Lipid flippases and their biological functions

The typically distinct phospholipid composition of the two leaflets of a membrane bilayer is generated and maintained by bi-directional transport (flip-flop) of lipids between the leaflets. Specific membrane proteins, termed lipid flippases, play an essential role in this transport process. Energy-independent flippases allow common phospholipids to equilibrate rapidly between the two monolayers and also play a role in the biosynthesis of a variety of glycoconjugates such as glycosphingolipids, N-glycoproteins, and glycosylphosphatidylinositol (GPI)-anchored proteins. ATP-dependent flippases, including members of a conserved subfamily of P-type ATPases and ATP-binding cassette transporters, mediate the net transfer of specific phospholipids to one leaflet of a membrane and are involved in the creation and maintenance of transbilayer lipid asymmetry of membranes such as the plasma membrane of eukaryotes. Energy-dependent flippases also play a role in the biosynthesis of glycoconjugates such as bacterial lipopolysaccharide. This review summarizes recent progress on the identification and characterization of the various flippases and the demonstration of their biological functions. Received 12 April 2006; received after revision 22 June 2006; accepted 30 August 2006     

Selective effect of burn injury on splenic CD11c+ dendritic cells and CD8α+CD4−CD11c+ dendritic cell subsets

Burn injury causes an immunosuppression associated with suppressed adaptive immune function. Dendritic cells (DCs) are APCs for which signaling via their Toll-like receptors (TLRs) induces their maturation and activation, which is essential for the adaptive immune response. In this study, we examined if burn injury alters the TLR activity of splenic DCs. After injury, we noticed that DC functions were impaired, characterized by a suppressed capacity to prime naive T cells when triggering the TLR4 signaling cascade using specific ligands (LPS or rHSP60). The observed perturbations on LPS-primed DCs isolated from burned mice exhibited significantly diminished IL-12p40 production and enhanced IL-10 secretion-associated impairment in mitogen-activated protein kinase activation. Interestingly, we observed a decrease of TLR4/MD-2 expression on the CD8α⁺ DC subset that persisted following LPS stimulation. The altered TLR4 expression on LPS-stimulated CD8α⁺ DCs was associated with reduced capacity to produce IL-12 after stimulation. Our results suggested that TLR4 reactivity on DCs, especially CD8α⁺ DCs, is disturbed after burn injury.     

Novel macrophage polarization model: from gene expression to identification of new anti-inflammatory molecules

Plasticity is a well-known property of macrophages that is controlled by different changes in environmental signals. Macrophage polarization is regarded as a spectrum of activation phenotypes adjusted from one activation extreme, the classic (M1), to the other, the alternative (M2) activation. Here we show, in vitro and in vivo, that both M1 and M2 macrophage phenotypes are tightly coupled to specific patterns of gene expression. Novel M2-associated markers were characterized and identified as genes controlling the extracellular metabolism of ATP to generate pyrophosphates (PPi). Stimulation of M1 macrophages with PPi dampens both NLR and TLR signaling and thus mediates cytokine production. In this context extracellular PPi enhanced the resolution phase of a murine peritonitis model via a decrease in pro-inflammatory cytokine production. Therefore, our study reveals an additional level of plasticity modulating the resolution of inflammation.     

Flippases: still more questions than answers

Our understanding of flippase-mediated lipid translocation and membrane vesiculation, and the involvement of P-type ATPases in these processes is just beginning to emerge. The results obtained so far demonstrate significant complexity within this field and point to major tasks for future research. Most importantly, biochemical characterization of P₄-ATPases is required in order to clarify whether these transporters indeed are capable of catalyzing transmembrane phospholipid flipping. The β-subunit of P₄-ATPases shows unexpected similarities between the β- and γ-subunits of the Na⁺/K⁺-ATPase. It is likely that these proteins provide a similar solution to similar problems, and might have adopted similar structures to accomplish these tasks. No P₄-ATPases have been identified in the endoplasmic reticulum and it remains an intriguing possibility that, in this compartment, P_5A-ATPases are functional homologues of P₄-ATPases. Received 19 June 2008; received after revision 31 July 2008; accepted 15 August 2008     

Redox-dependent thiol modifications: implications for the release of extracellular vesicles

