RESUMO
The DHCR7 enzyme converts 7-DHC into cholesterol. Mutations in DHCR7 can block cholesterol production, leading to abnormal accumulation of 7-DHC and causing Smith-Lemli-Opitz syndrome (SLOS). SLOS is an autosomal recessive disorder characterized by multiple malformations, including microcephaly, intellectual disability, behavior reminiscent of autism, sleep disturbances, and attention-deficit/hyperactivity disorder (ADHD)-like hyperactivity. Although 7-DHC affects neuronal differentiation in ex vivo experiments, the precise mechanism of SLOS remains unclear. We generated Dhcr7 deficient (dhcr7-/-) zebrafish that exhibited key features of SLOS, including microcephaly, decreased neural stem cell pools, and behavioral phenotypes similar to those of ADHD-like hyperactivity. These zebrafish demonstrated compromised myelination, synaptic anomalies, and neurotransmitter imbalances. The axons of the dhcr7-/- zebrafish showed increased lysosomes and attenuated autophagy, suggesting that autophagy-related neuronal homeostasis is disrupted.
Assuntos
Axônios , Colesterol , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Peixe-Zebra , Animais , Autofagia , Axônios/metabolismo , Colesterol/metabolismo , Lisossomos/metabolismo , Neurogênese , Neurônios/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Síndrome de Smith-Lemli-Opitz/metabolismo , Síndrome de Smith-Lemli-Opitz/genética , Síndrome de Smith-Lemli-Opitz/patologia , Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Tissue absorbance, light scattering, and autofluorescence are significantly lower in the near-infrared (NIR) range than in the visible range. Because of these advantages, NIR fluorescent proteins (FPs) are in high demand for in vivo imaging. Nevertheless, application of NIR FPs such as iRFP is still limited due to their dimness in mammalian cells. In contrast to GFP and its variants, iRFP requires biliverdin (BV) as a chromophore. The dimness of iRFP is at least partly due to rapid reduction of BV by biliverdin reductase-A (BLVRA). Here, we established biliverdin reductase-a knockout (Blvra-/-) mice to increase the intracellular BV concentration and, thereby, to enhance iRFP fluorescence intensity. As anticipated, iRFP fluorescence intensity was significantly increased in all examined tissues of Blvra-/- mice. Similarly, the genetically encoded calcium indicator NIR-GECO1, which is engineered based on another NIR FP, mIFP, exhibited a marked increase in fluorescence intensity in mouse embryonic fibroblasts derived from Blvra-/- mice. We expanded this approach to an NIR light-sensing optogenetic tool, the BphP1-PpsR2 system, which also requires BV as a chromophore. Again, deletion of the Blvra gene markedly enhanced the light response in HeLa cells. These results indicate that the Blvra-/- mouse is a versatile tool for the in vivo application of NIR FPs and NIR light-sensing optogenetic tools.Key words: in vivo imaging, near-infrared fluorescent protein, biliverdin, biliverdin reductase, optogenetic tool.
Assuntos
Biliverdina/metabolismo , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Animais , Biliverdina/genética , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismoRESUMO
Endothelial dysfunction accompanied by the loss of endothelial cell phenotype plays an essential role in cardiovascular diseases. Here, we report that knockdown of biliverdin reductase (BVR), the enzyme of the heme degradation pathway converting biliverdin to bilirubin, shifts endothelial phenotype of the primary human aortic endothelial cells (HAECs) to mesenchymal-like one. It is reflected by the loss of endothelial markers and angiogenic response, with concomitant acquiring of mesenchymal markers, increased migratory capacity and metalloproteinase activity. BVR-deficiency induces the activity of Nrf2 transcription factor and increases heme oxygenase-1 (HO-1) level, which is accompanied by the reduction of cellular heme content, increase in a free iron fraction and oxidative stress. Accordingly, the phenotype of BVR-deficient cells can be mimicked by hemin or iron overload. Depletion of HO-1 in BVR-deficient ECs abrogates the increase in intracellular free iron and oxidative stress, preventing the loss of endothelial markers. Treatment of BVR-deficient cells with bilirubin does not rescue the endothelial phenotype of HAECs. Unlike BLVRA mRNA level, the expression of HMOX1, HMOX1:BLVRA ratio and HO-1 protein level positively correlate with abdominal aortic aneurysm size in clinical samples. Collectively, the non-enzymatic activity of BVR contributes to the maintenance of healthy endothelial phenotype through the prevention of HO-1-dependent iron-overload, oxidative stress and subsequent endothelial-to-mesenchymal transition (EndMT).
