RESUMO
Small heterodimer partner (SHP), specifically expressed in liver and a limited number of other tissues, is an unusual orphan nuclear receptor that lacks the conventional DNA binding domain. In this work, we found that SHP expression is abundant in murine macrophage cell line RAW 264.7 but was suppressed by oxidized low density lipoprotein (oxLDL) and its constituent 13-hydroxyoctadecadienoic acid, a ligand for peroxisome proliferator-activated receptor gamma. Furthermore, SHP acted as a transcription coactivator of nuclear factor-kappa B (NF kappa B) and was essential for the previously described NF kappa B transactivation by palmitoyl lysophosphatidylcholine, one of the oxLDL constituents. Accordingly NF kappa B, which was transcriptionally active in the beginning, became progressively inert in oxLDL-treated RAW 264.7 cells as oxLDL decreased the SHP expression. Thus, SHP appears to be an important modulatory component to regulate the transcriptional activities of NF kappa B in oxLDL-treated, resting macrophage cells.
Assuntos
Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , NF-kappa B/química , NF-kappa B/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Fatores de Transcrição/química , Animais , Northern Blotting , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Receptor Constitutivo de Androstano , Dimerização , Relação Dose-Resposta a Droga , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Ligantes , Ácidos Linoleicos/farmacologia , Camundongos , Modelos Biológicos , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Maternally inherited mitochondrial DNA (mtDNA) has been suggested to be a genetic factor for diabetes. Reports have shown a decrease of mtDNA content in tissues of diabetic patients. We investigated the effects of mtDNA depletion on glucose metabolism by use of rho(0) SK-Hep1 human hepatoma cells, whose mtDNA was depleted by long-term exposure to ethidium bromide. The rho(0) cells failed to hyperpolarize mitochondrial membrane potential in response to glucose stimulation. Intracellular ATP content, glucose-stimulated ATP production, glucose uptake, steady-state mRNA and protein levels of glucose transporters, and cellular activities of glucose-metabolizing enzymes were decreased in rho(0) cells compared with parental rho(+) cells. Our results suggest that the quantitative reduction of mtDNA may suppress the expression of nuclear DNA-encoded glucose transporters and enzymes of glucose metabolism. Thus this may lead to diabetic status, such as decreased ATP production and glucose utilization.
Assuntos
DNA Mitocondrial/metabolismo , Glucose/farmacocinética , Proteínas de Transporte de Monossacarídeos/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Carcinoma Hepatocelular , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Inibidores Enzimáticos/farmacologia , Etídio/farmacologia , Glucosefosfato Desidrogenase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Neoplasias Hepáticas , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Proteínas de Transporte de Monossacarídeos/genética , Células Tumorais CultivadasRESUMO
OBJECTIVES: A healthy, intact coronary artery endothelium is important because most common coronary artery diseases result from loss of endothelial integrity. In this study, we explored the biological significance of the angiopoietin-Tie2 system in porcine coronary artery. METHODS: Cultured porcine coronary artery endothelial cells and explanted coronary arteries were used. RESULTS: Immunohistochemical analyses indicated that Ang1 is selectively expressed in vascular muscular cells, whereas angiopoietin-2 (Ang2) and Tie2 are selectively expressed in endothelial cells. Accordingly, Ang1 mRNA is mainly expressed in cultured porcine coronary artery vascular smooth muscle cells, whereas Ang2 and Tie2 mRNAs are mainly expressed in cultured porcine coronary artery endothelial cells (PCAECs). Ang1 (200 ng/ml) induced Tie2 phosphorylation, while Ang2 (200 ng/ml) did not produce Tie2 phosphorylation. Ang1 increased the survival of cultured PCAECs during apoptosis induced by oxidized low-density lipoprotein (OxLDL). This survival effect was does-dependent and PI. Furthermore, Ang1 also protected endothelial cells of explanted coronary artery against OxLDL-induced apoptosis artery. CONCLUSION: These results suggest that adult coronary artery contains Ang1-Tie2 components that enhance endothelial cell survival to help maintain the normal integrity of the coronary artery endothelium.
