Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol

We previously showed that macrophages from macrophage-specific ATP-binding cassette transporter A1 (ABCA1) knockout (Abca1^-M/-M) mice had an enhanced proinflammatory response to the Toll-like receptor (TLR) 4 agonist, lipopolysaccharide (LPS), compared with wild-type (WT) mice. In the present study, we demonstrate a direct association between free cholesterol (FC), lipid raft content, and hyper-responsiveness of macrophages to LPS in WT mice. Abca1^-M/-M macrophages were also hyper-responsive to specific agonists to TLR2, TLR7, and TLR9, but not TLR3, compared with WT macrophages. We hypothesized that ABCA1 regulates macrophage responsiveness to TLR agonists by modulation of lipid raft cholesterol and TLR mobilization to lipid rafts. We demonstrated that Abca1^-M/-M vs. WT macrophages contained 23% more FC in isolated lipid rafts. Further, mass spectrometric analysis suggested raft phospholipid composition was unchanged. Although cell surface expression of TLR4 was similar between Abca1^-M/-M and WT macrophages, significantly more TLR4 was distributed in membrane lipid rafts in Abca1^-M/-M macrophages. Abca1^-M/-M macrophages also exhibited increased trafficking of the predominantly intracellular TLR9 into lipid rafts in response to TLR9-specific agonist (CpG). Collectively, our data suggest that macrophage ABCA1 dampens inflammation by reducing MyD88-dependent TLRs trafficking to lipid rafts by selective reduction of FC content in lipid rafts.     

Increased cellular free cholesterol in macrophage-specific Abca1 knock-out mice enhances pro-inflammatory response of macrophages

Macrophage-specific Abca1 knock-out (Abca1(-)(M)(/-)(M)) mice were generated to determine the role of macrophage ABCA1 expression in plasma lipoprotein concentrations and the innate immune response of macrophages. Plasma lipid and lipoprotein concentrations in chow-fed Abca1(-)(M)(/-)(M) and wild-type (WT) mice were indistinguishable. Compared with WT macrophages, Abca1(-)(M)(/-)(M) macrophages had a >95% reduction in ABCA1 protein, failed to efflux lipid to apoA-I, and had a significant increase in free cholesterol (FC) and membrane lipid rafts without induction of endoplasmic reticulum stress. Lipopolysaccharide (LPS)-treated Abca1(-)(M)(/-)(M) macrophages exhibited enhanced expression of pro-inflammatory cytokines and increased activation of the NF-kappaB and MAPK pathways, which could be diminished by silencing MyD88 or by chemical inhibition of NF-kappaB or MAPK. In vivo LPS injection also resulted in a higher pro-inflammatory response in Abca1(-)(M)(/-)(M) mice compared with WT mice. Furthermore, cholesterol depletion of macrophages with methyl-beta-cyclodextrin normalized FC content between the two genotypes and their response to LPS; cholesterol repletion of macrophages resulted in increased cellular FC accumulation and enhanced cellular response to LPS. Our results suggest that macrophage ABCA1 expression may protect against atherosclerosis by facilitating the net removal of excess lipid from macrophages and dampening pro-inflammatory MyD88-dependent signaling pathways by reduction of cell membrane FC and lipid raft content.     

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.     

CFTR in a lipid raft-TNFR1 complex modulates gap junctional intercellular communication and IL-8 secretion

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause a chronic inflammatory response in the lung of patients with Cystic Fibrosis (CF). We have showed that TNF-alpha signaling through the Src family tyrosine kinases (SFKs) was defective as determined by an inability of TNF-alpha to regulate gap junctional communication (GJIC) in CF cells. Here, we sought to elucidate the mechanisms linking TNF-alpha signaling to the functions of CFTR at the molecular level. In a MDCKI epithelial cell model expressing wild-type (WtCFTR) or mutant CFTR lacking its PDZ-interacting motif (CFTR-DeltaTRL), TNF-alpha increased the amount of WtCFTR but not CFTR-DeltaTRL in detergent-resistant membrane microdomains (DRMs). This recruitment was modulated by SFK activity and associated with DRM localization of TNFR1 and c-Src. Activation of TNFR1 signaling also decreased GJIC and markedly stimulated IL-8 production in WtCFTR cells. In contrast, the absence of CFTR in DRMs was associated with abnormal TNFR1 signaling as revealed by no recruitment of TNFR1 and c-Src to lipid rafts in CFTR-DeltaTRL cells and loss of regulation of GJIC and IL-8 secretion. These results suggest that localization of CFTR in lipid rafts in association with c-Src and TNFR1 provides a responsive signaling complex to regulate GJIC and cytokine signaling.     

