RESUMEN
Staphylococcal nuclease and Tudor domain containing 1 (SND1) is an evolutionarily conserved protein present in eukaryotic cells from protozoa to mammals. SND1 has gained importance because it is overexpressed in aggressive cancer cells and diverse primary tumors. Indeed, it is regarded as a marker of cancer malignity. A broad range of molecular functions and the participation in many cellular processes have been attributed to SND1, mostly related to the regulation of gene expression. An increasing body of evidence points to a relevant relationship between SND1 and lipid metabolism. In this review, we summarize the knowledge about SND1 and its molecular and functional relationship with lipid metabolism. We highlight that SND1 plays a direct role in the regulation of cholesterol metabolism by affecting the activation of sterol response element-binding protein 2 (SREBP2) and we propose that that might have implications in the response of lipid homeostasis to stress situations.
Asunto(s)
Endonucleasas/genética , Metabolismo de los Lípidos/genética , Neoplasias/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Estrés Fisiológico/genética , Secuencias de Aminoácidos , Animales , Colesterol/metabolismo , Biología Computacional , Endonucleasas/metabolismo , Ácidos Grasos/metabolismo , Regulación Neoplásica de la Expresión Génica , Homeostasis/genética , Humanos , Neoplasias/metabolismo , Regiones Promotoras Genéticas/genética , Dominios Proteicos , Interferencia de ARN , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , ARN Mensajero/metabolismo , Empalmosomas/metabolismo , Transcripción GenéticaRESUMEN
The data contain information related to the research article entitled "Profiling of promoter occupancy by the SND1 transcriptional coactivator identifies downstream glycerolipid metabolic genes involved in TNFα response in human hepatoma cells" (DOI: 10.1093/nar/gkv858). In the article alluded to, we reported that tumor necrosis factor alpha (TNFα) increases notably the cellular content of the major glycerolipid phosphatidylcholine (PC). Here, accompanying lipidomic data determine the PC structural variants that have been identified in human hepatoma HepG2 cells and those whose relative abundance is modified by TNFα. We used ultrahigh performance liquid chromatography (UHPLC) coupled to electrospray ionization (ESI) tandem mass spectrometry (MS/MS)-based lipidomic profiling to analyze lipid extracts of control and TNFα-treated HepG2 cells. The identity of PC individual species was elucidated using the values of the retention time and molecular weight in addition to the fragmentation patterns. MS data were then processed and analyzed for the characterization of statistically significant differences in detected structural variants. We have annotated the dataset of PC species that characterize HepG2 cells' phenotype, both under normal and pro-inflammatory conditions.
RESUMEN
SND1 is a putative oncoprotein whose molecular function remains unclear. Its overexpression in hepatocellular carcinoma impairs cholesterol homeostasis due to the altered activation of the sterol regulatory element-binding protein (SREBP) 2, which results in the accumulation of cellular cholesteryl esters (CE). In this work, we explored whether high cholesterol synthesis and esterification originates changes in glycerolipid metabolism that might affect cell growth, given that acetyl-coenzyme A is required for cholesterogenesis and fatty acids (FA) are the substrates of acyl-coenzyme A:cholesterol acyltransferase (ACAT). SND1-overexpressing hepatoma cells show low triglyceride (TG) synthesis, but phospholipid biosynthesis or cell growth is not affected. Limited TG synthesis is not due to low acetyl-coenzyme A or NADPH availability. We demonstrate that the main factor limiting TG synthesis is the utilization of FAs for cholesterol esterification. These metabolic adaptations are linked to high Scd1 expression, needed for the de novo production of oleic acid, the main FA used by ACAT. We conclude that high cholesterogenesis due to SND1 overexpression might determine the channeling of FAs to CEs.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Ácidos Grasos/biosíntesis , Triglicéridos/metabolismo , Acilcoenzima A/metabolismo , Aciltransferasas/metabolismo , Animales , Línea Celular Tumoral , Colesterol/metabolismo , Ésteres del Colesterol/biosíntesis , Ésteres del Colesterol/metabolismo , Endonucleasas , Esterificación/fisiología , Hipercolesterolemia/metabolismo , Metabolismo de los Lípidos , Lipogénesis , Neoplasias Hepáticas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ácido Oléico/metabolismo , Ratas , Esterol O-Aciltransferasa/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Triglicéridos/biosíntesisRESUMEN
The staphylococcal nuclease and Tudor domain containing 1 gene (SND1), also known as Tudor-SN, TSN or p100, encodes an evolutionarily conserved protein with invariant domain composition. SND1 contains four repeated staphylococcal nuclease domains and a single Tudor domain, which confer it endonuclease activity and extraordinary capacity for interacting with nucleic acids, individual proteins and protein complexes. Originally described as a transcriptional coactivator, SND1 plays fundamental roles in the regulation of gene expression, including RNA splicing, interference, stability, and editing, as well as in the regulation of protein and lipid homeostasis. Recently, SND1 has gained attention as a potential disease biomarker due to its positive correlation with cancer progression and metastatic spread. Such functional diversity of SND1 marks this gene as interesting for further analysis in relation with the multiple levels of regulation of SND1 protein production. In this review, we summarize the SND1 genomic region and promoter architecture, the set of transcription factors that can bind the proximal promoter, and the evidence supporting transactivation of SND1 promoter by a number of signal transduction pathways operating in different cell types and conditions. Unraveling the mechanisms responsible for SND1 promoter regulation is of utmost interest to decipher the SND1 contribution in the realm of both normal and abnormal physiology.
