Sortilin调控ABCA1蛋白水平影响荷脂巨噬细胞内脂质流出

目的　探讨Sortilin对荷脂THP-1巨噬细胞ABCA1蛋白表达及胞内脂质流出的影响。 方法　采用Western blot和qRT-PCR检测细胞内ABCA1蛋白和mRNA表达水平;Co-IP实验检测Sortilin和ABCA1的结合情况;高效液相色谱法检测细胞内总胆固醇、游离胆固醇、胆固醇酯含量变化;油红O染色观察细胞内脂滴情况。 结果　Sortilin下调细胞内ABCA1蛋白水平,但对其mRNA水平无显著影响;Co-IP结果表明Sortilin能与ABCA1结合;与对照组相比,Sortilin过表达后荷脂巨噬细胞内胆固醇流出减少,胞内脂质含量增加且脂滴肥大,数量明显增多,Sortilin沉默后荷脂巨噬细胞胆固醇流出增加,胞内脂质含量减少,脂滴瘦小,数量减少;溶酶体抑制剂氯喹共处理可部分逆转Sortilin过表达对巨噬细胞ABCA1蛋白和胆固醇流出的抑制作用。 结论　Sortilin下调巨噬细胞ABCA1蛋白水平,抑制胆固醇流出,促进胞内脂质蓄积。     

Sortilin调控ABCA1蛋白水平影响荷脂巨噬细胞内脂质流出

目的　探讨Sortilin对荷脂THP-1巨噬细胞ABCA1蛋白表达及胞内脂质流出的影响。 方法　采用Western blot和qRT-PCR检测细胞内ABCA1蛋白和mRNA表达水平;Co-IP实验检测Sortilin和ABCA1的结合情况;高效液相色谱法检测细胞内总胆固醇、游离胆固醇、胆固醇酯含量变化;油红O染色观察细胞内脂滴情况。 结果　Sortilin下调细胞内ABCA1蛋白水平,但对其mRNA水平无显著影响;Co-IP结果表明Sortilin能与ABCA1结合;与对照组相比,Sortilin过表达后荷脂巨噬细胞内胆固醇流出减少,胞内脂质含量增加且脂滴肥大,数量明显增多,Sortilin沉默后荷脂巨噬细胞胆固醇流出增加,胞内脂质含量减少,脂滴瘦小,数量减少;溶酶体抑制剂氯喹共处理可部分逆转Sortilin过表达对巨噬细胞ABCA1蛋白和胆固醇流出的抑制作用。 结论　Sortilin下调巨噬细胞ABCA1蛋白水平,抑制胆固醇流出,促进胞内脂质蓄积。     

载脂蛋白E在ABCA1和ABCG1介导的胆固醇流出中的作用

目的:探讨载脂蛋白E(ApoE)在ATP结合盒转运蛋白A1(ABCA1)和ATP结合盒转运蛋白G1(ABCG1)介导的胆固醇流出中的作用及可能机制。方法:将RAW264.7细胞随机分为3组:空白对照组:用单纯培养基培养;低密度脂蛋白受体基因敲除(LDLr^-/-)组:用20mg/LLDLr^-/-小鼠脂蛋白处理细胞;ApoE基因敲除(ApoE^-/-)组:用20mg/LApoE^-/-小鼠脂蛋白处理细胞。采用透射电镜和酶法检测细胞内脂质含量,用液闪仪技术检测胆固醇流出变化,用免疫印迹技术和实时定量PCR技术检测ABCA1和ABCG1的表达水平。结果:与对照组相比,LDLr^-/-小鼠脂蛋白处理后细胞内脂质含量并未明显改变,而ApoE^-/-小鼠脂蛋白处理48h后,细胞内胆固醇酯增加了60%;同时,与LDLr^-/-小鼠脂蛋白处理组相比,用ApoE^-/-小鼠脂蛋白处理后,巨噬细胞胆固醇流出至载脂蛋白A-I(ApoA-I)和高密度脂蛋白(HDL)的量均明显降低。与对照组相比,LDLr^-/-小鼠脂蛋白和ApoE^-/-小鼠脂蛋白处理显著增加ABCA1和ABCG1的蛋白和mRNA水平,但是ApoE^-/-小鼠脂蛋白对ABCA1和ABCG1蛋白和mRNA水平的诱导程度明显低于LDLr^-/-小鼠脂蛋白。结论:ApoE在介导巨噬细胞胆固醇流出中发挥重要作用,该作用与其调节ABCA1和ABCG1的表达水平有关。     