Assuntos
Transdiferenciação Celular/genética , Células Endoteliais/citologia , Mesoderma/citologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Técnicas de Silenciamento de Genes , Humanos , FenótipoRESUMO
Apoptosis and the proper clearance of apoptotic cells play a central role in maintaining tissue homeostasis. Previous work in our laboratory has shown that when a high number of cells enters apoptosis in a tissue, the macrophages that engulf them produce retinoids to enhance their own phagocytic capacity by upregulating several phagocytic genes. Our data indicated that these retinoids might be dihydroretinoids, which are products of the retinol saturase (RetSat) pathway. In the present study, the efferocytosis of RetSat-null mice was investigated. We show that among the retinoid-sensitive phagocytic genes, only transglutaminase 2 responded in macrophages and in differentiating monocytes to dihydroretinol. Administration of dihydroretinol did not affect the expression of the tested genes differently between differentiating wild type and RetSat-null monocytes, despite the fact that the expression of RetSat was induced. However, in the absence of RetSat, the expression of numerous differentiation-related genes was altered. Among these, impaired production of MFG-E8, a protein that bridges apoptotic cells to the αvß3/ß5 integrin receptors of macrophages, resulted in impaired efferocytosis, very likely causing the development of mild autoimmunity in aged female mice. Our data indicate that RetSat affects monocyte/macrophage differentiation independently of its capability to produce dihydroretinol at this stage.
Assuntos
Envelhecimento/imunologia , Apoptose/imunologia , Doenças Autoimunes/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Envelhecimento/genética , Envelhecimento/patologia , Animais , Apoptose/genética , Doenças Autoimunes/enzimologia , Doenças Autoimunes/genética , Doenças Autoimunes/patologia , Feminino , Macrófagos/enzimologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Monócitos/enzimologia , Monócitos/patologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/imunologiaRESUMO
A large number of studies have demonstrated significance of polyunsaturated fatty acids (PUFAs) for human health. However, many aspects on signals translating PUFA-sensing into body homeostasis have remained enigmatic. To shed light on PUFA physiology, we have generated a mouse line defective in mitochondrial dienoyl-CoA reductase (Decr), which is a key enzyme required for ß-oxidation of PUFAs. Previously, we have shown that these mice, whose oxidation of saturated fatty acid is intact but break-down of unsaturated fatty acids is blunted, develop severe hypoglycemia during metabolic stresses and fatal hypothermia upon acute cold challenge. In the current work, indirect calorimetry and thermography suggested that cold intolerance of Decr-/- mice is due to failure in maintaining appropriate heat production at least partly due to failure of brown adipose tissue (BAT) thermogenesis. Magnetic resonance imaging, electron microscopy, mass spectrometry and biochemical analysis showed attenuation in activation of lipolysis despite of functional NE-signaling and inappropriate expression of genes contributing to thermogenesis in iBAT when the Decr-/- mice were exposed to cold. We hypothesize that the failure in turning on BAT thermogenesis occurs due to accumulation of unsaturated long-chain fatty acids or their metabolites in Decr-/- mice BAT suppressing down-stream propagation of NE-signaling.