Proteomic Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling

Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1^−/− macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1^+/+ and Abca1^−/− macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1^+/+ macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1^−/− rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response.Lipid rafts are cholesterol-enriched membrane microdomains, thought to be present in all cells, that concentrate and organize cell-surface signal transduction events in several signaling cascades, including those of the Toll-like receptors (TLRs) (1). The selectivity of rafts for particular proteins, and, consequently, the signal strength of pathways initiating from ligated raft-resident receptors, are thought to derive in large part from the high cholesterol content of raft microdomains (2–4). In vitro, altering raft cholesterol of living cells downward or upward with chemical tools (e.g. cyclodextrins) leads to parallel changes in raft protein abundance (3, 4). The relevance of cholesterol-driven alterations in the raft proteome to disease is suggested by reports that hypercholesterolemia cholesterol-loads macrophage rafts and amplifies their responsiveness to lipopolysaccharide (LPS) (3, 4). Proteomic strategies have recently been applied to raft isolates from a variety of cell types, aiming to better understand the identity of proteins tonically present in rafts, as well as proteins dynamically recruited to rafts upon cell stimulation (2, 5–8). To date, however, most reports have used cell lines of uncertain physiological relevance. In addition, although raft cholesterol levels are regulated in vivo by intracellular cholesterol trafficking (1), no reports to date have sought to define how the raft proteome is physiologically regulated by cholesterol trafficking proteins.ATP binding cassette (ABC) A1, a member of the ABC transporter superfamily, plays a key role in regulating levels of cholesterol in macrophages and other cells via promoting efflux of cellular cholesterol to extracellular acceptors, in particular lipid-free apolipoprotein (apo) A-I (9). The importance of ABCA1¹ to human health is clearly illustrated by Tangier disease, a rare ABCA1 mutation syndrome typified by severe HDL deficiency, widespread macrophage foam cells, and premature atherosclerosis (10). In addition, the large number of common ABCA1 polymorphisms that have been associated with human cardiovascular disease (10) suggest a broad-spanning impact of ABCA1 on human health. It remains somewhat controversial whether ABCA1-effluxed cholesterol derives from raft or extra-raft membranes (11). Nonetheless, both human Tangier disease cells and ABCA1-null murine macrophages have been shown to have greatly expanded lipid rafts that contain increased cholesterol and increased TLR4 (12, 13). These changes are associated with enhanced responsiveness to LPS that can be reversed by cholesterol depletion (13–15). Collectively, these findings indicate that ABCA1 may regulate the raft proteome and innate immune response through control of raft cholesterol. However, no proteomic analysis of rafts from ABCA1-deficient cells has been reported to date.Herein, we report a proteomic analysis of raft isolates from naive and LPS-stimulated Abca1^+/+ and Abca1^−/− primary murine macrophages. Unexpectedly, we found that ABCA1 deletion and LPS stimulation induced many similar changes in the raft proteome. Stomatin-like protein 2 (SLP-2), a lesser known member of the stomatin-prohibitin-flotillin-HflK/C (SPFH) family of membrane scaffolding proteins, was unique among SPFH proteins in being robustly up-regulated in rafts of unstimulated Abca1^−/− cells compared with Abca1^+/+ counterparts. We found that rafts of SLP-2 knockdown cells were abnormal, displaying increased binding of cholera toxin subunit B—a probe for the raft-specific ganglioside GM1—but markedly decreased protein, including flotillins-1 and -2, and CD14. Whereas SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux, it reduced macrophage responsiveness to LPS and multiple additional TLR ligands. Taken together, we report that ABCA1 regulates the macrophage raft proteome and identify SLP-2 as a novel ABCA1-dependent regulator of raft composition that controls the innate immune response.     

