SCP-2/SCP-x gene ablation alters lipid raft domains in primary cultured mouse hepatocytes

Although reverse cholesterol transport from peripheral cell types is mediated through plasma membrane microdomains termed lipid rafts, almost nothing is known regarding the existence, protein/lipid composition, or structure of these putative domains in liver hepatocytes, cells responsible for the net removal of cholesterol from the body. Lipid rafts purified from hepatocyte plasma membranes by a nondetergent affinity chromatography method were: i) present at 33 +/- 3% of total plasma membrane protein; ii) enriched in key proteins of the reverse cholesterol pathway [scavenger receptor class B type I (SR-B1), ABCA1, P-glycoprotein (P-gp), sterol carrier protein-2 (SCP-2)]; iii) devoid of caveolin-1; iv) enriched in cholesterol, sphingomyelin, GM1, and phospholipids low in polyunsaturated fatty acid and double bond index; and v) exhibited an intermediate liquid-ordered lipid phase with significant transbilayer fluidity gradient. Ablation of the gene encoding SCP-2 significantly altered lipid rafts to: i) increase the proportion of lipid rafts present, thereby increasing raft total content of ABCA1, P-gp, and SR-B1; ii) increase total phospholipids while decreasing GM1 in lipid rafts; iii) decrease the fluidity of lipid rafts, consistent with the increased intermediate liquid-ordered phase; and iv) abolish the lipid raft transbilayer fluidity gradient. Thus, despite the absence of caveolin-1 in liver hepatocytes, lipid rafts represented nearly one-third of the mouse hepatocyte plasma membrane proteins and displayed unique protein, lipid, and biophysical properties that were differentially regulated by SCP-2 expression.     

Regulation of intestinal NPC1L1 expression by dietary fish oil and docosahexaenoic acid

To address the effect of the n-3 fatty acid, docosahexaenoic acid (22:6), on proteins that play a role in cholesterol absorption, CaCo-2 cells were incubated with taurocholate micelles alone or micelles containing 22:6 or oleic acid (18:1). Compared with controls or 18:1, 22:6 did not interfere with the cellular uptake of micellar cholesterol. Apical cholesterol efflux was enhanced in cells incubated with 22:6. Cholesterol trafficking from the plasma membrane to the endoplasmic reticulum was decreased by 22:6. 22:6 decreased Niemann-Pick C1-Like 1 (NPC1L1) protein and mRNA levels without altering gene or protein expression of ACAT2, annexin-2, caveolin-1, or ABCG8. Peroxisome proliferator-activated receptor delta (PPARdelta) activation decreased NPC1L1 mRNA levels and cholesterol trafficking to the endoplasmic reticulum, suggesting that 22:6 may act through PPARdelta. Compared with hamsters fed a control diet or olive oil (enriched 18:1), NPC1L1 mRNA levels were decreased in duodenum and jejunum of hamsters ingesting fish oil (enriched 22:6). In an intestinal cell, independent of changes in ABCG8 expression, 22:6 increases the apical efflux of cholesterol. 22:6 interferes with cholesterol trafficking to the endoplasmic reticulum by the suppression of NPC1L1, perhaps through the activation of PPARdelta. Moreover, a diet enriched in n-3 fatty acids decreases the gene expression of NPC1L1 in duodenum and jejunum of hamster.     