Assuntos
Tecido Adiposo Marrom/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Termogênese/genética , Tecido Adiposo Branco/metabolismo , Animais , Ácidos Graxos/metabolismo , Expressão Gênica , Humanos , Lipólise , Redes e Vias Metabólicas , Camundongos , Camundongos Knockout , Oxirredução , Estresse Fisiológico , TermografiaAssuntos
Anormalidades Múltiplas , Hepatopatias/diagnóstico , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Erros Inatos do Metabolismo de Esteroides/diagnóstico , Criança , Diagnóstico Diferencial , Progressão da Doença , Feminino , Predisposição Genética para Doença , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hepatopatias/complicações , Hepatopatias/tratamento farmacológico , Sinvastatina/uso terapêutico , Erros Inatos do Metabolismo de Esteroides/complicações , Erros Inatos do Metabolismo de Esteroides/tratamento farmacológicoRESUMO
Biliverdin reductase-A (BVR-A) is a serine/threonine/tyrosine kinase involved in the regulation of insulin signaling. In vitro studies have demonstrated that BVR-A is a substrate of the insulin receptor and regulates IRS1 by avoiding its aberrant activation, and in animal model of obesity the loss of hepatic BVR-A has been associated with glucose/insulin alterations and fatty liver disease. However, no studies exist in humans. Here, we evaluated BVR-A expression levels and activation in peripheral blood mononuclear cells (PBMC) from obese subjects and matched lean controls and we investigated the related molecular alterations of the insulin along with clinical correlates. We showed that BVR-A levels are significantly reduced in obese subjects and associated with a hyper-activation of the IR/IRS1/Akt/GSK-3ß/AS160/GLUT4 pathway. Low BVR-A levels also associate with the presence of obesity, metabolic syndrome, NASH and visceral adipose tissue inflammation. These data suggest that the reduction of BVR-A may be responsible for early alterations of the insulin signaling pathway in obesity and in this context may represent a novel molecular target to be investigated for the comprehension of the process of insulin resistance development in obesity.
Assuntos
Regulação da Expressão Gênica , Resistência à Insulina/genética , Insulina/sangue , Obesidade/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Transdução de Sinais/genética , Adulto , Cirurgia Bariátrica/métodos , Estudos de Casos e Controles , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Feminino , Proteínas Ativadoras de GTPase/sangue , Proteínas Ativadoras de GTPase/genética , Transportador de Glucose Tipo 4/sangue , Transportador de Glucose Tipo 4/genética , Glicogênio Sintase Quinase 3 beta/sangue , Glicogênio Sintase Quinase 3 beta/genética , Humanos , Proteínas Substratos do Receptor de Insulina/sangue , Proteínas Substratos do Receptor de Insulina/genética , Gordura Intra-Abdominal/metabolismo , Gordura Intra-Abdominal/patologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , Obesidade/sangue , Obesidade/patologia , Obesidade/cirurgia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/sangue , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/sangue , Proteínas Proto-Oncogênicas c-akt/genética , Serina-Treonina Quinases TOR/sangue , Serina-Treonina Quinases TOR/genética , Triglicerídeos/sangueRESUMO
BACKGROUND: Cerebrotendinous xanthomatosis (CTX) and Lathosterolosis represent two treatable inherited disorders of cholesterol metabolism that are characterized by the accumulation of cholestanol and lathosterol, respectively. The age of the patients suspected of having these disorders is highly variable due to the very different phenotypes. The early diagnosis of these disorders is important because specific therapeutic treatment could prevent the disease progression. The biochemical diagnosis of these defects is generally performed analyzing the sterol profile. Since age-related levels of these sterols are lacking, this study aims to determine a preliminary comparison of plasma levels of cholestanol and lathosterol among Italian unaffected newborns, children and healthy adults. METHODS: The sterols were extracted from 130 plasma samples (24 newborns, 33 children and 73 adults) by a liquid-liquid separation method and quantified by gas chromatography coupled with a flame ionization detector. RESULTS: Cholesterol, cholestanol and lathosterol levels together with the cholestanol/cholesterol and lathosterol/cholesterol ratios are statistically different among the three groups. Cholesterol levels progressively increased from newborns to children and to adults, whereas cholestanol/cholesterol and cholestanol/lathosterol ratios progressively decreased from newborns to children and to adults. Lathosterol levels were higher in adults than in both newborns and children. In the total population a positive correlation was observed between cholesterol levels and both cholestanol (correlation coefficient = 0.290, p = 0.001) and lathosterol levels (correlation coefficient = 0.353, p < 0.0001). CONCLUSIONS: Although this study can only be considered an explorative experience due to the low number of analyzed samples, we revealed several differences of plasma cholestanol and lathosterol levels and their ratios to cholesterol levels among newborns, children and adults. These evidences indicate the need of age-related reference values of cholestanol and lathosterol concentrations, including also newborns and children.
