Apolipoprotein E4 Is Deficient in Inducing Macrophage ABCA1 Expression and Stimulating the Sp1 Signaling Pathway

ATP binding cassette A1 (ABCA1) is a membrane protein that promotes cellular cholesterol efflux. Using RAW 264.7 macrophages, we studied the relative effects of apolipoprotein (apo) E3 and apoE4 on ABCA1 and on the signaling pathway that regulates its expression. Both lipid-associated and lipid-free apoE4 forms induced ∼30% lower levels of ABCA1 protein and mRNA than apoE3 forms. Phosphorylated levels of phosphoinositol 3-kinase (PI3K), protein kinase Cζ (PKCζ) and specificity protein 1 (Sp1) were also lower when treated with apoE4 compared to apoE3. The reduced ability of apoE4 to induce ABCA1 expression, PKCζ and Sp1 phosphorylation were confirmed in human THP-1 monocytes/macrophages. Sequential phosphorylation of PI3K, PKCζ and Sp1 has been suggested as a mechanism for upregulation of ABCA1 expression. Both apoE3 and apoE4 reduced total cholesterol and cholesterol esters in lipid-laden RAW 264.7 cells, and induced apoAI-mediated cholesterol efflux. However, the cholesterol esters and cholesterol efflux in apoE4-treated cells were ∼50% and ∼24% lower, respectively, compared to apoE3-treated cells. Accumulation of cholesterol esters in macrophages is a mechanism for foam cell formation. Thus the reduced ability of apoE4 to activate the PI3K-PKCζ-Sp1 signaling pathway and induce ABCA1 expression likely impairs cholesterol ester removal, and increases foam cell formation.     

ATF6调节ABCA1基因的表达

ATP结合盒式运载蛋白A1(ATP-binding cassette transporter A1,ABCA1)是近年来发现的极其重要的脂质转运大分子膜蛋白,它可将过量胆固醇从细胞内向细胞外输送到载脂蛋白并包装成高密度脂蛋白(HDL)的膜蛋白,促进胆固醇的逆转运.初步研究转录因子ATF6对ABCA1的表达调控,结果发现,ATF6在人胚胎肾细胞HEK293内剂量依赖性地调节ABCA1基因转录及蛋白质表达. ATF6调节ABCA1与内质网应激信号通路无关. 启动子序列缺失与突变分析表明ATF6作用区位于ABCA1启动子上游-156～-928bp之间, 可能需要E-box的参与,但不需要DR4元件.进而,动物试验结果显示用腺病毒在C57小鼠肝脏过表达ATF6,在mRNA水平上调ABCA1. 本文的研究发现了ATF6新的功能以及调控ABCA1的新机制.     

The citrus flavonone naringenin reduces lipopolysaccharide-induced inflammatory pain and leukocyte recruitment by inhibiting NF-κB activation

Lipopolysaccharide (LPS) is the major structural component of Gram-negative bacteria cell wall and a highly pro-inflammatory toxin. Naringenin is found in Citrus fruits and exhibits antioxidant and anti-inflammatory properties through inhibition of NF-κB activation but its effects in LPS-induced inflammatory pain and leukocyte recruitment were not investigated yet. We investigated the effects of naringenin in mechanical hyperalgesia, thermal hyperalgesia and leukocyte recruitment induced by intraplantar injection of LPS in mice. We found that naringenin reduced hyperalgesia to mechanical and thermal stimuli, myeloperoxidase (MPO, a neutrophil and macrophage marker) and N-acetyl-β-D-glucosaminidase (NAG, a macrophage marker) activities, oxidative stress and cytokine (TNF-α, IL-1β, IL-6, and IL-12) production in the paw skin. In the peritoneal cavity, naringenin reduced neutrophil and mononuclear cell recruitment, and abrogated MPO and NAG activity, cytokine and superoxide anion production, and lipid peroxidation. In vitro, pre-treatment with naringenin inhibited superoxide anion and cytokine (TNF-α, IL-1β, IL-6, and IL-12) production by LPS-stimulated RAW 264.7 macrophages. Finally, we demonstrated that naringenin inhibited NF-κB activation in vitro and in vivo. Therefore, naringenin is a promising compound to treat LPS-induced inflammatory pain and leukocyte recruitment.     

