RESUMEN
Apolipoprotein A-I (apoA-I), the main protein component of High-Density Lipoprotein (HDL), is modified in plasma and the arterial wall by various enzymes. Myeloperoxidase (MPO), a leukocyte-derived peroxidase, is highly expressed during inflammation and associates with HDL reducing its functionality and contributing to atherosclerosis. In the present study we sought to explore further the effect of MPO on HDL structure and functionality in vivo using adenovirus-mediated gene transfer of human MPO combined with human apoA-I forms containing substitutions at MPO-sensitive sites or wild type apoA-I. We found that overexpression of MPO in mice significantly increased plasma apoA-I and HDL levels without affecting the expression of genes involved in HDL biogenesis or catabolism in the liver. Overexpression of MPO in the liver reduced the expression of pro-inflammatory genes and increased or did not affect the expression of anti-inflammatory genes suggesting that MPO had no toxic effects in this organ. In the plasma of mice overexpressing MPO, no significant alterations in HDL size or electrophoretic mobility was observed with the exception of mice expressing apoA-I (M148A) which showed enriched pre-ß relative to α HDL particles, suggesting that the apoA-I (M148A) mutation may interfere with HDL remodelling. Overexpression of MPO was associated with reduced anti-oxidant capacity of HDL particles in all mice. Interestingly, HDL particles bearing apoA-I (Y192A) showed enhanced ABCA1-dependent cholesterol efflux from macrophages which was not affected by MPO and these mice had reduced levels of LDL-c. These findings provide new insights on the role of specific amino acid residues of apoA-I in HDL structure and function following modification by MPO. This knowledge may facilitate the development of novel therapies based on improved HDL forms for patients with chronic diseases that are characterized by dysfunctional HDL.
Asunto(s)
Infecciones por Adenoviridae , Apolipoproteína A-I , Transportador 1 de Casete de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Animales , Apolipoproteína A-I/metabolismo , Humanos , Lipoproteínas HDL , Ratones , Peroxidasa/genética , Peroxidasa/metabolismoRESUMEN
The white adipose tissue (WAT) contributes to the metabolic imbalance observed in obesity and the metabolic syndrome (MetS) by mechanisms that are poorly understood. The aim of this study was to monitor changes in the transcriptome of epididymal WAT during the development of MetS. ApoE3L.CETP mice were fed a high fat (HFD) or a low-fat (LFD) diet for different time periods. Adipose RNA was analyzed by microarrays. We found an increasing number of differentially expressed transcripts during MetS development. In mice with MetS, 1396 transcripts were differentially expressed including transcripts related to immune/inflammatory responses and extracellular matrix enzymes, suggesting significant inflammation and tissue remodeling. The top list of pathways included focal adhesion, chemokine, B and T cell receptor and MAPK signaling. The data identify for the first time adipose gene signatures in apoE3L.CETP mice with diet-induced MetS and might open new avenues for investigation of potential biomarkers or therapeutic targets.
Asunto(s)
Tejido Adiposo Blanco/metabolismo , Síndrome Metabólico/genética , Algoritmos , Animales , Apolipoproteína E3/genética , Proteínas de Transferencia de Ésteres de Colesterol/genética , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Hígado/metabolismo , Redes y Vías Metabólicas/genética , Síndrome Metabólico/etiología , Síndrome Metabólico/metabolismo , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de SeñalRESUMEN
High-Density Lipoprotein cholesterol (HDL-C) levels do not correlate well with Coronary Artery Disease (CAD) risk, while HDL functionality affects atherogenesis and is a better prognostic marker for CAD. Often, the extreme HDL-C levels have a multigenic origin. Here, we searched for single-nucleotide polymorphisms (SNPs) in ten genes of HDL metabolism in a Greek cohort with very low (<10th percentile, n = 13) or very high (>90th percentile, n = 21) HDL-C. We also evaluated the association between HDL-C levels, HDL functionality (anti-oxidant capacity) and CAD in the subjects of this cohort. Individuals with low HDL-C levels had higher triglyceride levels, lower apoA-I levels, decreased HDL anti-oxidant capacity and higher incidence of CAD compared with individuals with control or high HDL-C levels. With next generation sequencing we identified 18 exonic SNPs in 6 genes of HDL metabolism and for selected amino acid changes we performed computer-aided structural analysis and modeling. A previously uncharacterized rare apolipoprotein A-IV variant, apoA-IV [V336M], present in a subject with low HDL-C (14 mg/dL) and CAD, was expressed in recombinant form and structurally and functionally characterized. ApoA-IV [V336M] had similar α-helical content to WT apoA-IV but displayed a small thermodynamic stabilization by chemical unfolding analysis. ApoA-IV [V336M] was able to associate with phospholipids but presented reduced kinetics compared to WT apoA-IV. Overall, we identified a rare apoA-IV variant in a subject with low HDL levels and CAD with altered biophysical and phospholipid binding properties and showed that subjects with very low HDL-C presented with HDL dysfunction and higher incidence of CAD in a Greek cohort.