Assuntos
Colestanol/sangue , Colesterol/sangue , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Erros Inatos do Metabolismo de Esteroides/sangue , Xantomatose Cerebrotendinosa/sangue , Adulto , Fatores Etários , Criança , Progressão da Doença , Diagnóstico Precoce , Feminino , Humanos , Recém-Nascido , Masculino , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/sangue , Fitosteróis/sangue , Erros Inatos do Metabolismo de Esteroides/patologia , Xantomatose Cerebrotendinosa/patologiaRESUMO
Obesity and increased lipid availability have been implicated in the development and progression of chronic kidney disease. One of the major sites of renal lipid accumulation is in the proximal tubule cells of the kidney, suggesting that these cells may be susceptible to lipotoxicity. We previously demonstrated that loss of hepatic biliverdin reductase A (BVRA) causes fat accumulation in livers of mice on a high-fat diet. To determine the role of BVRA in mouse proximal tubule cells, we generated a CRISPR targeting BVRA for a knockout in mouse proximal tubule cells (BVRA KO). The BVRA KO cells had significantly less metabolic potential and mitochondrial respiration, which was exacerbated by treatment with palmitic acid, a saturated fatty acid. The BVRA KO cells also showed increased intracellular triglycerides which were associated with higher fatty acid uptake gene cluster of differentiation 36 as well as increased de novo lipogenesis as measured by higher neutral lipids. Additionally, neutrophil gelatinase-associated lipocalin 1 expression, annexin-V FITC staining, and lactate dehydrogenase assays all demonstrated that BVRA KO cells are more sensitive to palmitic acid-induced lipotoxicity than wild-type cells. Phosphorylation of BAD which plays a role in cell survival pathways, was significantly reduced in palmitic acid-treated BVRA KO cells. These data demonstrate the protective role of BVRA in proximal tubule cells against saturated fatty acid-induced lipotoxicity and suggest that activating BVRA could provide a benefit in protecting from obesity-induced kidney injury.
Assuntos
Apoptose/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Lipogênese/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Ácido Palmítico/toxicidade , Animais , Antígenos CD36/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Metabolismo Energético/efeitos dos fármacos , Deleção de Genes , Edição de Genes/métodos , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/patologia , L-Lactato Desidrogenase/metabolismo , Lipocalina-2/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Ácido Palmítico/metabolismo , Fosforilação , Triglicerídeos/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Retinol saturase (RetSat) catalyzes the saturation of double bonds of all-trans-retinol leading to the production of dihydroretinoid metabolites. Beside its role in retinoid metabolism, there is evidence that RetSat modulates the cellular response to oxidative stress and plays critical roles in adipogenesis and the accumulation of lipids. Here, we explore the relationship between RetSat, lipid metabolism and oxidative stress using in vitro and in vivo models with altered expression of RetSat. Our results reveal that RetSat is a potent modulator of the cellular response to oxidative stress and the generation of reactive oxygen species (ROS). The levels of reactive aldehydes products of lipid peroxidation, as measured based on thiobarbituric acid reactivity, are increased in RetSat overexpressing cells and, conversely, reduced in cells and tissues with reduced or absent expression of RetSat compared to controls. Despite increased weight gain, neutral lipid accumulation and alterations in hepatic lipid composition, RetSat-/- mice exhibit normal responses to insulin. In conclusion, our findings further expand upon the role of RetSat in oxidative stress and lipid metabolism and could provide insight in the significance of alterations of RetSat expression as observed in metabolic disorders.
