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1.
PLoS One ; 18(11): e0293526, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37910485

RESUMO

Liver sinusoidal endothelial cells (LSECs) are fenestrated endothelial cells with a unique, high endocytic clearance capacity for blood-borne waste macromolecules and colloids. This LSEC scavenger function has been insufficiently characterized in liver disease. The Glmpgt/gt mouse lacks expression of a subunit of the MFSD1/GLMP lysosomal membrane protein transporter complex, is born normal, but soon develops chronic, mild hepatocyte injury, leading to slowly progressing periportal liver fibrosis, and splenomegaly. This study examined how LSEC scavenger function and morphology are affected in the Glmpgt/gt model. FITC-labelled formaldehyde-treated serum albumin (FITC-FSA), a model ligand for LSEC scavenger receptors was administered intravenously into Glmpgt/gt mice, aged 4 months (peak of liver inflammation), 9-10 month, and age-matched Glmpwt/wt mice. Organs were harvested for light and electron microscopy, quantitative image analysis of ligand uptake, collagen accumulation, LSEC ultrastructure, and endocytosis receptor expression (also examined by qPCR and western blot). In both age groups, the Glmpgt/gt mice showed multifocal liver injury and fibrosis. The uptake of FITC-FSA in LSECs was significantly reduced in Glmpgt/gt compared to wild-type mice. Expression of LSEC receptors stabilin-1 (Stab1), and mannose receptor (Mcr1) was almost similar in liver of Glmpgt/gt mice and age-matched controls. At the same time, immunostaining revealed differences in the stabilin-1 expression pattern in sinusoids and accumulation of stabilin-1-positive macrophages in Glmpgt/gt liver. FcγRIIb (Fcgr2b), which mediates LSEC endocytosis of soluble immune complexes was widely and significantly downregulated in Glmpgt/gt liver. Despite increased collagen in space of Disse, LSECs of Glmpgt/gt mice showed well-preserved fenestrae organized in sieve plates but the frequency of holes >400 nm in diameter was increased, especially in areas with hepatocyte damage. In both genotypes, FITC-FSA also distributed to endothelial cells of spleen and bone marrow sinusoids, suggesting that these locations may function as possible compensatory sites of clearance of blood-borne scavenger receptor ligands in liver fibrosis.


Assuntos
Células Endoteliais , Fígado , Camundongos , Animais , Células Endoteliais/metabolismo , Ligantes , Regulação para Baixo , Fluoresceína-5-Isotiocianato/metabolismo , Fígado/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Hepatócitos/metabolismo , Modelos Animais de Doenças , Colágeno/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo
2.
Int J Mol Sci ; 22(2)2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33467660

RESUMO

Multifactorial metabolic diseases, such as non-alcoholic fatty liver disease, are a major burden to modern societies, and frequently present with no clearly defined molecular biomarkers. Herein we used system medicine approaches to decipher signatures of liver fibrosis in mouse models with malfunction in genes from unrelated biological pathways: cholesterol synthesis-Cyp51, notch signaling-Rbpj, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling-Ikbkg, and unknown lysosomal pathway-Glmp. Enrichment analyses of Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome and TRANScription FACtor (TRANSFAC) databases complemented with genome-scale metabolic modeling revealed fibrotic signatures highly similar to liver pathologies in humans. The diverse genetic models of liver fibrosis exposed a common transcriptional program with activated estrogen receptor alpha (ERα) signaling, and a network of interactions between regulators of lipid metabolism and transcription factors from cancer pathways and the immune system. The novel hallmarks of fibrosis are downregulated lipid pathways, including fatty acid, bile acid, and steroid hormone metabolism. Moreover, distinct metabolic subtypes of liver fibrosis were proposed, supported by unique enrichment of transcription factors based on the type of insult, disease stage, or potentially, also sex. The discovered novel features of multifactorial liver fibrotic pathologies could aid also in improved stratification of other fibrosis related pathologies.


