PPARγ signal transduction pathway in the foam cell formation induced by visfatin

The aim of the present study was to investigate the role of peroxisome proliferator-activated receptor γ (PPARγ) signal transduction pathway in the expression of ATP binding cassette transporter A1 (ABCA1) and acyl-CoA:cholesterol acyltransferase 1 (ACAT1) induced by visfatin and to discuss the mechanism of foam cell formation induced by visfatin. THP-1 monocytes were induced into macrophages by 160 nmol/L phorbol myristate acetate (PMA) for 48 h, and then the macrophages were exposed to visfatin and PPARγ activator rosiglitazone, respectively. The expressions of PPARγ, ABCA1 and ACAT1 mRNA and protein were determined by RT-PCR and Western blot respectively. The contents of total cholesterol (TC) and free cholesterol (FC) were detected by enzyme fluorescence analysis. The content of cholesterol ester (CE) was calculated by the difference between TC and FC. The results showed that visfatin decreased the mRNA and protein expressions of PPARγ and ABCA1, increased the mRNA and protein expressions of ACAT1, and increased the contents of FC and CE in a concentration-dependent manner. These above effects of visfatin were inhibited by rosiglitazone in a concentration-dependent manner. These results suggest that visfatin may down-regulate the ABCA1 expression and up-regulate the ACAT1 expression via PPARγ signal transduction pathway, which decreases the outflow of FC, increases the content of CE, and then induces foam cell formation.     

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.     

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.     

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.     

Lycopene and the LXRα agonist T0901317 synergistically inhibit the proliferation of androgen-independent prostate cancer cells via the PPARγ-LXRα-ABCA1 pathway

In our previous study, we demonstrated that lycopene can inhibit the proliferation of androgen-dependent prostate LNCaP cancer cells through the activation of the peroxisome proliferator-activated receptor gamma (PPARγ)-liver X receptor alpha (LXRα)-ATP-binding cassette transporter 1 (ABCA1) pathway. However, it is still unclear whether lycopene possesses similar effects in androgen-independent prostate cancer cells DU145 and PC-3. As lycopene inhibited the proliferation of both cell types to a similar extent, we chose DU145 cells for most of the subsequent studies. We show that lycopene significantly increased protein and mRNA expression of PPARγ, LXRα and ABCA1 and cholesterol efflux (i.e., decreased cellular cholesterol and increased cholesterol in culture medium). Lycopene (10 μM) in the presence of a specific antagonist of PPARγ (GW9662) or of LXRα (GGPP) restored the proliferation of DU145 cells and significantly suppressed lycopene-induced protein and mRNA expression of PPARγ and LXRα and cholesterol efflux. Liver X receptor α knockdown by siRNA against LXRα significantly promoted the proliferation of DU145 cells, whereas si-LXRα knockdown followed by incubation with lycopene (10 μM) restored the proliferation to the control level. Furthermore, lycopene in combination with the LXRα agonist T0901317 exhibited synergistic effects on cell proliferation and protein expression of PPARγ, LXRα and ABCA1. These results demonstrate that lycopene can inhibit DU145 cell proliferation via PPARγ-LXRα-ABCA1 pathway and that lycopene and T0901317 exhibit synergistic effects.     

25-Hydroxycholesterol-3-sulfate attenuates inflammatory response via PPARγ signaling in human THP-1 macrophages

The nuclear receptor peroxisome proliferator-activated receptors (PPARs) are important in regulating lipid metabolism and inflammatory responses in macrophages. Activation of PPARγ represses key inflammatory response gene expressions. Recently, we identified a new cholesterol metabolite, 25-hydroxycholesterol-3-sulfate (25HC3S), as a potent regulatory molecule of lipid metabolism. In this paper, we report the effect of 25HC3S and its precursor 25-hydroxycholesterol (25HC) on PPARγ activity and on inflammatory responses. Addition of 25HC3S to human macrophages markedly increased nuclear PPARγ and cytosol IκB and decreased nuclear NF-κB protein levels. PPARγ response element reporter gene assays showed that 25HC3S significantly increased luciferase activities. PPARγ competitor assay showed that the K(i) for 25HC3S was ～1 μM, similar to those of other known natural ligands. NF-κB-dependent promoter reporter gene assays showed that 25HC3S suppressed TNFα-induced luciferase activities only when cotransfected with pcDNAI-PPARγ plasmid. In addition, 25HC3S decreased LPS-induced expression and release of IL-1β. In the PPARγ-specific siRNA transfected macrophages or in the presence of PPARγ-specific antagonist, 25HC3S failed to increase IκB and to suppress TNFα and IL-1β expression. In contrast to 25HC3S, its precursor 25HC, a known liver X receptor ligand, decreased nuclear PPARγ and cytosol IκB and increased nuclear NF-κB protein levels. We conclude that 25HC3S acts in macrophages as a PPARγ ligand and suppresses inflammatory responses via the PPARγ/IκB/NF-κB signaling pathway.     

oxHDL decreases the expression of CD36 on human macrophages through PPARγ and p38 MAP kinase dependent mechanisms

CD36, belongs to class B scavenger receptor family, is a macrophage receptor for oxidized low-density lipoprotein (oxLDL) and has been proven to play a critical role in atherosclerotic foam cell formation. In addition, CD36 expression is regulated by many factors including oxLDL and HDL. A recent study suggests that CD36 can also bind with oxidized high-density lipoprotein (oxHDL). However, the direct role of oxHDL in atherosclerosis is still not clear and it is not known whether oxHDL has any influence on the expression of CD36 in macrophages. Here, we performed experiments to investigate the effect of oxHDL on the expression of CD36 on human peripheral blood monocytes–macrophages and the possible mechanisms. Our results suggest that the uptake of oxHDL by CD36 on macrophages accelerates foam cell formation. In addition, oxHDL can down-regulate both the mRNA and surface protein expression of CD36 on human peripheral macrophages in vitro. oxHDL increased the mRNA expression and protein phosphorylation of peroxisome proliferators-activated receptor-γ (PPARγ). Using different mitogen-activated protein kinase (MAPK) inhibitors, we demonstrated that oxHDL regulated CD36 and PPARγ expression in a p38-MAP kinase dependent mechanism.     