Extracellular vesicles (EVs), including microvesicles and exosomes, are emerging as important regulators of homeostasis and pathophysiology. During pro-inflammatory and pro-oxidant conditions, EV release is induced. As EVs released under such conditions often exert pro-inflammatory and procoagulant effects, they may actively promote the pathogenesis of chronic diseases. There is evidence that thiol group-containing antioxidants can prevent EV induction by pro-inflammatory and oxidative stimuli, likely by protecting protein thiols of the EV-secreting cells from oxidation. As the redox state of protein thiols greatly impacts three-dimensional protein structure and, consequently, function, redox modifications of protein thiols may directly modulate EV release in response to changes in the cell’s redox environment. In this review article, we discuss targets of redox-dependent thiol modifications that are known or expected to be involved in the regulation of EV release, namely redox-sensitive calcium channels, N-ethylmaleimide sensitive factor, protein disulfide isomerase, phospholipid flippases, actin filaments, calpains and cell surface-exposed thiols. Thiol protection is proposed as a strategy for preventing detrimental changes in EV signaling in response to inflammation and oxidative stress. Identification of the thiol-containing proteins that modulate EV release in pro-oxidant environments could provide a rationale for broad application of thiol group-containing antioxidants in chronic inflammatory diseases.     

Exogenous or endogenous Toll-like receptor ligands: which is the MVP in tumorigenesis?

Toll-like receptors (TLRs) are a class of pattern recognition receptors sensing microbial components and triggering an immune response against pathogens. In addition to their role in anti-infection immunity, increasing evidence indicates that engagement of TLRs can promote cancer cell survival and proliferation, induce tumor immune evasion, and enhance tumor metastasis and chemoresistance. Recent studies have demonstrated that endogenous molecules or damage-associated molecular patterns released from damaged/necrotic tissues are capable of activating TLRs and that the endogenous ligands-mediated TLR signaling is implicated in the tumor development and affects the therapeutic efficacy of tumors. Since both exogenous and endogenous TLR ligands can initiate TLR signaling, which is the most valuable player in tumor development becomes an interesting question. Here, we summarize the effect of TLR signaling on the development and progression of tumors, and discuss the role of exogenous and endogenous TLR ligands in the tumorigenesis.     

慢性酒精性胰腺炎中CD14、TLR4、TNFα的表达

目的 研究酒精性慢性胰腺炎组织中白细胞分化抗原14(CD14)、钟样受体4(TLR4)、肿瘤坏死因子(TNFα)的表达,探讨酒精性慢性胰腺炎的发病机制.方法 24只1月龄雄性SD大鼠随机分为对照组、脂多糖组、酒精组、酒精联合脂多糖组(以下简称联合组)各6只.酒精组和联合组饲以25％酒精,饮酒12用后联合组和脂多糖组,反复腹腔注射脂多糖2 mg/kg·w,共4次.用免疫组化及RT-PCR检测CD14、TLR4、TNF在各组的表达.结果 酒精组CD14、TLR4和TNF表达较对照组和脂多糖组增加(P＜0.05),联合组CD14、TLR4和TNF表达较对照组、脂多糖组明显增加(P＜0.01),较酒精组增加(P＜0.05).结论 酒精性慢性胰腺炎组织中CD14、TLR4和TNF表达增加,脂多糖通路可能参与了慢性胰腺炎发生发展.     

Involvement of xanthine oxidase and hypoxia-inducible factor 1 in Toll-like receptor 7/8-mediated activation of caspase 1 and interleukin-1β

Inflammatory reactions to ssRNA viruses are induced by the endosomal Toll-like receptors (TLRs) 7 and 8. TLR7/8-mediated inflammatory reaction results in activation of the Nalp3 inflammasome via an unknown mechanism. Here we report for the first time that TLR7/8 mediate activation of xanthine oxidase (XOD) in an HIF-1α-dependent manner. XOD produces uric acid and reactive oxygen species, which could activate Nalp3 and therefore induce activation of caspase 1, known to convert inactive pro-IL-1β into active IL-1β. Specific inhibition of the XOD activity attenuates TLR7/8-mediated activation of caspase 1 and IL-1β release. These results were obtained using human THP-1 myeloid macrophages. The findings were verified by conducting in vivo experiments on mice.     

