Caveolin-1-mediated apolipoprotein A-I membrane binding sites are not required for cholesterol efflux

Caveolin-1 (Cav1), a structural protein required for the formation of invaginated membrane domains known as caveolae, has been implicated in cholesterol trafficking and homeostasis. Here we investigated the contribution of Cav1 to apolipoprotein A-I (apoA-I) cell surface binding and intracellular processing using mouse embryonic fibroblasts (MEFs) derived from wild type (WT) or Cav1-deficient (Cav1(-/-)) animals. We found that cells expressing Cav1 have 2.6-fold more apoA-I binding sites than Cav1(-/-) cells although these additional binding sites are not associated with detergent-free lipid rafts. Further, Cav1-mediated binding targets apoA-I for internalization and degradation and these processes are not correlated to cholesterol efflux. Despite lower apoA-I binding, cholesterol efflux from Cav1(-/-) MEFs is 1.7-fold higher than from WT MEFs. Stimulation of ABCA1 expression with an LXR agonist enhances cholesterol efflux from both WT and Cav1(-/-) cells without increasing apoA-I surface binding or affecting apoA-I processing. Our results indicate that there are at least two independent lipid binding sites for apoA-I; Cav1-mediated apoA-I surface binding and uptake is not linked to cholesterol efflux, indicating that membrane domains other than caveolae regulate ABCA1-mediated cholesterol efflux.     

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.     

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.     

HP1α mediates defective heterochromatin repair and accelerates senescence in Zmpste24-deficient cells

Heterochromatin protein 1 (HP1) interacts with various proteins, including lamins, to play versatile functions within nuclei, such as chromatin remodeling and DNA repair. Accumulation of prelamin A leads to misshapen nuclei, heterochromatin disorganization, genomic instability, and premature aging in Zmpste24-null mice. Here, we investigated the effects of prelamin A on HP1α homeostasis, subcellular distribution, phosphorylation, and their contribution to accelerated senescence in mouse embryonic fibroblasts (MEFs) derived from Zmpste24^−/− mice. The results showed that the level of HP1α was significantly increased in Zmpste24^−/− cells. Although prelamin A interacted with HP1α in a manner similar to lamin A, HP1α associated with the nuclease-resistant nuclear matrix fraction was remarkably increased in Zmpste24^−/− MEFs compared with that in wild-type littermate controls. In wild-type cells, HP1α was phosphorylated at Thr50, and the phosphorylation was maximized around 30 min, gradually dispersed 2 h after DNA damage induced by camptothecin. However, the peak of HP1α phosphorylation was significantly compromised and appeared until 2 h, which is correlated with the delayed maximal formation of γ-H2AX foci in Zmpste24^−/− MEFs. Furthermore, knocking down HP1α by siRNA alleviated the delayed DNA damage response and accelerated senescence in Zmpste24^−/− MEFs, evidenced by the rescue of the delayed γ-H2AX foci formation, downregulation of p16, and reduction of senescence-associated β-galactosidase activity. Taken together, these findings establish a functional link between prelamin A, HP1α, chromatin remodeling, DNA repair, and early senescence in Zmpste24-deficient mice, suggesting a potential therapeutic strategy for laminopathy-based premature aging via the intervention of HP1α.     

Protein Kinase Cε Modulates Insulin Receptor Localization and Trafficking in Mouse Embryonic Fibroblasts

We have previously shown that deletion of protein kinase C epsilon (PKCε) in mice results in protection against glucose intolerance caused by a high fat diet. This was in part due to reduced insulin uptake by hepatocytes and insulin clearance, which enhanced insulin availability. Here we employed mouse embryonic fibroblasts (MEFs) derived from wildtype (WT) and PKCε-deficient (PKCε^−/−) mice to examine this mechanistically. PKCε^−/− MEFs exhibited reduced insulin uptake which was associated with decreased insulin receptor phosphorylation, while downstream signalling through IRS-1 and Akt was unaffected. Cellular fractionation demonstrated that PKCε deletion changed the localization of the insulin receptor, a greater proportion of which co-fractionated with flotillin-1, a marker of membrane microdomains. Insulin stimulation resulted in redistribution of the receptor in WT cells, while this was markedly reduced in PKCε^−/− cells. These alterations in insulin receptor trafficking were associated with reduced expression of CEACAM1, a receptor substrate previously shown to modulate insulin clearance. Virally-mediated reconstitution of PKCε in MEFs increased CEACAM1 expression and partly restored the sensitivity of the receptor to insulin-stimulated redistribution. These data indicate that PKCε can affect insulin uptake in MEFs through promotion of receptor-mediated endocytosis, and that this may be mediated by regulation of CEACAM1 expression.     