RESUMEN
Upregulation of Staphylococcal nuclease and tudor domain containing 1 (SND1) is linked to cancer progression and metastatic spread. Increasing evidence indicates that SND1 plays a role in lipid homeostasis. Recently, it has been shown that SND1-overexpressing hepatocellular carcinoma cells present an increased de novo cholesterol synthesis and cholesteryl ester accumulation. Here we reveal that SND1 oncogene is a novel target for SREBPs. Exposure of HepG2 cells to the cholesterol-lowering drug simvastatin or to a lipoprotein-deficient medium triggers SREBP-2 activation and increases SND1 promoter activity and transcript levels. Similar increases in SND1 promoter activity and mRNA are mimicked by overexpressing nuclear SREBP-2 through expression vector transfection. Conversely, SREBP-2 suppression with specific siRNA or the addition of cholesterol/25-hydroxycholesterol to cell culture medium reduces transcriptional activity of SND1 promoter and SND1 mRNA abundance. Chromatin immunoprecipitation assays and site-directed mutagenesis show that SREBP-2 binds to the SND1 proximal promoter in a region containing one SRE and one E-box motif which are critical for maximal transcriptional activity under basal conditions. SREBP-1, in contrast, binds exclusively to the SRE element. Remarkably, while ectopic expression of SREBP-1c or -1a reduces SND1 promoter activity, knocking-down of SREBP-1 enhances SND1 mRNA and protein levels but failed to affect SND1 promoter activity. These findings reveal that SREBP-2 and SREBP-1 bind to specific sites in SND1 promoter and regulate SND1 transcription in opposite ways; it is induced by SREBP-2 activating conditions and repressed by SREBP-1 overexpression. We anticipate the contribution of a SREBPs/SND1 pathway to lipid metabolism reprogramming of human hepatoma cells.
RESUMEN
The NF-κB-inducible Staphylococcal nuclease and tudor domain-containing 1 gene (SND1) encodes a coactivator involved in inflammatory responses and tumorigenesis. While SND1 is known to interact with certain transcription factors and activate client gene expression, no comprehensive mapping of SND1 target genes has been reported. Here, we have approached this question by performing ChIP-chip assays on human hepatoma HepG2 cells and analyzing SND1 binding modulation by proinflammatory TNFα. We show that SND1 binds 645 gene promoters in control cells and 281 additional genes in TNFα-treated cells. Transcription factor binding site analysis of bound probes identified motifs for established partners and for novel transcription factors including HSF, ATF, STAT3, MEIS1/AHOXA9, E2F and p300/CREB. Major target genes were involved in gene expression and RNA metabolism regulation, as well as development and cellular metabolism. We confirmed SND1 binding to 21 previously unrecognized genes, including a set of glycerolipid genes. Knocking-down experiments revealed that SND1 deficiency compromises the glycerolipid gene reprogramming and lipid phenotypic responses to TNFα. Overall, our findings uncover an unexpected large set of potential SND1 target genes and partners and reveal SND1 to be a determinant downstream effector of TNFα that contributes to support glycerophospholipid homeostasis in human hepatocellular carcinoma during inflammation.