Assuntos
Ácidos Graxos/metabolismo , Cirrose Hepática/fisiopatologia , Fígado/fisiopatologia , Animais , Ácidos e Sais Biliares/química , Biomarcadores/metabolismo , Modelos Animais de Doenças , Feminino , Fibrose , Genoma , Humanos , Sistema Imunitário , Inflamação , Metabolismo dos Lipídeos , Lipídeos/química , Fígado/metabolismo , Cirrose Hepática/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais
3.
Elife ; 82019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31661432

RESUMO

Lysosomes are major sites for intracellular, acidic hydrolase-mediated proteolysis and cellular degradation. The export of low-molecular-weight catabolic end-products is facilitated by polytopic transmembrane proteins mediating secondary active or passive transport. A number of these lysosomal transporters, however, remain enigmatic. We present a detailed analysis of MFSD1, a hitherto uncharacterized lysosomal family member of the major facilitator superfamily. MFSD1 is not N-glycosylated. It contains a dileucine-based sorting motif needed for its transport to lysosomes. Mfsd1 knockout mice develop splenomegaly and severe liver disease. Proteomics of isolated lysosomes from Mfsd1 knockout mice revealed GLMP as a critical accessory subunit for MFSD1. MFSD1 and GLMP physically interact. GLMP is essential for the maintenance of normal levels of MFSD1 in lysosomes and vice versa. Glmp knockout mice mimic the phenotype of Mfsd1 knockout mice. Our data reveal a tightly linked MFSD1/GLMP lysosomal membrane protein transporter complex.


Assuntos
Fígado/fisiologia , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Homeostase , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Camundongos Knockout , Ligação Proteica
4.
Biomed Res Int ; 2019: 5496197, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31583245

RESUMO

BACKGROUND AND AIMS: Vascular endothelial growth factor (VEGF) receptors (VEGFR1 and VEGFR2) bind VEGF-A with high affinity. This study sought to determine the relative contributions of these two receptors to receptor-mediated endocytosis of VEGF-A and to clarify their endocytic itineraries in rat liver sinusoidal endothelial cells (LSECs). METHODS: Isolated LSECs and radiolabeled VEGF-A were used to examine surface binding and receptor-mediated endocytosis. Quantitative real time RT-PCR (Q-RT-PCR) and Western blotting were applied to demonstrate receptor expression. RESULTS: Q-RT-PCR analysis showed that VEGFR1 and VEGFR2 mRNA were expressed in LSECs. Ligand saturation analysis at 4°C indicated two different classes of [125I]-VEGFA binding sites on LSECs with apparent dissociation constants of 8 and 210 pM. At 37°C, LSECs efficiently took up and degraded [125I]-VEGF-A for at least 2 hours. Uptake of [125I]-VEGF-A by LSECs was blocked by dynasore that inhibits dynamin-dependent internalization, whereas inhibition of cysteine proteases by leupeptin inhibited degradation without affecting the uptake of [125I]-VEGF-A, suggesting that it is degraded following transport to lysosomes. Incubation of LSECs in the continued presence of a saturating concentration of unlabeled VEGF-A at 37°C was associated with a loss of as much as 75% of the total VEGFR2 within 30 min as shown by Western blot analysis, whereas there was no appreciable decrease in protein levels for VEGFR1 after 120 min incubation, suggesting that VEGF-A stimulation downregulates VEGFR2, but not VEGFR1, in LSECs. This possibility was supported by the observation that a hexapeptide that specifically blocks VEGF-A binding to VEGFR1 caused a marked reduction in the uptake of [125I]-VEGF-A, whereas a control peptide had no effect. Finally, live cell imaging studies using a fluorescently labeled anti-VEGFR2 antibody showed that VEGFR2 was transported via early and late endosomes to reach endolysosomes where degradation of the VEGFR2 takes place. CONCLUSION: Our studies suggest that, subsequent to VEGF-A binding and internalization, the unoccupied VEGFR1 may recycle to the cell surface allowing its reutilization, whereas the majority of the internalized VEGFR2 is targeted for degradation.