Wild-type p53-induced phosphatase 1 (Wip1) forestalls cellular premature senescence at physiological oxygen levels by regulating DNA damage response signaling during DNA replication

Wip1 (protein phosphatase Mg²⁺/Mn²⁺-dependent 1D, Ppm1d) is a nuclear serine/threonine protein phosphatase that is induced by p53 following the activation of DNA damage response (DDR) signaling. Ppm1d^−/− mouse embryonic fibroblasts (MEFs) exhibit premature senescence under conventional culture conditions; however, little is known regarding the role of Wip1 in regulating cellular senescence. In this study, we found that even at a representative physiological concentration of 3% O₂, Ppm1d^−/− MEFs underwent premature cellular senescence that depended on the functional activation of p53. Interestingly, Ppm1d^−/− MEFs showed increased H2AX phosphorylation levels without increased levels of reactive oxygen species (ROS) or DNA base damage compared with wild-type (Wt) MEFs, suggesting a decreased threshold for DDR activation or sustained DDR activation during recovery. Notably, the increased H2AX phosphorylation levels observed in Ppm1d^−/− MEFs were primarily associated with S-phase cells and predominantly dependent on the activation of ATM. Moreover, these same phenotypes were observed when Wt and Ppm1d^−/− MEFs were either transiently or chronically exposed to low levels of agents that induce replication-mediated double-stranded breaks. These findings suggest that Wip1 prevents the induction of cellular senescence at physiological oxygen levels by attenuating DDR signaling in response to endogenous double-stranded breaks that form during DNA replication.     

ABCG1 regulates mouse adipose tissue macrophage cholesterol levels and ratio of M1 to M2 cells in obesity and caloric restriction

In addition to triacylglycerols, adipocytes contain a large reserve of unesterified cholesterol. During adipocyte lipolysis and cell death seen during severe obesity and weight loss, free fatty acids and cholesterol become available for uptake and processing by adipose tissue macrophages (ATMs). We hypothesize that ATMs become cholesterol enriched and participate in cholesterol clearance from adipose tissue. We previously showed that ABCG1 is robustly upregulated in ATMs taken from obese mice and further enhanced by caloric restriction. Here, we found that ATMs taken from obese and calorie-restricted mice derived from transplantation of WT or Abcg1-deficient bone marrow are cholesterol enriched. ABCG1 levels regulate the ratio of classically activated (M1) to alternatively activated (M2) ATMs and their cellular cholesterol content. Using WT and Abcg1^−/− cultured macrophages, we found that Abcg1 is most highly expressed by M2 macrophages and that ABCG1 deficiency is sufficient to retard macrophage chemotaxis. However, changes in myeloid expression of Abcg1 did not protect mice from obesity or impaired glucose homeostasis. Overall, ABCG1 modulates ATM cholesterol content in obesity and weight loss regimes leading to an alteration in M1 to M2 ratio that we suggest is due to the extent of macrophage egress from adipose tissue.     

Syndecans reside in sphingomyelin-enriched low-density fractions of the plasma membrane isolated from a parathyroid cell line

Background

Heparan sulfate proteoglycans (HSPGs) are one of the basic constituents of plasma membranes. Specific molecular interactions between HSPGs and a number of extracellular ligands have been reported. Mechanisms involved in controlling the localization and abundance of HSPG on specific domains on the cell surface, such as membrane rafts, could play important regulatory roles in signal transduction.

Methodology/Principal Findings

Using metabolic radiolabeling and sucrose-density gradient ultracentrifugation techniques, we identified [³⁵S]sulfate-labeled macromolecules associated with detergent-resistant membranes (DRMs) isolated from a rat parathyroid cell line. DRM fractions showed high specific radioactivity ([³⁵S]sulfate/mg protein), implying the specific recruitment of HSPGs to the membrane rafts. Identity of DRM-associated [³⁵S]sulfate-labeled molecules as HSPGs was confirmed by Western blotting with antibodies that recognize heparan sulfate (HS)-derived epitope. Analyses of core proteins by SDS-PAGE revealed bands with an apparent MW of syndecan-4 (30–33 kDa) and syndecan-1 (70 kDa) suggesting the presence of rafts with various HSPG species. DRM fractions enriched with HSPGs were characterized by high sphingomyelin content and found to only partially overlap with the fractions enriched in ganglioside GM1. HSPGs could be also detected in DRMs even after prior treatment of cells with heparitinase.