Asunto(s)
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Factor de Necrosis Tumoral alfa/fisiología , Sitios de Unión , Carcinoma Hepatocelular/metabolismo , Inmunoprecipitación de Cromatina , Endonucleasas , Regulación Neoplásica de la Expresión Génica , Glicerofosfolípidos/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Proteínas Nucleares/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Staphyloccocal nuclease domain-containing protein 1 (SND1) is involved in the regulation of gene expression and RNA protection. While numerous studies have established that SND1 protein expression is modulated by cellular stresses associated with tumor growth, hypoxia, inflammation, heat-shock and oxidative conditions, little is known about the factors responsible for SND1 expression. Here, we have approached this question by analyzing the transcriptional response of human SND1 gene to pharmacological endoplasmic reticulum (ER) stress in liver cancer cells. RESULTS: We provide first evidence that SND1 promoter activity is increased in human liver cancer cells upon exposure to thapsigargin or tunicamycin or by ectopic expression of ATF6, a crucial transcription factor in the unfolded protein response triggered by ER stress. Deletion analysis of the 5'-flanking region of SND1 promoter identified maximal activation in fragment (-934, +221), which contains most of the predicted ER stress response elements in proximal promoter. Quantitative real-time PCR revealed a near 3 fold increase in SND1 mRNA expression by either of the stress-inducers; whereas SND1 protein was maximally upregulated (3.4-fold) in cells exposed to tunicamycin, a protein glycosylation inhibitor. CONCLUSION: Promoter activity of the cell growth- and RNA-protection associated SND1 gene is up-regulated by ER stress in human hepatoma cells.
Asunto(s)
Carcinoma Hepatocelular/patología , División Celular , Retículo Endoplásmico/metabolismo , Neoplasias Hepáticas/patología , Proteínas Nucleares/genética , ARN/genética , Línea Celular Tumoral , Endonucleasas , HumanosRESUMEN
Staphylococcal nuclease domain-containing protein 1 (SND1), also called Tudor-SN, is required for many biological events ranging from gene expression to cell growth regulation. Promoter regulation of SND1 gene and its molecular mechanism have remained elusive to date. In this work, we have identified SND1 as a new target gene for NF-κB, Sp1 and NF-Y transcription factors. We isolated and characterized a 3808 bp sequence corresponding to the human SND1 gene promoter (GenBank ID: EF690304). It lacks the typical TATA-box element and contains a CpG island with several Sp1 binding sites at the 3' end, and a highly conserved 300 bp segment with two inverted CCAAT boxes that bind NF-Y, in addition to NF-κB sites and other cis-regulatory elements. Electrophoretic mobility shift assays and chromatin immunoprecipitation experiments confirmed the ability of SND1 promoter to bind NF-κB, Sp1 and NF-Y in vitro and in vivo. Deletion analysis of the 5'-flanking region by luciferase reporter assays, showed the minimum promoter activity 112 base-pair upstream from the transcription start site, and an enhancer region between -112 and -274 bp responsible for the maximal transcriptional activity of the promoter. Site-directed mutagenesis of the CCAAT and GC boxes and the NF-κB elements within the proximal region substantially reduced SND1 promoter activity. Proinflammatory cytokine TNF-α caused an increase of SND1 promoter activity that is mediated, at least in part, via NF-κB as mutation in the NF-κB sites impaired the promoter stimulation. We provide for the first time the characterization of the human SND1 promoter activity and establish a transcriptional network associated to the key transcription factors NF-κB, Sp1 and NF-Y that operates in the control of the SND1 gene expression.
Asunto(s)
Factor de Unión a CCAAT/metabolismo , FN-kappa B/metabolismo , Proteínas Nucleares/genética , Factor de Transcripción Sp1/metabolismo , Secuencia de Bases , Sitios de Unión , Secuencia Conservada , Endonucleasas , Células Hep G2 , Humanos , Datos de Secuencia Molecular , Mutagénesis , Mutación , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Dyslipidemia and hepatic overproduction of very low density lipoprotein (VLDL) are hallmarks of the septic response, yet the underlying mechanisms are not fully defined. We evaluated the lipoprotein subclasses profile and hepatic VLDL assembly machinery over 24 h in fasted LPS-treated rats. The response of serum non-esterified fatty acids (NEFA) and glucose to endotoxin was biphasic, with increased levels of NEFA and hypoglycemia in the first 12 h-phase, and low NEFA and high glucose in the second 12 h-phase. Hypertriglyceridemia was more marked in the first 12 h (6.8-fold), when triglyceride abundance increased in all lipoprotein subclasses, and preferentially in large VLDL. The abundance of medium-sized VLDL and the increase in the number of VLDL particles was higher in the second phase (10-fold vs 5-fold in the first phase); however, apoB gene transcript abundance increased only in the second phase. Analysis of putative pre-translational mechanisms revealed that neither increased Apob transcription rate nor increased transcript binding to mRNA stabilizing HuR (Hu antigen R) protein paralleled the increase in apoB transcripts. In conclusion, endotoxin challenge induces increases in plasma NEFA and large, triglyceride-rich VLDL. After approximately 12 h, the triglyceride-rich VLDLs are replaced by medium-sized, triglyceride-poor VLDL particles. Hepatic apoB mRNA abundance also increases during the second period, suggesting a role for apoB protein expression in the acute reaction against sepsis.