Effect of isolated isoflavone supplementation on ABCA1-dependent cholesterol efflux potential in postmenopausal women

OBJECTIVE: Isoflavones may display beneficial health effects in postmenopausal women. We studied in a clinical trial whether isolated isoflavone treatment in postmenopausal women could affect reverse cholesterol transport as evaluated by adenosine triphosphate-binding cassette A1- (ABCA1), dependent cholesterol efflux from macrophages. In addition, various serum lipid and lipoprotein parameters were investigated. Furthermore, we separately assessed equol-producing and non-equol-producing women. DESIGN: Postmenopausal women (n=56) were treated with either isoflavone or placebo tablets for 3 months in a crossover design, separated by a 2-month washout period. Fifteen women were classified as equol producers, and 15 women were classified as non-equol producers. Serum samples were collected before and after each treatment period. [H]-Cholesterol-labeled J774 macrophage cells, with and without ABCA1 up-regulation, were incubated with the samples, and ABCA1-dependent cholesterol efflux and serum lipid and lipoprotein levels were assessed. RESULTS: Serum promoted 3.1%+/-1.1% and 3.2%+/-1.1% cholesterol efflux from macrophages after isoflavone and placebo treatment, respectively. Thus, isoflavone supplementation did not affect ABCA1-dependent cholesterol efflux to serum. However, as a novel finding, isoflavone treatment increased a subclass of high-density lipoprotein, the pre-beta high-density lipoprotein levels by 18% without affecting any other serum lipid concentrations. ABCA1-facilitated cholesterol efflux and lipid parameters did not differ between equol-producing and non-equol-producing women. CONCLUSION: In postmenopausal women, isolated isoflavone treatment does not affect ABCA1-dependent cholesterol efflux potential from macrophages but increases circulating pre-beta high-density lipoprotein level, which could provide beneficial vascular effects.     

The role of vesicular transport in ABCA1-dependent lipid efflux and its connection with NPC pathways

The membrane transporter ATP-binding cassette transporter A1 (ABCA1) has been shown to be the rate-limiting step in the initial formation of plasma high-density lipoprotein (HDL) particles. The mechanisms of action of ABCA1, including its role in the vesicular transport of lipids to the cell surface for the lipidation of HDL apolipoproteins, are not fully understood. Niemann–Pick type C (NPC) disease is most often caused by mutations in the NPC1 gene, whose protein product is believed to facilitate the egress of cholesterol and other lipids from late endosomes and lysosomes to other cellular compartments. This report reviews current knowledge regarding the role of ABCA1 in vesicular lipid transport mechanisms required for HDL particle formation, and the relationship between ABCA1 and NPC1 in this process.     

Impairment of the ABCA1 and SR-BI-mediated cholesterol efflux pathways and HDL anti-inflammatory activity in Alzheimer's disease

The aim of our study was to investigate the effect of Alzheimer's disease (AD) on the cholesterol efflux capacity and anti-inflammatory activity of HDL. HDL and apoA-I were isolated from 20 healthy subjects and from 39 AD patients. Our results showed that serum- and HDL-mediated cholesterol efflux is significantly impaired in AD patients. This impairment of serum and HDL cholesterol efflux capacity was significantly inversely correlated to the AD severity as evaluated by MMSE scores. Results obtained from SR-BI-enriched Fu5AH and ABCA1-enriched J774 cells revealed that AD impaired the interaction of HDL and apoA-I with both the ABCA1 transporter and SR-BI receptor. Purified apoA-I from AD patients also failed to remove free excess cholesterol from ABCA1-enriched J774 macrophages. Interestingly, the decrease in plasma α-tocopherol content and the increase in MDA formation and HDL relative electrophoretic mobility indicated that AD patients had higher levels of oxidative stress. The anti-inflammatory activity of HDL was also significantly lower in AD patients as measured by the level of ICAM-1 expression. In conclusion, our study provides evidence for the first time that the functionality of HDL is impaired in AD and that this alteration might be caused by AD-associated oxidative stress and inflammation.     