Asunto(s)
Apolipoproteínas A/genética , HDL-Colesterol/metabolismo , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/metabolismo , Lipoproteínas HDL/metabolismo , Adulto , Apolipoproteínas A/química , Arildialquilfosfatasa/metabolismo , Estudios de Cohortes , Femenino , Grecia , Humanos , Masculino , Persona de Mediana Edad , Modelos Moleculares , Polimorfismo de Nucleótido SimpleRESUMEN
Long-term exposure to excess dietary fat leads to obesity and the metabolic syndrome (MetS). The purpose of the present study was to identify global changes in liver gene expression and circulating miRNAs in a humanized mouse model of diet-induced MetS. Male apoE3L.CETP mice received a high-fat diet (HFD) or a low-fat diet (LFD) for different time periods and the progression of MetS pathology was monitored. A separate group of mice was divided into responders (R) or nonresponders (NR) and received HFD for 16 weeks. We found that mice receiving the HFD developed manifestations of MetS and displayed an increasing number of differentially expressed transcripts at 4, 8, and 12 weeks compared with mice receiving the LFD. Significantly changed genes were functionally annotated to metabolic diseases and pathway analysis revealed the downregulation of genes in cholesterol and fatty acid biosynthesis and upregulation of genes related to lipid droplet formation, which was in line with the development of hepatic steatosis. In the serum of the apoE3L.CETP mice we identified three miRNAs that were upregulated specifically in the HFD group. We found that responder mice have a distinct gene signature that differentiates them from nonresponders. Comparison of the two diet intervention studies revealed a limited number of common differentially expressed genes but the expression of these common genes was affected in a similar way in both studies. In conclusion, the characteristic hepatic gene signatures and serum miRNAs identified in the present study provide novel insights to MetS pathology and could be exploited for diagnostic or therapeutic purposes.
Asunto(s)
Dieta Alta en Grasa , Hígado/metabolismo , Síndrome Metabólico/genética , Síndrome Metabólico/metabolismo , Animales , MicroARN Circulante/genética , Dieta con Restricción de Grasas/efectos adversos , Dieta Alta en Grasa/efectos adversos , Grasas de la Dieta/metabolismo , Modelos Animales de Enfermedad , Hígado Graso/metabolismo , Perfilación de la Expresión Génica/métodos , Masculino , Ratones , Obesidad/metabolismoRESUMEN
BACKGROUND/OBJECTIVES: The incidence of obesity and metabolic syndrome (MetS) has rapidly increased worldwide. Roux-en-Y gastric bypass (RYGB) achieves long-term weight loss and improves MetS-associated comorbidities. Using a mouse model with a humanized lipoprotein metabolism, we elucidated whether improvements in lipid and glucose metabolism after RYGB surgery are body weight loss-dependent or not. SUBJECTS/METHODS: Male ApoE*3Leiden.CETP (ApoE3L.CETP) mice fed Western type diet for 6 weeks underwent RYGB or Sham surgery. Sham groups were either fed ad libitum or were body weight-matched (BWm) to the RYGB mice to discriminate surgical effects from body weight loss-associated effects. Before and after surgery, plasma was collected to assess the metabolic profile, and glucose tolerance and insulin sensitivity were tested. Twenty days after surgery, mice were sacrificed, and liver was collected to assess metabolic, histological and global gene expression changes after surgery. RESULTS: RYGB induced a marked reduction in body weight, which was also achieved by severe food restriction in BWm mice, and total fat mass compared to Sham ad libitum mice (Sham AL). Total cholesterol, non-high-density lipoprotein cholesterol (non-HDL-C) and ceramide were strongly reduced 20 days after surgery in RYGB compared to BWm mice. Glucose tolerance and insulin sensitivity improved 13 days after surgery similarly in RYGB and BWm mice. Liver histology confirmed lipid reduction in RYGB and BWm mice while the transcriptomics data indicated altered genes expression in lipid metabolism. CONCLUSIONS: RYGB surgery improves glucose metabolism and greatly ameliorates lipid metabolism in part in a body weight-dependent manner. Given that ApoE3L.CETP mice were extensively studied to describe the MetS, and given that RYGB improved ceramide after surgery, our data confirmed the usefulness of ApoE3L.CETP mice after RYGB in deciphering the metabolic improvements to treat the MetS.