Asunto(s)
Apolipoproteínas B/metabolismo , VLDL-Colesterol/metabolismo , Endotoxemia/metabolismo , Bacterias Grampositivas/fisiología , Hígado/metabolismo , Animales , Apolipoproteínas B/genética , Proteínas ELAV/metabolismo , Endotoxemia/complicaciones , Endotoxemia/fisiopatología , Ácidos Grasos/metabolismo , Femenino , Glucosa/metabolismo , Bacterias Grampositivas/patogenicidad , Humanos , Hipertrigliceridemia/etiología , Hígado/inmunología , Hígado/microbiología , Ratas , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
Hepatic steatosis is a risk factor for the progression of non-alcoholic fatty liver disease. The role of pro-inflammatory interleukin (IL)-6 in hepatic steatosis etiology is controversial. We investigated in vivo and in primary hepatocyte cultures whether IL-6 has a modulator role in liver and mitochondria lipid composition and cell death in a choline-deficient (CD) diet rat model of hepatic steatosis. Dietary choline deficiency increased triglycerides and cholesterol, and reduced phosphatidylcholine (PC), phosphatidylethanolamine (PE) and the membrane integrity marker PC:PE ratio in liver. Choline-deficient diet enhanced systemic IL-6, and IL-6 receptor expression and cell death vulnerability in hepatocytes. Derangement of the mitochondrial electron transport chain and of its phospholipid environment was found in CD rat liver mitochondria, which exhibited elevated concentrations of triglycerides, cardiolipin and PC and elevated PC:PE ratio. The cell treatment with IL-6, but not PC, eliminated much of the CD-promoted lipid imbalance in mitochondria but not tumor-necrosis factor (TNF)-alpha-induced cell death. However, PC supplementation prevented the TNF-alpha-induced DNA fragmentation, cytochrome-c release and caspase-3 activity in control and CD hepatocytes. In conclusion, IL-6 ameliorated the mitochondria lipid disturbance in hepatocytes isolated from steatotic animals. Furthermore, PC is identified as a new survival agent that reverses several TNFalpha-inducible responses that are likely to promote steatosis and necrosis.
Asunto(s)
Deficiencia de Colina/inmunología , Hígado Graso/inmunología , Hepatocitos/metabolismo , Interleucina-6/metabolismo , Receptores de Interleucina-6/biosíntesis , Animales , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/inmunología , Células Cultivadas , Deficiencia de Colina/tratamiento farmacológico , Deficiencia de Colina/fisiopatología , Dieta , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Transporte de Electrón/efectos de los fármacos , Transporte de Electrón/inmunología , Hígado Graso/tratamiento farmacológico , Hígado Graso/metabolismo , Hígado Graso/patología , Hígado Graso/fisiopatología , Hepatocitos/efectos de los fármacos , Hepatocitos/inmunología , Hepatocitos/patología , Interleucina-6/farmacología , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/fisiología , Fosfatidilcolinas/farmacología , Ratas , Ratas Wistar , Receptores de Interleucina-6/genética , Triglicéridos/biosíntesis , Triglicéridos/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Plasma VLDL accumulation in Gram-negative sepsis is partly ascribed to an increased hepatic VLDL production driven by pro-inflammatory cytokines. We previously showed that hepatocytes of the Kupffer cell (KC)-rich periportal area are major contributors to enhanced VLDL production in lipopolysaccharide (LPS)-injected rats. However, it remains to be established whether KC generated products directly affect the number (apoB) and composition of secreted VLDL. Using rat primary cells, we show here that hepatocytes respond to stimulation by soluble mediators released by LPS-stimulated Kupffer cells with enhanced secretion of apoB and triglycerides in phospholipid-rich VLDL particles. Unstimulated KC products also augmented the secretion of normal VLDL, doubling apoB mRNA abundance. IL-1beta treatment resulted in concentration-dependent increases of hepatocyte apoB mRNA and protein secretion, increases that were greater, but not additive, when combined with IL-6 and TNF-alpha. Lipid secretion and MTP mRNA levels were unaffected by cytokines. In summary: (i) enhanced secretion of phospholipid-rich VLDL particles is a net hepatocyte response to LPS-stimulated KC products, which gives a clue about the local role of Kupffer cells in septic dyslipidemia induction; and (ii) pro-inflammatory cytokines act redundantly to enhance apoB secretion involving translational apoB up-regulation, but other humoral components or KC mediators are necessary to accomplish increased lipid association.