脂筏与精子膜信号传导

脂筏是细胞膜内由特殊脂质与蛋白质构成的微域。小窝是脂筏的一种形式,小窝标记蛋白有小窝蛋白和小窝舟蛋白。脂筏或小窝与生物信号传导、细胞蛋白转运和胆固醇平衡有关。最近实验证实哺乳动物精子膜具有脂筏结构,脂筏与膜胆固醇外逸对于启动受精的信号传导具有重要作用。     

Involvement of CD14 in the inhibitory effects of dimethyl-alpha-cyclodextrin on lipopolysaccharide signaling in macrophages

The potential use of alpha-cyclodextrin and its hydrophilic alpha-cyclodextrin derivatives (alpha-CyDs) as antagonists against lipopolysaccharide (LPS), which stimulates the nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production as well as nuclear factor-kappaB (NF-kappaB) activation in macrophages was examined. Of three alpha-CyDs used in the present study, 2,6-di-O-methyl-alpha-CyD (DM-alpha-CyD) had greater inhibitory activity than did the other CyDs against NO and TNF-alpha production through an impairment of gene expression in macrophage cell lines and primary macrophages stimulated with LPS and lipid A in a concentration-dependent manner. Concomitantly, DM-alpha-CyD inhibited NF-kappaB translocation into nucleus. These inhibitory effects of DM-alpha-CyD could be attributed to the release of CD14 from lipid rafts caused by an efflux of phospholipids, but not cholesterol. These results suggest that DM-alpha-CyD may have promise as a potent and unique antagonist for excess activation of macrophages stimulated with LPS.     

Salicylate improves macrophage cholesterol homeostasis via activation of Ampk

Atherosclerosis stems from imbalances in lipid metabolism and leads to maladaptive inflammatory responses. The AMP-activated protein kinase (Ampk) is a highly conserved serine/threonine kinase that regulates many aspects of lipid and energy metabolism, although its specific role in controlling macrophage cholesterol homeostasis remains unclear. We sought to address this question by testing the effects of direct Ampk activators in primary bone marrow-derived macrophages from Ampk β1-deficient (β1^−/−) mice. Macrophages from Ampk β1^−/− mice had enhanced lipogenic capacity and diminished cholesterol efflux, although cholesterol uptake was unaffected. Direct activation of Ampk β1 via salicylate (the unacetylated form of aspirin) or A-769662 (a small molecule activator), decreased the synthesis of FAs and sterols in WT but not Ampk β1^−/− macrophages. In lipid-laden macrophages, Ampk activation decreased cholesterol content (foam cell formation) and increased cholesterol efflux to HDL and apoA-I, effects that occurred in an Ampk β1-dependent manner. Increased cholesterol efflux was also associated with increased gene expression of the ATP binding cassette transporters, Abcg1 and Abca1. Moreover, in vivo reverse cholesterol transport was suppressed in mice that received Ampk β1^−/− macrophages compared with the WT control. Our data highlight the therapeutic potential of targeting macrophage Ampk with new or existing drugs for the possible reduction in foam cell formation during the early stages of atherosclerosis.     

Membrane lipid domains distinct from cholesterol/sphingomyelin-rich rafts are involved in the ABCA1-mediated lipid secretory pathway

Efflux of excess cellular cholesterol mediated by lipid-poor apolipoproteins occurs by an active mechanism distinct from passive diffusion and is controlled by the ATP-binding cassette transporter ABCA1. Here we examined whether ABCA1-mediated lipid efflux involves the selective removal of lipids associated with membrane rafts, plasma membrane domains enriched in cholesterol and sphingomyelin. ABCA1 was not associated with cholesterol and sphingolipid-rich membrane raft domains based on detergent solubility and lack of colocalization with marker proteins associated with raft domains. Lipid efflux to apoA-I was accounted for by decreases in cellular lipids not associated with cholesterol/sphingomyelin-rich membranes. Treating cells with filipin, to disrupt raft structure, or with sphingomyelinase, to digest plasma membrane sphingomyelin, did not impair apoA-I-mediated cholesterol or phosphatidylcholine efflux. In contrast, efflux of cholesterol to high density lipoproteins (HDL) or plasma was partially accounted for by depletion of cholesterol from membrane rafts. Additionally, HDL-mediated cholesterol efflux was partially inhibited by filipin and sphingomyelinase treatment. Apo-A-I-mediated cholesterol efflux was absent from fibroblasts with nonfunctional ABCA1 (Tangier disease cells), despite near normal amounts of cholesterol associated with raft domains and normal abilities of plasma and HDL to deplete cholesterol from these domains. Thus, the involvement of membrane rafts in cholesterol efflux applies to lipidated HDL particles but not to lipid-free apoA-I. We conclude that cholesterol and sphingomyelin-rich membrane rafts do not provide lipid for efflux promoted by apolipoproteins through the ABCA1-mediated lipid secretory pathway and that ABCA1 is not associated with these domains.     