Assuntos
Fígado/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Membrana Celular/genética , Endocitose/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Regulação da Expressão Gênica/genética , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/patologia , RNA Mensageiro/genética , Ratos , Transdução de Sinais/genética
5.
Artigo em Inglês | MEDLINE | ID: mdl-27141234

RESUMO

BACKGROUND: Mice lacking glycosylated lysosomal membrane protein (Glmp (gt/gt) mice) have liver fibrosis as the predominant phenotype due to chronic liver injury. The Glmp (gt/gt) mice grow and reproduce at the same rate as their wild-type siblings. Life expectancy is around 18 months. METHODS: Wild-type and Glmp (gt/gt) mice were studied between 1 week and 18 months of age. Livers were analyzed using histological, immunohistochemical, biochemical, and qPCR analyses. RESULTS: It was shown that Glmp (gt/gt) mice were not born with liver injury; however, it appeared shortly after birth as indicated by excess collagen expression, deposition of fibrous collagen in the periportal areas, and increased levels of hydroxyproline in Glmp (gt/gt) liver. Liver functional tests indicated a chronic, mild liver injury. Markers of inflammation, fibrosis, apoptosis, and modulation of extracellular matrix increased from an early age, peaking around 4 months of age and followed by attenuation of these signals. To compensate for loss of hepatocytes, the oval cell compartment was activated, with the highest activity of the oval cells detected at 3 months of age, suggesting insufficient hepatocyte proliferation in Glmp (gt/gt) mice around this age. Although constant proliferation of hepatocytes and oval cells maintained adequate hepatic function in Glmp (gt/gt) mice, it also resulted in a higher frequency of liver tumors in older animals. CONCLUSIONS: The Glmp (gt/gt) mouse is proposed as a model for slowly progressing liver fibrosis and possibly as a model for a yet undescribed human lysosomal disorder.

6.
Arch Physiol Biochem ; 122(1): 36-45, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26707125

RESUMO

Glycosylated lysosomal membrane protein (GLMP) has been reported to enhance the expression from a peroxisome proliferator-activated receptor alpha (PPARα) responsive promoter, but also to be an integral lysosomal membrane protein. Using myotubes established from wild-type and Glmp(gt/gt) mice, the importance of GLMP in skeletal muscle was examined. Glmp(gt/gt) myotubes expressed a more glycolytic phenotype than wild-type myotubes. Myotubes from Glmp(gt/gt) mice metabolized glucose faster and had a larger pool of intracellular glycogen, while oleic acid uptake, storage and oxidation were significantly reduced. Gene expression analyses indicated lower expression of three PPAR-isoforms, a co-regulator of PPAR (PGC1α) and several genes important for lipid metabolism in Glmp(gt/gt) myotubes. However, ablation of GLMP did not seem to substantially impair the response to PPAR agonists. In conclusion, myotubes established from Glmp(gt/gt) mice were more glycolytic than myotubes from wild-type animals, in spite of no differences in muscle fiber types in vivo.


Assuntos
Ácidos Graxos/metabolismo , Deleção de Genes , Glucose/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Masculino , Proteínas de Membrana/deficiência , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Ácido Oleico/metabolismo , Oxirredução/efeitos dos fármacos , Receptores Ativados por Proliferador de Peroxissomo/agonistas , Receptores Ativados por Proliferador de Peroxissomo/metabolismo
7.
PLoS One ; 10(6): e0129402, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26047317