CD45-mediated signals can trigger shedding of lymphocyte L-selectin

The adhesion molecule L-selectin is proteolytically cleaved from the surface of lymphocytes and neutrophils within minutes after stimulation by phorbol ester or calcium ionophores. In contrast to neutrophils, soluble factors have not been shown to induce down-regulation of L- selectin on lymphocytes. We therefore examined whether signals generated by interaction with cell surface receptors could deliver physiological stimuli inducing this regulatory mechanism. While cross- linking of several adhesion molecules (CD2, CD44, alpha 4-integrin, LFA- 1) by antibody did not result in a significant reduction of the expression of L-selectin, antibodies against CD45 and Thy-1.2, both involved in the regulation of lymphocyte activation, induced loss of cell surface L-selectin within minutes, even at 4 degrees C, by shedding into the supernatant. Cross-linking of these molecules was shown to be essential, but Fc interactions or adherent cells were not required. A similar response, albeit less effective, was found after cross-linking of CD3. Interestingly, initiation of shedding only occurred in the presence of cell-cell contact, pointing to a second, as yet unknown, signal required. Loss of L-selectin induced by CD45 cross- linking is followed by a rapid re-expression of the molecule upon incubation at 37 degrees C. This reaction is also dependent on specific triggering signals as rapid re-expression was not observed after removal of L-selectin by trypsin. The data indicate that the protein phosphatase CD45 as well as the TCR complex itself in combination with a further, as yet unknown, cell-cell contact-dependent stimulus have a regulatory role in the dynamic control of L-selectin expression in lymphocytes.      

ABCA3 missense mutations causing surfactant dysfunction disorders have distinct cellular phenotypes

Mutations in the ATP‐binding cassette subfamily A member 3 (ABCA3) gene are the most common monogenetic cause of surfactant dysfunction disorders in newborns and interstitial lung diseases in children and young adults. Although the effect of mutations resulting in truncated or incomplete proteins can be predicted, the consequences of missense variants cannot be as easily. Our aim was to investigate the intracellular handling and disturbance of the cellular surfactant system in a stable cell model with several different clinically relevant ABCA3 missense mutations. We found that the investigated missense mutations within the ABCA3 gene affect surfactant homeostasis in different ways: first by disrupting intracellular ABCA3 protein localization (c.643C > A, p.Q215K; c.2279T > G, p.M760R), second by impairing the lipid transport of ABCA3 protein (c.875A > T, p.E292V; c.4164G > C, p.K1388N), and third by yet undetermined mechanisms predisposing for the development of interstitial lung diseases despite correct localization and normal lipid transport of the variant ABCA3 protein (c.622C > T, p.R208W; c.863G > A, p.R288K; c.2891G > A, p.G964D). In conclusion, we classified cellular consequences of missense ABCA3 sequence variations leading to pulmonary disease of variable severity. The corresponding molecular pathomechanisms of such ABCA3 variants may specifically be addressed by targeted treatments.     

Effects of ouabain and furosemide on basolateral membrane Na efflux of frog skin

The kinetics of Na tracer fluxes were reinvestigated in isolated epithelia of frog skin in which the unstirred layers of the corium were removed. The rate of appearance of 22Na in the basolateral solution (JNa13) conformed to a single exponential for tracer buildup with a mean t1/2 of 1.9 min. The Na transport pool labeled isotopically from the apical solution was about 40 neq/cm2 for epithelia bathed in either a chloride or sulfate Ringer solution. Basolateral-to-apical solution unidirectional Na flux (JNa31) was low, averaging 0.7 microA/cm2 and remained low for 40 min of ouabain treatment of the epithelia. In agreement with electrophysiological data, ouabain at 10(-4) M caused an acute inhibition (less than 1 min) of the Na efflux at the basolateral membrane (JNa23), falling to 40.6 and 26.7% of control for epithelia bathed in Cl and SO4 Ringer, respectively. Although furosemide exerted little or no consistent effect on the Na flux of control epithelia, this drug in ouabain-poisoned epithelia caused a substantial inhibition of a neutral, chloride-dependent, ouabain-insensitive Na efflux. It is suggested that ouabain "induces" a neutral mechanism of Na transport at the basolateral membrane of the cells that is chloride dependent and furosemide sensitive.     