Assuntos
Ácidos Graxos/metabolismo , Fibroblastos/enzimologia , Metabolismo dos Lipídeos/genética , Fígado/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Espécies Reativas de Oxigênio/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Embrião de Mamíferos , Fibroblastos/citologia , Expressão Gênica , Insulina/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Estresse Oxidativo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
The lower risk of some internal cancers at lower latitudes has been linked to greater sun exposure and consequent higher levels of ultraviolet radiation (UVR)-produced vitamin D3 (D3). To separate the experimental effects of sunlight and of all forms of D3, a mouse in which UVR does not produce D3 would be useful. To this end we have generated mice carrying a modified allele of sterol C5-desaturase (Sc5d), the gene encoding the enzyme that converts lathosterol to 7-dehydrocholesterol (7-DHC), such that Sc5d expression can be inactivated using the Cre/lox site-specific recombination system. By crossing to mice with tissue-specific expression of Cre or CreER2 (Cre/estrogen receptor), we generated two lines of transgenic mice. One line has constitutive keratinocyte-specific inactivation of Sc5d (Sc5dk14KO). The other line (Sc5dk14KOi) has tamoxifen-inducible keratinocyte-specific inactivation of Sc5d. Mice deleted for keratinocyte Sc5d lose the ability to increase circulating D3 following UVR exposure of the skin. Thus, unlike in control mice, acute UVR exposure did not affect circulating D3 level in inducible Sc5dk14KOi mice. Keratinocyte-specific inactivation of Sc5d was proven by sterol measurement in hair - in control animals lathosterol and cholesta-7,24-dien-3ß-ol, the target molecules of SC5D in the sterol biosynthetic pathways, together constituted a mean of 10% of total sterols; in the conditional knockout mice these sterols constituted a mean of 56% of total sterols. The constitutive knockout mice had an even greater increase, with lathosterol and cholesta-7,24-dien-3ß-ol accounting for 80% of total sterols. In conclusion, the dominant presence of the 7-DHC precursors in hair of conditional animals and the lack of increased circulating D3 following exposure to UVR reflect attenuated production of the D3 photochemical precursor 7-DHC and, consequently, of D3 itself. These animals provide a useful new tool for investigating the role of D3 in UVR-induced physiological effects and, more broadly, for investigations of the cholesterol synthetic pathway in the skin and other targeted tissues.
Assuntos
Colecalciferol/sangue , Modelos Animais de Doenças , Queratinócitos/metabolismo , Erros Inatos do Metabolismo/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Pele/metabolismo , Animais , Colecalciferol/biossíntese , Colesterol/metabolismo , Cruzamentos Genéticos , Desidrocolesteróis/metabolismo , Feminino , Cabelo/metabolismo , Heterozigoto , Estimativa de Kaplan-Meier , Queratinócitos/patologia , Queratinócitos/efeitos da radiação , Masculino , Erros Inatos do Metabolismo/sangue , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Gravidez , Distribuição Aleatória , Pele/patologia , Pele/efeitos da radiação , Raios UltravioletaRESUMO
The formation of lipid electrophile-protein adducts is associated with many disorders that involve perturbations of cellular redox status. The identities of adducted proteins and the effects of adduction on protein function are mostly unknown and an increased understanding of these factors may help to define the pathogenesis of various human disorders involving oxidative stress. 7-Dehydrocholesterol (7-DHC), the immediate biosynthetic precursor to cholesterol, is highly oxidizable and gives electrophilic oxysterols that adduct proteins readily, a sequence of events proposed to occur in Smith-Lemli-Opitz syndrome (SLOS), a human disorder resulting from an error in cholesterol biosynthesis. Alkynyl lanosterol (a-Lan) was synthesized and studied in Neuro2a cells, Dhcr7-deficient Neuro2a cells and human fibroblasts. When incubated in control Neuro2a cells and control human fibroblasts, a-Lan completed the sequence of steps involved in cholesterol biosynthesis and alkynyl-cholesterol (a-Chol) was the major product formed. In Dhcr7-deficient Neuro2a cells or fibroblasts from SLOS patients, the biosynthetic transformation was interrupted at the penultimate step and alkynyl-7-DHC (a-7-DHC) was the major product formed. When a-Lan was incubated in Dhcr7-deficient Neuro2a cells and the alkynyl tag was used to ligate a biotin group to alkyne-containing products, protein-sterol adducts were isolated and identified. In parallel experiments with a-Lan and a-7-DHC in Dhcr7-deficient Neuro2a cells, a-7-DHC was found to adduct to a larger set of proteins (799) than a-Lan (457) with most of the a-Lan protein adducts (423) being common to the larger a-7-DHC set. Of the 423 proteins found common to both experiments, those formed from a-7-DHC were more highly enriched compared to a DMSO control than were those derived from a-Lan. The 423 common proteins were ranked according to the enrichment determined for each protein in the a-Lan and a-7-DHC experiments and there was a very strong correlation of protein ranks for the adducts formed in the parallel experiments.
