Effect of TNFα on activities of different promoters of human apolipoprotein A-I gene

Human apolipoprotein A-I (ApoA-I) is a major structural and functional protein component of high-density lipoproteins. The expression of the apolipoprotein A-I gene (apoA-I) in hepatocytes is repressed by pro-inflammatory cytokines such as IL-1β and TNFα. Recently, two novel additional (alternative) promoters for human apoA-I gene have been identified. Nothing is known about the role of alternative promoters in TNFα-mediated downregulation of apoA-I gene. In this article we report for the first time about the different effects of TNFα on two alternative promoters of human apoA-I gene. Stimulation of HepG2 cells by TNFα leads to activation of the distal alternative apoA-I promoter and downregulation of the proximal alternative and the canonical apoA-I promoters. This effect is mediated by weakening of the promoter competition within human apoA-I 5′-regulatory region (apoA-I promoter switching) in the cells treated by TNFα. The MEK1/2-ERK1/2 cascade and nuclear receptors PPARα and LXRs are important for TNFα-mediated apoA-I promoter switching.     

Autoregulation of the human liver X receptor alpha promoter

Previous work has implicated the nuclear receptors liver X receptor alpha (LXR alpha) and LXR beta in the regulation of macrophage gene expression in response to oxidized lipids. Macrophage lipid loading leads to ligand activation of LXRs and to induction of a pathway for cholesterol efflux involving the LXR target genes ABCA1 and apoE. We demonstrate here that autoregulation of the LXR alpha gene is an important component of this lipid-inducible efflux pathway in human macrophages. Oxidized low-density lipoprotein, oxysterols, and synthetic LXR ligands induce expression of LXR alpha mRNA in human monocyte-derived macrophages and human macrophage cell lines but not in murine peritoneal macrophages or cell lines. This is in contrast to peroxisome proliferator-activated receptor gamma (PPAR gamma)-specific ligands, which stimulate LXR alpha expression in both human and murine macrophages. We further demonstrate that LXR and PPAR gamma ligands cooperate to induce LXR alpha expression in human but not murine macrophages. Analysis of the human LXR alpha promoter led to the identification of multiple LXR response elements. Interestingly, the previously identified PPAR response element (PPRE) in the murine LXR alpha gene is not conserved in humans; however, a different PPRE is present in the human LXR 5'-flanking region. These results have implications for cholesterol metabolism in human macrophages and its potential to be regulated by synthetic LXR and/or PPAR gamma ligands. The ability of LXR alpha to regulate its own promoter is likely to be an integral part of the macrophage physiologic response to lipid loading.     

Liver X Receptor (LXR) activation negatively regulates visfatin expression in macrophages

Adipose tissue macrophages (ATM) are the major source of visfatin, a visceral fat adipokine upregulated during obesity. Also known to play a role in B cell differentiation (pre-B cell colony-enhancing factor (PBEF)) and NAD biosynthesis (nicotinamide phosphoribosyl transferase (NAMPT)), visfatin has been suggested to play a role in inflammation.Liver X Receptor (LXR) and Peroxisome Proliferator-Activated Receptor (PPAR)γ are nuclear receptors expressed in macrophages controlling the inflammatory response. Recently, we reported visfatin as a PPARγ target gene in human macrophages. In this study, we examined whether LXR regulates macrophage visfatin expression. Synthetic LXR ligands decreased visfatin gene expression in a LXR-dependent manner in human and murine macrophages. The decrease of visfatin mRNA was paralleled by a decrease of protein secretion. Consequently, a modest and transient decrease of NAD⁺ concentration was observed. Interestingly, LXR activation decreased the PPARγ-induced visfatin gene and protein secretion in human macrophages.Our results identify visfatin as a gene oppositely regulated by the LXR and PPARγ pathways in human macrophages.     

TNFα activation of PKCδ, mediated by NFκB and ER stress,cross-talks with the insulin signaling cascade