RESUMO

Ablation of glycosylated lysosomal membrane protein (GLMP, formerly known as NCU-G1) has been shown to cause chronic liver injury which progresses into liver fibrosis in mice. Both lysosomal dysfunction and chronic liver injury can cause metabolic dysregulation. Glmp gt/gt mice (formerly known as Ncu-g1gt/gt mice) were studied between 3 weeks and 9 months of age. Body weight gain and feed efficiency of Glmp gt/gt mice were comparable to wild type siblings, only at the age of 9 months the Glmp gt/gt siblings had significantly reduced body weight. Reduced size of epididymal fat pads was accompanied by hepatosplenomegaly in Glmp gt/gt mice. Blood analysis revealed reduced levels of blood glucose, circulating triacylglycerol and non-esterified fatty acids in Glmp gt/gt mice. Increased flux of glucose, increased de novo lipogenesis and lipid accumulation were detected in Glmp gt/gt primary hepatocytes, as well as elevated triacylglycerol levels in Glmp gt/gt liver homogenates, compared to hepatocytes and liver from wild type mice. Gene expression analysis showed an increased expression of genes involved in fatty acid uptake and lipogenesis in Glmp gt/gt liver compared to wild type. Our findings are in agreement with the metabolic alterations observed in other mouse models lacking lysosomal proteins, and with alterations characteristic for advanced chronic liver injury.


Assuntos
Lipogênese/genética , Fígado/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/genética , Tecido Adiposo/metabolismo , Animais , Glicemia/metabolismo , Western Blotting , Células Cultivadas , Epididimo/metabolismo , Ácidos Graxos/sangue , Expressão Gênica , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Hepatócitos/metabolismo , Hepatomegalia/sangue , Hepatomegalia/genética , Metabolismo dos Lipídeos/genética , Fígado/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esplenomegalia/sangue , Esplenomegalia/genética , Triglicerídeos/sangue , Aumento de Peso/genética
8.
Dis Model Mech ; 7(3): 351-62, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24487409

RESUMO

Human kidney predominant protein, NCU-G1, is a highly conserved protein with an unknown biological function. Initially described as a nuclear protein, it was later shown to be a bona fide lysosomal integral membrane protein. To gain insight into the physiological function of NCU-G1, mice with no detectable expression of this gene were created using a gene-trap strategy, and Ncu-g1(gt/gt) mice were successfully characterized. Lysosomal disorders are mainly caused by lack of or malfunctioning of proteins in the endosomal-lysosomal pathway. The clinical symptoms vary, but often include liver dysfunction. Persistent liver damage activates fibrogenesis and, if unremedied, eventually leads to liver fibrosis/cirrhosis and death. We demonstrate that the disruption of Ncu-g1 results in spontaneous liver fibrosis in mice as the predominant phenotype. Evidence for an increased rate of hepatic cell death, oxidative stress and active fibrogenesis were detected in Ncu-g1(gt/gt) liver. In addition to collagen deposition, microscopic examination of liver sections revealed accumulation of autofluorescent lipofuscin and iron in Ncu-g1(gt/gt) Kupffer cells. Because only a few transgenic mouse models have been identified with chronic liver injury and spontaneous liver fibrosis development, we propose that the Ncu-g1(gt/gt) mouse could be a valuable new tool in the development of novel treatments for the attenuation of fibrosis due to chronic liver damage.


Assuntos
Ferro/metabolismo , Células de Kupffer/metabolismo , Lipofuscina/metabolismo , Cirrose Hepática/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Animais , Catepsina D/metabolismo , Morte Celular , Colágeno/metabolismo , Feminino , Fluorescência , Marcação de Genes , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Inflamação/patologia , Células de Kupffer/patologia , Células de Kupffer/ultraestrutura , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/patologia , Masculino , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Fenótipo , Reprodutibilidade dos Testes , Esplenomegalia/metabolismo , Esplenomegalia/patologia
9.
BMC Mol Biol ; 8: 106, 2007 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-18021396