Assuntos
Lanosterol/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Proteoma/análise , Síndrome de Smith-Lemli-Opitz/metabolismo , Animais , Linhagem Celular , Colesterol/biossíntese , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Lanosterol/síntese química , Lanosterol/química , Peroxidação de Lipídeos , Camundongos , Modelos Biológicos , Esteróis/químicaRESUMO
Epithelial-to-mesenchymal transition (EMT) plays an important role in the development of the invasive and metastatic potentials of breast cancer cells during progression. Human biliverdin reductase (hBVR), an enzyme in the heme metabolism pathway, is involved in hypoxia-induced renal tubular EMT. However, whether hBVR contributes to the EMT of breast cancer remains unclear. Here, we used breast cancer cell lines (MCF-7, T-47D) and normal breast epithelial cells (MCF-10A) to explore the potential role of hBVR in the EMT of breast cancer. Western blot, RT-PCR and immunofluorescence were employed to test the expression and location of hBVR in the cell lines. Small interfering RNA of hBVR (si-hBVR) was used to knockdown the expression of hBVR, and U0126 was applied to inhibit the ERK1/2 signaling in MCF-7, T-47D cells. We found that hBVR highly expressed in MCF-7 and T-47D cells compared with MCF-10A cells, and had different cellular locations between them. Our results revealed that EMT occurred in tissues from breast cancer patients and breast cancer cell lines. However, the EMT in MCF-7 and T-47D cells was suppressed by si-hBVR and U0126. Furthermore, the expression of phosphorylated ERK1/2 was down-regulated by si-hBVR. In addition, hBVR regulated EMT through the ERK1/2 signaling, but bilirubin, which is a product of hBVR in the heme metabolism pathway in breast cancer, did not. Taken together, these findings provide new evidence that hBVR plays an important role in promoting EMT in human breast cancer through the ERK1/2 signaling pathway, and hBVR may be a therapeutic target for this disease.
Assuntos
Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal , Sistema de Sinalização das MAP Quinases , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Terapia de Alvo Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , RNA Interferente Pequeno/genética , Regulação para CimaRESUMO
Well-established cell culture models were combined with new analytical methods to assess the effects of small molecules on the cholesterol biosynthesis pathway. The analytical protocol, which is based on sterol derivation with the dienolphile PTAD, was found to be reliable for the analysis of 7-DHC and desmosterol. The PTAD method was applied to the screening of a small library of pharmacologically active substances, and the effect of compounds on the cholesterol pathway was determined. Of some 727 compounds, over 30 compounds decreased 7-DHC in Dhcr7-deficient Neuro2a cells. The examination of chemical structures of active molecules in the screen grouped the compounds into distinct categories. In addition to statins, our screen found that SERMs, antifungals, and several antipsychotic medications reduced levels of 7-DHC. The activities of selected compounds were verified in human fibroblasts derived from Smith-Lemli-Opitz syndrome (SLOS) patients and linked to specific transformations in the cholesterol biosynthesis pathway.
Assuntos
Desidrocolesteróis/metabolismo , Fibroblastos/metabolismo , Homeostase/efeitos dos fármacos , Neurônios/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Bibliotecas de Moléculas Pequenas/farmacologia , Esteróis/metabolismo , Animais , Linhagem Celular , Desidrocolesteróis/análise , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Humanos , Camundongos , Conformação Molecular , Neurônios/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND AND PURPOSE: 3ß-Hydroxysteroid-Δ24 reductase (DHCR24) or selective alzheimer disease indicator 1 (seladin-1), an enzyme of cholesterol biosynthetic pathway, has been implicated in neuroprotection, oxidative stress, and inflammation. However, its role in ischemic stroke remains unexplored. The aim of this study was to characterize the effect of seladin-1/DHCR24 using an experimental stroke model in mice. METHODS: Dhcr24(+/-) and wild-type (WT) mice were subjected to permanent middle cerebral artery occlusion. In another set of experiments, WT mice were treated intraperitoneally either with vehicle or U18666A (seladin-1/DHCR24 inhibitor, 10 mg/kg) 30 minutes after middle cerebral artery occlusion. Brains were removed 48 h after middle cerebral artery occlusion for infarct volume determination. For protein expression determination, peri-infarct region was obtained 24 h after ischemia, and Western blot or cytometric bead array was performed. RESULTS: Dhcr24(+/-) mice displayed larger infarct volumes after middle cerebral artery occlusion than their WT littermates. Treatment of WT mice with the seladin-1/DHCR24 inhibitor U18666A also increased ischemic lesion. Inflammation-related mediators were increased after ischemia in Dhcr24(+/-) mice compared with WT counterparts. Consistent with a role of cholesterol in proper function of glutamate transporter EAAT2 in membrane lipid rafts, we found a decreased association of EAAT2 with lipid rafts after ischemia when DHCR24 is genetically deleted or pharmacologically inhibited. Accordingly, treatment with U18666A decreases [(3)H]-glutamate uptake in cultured astrocytes. CONCLUSIONS: These results support the idea that lipid raft integrity, ensured by seladin-1/DHCR24, plays a crucial protective role in the ischemic brain by guaranteeing EAAT2-mediated uptake of glutamate excess.
