Altered 5'-nucleotidase specific activity and distribution between two plasma membrane domains of ascites tumor cells with modified lipid composition

1. The cholesterol and phospholipid content of the surface membranes of ascites tumor cells cultivated in lipid-depleted medium was reduced to about 60(70)% of the control, but the relative composition of the individual phospholipids was not altered. 2. Differences in lipid composition were also observed between the two plasma membrane domains isolated from the cells cultured in normal and lipid-depleted medium respectively. 3. The fatty acid spectrum of the lipid-depleted membranes showed a greater fraction of saturated vs unsaturated acids. 4. The membrane lipid fluidity measured by fluorescence polarization was decreased in the modified surface membranes. 5. The 5'-nucleotidase specific activity was drastically reduced (46-66%) in the lipid-deleted membranes, and in addition its distribution between the two vesicle fractions was altered.     

Induction of an endothermic transition in the Arrhenius plot of fatty acid uptake by lipid-depleted ascites tumor cells

Cultured ascites tumor cells and their lipid-depleted variants containing 35-40% less membrane phospholipid and cholesterol were used to study uptake and metabolism of fatty acids complexed to albumin. Uptake of stearate and oleate at 37 degrees C was considerably higher in the lipid-depleted cells, but no significant difference in the affinity constants for stearate uptake of 3.70 microM for the lipid-depleted and 2.50 microM for the control cells was observed. Similar rates of uptake of both cultures were observed at lower temperatures up to 30 degrees C. The drastic increase in stearate uptake above 30 degrees C resulted in an endothermic transition in the Arrhenius plot with an activation energy of 20.8 kJ/mol versus 6.5 kJ/mol for the control cells. Uptake of stearate and oleate of the control cells was only slightly reduced by metabolic inhibitors, which was similar to stearic acid transport in the lipid-depleted variants. However, oleate uptake was substantially decreased in these variants. Incorporated stearate was esterified to about 50% in both cultures, and oleate between 85 and 90%. Mainly triacylglycerols and phospholipids with phosphatidylcholine (41%) and phosphatidylethanolamine (35%) as major polar lipid components, and also lower acylglycerols and cholesterol were found to be labeled. Under lipid-depleted conditions, a pronounced increase in the relative proportion of oleate incorporation into triacylglycerols was determined. It is suggested that fatty acid uptake is controlled by the number of active sites of the putative transport protein, which increases upon lipid depletion as shown from the V values. This increase may result from the segregation of membrane-bound proteins into domains (Haeffner et al. (1986) Cell Mol. Biol. 32, 359-368), which are known to be formed as a consequence of lipid phase separation in the lipid-depleted cells.     

Estrogens induce low-density lipoprotein receptor activity and decrease intracellular cholesterol in human hepatoma cell line Hep G2

Administration of estrogens in pharmacologic doses to rats and rabbits induces hepatic low-density lipoprotein (LDL) receptor activity. To determine if estrogens can regulate LDL receptor activity in human cells, 125I-LDL binding and ligand blotting studies were performed with the cell line Hep G2, well-differentiated cells derived from a human hepatoma, and with normal human fibroblasts. Addition of estradiol to Hep G2 cells growing in lipoprotein-deficient medium increased cell surface receptor activity by 141%, whereas fibroblast receptors were slightly reduced. Measurement of LDL internalization and degradation showed that estradiol induced the entire LDL receptor pathway and not simply surface receptors for LDL. Scatchard analysis of specific binding data in Hep G2 cells revealed that increased LDL receptor activity was due to high-affinity binding. When Hep G2 cells were incubated with LDL as well as estradiol, estradiol induction of LDL receptor activity did not occur. Estrogen treatment reduced Hep G2 free cholesterol content by 24% as determined by gas-liquid chromatography but had no significant effect on fibroblast free cholesterol, suggesting that estrogens may induce Hep G2 LDL receptor activity indirectly by lowering intracellular cholesterol. LDL receptor activity in Hep G2 cells grown in the absence of estradiol was resistant to down-regulation by LDL; incubation of cells with LDL for 48 h reduced receptor activity by only 25.8% in Hep G2 cells compared to 80.3% in fibroblasts. The Hep G2 LDL receptor was shown to be biochemically similar to the fibroblast receptor by ligand blotting and immunoblotting with IgG-C7, a monoclonal antibody to the extrahepatic LDL receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence-polarization measurements on normal and mutant human skin fibroblasts

