoE-induced hypertriglyceridemia.


Asunto(s)
Apolipoproteínas E/metabolismo , Colesterol/metabolismo , Homeostasis , Triglicéridos/metabolismo , Adenoviridae/genética , Animales , Apolipoproteína E4 , Apolipoproteínas E/sangre , Apolipoproteínas E/química , Apolipoproteínas E/genética , Secuencia de Bases , Colesterol/sangre , Cromatografía Liquida , Cartilla de ADN , Femenino , Humanos , Hígado/metabolismo , Ratones , Ratones Noqueados , ARN Mensajero/genética , ARN Mensajero/metabolismo , Triglicéridos/sangre , Células Tumorales Cultivadas
11.
Biochemistry ; 39(51): 15910-9, 2000 Dec 26.
Artículo en Inglés | MEDLINE | ID: mdl-11123918

RESUMEN

To probe the secondary structure of the C-terminus (residues 165-243) of lipid-free human apolipoprotein A-I (apoA-I) and its role in protein stability, recombinant wild-type and seven site-specific mutants have been produced in C127 cells, purified, and studied by circular dichroism and fluorescence spectroscopy. A double substitution (G185P, G186P) increases the protein stability without altering the secondary structure, suggesting that G185 and G186 are located in a loop/disordered region. A triple substitution (L222K, F225K, F229K) leads to a small increase in the alpha-helical content and stability, indicating that L222, F225, and F229 are not involved in stabilizing hydrophobic core contacts. The C-terminal truncation Delta(209-243) does not change the alpha-helical content but reduces the protein stability. Truncation of a larger segment, Delta(185-243), does not affect the secondary structure or stability. In contrast, an intermediate truncation, Delta(198-243), leads to a significant reduction in the alpha-helical content, stability, and unfolding cooperativity. The internal 11-mer deletion Delta(187-197) has no significant effect on the conformation or stability, whereas another internal 11-mer deletion, Delta(165-175), dramatically disrupts and destabilizes the protein conformation, suggesting that the presence of residues 165-175 is crucial for proper apoA-I folding. Overall, the findings suggest the presence of stable helical structure in the C-terminal region 165-243 of lipid-free apoA-I and the involvement of segment 209-243 in stabilizing interactions in the molecule. The effect of the substitution (G185P, G186P) on the exposure of tryptophans located in the N-terminal half suggests an apoA-I tertiary conformation with the C-terminus located close to the N-terminus.


Asunto(s)
Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Mutagénesis Sitio-Dirigida , Sustitución de Aminoácidos/genética , Animales , Apolipoproteína A-I/metabolismo , Dicroismo Circular , Guanidina , Humanos , Metabolismo de los Lípidos , Ratones , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Espectrometría de Fluorescencia , Temperatura , Células Tumorales Cultivadas
12.
J Biol Chem ; 275(52): 41405-14, 2000 Dec 29.
Artículo en Inglés | MEDLINE | ID: mdl-10995777

RESUMEN

Cotransfection of HepG2 cells with SMADs established that SMAD3 and SMAD3-SMAD4 transactivated (15-70-fold) the -890/+24 apoCIII promoter and shorter promoter segments, whereas cotransfection with a dominant negative SMAD4 mutant repressed the apoCIII promoter activity by 50%, suggesting that SMAD proteins participate in apoCIII gene regulation. Transactivation required the presence of a hormone response element, despite the fact that SMADs could not bind directly to it. Cotransfection of SMAD3-SMAD4 along with hepatocyte nuclear factor-4 resulted in a strong synergistic transactivation of the -890/+24 apoCIII promoter, proximal promoter segments, or synthetic promoters containing either the apoCIII enhancer or the proximal apoCIII hormone response element. Inhibition of endogenous hepatocyte nuclear factor-4 synthesis by an antisense ribozyme construct reduced the constitutive activity of the apoCIII promoter in HepG2 cells to 10% and abolished the SMAD-mediated transactivation. Co-immunoprecipitation and GST pull-down assays provided evidence for physical interactions between SMAD3, SMAD4, and hepatic nuclear factor-4. Our findings indicate that transforming growth factor beta and its signal transducer SMAD proteins can modulate gene transcription by novel mechanisms that involve their physical and functional interaction with hepatocyte nuclear factor-4, suggesting that SMAD proteins may play an important role in apolipoprotein gene expression and lipoprotein metabolism.