HIV-1 Nef mobilizes lipid rafts in macrophages through a pathway that competes with ABCA1-dependent cholesterol efflux

HIV infection, through the actions of viral accessory protein Nef, impairs activity of cholesterol transporter ABCA1, inhibiting cholesterol efflux from macrophages and elevating the risk of atherosclerosis. Nef also induces lipid raft formation. In this study, we demonstrate that these activities are tightly linked and affect macrophage function and HIV replication. Nef stimulated lipid raft formation in macrophage cell line RAW 264.7, and lipid rafts were also mobilized in HIV-1-infected human monocyte-derived macrophages. Nef-mediated transfer of cholesterol to lipid rafts competed with the ABCA1-dependent pathway of cholesterol efflux, and pharmacological inhibition of ABCA1 functionality or suppression of ABCA1 expression by RNAi increased Nef-dependent delivery of cholesterol to lipid rafts. Nef reduced cell-surface accessibility of ABCA1 and induced ABCA1 catabolism via the lysosomal pathway. Despite increasing the abundance of lipid rafts, expression of Nef impaired phagocytic functions of macrophages. The infectivity of the virus produced in natural target cells of HIV-1 negatively correlated with the level of ABCA1. These findings demonstrate that Nef-dependent inhibition of ABCA1 is an essential component of the viral replication strategy and underscore the role of ABCA1 as an innate anti-HIV factor.     

ABCA1 mediates concurrent cholesterol and phospholipid efflux to apolipoprotein A-I

Prior studies provide data supporting the notion that ATP binding cassette transporter A1 (ABCA1) promotes lipid efflux to extracellular acceptors in a two-step process: first, ABCA1 mediates phospholipid efflux to an apolipoprotein, and second, this apolipoprotein-phospholipid complex accepts free cholesterol in an ABCA1-independent manner. In the current study using RAW264.7 cells, ABCA1-mediated free cholesterol and phospholipid efflux to apolipoprotein A-I (apoA-I) were tightly coupled to each other both temporally and after treatment with ABCA1 inhibitors. The time course and temperature dependence of ABCA1-mediated lipid efflux to apoA-I support a role for endocytosis in this process. Cyclodextrin treatment of RAW264.7 cells partially inhibited 8Br-cAMP-induced efflux of free cholesterol and phospholipid to apoA-I. ABCA1-expressing cells are more sensitive to cell damage by high-dose cyclodextrin and vanadate, leading to increased lactate dehydrogenase leakage and phospholipid release even in the absence of the acceptor apoA-I. Finally, we could not reproduce a two-step effect on lipid efflux using conditioned medium from ABCA1-expressing cells pretreated with cyclodextrin.     

Kaempferol Inhibits Endoplasmic Reticulum Stress-Associated Mucus Hypersecretion in Airway Epithelial Cells And Ovalbumin-Sensitized Mice

Mucus hypersecretion is an important pathological feature of chronic airway diseases, such as asthma and pulmonary diseases. MUC5AC is a major component of the mucus matrix forming family of mucins in the airways. The initiation of endoplasmic reticulum (ER)-mediated stress responses contributes to the pathogenesis of airway diseases. The present study investigated that ER stress was responsible for airway mucus production and this effect was blocked by the flavonoid kaempferol. Oral administration of ≥10 mg/kg kaempferol suppressed mucus secretion and goblet cell hyperplasia observed in the bronchial airway and lung of BALB/c mice sensitized with ovalbumin (OVA). TGF-β and tunicamycin promoted MUC5AC induction after 72 h in human bronchial airway epithelial BEAS-2B cells, which was dampened by 20 μM kaempferol. Kaempferol inhibited tunicamycin-induced ER stress of airway epithelial cells through disturbing the activation of the ER transmembrane sensor ATF6 and IRE1α. Additionally, this compound demoted the induction of ER chaperones such as GRP78 and HSP70 and the splicing of XBP-1 mRNA by tunicamycin. The in vivo study further revealed that kaempferol attenuated the induction of XBP-1 and IRE1α in epithelial tissues of OVA-challenged mice. TGF-β and tunicamycin induced TRAF2 with JNK activation and such induction was deterred by kaempferol. The inhibition of JNK activation encumbered the XBP-1 mRNA splicing and MUC5AC induction by tunicamycin and TGF-β. These results demonstrate that kaempferol alleviated asthmatic mucus hypersecretion through blocking bronchial epithelial ER stress via the inhibition of IRE1α-TRAF2-JNK activation. Therefore, kaempferol may be a potential therapeutic agent targeting mucus hypersecretion-associated pulmonary diseases.     