Primary cilia elongation in response to interleukin-1 mediates the inflammatory response

Primary cilia are singular, cytoskeletal organelles present in the majority of mammalian cell types where they function as coordinating centres for mechanotransduction, Wnt and hedgehog signalling. The length of the primary cilium is proposed to modulate cilia function, governed in part by the activity of intraflagellar transport (IFT). In articular cartilage, primary cilia length is increased and hedgehog signaling activated in osteoarthritis (OA). Here, we examine primary cilia length with exposure to the quintessential inflammatory cytokine interleukin-1 (IL-1), which is up-regulated in OA. We then test the hypothesis that the cilium is involved in mediating the downstream inflammatory response. Primary chondrocytes treated with IL-1 exhibited a 50% increase in cilia length after 3 h exposure. IL-1-induced cilia elongation was also observed in human fibroblasts. In chondrocytes, this elongation occurred via a protein kinase A (PKA)-dependent mechanism. G-protein coupled adenylate cyclase also regulated the length of chondrocyte primary cilia but not downstream of IL-1. Chondrocytes treated with IL-1 exhibit a characteristic increase in the release of the inflammatory chemokines, nitric oxide and prostaglandin E2. However, in cells with a mutation in IFT88 whereby the cilia structure is lost, this response to IL-1 was significantly attenuated and, in the case of nitric oxide, completely abolished. Inhibition of IL-1-induced cilia elongation by PKA inhibition also attenuated the chemokine response. These results suggest that cilia assembly regulates the response to inflammatory cytokines. Therefore, the cilia proteome may provide a novel therapeutic target for the treatment of inflammatory pathologies, including OA.     

Transmembrane movements of lipids

Summary Membranes allow the rapid passage of uncharged lipids. Phospholipids on the other hand diffuse very slowly from one monolayer to another with a half-time of several hours. This slow spontaneous movement in a pure lipid bilayer can be selectively modulated in biological membranes by intrinsic proteins. In microsomes, and probably in bacterial membranes, non-specific phospholipid flippases allow the rapid redistribution of newly synthesized phospholipids. In eukaryotic plasma membranes, aminophospholipid translocase selectively pumps phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the outer to the inner leaflet and establishes a permanent lipid asymmetry. The discovery of an aminophospholipid translocase in chromaffin granules proves that eukaryotic organelles may also contain lipid translocators.     

Lipid transport pathways in mammalian cells

Summary A major deficit in our understanding of membrane biogenesis in eukaryotes is the definition of mechanisms by which the lipid constituents of cell membranes are transported from their sites of intracellular synthesis to the multiplicity of membranes that constitute a typical cell. A variety of approaches have been used to examine the transport of lipids to different organelles. In many cases the development of new methods has been necessary to study the problem. These methods include cytological examination of cells labeled with fluorescent lipid analogs, improved methods of subcellular fractionation, in situ enzymology that demonstrates lipid translocation by changes in lipid structure, and cell-free reconstitution with isolated organelles. Several general patterns of lipid transport have emerged but there does not appear to be a unifying mechanism by which lipids move among different organelles. Significant evidence now exists for vesicular and metabolic energy-dependent mechanisms as well as mechanisms that are clearly independent of cellular ATP content.     

The odd couple: signal transduction and endocytosis

Cell surface receptors are used to transmit extracellular information. The activation of cell surface receptors initiates signal transduction and receptor endocytosis. Signal transduction and the endosomal transport of activated receptors require precise regulation. New concepts for the integration of endocytosis and signaling arise from recent findings that suggest bidirectional interplay of these two processes. This review discusses the following questions: (i) do activated cell surface receptors modify the endosomal system to promote internalization and endosomal traffic, and (ii) do internalized cell surface receptors use specifically localized signaling complexes to generate specific biological signals?     

DiC14-amidine confers new anti-inflammatory properties to phospholipids

The inflammatory effect of unmethylated CpG DNA sequences represents a major obstacle to the use of cationic lipids for in vivo gene therapy. Although the mechanism of CpG-induced inflammatory response is rather well understood nowadays, few solutions have been designed to circumvent this effect in gene therapy experiments. Our previous work has shown that a refractory state towards inflammation can be elicited by preinjecting cationic liposomes. Here, we present evidence that diC14-amidine liposomes confer new anti-inflammatory properties to phospholipids from low-density lipoprotein (LDL) and even to synthetic phospholipids for which such an observation has not been reported so far. Whereas oxidation of LDL lipids was a prerequisite for any anti-inflammatory activity, lipid oxidation is no longer required in our experiments, suggesting that cationic lipids transport phospholipids through a different route and affect different pathways.This opens up new possibilities for manipulating inflammatory responses in gene therapy protocols but also in a general manner in immunological experiments. Received 12 November 2007; received after revision 4 December 2007; accepted 4 December 2007     