脂筏在病毒感染中的作用

脂筏是细胞膜上富含鞘脂和胆固醇的微区结构,广泛分布于细胞的膜系统.脂筏中含有诸多信号分子和免疫受体,在细胞的生命活动中扮演非常重要的角色.更为重要的是,脂筏为细胞表面发生的蛋白质-蛋白质和蛋白质-脂类分子间的相互作用提供了平台.研究表明,很多病毒可以利用细胞膜表面的脂筏结构介导其侵入宿主细胞,一些病毒可以借助脂筏结构完成病毒颗粒的组装和出芽.本文将综述不同类型的病毒如SV40、HIV等借助脂筏完成入侵以及流感病毒等利用脂筏完成组装和出芽的证据及机理,并概述目前研究病毒与脂筏相互作用的方法及存在的问题.深入研究脂筏在病毒感染中的作用,将有助于对病毒与宿主细胞的相互作用的理解,从而可能发现新的、有效的对抗病毒的方法。     

Beta-cell ABCA1 influences insulin secretion, glucose homeostasis and response to thiazolidinedione treatment

Type 2 diabetes is characterized by both peripheral insulin resistance and reduced insulin secretion by beta-cells. The reasons for beta-cell dysfunction in this disease are incompletely understood but may include the accumulation of toxic lipids within this cell type. We examined the role of Abca1, a cellular cholesterol transporter, in cholesterol homeostasis and insulin secretion in beta-cells. Mice with specific inactivation of Abca1 in beta-cells had markedly impaired glucose tolerance and defective insulin secretion but normal insulin sensitivity. Islets isolated from these mice showed altered cholesterol homeostasis and impaired insulin secretion in vitro. We found that rosiglitazone, an activator of the peroxisome proliferator-activated receptor-gamma, which upregulates Abca1 in beta-cells, requires beta-cell Abca1 for its beneficial effects on glucose tolerance. These experiments establish a new role for Abca1 in beta-cell cholesterol homeostasis and insulin secretion, and suggest that cholesterol accumulation may contribute to beta-cell dysfunction in type 2 diabetes.     

Rafts defined: a report on the Keystone Symposium on Lipid Rafts and Cell Function

The recent Keystone Symposium on Lipid Rafts and Cell Function (March 23-28, 2006 in Steamboat Springs, CO) brought together biophysicists, biochemists, and cell biologists to discuss the structure and function of lipid rafts. What emerged from the meeting was a consensus definition of a membrane raft: "Membrane rafts are small (10-200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Small rafts can sometimes be stabilized to form larger platforms through protein-protein and protein-lipid interactions." This definition helps to clarify current thinking in a field that has been plagued by the heterogeneous and sometimes ephemeral nature of its subject.     

Evidence that HIV budding in primary macrophages occurs through the exosome release pathway

Lipid rafts are specialized regions of cell membranes enriched in cholesterol and sphingolipids that are involved in immune activation and signaling. Studies in T-cells indicate that these membrane domains serve as sites for release of human immunodeficiency virus (HIV). By budding through lipid rafts in T-cells, HIV selectively incorporates raft markers and excludes non-raft proteins. This process has been well studied in T-cells, but it is unknown whether lipid rafts serve as budding sites for HIV in macrophages. Recently, we proposed a new model of retroviral biogenesis called the Trojan exosome hypothesis (Gould, S. J., Booth, A., and Hildreth, J. E. K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10592-10597). This model proposes that retroviruses coopt the existing cellular machinery for exosomal release. Here, we performed the first test designed to differentiate between the lipid raft hypothesis of retroviral biogenesis and the Trojan exosome hypothesis. Using macrophages, we examined the relative abundance of several host proteins on the cell surface, in lipid rafts, and on both HIV particles and exosomes derived from these cells. Our results show significant differences in the abundance of host proteins on the cell surface and in HIV. Moreover, our data demonstrate discordance in the abundance of some proteins in lipid rafts and in HIV. Finally, our data reveal a strong concordance between the host cell protein profile of exosomes and that of HIV. These results strongly support the Trojan exosome hypothesis and its prediction that retroviral budding represents exploitation of a pre-existing cellular pathway of intercellular vesicle trafficking.     