Asunto(s)
Apolipoproteínas C/genética , Proteínas de Unión al ADN/fisiología , Fosfoproteínas/fisiología , Regiones Promotoras Genéticas , Transactivadores/fisiología , Factores de Transcripción/fisiología , Activación Transcripcional , Animales , Apolipoproteína C-III , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Sitios de Unión , Células COS , Factor Nuclear 4 del Hepatocito , Humanos , Elementos de Respuesta , Proteína smad3 , Proteína Smad4 , Factor de Crecimiento Transformador beta/fisiología , Células Tumorales Cultivadas
13.
J Biol Chem ; 275(28): 21262-71, 2000 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-10801839

RESUMEN

The binding of apoA-I-containing ligands to the HDL receptor scavenger receptor class B type I (SR-BI) was characterized using two different assays. The first employed conventional binding or competition assays with (125)I-labeled ligands. The second is a new nonradioactive ligand binding assay, in which the receptor-associated ligand is detected by quantitative immunoblotting ("immunoreceptor assay"). Using both methods, we observed that the K(d) value for spherical HDL (density = 1.1-1.13 g/ml) was approximately 16 microgram of protein/ml, while the values for discoidal reconstituted HDL (rHDL) containing proapoA-I or plasma apoA-I were substantially lower (approximately 0.4-5 microgram of protein/ml). We also observed reduced affinity and/or competition for spherical (125)I-HDL cell association by higher relative to lower density HDL and very poor competition by lipid-free apoA-I and pre-beta-1 HDL. Deletion of either 58 carboxyl-terminal or 59 amino-terminal residues from apoA-I, relative to full-length control apoA-I, resulted in little or no change in the affinity of corresponding rHDL particles. However, rHDL particles containing a double mutant lacking both terminal domains competed poorly with spherical (125)I-HDL for binding to SR-BI. These findings suggest an important role for apoA-I and its conformation/organization within particles in mediating HDL binding to SR-BI and indicate that the NH(2) and COOH termini of apoA-I directly or indirectly contribute independently to binding to SR-BI.


Asunto(s)
Apolipoproteína A-I/metabolismo , Antígenos CD36/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de la Membrana , Receptores Inmunológicos , Animales , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Sitios de Unión , Antígenos CD36/química , Exones , Humanos , Radioisótopos de Yodo , Cinética , Ligandos , Ratones , Mutagénesis Sitio-Dirigida , Ensayo de Unión Radioligante , Receptores de Lipoproteína/metabolismo , Receptores Depuradores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Receptores Depuradores de Clase B , Eliminación de Secuencia , Transfección , Células Tumorales Cultivadas
15.
Biochemistry ; 38(28): 8918-25, 1999 Jul 13.
Artículo en Inglés | MEDLINE | ID: mdl-10413465