Conclusions/Significance

Both syndecan-1 and syndecan-4 have been found to specifically associate with membrane rafts and their association seemed independent of intact HS chains. Membrane rafts in which HSPGs reside were also enriched with sphingomyelin, suggesting their possible involvement in FGF signaling. Further studies, involving proteomic characterization of membrane domains containing HSPGs might improve our knowledge on the nature of HSPG-ligand interactions and their role in different signaling platforms.     

ABCD2 is abundant in adipose tissue and opposes the accumulation of dietary erucic acid (C22:1) in fat

The ATP binding cassette transporter, ABCD2 (D2), is a peroxisomal protein whose mRNA has been detected in the adrenal, brain, liver, and fat. Although the role of this transporter in neural tissues has been studied, its function in adipose tissue remains unexplored. The level of immunoreactive D2 in epididymal fat is >50-fold of that found in brain or adrenal. D2 is highly enriched in adipocytes and is upregulated during adipogenesis but is not essential for adipocyte differentiation or lipid accumulation in day 13.5 mouse embryonic fibroblasts isolated from D2-deficient (D2^−/−) mice. Although no differences were appreciated in differentiation percentage, total lipid accumulation was greater in D2^−/− adipocytes compared with the wild type. These results were consistent with in vivo observations in which no significant differences in adiposity or adipocyte diameter between wild-type and D2^−/− mice were observed. D2^−/− adipose tissue showed an increase in the abundance of 20:1 and 22:1 fatty acids. When mice were challenged with a diet enriched in erucic acid (22:1), this lipid accumulated in the adipose tissue in a gene-dosage-dependent manner. In conclusion, D2 is a sterol regulatory element binding protein target gene that is highly abundant in fat and opposes the accumulation of dietary lipids generally absent from the triglyceride storage pool within adipose tissue.     

Caveolin-1 is required for fatty acid translocase (FAT/CD36) localization and function at the plasma membrane of mouse embryonic fibroblasts

Several lines of evidence suggest that lipid rafts are involved in cellular fatty acid uptake and influence fatty acid translocase (FAT/CD36) function. However, it remains unknown whether caveolae, a specialized raft type, are required for this mechanism. Here, we show that wild-type (WT) mouse embryonic fibroblasts (MEFs) and caveolin-1 knockout (KO) MEFs, which are devoid of caveolae, have comparable overall expression of FAT/CD36 protein but altered subcellular FAT/CD36 localization and function. In WT MEFs, FAT/CD36 was isolated with both lipid raft enriched detergent-resistant membranes (DRMs) and detergent-soluble membranes (DSMs), whereas in cav-1 KO cells it was exclusively associated with DSMs. Subcellular fractionation demonstrated that FAT/CD36 in WT MEFs was localized intracellularly and at the plasma membrane level while in cav-1 KO MEFs it was absent from the plasma membrane. This mistargeting of FAT/CD36 in cav-1 KO cells resulted in reduced fatty acid uptake compared to WT controls. Adenoviral expression of caveolin-1 in KO MEFs induced caveolae formation, redirection of FAT/CD36 to the plasma membrane and rescue of fatty acid uptake. In conclusion, our data provide evidence that caveolin-1 is necessary to target FAT/CD36 to the plasma membrane. Caveolin-1 may influence fatty acid uptake by regulating surface availability of FAT/CD36.     

Brij detergents reveal new aspects of membrane microdomain in erythrocytes

Membrane microdomains enriched in cholesterol, sphingolipids (rafts), and specific proteins are involved in important physiological functions. However their structure, size and stability are still controversial. Given that detergent-resistant membranes (DRMs) are in the liquid-ordered state and are rich in raft-like components, they might correspond to rafts at least to some extent. Here we monitor the lateral order of biological membranes by characterizing DRMs from erythrocytes obtained with Brij-98, Brij-58, and TX-100 at 4?°C and 37?°C. All DRMs were enriched in cholesterol and contained the raft markers flotillin-2 and stomatin. However, sphingomyelin (SM) was only found to be enriched in TX-100-DRMs – a detergent that preferentially solubilizes the membrane inner leaflet – while Band 3 was present solely in Brij-DRMs. Electron paramagnetic resonance spectra showed that the acyl chain packing of Brij-DRMs was lower than TX-100-DRMs, providing evidence of their diverse lipid composition. Fatty acid analysis revealed that the SM fraction of the DRMs was enriched in lignoceric acid, which should specifically contribute to the resistance of SM to detergents. These results indicate that lipids from the outer leaflet, particularly SM, are essential for the formation of the liquid-ordered phase of DRMs. At last, the differential solubilization process induced by Brij-98 and TX-100 was monitored using giant unilamellar vesicles. This study suggests that Brij and TX-100-DRMs reflect different degrees of lateral order of the membrane microdomains. Additionally, Brij DRMs are composed by both inner and outer leaflet components, making them more physiologically relevant than TX-100-DRMs to the studies of membrane rafts.     