Asunto(s)
Peso Corporal/fisiología , Derivación Gástrica , Metabolismo de los Lípidos/fisiología , Pérdida de Peso/fisiología , Animales , Apolipoproteínas E/genética , Glucemia/metabolismo , Modelos Animales de Enfermedad , Ingestión de Alimentos/fisiología , Hígado/química , Hígado/fisiología , Masculino , Síndrome Metabólico/fisiopatología , Ratones , Ratones TransgénicosRESUMEN
Rho GTPases are highly conserved proteins that play critical roles in many cellular processes including actin dynamics, vesicular trafficking, gene transcription, cell-cycle progression, and cell adhesion. The main mode of regulation of Rho GTPases is through guanine nucleotide binding (cycling between an active GTP-bound form and an inactive GDP-bound form), but transcriptional, post-transcriptional, and post-translational modes of Rho regulation have also been described. In the present review, we summarize recent progress on the mechanisms that control the expression of the three members of the Rho-like subfamily (RhoA, RhoB, and RhoC) at the level of gene transcription as well as their post-transcriptional regulation by microRNAs. We also discuss the progress made in deciphering the mechanisms of cross-talk between Rho proteins and the transforming growth factor ß signaling pathway and their implications for the pathogenesis of human diseases such as cancer metastasis and fibrosis.
Asunto(s)
Regulación de la Expresión Génica , MicroARNs/genética , Activación Transcripcional , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoB/genética , Proteína rhoC de Unión a GTP/genética , Animales , Humanos , MicroARNs/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoB/metabolismo , Proteína rhoC de Unión a GTP/metabolismoRESUMEN
Apolipoprotein E (apoE) is mainly secreted by hepatocytes and incorporated into most plasma lipoproteins. Macrophages, which accumulate cholesterol and are critical for the development of the atherosclerotic plaque, are also an important, albeit smaller, apoE source. Distal regulatory elements control cell-specific activity of the apoE promoter: multienhancers (ME.1/2) in macrophages and hepatic control regions (HCR-1/2) in hepatocytes. A member of AP-1 cell growth regulator, c-Jun regulates the transcription of various apolipoproteins and proinflammatory molecules implicated in atherosclerosis. We aimed to investigate the effect of c-Jun on apoE expression in macrophages versus hepatocytes and to reveal the underlying molecular mechanisms. Herein we show that c-Jun had an opposite, cell-specific effect on apoE expression: downregulation in macrophages but upregulation in hepatocytes. Transient transfections using ME.2 deletion mutants and DNA pull-down (DNAP) assays showed that the inhibitory effect of c-Jun on the apoE promoter in macrophages was mediated by a functional c-Jun binding site located at 301/311 on ME.2. In hepatocytes, c-Jun overexpression strongly increased apoE expression, and this effect was due to c-Jun binding at the canonical site located at -94/-84 on the apoE proximal promoter, identified by transient transfections using apoE deletion mutants, DNAP, and chromatin immunoprecipitation assays. Overall, the dual effect of c-Jun on apoE gene expression led to decreased cholesterol efflux in macrophages resident in the atherosclerotic plaque synergized with an increased level of systemic apoE secreted by the liver to exacerbate atherogenesis.
Asunto(s)
Apolipoproteínas E/genética , Regulación de la Expresión Génica , Hepatocitos/metabolismo , Macrófagos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Animales , Elementos de Facilitación Genéticos , Hepatocitos/inmunología , Macrófagos/inmunología , Ratones , Modelos Biológicos , Regiones Promotoras Genéticas , Células RAW 264.7 , Factor de Transcripción AP-1/metabolismoRESUMEN
Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent, acute, and self-limiting attacks of fever. Mutations in MEFV gene encoding pyrin account for FMF, but the high number of heterozygote patients with typical symptoms of the disease has driven a number of alternative aetiopathogenic hypotheses. The MEFV gene was knocked down in human myelomonocytic cells that express endogenous pyrin to identify deregulated microRNAs (miRNAs). Microarray analyses revealed 29 significantly differentially expressed miRNAs implicated in pathways associated with cellular integrity and survival. Implementation of in silico gene network prediction algorithms and bioinformatics analyses showed that miR-4520a is predicted to target genes implicated in autophagy through regulation of RHEB/mTOR signaling. Differential expression levels of RHEB were confirmed by luciferase reporter gene assays providing further evidence that is directly targeted by miR-4520a. Although the relative expression levels of miR-4520a were variable among FMF patients, the statistical expression of miR-4520a was different between FMF mutation carriers and controls (P = 0.0061), indicating an association between miR-4520a expression and MEFV mutations. Comparison between FMF patients bearing the M694V mutation, associated with severe disease, and healthy controls showed a significant increase in miR-4520a expression levels (P = 0.00545). These data suggest that RHEB, the main activator of mTOR signaling, is a valid target of miR-4520a with the relative expression levels of the latter being significantly deregulated in FMF patients and highly dependent on the presence of pyrin mutations, especially of the M694V type. These results suggest a role of deregulated autophagy in the pathogenesis of FMF. J. Cell. Physiol. 232: 1326-1336, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Fiebre Mediterránea Familiar/genética , Perfilación de la Expresión Génica , MicroARNs/genética , Mutación/genética , Pirina/genética , Adulto , Estudios de Casos y Controles , Línea Celular , Femenino , Redes Reguladoras de Genes , Humanos , Luciferasas/metabolismo , Masculino , MicroARNs/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína Homóloga de Ras Enriquecida en el CerebroRESUMEN