TNFα plays key roles in the regulation of inflammation, cell death, and proliferation and its signaling cascade cross-talks with the insulin signaling cascade. PKCδ, a novel PKC isoform, is known to participate in proximal TNFα signaling events. However, it has remained unclear whether PKCδ plays a role in distal TNFα signaling events. Here we demonstrate that PKCδ is activated by TNFα in a delayed fashion that is temporally associated with JNK activation. To investigate the signaling pathways activating PKCδ and JNK, we used pharmacological and genetic inhibitors of NFκB. We found that inhibition of NFκB attenuated PKCδ and JNK activations. Further analysis revealed that ER stress contributes to TNFα-stimulated PKCδ and JNK activations. To investigate the role of PKCδ in TNFα action, we used 29-mer shRNAs to silence PKCδ expression. A reduction of ~90% in PKCδ protein levels reduced TNFα-stimulated stress kinase activation, including JNK. Further, PKCδ was necessary for thapsigargin-stimulated JNK activation. Because thapsigargin is a potent inducer of ER stress, we determined whether PKCδ was necessary for induction of the UPR. Indeed, a reduction in PKCδ protein levels reduced thapsigargin-stimulated CHOP induction, a hallmark of the UPR, but not BiP/GRP78 induction, suggesting that PKCδ does not globally regulate the UPR. Next, the role of PKCδ in TNFα mediated cross-talk with the insulin signaling pathway was investigated in cells expressing human IRS-1 and a 29-mer shRNA to silence PKCδ expression. We found that a reduction in PKCδ protein levels reversed the TNFα-mediated reduction in insulin-stimulated IRS-1 Tyr phosphorylation, Akt activation, and glycogen synthesis. In addition, TNFα-stimulated IRS protein Ser/Thr phosphorylation and degradation were blocked. Our results indicate that: 1) NFκB and ER stress contribute in part to PKCδ activation; 2) PKCδ plays a key role in the propagation of the TNFα signal; and 3) PKCδ contributes to TNFα-induced inhibition of insulin signaling events.     

PPARα-LXR as a novel metabolostatic signalling axis in skeletal muscle that acts to optimize substrate selection in response to nutrient status

LXR (liver X receptor) and PPARα (peroxisome-proliferator-activated receptor α) are nuclear receptors that control the expression of genes involved in glucose and lipid homoeostasis. Using wild-type and PPARα-null mice fed on an LXR-agonist-supplemented diet, the present study analysed the impact of pharmacological LXR activation on the expression of metabolically important genes in skeletal muscle, testing the hypothesis that LXR activation can modulate PPAR action in skeletal muscle in a manner dependent on nutritional status. In the fed state, LXR activation promoted a gene profile favouring lipid storage and glucose oxidation, increasing SCD1 (stearoyl-CoA desaturase 1) expression and down-regulating PGC-1α (PPARγ co-activator-1α) and PDK4 (pyruvate dehydrogenase kinase 4) expression. PPARα deficiency enhanced LXR stimulation of SCD1 expression, and facilitated elevated SREBP-1 (sterol-regulatory-element-binding protein-1) expression. However, LXR-mediated down-regulation of PGC-1α and PDK4 was opposed and reversed by PPARα deficiency. During fasting, prior LXR activation augmented PPARα signalling to heighten FA (fatty acid) oxidation and decrease glucose oxidation by augmenting fasting-induced up-regulation of PGC-1α and PDK4 expression, effects opposed by PPARα deficiency. Starvation-induced down-regulation of SCD1 expression was opposed by antecedent LXR activation in wild-type mice, an effect enhanced further by PPARα deficiency, which may elicit increased channelling of FA into triacylglycerol to limit lipotoxicity. Our results also identified potential regulatory links between the protein deacetylases SIRT1 (sirtuin 1) and SIRT3 and PDK4 expression in muscle from fasted mice, with a requirement for PPARα. In summary, we therefore propose that a LXR-PPARα signalling axis acts as a metabolostatic regulatory mechanism to optimize substrate selection and disposition in skeletal muscle according to metabolic requirement.     

Lycopene inhibits the proliferation of androgen-dependent human prostate tumor cells through activation of PPARγ-LXRα-ABCA1 pathway

The activation of nuclear receptors, peroxisome proliferator-activated receptor gamma (PPARγ) and liver X receptor alpha (LXRα), has been shown to inhibit the growth of prostate cancer cells. This study examined whether the anti-proliferative effect of lycopene on androgen-dependent human prostate cancer (LNCaP) cells involves the up-regulation of the expression of PPARγ and LXRα. As expected, lycopene treatment (2.5-10 μM) significantly inhibited the proliferation of LNCaP cells during incubation for 96 h. Lycopene significantly increased the protein and mRNA expression of PPARγ and LXRα at 24 and 48 h, while the increased in the expression of ATP-binding cassette transporter 1 (ABCA1) was only evident 96 h. In addition, lycopene significantly decreased cellular total cholesterol levels and increased apoA1 protein expression at 96 h. Incubation of LNCaP cells with lycopene (10 μM) in the presence (20 μM) of a specific antagonist of PPARγ (GW9662) and LXRα (GGPP) restored the proliferation of LNCaP cells to the control levels and significantly suppressed protein expression of PPARγ and LXRα as well as increased cellular total cholesterol levels. LXRα knockdown by siRNA against LXRα significantly enhanced the proliferation of LNCaP cells, whereas si-LXRα knockdown followed by incubation with lycopene (10 μM) restored the proliferation to the control level. The present study is the first to demonstrate that the anti-proliferative effect of lycopene on LNCaP cells involves the activation of the PPARγ-LXRα-ABCA1 pathway, leading to reduced cellular total cholesterol levels.     