NF-κB在AngⅡ介导THP-1巨噬细胞源性泡沫细胞胆固醇流出及ABCA1表达调控过程中的作用研究

目的：探讨核因子NF-κB活化对AngⅡ诱导的THP-1巨噬细胞源性泡沫细胞ABCA1基因表达、胆固醇含量的影响。方法：体外培养THP-1细胞以构建泡沫细胞模型，以AngⅡ和NF-κB活化抑制剂TPCK孵育细胞；以RT-PCR法、Western blot法测定THP-1源泡沫细胞ABCA1 mRNA和蛋白表达状态；采用酶法，通过荧光分光光度计检测细胞内胆固醇含量，应用液体闪烁技术仪检测胆固醇流出的变化；借助Sandwich ELISA法测定核因子NF-κB活化／核易位情况。结果：AngⅡ可即刻激活NF-κB表达活性，并导致干预24小时后泡沫细胞ABCA1 mRNA和蛋白表达下调；若预先用TPCK孵育，则TPCK可抑制AngⅡ对NF-κB的激活，且24小时后泡沫细胞ABCA1 mRNA和蛋白表达下调幅度减小。结论：AngⅡ能引起THP-1源性泡沫细胞胆固醇含量显著增高（P〈0．05）、ABCA1表达显著减少（P〈0．05）。机制可能与AngⅡ即刻激活NF-κB信号途径，早期活化的NF-κB可阻遏THP-1源泡沫细胞ABCA1基因和蛋白的表达，继而影响泡沫细胞内胆固醇的流出有关。     

Potassium efflux fires the canon: Potassium efflux as a common trigger for canonical and noncanonical NLRP3 pathways

Murine caspase‐11 and its human orthologues, caspase‐4 and caspase‐5, activate an inflammatory response following cytoplasmic recognition of cell wall constituents from Gram‐negative bacteria, such as LPS. This inflammatory response involves pyroptotic cell death and the concomitant release of IL‐1α, as well as the production of IL‐1β and IL‐18 through the noncanonical NLR family, pyrin domain containing 3 (NLRP3) pathway. This commentary discusses three papers in this issue of the European Journal of Immunology that advance our understanding of the roles of caspase‐11, ‐4, and ‐5 in the noncanonical pathway. By utilizing the new gene editing technique, clustered regularly interspaced short palindromic repeats (CRISPR), as well as sensitive cell imaging techniques, these papers establish that cytoplasmic LPS‐dependent IL‐1β production requires the NLRP3 inflammasome and that its activation is dependent on K⁺ efflux, whereas IL‐1α release and pyroptotic cell death pathways are NLRP3‐independent. These findings expand on previous research implicating K⁺ efflux as the principal trigger for NLRP3 activation and suggest that canonical and noncanonical NLRP3 pathways are not as dissimilar as first thought.     

ABCA1 polymorphisms and Alzheimer's disease

In our search for genetic factors related to the development of Alzheimer's disease, we have genotyped 332 pedigrees for three coding polymorphisms in the ABCA1 gene, two of which are known to alter plasma cholesterol levels, as well as a non-coding polymorphism within the promoter. We show an apparent weak association of rs2230806 (p-value=0.01) with the disease in a sibpair series of Alzheimer's disease that had shown previously evidence for linkage to the chromosome 9 locus where ABCA1 maps.     

Titanium dioxide nanoparticles trigger p53-mediated damage response in peripheral blood lymphocytes

Titanium dioxide nanoparticles (nano-TiO2) are widely used as a photocatalyst in air and water remediation. These nanoparticles are known to induce toxicity; however, their cytotoxic mechanism is not fully understood. In this study, we investigated the underlying mechanism of nano-TiO2-induced cytotoxicity in peripheral blood lymphocytes. We examined the genotoxic effects of nano-TiO2 in lymphocytes using alkaline single-cell gel electrophoresis (Comet) and cytokinesis-block micronucleus (CBMN) assays. Lymphocytes treated with nano-TiO2 showed significantly increased micronucleus formation and DNA breakage. Western-blot analysis to identify proteins involved in the p53-mediated response to DNA damage revealed the accumulation of p53 and activation of DNA damage checkpoint kinases in nano-TiO2-treated lymphocytes. However, p21 and bax, downstream targets of p53, were not affected, indicating that nano-TiO2 does not stimulate transactivational activity of p53. The generation of reactive oxygen species (ROS) in nano-TiO2-treated cells was also observed, andN-acetylcysteine (NAC) supplementation inhibited the level of nano-TiO2-induced DNA damage. Given that ROS-induced DNA damage leads to p53 activation in the DNA damage response, our results suggest that nano-TiO2 induces ROS generation in lymphocytes, thereby activating p53-mediated DNA damage checkpoint signals.     