Small GTPases of the Rho subfamily have been implicated in many physiological and pathological processes in various cell types including embryonic stem cells (ESCs). In the present study we performed a functional analysis of the promoters of the RhoA and the RhoB genes in order to identify regulatory elements that are important for their transcriptional control in ESCs. We show that RhoA mRNA levels were significantly higher compared with the RhoB mRNA levels in ESCs as well in various cancer cell lines and this difference could be accounted for by differences in the activities of the corresponding promoters. Deletion analysis of the RhoA and RhoB promoters in ESCs revealed that the proximal regions contain regulatory elements that are critical for their activity. Both proximal promoters contain CCAAT boxes and mutagenesis of these elements decreased significantly the activity of both promoters suggesting a coordinated regulation of the two genes by CCAAT box binding factors. Finally, we show that both genes are subjects to autoregulation in ESCs and in the case of RhoB, this autoregulation requires the GTPase activity of the Rho proteins. Understanding the mechanisms that control the transcription of Rho GTPases in ESCs may shed new light into the still unknown roles of these proteins in stem cell functions.
Asunto(s)
Células Madre Embrionarias/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Regiones Promotoras Genéticas/genética , Elementos Reguladores de la Transcripción/genética , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoB/genética , Animales , Línea Celular , RatonesRESUMEN
Aberrant levels and function of the potent anti-inflammatory high-density lipoprotein (HDL) and accelerated atherosclerosis have been reported in patients with autoimmune inflammatory diseases. Whether HDL affects the development of an autoimmune response remains elusive. In this study, we used apolipoprotein A-I-deficient (apoA-I(-/-)) mice, characterized by diminished circulating HDL levels, to delineate the role of HDL in autoimmunity. ApoA-I(-/-) mice exhibited increased severity of Ag-induced arthritis compared with wild-type mice, and this was associated with elevated Th1 and Th17 cell reactivity in the draining lymph nodes. Furthermore, reconstituted HDL (rHDL) attenuated IFN-γ and IL-17 secretion by Ag-specific T cells upon stimulation of draining lymph nodes in vitro. The suppressive effects of rHDL were mediated through modulation of dendritic cell (DC) function. Specifically, rHDL-treated DCs demonstrated an immature phenotype characterized by downregulated costimulatory molecules, the release of low amounts of proinflammatory cytokines, and failure to promote T cell proliferation in vitro. The mechanism of action involved the inhibition of NF-κB nuclear translocation and the decrease of Myd88 mRNA levels by rHDL. Finally, modulation of DC function by rHDL was critically dependent on the presence of scavenger receptor class B type I and ATP Binding Cassette Transporter A1, but not the ATP Binding Cassette Transporter G1. These findings reveal a novel role of HDL in the regulation of adaptive inflammatory responses through suppression of DC function that could be exploited therapeutically in autoimmune inflammatory diseases.
Asunto(s)
Autoinmunidad/inmunología , Células Dendríticas/inmunología , Lipoproteínas HDL/inmunología , Células TH1/inmunología , Células Th17/inmunología , Animales , Artritis Experimental/inmunología , Western Blotting , Diferenciación Celular/inmunología , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Linfocitos T/inmunologíaRESUMEN
Liver X Receptors (LXRs) are sterol-activated transcription factors that play major roles in cellular cholesterol homeostasis, HDL biogenesis and reverse cholesterol transport. The aim of the present study was to investigate the mechanisms that control the expression of the human LXRα gene in hepatic cells. A series of reporter plasmids containing consecutive 5' deletions of the hLXRα promoter upstream of the luciferase gene were constructed and the activity of each construct was measured in HepG2 cells. This analysis showed that the activity of the human LXRα promoter was significantly reduced by deleting the -111 to -42 region suggesting the presence of positive regulatory elements in this short proximal fragment. Bioinformatics data including motif search and ChIP-Seq revealed the presence of a potential binding motif for Hepatocyte Nuclear Factor 4 α (HNF-4α) in this area. Overexpression of HNF-4α in HEK 293T cells increased the expression of all LXRα promoter constructs except -42/+384. In line, silencing the expression of endogenous HNF-4α in HepG2 cells was associated with reduced LXRα protein levels and reduced activity of the -111/+384 LXRα promoter but not of the -42/+384 promoter. Using ChiP assays in HepG2 cells combined with DNAP assays we mapped the novel HNF-4α specific binding motif (H4-SBM) in the -50 to -40 region of the human LXRα promoter. A triple mutation in this H4-SBM abolished HNF-4α binding and reduced the activity of the promoter to 65% relative to the wild type. Furthermore, the mutant promoter could not be transactivated by HNF-4α. In conclusion, our data indicate that HNF-4α may have a wider role in cell and plasma cholesterol homeostasis by controlling the expression of LXRα in hepatic cells.