Measurements of fluorescence polarization in intact diploid skin fibroblasts after exposure to 1,6-diphenyl-1,3,5-hexatriene were used to estimate the fluidity of the lipid phase(s) of cellular membranes. The membrane lipids of cells derived from four patients with homozygous familial hypercholesterolemia were in a more fluid state than those of cells obtained from 13 other individuals of normal and nonrelated mutant genotypes when all cultures were grown on medium with native serum. The only other cell type having membrane lipids of increased fluidity under these conditions was one fibroblast line derived from a patient with the Lesch-Nyhan syndrome. Examination of two additional nonconsanguinous lines of Lesch-Nyhan fibroblasts, however, revealed that an abnormally high level of lipid fluidity was not a common property of the membranes of cells of this genotype. Incubation of cultures in medium containing lipid-depleted serum (virtually devoid of lipoprotein-bound sterol) caused a reversible increase in the fluidity of the membranes of normal cells to values similar to those of the hypercholesterolemic cells, but had no effect on the membranelipid fluidity of the latter. By contrast, exposure of cultures to cholesterol not bound to lipoprotein in serum-free medium resulted in a decrease in the lipid fluidity of the membranes of both normo- and hypercholesterolemic fibroblasts.     

Resistance to streptolysin O in mammalian cells treated with oxygenated derivatives of cholesterol cholesterol content of resistant cells and recovery of streptolysin O sensitivity

Cultures of L cells and HeLa cells were made resistant to the cytolytic toxin, streptolysin O, by incubating them in the presence of 20α-hydroxycholesterol or 25-hydroxycholesterol. Such cells were also found to be more resistant to the cytotoxic effects of saponin and digitonin, agents known to interact with membrane cholesterol. Sterol synthesis in L cells that had been treated with either of the oxygenated derivatives of cholesterol was reduced by almost 90%, and the free cholesterol content of streptolysin O-resistant HeLa and L cells fell to approx. 50% of control cell levels. Significant recovery of sensitivity to streptolysin O occurred in about 6 h when refractory L cells were incubated in serum or cholesterol. Partial recovery was observed when the cultures were incubated for 24 h in mevalonate or lipid-depleted serum. The results provide further support for the role of membrane cholesterol in the cytotoxic action of streptolysin O on mammalian cells.     

Fusion of lipid vesicles with ascites tumor cells and their lipid-depleted variants. Studies with radioactive- and fluorescent-labeled vesicles

Cultured ascites tumor cells and their lipid-depleted variants, which contained 35-40% less membrane phospholipid and cholesterol, were used for fusion experiments with unilamellar lipid vesicles which were between 300 and 600 nm in diameter. Vesicle-cell interaction was followed by tracer studies using vesicles double-labeled in the lipid moiety, by vesicle-encapsulated [3H] dextran, and by measurements of energy transfer between N-(10-[1-pyrene]decanoyl)sphingomyelin-labeled vesicles and alpha-parinaric acid-labeled cells in the presence of poly(ethylene glycol) (PEG) as fusogen. The reaction rates measured with the radiolabeled vesicles were found to follow patterns similar to those obtained with the resonance energy transfer assay. This latter method revealed a vesicle-cell membrane fusion reaction, which was substantiated by radiolabeling the internal cellular compartment after treatment of the cells with [3H]dextran-encapsulated vesicles as shown by electron microscopic autoradiography on semi-thin sections. Endocytosis as a reaction mechanism can be excluded, since no energy transfer was observed at 25 degrees C in the absence of PEG. Investigations of vesicle bilayer order and fluidity on vesicle-cell interaction revealed optimal reactivity, with intermediate fluidity corresponding to cholesterol/phospholipid ratios between 0.7 and 1.0 and fluorescence depolarization (P) values of 0.18 and 0.21. Lipid depletion decreased the reaction velocity between cells and vesicles by about 20%, exhibiting V values of 33.2 mumol/min, as compared to the control of 41.4 mumol/min determined for 10(7) cells. The affinity constants for vesicle lipid were affected only slightly with Km values of 0.195 mM (0.210 mM). The activation energies for the reaction were calculated to give values of EA = 22.44 kJ/mol for the control and of EA = 20.4 kJ/mol for the modified cells. These data indicate that the decrease in membrane lipid content apparently has no major influence on the extent of the interaction.     