Assuntos
Transportador 2 de Aminoácido Excitatório/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/deficiência , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/prevenção & controle , Androstenos/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Transportador 2 de Aminoácido Excitatório/genética , Ácido Glutâmico/metabolismo , Masculino , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Acidente Vascular Cerebral/genéticaRESUMO
Smith-Lemli-Opitz syndrome is an autosomal recessive mental retardation and multiple malformation syndrome caused by deficiency of the 7-dehydrocholesterol reductase, the enzyme catalyzing the last step in cholesterol biosynthesis. The authors summarize the pathophysiology, epidemiology, clinical picture, diagnostics and therapy of the disease based on a review of the international literature. Since 2004, fourteen patients have been diagnosed with Smith-Lemli-Opitz syndrome in Hungary, which suggests an underdiagnosis of the disease based upon estimated incidence data. Due to deficiency of the 7-dehydrocholesterol reductase, serum cholesterol concentration is low and 7-dehydrocholesterol concentration is elevated in blood and tissues; the latter being highly specific for the syndrome. Detection of disease-causing mutations makes the prenatal diagnosis possible. The clinical spectrum is wide, the most common symptom is syndactyly of the second and third toes. Standard therapy is cholesterol supplementation. Recent publications suggest that oxidative compounds of 7-dehydrocholesterol may play a role in the pathophysiology of the disease as well.
Assuntos
Colesterol/administração & dosagem , Colesterol/biossíntese , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Síndrome de Smith-Lemli-Opitz , Colesterol/sangue , Ensaios Clínicos como Assunto , Anormalidades Congênitas/diagnóstico , Desidrocolesteróis/metabolismo , Aconselhamento Genético , Genótipo , Humanos , Hungria/epidemiologia , Diagnóstico Pré-Natal , Índice de Gravidade de Doença , Síndrome de Smith-Lemli-Opitz/sangue , Síndrome de Smith-Lemli-Opitz/diagnóstico , Síndrome de Smith-Lemli-Opitz/tratamento farmacológico , Síndrome de Smith-Lemli-Opitz/epidemiologia , Síndrome de Smith-Lemli-Opitz/genética , Sindactilia , Falha de TratamentoRESUMO
Cholesterol is essential to human health, and its levels are tightly regulated by a balance of synthesis, uptake, and efflux. Cholesterol synthesis requires the actions of more than twenty enzymes to reach the final product, through two alternate pathways. Here we describe a physical and functional interaction between the two terminal enzymes. 24-Dehydrocholesterol reductase (DHCR24) and 7-dehydrocholesterol reductase (DHCR7) coimmunoprecipitate, and when the DHCR24 gene is knocked down by siRNA, DHCR7 activity is also ablated. Conversely, overexpression of DHCR24 enhances DHCR7 activity, but only when a functional form of DHCR24 is used. DHCR7 is important for both cholesterol and vitamin D synthesis, and we have identified a novel layer of regulation, whereby its activity is controlled by DHCR24. This suggests the existence of a cholesterol "metabolon", where enzymes from the same metabolic pathway interact with each other to provide a substrate channeling benefit. We predict that other enzymes in cholesterol synthesis may similarly interact, and this should be explored in future studies.