Assuntos
Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Glicoproteínas de Membrana/farmacologia , Músculo Liso Vascular/metabolismo , Proteínas/farmacologia , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Análise de Variância , Angiopoietina-1 , Angiopoietina-2 , Animais , Apoptose , Células Cultivadas , LDL-Colesterol/farmacologia , Vasos Coronários , Endotélio Vascular/citologia , Imuno-Histoquímica , Glicoproteínas de Membrana/genética , Microscopia de Contraste de Fase , Músculo Liso Vascular/citologia , Proteínas de Neoplasias/metabolismo , Fosforilação , Proteínas/genética , RNA Mensageiro/análise , Receptores Proteína Tirosina Quinases/genética , Receptor TIE-2 , SuínosRESUMO
Oxidized low-density lipoprotein (oxLDL) has been shown to modulate transactivation by the peroxisome proliferator-activated receptor (PPAR)-gamma and by nuclear factor-kappaB (NF-kappaB). In the present study, the oxLDL signalling pathways involved in NF-kappaB transactivation were investigated by utilizing a reporter construct driven by three upstream NF-kappaB binding sites, and various pharmacological inhibitors. OxLDL and its constituent lysophophatidylcholine (lysoPC) induced a rapid and transient increase in intracellular calcium and stimulated NF-kappaB transactivation in resting RAW264.7 macrophage cells in an oxidation-dependent manner. NF-kappaB activation by oxLDL or lysoPC was inhibited by inhibitors of protein kinase C or by a chelator of intracellular calcium. Tyrosine kinase or phosphatidylinositol 3-kinase inhibitors did not block NF-kappaB transactivation. Furthermore, oxLDL-induced NF-kappaB activity was abolished by PPAR-gamma ligands. When the endocytosis of oxLDL was blocked by cytochalasin B, NF-kappaB transactivation by oxLDL was synergistically increased, while PPAR transactivation was blocked. These results suggest that oxLDL activates NF-kappaB in resting macrophages via protein kinase C- and/or calcium-dependent pathways, and that this does not involve the endocytic processing of oxLDL. The endocytosis-dependent activation of PPAR-gamma by oxLDL may function as a route of inactivation of the oxLDL-induced NF-kappaB signal.
Assuntos
Endocitose , Lipoproteínas LDL/fisiologia , NF-kappa B/genética , Ativação Transcricional/fisiologia , Animais , Cálcio/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Humanos , Macrófagos/metabolismo , Camundongos , NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Lipopolysaccharide (LPS) increases the production of interleukin-12 (IL-12) from mouse macrophages via a kappaB site within the IL-12 p40 promoter. In this study, we found that oxidized low density lipoprotein (oxLDL) inhibited this LPS-stimulated production of IL-12 in a dose-dependent manner while native LDL did not. OxLDL inhibited p40 promoter activation in monocytic RAW264.7 cells transiently transfected with p40 promoter/reporter constructs, and the repressive effect mapped to a region in the p40 promoter containing a binding site for nuclear factor-kappaB (NF-kappaB) (p40-kappaB). Activation of macrophages by LPS in the presence of oxLDL resulted in markedly reduced binding to the kappaB site, as demonstrated by the electrophoretic mobility shift assays. In contrast, native LDL did not inhibit the IL-12 p40 promoter activation and NF-kappaB binding to the kappaB sites, suggesting that oxidative modification of LDL was crucial for the inhibition of NF-kappaB-mediated IL-12 production. 9-Hydroxyoctadecadienoic acid, a major oxidized lipid component of oxLDL, significantly inhibited IL-12 production in LPS-stimulated mouse macrophages and also suppressed NF-kappaB-mediated activation in IL-12 p40 promoter. The NF-kappaB components p50 and p65 directly bound peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in vitro. In cotransfections of CV-1 and HeLa cells, PPAR-gamma inhibited the NF-kappaB transactivation in an oxLDL-dependent manner. From these results, we propose that oxLDL-mediated suppression of the IL-12 production from LPS-activated mouse macrophages may, at least in part, involve both inhibition of the NF-kappaB-DNA interactions and physical interactions between NF-kappaB and PPAR-gamma.
Assuntos
Regulação da Expressão Gênica/imunologia , Interleucina-12/genética , Lipopolissacarídeos/farmacologia , Lipoproteínas LDL/farmacologia , Macrófagos/fisiologia , NF-kappa B/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Tiazolidinedionas , Fatores de Transcrição/fisiologia , Animais , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Hipoglicemiantes/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos DBA , Baço/imunologia , Tiazóis/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/imunologia , TransfecçãoRESUMO
Oxidized low-density lipoprotein (oxLDL) induces a wide range of cellular responses to produce atherosclerotic lesion, but key factors determining the response are not understood. In this study, purified LDL was oxidized with copper sulfate, and its physical properties and the related biological responses were investigated. The average hydrodynamic diameter of the lightly oxidized LDL was approximately 25 nm and its Rf value relative to nLDL on agarose gel was between 1.0 and 1.25. The diameter of the extensively oxidized LDL was over 30 nm, the Rf value was over 2.0. A 24 h-exposure of resting RAW264.7 macrophage cells to 100 microg/ml of the lightly oxidized LDL induced proliferation and macrophage activation whereas the extensively oxidized LDL induced cell death at the same concentration. In contrast, 200 microg/ml of oxLDL caused cell death regardless of oxidation degree. Short incubation (4-6 h) of the highly oxidized LDL (100 microg/ml) also resulted in cell proliferation. OxLDL-induced cell death showed mixed characteristics of apoptosis and/or necrosis depending on the strength and duration of the insult. These results suggest that cellular responses induced by oxLDL be dependent on the oxidation degree, the duration of exposure, and the concentration of oxLDL.