Cholesterol independent effect of LXR agonist TO-901317 on gamma-secretase

The balance of intracellular cholesterol has proven to be critical to the production of beta-amyloid (A beta). Reducing cholesterol in vitro leads to decreased production of A beta, whereas an increase in cellular cholesterol induces A beta production. Liver X Receptor (LXR) agonists are known to increase cholesterol efflux from cells, but there are conflicting reports as to the effects of these agonists on A beta production. We therefore examined the effects of efflux-inducing agents on A beta production in vitro. We used methyl-beta-cyclodextrin and an LXR agonist (TO-901317) to induce cholesterol efflux and studied the resulting A beta production in a stable amyloid precursor protein (APP) -transfected cell line. When cholesterol efflux was induced with methyl-beta-cyclodextrin there was a >60% decrease in A beta(40) and A beta(42) production. However, while activation of LXR using TO-901317-induced cholesterol efflux in the presence of a cholesterol acceptor, no changes in A beta levels were recorded. When cells were incubated with TO-901317 above the concentration required for maximal cholesterol efflux, there was a 150% increase in A beta(42) levels. The absence of a cholesterol acceptor from the culture media (preventing cholesterol efflux) did not blunt this increase in A beta(42), suggesting that the effects of TO-901317 on A beta(42) are efflux independent. These results were confirmed in APP stably transfected human H4 cells, which revealed in addition to a 200% increase in A beta(42) levels, a concomitant 80% reduction in A beta(38). A cell-free gamma-secretase assay confirmed that TO-901317 can directly alter gamma-secretase activity. These data demonstrate that TO-901317 can directly modulate the site of cleavage of APP by gamma-secretase in vitro.     

A specific inhibitor of cholesterol biosynthesis, BM15.766, reduces the expression of beta-secretase and the production of amyloid-beta in vitro

We have previously shown that statins reduce the production of amyloid-beta (Abeta) by both isoprenoid- and cholesterol-dependent mechanisms. These pathways contribute to the regulation of the dimerisation of BACE into its physiologically active form. Statins reduce cellular cholesterol levels by 20-40%; therefore, it is possible that the remaining cholesterol within the cell may play a significant role in the production of Abeta. Incubation of cells with the specific cholesterol biosynthesis inhibitor BM15.766 together with 50 micromol/L simvastatin and 400 micromol/L mevalonate reduced cellular cholesterol levels in a dose-dependent manner with increasing BM15.766 concentration (r = -0.9736, p = 0.0264). Furthermore, decreases in cellular cholesterol levels correlated with reductions in total Abeta production (r = 0.9683, p = 0.0317). A total of 2.5 micromol/L BM15.766 inhibited the dimerisation of BACE, whilst the expression of BACE monomer was reduced by 5 micromol/L BM15.766. BM15.766 treatment localised BACE predominantly within the Golgi, and reduced total BACE expression per cell. Similar changes were observed in the expression of the Golgi marker golgin-97, suggesting that reduced BACE expression may arise from a decrease in protein trafficking and an increase in degradation. By targeting cholesterol synthesis using specific cholesterol biosynthesis inhibitors, it is possible to reduce Abeta production without reducing protein isoprenylation.     

Characterisation of three pathways for osmolyte efflux in human erythroleukemia cells

Cell volume decrease is a key step during differentiation of erythroid cells. This could arise from membrane transporter activation leading to a loss of cell osmolytes; however, the pathways involved are poorly understood. We have characterised Cl(-)-independent K(+) and (3)H-taurine efflux from the erythroleukemia cell line, K562. K(+) efflux (measured using (86)Rb(+)) from pre-loaded cells subjected to hypo-osmotic challenge demonstrated two phases, a rapid increase in K(+) efflux followed by a smaller slower increase. Swelling-activated taurine efflux only demonstrated a single phase. Both phases of K(+) efflux were significantly (P<0.05) blocked by anion channel inhibitor 5-nitro-2-(3-phenypropylamino)-benzoic acid (NPPB). However the antiestrogen, tamoxifen, only inhibited the slow late phase. The initial rapid phase had a higher IC(50) for NPPB inhibition than the slow phase, and was insensitive to protein kinases inhibitors KN-62, wortmannin and PD98059. For the slow K(+) efflux phase, the IC(50) for NPPB inhibition and the inhibition by KN-62, wortmannin, genistein or PD98059, were very similar to those measured for the hypo-osmotically-activated taurine efflux. With NPPB (100 microM) present, the slow K(+) efflux phase was further significantly decreased by the Ca(2+) chelator BAPTA-AM or by the Ca(2+)-activated K(+) channel blockers clotrimazole and charybdotoxin but not by apamin. Thus, at least 3 Cl(-)-independent pathways are involved: (a) a tamoxifen-sensitive and taurine-permeable anion channel; (b) a tamoxifen-insensitive and taurine-impermeable K(+) efflux pathway; and (c) a subtype of Ca(2+)-activated K(+) channel. Any or all of these could be involved in the cell volume decrease associated with differentiation in K562 cells.     

