BACKGROUND: Cholesterol is a major component of specialized membrane microdomains known as lipid rafts or caveolae, which modulate the fluidity of biological membranes. Membrane cholesterol therefore plays an important role in cell signaling and vesicular transport. OBJECTIVE: In this study, we investigated the effects of cholesterol on matrix metalloproteinase-1 (MMP-1) expression in human dermal fibroblasts. METHODS: MMP-1 mRNA and protein expression were determined by RT-PCR and Western blotting, respectively. AP-1 DNA binding activity was detected by electrophoretic mobility shift assays. The amount of cholesterol was analyzed by cholesterol assay kit. RESULTS: We observed that MMP-1 mRNA and protein expression was dose-dependently decreased by cholesterol treatment. In contrast, cholesterol depletion by a cholesterol depletion agent, methyl-beta-cyclodextrin (M beta CD) in human dermal fibroblasts, increased MMP-1 mRNA and protein expression in a dose-dependent manner. Also, we investigated the regulatory mechanism of M beta CD-induced MMP-1 expression: cholesterol depletion by M beta CD, activated ERK1/2 and JNK, but not p38 MAPK cascade, and it also significantly increased c-Jun phosphorylation, c-Fos expression and activator protein-1 binding activity. Furthermore, the inhibition of ERK or JNK with specific chemical inhibitors prevented M beta CD-induced MMP-1 expression, which indicates that ERK and JNK play an important role in cholesterol depletion-mediated MMP-1 induction. In addition, M beta CD-induced phosphorylation of ERK and JNK and MMP-1 expression were suppressed by cholesterol repletion. CONCLUSION: Our results suggest that cholesterol regulates MMP-1 expression through the control of ERK and JNK activity in human dermal fibroblasts.
beta-Cyclodextrins ; Blotting, Western ; Caveolae ; Cholesterol ; DNA ; Electrophoretic Mobility Shift Assay ; Fibroblasts ; Humans ; Matrix Metalloproteinase 1 ; Membrane Microdomains ; Membranes ; p38 Mitogen-Activated Protein Kinases ; Phosphorylation ; RNA, Messenger ; Transcription Factor AP-1

Functional membrane microdomains (FMMs) that are mainly composed of scaffold proteins and polyisoprenoids play important roles in diverse cellular physiological processes in bacteria. The aim of this study was to identify the correlation between MK-7 and FMMs and then regulate the MK-7 biosynthesis through FMMs. Firstly, the relationship between FMMs and MK-7 on the cell membrane was determined by fluorescent labeling. Secondly, we demonstrated that MK-7 is a key polyisoprenoid component of FMMs by analyzing the changes in the content of MK-7 on cell membrane and the changes in the membrane order before and after destroying the integrity of FMMs. Subsequently, the subcellular localization of some key enzymes in MK-7 synthesis was explored by visual analysis, and the intracellular free pathway enzymes Fni, IspA, HepT and YuxO were localized to FMMs through FloA to achieve the compartmentalization of MK-7 synthesis pathway. Finally, a high MK-7 production strain BS3AT was successfully obtained. The production of MK-7 reached 300.3 mg/L in shake flask and 464.2 mg/L in 3 L fermenter.
Bacillus subtilis/metabolism* ; Vitamin K 2/metabolism* ; Bioreactors/microbiology* ; Membrane Microdomains/metabolism*

Rafts, cholesterol- and sphingolipid-rich membrane microdomains, have been shown to play an important role in immune cell activation. More recently rafts were implicated in the signal transduction by members of the TNF receptor (TNFR) family. In this study, we provide evidences that the raft microdomain has a crucial role in RANK (receptor activator of NF-kappaB) signaling. We found that the majority of the ectopically expressed RANK and substantial portion of endogenous TRAF2 and TRAF6 were detected in the low-density raft fractions. In addition, TRAF6 association with rafts was increased by RANKL stimulation. The disruption of rafts blocked the TRAF6 translocation by RANK ligand and impeded the interaction between RANK and TRAF6. Our observations demonstrate that proper RANK signaling requires the function of raft membrane microdomains.
Carrier Proteins/metabolism ; Glycoproteins/*metabolism ; Human ; Membrane Glycoproteins/metabolism ; Membrane Microdomains/*metabolism ; Protein Transport/physiology ; Proteins/*metabolism ; Receptors, Cytoplasmic and Nuclear/*metabolism