RESUMEN

Recent studies have shown that at physiological conditions (pH 7.6, 37 degrees C), the reactivity of recombinant apoE isoforms secreted by mammalian cells toward amyloid peptide beta (Abeta40) follows the order apoE2 > apoE3 > apoE4 for the apoE monomer and apoE2 > apoE3 for apoE dimer that is formed via that intramolecular disulfide bridges. Different Abeta binding properties have been reported for the plasma-derived apoE and commercially available apoE preparations that differ from the native apoE forms in the degree of their O-glycosylation. To define structural elements of apoE involved in the interaction with Abeta, we have introduced point mutations as well as amino- and carboxy-terminal deletions in the apoE structure. The mutant apoE forms were expressed transiently using the Semliki Forest Virus system, and the culture medium was utilized to study the reactivity of the mutated proteins with Abeta 40. This analysis showed that a mutation in the O-glycosylation site of apoE2 (Thr194-Ala) did not affect the SDS-stable binding of apoE to Abeta. In contrast, introduction of cysteine at position 158 of apoE4 (Arg112, Cys158) increased the SDS-stable binding of apoE to Abeta to the levels similar to those observed in apoE2. Similar analysis showed that apoE truncated at residues 259, 249, 239, and 229 retains the SDS-stable binding to Abeta40, whereas apoE truncated at residues 185 and 165 does not bind to Abeta. The deletion of aminoterminal residues 2-19 reduced the SDS-stable binding of apoE2 to Abeta and deletion of residues 2-81 abolished binding to Abeta. It is also noteworthy that the (Delta2-81) apoE mutant exists predominantly as a dimer, suggesting that removal of residues 2-81 promoted dimerization of apoE. These findings suggest that the amino- and carboxy-terminal residues of apoE are required for SDS-stable binding of apoE to Abeta and that the presence of at least one cysteine contributes to the efficient Abeta binding.


Asunto(s)
Péptidos beta-Amiloides/metabolismo , Apolipoproteínas E/metabolismo , Cisteína/metabolismo , Fragmentos de Péptidos/metabolismo , Treonina/metabolismo , Sustitución de Aminoácidos/genética , Péptidos beta-Amiloides/química , Animales , Apolipoproteínas E/química , Apolipoproteínas E/genética , Arginina/genética , Sitios de Unión/genética , Conformación de Carbohidratos , Línea Celular , Cricetinae , Cisteína/química , Cisteína/genética , Electroforesis en Gel de Poliacrilamida , Glicosilación , Humanos , Riñón/citología , Sustancias Macromoleculares , Fragmentos de Péptidos/química , Mutación Puntual , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Treonina/química , Treonina/genética
16.
Arterioscler Thromb Vasc Biol ; 19(6): 1456-69, 1999 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-10364076

RESUMEN

-Screening of an expression human liver cDNA library resulted in the isolation of several cDNA clones homologous to sterol regulatory element-binding protein-1 (SREBP-1) that recognize the regulatory element AIIAB and AIIK of the human apoA-II promoter. DNaseI footprinting of the apoA-II promoter using SREBP-1 (1 to 460) expressed in bacteria identified 5 overall protected regions designated AIIAB (-64 to -48), AIICD (-178 to -154), AIIDE (-352 to -332), AIIHI (-594 to -574), and AIIK (-760 to -743). These regions contain inverted E-box palindromic or direct repeat motifs and bind SREBP-1 with different affinities. Transient cotransfection experiments in HepG2 cells showed that SREBP-1 transactivated the -911/29 apoA-II promoter 3.5-fold as well as truncated apoA-II promoter segments that contain 1, 2, 3, or 4 SREBP binding sites. Mutagenesis analysis showed that transactivation by SREBP was mainly affected by mutations in element AIIAB. Despite the strong transactivation of the apoA-II promoter by SREBP-1 we could not demonstrate significant changes on the endogenous apoA-II mRNA levels of HepG2 cells after cotransfection with SREBP-1 or in the presence or absence of cholesterol and 25-OH-cholesterol. An SREBP-1 mutant lacking the amino-terminal activation domain bound normally to its cognate sites and repressed the apoA-II promoter activity. Repression was also caused by specific amino acid substitutions of Leu, Val, or Gly for Lys359, which affected DNA binding. Repression by the DNA binding-deficient mutants was abolished by deletion of the amino-terminal activation domain (1 to 90) of SREBP-1. Overall, the findings suggest that the wild-type SREBP-1 can bind and transactivate efficiently the apoA-II promoter in cell culture. SREBP-1 mutants lacking the activation domain bind to their cognate sites and directly repress the apoA-II promoter whereas mutants defective in DNA binding indirectly repress the apoA-II promoter activity, possibly by a squelching mechanism.