Detergent-resistant membrane fractions contribute to the total 1H NMR-visible lipid signal in cells.

Leukocytes and other cells show an enhanced intensity of mobile lipid in their 1H NMR spectra under a variety of conditions. Such conditions include stimulation, which has recently been shown to involve detergent-resistant, plasma membrane domains (DRMs) often called lipid rafts. As there is much speculation surrounding the origin of cellular NMR-visible lipid, we analysed subcellular fractions, including DRMs, by NMR spectroscopy. We demonstrated that DRMs isolated by density gradient centrifugation from lymphoid (CEM-T4, stimulated Jurkat cells), and monocytoid (THP-1) cells produced NMR-visible, lipid signals. Large scale subfractionation of THP-1 cells determined that while cytoplasmic lipid droplets constituted much of the total NMR-visible lipid, the contribution of DRMs was significant. Qualitative and quantitative lipid analyses revealed that DRMs and lipid droplets differed in their lipid composition. DRMs were enriched in cholesterol and ganglioside GM1, and contained relatively unsaturated fatty acids compared with the lipid droplets. Both lipid droplets and DRMs contained neutral lipids (triacylgycerols, cholesterol ester, fatty acids in THP-1 cells) that could, in addition to phospholipids, contribute to the NMR-visible lipid. The lipid droplets also exhibited different protein profiles and contained 500-fold less protein than DRMs, confirming that DRMs and droplets were fractionated as separate entities. The NMR-visible lipid in DRMs is therefore unlikely to be a contaminant from lipid droplets. We propose a micropartitioning of the NMR-visible mobile lipid of whole cells between intracellular lipid droplets, where most of this lipid resides, and detergent-resistant plasma membrane domains.     

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

The formation of the neuromuscular junction is characterized by the progressive accumulation of nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane facing the nerve terminal, induced predominantly through the agrin/muscle-specific kinase (MuSK) signaling cascade. However, the cellular mechanisms linking MuSK activation to AChR clustering are still poorly understood. Here, we investigate whether lipid rafts are involved in agrin-elicited AChR clustering in a mouse C2C12 cell line. We observed that in C2C12 myotubes, both AChR clustering and cluster stability were dependent on cholesterol, because depletion by methyl-beta-cyclodextrin inhibited cluster formation or dispersed established clusters. Importantly, AChR clusters resided in ordered membrane domains, a biophysical property of rafts, as probed by Laurdan two-photon fluorescence microscopy. We isolated detergent-resistant membranes (DRMs) by three different biochemical procedures, all of which generate membranes with similar cholesterol/GM1 ganglioside contents, and these were enriched in several postsynaptic components, notably AChR, syntrophin, and raft markers flotillin-2 and caveolin-3. Agrin did not recruit AChRs into DRMs, suggesting that they are present in rafts independently of agrin activation. Consequently, in C2C12 myotubes, agrin likely triggers AChR clustering or maintains clusters through the coalescence of lipid rafts. These data led us to propose a model in which lipid rafts play a pivotal role in the assembly of the postsynaptic membrane at the neuromuscular junction upon agrin signaling.     

The sensitivity of lipid domains to small perturbations demonstrated by the effect of Triton

The hypothesis of lipid rafts describes functional domains in biological membranes. It is often assumed that rafts form by spontaneous de-mixing of certain lipids and that they can be isolated as detergent-resistant membrane particles (DRMs) using the detergent Triton X-100 (TX). Here, we present a model that describes the process of domain formation in membranes in the presence and in the absence of TX. We measure the interactions between TX and an equimolar mixture of sphingomyelin (SM), cholesterol (Cho), and 1-palmitoyl-2-oleoyl-3-sn-glycero-phosphatidylcholine (POPC) (1:1:1, mol) by means of isothermal titration calorimetry. Comparison with pure POPC membranes reveals a very unfavorable interaction between TX and SM/Cho, which causes a substantial tendency to segregate these molecules into separate, DRM-like (SM-rich) and fluid (TX-rich), domains. If rafts are indeed formed by spontaneous de-mixing of PC and SM/Cho, they must be very sensitive, and perturbations caused by techniques used to study rafts could lead to misleading results. If, however, rafts are much more stable than PC-SM-Cho domains, there must be an unknown raft stabilizer. Subtle changes of such a promoter could serve to modulate raft function.     

