RESUMO
Both estrogen and selective estrogen receptor modulators (SERMs) such as tamoxifen and raloxifene have been demonstrated to lower plasma low density lipoprotein (LDL)-cholesterol concentrations by stimulation of LDL receptor gene expression. To determine the molecular mechanisms of estradiol- and tamoxifen-induced LDL receptor expression, we performed transient transfection experiments with luciferase reporter gene-constructs under transcriptional control of the human LDL receptor promoter. We demonstrate, that estradiol and tamoxifen stimulate LDL receptor gene expression in human HepG2 hepatoma cells only when estrogen receptor (ER)-alpha but not when ER-beta is cotransfected. Deletion mutants and point mutations of the LDL receptor promoter reveal that estradiol- and tamoxifen-stimulated expression of this gene depends on an intact repeat 3 in the LDL receptor promoter, a cis-element previously shown to interact with Sp1. Gel mobility analyses demonstrated estradiol- and tamoxifen-stimulated binding of nuclear proteins to repeat 3 (bp -56 to bp -36) of the LDL receptor promoter. These data provide an alternative mechanism of LDL receptor gene expression by non-classical estradiol- and tamoxifen-stimulated induction through an ER-alpha/Sp1 complex.
Assuntos
Receptores de Estrogênio/fisiologia , Receptores de LDL/biossíntese , Fator de Transcrição Sp1/fisiologia , Linhagem Celular Tumoral , Estradiol/farmacologia , Receptor alfa de Estrogênio , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mutação , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Elementos de Resposta , Fator de Transcrição Sp1/metabolismo , Tamoxifeno/farmacologiaRESUMO
BACKGROUND: Congenital generalized lipodystrophy (CGL) or Berardinelli-Seip congenital lipodystrophy (BSCL) is a rare genetic syndrome characterized by the absence of adipose tissue. As CGL is thought to be related to malfunctions in adipocyte development, genes involved in the mechanisms of adipocyte biology and maintenance or differentiation of adipocytes, especially transcription factors are candidates. Several genes (BSCL1-4) were found to be associated to the syndrome but not all CGL patients carry mutations in these genes. METHODS AND RESULTS: In a patient with CGL and insulin resistance we investigated the known candidate genes but the patient did not carry a relevant mutation. Analyses of the insulin activated signal transduction pathways in isolated fibroblasts of the patient revealed a postreceptor defect altering expression of the immediate early gene c-fos. Sequence analyses revealed a novel homozygous point mutation (c.-439, TâA) in the patients' c-fos promoter. The point mutation was located upstream of the well characterized promoter elements in a region with no homology to any known cis-elements. The identified mutation was not detected in a total of n=319 non lipodystrophic probands. In vitro analyses revealed that the mutation facilitates the formation of a novel and specific protein/DNA complex. Using mass spectrometry we identified the proteins of this novel complex. Cellular investigations demonstrate that the wild type c-fos promoter can reconstitute the signaling defect in the patient, excluding further upstream signaling alterations, and vice versa the investigations with the c-fos promoter containing the identified mutation generally reduce basal and inducible c-fos transcription activity. As a consequence of the identified point mutation gene expression including c-Fos targeted genes is significantly altered, shown exemplified in cells of the patient. CONCLUSION: The immediate-early gene c-fos is one essential transcription factor to initiate adipocyte differentiation. According to the role of c-fos in adipocyte differentiation our findings of a mutation that initiates a repression mechanism at c-fos promoter features the hypothesis that diminished c-fos expression might play a role in CGL by interfering with adipocyte development.
Assuntos
Genes fos/genética , Lipodistrofia Generalizada Congênita/genética , Mutação Puntual , Adipócitos/citologia , Adipócitos/fisiologia , Adulto , Diferenciação Celular , Feminino , Expressão Gênica , Genes Precoces , Genes fos/fisiologia , HumanosRESUMO
OBJECTIVE: Increased lipid accumulation in cells and tissues is a key phenomenon in the development of obesity, insulin resistance, and atherosclerosis. In the regulation of lipid content of cells and tissues the SREBPs play a dominant role as transcription factors. METHODS: Since growth hormone (GH) affects lipid metabolism and function of fat as well as liver cells, we have investigated the role of SREBP-1a, SREBP-1c and SREBP-2 in the gene regulatory action of GH in the human liver cell line HepG2 and primary mouse hepatocytes. RESULTS: These experiments showed that SREBP-1a couples the stimulatory effect of GH on cholesterol regulated genes, e.g. LDL receptor gene, via sterol sensitive binding cis-element (sre-1). This effect was not depending on RNA expression, but related to phosphorylation of SREBP-1a protein. The result was supported by experiments with the mutant variant SREBP-1a S117A, which is not phosphorylated by Erk-MAP kinases. To analyse a possible role of GH-induced SREBP-1a phosphorylation in cellular physiology we investigated an expression profile of genes coding for central players in lipid transport or lipid metabolism as well as for transcription factors by real time PCR in primary mouse hepatocytes and human hepatoma cell line stably overexpressing the mature form of SREBP-1a or mutated form. CONCLUSION: These experiments emphasize the role SREBP-1a and its phosphorylation for gene regulatory effects of GH.