RESUMO

BACKGROUND: Novel, uncharacterised proteins represent a challenge in biochemistry and molecular biology. In this report we present an initial functional characterization of human kidney predominant protein, NCU-G1. RESULTS: NCU-G1 was found to be a highly conserved nuclear protein rich in proline with a molecular weight of approximately 44 kDa. It is localized on chromosome 1 and consists of 6 exons. Analysis of the amino acid sequence revealed no known transcription activation domains or DNA binding regions, however, four nuclear receptor boxes (LXXLL), and four SH3-interaction motives in addition to numerous potential phosphorylation sites were found. Two nuclear export signals were identified, but no nuclear localization signal. In man, NCU-G1 was found to be widely expressed at the mRNA level with especially high levels detected in prostate, liver and kidney. Electrophoretic mobility shift analysis showed specific binding of NCU-G1 to an oligonucleotide representing the footprint 1 element of the human cellular retinol-binding protein 1 gene promoter. NCU-G1 was found to activate transcription from this promoter and required presence of the footprint 1 element. In transiently transfected Drosophila Schneider S2 cells, we demonstrated that NCU-G1 functions as a co-activator for ligand-activated PPAR-alpha, resulting in an increased expression of a CAT reporter gene under control of the peroxisome proliferator-activated receptor-alpha responsive acyl-CoA oxidase promoter. CONCLUSION: We propose that NCU-G1 is a dual-function protein capable of functioning as a transcription factor as well as a nuclear receptor co-activator.


Assuntos
Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Clonagem Molecular , Perfilação da Expressão Gênica , Células HeLa , Humanos , Ligantes , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Oligonucleotídeos/metabolismo , Especificidade de Órgãos , PPAR alfa/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Celulares de Ligação ao Retinol/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transcrição Gênica , Ativação Transcricional/genética
10.
BMC Mol Biol ; 8: 5, 2007 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-17241464

RESUMO

BACKGROUND: Scavenger receptor type B class I (SR-BI), ABC transporter A1 (ABCA1) -and G1 (ABCG1) all play important roles in the reverse cholesterol transport. Reverse cholesterol transport is a mechanism whereby the body can eliminate excess cholesterol. Here, the regulation of SR-BI, ABCA1, and ABCG1 by dexamethasone (a synthetic glucocorticoid) and insulin were studied in order to gain more insight into the role of these two hormones in the cholesterol metabolism. RESULTS: By use of real time RT-PCR and Western blotting we examined the expression of our target genes. The results show that SR-BI, ABCA1 and ABCG1 mRNA expression increased in response to dexamethasone while insulin treatment reduced the expression in primary rat hepatocytes. The stimulatory effect of dexamethasone was reduced by the addition of the anti-glucocorticoid mifepristone. In HepG2 cells and THP-1 macrophages, however, the effect of dexamethasone was absent or inhibitory with no significant change in the presence of mifepristone. The latter observation may be a result of the low protein expression of glucocorticoid receptor (GR) in these cell lines. CONCLUSION: Our results illustrates that insulin and glucocorticoids, two hormones crucial in the carbohydrate metabolism, also play an important role in the regulation of genes central in reverse cholesterol transport. We found a marked difference in mRNA expression between the primary cells and the two established cell lines when studying the effect of dexamethasone which may result from the varying expression levels of GR.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Dexametasona/metabolismo , Hepatócitos/metabolismo , Insulina/metabolismo , Receptores Depuradores Classe B/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Colesterol/metabolismo , Humanos , Macrófagos/metabolismo , Ratos , Ratos Wistar , Receptores de Glucocorticoides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
11.
Biochem Biophys Res Commun ; 336(4): 1096-105, 2005 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-16168958

RESUMO

Hepatic SR-BI mediates uptake of circulating cholesterol into liver hepatocytes where a part of the cholesterol is metabolised to bile acids. In the hepatocytes, bile acids reduce their own synthesis by a negative feedback loop to prevent toxic high levels of bile acids. Bile acid-activated FXR/RXR represses expression of CYP7A1, the rate-limiting enzyme during bile acid synthesis, by inducing the expression of SHP, which inhibits LXR/RXR and LRH-1-transactivation of CYP7A1. The present paper presents data indicating that CDCA suppresses SR-BI expression by the same pathway. As previously reported, LRH-1 induces SR-BI promoter activity. Here we show that CDCA or over-expression of SHP inhibit this transactivation. No FXR-response element was identified in the bile acid-responsive region of the SR-BI promoter (-1200bp/-937bp). However, a binding site for LRH-1 was characterised and shown to specifically bind LRH-1. The present study shows that also the SR-BI-mediated supply of cholesterol, the substrate for bile acid synthesis, is feedback regulated by bile acids.