The skin-reactive antigens of Mycoplasma pneumoniae.

Guinea pigs experimentally infected with Mycoplasma pneumoniae or immunized with the orgnaism in combination with Freund's complete adjuvant developed a delayed hypersensitive skin reaction following on intradermal injection of the M. pneumoniae antigen. The amount of protein necessary to produce the delayed skin reaction was as low as 0.01 mug. When the sonicated whole cells were extracted with aqueous acetone, the delayed skin reactivity was found mostly in the acetone insoluble (lipid-depleted) fraction. On the other hand, the lipid fraction which was isolated by a chloroform-methanol extraction of the acetone-soluble fraction and had a high titer of complement-fixing activity, exhibited little delayed skin reactivity. The lipid-depleted antigens as the whole cell antigens produced delayed skin reactivities in human patients.     

CXCR4 function requires membrane cholesterol: implications for HIV infection

HIV requires cholesterol and lipid rafts on target cell membranes for infection. To elucidate a possible mechanism, we determined that cholesterol extraction by hydroxypropyl-beta-cyclodextrin (BCD) inhibits stromal cell-derived factor 1alpha (SDF-1alpha) binding to CXCR4 on T cell lines and PBMCs. Intracellular calcium responses to SDF-1alpha, as well as receptor internalization, were impaired in treated T cells. Loss in ligand binding is likely due to conformational changes in CXCR4 and not increased sensitivity to internalization. SDF-1alpha binding and calcium responses were effectively restored by reloading cholesterol. Immunofluorescence microscopy revealed that SDF-1alpha binding occurred in lipid raft microdomains that contained GM1. CXCR4 surface expression, on the other hand, only partially colocalized with GM1. HIV-1(IIIB) infection assays confirmed the functional loss of CXCR4 in the cell lines tested, Sup-T1 and CEM-NKR-CCR5. These data suggest that cholesterol is essential for CXCR4 conformation and function and that lipid rafts may play a regulatory role in SDF-1alpha signaling.     

Up-regulation of high density lipoprotein receptor activity by gamma-interferon associated with inhibition of cell proliferation

High density lipoprotein (HDL) binds to cell surface receptors and promotes selective removal of excess cholesterol from intracellular pools. The activity of this receptor is up-regulated when cells become loaded with cholesterol, but the relative degree of up-regulation depends on the growth state of the cells. The current study demonstrates that treatment of proliferating fibroblasts with gamma-interferon (IFN) increases the activity of the HDL receptor in association with a decrease in the rate of cell proliferation. Addition of IFN during the growth phase reduced the number of cells but had little effect on total cell protein, indicating that IFN inhibited cell proliferation but produced larger cells. IFN treatment increased the number of high affinity receptors for HDL on the surface of cholesterol-loaded fibroblasts, whether receptor binding was expressed per cell or per unit of cell surface area, cell protein, or cell cholesterol. IFN treatment also appeared to increase the amount of 110-kDa HDL binding protein in fibroblast membranes that has been postulated to represent the HDL receptor molecule. The IFN-induced increase in HDL receptor activity was associated with an enhanced ability of HDL3 to remove cholesterol from intracellular pools. These results are consistent with the hypothesis that inhibition of cell proliferation increases HDL receptor-mediated transport of excess cholesterol from cells, possibly to rid cells of cholesterol that accumulates in response to a reduced rate of membrane synthesis.     

