Cellular cholesterol efflux is modulated by phospholipid-derived signaling molecules in familial HDL deficiency/Tangier disease fibroblasts

Familial HDL deficiency (FHD) is the heterozygous form of Tangier disease (TD). Mutations of the ABCA1 gene cause FHD and TD. FHD/TD cells are unable to normally efflux cholesterol onto nascent HDL particles, which are rapidly catabolized. TD fibroblasts have an abnormal pattern of PLC and PLD activation following cell stimulation with HDL(3) or apolipoprotein A-I (apoA-I). We examined cellular cholesterol efflux in FHD and TD fibroblasts by phospholipid-derived-molecules, compared with that of normal cells. We used the PKC agonist 1,2-dioctanoylglycerol (DOG) and phorbol myristate acetate (PMA) to activate PKC, calphostin C, and GO 6976, as inhibitors of PKC; phosphatidic acid (PA), which is the product of PLD, and lysophosphatidic acid (LPA), phosphatidylcholine, sphingomyelin, and beta-cyclodextrin to investigate their potential effect in modulating cellular cholesterol efflux in [(3)H]cholesterol-labeled and cholesterol-loaded fibroblasts. Phosphatidylcholine, sphingomyelin, and beta-cyclodextrin promoted cholesterol efflux in an identical fashion in control, FHD, or TD fibroblasts. In a dose-dependent fashion, DOG (0-200 microM) increased apoA-I-mediated cellular cholesterol efflux by 40% in controls, 71% in FHD, and 242% in TD cells. PMA similarly increased cholesterol efflux to a maximum of 256% in controls, 182% in FHD, and 191% in TD cells. This effect was inhibited by calphostin C. PA (100 microM) also increased cholesterol efflux by 25% in control, 44% in FHD, and 100% in TD cells. Conversely, LPA reduced cholesterol efflux in a dose-dependent fashion in control and FHD cells (-50%, 200 microM) but not in TD cells, where efflux was increased by 140%. Propranolol (100 microM) significantly increased cholesterol efflux at 24 h in all three cell lines. n-Butanol partially decreased the DOG-mediated increase in cholesterol efflux. The inhibitory effect of calphostin C on DOG-stimulated cholesterol efflux could be partially overcome by propranolol, suggesting that PA is a downstream mediator of PKC-stimulated cholesterol efflux.We conclude that PLC and PLD activities are required for apoA-I-mediated cellular cholesterol efflux, and modulating cellular PA concentration can correct, at least partially, the cholesterol efflux defect in FHD and TD.     

Uncoupling of the cholera toxin-G(M1) ganglioside receptor complex from endocytosis, retrograde Golgi trafficking, and downstream signal transduction by depletion of membrane cholesterol

To induce toxicity, cholera toxin (CT) must first bind ganglioside G(M1) at the plasma membrane, enter the cell by endocytosis, and then traffic retrograde into the endoplasmic reticulum. We recently proposed that G(M1) provides the sorting motif necessary for retrograde trafficking into the biosynthetic/secretory pathway of host cells, and that such trafficking depends on association with lipid rafts and lipid raft function. To test this idea, we examined whether CT action in human intestinal T84 cells depends on membrane cholesterol. Chelation of cholesterol with 2-hydroxypropyl beta-cyclodextrin or methyl beta-cyclodextrin reversibly inhibited CT-induced chloride secretion and prolonged the time required for CT to enter the cell and induce toxicity. These effects were specific to CT, as identical conditions did not alter the potency or toxicity of anthrax edema toxin that enters the cell by another mechanism. We found that endocytosis and trafficking of CT into the Golgi apparatus depended on membrane cholesterol. Cholesterol depletion also changed the density and specific protein content of CT-associated lipid raft fractions but did not entirely displace the CT-G(M1) complex from these lipid raft microdomains. Taken together these data imply that cholesterol may function to couple the CT-G(M1) complex with raft domains and with other membrane components of the lipid raft required for CT entry into the cell.     

ATP-binding cassette transporter A1 (ABCA1) functions as a cholesterol efflux regulatory protein