SCP-2/SCP-x gene ablation alters lipid raft domains in primary cultured mouse hepatocytes

Although reverse cholesterol transport from peripheral cell types is mediated through plasma membrane microdomains termed lipid rafts, almost nothing is known regarding the existence, protein/lipid composition, or structure of these putative domains in liver hepatocytes, cells responsible for the net removal of cholesterol from the body. Lipid rafts purified from hepatocyte plasma membranes by a nondetergent affinity chromatography method were: i) present at 33 +/- 3% of total plasma membrane protein; ii) enriched in key proteins of the reverse cholesterol pathway [scavenger receptor class B type I (SR-B1), ABCA1, P-glycoprotein (P-gp), sterol carrier protein-2 (SCP-2)]; iii) devoid of caveolin-1; iv) enriched in cholesterol, sphingomyelin, GM1, and phospholipids low in polyunsaturated fatty acid and double bond index; and v) exhibited an intermediate liquid-ordered lipid phase with significant transbilayer fluidity gradient. Ablation of the gene encoding SCP-2 significantly altered lipid rafts to: i) increase the proportion of lipid rafts present, thereby increasing raft total content of ABCA1, P-gp, and SR-B1; ii) increase total phospholipids while decreasing GM1 in lipid rafts; iii) decrease the fluidity of lipid rafts, consistent with the increased intermediate liquid-ordered phase; and iv) abolish the lipid raft transbilayer fluidity gradient. Thus, despite the absence of caveolin-1 in liver hepatocytes, lipid rafts represented nearly one-third of the mouse hepatocyte plasma membrane proteins and displayed unique protein, lipid, and biophysical properties that were differentially regulated by SCP-2 expression.     

ABCA1 mutants reveal an interdependency between lipid export function, apoA-I binding activity, and Janus kinase 2 activation

ABCA1 exports cholesterol and phospholipids from cells by a multistep pathway that involves forming cell surface lipid domains, solubilizing these lipids by apolipoproteins, binding of apolipoproteins to ABCA1, and activating signaling processes. Here we used a mutational analysis approach to evaluate the relationship between these events. We prepared seven naturally occurring mutants and one artificial missense mutant of ABCA1 with varying degrees of impaired function, expressed them to similar levels as wild-type ABCA1 on the cell surface of BHK cells, and measured ABCA1-dependent lipid export, apolipoprotein A-I (apoA-I) binding, and signaling activities. Linear regression analyses showed that cholesterol and phospholipid efflux and cellular apoA-I binding correlated significantly with the ability of ABCA1 to form cell surface lipid domains. Lipid export and cellular apoA-I binding activities and formation of lipid domains also correlated with the amount of apoA-I that could be cross-linked to ABCA1. Moreover, each of these lipid export and apoA-I binding activities correlated with apoA-I-induced Janus kinase 2 (JAK2) activation. Thus, these missense mutations in ABCA1 impair lipid export, apoA-I binding, and apoA-I-stimulated JAK2 activities to similar extents, indicating that these processes are highly interactive components of a pathway that functions to export lipids from cells.     

Francisella targets cholesterol-rich host cell membrane domains for entry into macrophages

Francisella tularensis is a pathogen optimally adapted to efficiently invade its respective host cell and to proliferate intracellularly. We investigated the role of host cell membrane microdomains in the entry of F. tularensis subspecies holarctica vaccine strain (F. tularensis live vaccine strain) into murine macrophages. F. tularensis live vaccine strain recruits cholesterol-rich lipid domains ("lipid rafts") with caveolin-1 for successful entry into macrophages. Interference with lipid rafts through the depletion of plasma membrane cholesterol, through induction of raft internalization with choleratoxin, or through removal of raft-associated GPI-anchored proteins by treatment with phosphatidylinositol phospholipase C significantly inhibited entry of Francisella and its intracellular proliferation. Lipid raft-associated components such as cholesterol and caveolin-1 were incorporated into Francisella-containing vesicles during entry and the initial phase of intracellular trafficking inside the host cell. These findings demonstrate that Francisella requires cholesterol-rich membrane domains for entry into and proliferation inside macrophages.     

