Effect of docosahexaenoic acid on interleukin-2 receptor signaling pathway in lipid rafts

PUFAs have been shown to mediate immune re-sponse especially the functions of T cells[1]. Recent researches have demonstrated that PUFAs can in-crease membrane fluidity and modify the functions of membrane receptors and enzymes in T cell membra-ne[2,3]. M…     

Dietary docosahexaenoic acid suppresses T cell protein kinase C theta lipid raft recruitment and IL-2 production

To date, the proximal molecular targets through which dietary n-3 polyunsaturated fatty acids (PUFA) suppress the inflammatory process have not been elucidated. Because cholesterol and sphingolipid-enriched rafts have been proposed as platforms for compartmentalizing dynamically regulated signaling assemblies at the plasma membrane, we determined the in vivo effects of fish oil and highly purified docosahexaenoic acid (DHA; 22:6n-3) on T cell microdomain lipid composition and the membrane subdomain distribution of signal-transducing molecules (protein kinase C (PKC)theta;, linker for activation of T cells, and Fas/CD95), before and after stimulation. Mice were fed diets containing 5 g/100 g corn oil (control), 4 g/100 g fish oil (contains a mixture of n-3 PUFA) plus 1 g/100 g corn oil, or 4 g/100 g corn oil plus 1 g/100 g DHA ethyl ester for 14 days. Dietary n-3 PUFA were incorporated into splenic T cell lipid raft and soluble membrane phospholipids, resulting in a 30% reduction in raft sphingomyelin content. In addition, polyclonal activation-induced colocalization of PKCtheta; with lipid rafts was reduced by n-3 PUFA feeding. With respect to PKCtheta; effector pathway signaling, both AP-1 and NF-kappaB activation, IL-2 secretion, and lymphoproliferation were inhibited by fish oil feeding. Similar results were obtained when purified DHA was fed. These data demonstrate for the first time that dietary DHA alters T cell membrane microdomain composition and suppresses the PKCtheta; signaling axis.     

n-3 PUFA and membrane microdomains: a new frontier in bioactive lipid research

In recent years, our understanding of the plasma membrane has changed considerably as our knowledge of lipid microdomains has expanded. Lipid microdomains include structures known as lipid rafts and caveolae, which are readily identified by their unique lipid constituents. Cholesterol, sphingolipids and phospholipids with saturated fatty acyl chain moieties are highly enriched in these lipid microdomains. Lipid rafts and caveolae have been shown to play an important role in the compartmentalization, modulation and integration of cell signaling. Therefore, these microdomains may have an influential role in human disease. Dietary n-3 polyunsaturated fatty acids (PUFA) ameliorate a number of human diseases including coronary heart disease, autoimmune and inflammatory disorders, diabetes, obesity and cancer, which has been generally linked to its membrane remodeling properties. Recent in vitro evidence suggests that perturbations in membrane composition alter the function of resident proteins and, consequently, cellular responses. This review examines the role of n-3 PUFA in modulating the lipid composition and functionality of lipid microdomains and its potential significance to human health.     

Plasma membrane rafts and chaperones in cytokine/STAT signaling

We and others have recently obtained data suggesting that cytokine-STAT signaling in many different cell-types is a chaperoned pathway initiated at the level of specialized plasma membrane microdomains called "rafts" (the "raft-STAT signaling hypothesis"). These findings are of broad significance in that all cytokines and growth factors initiate signaling in target cells by interacting with respective cell-surface receptors. The new data suggest that raft microdomains represent the units of function at the cell-surface through which ligand-stimulated STAT signaling is initiated. Moreover, recent evidence shows the involvement of chaperone proteins in regulating the STAT signaling pathway. These chaperones include the human homolog of the tumorous imaginal disc 1 protein (hTid1) which associates with Janus kinase 2 (JAK2) at the level of the plasma membrane, heat shock protein 90 (HSP90) which associates with STAT3 and STAT1 proteins in caveolin-1-containing raft and cytoplasmic complexes, and glucose regulated protein 58 (GRP58/ER-60/ERp57), a thiol dependent protein-disulfide isomerase, found in association with STAT3 "statosome" complexes in the cytosol and in the raft fraction. We suggest a function of the HSP90 chaperone system in preserving IL-6/STAT3 signaling in liver cells in the context of fever. The identification and function of protein partners associated with specific STAT species in rafts and in cytosolic complexes, and in the efficient departure of cytokine-activated STATs from the cytosolic face of rafts towards the cell nucleus are now areas of active investigation.     