Asunto(s)
Regulación de la Expresión Génica/genética , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Receptores Nucleares Huérfanos/genética , Receptores Nucleares Huérfanos/metabolismo , Activación Transcripcional/genética , Sitios de Unión , Células Hep G2 , Factor Nuclear 4 del Hepatocito/química , Humanos , Receptores X del Hígado , Receptores Nucleares Huérfanos/química , Unión ProteicaRESUMEN
We have investigated how the natural LCAT[T147I] and LCAT[P274S] mutations affect the pathway of biogenesis of HDL. Gene transfer of WT LCAT in LCAT(-/-) mice increased 11.8-fold the plasma cholesterol, whereas the LCAT[T147I] and LCAT[P274S] mutants caused a 5.2- and 2.9-fold increase, respectively. The LCAT[P274S] and the WT LCAT caused a monophasic distribution of cholesterol in the HDL region, whereas the LCAT[T147I] caused a biphasic distribution of cholesterol in the LDL and HDL region. Fractionation of plasma showed that the expression of WT LCAT increased plasma apoE and apoA-IV levels and shifted the distribution of apoA-I to lower densities. The LCAT[T147I] and LCAT[P274S] mutants restored partially apoA-I in the HDL3 fraction and LCAT[T147I] increased apoE in the VLD/IDL/LDL fractions. The in vivo functionality of LCAT was further assessed based on is its ability to correct the aberrant HDL phenotype that was caused by the apoA-I[L159R]FIN mutation. Co-infection of apoA-I(-/-) mice with this apoA-I mutant and either of the two mutant LCAT forms restored only partially the HDL biogenesis defect that was caused by the apoA-I[L159R]FIN and generated a distinct aberrant HDL phenotype.
Asunto(s)
Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Mutación Puntual , Animales , Apolipoproteína A-I/sangre , Apolipoproteína A-I/metabolismo , Apolipoproteínas A/sangre , Apolipoproteínas A/metabolismo , Apolipoproteínas E/sangre , Apolipoproteínas E/metabolismo , Línea Celular , Colesterol/sangre , Humanos , Lípidos/sangre , Lipoproteínas HDL/sangre , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
ATP binding cassette transporter A1 (ABCA1) plays a key role in the biogenesis of HDL by promoting the efflux of cellular cholesterol and phospholipids to lipid free apoA-I. Mutations in the ABCA1 gene cause Tangier disease which is characterized by near or complete absence of circulating plasma HDL. In the present study we show that the winged helix/forkhead box containing transcription factor A2 (FOXA2) shown previously to play a role in glucose and bile acid homeostasis in the liver and in energy utilization in adipose tissue is a negative modulator of ABCA1 gene expression in hepatic cells. We show that the ABCA1 promoter contains three FOXA2 binding elements in the proximal region. Two of the sites are localized in a region of the ABCA1 promoter enriched in binding elements for transcriptional repressor proteins whereas the third site is the core of the TATA element of the ABCA1 promoter. Inhibition of FOXA2 binding to the ABCA1 promoter by site-directed mutagenesis or FOXA2 gene expression by siRNA was associated with increased ABCA1 promoter activity and protein levels. Overexpression of FOXA2 inhibited both the constitutive ABCA1 gene expression as well as ABCA1 gene induction by oxysterols and retinoids via nuclear receptors LXRα/RXRα. In summary, the present study identifies transcription factor FOXA2 as a negative modulator of ABCA1 gene expression in hepatic cells and reveals a novel mechanism of transcriptional repression by FOXA2 which involves the TATA element of the ABCA1 gene.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/genética , Carcinoma Hepatocelular/genética , Regulación de la Expresión Génica/efectos de los fármacos , Factor Nuclear 3-beta del Hepatocito/metabolismo , Neoplasias Hepáticas/genética , Transportador 1 de Casete de Unión a ATP/metabolismo , Carcinoma Hepatocelular/metabolismo , Células Cultivadas , Inmunoprecipitación de Cromatina , Técnica del Anticuerpo Fluorescente Indirecta , Células HEK293 , Factor Nuclear 3-beta del Hepatocito/genética , Humanos , Hidroxicolesteroles/farmacología , Neoplasias Hepáticas/metabolismo , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Retinoides/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacosRESUMEN
Krüppel-like factor 4 (KLF4) is a critical regulator of monocyte differentiation and macrophage polarization, and it also plays an important role in several vascular diseases, including atherosclerosis. Apolipoprotein E (apoE) is an essential anti-atherosclerotic glycoprotein involved in lipid metabolism, expressed by the liver, macrophages and other cell types. We hypothesized that KLF4 is involved in apoE gene regulation in macrophages. Our experiments showed that differentiation of THP-1 monocytes to macrophages using PMA was associated with a robust induction of both KLF4 and apoE genes. KLF4 bound to the apoE promoter, as revealed by chromatin immunoprecipitation and DNA pull-down (DNAP) assays, and transactivated the apoE promoter in a dose-dependent manner. Using a series of apoE promoter deletion mutants we revealed the