Assuntos
Hormônio do Crescimento/metabolismo , Hepatócitos/metabolismo , Metabolismo dos Lipídeos , Lipídeos/química , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Animais , Linhagem Celular , Colesterol/metabolismo , Hepatócitos/citologia , Humanos , Resistência à Insulina , Camundongos , Modelos Biológicos , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Sterol regulatory element binding protein-1a (SREBP-1a) is a transcription factor that is a major player in lipid metabolism and insulin action. We have generated human liver cells (HepG2) overexpressing active SREBP-1a constitutively called SREBP-1a (+). These cells show massive intracellular lipid accumulation. To elucidate the effect of SREBP-1a on lipid metabolism at the level of the cellular protein network, we have analyzed the protein pattern of mitochondria using the novel technique two-dimensional difference gel electrophoresis (2D-DIGE). Mitochondria were enriched by subcellular fractionation using differential and isopyknic centrifugation. Proteins of isolated organelles were labeled with Cy dyes and separated on 2D gels. These gels revealed more than 100 protein spots, which were significantly different in their abundance between wild-type and SREBP-1a (+) cells. MALDI mass spectrometry showed that 68% of identified proteins belong to mitochondria. In SREBP-1a (+) cells, several enzymes involved in lipid metabolism were significantly altered, suggesting that cellular lipid metabolism is triggered by accumulation of fatty acids rather than by its degradation. To test the possible functional relevance of this finding, intracellular fatty acid (FA) patterns were analyzed by gas chromatography. The results showed a significant increase in total fatty acid content with a shift in composition to long-chain unsaturated FAs. Therefore, the detected protein differences might be an explanation for the observed intracellular lipid accumulation and might link SREBP-1a to features like steatosis hepatis.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/metabolismo , Hepatócitos/metabolismo , Proteínas Mitocondriais/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT/genética , Linhagem Celular , DNA/genética , Proteínas de Ligação a DNA/genética , Eletroforese em Gel Bidimensional , Expressão Gênica , Hepatócitos/ultraestrutura , Humanos , Metabolismo dos Lipídeos , Microscopia Eletrônica , Proteínas Mitocondriais/classificação , Proteínas Mitocondriais/isolamento & purificação , RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteína de Ligação a Elemento Regulador de Esterol 1 , Fatores de Transcrição/genéticaRESUMO
The transcription factor sterol regulatory element binding protein (SREBP)-2 plays a pivotal role in lipid metabolism. Previously, we have shown that the mature form of SREBP-2 is a substrate of Erk-mitogen-activated protein kinases (MAPK). The aim of the present study was to identify Erk-specific phosphorylation sites. Using a protein chemistry approach, we could identify Ser-432 and Ser-455 as major phosphorylation sites. Further characterization by electrophoretic mobility shift assay and promoter reporter gene analyses revealed that phosphorylation does not influence protein/DNA interaction, but enhances trans-activity. In intact cells, SREBP-2 is phosphorylated by insulin, which seems to be related to their bio-responses on low density lipoprotein receptor activity. These results suggest that activation of Erk-MAPK pathways by hormones such as insulin might be related to a novel regulatory principle of SREBP-2.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Insulina/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Serina/química , Fatores de Transcrição/metabolismo , Sítios de Ligação , Western Blotting , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA/metabolismo , Genes Reporter , Humanos , Luciferases/metabolismo , Sistema de Sinalização das MAP Quinases , Mutagênese Sítio-Dirigida , Peptídeos/química , Fosforilação , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteína de Ligação a Elemento Regulador de Esterol 2 , Ativação Transcricional , TransfecçãoRESUMO
Gab1 (Grb2-associated binder1) belongs to a family of multifunctional docking proteins that play a central role in the integration of receptor tyrosine kinase (RTK) signaling, i.e., mediating cellular growth response, transformation, and apoptosis. In addition to RTK-specific tyrosine phosphorylation, these docking proteins also can be phosphorylated on serine/threonine residues affecting signal transduction. Since serine and threonine phosphorylation are capable of modulating the initial signal one major task to elucidate signal transduction via Gab1 is to determine the exact localization of distinct phosphorylation sites. To address this question in this report we examined extracellular signal-regulated kinases 1/2 (ERK) specific serine/threonine phosphorylation of the entire Gab1 engaged in insulin signaling in more detail in vitro. To elucidate the ERK1/2-specific phosphorylation pattern of Gab1, we used phosphopeptide mapping by two-dimensional HPLC analysis. Subsequently, phosphorylated serine/threonine residues were identified by sequencing the separated phosphopeptides using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and Edman degradation. Our results demonstrate that ERK1/2 phosphorylate Gab1 at six serine/threonine residues (T312, S381, S454, T476, S581, S597) in consensus motifs for MAP kinase phosphorylation. Serine residues S454, S581, S597, and threonine residue T476 represent nearly 80% of overall incorporated phosphate. These sites are located adjacent to src homology region-2 (SH2) binding motifs (YVPM-motif: Y447, Y472, Y619) specific for the phosphatidylinositol 3kinase (PI3K). The biological role of identified phosphorylation sites was proven by PI3K and Akt activity in intact cells. These data demonstrate that ERK1/2 modulate insulin action via Gab1 by targeting serine and threonine residues beside YXXM motifs. Accordingly, insulin signaling is blocked at the level of PI3K.