Liver X receptor and retinoic X receptor mediated ABCA1 regulation and cholesterol efflux in macrophage cells-messenger RNA measured by branched DNA technology

ABCA1 is an ATP binding cassette transporter that plays an essential role in cholesterol and phospholipid efflux and HDL biogenesis. ABCA1 expression in macrophage cells is subject to regulation by cAMP, cholesterol loading, and ligands of the nuclear receptors liver X receptor (LXR) and retinoid X receptor (RXR). We report here the development of a rapid and high volume branched DNA (bDNA) method to measure ABCA1 mRNA. By using the bDNA method, we show that both LXR and RXR ligands effectively regulate ABCA1 expression in three macrophage cell types: mouse RAW264.7 cell line, mouse peritoneal macrophage cells, and human macrophage THP-1 cells and their regulation is additive. Furthermore, by using a radiolabeled cholesterol efflux assay, we show that both LXR and RXR ligands are sufficient to mediate cholesterol efflux in macrophage cells and their efficacy correlates with ABCA1 regulation. These studies strengthen further the notion that LXR and RXR mediate ABCA1 expression and cholesterol efflux in macrophage cells as a permissive heterodimer and development of small molecule ligands of these nuclear receptors may represent a promising approach to modulating cholesterol efflux and plasma HDL cholesterol level in humans.     

ABCA1在脂质沉积中的研究进展

ATP结合盒转运蛋白A1(ABCA1)是一种以ATP为能源进行物质转运的膜蛋白,肝脏X受体(LXRs)、维甲酸X受体(RXRs)、固醇调节元结合蛋白(SREBPs)和microRNAs等因子对其表达有重要影响。ABCA1作用于脂质沉积的多个信号通路,上调其表达可负向调控脂质沉积过程,为代谢性疾病带来了新的治疗方向。     

ABCA1, from pathology to membrane function

The ABCA1 transporter is the prototype of the A class of mammalian adenosine triphosphate binding cassette transporters and one of the largest members of this family. ABCA1 has been originally identified as an engulfment receptor on macrophages and, more recently, it has been shown to play an essential role in the handling of cellular lipids. Indeed by promoting the effluxes of membrane phospholipids and cholesterol to lipid-poor apoprotein acceptors, ABCA1 controls the formation of high-density lipoproteins and thus the whole process of reverse cholesterol transport. A number of additional phenotypes have been found in the mouse model of invalidation of the ABCA1 gene. In spite of their clinical diversity, they all are extremely sensitive to variations in the physicochemical properties of the cell membrane, which ABCA1 controls as a lipid translocator.     

Endomorphins exit the brain by a saturable efflux system at the basolateral surface of cerebral endothelial cells

Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are two highly selective mu-opiate receptor agonists. We recently demonstrated that EM-1 and EM-2 have a saturable transport system from brain-to-blood in vivo. Since the endothelial cells are the main component of the non-fenestrated microvessels of the blood-brain barrier (BBB), we examined whether these endogenous tetrapeptides have a saturable transport system in cultured cerebral endothelial cells. EM-1 and EM-2 binding and transport were studied in a transwell system in which primary mouse endothelial cells were co-cultured with rat glioma cells. We found that binding of both endomorphins was greater on the basolateral than the apical cell surface. Flux of EM-1 and EM-2 occurred predominantly in the basolateral to apical direction, each showing self-inhibition with an excess of the respective endomorphin. Transport was not influenced by the addition of the P-glycoprotein inhibitor, cyclosporin A. Neither the mu-opiate receptor agonist DAMGO nor the delta-opiate receptor agonist DPDPE had any effect on the transport. Thus, the results show that a saturable transport system for EM-1 and EM-2 occurs at the level of endothelial cells of the BBB, and unlike ß-endorphin and morphine, P-glycoprotein is not needed for the brain-to-blood transport. Cross-inhibition of the transport of each endomorphin by the other suggests a shared transport system that is different from mu- or delta-opiate receptors. As endormorphins are mainly produced in the CNS, the presence of the efflux system at the BBB could play an important role in pain modulation and neuroendocrine control.     

氧化型胆固醇下调血管平滑肌细胞金属蛋白酶组织抑制剂基因表达

目的:研究氧化型胆固醇对血管平滑肌细胞MMP-9及TIMP-1表达的影响。方法:离体培养兔主动脉血管平滑肌细胞,分别用胆固醇、Triol与25-OH负载细胞,狭缝杂交测定TIMP-1mRNA表达,细胞免疫化学测定MMP-9与TIMP-1蛋白表达。结果:Triol与25-OH(1 mg/L,24 h)抑制TIMP-1 mRNA及蛋白表达,对MMP-9蛋白表达无影响。结论:氧化型胆固醇可以下调血管平滑肌细胞TIMP-1基因表达。