Docosahexaenoic acid alters the size and distribution of cell surface microdomains

We recently generated nutritional data suggesting that chemoprotective dietary n-3 polyunsaturated fatty acids (n-3 PUFA) are capable of displacing acylated proteins from lipid raft microdomains in vivo [D.W. Ma, J. Seo, L.A. Davidson, E.S. Callaway, Y.Y. Fan, J.R. Lupton, R.S. Chapkin, n-3 PUFA alter caveolae lipid composition and resident protein localization in mouse colon, FASEB J. 18 (2004) 1040-1042; Y.Y. Fan, L.H. Ly, R. Barhoumi, D.N. McMurray, R.S. Chapkin, Dietary docosahexaenoic acid suppresses T cell protein kinase Ctheta lipid raft recruitment and IL-2 recruitment, J. Immunol. 173 (2004) 6151-6160]. A primary source of very long chain n-3 PUFA in the diet is derived from fish enriched with docosahexaenoic acid (DHA, 22:6n-3). In this study, we sought to determine the effect of DHA on cell surface microdomain organization in situ. Using immuno-gold electron microscopy of plasma membrane sheets coupled with spatial point analysis of validated microdomain markers, morphologically featureless microdomains were visualized in HeLa cells at high resolution. Clustering of probes within cholesterol-dependent (GFP-tH) versus cholesterol-independent (GFP-tK) nanoclusters was differentially sensitive to n-3 PUFA treatment of cells. Univariate K-function analysis of GFP-tH (5 nm gold) revealed a significant increase in clustering (p<0.05) by pre-treatment with DHA and linoleic acid (LA, 18:2(Delta9,12)) compared to control fatty acids; whereas LA significantly (p<0.05) reduced GFP-tK clustering. These novel data suggest that the plasma membrane organization of inner leaflets is fundamentally altered by PUFA-enrichment. We speculate that our findings may help define a new paradigm to better understand the complexity of n-3 PUFA modulation of signaling networks.     

Leptin介导的JAK/STAT信号通路对脂类代谢调节的研究进展

Leptin介导的JAK/STAT信号通路主要参与脂类代谢的调节。JAK/STAT信号通路激活后,CPT-1的表达水平升高,通过促进脂肪酸分解而参与脂类代谢的调节。本文主要介绍了近年来关于leptin介导的JAK/STAT信号通路的组成、作用机制、活性调节和leptin与受体结合激活细胞内多个信号通路如JAK/STAT、PI3K/Akt、MAPK等,以及这些信号通路对脂类代谢调节的最新研究进展。     

Diet-induced docosahexaenoic acid non-raft domains and lymphocyte function

Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid (PUFA) that generally suppresses the function of T lymphocytes and antigen presenting cells (APCs). An emerging mechanism by which DHA modifies lymphocyte function is through changes in the organization of sphingolipid/cholesterol lipid raft membrane domains. Two contradictory models have been proposed to explain how DHA exerts its effects through changes in raft organization. The biophysical model, developed in model membranes, shows that DHA-containing phospholipids form unique non-raft membrane domains, that are organizationally distinct from lipid rafts, which serve to alter the conformation and/or lateral organization of lymphocyte proteins. In contrast, the cellular model on DHA and rafts shows that DHA suppresses lymphocyte function, in part, by directly incorporating into lipid rafts and altering protein activity. To reconcile opposing biophysical and cellular viewpoints, a major revision to existing models is presented herein. Based largely on quantitative microscopy data, it is proposed that DHA, consumed through the diet, modifies lymphocyte function, in part, through the formation of nanometer scale DHA-rich domains. These nano-scale domains disrupt the optimal raft-dependent clustering of proteins necessary for initial signaling. The data covered in this review highlights the importance of understanding how dietary n-3 PUFAs modify lymphocyte membranes, which is essential toward developing these fatty acids as therapeutic agents for treating inflammatory diseases.     