Asunto(s)
Apolipoproteína B-100/genética , Hepatocitos/metabolismo , Interleucina-1beta/farmacología , Macrófagos del Hígado/metabolismo , Lipoproteínas VLDL/metabolismo , Fagocitosis/efectos de los fármacos , Animales , Apolipoproteína B-100/biosíntesis , Apolipoproteína B-100/inmunología , Técnicas de Cultivo de Célula , Medios de Cultivo Condicionados/metabolismo , Medios de Cultivo Condicionados/farmacología , Endotoxemia , Femenino , Hepatocitos/efectos de los fármacos , Humanos , Macrófagos del Hígado/inmunología , Lipopolisacáridos/administración & dosificación , Lipoproteínas VLDL/agonistas , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Tumor necrosis factor-alpha (TNF-alpha) plays a pivotal role in the host response to infection. Rapidly liberated to the bloodstream, TNF-alpha triggers the production of other cytokines and the acute-phase response. Hypertriglyceridemia is a sepsis hallmark associated with high plasma levels of very low-density lipoprotein (VLDL) particles, partly ascribed to increased hepatic production. The kinetics of the hepatocyte response, the cytokine/s responsible, and the underlying mechanisms are not fully elucidated. VLDL biogenesis is a complex, time-consuming process that depends on lipid availability and microsomal triglyceride transfer protein (MTP) activity for correct apolipoprotein B (apoB) lipidation. Studies were performed to define the direct effect of TNF-alpha on VLDL secretion rate and composition in rat hepatocytes cultured in conditions resembling the fed situation. Increases of 17-24% in the number of VLDL particles secreted and of 44-88% in the cellular levels of apoB mRNA were caused by 5, 20, or 100 ng/mL TNF-alpha in 8 h. Lipoprotein secretion returned to baseline levels in 16 h, whereas TNF-alpha-treated cells continued to exhibit higher apoB transcript levels. The mass of each lipid class in secreted VLDL and of MTP mRNA in cells was not affected by any of the tested TNF-alpha doses or treatment periods. These findings indicate that over a wide range of concentrations, TNF-alpha was capable of inducing sustained upregulation of apoB mRNA expression and transient increase in secretion of its protein, but, apparently, VLDL triglyceride secretion was not a TNF-alpha target under conditions in which fatty acids were not extracellularly provided.
Asunto(s)
Apolipoproteínas B/metabolismo , Ácidos Grasos/metabolismo , Hepatocitos/citología , Lípidos/química , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba , Animales , Proteínas Portadoras/metabolismo , Femenino , Cinética , Lipoproteínas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Triglicéridos/metabolismoRESUMEN
In the liver, prostaglandins (PG) generated mainly by activated non-parenchymal cells can modulate the parenchymal cell function during homeostasis and inflammation. Whether prostaglandins regulate the hepatocyte VLDL assembling/secretor phenotype in both conditions remains unresolved. We sought to determine whether and how PGE2, PGD2, and PGF2alpha (5 and 50 microM) have a role in VLDL secretion regulation in resting and interleukin-6 (IL-6) stimulated rat hepatocytes. Prostaglandins led to comparable, concentration-dependent reductions in the secretion of VLDL apoB and lipids by resting, 24 h-cultured cells. Moreover, each apoB copy recruited less of each lipid class, correlating with reduced particle size, lipogenesis and cholesterogenesis, and impaired cellular triacylglycerol recycling. Triacylglycerol output reduction occurred early, as the transient PGD2- and PGF2alpha -promoted apoB mRNA decreases. IL-6 markedly increased the apoB mRNA expression and the secretion of its protein in triacylglycerol-poor VLDL. The latter was uniquely blunted by PGE2, which unaffected basal or IL-6-activated apoB gene expression. Collectively, our findings show inflammation condition-based roles for 2-series-prostaglandins in VLDL secretion modulation. Whereas in non-stimulated hepatocytes, they all inhibited VLDL-apoB output, interfered with lipid provision for lipoprotein assembly and may be regarded as pro-steatotic, the anti-inflammatory PGE2 antagonized the IL-6-promoted VLDL secretion contributing in restoring liver homeostasis.