Adiponectin prevents atherosclerosis by increasing cholesterol efflux from macrophages

Plasma high density lipoprotein (HDL)-cholesterol levels are inversely correlated to the risk of atherosclerotic cardiovascular diseases. Reverse cholesterol transport (RCT) is one of the major protective systems against atherosclerosis, in which HDL particles play a crucial role to carry cholesterol derived from peripheral tissues to the liver. Recently, ATP-binding cassette transporters (ABCA1, ABCG1) and scavenger receptor (SR-BI) have been identified as important membrane receptors to generate HDL by removing cholesterol from foam cells. Adiponectin (APN) secreted from adipocytes is one of the important molecules to inhibit the development of atherosclerosis. Epidemiological studies have revealed a positive correlation between plasma HDL-cholesterol and APN concentrations in humans, although its mechanism has not been clarified. Therefore, in the present study, we investigated the role of APN on RCT, in particular, cellular cholesterol efflux from human monocyte-derived and APN-knockout (APN-KO) mice macrophages. APN up-regulated the expression of ABCA1 in human macrophages, respectively. ApoA-1-mediated cholesterol efflux from macrophages was also increased by APN treatment. Furthermore, the mRNA expression of LXRα and PPARγ was increased by APN. In APN-KO mice, the expression of ABCA1, LXRα, PPARγ, and apoA-I-mediated cholesterol efflux was decreased compared with wild-type mice. In summary, APN might protect against atherosclerosis by increasing apoA-I-mediated cholesterol efflux from macrophages through ABCA1-dependent pathway by the activation of LXRα and PPARγ.     

22(R)-hydroxycholesterol and pioglitazone synergistically decrease cholesterol ester via the PPARγ–LXRα–ABCA1 pathway in cholesterosis of the gallbladder

Cholesterosis is a disease of cholesterol metabolism characterized by the presence of excessive lipid droplets in the cytoplasm. These lipid droplets are mainly composed of cholesterol esters derived from free cholesterol. The removal of excess cholesterol from gallbladder epithelial cells (GBECs) is very important for the maintenance of intracellular cholesterol homeostasis and the preservation of gallbladder function. Several lines of evidence have indicated that the activation of either peroxisome proliferator-activated receptor gamma (PPARγ) or liver X receptor α (LXRα) relates to cholesterol efflux. While pioglitazone can regulate the activation of PPARγ, 22(R)-hydroxycholesterol can activate LXRα and is a metabolic intermediate in the biosynthesis of steroid hormones. However, the effect of 22(R)-hydroxycholesterol in combination with pioglitazone on cholesterosis of the gallbladder is unclear. GBECs were treated with pioglitazone, 22(R)-hydroxycholesterol or PPARγ siRNA followed by Western blot analysis for ATP-binding cassette transporter A1 (ABCA1), PPARγ and LXRα. Cholesterol efflux to apoA-I was determined, and Oil Red O staining was performed to monitor variations in lipid levels in treated GBECs. Our data showed that 22(R)-hydroxycholesterol can modestly up-regulate LXRα while simultaneously increasing ABCA1 by 56%. The combination of 22(R)-hydroxycholesterol and pioglitazone resulted in a 3.64-fold increase in ABCA1 expression and a high rate of cholesterol efflux. Oil Red O staining showed an obvious reduction in the lipid droplets associated with cholesterosis in GBECs. In conclusion, the present findings indicate that the anti-lipid deposition action of 22(R)-hydroxycholesterol combined with pioglitazone involves the activation of the PPARγ–LXRα–ABCA1 pathway, increased ABCA1 expression and the efflux of cholesterol from GBECs. Thus, 22(R)-hydroxycholesterol synergistically combined with pioglitazone to produce a remarkable effect on lipid deposition in cholesterosis GBECs.     

The atypical interaction of peroxisome proliferator-activated receptor α with liver X receptor α antagonizes the stimulatory effect of their respective ligands on the murine cholesterol 7α-hydroxylase gene promoter

Cholesterol 7α-hydroxylase (cyp7a) mediates cholesterol elimination in the liver by catalyzing the first and rate-limiting step in the conversion of cholesterol into bile acids. Peroxisome proliferator-activated receptor α (PPARα; NR1C1) and liver X receptor α (LXRα; NR1H3) are two nuclear receptors that stimulate the murine Cyp7a1 gene. Here we report that co-expression of PPARα and LXRα in hepatoma cells abolishes the stimulation of Cyp7a1 gene promoter in response to their respective agonists. PPARα and LXRα form an atypical heterodimer that binds to two directly adjacent hexameric sequences localized within overlapping PPARα and LXRα response elements (termed Site I), antagonizing the interaction of PPARα:retinoid X receptor α (RXRα) or RXRα:LXRα with the Cyp7a1 gene promoter. Mutations within either hexameric sequences that specifically abolished LXRα:PPARα heterodimer binding to the murine Cyp7a1 Site I also relieved promoter inhibition. The LXRα:PPARα heterodimer may be important in coordinating the expression of genes that encode proteins involved in metabolism of fats and cholesterol.