biological activity of multiple KLF4 binding sites located in the [-500/-100] region of apoE promoter. Moreover, overexpression of cAMP-response-element-binding protein (CREB) further increased KLF4 up-regulatory effect on apoE promoter. Despite the fact that no putative CREB binding sites were predicted in silico, we found that in macrophages CREB bound to apoE proximal promoter in the region -200/+4 and even more strongly on -350/-274 region. In similar DNAP experiments using cell extracts obtained from monocytes (lacking KLF4), a very weak binding of CREB was detected, indicating that interaction of CREB with apoE promoter takes place indirectly. In conclusion our results show: (i) a robust synchronized induction of KLF4 and apoE expression during differentiation of monocytes to macrophages; (ii) KLF4 up-regulates apoE gene in a dose-dependent manner; (iii) biologically active KLF4 binding sites are present on apoE promoter and (iv) the interaction of KLF4 with CREB results in an enhanced up-regulatory effect of KLF4 on apoE promoter. Taken together these data provide novel knowledge on apoE gene regulation mechanism in macrophages, and offer insight into the therapeutic potential of KLF4 in atherosclerosis.
Asunto(s)
Apolipoproteínas E/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Macrófagos/citología , Monocitos/citología , Regiones Promotoras Genéticas , Secuencia de Bases , Diferenciación Celular , Línea Celular , Humanos , Factor 4 Similar a Kruppel , Macrófagos/metabolismo , Datos de Secuencia Molecular , Monocitos/metabolismo , Regulación hacia ArribaRESUMEN
Apolipoprotein E (apoE), a protein mainly involved in lipid metabolism, is associated with several neurodegenerative disorders including Alzheimer's disease. Despite numerous attempts to elucidate apoE gene regulation in the brain, the exact mechanism is still uncovered. The mechanism of apoE gene regulation in the brain involves the proximal promoter and multienhancers ME.1 and ME.2, which evolved by gene duplication. Herein we questioned whether thyroid hormones and their nuclear receptors have a role in apoE gene regulation in astrocytes. Our data showed that thyroid hormones increase apoE gene expression in HTB14 astrocytes in a dose-dependent manner. This effect can be intermediated by the thyroid receptor ß (TRß) which is expressed in these cells. In the presence of triiodothyronine (T3) and 9-cis retinoic acid, in astrocytes transfected to overexpress TRß and retinoid X receptor α (RXRα), apoE promoter was indirectly activated through the interaction with ME.2. To determine the location of TRß/RXRα binding site on ME.2, we performed DNA pull down assays and found that TRß/RXRα complex bound to the region 341-488 of ME.2. This result was confirmed by transient transfection experiments in which a series of 5'- and 3'-deletion mutants of ME.2 were used. These data support the existence of a biologically active TRß binding site starting at 409 in ME.2. In conclusion, our data revealed that ligand-activated TRß/RXRα heterodimers bind with high efficiency on tissue-specific distal regulatory element ME.2 and thus modulate apoE gene expression in the brain.
Asunto(s)
Apolipoproteínas E/genética , Astrocitos/metabolismo , Hormonas Tiroideas/metabolismo , Regulación hacia Arriba , Alitretinoína , Sitios de Unión , Línea Celular , Humanos , Regiones Promotoras Genéticas , Receptores de Hormona Tiroidea/metabolismo , Receptor alfa X Retinoide/metabolismo , Receptores X Retinoide/metabolismo , Receptores beta de Hormona Tiroidea/genética , Tretinoina/metabolismo , Triyodotironina/metabolismoRESUMEN
HDL regulation is exerted at multiple levels including regulation at the level of transcription initiation by transcription factors and signal transduction cascades; regulation at the posttranscriptional level by microRNAs and other noncoding RNAs which bind to the coding or noncoding regions of HDL genes regulating mRNA stability and translation; as well as regulation at the posttranslational level by protein modifications, intracellular trafficking, and degradation. The above mechanisms have drastic effects on several HDL-mediated processes including HDL biogenesis, remodeling, cholesterol efflux and uptake, as well as atheroprotective functions on the cells of the arterial wall. The emphasis is on mechanisms that operate in physiologically relevant tissues such as the liver (which accounts for 80% of the total HDL-C levels in the plasma), the macrophages, the adrenals, and the endothelium. Transcription factors that have a significant impact on HDL regulation such as hormone nuclear receptors and hepatocyte nuclear factors are extensively discussed both in terms of gene promoter recognition and regulation but also in terms of their impact on plasma HDL levels as was revealed by knockout studies. Understanding the different modes of regulation of this complex lipoprotein may provide useful insights for the development of novel HDL-raising therapies that could be used to fight against atherosclerosis which is the underlying cause of coronary heart disease.