Neuronal apoptosis induced by amyloid beta-peptide (A beta) plays an important role in the pathophysiology of Alzheimer's disease (AD). However, the molecular mechanism underlying A beta-induced apoptosis remains undetermined. The disialoganglioside GD3 involves ceramide-, Fas- and TNF-alpha-mediated apoptosis in lymphoid cells and hepatocytes. Although the implication of GD3 has been suggested, the precise role of GD3 in A beta-induced apoptosis is still unclear. Here, we investsigated the changes of GD3 metabolism and characterized the distribution and trafficking of GD3 during A beta-induced apoptosis using human brain-derived TE671 cells. Extracellular A beta induced apoptosis in a mitochondrial-dependent manner. GD3 level was negligible in the basal condition. However, in response to extracellular A beta, both the expression of GD3 synthase mRNA and the intracellular GD3 level were dramatically increased. Neosynthesized GD3 rapidly accumulated in cell surface lipid microdomains, and was then translocated to mitochondria to execute the apoptosis. Disruption of membrane lipid microdomains with methyl-beta-cyclodextrin significantly prevented both GD3 accumulation in cell surface and A beta-induced apoptosis. Our data suggest that rapidly accumulated GD3 in plasma membrane lipid microdomains prior to mitochondrial translocation is one of the key events in A beta-induced apoptosis.
Amyloid beta-Peptides/*pharmacology ; *Apoptosis ; Cell Line ; Gangliosides/*metabolism/physiology ; Humans ; Membrane Microdomains/*metabolism ; Mitochondria/*metabolism ; Sialyltransferases/genetics/metabolism ; beta-Cyclodextrins/pharmacology

To explore the role of lipid raft in assembly of human herpesvirus 6, the HHV-6 GS strain was applied to infect the HSB2 cells and then the lipid raft composition was extracted from the cells with non-ionic detergent Triton-X 100. The relationship between the HHV-6 envelope glycoprotein and lipid raft was analyzed by Western Blot. Immunofluorescence double-staining was used to study the colocalization of the HHV-6 glycoprotein B(gB) with GPI anchored protein CD59 and ganglioside GM respectively. HHV-6 envelope glycoprotein B, H, L, Q1 and Q2 (gB, gH, gL, gQ1 and gQ2) were all existed in the lipid raft. Moreover, CD59 and HHV-6 envelope glycoprotein B showed the same localization through the confocal microscope. We concluded the lipid raft provided the platform for HHV-6 assembly. This is the first report concerning to the role of lipid raft in assembly of human Herpesvirus 6.
CD59 Antigens ; analysis ; Fluorescent Antibody Technique ; G(M1) Ganglioside ; analysis ; Herpesvirus 6, Human ; physiology ; Membrane Microdomains ; physiology ; Viral Envelope Proteins ; analysis ; Virus Assembly

OBJECTIVETo reveal the transmembrane signal pathway participating in regulating neuron functions of treating Alzheimer's disease (AD) by acupuncture.

METHODSSAMP8 mice was used for AD animal model. The effect of acupuncture method for qi benefiting, blood regulating, health supporting, and root strengthening on the amount and varieties of transmembrane signal proteins from hippocampal lipid rafts in SAMP8 mice was detected using HPLC MS/MS proteomics method.

RESULTSCompared with the control group, acupuncture increased 39 transmembrane signal proteins from hippocampal lipid rafts in SAMP8 mice, of them, 14 belonged to ionophorous protein, 8 to G protein, 8 to transmembrane signal receptor, and 9 to kinase protein. Totally 3 main cell signal pathways were involved, including G-protein-coupled receptors signal, enzyme linked receptor signal, and ion-channel mediated signal. Compared with the sham-acupuncture group, acupuncture resulted in significant increase of kinase signal protein amount. From the aspect of functions, they were dominant in regulating synapse functions relevant to cytoskeleton and secreting neurotransmitters.

CONCLUSIONThe cell biological mechanism for treating AD by acupuncture might be achieved by improving synapse functions and promoting the secretion of neurotransmitters through transmembrane signal transduction, thus improving cognitive function of AD patients.

Acupuncture Therapy ; Alzheimer Disease ; metabolism ; Animals ; Disease Models, Animal ; Female ; Male ; Membrane Microdomains ; metabolism ; Mice ; Proteomics ; Signal Transduction ; Tandem Mass Spectrometry

Galectin-4, a tandem repeat member of the β-galactoside-binding proteins, possesses two carbohydrate-recognition domains (CRD) in a single peptide chain. This lectin is mostly expressed in epithelial cells of the intestinal tract and secreted to the extracellular. The two domains have 40% similarity in amino acid sequence, but distinctly binding to various ligands. Just because the two domains bind to different ligands simultaneously, galectin-4 can be a crosslinker and crucial regulator in a large number of biological processes. Recent evidence shows that galectin-4 plays an important role in lipid raft stabilization, protein apical trafficking, cell adhesion, wound healing, intestinal inflammation, tumor progression, etc. This article reviews the physiological and pathological features of galectin-4 and its important role in such processes.
Animals ; Axons ; metabolism ; Endocytosis ; Galectin 4 ; blood ; genetics ; metabolism ; Humans ; Inflammatory Bowel Diseases ; metabolism ; pathology ; Membrane Microdomains ; metabolism ; Neoplasms ; metabolism ; pathology ; Neurons ; metabolism ; Wound Healing