ABCA1, an ATP-binding cassette transporter mutated in Tangier disease, promotes cellular phospholipid and cholesterol efflux by loading free apoA-I with these lipids. This process involves binding of apoA-I to the cell surface and phospholipid translocation by ABCA1. The goals of this study were to examine the relationship between ABCA1-mediated lipid efflux and apolipoprotein binding and to determine whether phospholipid and cholesterol efflux are coupled. Inhibition of lipid efflux by glybenclamide treatment or by mutation of the ATP-binding cassette of ABCA1 showed a close correlation between lipid efflux, the binding of apoA-I to cells, and cross-linking of apoA-I to ABCA1. The data suggest that a functionally important apoA-I binding site exists on ABCA1 and that the binding site could also involve lipids. After using cyclodextrin preincubation to deplete cellular cholesterol, ABCA1-mediated cholesterol efflux was abolished but phospholipid efflux and the binding of apoA-I were unaffected. The conditioned media from cyclodextrin-pretreated, ABCA1-expressing cells readily promoted cholesterol efflux when added to fresh cells not expressing ABCA1, indicating that cholesterol efflux can be dissociated from phospholipid efflux. Further, using a photoactivatable cholesterol analog, we showed that ABCA1 did not bind cholesterol directly, even though several other cholesterol-binding proteins specifically bound the cholesterol analog. The data suggest that the binding of apoA-I to ABCA1 leads to the formation of phospholipid-apoA-I complexes, which subsequently promote cholesterol efflux in an autocrine or paracrine fashion.     

Changes in neutral amino acid efflux and membrane potential associated with the expression of CFTR protein

Summary The expression of wild type CFTR facilitates the efflux of neutral amino acids (Rotoli et al., Biochem. Biophys. Res. Commun. 204: 653–658, 1994); as a result, after an extensive depletion of intracellular amino acid pool obtained through an incubation in saline solution, the intracellular leucine levels were lower in murine C127 cells transfected with the wild type CF gene (C127 CFTRw/t) than in cells transfected with either mutant CF (C127 CFTRF508 cells) or mock vector only. No change in amino acid efflux was detected when C127 CFTRw/t and C127 CFTRw/t and C127 CFTRF508 cells were studied under conditions known to activate protein kinase A. Upon an incubation in Cl^– free medium, a permeant analogue of cAMP caused a marked cell depolarization of C127 CFTRw/t cells but not of C127 CFTRF508 cells, thus showing a functional expression of CFTR protein in the former cell line. However, we found that, upon a Cl^– free incubation and in the absence of exogenous cAMP, C127 CFTRw/t cells developed a marked hyperpolarization that was not detected in C127 CFTRF508 cells. It is concluded that the expression of normal CFTR accelerates amino acid efflux and enhances cell hyperpolarization in Cl^– free media; both these effects appear to be independent from PKA stimulation of CFTR.Abbreviations 8-Br-cAMP 8-bromoadenosine 3,5-cyclic monophosphate - C127 CFTRw/t C127i cells expressing CFTR wild type - C127 CFTRF508 C127i cells expressing CFTR bearing F508 mutation - C127 mock C127i cells transfected with the mock vector - CF cystic fibrosis - CFTR cystic fibrosis transmembrane conductance regulator - DMEM Dulbecco's modified Eagle medium - EBSS Earle's balanced salt solution - FBS fetal bovine serum - PKA protein kinase A     

The lipidation status of acute-phase protein serum amyloid A determines cholesterol mobilization via scavenger receptor class B, type I

During the acute-phase reaction, SAA (serum amyloid A) replaces apoA-I (apolipoprotein A-I) as the major HDL (high-density lipoprotein)-associated apolipoprotein. A remarkable portion of SAA exists in a lipid-free/lipid-poor form and promotes ABCA1 (ATP-binding cassette transporter A1)-dependent cellular cholesterol efflux. In contrast with lipid-free apoA-I and apoE, lipid-free SAA was recently reported to mobilize SR-BI (scavenger receptor class B, type I)-dependent cellular cholesterol efflux [Van der Westhuyzen, Cai, de Beer and de Beer (2005) J. Biol. Chem. 280, 35890-35895]. This unique property could strongly affect cellular cholesterol mobilization during inflammation. However, in the present study, we show that overexpression of SR-BI in HEK-293 cells (human embryonic kidney cells) (devoid of ABCA1) failed to mobilize cholesterol to lipid-free or lipid-poor SAA. Only reconstituted vesicles containing phospholipids and SAA promoted SR-BI-mediated cholesterol efflux. Cholesterol efflux from HEK-293 and HEK-293[SR-BI] cells to lipid-free and lipid-poor SAA was minimal, while efficient efflux was observed from fibroblasts and CHO cells (Chinese-hamster ovary cells) both expressing functional ABCA1. Overexpression of SR-BI in CHO cells strongly attenuated cholesterol efflux to lipid-free SAA even in the presence of an SR-BI-blocking IgG. This implies that SR-BI attenuates ABCA1-mediated cholesterol efflux in a way that is not dependent on SR-BI-mediated re-uptake of cholesterol. The present in vitro experiments demonstrate that the lipidation status of SAA is a critical factor governing cholesterol acceptor properties of this amphipathic apolipoprotein. In addition, we demonstrate that SAA mediates cellular cholesterol efflux via the ABCA1 and/or SR-BI pathway in a similar way to apoA-I.     

Comparison of the capacity of beta-cyclodextrin derivatives and cyclophanes to shuttle cholesterol between cells and serum lipoproteins.