乙型肝炎病毒表面抗原对细胞脂质合成作用的研究

乙型肝炎病毒(hepatitis B virus,HBV)合成的蛋白调节细胞脂质代谢的研究不断被报道,但乙型肝炎病毒表面抗原(hepatitis B virus surface antigen,HBsAg)与脂质代谢的相互调控研究较少,且机制尚不明确。本研究通过对细胞转录组学的分析,揭示HBsAg对脂质代谢的调控机制。选用稳定表达HBsAg的细胞系HepG2-S-G2与其对照细胞系HepG2-neo-F4进行转录组学分析。利用定量聚合酶链反应(polymerase chain reaction,PCR)、蛋白质印迹法(Western blot,WB)分别检测重要差异基因OXCT1和CYP4F3在mRNA水平和蛋白水平的表达差异。为验证HBsAg促进脂质合成上调的表型,对两种细胞系进行油红O染色并检测细胞脂肪酸、总胆固醇水平。进一步对稳定转染HBV的细胞系HepG2.2.15进行降脂处理,以观察细胞上清液中HBsAg与脂质合成之间是否存在相互调控。结果显示,参与脂质代谢的差异基因发生显著变化,提示HBsAg引起了宿主细胞脂质合成途径的上调和消耗途径下调。定量PCR结果显示,相对于HepG2-neo-F4细胞,HepG2-S-G2细胞的3-酮酸辅酶A转移酶1(3-oxoacid CoA-transferase 1,OXCT1)mRNA水平升高约9倍,与转录组测序结果基本一致;CYP4F3基因在HepG2-S-G2细胞中转录相对下调。 WB结果显示,OXCT1和CYP4F3蛋白表达均出现相应的显著上调或下调,并且趋势与转录组分析一致。油红O染色以及细胞脂肪酸、总胆固醇水平检测结果证实HepG2-S-G2细胞中脂滴更明显,且游离脂肪酸和总胆固醇均显著升高。降脂处理结果显示细胞上清液中HBsAg显著降低。上述结果表明,HBsAg可上调脂质代谢、促进脂质合成,提示降脂可能成为抑制HBsAg的潜在有效途径。     

The Skin-Reactive Antigens of Mycoplasma pneumoniae

Guinea pigs experimentally infected with Mycoplasma pneumoniae or immunized with the organism in combination with Freund's complete adjuvant developed a delayed hypersensitive skin reaction following on intradermal injection of the M. pneumoniae antigen. The amount of protein necessary to produce the delayed skin reaction was as low as 0.01 μg. When the sonicated whole cells were extracted with aqueous acetone, the delayed skin reactivity was found mostly in the acetone insoluble (lipid-depleted) fraction. On the other hand, the lipid fraction which was isolated by a chloroform–methanol extraction of the acetone-soluble fraction and had a high titer of complement-fixing activity, exhibited little delayed skin reactivity. The lipid-depleted antigens as the whole cell antigens produced delayed skin reactivities in human patients.     

Isolation and characterization of Chinese hamster ovary cell mutants deficient in acyl-coenzyme A:cholesterol acyltransferase activity