Docosahexaenoic acid alters the size and distribution of cell surface microdomains

We recently generated nutritional data suggesting that chemoprotective dietary n-3 polyunsaturated fatty acids (n-3 PUFA) are capable of displacing acylated proteins from lipid raft microdomains in vivo [D.W. Ma, J. Seo, L.A. Davidson, E.S. Callaway, Y.Y. Fan, J.R. Lupton, R.S. Chapkin, n-3 PUFA alter caveolae lipid composition and resident protein localization in mouse colon, FASEB J. 18 (2004) 1040-1042; Y.Y. Fan, L.H. Ly, R. Barhoumi, D.N. McMurray, R.S. Chapkin, Dietary docosahexaenoic acid suppresses T cell protein kinase Cθ lipid raft recruitment and IL-2 recruitment, J. Immunol. 173 (2004) 6151-6160]. A primary source of very long chain n-3 PUFA in the diet is derived from fish enriched with docosahexaenoic acid (DHA, 22:6n-3). In this study, we sought to determine the effect of DHA on cell surface microdomain organization in situ. Using immuno-gold electron microscopy of plasma membrane sheets coupled with spatial point analysis of validated microdomain markers, morphologically featureless microdomains were visualized in HeLa cells at high resolution. Clustering of probes within cholesterol-dependent (GFP-tH) versus cholesterol-independent (GFP-tK) nanoclusters was differentially sensitive to n-3 PUFA treatment of cells. Univariate K-function analysis of GFP-tH (5 nm gold) revealed a significant increase in clustering (p < 0.05) by pre-treatment with DHA and linoleic acid (LA, 18:2^Δ9,12) compared to control fatty acids; whereas LA significantly (p < 0.05) reduced GFP-tK clustering. These novel data suggest that the plasma membrane organization of inner leaflets is fundamentally altered by PUFA-enrichment. We speculate that our findings may help define a new paradigm to better understand the complexity of n-3 PUFA modulation of signaling networks.     

Glucocorticoids alter the lipid and protein composition of membrane rafts of a murine T cell hybridoma

Glucocorticoids (GC) are widely used anti-inflammatory agents known to suppress T cell activation by interfering with the TCR activation cascade. The attenuation of early TCR signaling events by these compounds has been recently attributed to a selective displacement of key signaling proteins from membrane lipid rafts. In this study, we demonstrate that GC displace the acyl-bound adaptor proteins linker for activation of T cells and phosphoprotein associated with glycosphingolipid-enriched microdomains from lipid rafts of murine T cell hybridomas, possibly by inhibiting their palmitoylation status. Analysis of the lipid content of the membrane rafts revealed that GC treatment led to a significant decrease in palmitic acid content. Moreover, we found an overall decrease in the proportion of raft-associated saturated fatty acids. These changes were consistent with a decrease in fluorescence anisotropy of isolated lipid rafts, indicating an increase in their fluidity. These findings identify the mechanisms underlying the complex inhibitory effects of glucocorticoids on early TCR signaling and suggest that some of the inhibitory properties of GC on T cell responses may be related to their ability to affect the membrane lipid composition and the palmitoylation status of important signaling molecules.     