Asunto(s)
Apolipoproteína A-II/genética , Proteínas Potenciadoras de Unión a CCAAT , Proteínas de Unión al ADN/fisiología , ADN/metabolismo , Proteínas Nucleares/fisiología , Regiones Promotoras Genéticas , Factores de Transcripción/fisiología , Activación Transcripcional , Secuencia de Aminoácidos , Apolipoproteína C-III , Apolipoproteínas C/genética , Secuencia de Bases , Sitios de Unión , Humanos , Datos de Secuencia Molecular , Elementos de Respuesta , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Relación Estructura-Actividad , Factores de Transcripción/metabolismo , Factores Estimuladores hacia 5'
17.
Nucleic Acids Res ; 27(4): 1104-17, 1999 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-9927745

RESUMEN

DNase I footprinting of the apoA-II promoter using sterol regulatory element binding protein-2 [(SREBP-2 (1-458)] expressed in bacteria identified four protected regions, designated AIIAB (-64 to -48), AIICD (-178 to -154), AIIDE (-352 to -332) and AIIK (-760 to -743), which bind SREBP-2 and contain either palindromic or direct repeat motifs. Potassium permanganate and dimethyl sulfate interference experiments using the AIIAB region as probe showed that the nucleotides of a decameric palindromic repeat RTCAMVTGMY and two 5' T residues participate in DNA-protein interactions. SREBP-2 transactivated the intact (-911/+29) apoA-II promoter 1.7-fold and truncated apoA-II promoter segments which contain one, two or three SREBP-2 sites 11- to 17-fold in HepG2 cells. Transactivation of a promoter construct containing the binding site AIIAB and the apoA-II enhancer, which includes the binding site AIIK, was abolished by mutations in element AIIAB. An SREBP-2 mutant defective in DNA binding caused a dose-dependent repression of the apoA-II promoter activity. Repression was also caused by an SREBP-2 mutant which lacks the N-terminal activation domain (residues 1-93) but binds normally to its cognate sites. In contrast, a double SREBP-2 mutant which lacks both the DNA binding and the activation domains has no effect on the apoA-II promoter activity. Overall, the findings suggest that SREBP-2 can transactivate the apoA-II promoter by binding to multiple sites. Furthermore, the repression caused by the DNA binding deficient mutants results from squelching of positive activator(s) which appear to recognize the activation domain of SREBP-2.


Asunto(s)
Apolipoproteína A-II/genética , Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Activación Transcripcional , Animales , Secuencia de Bases , Sitios de Unión , ADN Complementario , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Proteína 2 de Unión a Elementos Reguladores de Esteroles , Factores de Transcripción/genética , Células Tumorales Cultivadas
18.
Biochemistry ; 38(3): 964-75, 1999 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-9893992