Asunto(s)
Lipoproteínas HDL/genética , Lipoproteínas HDL/metabolismo , Procesamiento Proteico-Postraduccional , Procesamiento Postranscripcional del ARN , Transcripción Genética , Animales , Regulación de la Expresión Génica , Humanos , Metabolismo de los Lípidos/genética , Lipoproteínas HDL/sangreRESUMEN
In this chapter, we review how HDL is generated, remodeled, and catabolized in plasma. We describe key features of the proteins that participate in these processes, emphasizing how mutations in apolipoprotein A-I (apoA-I) and the other proteins affect HDL metabolism. The biogenesis of HDL initially requires functional interaction of apoA-I with the ATP-binding cassette transporter A1 (ABCA1) and subsequently interactions of the lipidated apoA-I forms with lecithin/cholesterol acyltransferase (LCAT). Mutations in these proteins either prevent or impair the formation and possibly the functionality of HDL. Remodeling and catabolism of HDL is the result of interactions of HDL with cell receptors and other membrane and plasma proteins including hepatic lipase (HL), endothelial lipase (EL), phospholipid transfer protein (PLTP), cholesteryl ester transfer protein (CETP), apolipoprotein M (apoM), scavenger receptor class B type I (SR-BI), ATP-binding cassette transporter G1 (ABCG1), the F1 subunit of ATPase (Ecto F1-ATPase), and the cubulin/megalin receptor. Similarly to apoA-I, apolipoprotein E and apolipoprotein A-IV were shown to form discrete HDL particles containing these apolipoproteins which may have important but still unexplored functions. Furthermore, several plasma proteins were found associated with HDL and may modulate its biological functions. The effect of these proteins on the functionality of HDL is the topic of ongoing research.
Asunto(s)
Lipoproteínas HDL/metabolismo , Animales , Biomarcadores/metabolismo , Humanos , Metabolismo de los Lípidos , Lipoproteínas HDL/biosíntesis , Lipoproteínas HDL/sangre , Lipoproteínas HDL/química , Lipoproteínas HDL/clasificación , Conformación Proteica , Relación Estructura-ActividadRESUMEN
The K146N/R147W substitutions in apoE3 were described in patients with a dominant form of type III hyperlipoproteinemia. The effects of these mutations on the in vivo functions of apoE were studied by adenovirus-mediated gene transfer in different mouse models. Expression of the apoE3[K146N/R147W] mutant in apoE-deficient (apoE(-/-)) or apoA-I-deficient (apoA-I(-/-))×apoE(-/-) mice exacerbated the hypercholesterolemia and increased plasma apoE and triglyceride levels. In apoE(-/-) mice, the apoE3[K146N/R147W] mutant displaced apoA-I from the VLDL/LDL/HDL region and caused the accumulation of discoidal apoE-containing HDL. The WT apoE3 cleared the cholesterol of apoE(-/-) mice without induction of hypertriglyceridemia and promoted formation of spherical HDL. A unique property of the truncated apoE3[K146N/R147W]202 mutant, compared with similarly truncated apoE forms, is that it did not correct the hypercholesterolemia. The contribution of LPL and LCAT in the induction of the dyslipidemia was studied. Treatment of apoE(-/-) mice with apoE3[K146N/R147W] and LPL corrected the hypertriglyceridemia, but did not prevent the formation of discoidal HDL. Treatment with LCAT corrected hypertriglyceridemia and generated spherical HDL. The combined data indicate that the K146N/R147W substitutions convert the full-length and the truncated apoE3[K146N/R147W] mutant into a dominant negative ligand that prevents receptor-mediated remnant clearance, exacerbates the dyslipidemia, and inhibits the biogenesis of HDL.