Estimation of autotrophic and heterotrophic components of soil respiration by trenching is sensitive to corrections for root decomposition and changes in soil water content

This study aims to assess the effects of corrections for disturbances such as an increased amount of dead roots and an increase in volumetric soil water content on the calculation of soil CO₂ efflux partitioning. Soil CO₂ efflux, soil temperature and superficial soil water content were monitored in two young beech sites (H1 and H2) during a trenching experiment. Trenching induced a significant input of dead root mass that participated in soil CO₂ efflux and reduced the soil dissolved organic carbon content, while it increased superficial soil water content within the trenched plot. Annual soil CO₂ efflux in control plots was 528 g C m⁻² year⁻¹ at H1 and 527 g C m⁻² year⁻¹ at H2. The annual soil CO₂ efflux in trenched plots was 353 g C m⁻² year⁻¹ at H1 and 425 g C m⁻² year⁻¹ at H2. By taking into account annual CO₂ efflux from decaying trenched roots, the autotrophic contribution to total soil CO₂ efflux reached 69% at H1 and 54% at H2. The partitioning calculation was highly sensitive to the initial root mass estimated within the trenched plots. Uncertainties in the remaining root mass, the fraction of root C that is incorporated into soil organic matter during root decomposition, and the root decomposition rate constant had a limited impact on the partitioning calculation. Corrections for differences in superficial soil water content had a significant impact on annual respired CO₂ despite a limited effect on partitioning.     

The expression of genes coding for auxin carriers in different tissues and along the organ can explain variations in auxin transport and the growth pattern in etiolated lupin hypocotyls

Novel cDNA clones encoding putative auxin influx and efflux carriers have been isolated and characterized from etiolated lupin (Lupinus albus L) hypocotyls. The full length of LaAUX1 and LaPIN1 and the partial length of LaPIN3 were obtained and the deduced amino acid sequence revealed a high degree of identity with the corresponding auxin carrier proteins from several species. The expression of these genes depended on the tissue, the hypocotyl zone and seedling age. LaAUX1 and LaPIN3 were expressed in stele and outer tissues, while LaPIN1 was restricted to the stele. From the above-mentioned results and taking into account the role proposed for the efflux carrier PIN1, it is suggested that LaPIN1 could mediate the basipetal auxin transport already described in this organ. LaAUX1 might facilitate auxin influx in the transport cells. The expression of the three genes decreased down the hypocotyl. The basipetally decreasing gradient in the expression of LaPIN1 coincides with previous results showing a similar gradient in the intensity and polarity of auxin transport. The decisive role ascribed to PIN1 in polar auxin transport due to its localization in the basal end of transporting cells and the existence of such a gradient in the expression of LaPIN1 support the hypothesis of a barrier effect (generated by decreasing auxin transport) previously proposed by our research group as being responsible for the auxin gradient, which controls the growth pattern in etiolated lupin hypocotyls. José Sánchez-Bravo, Manuel Acosta and Carlos Nicolás contributed equally to the paper. Nucleotide sequence database accession numbers: LaAUX1: AM235387; LaPIN1: AM235388; LaPIN3: AM407405.     

Multidrug resistance protein 1 is not associated to detergent-resistant membranes

Multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette superfamily. Using the energy provided by ATP hydrolysis, it transports a broad spectrum of substrates across the plasma membrane, including hormones, leukotriene C(4), bile salts, and anti-cancer drugs. Recent works have suggested that P-glycoprotein is associated to cholesterol and sphingolipid-rich membrane microdomains and that cholesterol upregulates its ATPase and drug transport activities. Confocal microscopy experiments and Triton X-100 extraction of detergent-resistant membranes provide evidence that MRP1 is not located in raft-like structures and that its activity is downregulated by cholesterol. The data are discussed in terms of cholesterol-protein interaction and topology.