RESUMEN

Human apolipoprotein CIII (apoCIII) is a major determinant of plasma triglyceride metabolism. The regulatory elements that control both hepatic and intestinal transcription of the human apoCIII gene are localized between nucleotides -792 and -25 of the apoCIII promoter. Elements important for apoCIII promoter activity are three hormone response elements (HREs) and three SP1-binding sites. Orphan members of the nuclear hormone receptor superfamily can bind the HREs and strongly enhance or repress apoCIII promoter activity. In the present study we have investigated the ability of ligand-dependent nuclear hormone receptors to bind and modulate the human apoCIII promoter activity. Experiments using DNA binding and competition assays showed that the proximal element B (-87/-72) binds strongly, in addition to HNF-4, ARP-1, EAR-2, and EAR-3, heterodimers of RXRalpha with RARalpha, and less efficiently, homodimers of RARalpha and heterodimers of RXRalpha with T3Rbeta or PPARalpha. Element G (-669/-648), which was shown previously to bind ARP-1 and EAR-3 but not HNF-4, binds strongly heterodimers of RXRalpha with either RARalpha or T3Rbeta. Finally element I4 (-732/-712), which was shown to bind HNF-4, also binds strongly ARP-1 and EAR-3, as well as RXRalpha/RARalpha heterodimers and less efficiently, RXRalpha/T3Rbeta heterodimers. Methylation interference experiments have identified the protein-DNA interactions between different nuclear receptors and the respective HREs on the apoCIII promoter. RXRalpha/RARalpha heterodimers and HNF-4 homodimers bind to DR-1 motifs on elements B and I4, respectively. RXRalpha/T3Rbeta heterodimers and ARP-1 bind to DR-5 and DR-0 motifs respectively on element G. Cotransfection experiments in HepG2 cells showed that RXRalpha or a combination of RXRalpha and RARalpha increased the apoCIII promoter activity approximately 2-fold in the presence of the ligands 9-cis or all-trans RA. In contrast, a combination of RXRalpha and T3Rbeta transactivated the apoCIII promoter 1.5-fold in the presence of 9-cis RA but it repressed the apoCIII promoter activity in the presence of T3. Mutations in the HREs of elements B, G, or I4 or in the SP1-binding site of element H, which abolished the binding of nuclear hormone receptors or SP1 to their cognate site, reduced the promoter strength and exhibited different responses to the ligand-dependent nuclear receptors. The findings suggest that modulation of the apoCIII promoter activity by orphan and ligand-dependent nuclear receptors involves complex interactions among nuclear receptors, SP1 and possibly other factors bound to the enhancer and the proximal promoter region.


Asunto(s)
Apolipoproteínas C/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores de Esteroides , Animales , Apolipoproteína C-III , Apolipoproteínas C/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Células COS , Factor de Transcripción COUP II , Factores de Transcripción COUP , Proteínas de Unión al ADN/genética , Dimerización , Factor Nuclear 4 del Hepatocito , Humanos , Ligandos , Hígado , Fosfoproteínas/metabolismo , Ratas , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Receptores X Retinoide , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas
19.
Biochemistry ; 37(40): 14078-87, 1998 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-9760243

RESUMEN

It was shown previously that cytokines such as tumor necrosis factor-alpha that stimulate signal transduction pathways involving transcription factors ATF-2 and Jun repress apoCIII promoter activity in HepG2 cells. In the present study, DNase I footprinting analysis established that ATF-2 protected three regions in the apoCIII promoter. One region (-747/-726) present in the apoCIII enhancer is within the previously identified footprint I and has overlapping boundaries with the binding sites of Sp1 (-764/-742) and HNF-4 (-736/-714). The other two regions represent new footprints and have been designated D/E (-219/-199) and B/C (-102/-75). The B/C region overlaps with the previously identified footprint B which contains an HNF-4 binding site (-87/-63). Cotransfection experiments in HepG2 cells showed that ATF-2 transactivated the -890/+24 apoCIII promoter 1.6-fold. In addition, mutations in the proximal D/E (-219/-199) and distal I (-747/-726) ATF-2-binding sites reduced the apoCIII promoter strength to 33 and 9% of control, respectively, indicating that ATF-2 is a positive regulator of apoCIII gene transcription. Cotransfections with ATF-2 and HNF-4 expression plasmids resulted in additive transactivation of the apoCIII promoter. Furthermore, apoCIII promoter constructs bearing mutations in the D/E and I ATF-2 binding sites were efficiently transactivated by HNF-4, suggesting that these two factors contribute independently to the apoCIII promoter strength. Members of the Jun family (c-Jun, JunB, and JunD) caused a dose-dependent inhibition of the -890/+24 apoCIII promoter activity. A synthetic promoter containing the apoCIII enhancer in front of the minimal AdML promoter was also repressed by Jun. In contrast, apoCIII promoter segments lacking the enhancer region were transactivated by Jun. The findings suggest that homodimers of Jun or heterodimers of Jun with other AP-1 subunits could be responsible for the observed repression by interfering with the function(s) of the apoCIII enhancer. Repression by Jun could be reversed in the presence of ATF-2 and HNF-4, suggesting that ATF2 and possibly Jun/ATF-2 heterodimers exert a positive effect on apoCIII gene transcription, as opposed to Jun homodimers or heterodimers with other AP-1 members. These findings suggest a role for members of the Jun family and ATF-2 that participate in signal transduction pathways in basal or induced apoCIII promoter activity in cells of hepatic origin.