G protein-coupled receptor kinase 6 acts as a critical regulator of cytokine-induced hyperalgesia by promoting phosphatidylinositol 3-kinase and inhibiting p38 signaling

The molecular mechanisms determining magnitude and duration of inflammatory pain are still unclear. We assessed the contribution of G protein–coupled receptor kinase (GRK)-6 to inflammatory hyperalgesia in mice. We showed that GRK6 is a critical regulator of severity and duration of cytokine-induced hyperalgesia. In GRK6^−/− mice, a significantly lower dose (100 times lower) of intraplantar interleukin (IL)-1β was sufficient to induce hyperalgesia compared with wild-type (WT) mice. In addition, IL-1β hyperalgesia lasted much longer in GRK6^−/− mice than in WT mice (8 d in GRK6^−/− versus 6 h in WT mice). Tumor necrosis factor (TNF)-α–induced hyperalgesia was also enhanced and prolonged in GRK6^−/− mice. In vitro, IL-1β–induced p38 phosphorylation in GRK6^−/− dorsal root ganglion (DRG) neurons was increased compared with WT neurons. In contrast, IL-1β only induced activation of the phosphatidylinositol (PI) 3-kinase/Akt pathway in WT neurons, but not in GRK6^−/− neurons. In vivo, p38 inhibition attenuated IL-1β– and TNF-α–induced hyperalgesia in both genotypes. Notably, however, whereas PI 3-kinase inhibition enhanced and prolonged hyperalgesia in WT mice, it did not have any effect in GRK6-deficient mice. The capacity of GRK6 to regulate pain responses was also apparent in carrageenan-induced hyperalgesia, since thermal and mechanical hypersensitivity was significantly prolonged in GRK6^−/− mice. Finally, GRK6 expression was reduced in DRGs of mice with chronic neuropathic or inflammatory pain. Collectively, these findings underline the potential role of GRK6 in pathological pain. We propose the novel concept that GRK6 acts as a kinase that constrains neuronal responsiveness to IL-1β and TNF-α and cytokine-induced hyperalgesia via biased cytokine-induced p38 and PI 3-kinase/Akt activation.     

Dissecting the roles of ROCK isoforms in stress-induced cell detachment

The homologous Rho kinases, ROCK1 and ROCK2, are involved in stress fiber assembly and cell adhesion and are assumed to be functionally redundant. Using mouse embryonic fibroblasts (MEFs) derived from ROCK1^−/− and ROCK2^−/− mice, we have recently reported that they play different roles in regulating doxorubicin-induced stress fiber disassembly and cell detachment: ROCK1 is involved in destabilizing the actin cytoskeleton and cell detachment, whereas ROCK2 is required for stabilizing the actin cytoskeleton and cell adhesion. Here, we present additional insights into the roles of ROCK1 and ROCK2 in regulating stress-induced impairment of cell-matrix and cell-cell adhesion. In response to doxorubicin, ROCK1^−/− MEFs showed significant preservation of both focal adhesions and adherens junctions, while ROCK2^−/− MEFs exhibited impaired focal adhesions but preserved adherens junctions compared with the wild-type MEFs. Additionally, inhibition of focal adhesion or adherens junction formations by chemical inhibitors abolished the anti-detachment effects of ROCK1 deletion. Finally, ROCK1^−/− MEFs, but not ROCK2^−/− MEFs, also exhibited preserved central stress fibers and reduced cell detachment in response to serum starvation. These results add new insights into a novel mechanism underlying the anti-detachment effects of ROCK1 deletion mediated by reduced peripheral actomyosin contraction and increased actin stabilization to promote cell-cell and cell-matrix adhesion. Our studies further support the differential roles of ROCK isoforms in regulating stress-induced loss of central stress fibers and focal adhesions as well as cell detachment.     