A protocol has been developed for isolating cholesterol ester-deficient cells from the Chinese hamster ovary cell clone 25-RA. This cell line previously was shown to be partially resistant to suppression of cholesterogenic enzyme activities by 25-hydroxycholesterol and to accumulate a large amount of intracellular cholesterol ester when grown in medium containing 10% fetal calf serum (Chang, T. Y., and Limanek, J. S. (1980) J. Biol. Chem. 255, 7787-7795). The higher cholesterol ester content of 25-RA is due to an increase in the rate of cholesterol biosynthesis and low density lipoprotein receptor activity compared to wild-type Chinese hamster ovary cells, and not due to an abnormal acyl-CoA:cholesterol acyltransferase enzyme. The procedure to isolate cholesterol ester-deficient mutants utilizes amphotericin B, a polyene antibiotic known to bind to cholesterol and to form pore complexes in membranes. After incubation in cholesterol-free medium plus an inhibitor of endogenous cholesterol biosynthesis, 25-RA cells were found to be 50-500 times more sensitive to amphotericin B killing than were mutant cells containing reduced amounts of cholesterol ester. Twelve amphotericin B-resistant mutants were isolated which retained the 25-hydroxycholesterol-resistant phenotype. These mutants did not exhibit the perinuclear lipid droplets characteristic of 25-RA cells, and lipid analysis revealed a large (up to 40-fold) reduction in cellular cholesterol ester. The acyl-CoA:cholesterol acyltransferase activities of these cholesterol ester-deficient mutants were markedly lower than 25-RA when assayed in intact cells or in an in vitro reconstitution assay. The tightest mutant characterized, AC29, was found to have less than 1% of the parental acyl-CoA:cholesterol acyltransferase activity. These mutants all have reduced rates of sterol synthesis and lower low density lipoprotein receptor activity compared to 25-RA, probably as a consequence of their reduced enzyme activities. Cell fusion experiments revealed that the phenotypes of all the mutants examined are not dominant and that the mutants all belong to the same complementation group. We conclude that these mutants contain a lesion in the gene encoding acyl-CoA:cholesterol acyltransferase or in a gene encoding a factor needed for enzyme production.     

Interleukin-10 Facilitates Both Cholesterol Uptake and Efflux in Macrophages

Foam cell formation is a hallmark event during atherosclerosis. The current paradigm is that lipid uptake by scavenger receptor in macrophages initiates the chronic proinflammatory cascade and necrosis core formation that characterize atherosclerosis. We report here that a cytokine considered to be anti-atherogenic, interleukin-10 (IL10), promotes cholesterol uptake from modified lipoproteins in macrophages and its transformation into foam cells by increasing the expression of scavenger receptor CD36 and scavenger receptor A. Although uptake of modified lipoproteins is considered proatherogenic, we found that IL10 also increases cholesterol efflux from macrophages to protect against toxicity of free cholesterol accumulation in the cell. This process was PPARγ-dependent and was mediated through up-regulation of ABCA1 (ATP-binding cassette transporter A1) protein expression. Importantly, expression of inflammatory molecules, such as tumor necrosis factor-α, intercellular adhesion molecule-1, and MMP9 as well as apoptosis were dramatically suppressed in lipid-laden foam cells treated with IL10. The notion that IL10 can mediate both the uptake of cholesterol from modified lipoproteins and the efflux of stored cholesterol suggests that the process of foam cell formation is not necessarily detrimental as long as mechanisms of cholesterol efflux and transfer to an exogenous acceptor are functioning robustly. Our results present a comprehensive antiatherogenic role of IL10 in macrophages, including enhanced disposal of harmful lipoproteins, inhibition of inflammatory molecules, and reduced apoptosis.     

Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1

Mutations of the ABC1 transporter have been identified as the defect in Tangier disease, characterized by low HDL and cholesterol ester accumulation in macrophages. A full-length mouse ABC1 cDNA was used to investigate the mechanisms of lipid efflux to apoA-I or HDL in transfected 293 cells. ABC1 expression markedly increased cellular cholesterol and phospholipid efflux to apoA-I but had only minor effects on lipid efflux to HDL. The increased lipid efflux appears to involve a direct interaction between apoA-I and ABC1, because ABC1 expression substantially increased apoA-I binding at the cell surface, and chemical cross-linking and immunoprecipitation analysis showed that apoA-I binds directly to ABC1. In contrast to scavenger receptor BI (SR-BI), another cell surface molecule capable of facilitating cholesterol efflux, ABC1 preferentially bound lipid-free apoA-I but not HDL. Immunofluorescence confocal microscopy showed that ABC1 is primarily localized on the cell surface. In the absence of apoA-I, cells overexpressing ABC1 displayed a distinctive morphology, characterized by plasma membrane protrusions and resembling echinocytes that form when there are excess lipids in the outer membrane hemileaflet. The studies provide evidence for a direct interaction between ABC1 and apoA-I, but not HDL, indicating that free apoA-I is the metabolic substrate for ABC1. Plasma membrane ABC1 may act as a phospholipid/cholesterol flippase, providing lipid to bound apoA-I, or to the outer membrane hemileaflet.     

ABCG1 redistributes cell cholesterol to domains removable by high density lipoprotein but not by lipid-depleted apolipoproteins

ATP binding cassette transporter G1 (ABCG1) mediates the transport of cholesterol from cells to high density lipoprotein (HDL) but not to lipid-depleted apolipoprotein A-I. Here we show that human ABCG1 overexpressed in baby hamster kidney cells in the absence of lipoproteins traffics to the plasma membrane and redistributes membrane cholesterol to cell-surface domains accessible to treatment with the enzyme cholesterol oxidase. Cholesterol removed by HDL was largely derived from these domains in ABCG1 transfectants but not in cells lacking ABCG1. Overexpression of ABCG1 also increased cholesterol esterification, which was decreased by the addition of HDL, suggesting that a proportion of the cell-surface cholesterol not removed by HDL is transported to the intracellular esterifying enzyme acyl-CoA:cholesterol acyltransferase. A 638-amino acid ABCG1, which lacked the 40 N-terminal amino acids of the predicted full-length protein, was fully functional and of a similar size to ABCG1 expressed by cholesterol-loaded human monocyte-derived macrophages. Mutating an essential glycine residue in the Walker A motif abolished ABCG1-dependent cholesterol efflux and esterification and prevented localization of ABCG1 to the cell surface, indicating that the ATP binding domain in ABCG1 is essential for both lipid transport activity and protein trafficking. These studies show that ABCG1 redistributes cholesterol to cell-surface domains where it becomes accessible for removal by HDL, consistent with a direct role of ABCG1 in cellular cholesterol transport.     

Role of lipid rafts in porcine reproductive and respiratory syndrome virus infection in MARC-145 cells

Lipid rafts play an important role in the life cycle of many viruses. Cholesterol is a critical structural component of lipid rafts. Although the porcine reproductive and respiratory syndrome virus (PRRSV) has restricted cell tropism for cells of the monocyte/macrophage lineage, a non-macrophage cell MARC-145 was susceptible to PRRSV because of the expression of virus receptor CD163 on the cell surface, therefore MARC-145 cells is used as model cell for PRRSV studies. In order to determine if cholesterol is involved in PRRSV infection in MARC-145 cells, we used three pharmacological agents: methyl-β cyclodextrin (MβCD), mevinolin, and filipin complex to deplete cholesterol in MARC-145. Although these agents act by different mechanisms, they all significantly inhibited PRRSV infection. The inhibition could be prevented by addition of exogenous cholesterol. Cell membrane cholesterol depletion after virus infection had no effect on PRRSV production and cholesterol depletion pre-infection did not reduce the virus attachment, suggesting cholesterol is involved in virus entry. Further results showed that cholesterol depletion did not change expression levels of the PRRSV receptor CD163 in MARC-145, had no effect on clathrin-mediated endocytosis, but disturbed lipid-raft-dependent endocytosis. Collectively, these studies suggest that cholesterol is critical for PRRSV entry, which is likely to be mediated by a lipid-raft-dependent pathway.     