Asunto(s)
Apolipoproteínas C/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-jun/fisiología , Proteínas Represoras/fisiología , Factores de Transcripción/fisiología , Activación Transcripcional , Factor de Transcripción Activador 2 , Apolipoproteína C-III , Apolipoproteínas C/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Carcinoma Hepatocelular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Regulación hacia Abajo , Elementos de Facilitación Genéticos/fisiología , Humanos , Datos de Secuencia Molecular , Factores de Transcripción/genética , Células Tumorales Cultivadas
20.
J Biol Chem ; 273(28): 17810-6, 1998 Jul 10.
Artículo en Inglés | MEDLINE | ID: mdl-9651383

RESUMEN

The regulatory elements CIIC (-159/-116) and CIIB (-102/-81) of the apolipoprotein CII (apoCII) promoter have distinct specificities for orphan nuclear receptors (Vorgia, P., Zannis, V. I., and Kardassis, D. (1998) J. Biol. Chem. 273, 4188-4199). In this communication we investigated the contribution of ligand-dependent and orphan nuclear receptors on the transcriptional regulation of the human apoCII gene. It was found that element CIIC in addition to ARP-1 and EAR-2 binds RXRalpha/T3Rbeta heterodimers strongly, whereas element CIIB binds hepatic nuclear factor 4 (HNF-4) exclusively. Binding is abolished by mutations that alter the HRE binding motifs. Transient cotransfection experiments showed that in the presence of T3, RXRalpha/T3Rbeta heterodimers transactivated the -205/+18 apoCII promoter 1.6- and 11-fold in HepG2 and COS-1 respectively. No transactivation was observed in the presence of 9-cis-retinoic acid. Transactivation requires the regulatory element CIIC, suggesting that this element contains a thyroid hormone response element. HNF-4 did not affect the apoCII promoter activity in HepG2 cells. However, mutations in the HNF-4 binding site on element CIIB and inhibition of HNF-4 synthesis in HepG2 cells by antisense HNF-4 constructs decreased the apoCII promoter activity to 25-40% of the control, indicating that HNF-4 is a positive regulator of the apoCII gene. ARP-1 repressed the -205/+18 but not the -104/+18 apoCII promoter activity in HepG2 cells, indicating that the repression depends on the regulatory element CIIC. In contrast, combination of ARP-1 and HNF-4 transactivated different apoCII promoter segments as well as a minimal adenovirus major late promoter driven by the regulatory element CIIB. Mutagenesis or deletion of elements CIIB or CIIC established that the observed transactivation requires DNA binding of one of the two factors and may result from HNF-4-ARP-1 interactions that elicit the transactivation functions of HNF-4. The combined data indicate that RXRalpha/T3Rbeta in the presence of T3 and HNF-4 can upregulate the apoCII promoter activity by binding to the regulatory elements CIIC and CIIB, respectively. In addition, ARP-1 can either have inhibitory or stimulatory effects on the apoCII promoter activity via different mechanisms.


Asunto(s)
Apolipoproteínas C/genética , Regiones Promotoras Genéticas , Receptores de Ácido Retinoico/metabolismo , Receptores de Esteroides , Receptores de Hormona Tiroidea/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Animales , Apolipoproteína C-II , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Células COS , Factor de Transcripción COUP II , Factores de Transcripción COUP , Línea Celular , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Dimerización , Factor Nuclear 4 del Hepatocito , Humanos , Fosfoproteínas/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Receptores X Retinoide
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