Assuntos
Ácido Quenodesoxicólico/fisiologia , Proteínas de Ligação a DNA/metabolismo , Hepatócitos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptor X Retinoide alfa/metabolismo , Receptores Depuradores Classe B/biossíntese , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Chlorocebus aethiops , HDL-Colesterol/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Humanos , Masculino , Camundongos , Regiões Promotoras Genéticas , Ratos , Ratos Wistar , Receptor X Retinoide alfa/agonistas , Transdução de Sinais
12.
Biochem Biophys Res Commun ; 312(3): 716-24, 2003 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-14680824

RESUMO

The liver X receptors alpha and beta (LXRalpha and LXRbeta) are members of the nuclear receptor superfamily of proteins which are highly expressed in metabolically active tissues. They regulate gene expression of critical genes involved in cholesterol catabolism and transport, lipid and triglyceride biosynthesis, and carbohydrate metabolism in response to distinct oxysterol intermediates in the cholesterol metabolic pathway. Several LXR target genes have been identified, but there is limited information on how expression of the LXRs themselves is controlled. In this study we have characterized the upstream flanking region of the mouse LXRalpha gene. Transient transfections show that the LXRalpha promoter is able to drive transcription of a luciferase reporter gene, however, the transcriptional potential of the promoter in the cell lines used was low. The -2143 to -1513 region of the promoter mediates repression of reporter gene activity in all cells analyzed and multiple DNA-protein interactions were detected in this region by DNase I footprinting. The Zta, Ets, and Hes1 transcription factors were all shown to mediate alterations in reporter gene activity driven by LXRalpha promoter deletion constructs. These factors have been linked to cell cycle and differentiation processes suggesting that expression of LXRalpha might be under control of signalling mechanisms regulating cell proliferation. Several putative binding sites of the glucocorticoid receptor (GR) were identified in the LXRalpha promoter and transient cotransfections of the GR and LXRalpha promoter deletion constructs induced reporter gene activity. Addition of dexamethasone, a GR agonist, abolished this effect suggesting cross talk between GR and LXR signalling.


Assuntos
Regulação da Expressão Gênica/fisiologia , Regiões Promotoras Genéticas/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Pegada de DNA , Proteínas de Ligação a DNA , Humanos , Fígado/metabolismo , Receptores X do Fígado , Camundongos , Dados de Sequência Molecular , Receptores Nucleares Órfãos , Homologia de Sequência , Especificidade da Espécie , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
13.
Mol Endocrinol ; 17(12): 2630-8, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14525951

RESUMO

Testicular tumors in humans are reported to be significantly increasing in incidence. Embryo exposure to environmental estrogens has been proposed as one of the possible underlying causes. In mice, genetic, immunological, and experimental evidence suggest that germ cell testicular tumors may derive from primordial germ cells (PGCs), the embryonic precursors of gametes. Here we show that relatively high concentrations of estrogens stimulate mouse PGC growth in vitro through the somatic cells of the gonadal ridges. Moreover, we found that estrogens stimulate the transcription of the Steel gene and the production of c-Kit ligand in gonadal somatic cells, and that this growth factor is likely to be responsible for the observed stimulation of PGC growth via an Akt/PTEN pathway. Finally, we show that estrogen stimulation of gonadal somatic cells in culture, in combination with PTEN down-regulation in PGCs and the presence of leukemia inhibitory factor in the culture medium, result in high frequency of PGC transformation in tumorigenic cells. Based on these results, we present a novel experimental in vitro model for tumorigenic germ cell transformation and identify molecular pathways likely involved in development of germ cell tumors after estrogen exposure.