Assuntos
Colesterol/biossíntese , Proteínas do Tecido Nervoso/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Imunoprecipitação , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Ligação Proteica , Proteômica , RNA Interferente Pequeno/genéticaRESUMO
Cholesterol plays a key role in many cellular processes, and is generated by cells through de novo biosynthesis or acquired from exogenous sources through the uptake of low-density lipoproteins. Cholesterol biosynthesis is a complex, multienzyme-catalyzed pathway involving a series of sequentially acting enzymes. Inherited defects in genes encoding cholesterol biosynthetic enzymes or other regulators of cholesterol homeostasis result in severe metabolic diseases, many of which are rare in the general population and currently without effective therapy. Historically, these diseases have been viewed as discrete disorders, each with its own genetic cause and distinct pathogenic cascades that lead to its specific clinical features. However, studies have recently shown that three of these diseases have an unanticipated mechanistic convergence. This surprising finding is not only shedding light on details of cellular cholesterol homeostasis but also suggesting novel approaches to therapy.
Assuntos
Colesterol/metabolismo , Homeostase , Lipoproteínas LDL/metabolismo , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Fenótipo de Síndrome de Antley-Bixler/genética , Fenótipo de Síndrome de Antley-Bixler/patologia , Colesterol/biossíntese , Colesterol/genética , Condrodisplasia Punctata/genética , Condrodisplasia Punctata/patologia , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Humanos , Eritrodermia Ictiosiforme Congênita/genética , Eritrodermia Ictiosiforme Congênita/patologia , Deformidades Congênitas dos Membros/genética , Deformidades Congênitas dos Membros/patologia , Erros Inatos do Metabolismo Lipídico/genética , Erros Inatos do Metabolismo Lipídico/patologia , Lipoproteínas LDL/genética , Osteocondrodisplasias/genética , Osteocondrodisplasias/patologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Erros Inatos do Metabolismo de Esteroides/genética , Erros Inatos do Metabolismo de Esteroides/patologiaRESUMO
Dienoyl-CoA reductase (DECR) deficiency with hyperlysinemia is a rare disorder affecting the metabolism of polyunsaturated fatty acids and lysine. The molecular basis of this condition is currently unknown. We describe a new case with failure to thrive, developmental delay, lactic acidosis and severe encephalopathy suggestive of a mitochondrial disorder. Exome sequencing revealed a causal mutation in NADK2. NADK2 encodes the mitochondrial NAD kinase, which is crucial for NADP biosynthesis evidenced by decreased mitochondrial NADP(H) levels in patient fibroblasts. DECR and also the first step in lysine degradation are performed by NADP-dependent oxidoreductases explaining their in vivo deficiency. DECR activity was also deficient in lysates of patient fibroblasts and could only be rescued by transfecting patient cells with functional NADK2. Thus NADPH is not only crucial as a cosubstrate, but can also act as a molecular chaperone that activates and stabilizes enzymes. In addition to polyunsaturated fatty acid oxidation and lysine degradation, NADPH also plays a role in various other mitochondrial processes. We found decreased oxygen consumption and increased extracellular acidification in patient fibroblasts, which may explain why the disease course is consistent with clinical criteria for a mitochondrial disorder. We conclude that DECR deficiency with hyperlysinemia is caused by mitochondrial NADP(H) deficiency due to a mutation in NADK2.
Assuntos
Hiperlisinemias/genética , Proteínas Mitocondriais/genética , NADP/deficiência , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fibroblastos/metabolismo , Humanos , Hiperlisinemias/fisiopatologia , Mutação , Análise de Sequência de DNA , Estresse FisiológicoRESUMO
We report the outcome of liver transplantation (LT) in the only surviving patient with lathosterolosis, a defect of cholesterol biosynthesis characterized by high lathosterol levels associated with progressive cholestasis, multiple congenital anomalies and mental retardation. From her diagnosis at age 2 she had shown autistic behavior, was unable to walk unaided and her sight was impaired by cataracts. By age 7 she developed end-stage liver disease. After a soul-searching discussion within the transplantation team, she was treated with LT as this represented her only lifesaving option. At 1-year follow-up, her lathosterol levels had returned to normal (0.61 mg/dL from 13.04 ± 2.65) and her nutrition improved. She began exploring her environment and walking by holding onto an adult's hand and then independently. Her brain magnetic resonance imaging (MRI) had shown a normal picture at age 1, whereas a volume reduction of white matter with ex vacuo ventricular dilatation and defective myelinization were observed before transplant. At 5-year follow-up, a complete biochemical recovery, an arrest of mental deterioration and a stable MRI picture were achieved, with a return to her every day life albeit with limitations. Timely liver transplant in defects of cholesterol biosynthesis might arrest the progression of neurological damage.