Altered renal lipid metabolism and renal lipid accumulation in human diabetic nephropathy

Animal models link ectopic lipid accumulation to renal dysfunction, but whether this process occurs in the human kidney is uncertain. To this end, we investigated whether altered renal TG and cholesterol metabolism results in lipid accumulation in human diabetic nephropathy (DN). Lipid staining and the expression of lipid metabolism genes were studied in kidney biopsies of patients with diagnosed DN (n = 34), and compared with normal kidneys (n = 12). We observed heavy lipid deposition and increased intracellular lipid droplets. Lipid deposition was associated with dysregulation of lipid metabolism genes. Fatty acid β-oxidation pathways including PPAR-α, carnitine palmitoyltransferase 1, acyl-CoA oxidase, and L-FABP were downregulated. Downregulation of renal lipoprotein lipase, which hydrolyzes circulating TGs, was associated with increased expression of angiopoietin-like protein 4. Cholesterol uptake receptor expression, including LDL receptors, oxidized LDL receptors, and acetylated LDL receptors, was significantly increased, while there was downregulation of genes effecting cholesterol efflux, including ABCA1, ABCG1, and apoE. There was a highly significant correlation between glomerular filtration rate, inflammation, and lipid metabolism genes, supporting a possible role of abnormal lipid metabolism in the pathogenesis of DN. These data suggest that renal lipid metabolism may serve as a target for specific therapies aimed at slowing the progression of glomerulosclerosis.     

Loss of functional MYO1C/myosin 1c,a motor protein involved in lipid raft trafficking,disrupts autophagosome-lysosome fusion

MYO1C, a single-headed class I myosin, associates with cholesterol-enriched lipid rafts and facilitates their recycling from intracellular compartments to the cell surface. Absence of functional MYO1C disturbs the cellular distribution of lipid rafts, causes the accumulation of cholesterol-enriched membranes in the perinuclear recycling compartment, and leads to enlargement of endolysosomal membranes. Several feeder pathways, including classical endocytosis but also the autophagy pathway, maintain the health of the cell by selective degradation of cargo through fusion with the lysosome. Here we show that loss of functional MYO1C leads to an increase in total cellular cholesterol and its disrupted subcellular distribution. We observe an accumulation of autophagic structures caused by a block in fusion with the lysosome and a defect in autophagic cargo degradation. Interestingly, the loss of MYO1C has no effect on degradation of endocytic cargo such as EGFR, illustrating that although the endolysosomal compartment is enlarged in size, it is functional, contains active hydrolases, and the correct pH. Our results highlight the importance of correct lipid composition in autophagosomes and lysosomes to enable them to fuse. Ablating MYO1C function causes abnormal cholesterol distribution, which has a major selective impact on the autophagy pathway.     

An LXR–NCOA5 gene regulatory complex directs inflammatory crosstalk‐dependent repression of macrophage cholesterol efflux

LXR–cofactor complexes activate the gene expression program responsible for cholesterol efflux in macrophages. Inflammation antagonizes this program, resulting in foam cell formation and atherosclerosis; however, the molecular mechanisms underlying this antagonism remain to be fully elucidated. We use promoter enrichment‐quantitative mass spectrometry (PE‐QMS) to characterize the composition of gene regulatory complexes assembled at the promoter of the lipid transporter Abca1 following downregulation of its expression. We identify a subset of proteins that show LXR ligand‐ and binding‐dependent association with the Abca1 promoter and demonstrate they differentially control Abca1 expression. We determine that NCOA5 is linked to inflammatory Toll‐like receptor (TLR) signaling and establish that NCOA5 functions as an LXR corepressor to attenuate Abca1 expression. Importantly, TLR3–LXR signal crosstalk promotes recruitment of NCOA5 to the Abca1 promoter together with loss of RNA polymerase II and reduced cholesterol efflux. Together, these data significantly expand our knowledge of regulatory inputs impinging on the Abca1 promoter and indicate a central role for NCOA5 in mediating crosstalk between pro‐inflammatory and anti‐inflammatory pathways that results in repression of macrophage cholesterol efflux.