Asunto(s)
Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Lipoproteínas VLDL/metabolismo , Prostaglandinas/farmacología , Animales , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Secuencia de Bases , Proteínas Portadoras/genética , Células Cultivadas , Dinoprost/farmacología , Dinoprostona/farmacología , Ácido Graso Sintasas/genética , Homeostasis/efectos de los fármacos , Inflamación/metabolismo , Interleucina-6/farmacología , Prostaglandina D2/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , RatasRESUMEN
Prostaglandins (PG) produced by activated non-parenchymal liver cells regulate the function of parenchymal cells through six classes of G-protein-coupled receptors (-R): four receptor subtypes for PGE2, EP1-R-EP4-R and one type each for PGD2, DP-R, and PGF2alpha, FP-R. The mechanisms by which prostaglandins modulate the hepatocyte responding phenotype are poorly characterized. We have studied the concentration and time effect of PGE2, PGD2 and PGF2alpha on the mRNA expression level of their own receptors in the presence or absence of the inflammatory signal interleukin 6 (IL-6). The mRNA levels were determined in primary adult rat hepatocytes upon treatment with either prostaglandin (5 microM or 50 microM), or IL-6 (100 ng/ml), or both, for 4-24 h. A marked, concentration-dependent induction of EP2-R mRNA expression was promoted by PGE2, PGD2 or PGF2alpha after 4 h, whereas EP1-R, EP3-R and EP4-R transcript levels were unaffected. This expression pattern changed substantially upon 24 h. The induction of EP2-R mRNA, persisted, though attenuated. Furthermore, EP1-R mRNA upregulated two-three fold and EP3-R mRNA decreased modestly by 50 microM prostaglandin. None of the treatments affected the FP-R mRNA level, while that of DP-R mRNA was undetectable. In the presence of IL-6, prostaglandins had no such effects, but they did attenuate the IL-6-mediated changes in prostaglandin receptor mRNA expression. The findings indicate that prostaglandins modulate the prostaglandin receptor gene expression programme in primary adult rat hepatocytes in a manner that is specific to the receptor, the concentration and time of exposure, and the inflammatory condition of the cell.
Asunto(s)
Hepatocitos/metabolismo , Interleucina-6/farmacología , Prostaglandinas/farmacología , ARN Mensajero/biosíntesis , Receptores de Prostaglandina/biosíntesis , Regulación hacia Arriba/efectos de los fármacos , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Inflamación/metabolismo , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Receptores de Prostaglandina/genéticaRESUMEN
To address the role of cell membrane neutral sphingomyelinase (EC 3.1.4.12; SMase) in the regulation of cholesterol metabolism in the liver parenchymal cell, we examined the effect of exogenous neutral SMase on the metabolism of cholesteryl esters and the secretion of VLDL and biliary lipids in isolated rat hepatocytes. We show that treatment of hepatocytes with SMase (20 mU/mL) resulted in the intracellular buildup of cholesteryl esters, increased ACAT (EC 2.3.1.26) activity without affecting the ACAT2 mRNA level, and increased cytosolic and microsomal cholesteryl ester hydrolase (EC 3.1.1.13) activity. This was accompanied by increases in the secretion of biliary bile acid, phospholipid, and cholesterol and in increased cholesterol 7alpha-hydroxylase (EC 1.14.13.17) activity and levels of mRNA, as well as decreased levels of apoB mRNA and a decreased secretion of VLDL apoB (apoB-48, approximately 45%; apoB-100, approximately 32%) and lipids (approximately 55%). Moreover, the VLDL particles secreted had an abnormal size and lipid composition; they were larger than controls, were relatively enriched in cholesteryl ester, and depleted in TG and cholesterol. Cell-permeable ceramides did not replicate any of the reported effects. These findings demonstrate that the increased cholesteryl ester turnover, oversecretion of biliary cholesterol and bile acids, and undersecretion of VLDL cholesterol and particles are concerted responses of the primary hepatocytes to exogenous neutral SMase brought about by regulation at several levels. We suggest that plasma membrane neutral SMase may have a specific, ceramide-independent effect in the regulation of cholesterol output pathways in hepatocytes.