PPARγ stabilizes HO-1 mRNA in monocytes/macrophages which affects IFN-β expression

NADPH oxidase activation in either RAW264.7 cells or peritoneal macrophages (PM) derived from PPARγ wild-type mice increased reactive oxygen species (ROS) formation, caused PPARγ activation, heme oxygenase-1 (HO-1) induction, and concomitant IFN-β expression. In macrophages transduced with a dominant negative (d/n) mutant of PPARγ (RAW264.7 AF2) as well as PPARγ negative PM derived from Mac-PPARγ-KO mice, NADPH oxidase-dependent IFN-β expression was attenuated. As the underlying mechanism, we noted decreased HO-1 mRNA stability in RAW264.7 AF2 cells as well as PPARγ negative PM, compared to either parent RAW264.7 cells or wild-type PM. Assuming mRNA stabilization of HO-1 by PPARγ we transfected macrophages with a HO-1 3′-UTR reporter construct. The PPARγ agonist rosiglitazone significantly up-regulated luciferase expression in RAW264.7 cells, while it remained unaltered in RAW264.7 AF2 macrophages. Deletion of each of two AU-rich elements in the 3′-UTR HO-1 decreased luciferase activity in RAW264.7 cells. Using LPS as a NADPH oxidase activator, PM from Mac-PPARγ-KO mice showed a decreased HO-1 mRNA half-life in vitro and in vivo compared to PPARγ wild-type mice. These data identified a so far unappreciated role of PPARγ in stabilizing HO-1 mRNA, thus, contributing to the expression of the HO-1 target gene IFN-β.     

Regulation of peroxisome proliferator-activated receptor gamma on milk fat synthesis in dairy cow mammary epithelial cells

Peroxisome proliferator-activated receptor gamma (PPARγ) participates in lipogenesis in rats, goats, and humans. However, the exact mechanism of PPARγ regulation on milk fat synthesis in dairy cow mammary epithelial cells (DCMECs) remains largely unexplored. The aim of this study was to investigate the role of PPARγ regarding milk fat synthesis in DCMECs and to ascertain whether milk fat precursor acetic acid and palmitic acid could interact with PPARγ signaling to regulate milk fat synthesis. For this study, we examined the effects of PPARγ overexpression and gene silencing on cell growth, triacylglycerol synthesis, and the messenger RNA (mRNA) and protein expression levels of genes involved in milk fat synthesis in DCMECs. In addition, we investigated the influences of acetic acid and palmitic acid on the mRNA and protein levels of milk lipogenic genes and triacylglycerol synthesis in DCMECs transfected with PPARγ small interfering RNA (siRNA) and PPARγ expression vector. The results showed that when PPARγ was silenced, cell viability, proliferation, and triacylglycerol secretion were obviously reduced. Gene silencing of PPARγ significantly downregulated the expression levels of milk fat synthesis-related genes in DCMECs. PPARγ overexpression improved cell viability, proliferation, and triacylglycerol secretion. The expression levels of milk lipogenic genes were significantly increased when PPARγ was overexpressed. Acetic acid and palmitic acid could markedly improve triacylglycerol synthesis and upregulate the expression levels of PPARγ and other lipogenic genes in DCMECs. These results suggest that PPARγ is a positive regulator of milk fat synthesis in DCMECs and that acetic acid and palmitic acid could partly regulate milk fat synthesis in DCMECs via PPARγ signaling.     

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.     

Peroxisome proliferator-activated receptor gamma is required for CD4+ T cell-mediated lymphopenia-associated autoimmunity

The nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) was shown to play an immunoregulatory role in many immune-related cell types, and activation of PPARγ was reported to be an effective therapeutic approach in murine and human autoimmune disease. However, despite an association between lymphopenia and autoimmunity, there has been no study on the role of T cell PPARγ in lymphopenia-associated autoimmunity. In the present studies, we examined the role of PPARγ in CD4(+) T cells in two murine models of lymphopenia-associated autoimmunity. Surprisingly, we found that PPARγ expression in CD4(+) CD25(-) T cells (T effector cells [Teffs]) is actually required for development of autoimmunity under lymphopenic conditions. Mechanistically, the inability of PPARγ-deficient (T-PPAR) Teffs to mediate lymphopenic autoimmunity is associated with a significant decrease in accumulation of Teffs in the spleen, lymph nodes, and tissues after adoptive transfer. This abnormal accumulation of T-PPAR Teffs was associated with defects in both in vivo proliferation and survival. Additionally, T-PPAR Teffs demonstrated decreased cytokine production in inflammatory sites and decreased expression of the homing receptor α4β7. Finally, these abnormalities in T-PPAR Teff function were not elicited by lymphopenia alone but also required the additional activation involved in the mediation of autoimmunity. Thus, in contrast to its documented immunosuppressive role, we identified an unexpected function for PPARγ in Teffs: a role in Teff proliferation and survival in lymphopenia-associated autoimmunity. These findings highlight both the multifunctional role of PPARγ in T cells and the complexity of PPARγ as a potential therapeutic target in autoimmunity.