Assuntos
Morte Celular/fisiologia , Lipoproteínas LDL/metabolismo , Macrófagos/citologia , Animais , Apolipoproteínas B/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Divisão Celular/fisiologia , Sulfato de Cobre/metabolismo , Relação Dose-Resposta a Droga , Humanos , Metabolismo dos Lipídeos , Peroxidação de Lipídeos , Lipoproteínas LDL/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Necrose , Oxirredução , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
The effect of recombinant human hepatocyte growth factor (HGF) on low density lipoprotein (LDL) receptor gene expression was studied in the human hepatoma cell line HepG2. HepG2 cells were incubated with serum-free media in the presence and absence of HGF for various times and 125I-labeled LDL specific binding at 4 degrees C, uptake at 37 degrees C, and the levels of LDL receptor mRNA were measured. Incubation with HGF produced time- and concentration-dependent increases in 125I-labeled LDL binding (2-fold), uptake (2.5-fold), and LDL receptor mRNA (6-fold). HGF increased the rate of LDL receptor gene transcription 4- to 5-fold relative to that of several "house-keeping" genes as measured by nuclear run-on transcription. The half-life of LDL receptor mRNA, measured with actinomycin D, was not increased in HGF-treated cells. The stimulation of LDL receptor expression occurred independently of changes in cellular cholesterol or DNA biosynthesis or total cell protein. HepG2 cells were transiently transfected with plasmids bearing either three copies of repeats 2 and 3 (pLDLR(23)3LUC) or one copy of the LDL receptor promoter from -556 to +53 (pLDLR600LUC) linked to firefly luciferase. Incubation of pLDLR(23)3LUC, or pLDLR600LUC-transfected cells with HGF for 4 or 24 h at 37 degrees C produced a concentration-dependent increase in luciferase activity. A maximal stimulation of 3 to 6-fold was achieved for each construct at an HGF concentration of 100 ng/ml. In contrast, HGF had little or no effect on reporter activity in HepG2 cells transfected with a luciferase reporter plasmid bearing the HMG-CoA reductase promoter extending from -325 to +22. Thus, when compared to the native LDL receptor promoter, multiple copies of repeats 2 and 3 of the LDL receptor promoter can fully support activation of the luciferase reporter gene by HGF, demonstrating that the effect of HGF is mediated through the SRE-1. The lack of HGF effects mediated through the HMG-CoA reductase sterol regulatory element suggests, however, that sterol depletion may not be responsible for the induction of the LDL receptor promoter by growth factors. The signalling pathways or effectors responsible for activation of the LDL receptor and HMG-CoA reductase genes thus differ in their response to HGF. These data suggest that the level of SREBP's reaching the nucleus may be determined by as yet unidentified second messengers as well as by sterols.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Receptores de LDL/genética , Células Cultivadas , Cloranfenicol O-Acetiltransferase/genética , Humanos , Radioisótopos do Iodo , Luciferases/genética , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Sequências Repetitivas de Ácido Nucleico , Esteróis/farmacologia , Células Tumorais CultivadasRESUMO
Serum factors stimulate low density lipoprotein receptor (LDLR) gene expression in HepG2 cells through sterol-independent pathways. Promoter element other than sterol regulatory element-1 (SRE-1) seems to be necessary. Protein binding activity of the human LDLR promoter fragment (550bp) beyond the SRE-1 was determined by DNase I footprint assay. Five different promoter regions were protected from DNase I digestion; -226 to -258, -291 to -304, -324 to -336, -360 to -373, and -521 to -528. The regions of -324 to -336 and -521 to -528 showed serum response element (SRE)-like consensus sequence of CC(A/T)6GG. Serum incubation affected the protection degree of the SRE-like elements, but 25-hydroxycholesterol did not. It is proposed, therefore, that the promoter region of -324 to -336 and/or -521 to -528 showed serum response elements, but 25-hydroxycholesterol did not. It is proposed, therefore, that the promoter region of -324 to -336 and/or -521 to -528 in human LDLR gene may be responsible for the rapid activation of the gene transcription by serum factor in a sterol-independent manner.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Receptores de LDL/genética , Ativação Transcricional , Sequência de Bases , Sequência Consenso/genética , Pegada de DNA , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Humanos , Hidroxicolesteróis/farmacologia , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Ligação Proteica , Fator de Resposta Sérica , Proteína de Ligação a Elemento Regulador de Esterol 1 , Esteróis/farmacologia , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
The expression, distribution, and some aspects of the regulation of low-density lipoprotein (LDL) receptors in rat intestinal epithelial cells were examined. Cells prepared by a perfusion technique provided a pure preparation of epithelial cells and could be manipulated to produce crypt-villus units or villi alone. On a total protein basis, the abundance of LDL receptors in villus cell membranes was half that in hepatic membranes. The level of receptors in both tissues was reduced by feeding an atherogenic diet but was increased only in the liver by ethinyl estradiol-induced hypocholesterolemia. The level of LDL receptor mRNA in intestinal epithelial cells was somewhat lower than in liver. Regulation of LDL receptor mRNA was similar to that of protein. Judged by the ratio of mRNA in villus cells to the villus-crypt unit and nuclear run-on assay for LDL receptor gene transcription, we conclude that LDL receptor mRNA is produced in the villus cells. The effect of fat feeding was regulated at the level of transcription. Expression in villus cells in ileum was severalfold higher than in jejunum and higher than in the liver. Together the results suggest serum cholesterol level is not the prime determinant of LDL receptor level in intestine, but LDL degradation in this organ may be regulated by factors in the lumen.