Vaccinia virus replication is not affected by APOBEC3 family members

Background

HIV-1 entry into cells is a multifaceted process involving target cell CD4 and the chemokine receptors, CXCR4 or CCR5. The lipid composition of the host cell plays a significant role in the HIV fusion process as it orchestrates the appropriate disposition of CD4 and co-receptors required for HIV-1 envelope glycoprotein (Env)-mediated fusion. The cell membrane is primarily composed of sphingolipids and cholesterol. The effects of lipid modulation on CD4 disposition in the membrane and their role in HIV-1 entry have extensively been studied. To focus on the role of lipid composition on chemokine receptor function, we have by-passed the CD4 requirement for HIV-1 Env-mediated fusion by using a CD4-independent strain of HIV-1 Env.

Results

Cell fusion mediated by a CD4-independent strain of HIV-1 Env was monitored by observing dye transfer between Env-expressing cells and NIH3T3 cells bearing CXCR4 or CCR5 in the presence or absence of CD4. Chemokine receptor signaling was assessed by monitoring changes in intracellular [Ca²⁺] mobilization induced by CCR5 or CXCR4 ligand. To modulate target membrane cholesterol or sphingolipids we used Methyl-β-cyclodextrin (MβCD) or 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP), respectively. Treatment of the target cells with these agents did not change the levels of CD4 or CXCR4, but reduced levels of CCR5 on the cell surface. Chemokine receptor signalling was inhibited by cholesterol removal but not by treatment with PPMP. HIV-1 Env mediated fusion was inhibited by >50% by cholesterol removal. Overall, PPMP treatment appeared to slow down the rates of CD4-independent HIV-1 Env-mediated Fusion. However, in the case of CXCR4-dependent fusion, the differences between untreated and PPMP-treated cells did not appear to be significant.

Conclusion

Although modulation of cholesterol and sphingolipids has similar effects on CD4 -dependent HIV-1 Env-mediated fusion, sphingolipid modulation had little effect on CD4-independent HIV-1 Env-mediated fusion. Chemokine receptor function remained intact following treatment of cells with PPMP. Therefore such treatment may be considered a more suitable agent to inhibit CD4 dependent HIV-1 infection.     

Metabolism of cationized lipoproteins by human fibroblasts: biochemical and morphologic correlations

Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 μg/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester.     

Shedding of collagen XVII ectodomain depends on plasma membrane microenvironment

Collagen XVII, a hemidesmosomal component, mediates the adhesion of epidermal keratinocytes to the underlying basement membrane. It exists as a full-length transmembrane protein and a soluble ectodomain that is proteolytically released from the cell surface by sheddases of a disintegrin and metalloproteinase (ADAM) family; TACE, the tumor necrosis factor-alpha-converting enzyme, is the major physiological proteinase. Because both collagen XVII and the ADAMs are transmembrane proteins, their plasma membrane microenvironment can influence shedding. Lipid rafts, assemblies of sphingolipids and cholesterol within the plasma membrane, are responsible for the separation of membrane proteins and are thought to regulate shedding of cell surface proteins. In this study we analyzed the influence of the cholesterol-depleting agent methyl-beta-cyclodextrin (MbetaCD), which disintegrates lipid rafts, on the shedding of collagen XVII in HaCaT keratinocytes and in transfected COS-7 cells. Increasing concentrations of MbetaCD led to a dose-dependent decrease of membrane cholesterol levels and to stimulation of collagen XVII shedding. The stimulation was completely inhibited by sheddase inhibitors, and experiments with COS-7 cells co-transfected with TACE and collagen XVII demonstrated that TACE mediated the low cholesterol-dependent shedding. Co-patching analysis by double immunofluorescence staining revealed co-localization of collagen XVII with the raft resident phosphatidylinositol-linked placental alkaline phosphatase and segregation from the non-raft protein human transferrin receptor, indicating that a majority of collagen XVII molecules was incorporated into lipid rafts. These data deliver the first evidence for the role of plasma membrane lipid organization in the regulation of collagen XVII shedding and, therefore, in the regulation of keratinocyte migration and differentiation.