Alteration of gene expression profile in Niemann-Pick type C mice correlates with tissue damage and oxidative stress

Background

Niemann-Pick type C disease (NPC) is a neurovisceral lipid storage disorder mainly characterized by unesterified cholesterol accumulation in lysosomal/late endosomal compartments, although there is also an important storage for several other kind of lipids. The main tissues affected by the disease are the liver and the cerebellum. Oxidative stress has been described in various NPC cells and tissues, such as liver and cerebellum. Although considerable alterations occur in the liver, the pathological mechanisms involved in hepatocyte damage and death have not been clearly defined. Here, we assessed hepatic tissue integrity, biochemical and oxidative stress parameters of wild-type control (Npc1 ^+/+; WT) and homozygous-mutant (Npc1 ^−/−; NPC) mice. In addition, the mRNA abundance of genes encoding proteins associated with oxidative stress, copper metabolism, fibrosis, inflammation and cholesterol metabolism were analyzed in livers and cerebella of WT and NPC mice.

Methodology/Principal Findings

We analyzed various oxidative stress parameters in the liver and hepatic and cerebellum gene expression in 7-week-old NPC1-deficient mice compared with control animals. We found signs of inflammation and fibrosis in NPC livers upon histological examination. These signs were correlated with increased levels of carbonylated proteins, diminished total glutathione content and significantly increased total copper levels in liver tissue. Finally, we analyzed liver and cerebellum gene expression patterns by qPCR and microarray assays. We found a correlation between fibrotic tissue and differential expression of hepatic as well as cerebellar genes associated with oxidative stress, fibrosis and inflammation in NPC mice.

Conclusions/Significance

In NPC mice, liver disease is characterized by an increase in fibrosis and in markers associated with oxidative stress. NPC is also correlated with altered gene expression, mainly of genes involved in oxidative stress and fibrosis. These findings correlate with similar parameters in cerebellum, as has been previously reported in the NPC mice model.     

Further evidence that paroxysmal nocturnal haemoglobinuria is a disorder of defective cell membrane lipid rafts

The glycolipid glycosylphosphatidylinositol anchor (GPI-A) plays an important role in lipid raft formation, which is required for proper expression on the cell surface of two inhibitors of the complement cascade, CD55 and CD59. The absence of these markers from the surface of blood cells, including erythrocytes, makes the cells susceptible to complement lysis, as seen in patients suffering from paroxysmal nocturnal haemoglobinuria (PNH). However, the explanation for why PNH-affected hematopoietic stem/progenitor cells (HSPCs) expand over time in BM is still unclear. Here, we propose an explanation for this phenomenon and provide evidence that a defect in lipid raft formation in HSPCs leads to defective CXCR4- and VLA-4-mediated retention of these cells in BM. In support of this possibility, BM-isolated CD34⁺ cells from PNH patients show a defect in the incorporation of CXCR4 and VLA-4 into membrane lipid rafts, respond weakly to SDF-1 stimulation, and show defective adhesion to fibronectin. Similar data were obtained with the GPI-A⁻ Jurkat cell line. Moreover, we also report that chimeric mice transplanted with CD55^−/− CD59^−/− BM cells but with proper GPI-A expression do not expand over time in transplanted hosts. On the basis of these findings, we propose that a defect in lipid raft formation in PNH-mutated HSPCs makes these cells more mobile, so that they expand and out-compete normal HSPCs from their BM niches over time.     

Regulation of PI(3)K/Akt signalling and cellular transformation by inositol polyphosphate 4‐phosphatase‐1

Akt is a crucial phosphoinositide 3-kinase (PI(3)K) effector that regulates cell proliferation and survival. PI(3)K-generated signals, PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂, direct Akt plasma membrane engagement. Pathological Akt plasma membrane association promotes oncogenesis. PtdIns(3,4)P₂ is degraded by inositol polyphosphate 4-phosphatase-1 (4-ptase-1) forming PtdIns(3)P; however, the role of 4-ptase-1 in regulating the activation and function of Akt is unclear. In mouse embryonic fibroblasts lacking 4-ptase-1 (^−/−MEFs), the Akt-pleckstrin homology (PH) domain was constitutively membrane-associated both in serum-starved and agonist-stimulated cells, in contrast to ^+/+MEFs, in which it was detected only at the plasma membrane following serum stimulation. Epidermal growth factor (EGF) stimulation resulted in increased Ser⁴⁷³ and Thr³⁰⁸-Akt phosphorylation and activation of Akt-dependent signalling in ^−/−MEFs, relative to ^+/+MEFs. Significantly, loss of 4-ptase-1 resulted in increased cell proliferation and decreased apoptosis. SV40-transformed ^−/−MEFs showed increased anchorage-independent cell growth and formed tumours in nude mice. This study provides the first evidence, to our knowledge, that 4-ptase-1 controls the activation of Akt and thereby cell proliferation, survival and tumorigenesis.