We investigated the co-stimulatory role of a cell-surface protein, CD99. Co-ligation of CD99 and suboptimal CD3 induced T-cell activation to a level comparable to that obtained with optimal CD3 or CD3+CD28. We also noted concomitant enhancement of the earliest T-cell receptor (TCR) signaling events. In addition, co-ligation of CD99 and CD3 led to translocation of TCR complexes into the lipid raft, without concomitant migration of CD99 to the raft, and consequent enhancement of TCR zeta-mediated signal 1. These data demonstrate the unique properties of CD99 co-stimulation that distinguish this molecule from CD28 and other raft-resident co-stimulatory factors.
Antigens, CD/*immunology ; Antigens, CD3/immunology ; Cell Adhesion Molecules/*immunology ; Down-Regulation ; Humans ; Jurkat Cells ; Lymphocyte Activation/*immunology ; Membrane Microdomains/*immunology ; Membrane Proteins/*immunology ; Phosphorylation ; Phosphotyrosine/*metabolism ; Protein Transport ; Receptors, Antigen, T-Cell/*immunology ; T-Lymphocytes/*immunology

OBJECTIVETo investigate the relationship among a 50 Hz magnetic field (MF)-induced epidermal growth factor receptor (EGFR) clustering,lipid rafts and acid sphingomyelinase (ASM), and to explore its possible mechanism.

METHODSHuman amnion FL cells were exposed to 50 Hz, 0.4 mT MF for 15 min. EGF treatment was used as positive control. Nystatin was employed to study lipid rafts since it could disrupt lipid rafts structure.The EGF receptors, ASM and lipid rafts were labeled with polyclonal anti-EGFR antibody, anti-ASM antibody and FITC-Cholera toxin B, respectively. The images were observed by laser confocal scanning microscope.

RESULTBoth EGF treatment and 50 Hz MF exposure could induce EGFR clustering; however, nystatin pretreatment disrupted this effect. MF exposure turned ASM (labeled with Cy3) from a diffused state in the sham exposure group to a concentrated state on the cell membrane, which co-localized with lipid rafts (labeled with FITC).

CONCLUSIONThe results suggest that the EGFR clustering induced by 50 Hz MF depends on intact lipid rafts on cellular membrane, and the ASM might participate in the process of EGFR clustering.

Cell Membrane ; radiation effects ; Cells, Cultured ; Electromagnetic Fields ; Epidermal Growth Factor ; metabolism ; Humans ; Membrane Microdomains ; radiation effects ; Receptor, Epidermal Growth Factor ; metabolism ; radiation effects ; Signal Transduction ; physiology ; radiation effects ; Sphingomyelin Phosphodiesterase ; metabolism

Mitochondrial biogenesis is known to accompany adipogenesis to complement ATP and acetyl-CoA required for lipogenesis. Here, we demonstrated that mitochondrial proteins such as ATP synthase alpha and beta, and cytochrome c were highly expressed during the 3T3-L1 differentiation into adipocytes. Fully-differentiated adipocytes showed a significant increase of mitochondria under electron microscopy. Analysis by immunofluorescence, cellular fractionation, and surface biotinylation demonstrated the elevated levels of ATP synthase complex found not only in the mitochondria but also on the cell surface (particularly lipid rafts) of adipocytes. High rate of ATP (more than 30 micrometer) synthesis from the added ADP and Pi in the adipocyte media suggests the involvement of the surface ATP synthase complex for the exracellular ATP synthesis. In addition, this ATP synthesis was significantly inhibited in the presence of oligomycin, an ATP synthase inhibitor, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an ATP synthase uncoupler. Decrease of extracellular ATP synthesis in acidic but not in basic media further indicates that the surface ATP synthase may also be regulated by proton gradient through the plasma membrane.
Adenosine Triphosphate/analysis/*biosynthesis ; Adipocytes/*enzymology/ultrastructure ; Animals ; Cell Differentiation/physiology ; Cell Membrane/chemistry ; Cells, Cultured ; Humans ; Membrane Microdomains/chemistry/*enzymology ; Mice ; Mitochondria/metabolism/ultrastructure ; Mitochondrial Proton-Translocating ATPases/analysis/*physiology ; Research Support, Non-U.S. Gov't