Increased membrane cholesterol in lymphocytes diverts T-cells toward an inflammatory response

Cell signaling for T-cell growth, differentiation, and apoptosis is initiated in the cholesterol-rich microdomains of the plasma membrane known as lipid rafts. Herein, we investigated whether enrichment of membrane cholesterol in lipid rafts affects antigen-specific CD4 T-helper cell functions. Enrichment of membrane cholesterol by 40-50% following squalene administration in mice was paralleled by an increased number of resting CD4 T helper cells in periphery. We also observed sensitization of the Th1 differentiation machinery through co-localization of IL-2Rα, IL-4Rα, and IL-12Rβ2 subunits with GM1 positive lipid rafts, and increased STAT-4 and STAT-5 phosphorylation following membrane cholesterol enrichment. Antigen stimulation or CD3/CD28 polyclonal stimulation of membrane cholesterol-enriched, resting CD4 T-cells followed a path of Th1 differentiation, which was more vigorous in the presence of increased IL-12 secretion by APCs enriched in membrane cholesterol. Enrichment of membrane cholesterol in antigen-specific, autoimmune Th1 cells fostered their organ-specific reactivity, as confirmed in an autoimmune mouse model for diabetes. However, membrane cholesterol enrichment in CD4(+)Foxp3(+) T-reg cells did not alter their suppressogenic function. These findings revealed a differential regulatory effect of membrane cholesterol on the function of CD4 T-cell subsets. This first suggests that membrane cholesterol could be a new therapeutic target to modulate the immune functions, and second that increased membrane cholesterol in various physiopathological conditions may bias the immune system toward an inflammatory Th1 type response.     

Polyunsaturated eicosapentaenoic acid displaces proteins from membrane rafts by altering raft lipid composition

Polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (20:5 (n-3)) inhibit T lymphocyte activation probably by displacing acylated signaling proteins from membrane lipid rafts. Under physiological conditions, saturated fatty acyl residues of such proteins partition into the cytoplasmic membrane lipid leaflet with high affinity for rafts that are enriched in saturated fatty acyl-containing lipids. However, the biochemical alteration causing displacement of acylated proteins from rafts in PUFA-treated T cells is still under debate but could principally be attributed to altered protein acylation or changes in raft lipid composition. We show that treatment of Jurkat T cells with polyunsaturated eicosapentaenoic acid (20:5 (n-3)) results in marked enrichment of PUFAs (20:5; 22:5) in lipids from isolated rafts. Moreover, PUFAs were significantly incorporated into phosphatidylethanolamine that predominantly resides in the cytoplasmic membrane lipid leaflet. Notably, palmitate-labeled Src family kinase Lck and the linker for activation of T cells (LAT) were both displaced from lipid rafts indicating that acylation by PUFAs is not required for protein displacement from rafts in PUFA-treated T cells. In conclusion, these data provide strong evidence that displacement of acylated proteins from rafts in PUFA-treated T cells is predominantly due to altered raft lipid composition.     

The temperature-pressure phase diagram of a DPPC-ergosterol fungal model membrane -- a SAXS and FT-IR spectroscopy study

What distinguishes polyunsaturated fatty acids (PUFAs) from less unsaturated fatty acids is the presence of a repeating CH–CH₂–CH unit that produces an extremely flexible structure rapidly isomerizing through conformational states. Docosahexaenoic acid (DHA) with 6 double bonds is the most extreme example. The focus of this review is the profound impact that the high disorder of DHA has on its interaction with cholesterol when the PUFA is incorporated into membrane phospholipids. Results from a battery of biophysical techniques are described. They demonstrate an aversion of DHA for the sterol that drives the lateral segregation of DHA-containing phospholipids into liquid disordered (l_d) domains that are depleted in cholesterol. These domains are compositionally and organizationally the antithesis of lipid rafts, the much-studied liquid ordered (l_o) domain that is enriched in predominantly saturated sphingolipids and cholesterol. We hypothesize that the introduction of DHA-rich domains into the plasma membrane where they coexist with lipid rafts is the origin, in part, of the astonishing diversity of health benefits that accrue from dietary consumption of DHA. According to our model, changes in the conformation of signaling proteins when they move between these disparate domains have the potential to modulate cell function.     