Transmembrane movements of lipids

Membranes allow the rapid passage of unchanged lipids. Phospholipids on the other hand diffuse very slowly from one monolayer to another with a half-time of several hours. This slow spontaneous movement in a pure lipid bilayer can be selectively modulated in biological membranes by intrinsic proteins. In microsomes, and probably in bacterial membranes, non-specific phospholipid flippases allow the rapid redistribution of newly synthesized phospholipids. In eukaryotic plasma membranes, aminophospholipid translocase selectively pumps phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the outer to the inner leaflet and establishes a permanent lipid asymmetry. The discovery of an aminophospholipid translocase in chromaffin granules proves that eukaryotic organelles may also contain lipid translocators.     

Response of vascular mesenchymal stem/progenitor cells to hyperlipidemia

Hyperlipidemia is a risk factor for atherosclerosis that is characterized by lipid accumulation, inflammatory cell infiltration, and smooth muscle cell proliferation. It is well known that hyperlipidemia is a stimulator for endothelial dysfunction and smooth muscle cell migration during vascular disease development. Recently, it was found that vessel wall contains a variable number of mesenchymal stem cells (MSCs) that are quiescent in physiological conditions, but can be activated by a variety of stimuli, e.g., increased lipid level or hyperlipidemia. Vascular MSCs displayed characteristics of stem cells which can differentiate into several types of cells, e.g., smooth muscle cells, adipocytic, chondrocytic, and osteocytic lineages. In vitro, lipid loading can induce MSC migration and chemokines secretion. After MSC migration into the intima, they play an essential role in inflammatory response and cell accumulation during the initiation and progression of atherosclerosis. In addition, MSC transplantation has been explored as a therapeutic approach to treat atherosclerosis in animal models. In this review, we aim to summarize current progress in characterizing the identity of vascular MSCs and to discuss the mechanisms involved in the response of vascular stem/progenitor cells to lipid loading, as well as to explore therapeutic strategies for vascular diseases and shed new light on regenerative medicine.     

Endoplasmic reticulum stress responses

In homeostasis, cellular processes are in a dynamic equilibrium. Perturbation of homeostasis causes stress. In this review I summarize how perturbation of three major functions of the endoplasmic reticulum (ER) in eukaryotic cells–protein folding, lipid and sterol biosynthesis, and storing intracellular Ca²⁺ – causes ER stress and activates signaling pathways collectively termed the unfolded protein response (UPR). I discuss how the UPR reestablishes homeostasis, and summarize our current understanding of how the transition from protective to apoptotic UPR signaling is controlled, and how the UPR induces inflammatory signaling. Received 21 August 2007; received after revision 26 October 2007; accepted 29 October 2007     

Purinergic systems in microglia

Accumulating findings indicate that nucleotides play an important role in microglia through P2 purinoceptors. P2 purinoceptors are divided into two families, ionotropic receptors (P2X) and metabotropic receptors (P2Y). P2X receptors (7 types; P2X₁ – P2X₇) contain intrinsic pores that open by binding with ATP. P2Y receptors (8 types; P2Y_{1, 2, 4, 6, 11, 12, 13 and 14}) are activated by nucleotides and couple to intracellular second-messenger systems through heteromeric G-proteins. Nucleotides are released or leaked from non-excitable cells as well as neurons in physiological and pathophysiological conditions. Microglia express many types of P2 purinoceptors and are known as resident macrophages in the CNS. ATP and other nucleotides work as ‘warning molecules’ especially through activating microglia in pathophysiological conditions. Microglia play a key role in neuropathic pain, chemotaxis and phagocytosis through nucleotide-evoked activation of P2X₄, P2Y₁₂ and P2Y₆ receptors, respectively. These findings indicate that extracellular nucleotides are important players in the central stage of microglial function. Received 19 April 2008; received after revision 20 May 2008; accepted 23 May 2008     

Purinergic regulation of neutrophil chemotaxis

Chemotaxis allows polymorphonuclear neutrophils (PMN) to rapidly reach infected and inflamed sites. However, excessive influx of PMN damages host tissues. Better knowledge of the mechanisms that control PMN chemotaxis may lead to improved treatments of inflammatory diseases. Recent findings suggest that ATP and adenosine are involved in PMN chemotaxis. Therefore, these purinergic signaling processes may be suitable targets for novel therapeutic approaches to ameliorate host tissue damage.