Asunto(s)
Apolipoproteína E3/metabolismo , Lipoproteínas HDL/biosíntesis , Mutación Missense , Sustitución de Aminoácidos , Animales , Apolipoproteína E3/genética , Femenino , Hipertrigliceridemia/genética , Hipertrigliceridemia/metabolismo , Hipertrigliceridemia/patología , Lipoproteína Lipasa/genética , Lipoproteína Lipasa/metabolismo , Lipoproteínas HDL/genética , Masculino , Ratones , Ratones Noqueados , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismoRESUMEN
RATIONALE: RhoA and Rho kinase contribute to pulmonary vasoconstriction and vascular remodeling in pulmonary hypertension. RhoB, a protein homologous to RhoA and activated by hypoxia, regulates neoplastic growth and vasoconstriction but its role in the regulation of pulmonary vascular function is not known. OBJECTIVE: To determine the role of RhoB in pulmonary endothelial and smooth muscle cell responses to hypoxia and in pulmonary vascular remodeling in chronic hypoxia-induced pulmonary hypertension. METHODS AND RESULTS: Hypoxia increased expression and activity of RhoB in human pulmonary artery endothelial and smooth muscle cells, coincidental with activation of RhoA. Hypoxia or adenoviral overexpression of constitutively activated RhoB increased actomyosin contractility, induced endothelial permeability, and promoted cell growth; dominant negative RhoB or manumycin, a farnesyltransferase inhibitor that targets the vascular function of RhoB, inhibited the effects of hypoxia. Coordinated activation of RhoA and RhoB maximized the hypoxia-induced stress fiber formation caused by RhoB/mammalian homolog of Drosophila diaphanous-induced actin polymerization and RhoA/Rho kinase-induced phosphorylation of myosin light chain on Ser19. Notably, RhoB was specifically required for hypoxia-induced factor-1α stabilization and for hypoxia- and platelet-derived growth factor-induced cell proliferation and migration. RhoB deficiency in mice markedly attenuated development of chronic hypoxia-induced pulmonary hypertension, despite compensatory expression of RhoA in the lung. CONCLUSIONS: RhoB mediates adaptational changes to acute hypoxia in the vasculature, but its continual activation by chronic hypoxia can accentuate vascular remodeling to promote development of pulmonary hypertension. RhoB is a potential target for novel approaches (eg, farnesyltransferase inhibitors) aimed at regulating pulmonary vascular tone and structure.
Asunto(s)
Células Endoteliales/enzimología , Hipertensión Pulmonar/etiología , Hipoxia/complicaciones , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Proteína de Unión al GTP rhoB/metabolismo , Actomiosina/genética , Actomiosina/metabolismo , Animales , Permeabilidad Capilar , Hipoxia de la Célula , Movimiento Celular , Proliferación Celular , Células Cultivadas , Enfermedad Crónica , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Hipertensión Pulmonar Primaria Familiar , Farnesiltransferasa/antagonistas & inhibidores , Farnesiltransferasa/metabolismo , Humanos , Hipertensión Pulmonar/tratamiento farmacológico , Hipertensión Pulmonar/enzimología , Hipertensión Pulmonar/genética , Hipoxia/enzimología , Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Polienos/farmacología , Alcamidas Poliinsaturadas/farmacología , Arteria Pulmonar/enzimología , Interferencia de ARN , Serina , Fibras de Estrés/enzimología , Factores de Tiempo , Transfección , Vasoconstricción , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoB/deficiencia , Proteína de Unión al GTP rhoB/genéticaRESUMEN
BACKGROUND: ADP and ATP are importantly involved in vascular and thrombotic homeostasis, via multiple receptor pathways. Blockade of ADP P2Y12 receptors inhibits platelet aggregation and represents an effective cardiovascular disease prevention strategy. AZD3366 (APT102), a long-acting recombinant form of an optimized CD39L3 human apyrase, has effectively reduced ATP, ADP, and platelet aggregation and provided tissue protection in preclinical models, features that could be very beneficial in treating patients with cardiovascular disease. METHODS AND RESULTS: We conducted this phase 1, first-in-human study of single ascending doses of intravenous AZD3366 or placebo, including doses added to dual antiplatelet therapy with ticagrelor and acetylsalicylic acid. The primary objective was safety and tolerability; secondary and exploratory objectives included pharmacokinetics, pharmacodynamics (measured as inhibition of platelet aggregation), adenosine diphosphatase (ADPase) activity, and ATP/ADP metabolism. In total, 104 participants were randomized. AZD3366 was generally well tolerated, with no major safety concerns observed. ADPase activity increased in a dose-dependent manner with a strong correlation to AZD3366 exposure. Inhibition of ADP-stimulated platelet aggregation was immediate, substantial, and durable. In addition, there was a prompt decrease in systemic ATP concentration and an increase in adenosine monophosphate concentrations, whereas ADP concentration appeared generally unaltered. At higher doses, there was a prolongation of capillary bleeding time without detectable changes in the ex vivo thromboelastometric parameters. CONCLUSIONS: AZD3366 was well tolerated in healthy participants and demonstrated substantial and durable inhibition of platelet aggregation after single dosing. Higher doses prolonged capillary bleeding time without detectable changes in ex vivo thromboelastometric parameters. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique Identifier: NCT04588727.