Assuntos
Células Germinativas/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Divisão Celular/efeitos dos fármacos , Estradiol/farmacologia , Feminino , Genes Reporter , Células Germinativas/citologia , Células Germinativas/efeitos dos fármacos , Técnicas In Vitro , Masculino , Camundongos , Gravidez , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Testículo/citologia , Testículo/efeitos dos fármacos , Testículo/embriologia , Zearalenona/farmacologia
14.
Biochem Biophys Res Commun ; 299(5): 916-23, 2002 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-12470667

RESUMO

SR-BI mediates exchange of cholesterol between HDL and cells, and is a crucial factor in the transport of excessive cellular cholesterol from extrahepatic tissues to the liver ("reverse cholesterol transport") and, therefore, also for cholesterol homeostasis. Hepatic SR-BI mediates transfer of HDL-cholesterol to the hepatocytes where cholesterol may be metabolised to bile acids. LXR and SREBP are key factors in the regulation of cholesterol metabolism. The purpose of the present study was to determine whether these transcription factors are involved in the regulation of SR-BI. Here we show that LXRalpha/RXR and LXRbeta/RXR induce SR-BI transcription in human and murine hepatoma cell lines, and in 3T3-L1 preadipocytes independently of SREBP-1. The LXR/RXR response was mapped within -1,200 to -937 of the promoter region. Gel mobility shift analysis confirmed that the putative LXR response element bound LXRalpha/RXR and LXRbeta/RXR heterodimers.


Assuntos
Adipócitos/metabolismo , Antígenos CD36/genética , Hidroxicolesteróis/farmacologia , Proteínas de Membrana , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Imunológicos , Receptores de Lipoproteínas , Ativação Transcricional , Animais , Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Antígenos CD36/biossíntese , Células COS , Carcinoma Hepatocelular , Linhagem Celular , Proteínas de Ligação a DNA/fisiologia , Vetores Genéticos , Receptores X do Fígado , Camundongos , Receptores Nucleares Órfãos , Receptores Citoplasmáticos e Nucleares/genética , Receptores do Ácido Retinoico/metabolismo , Receptores Depuradores , Elementos de Resposta , Receptores X de Retinoides , Retroviridae/genética , Receptores Depuradores Classe B , Deleção de Sequência , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1 , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas
15.
Biochem Biophys Res Commun ; 293(5): 1333-40, 2002 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-12054659

RESUMO

LXRs have recently been shown to regulate key enzymes in cholesterol degradation, reverse transport of cholesterol from peripheral cells, cholesterol uptake and lipogenesis. The LXRalpha promoter was thus studied to investigate if LXRalpha gene expression is under the regulation of transcription factors involved in adipogenesis. We report that the C/EBP transcription factor interacts with the promoter of the LXRalpha gene. In in vitro footprinting experiments, protein extracts from several tissues gave footprints covering a putative C/EBP recognition site. Transfection experiments and EMSA showed a direct effect of these transcription factors on the LXRalpha promoter. C/EBPalpha upregulated expression of the reporter gene in an NIH 3T3-L1 preadipocyte cell line, while C/EBPbeta and C/EBPdelta had no effect. In liver hepatoma Fao II and Cos-7 kidney cells, both C/EBPalpha and C/EBPbeta downregulated expression of the reporter gene while C/EBPdelta induced activity, indicating that the functional consequences of C/EBP isoform interactions with the LXRalpha promoter are dependent on the cellular context. Monitoring of the LXR mRNA levels during adipose tissue differentiation showed that LXRbeta is constitutively expressed during the entire differentiation process while LXRalpha is induced upon addition of differentiation mix.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Células 3T3 , Adipócitos/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Células COS , Células Cultivadas , Colesterol/metabolismo , Proteínas de Ligação a DNA , Regulação para Baixo , Fibroblastos/metabolismo , Humanos , Leucina/química , Receptores X do Fígado , Camundongos , Dados de Sequência Molecular , Oligonucleotídeos/metabolismo , Receptores Nucleares Órfãos , Plasmídeos/metabolismo , Ligação Proteica , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Homologia de Sequência do Ácido Nucleico , Fatores de Tempo , Transcrição Gênica , Transfecção , Regulação para Cima
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