SREBP-2-driven transcriptional activation of human SND1 oncogene.

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.

Increased Circulating Levels of Alpha-Ketoglutarate in Morbidly Obese Women with Non-Alcoholic Fatty Liver Disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) causes a wide spectrum of liver damage, ranging from simple steatosis to cirrhosis. However, simple steatosis (SS) and steatohepatitis (NASH) cannot yet be distinguished by clinical or laboratory features. The aim of this study was to assess the relationship between alpha-ketoglutarate and the degrees of NAFLD in morbidly obese patients. MATERIALS AND METHODS: We used a gas chromatography-quadruple time-of-flight-mass spectrometry analysis to quantify alpha-ketoglutarate in serum from normal-weight subjects (n = 30) and morbidly obese women (n = 97) with or without NAFLD. RESULTS: We found that serum levels of alpha-ketoglutarate were significantly higher in morbidly obese women than in normal-weight women. We showed that circulating levels of alpha-ketoglutarate were lower in lean controls and morbidly obese patients without NAFLD. We also found that alpha-ketoglutarate serum levels were higher in both SS and NASH than in normal liver of morbidly obese patients. However, there was no difference between SS and NASH. Moreover, we observed that circulating levels of alpha-ketoglutarate were associated with glucose metabolism parameters, lipid profile, hepatic enzymes and steatosis degree. In addition, diagnostic performance of alpha-ketoglutarate has been analyzed in NAFLD patients. The AUROC curves from patients with liver steatosis exhibited an acceptable clinical utility. Finally, we showed that the combination of biomarkers (AST, ALT and alpha-ketoglutarate) had the highest accuracy in diagnosing liver steatosis. CONCLUSION: These findings suggest that alpha-ketoglutarate can determine the presence of non-alcoholic fatty liver in morbidly obese patients but it is not valid a biomarker for NASH.

PNPLA3 Expression Is Related to Liver Steatosis in Morbidly Obese Women with Non-Alcoholic Fatty Liver Disease.

Recent reports suggest a role for the Patatin-like phospholipase domain-containing protein 3 (PNPLA3) in the pathology of non-alcoholic fatty liver disease (NAFLD). Lipid deposition in the liver seems to be a critical process in the pathogenesis of NAFLD. The aim of the present work was to evaluate the association between the liver PNPLA3 expression, key genes of lipid metabolism, and the presence of NAFLD in morbidly obese women. We used real-time polymerase chain reaction (PCR) analysis to analyze the hepatic expression of PNPLA3 and lipid metabolism-related genes in 55 morbidly obese subjects with normal liver histology (NL, n = 18), simple steatosis (SS, n = 20), and non-alcoholic steatohepatitis (NASH, n = 17). Liver biopsies were collected during bariatric surgery. We observed that liver PNPLA3 expression was increased in NAFLD than in NL. It was also upregulated in SS than in NL. Interestingly, we found that the expression of PNPLA3 was significantly higher in severe than mild SS group. In addition, the expression of the transcription factors LXRα, PPARα, and SREBP2 was positively correlated with PNPLA3 liver expression. Regarding rs738409 polymorphism, GG genotype was positive correlated with the presence of NASH. In conclusion, our results show that PNPLA3 could be related to lipid accumulation in liver, mainly in the development and progression of simple steatosis.

Downregulation of de Novo Fatty Acid Synthesis in Subcutaneous Adipose Tissue of Moderately Obese Women.

The purpose of this work was to evaluate the expression of fatty acid metabolism-related genes in human adipose tissue from moderately obese women. We used qRT-PCR and Western Blot to analyze visceral (VAT) and subcutaneous (SAT) adipose tissue mRNA expression involved in de novo fatty acid synthesis (ACC1, FAS), fatty acid oxidation (PPARα, PPARδ) and inflammation (IL6, TNFα), in normal weight control women (BMI < 25 kg/m², n = 35) and moderately obese women (BMI 30-38 kg/m², n = 55). In SAT, ACC1, FAS and PPARα mRNA expression were significantly decreased in moderately obese women compared to controls. The downregulation reported in SAT was more pronounced when BMI increased. In VAT, lipogenic-related genes and PPARα were similar in both groups. Only PPARδ gene expression was significantly increased in moderately obese women. As far as inflammation is concerned, TNFα and IL6 were significantly increased in moderate obesity in both tissues. Our results indicate that there is a progressive downregulation in lipogenesis in SAT as BMI increases, which suggests that SAT decreases the synthesis of fatty acid de novo during the development of obesity, whereas in VAT lipogenesis remains active regardless of the degree of obesity.

Profiling of promoter occupancy by the SND1 transcriptional coactivator identifies downstream glycerolipid metabolic genes involved in TNFα response in human hepatoma cells.

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.

The promoter of cell growth- and RNA protection-associated SND1 gene is activated by endoplasmic reticulum stress in human hepatoma cells.

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.

NF-κB, Sp1 and NF-Y as transcriptional regulators of human SND1 gene.

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.

Inhibition of macrophage inflammatory cytokine secretion by chylomicron remnants is dependent on their uptake by the low density lipoprotein receptor.

Secretion of pro-inflammatory chemokines and cytokines by macrophages is a contributory factor in the pathogenesis of atherosclerosis. In this study, the effects of chylomicron remnants (CMR), the lipoproteins which transport dietary fat in the blood, on the production of pro-inflammatory chemokine and cytokine secretion by macrophages was investigated using CMR-like particles (CRLPs) together with THP-1 macrophages or primary human macrophages (HMDM). Incubation of CRLPs or oxidized CRLPs (oxCRLPs) with HMDM or THP-1 macrophages for up to 24h led to a marked decrease in the secretion of the pro-inflammatory chemokine monocyte chemoattractant protein-1 (MCP-1) and the pro-inflammatory cytokines tumour necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1ß (-50-90%), but these effects were reduced or abolished when CRLPs protected from oxidation by incorporation of the antioxidant drug, probucol, (pCRLPs) were used. In macrophages transfected with siRNA targeted to the low density lipoprotein receptor (LDLr), neither CRLPs nor pCRLPs had any significant effect on chemokine/cytokine secretion, but in cells transfected with siRNA targeted to the LDLr-related protein 1 (LRP1) both types of particles inhibited secretion to a similar extent to that observed with CRLPs in mock transfected cells. These findings demonstrate that macrophage pro-inflammatory chemokine/cytokine secretion is down-regulated by CMR, and that these effects are positively related to the lipoprotein oxidative state. Furthermore, uptake via the LDLr is required for the down-regulation, while uptake via LRP1 does not bring about this effect. Thus, the receptor-mediated route of uptake of CMR plays a crucial role in modulating their effects on inflammatory processes in macrophages.