Chemical–Physical Changes in Cell Membrane Microdomains of Breast Cancer Cells After Omega-3 PUFA Incorporation

Epidemiologic and experimental studies suggest that dietary fatty acids influence the development and progression of breast cancer. However, no clear data are present in literature that could demonstrate how n?-?3 PUFA can interfere with breast cancer growth. It is suggested that these fatty acids might change the structure of cell membrane, especially of lipid rafts. During this study we treated MCF-7 and MDA-MB-231 cells with AA, EPA, and DHA to assess if they are incorporated in lipid raft phospholipids and are able to change chemical and physical properties of these structures. Our data demonstrate that PUFA and their metabolites are inserted with different yield in cell membrane microdomains and are able to alter fatty acid composition without decreasing the total percentage of saturated fatty acids that characterize these structures. In particular in MDA-MB-231 cells, that displays the highest content of Chol and saturated fatty acids, we observed the lowest incorporation of DHA, probably for sterical reasons; nevertheless DHA was able to decrease Chol and SM content. Moreover, PUFA are incorporated in breast cancer lipid rafts with different specificity for the phospholipid moiety, in particular PUFA are incorporated in PI, PS, and PC phospholipids that may be relevant to the formation of PUFA metabolites (prostaglandins, prostacyclins, leukotrienes, resolvines, and protectines) of phospholipids deriving second messengers and signal transduction activation. The bio-physical changes after n?-?3 PUFA incubation have also been highlighted by atomic force microscopy. In particular, for both cell lines the DHA treatment produced a decrease of the lipid rafts in the order of about 20-30?%. It is worth noticing that after DHA incorporation lipid rafts exhibit two different height ranges. In fact, some lipid rafts have a higher height of 6-6.5?nm. In conclusion n?-?3 PUFA are able to modify lipid raft biochemical and biophysical features leading to decrease of breast cancer cell proliferation probably through different mechanisms related to acyl chain length and unsaturation. While EPA may contribute to cell apoptosis mainly through decrease of AA concentration in lipid raft phospholipids, DHA may change the biophysical properties of lipid rafts decreasing the content of cholesterol and probably the distribution of key proteins.     

Constitutive localization of the gonadotropin-releasing hormone (GnRH) receptor to low density membrane microdomains is necessary for GnRH signaling to ERK

Specialized membrane microdomains known as lipid rafts are thought to contribute to G-protein coupled receptor (GPCR) signaling by organizing receptors and their cognate signaling molecules into discrete membrane domains. To determine if the GnRHR, an unusual member of the GPCR superfamily, partitions into lipid rafts, homogenates of alpha T3-1 cells expressing endogenous GnRHR or Chinese hamster ovary cells expressing an epitope-tagged GnRHR were fractionated through a sucrose gradient. We found the GnRHR and c-raf kinase constitutively localized to low density fractions independent of hormone treatment. Partitioning of c-raf kinase into lipid rafts was also observed in whole mouse pituitary glands. Consistent with GnRH induced phosphorylation and activation of c-raf kinase, GnRH treatment led to a decrease in the apparent electrophoretic mobility of c-raf kinase that partitioned into lipid rafts compared with unstimulated cells. Cholesterol depletion of alpha T3-1 cells using methyl-beta-cyclodextrin disrupted GnRHR but not c-raf kinase association with rafts and shifted the receptor into higher density fractions. Cholesterol depletion also significantly attenuated GnRH but not phorbol ester-mediated activation of extracellular signal-related kinase (ERK) and c-fos gene induction. Raft localization and GnRHR signaling to ERK and c-Fos were rescued upon repletion of membrane cholesterol. Thus, the organization of the GnRHR into low density membrane microdomains appears critical in mediating GnRH induced intracellular signaling.     

Leptin介导JAK/STAT信号通路对猪皮下前脂肪细胞脂类代谢调控的研究

以猪原代皮下前脂肪细胞为研究材料,检测Leptin介导JAK/STAT信号通路中基因表达水平,旨在阐明Leptin介导JAK/STAT信号通路对脂肪代谢的分子机制.用0和100 ng/mL Leptin分别处理脂肪细胞48 h,油红O染色鉴定脂肪细胞,试剂盒测定细胞中甘油三酯和游离脂肪酸含量,Real-time PCR...