Endoplasmic reticulum stress impairs cholesterol efflux and synthesis in hepatic cells

Metabolic disorders such as type 2 diabetes cause hepatic endoplasmic reticulum (ER) stress, which affects neutral lipid metabolism. However, the role of ER stress in cholesterol metabolism is incompletely understood. Here, we show that induction of acute ER stress in human hepatic HepG2 cells reduced ABCA1 expression and caused ABCA1 redistribution to tubular perinuclear compartments. Consequently, cholesterol efflux to apoA-I, a key step in nascent HDL formation, was diminished by 80%. Besides ABCA1, endogenous apoA-I expression was reduced upon ER stress induction, which contributed to reduced cholesterol efflux. Liver X receptor, a key regulator of ABCA1 in peripheral cells, was not involved in this process. Despite reduced cholesterol efflux, cellular cholesterol levels remained unchanged during ER stress. This was due to impaired de novo cholesterol synthesis by reduction of HMG-CoA reductase activity by 70%, although sterol response element-binding protein-2 activity was induced. In mice, ER stress induction led to a marked reduction of hepatic ABCA1 expression. However, HDL cholesterol levels were unaltered, presumably because of scavenger receptor class B, type I downregulation under ER stress. Taken together, our data suggest that ER stress in metabolic disorders reduces HDL biogenesis due to impaired hepatic ABCA1 function.     

Overexpression of stearoyl-coenzyme A desaturase 1 in macrophages promotes reverse cholesterol transport

Stearoyl-coenzyme A desaturase 1 (SCD1) is the rate-limiting enzyme in the synthesis of monounsaturated fatty acids. However, the impact of SCD1 on atherosclerosis remains unclear. The aim of this study was to determine whether SCD1 affects macrophage reverse cholesterol transport (RCT) in mice. Compared to the control, adenoviral-mediated SCD1 overexpression in RAW264.7 macrophages increased cholesterol efflux to HDL, but not to apoA-I, without clear changes in ABCA1, ABCG1 and SR-BI expressions. While knockdown of ABCG1 and SR-BI did not affect the SCD1-induced cholesterol efflux to HDL, SCD1-overexpressing macrophages promoted the formation of both normal- and large-sized HDL in media, accompanying increased apolipoprotein A-I levels in HDL fractions. Transformation to larger particles of HDL was independently confirmed by nuclear magnetic resonance-based lipoprotein analysis. Interestingly, media transfer assays revealed that HDL generated by SCD1 had enhanced cholesterol efflux potential, indicating that SCD1 transformed HDL to a more anti-atherogenic phenotype. To study macrophage RCT in vivo, ³H-cholesterol-labeled RAW264.7 cells overexpressing SCD1 or the control were intraperitoneally injected into mice. Supporting the in vitro data, injection of SCD1-macrophages resulted in significant increases in ³H-tracer in plasma, liver, and feces compared to the control. Moreover, there was a shift towards larger particles in the ³H-tracer distribution of HDL fractions obtained from the mice.     

Nicotine suppresses tunicamycin-induced, but not thapsigargin-induced, expression of GRP78 during ER stress-mediated apoptosis in PC12 cells

We previously reported that nicotine protected against tunicamycin (Tm)-induced ER stress-mediated apoptosis, but not thapsigargin (Tg)-induced apoptosis in PC12 cells. In the present study, we report that the expression of glucose-regulated protein 78 (GRP78) was suppressed by nicotine in Tm-treated PC12 cells. Interestingly, the GRP78 expression was not changed by nicotine in Tg-treated cells. Moreover, nicotine reduced the activation of caspase-12 in Tm-treated cells, but not in Tg-treated cells. These results suggest that nicotine prevented Tm-induced ER stress-mediated apoptosis by attenuating an early stage of Tm-induced ER stress. It was possible that the suppression of GRP78 expression by nicotine was achieved through the suppression of the Ire1-XBP1 and/or ATF6 pathways. We observed that nicotine suppressed the Tm-induced, but not Tg-induced, splicing of XBP1 mRNA, and also suppressed the Tm-induced, but not Tg-induced, production of cleaved ATF6 in PC12 cells. These results indicate that the suppression of Ire1-XBP1 and ATF6 pathways contributes to the suppression of GRP78 expression by nicotine in Tm-treated PC12 cells, suggesting that nicotine suppresses a common step upstream of both the Ire1-XBP1 and ATF6 pathways which are required for the expression of GRP78 during Tm-induced ER stress.