Previous studies from this laboratory have demonstrated that low concentrations of cyclodextrins (<1.0 mm), when added to serum, act catalytically as cholesterol shuttles to accelerate the exchange of free cholesterol between cells and serum lipoproteins. As cholesterol shuttles, cyclodextrins have the potential to serve as pharmacological agents for modifying cholesterol metabolism. In the present study, we have quantitated the cholesterol-shuttling capacity of a series of newly synthesized beta-cyclodextrin derivatives (betaCDs), with varying structure, and two double-decker cyclophanes. The general protocol is as follows. [(3)H]cholesterol-labeled CHOK1 cells are incubated for 2 h with the test compounds alone or together with 5% human serum, and efflux of the cellular [(3)H]cholesterol is measured. As methyl beta-cyclodextrin (MbetaCD) served as the basis for comparison, initial experiments were conducted that demonstrated there was a dose-dependent stimulation of cell cholesterol efflux as the concentration of MbetaCD increased, with an EC(50) that was calculated to be 0.05 mm. To determine the cholesterol-shuttling capacity of the newly synthesized compounds, cell cholesterol efflux is measured when the compounds are present alone, at a concentration of 0.05 mm, or together with 5% human serum. Our results demonstrate that the double-decker cyclophanes are the most efficient cholesterol shuttles. Under our experimental conditions, methyl beta-cyclodextrin (MbetaCD) approximately doubles the efflux of cell cholesterol to serum, whereas one of the double-decker cyclophanes produces a 4-fold stimulation in efflux. Four of the beta-cyclodextrin derivatives (betaCDs) display shuttling ability similar to that of MbetaCD. Furthermore, there does not appear to be a structural pattern among the other betaCDs which could explain their shuttling capacity.     

cAMP induces ABCA1 phosphorylation activity and promotes cholesterol efflux from fibroblasts

ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in apoA-I lipidation, a key step in reverse cholesterol transport. cAMP induces apoA-I binding activity and promotes cellular cholesterol efflux. We investigated the role of the cAMP/protein kinase A (PKA) dependent pathway in the regulation of cellular cholesterol efflux. Treatment of normal fibroblasts with 8-bromo-cAMP (8-Br-cAMP) increased significantly apoA-I-mediated cholesterol efflux, with specificity for apoA-I, but not for cyclodextrin. Concomitantly, 8-Br-cAMP increased ABCA1 phosphorylation in a time-dependent manner. Maximum phosphorylation was reached in <10 min, representing a 260% increase compared to basal ABCA1 phosphorylation level. Forskolin, a known cAMP regulator, increased both cellular cholesterol efflux and ABCA1 phosphorylation. In contrast, H-89 PKA inhibitor reduced cellular cholesterol efflux by 70% in a dose-dependent manner and inhibited almost completely ABCA1 phosphorylation. To determine whether naturally occurring mutants of ABCA1 may affect its phosphorylation activity, fibroblasts from subjects with familial HDL deficiency (FHD, heterozygous ABCA1 defect) and Tangier disease (TD, homozygous/compound heterozygous ABCA1 defect) were treated with 8-Br-cAMP or forskolin. Cellular cholesterol efflux and ABCA1 phosphorylation were increased in FHD but not in TD cells. Taken together, these findings provide evidence for a link between the cAMP/PKA-dependent pathway, ABCA1 phosphorylation, and apoA-I mediated cellular cholesterol efflux.     

Resistance of smooth muscle cells to assembly of high density lipoproteins with extracellular free apolipoproteins and to reduction of intracellularly accumulated cholesterol.

Removal of intracellularly accumulated cholesterol by lipid-free human apolipoproteins (apo) A-I and A-II was studied for aortic smooth muscle cells (SMC) of rat, monkey and rabbit, human skin fibroblasts (FB), and mouse peritoneal macrophages (MP). The reaction generated high density lipoprotein (HDL)-like lipoproteins as did those and other helical apolipoproteins with MP, causing efflux of cellular cholesterol. From FB and MP, the maximum efflux rates with apoA-I and A-II per 24 h were as much as 30% of the apparent maximum efflux rate of prelabeled cellular cholesterol to human HDL. From rat SMC these rates were 7.2 and 6.8%, respectively, being independent of cellular cholesterol content. Those from monkey and rabbit SMC were also very low. When standardized for the initial cellular unesterified cholesterol pool size, the maximum efflux rates/24 h were 5.4 and 5.0% for apoA-I and A-II from rat SMC and even less from monkey and rabbit SMC in contrast to 42.4 and 39.7% from FB, and 53.0 and 45.5% from MP, respectively. The standardized apparent maximum efflux to HDL was 76% from rat SMC, 45 and 31% from monkey and rabbit SMC, 139% from FB and 166% from MP. Accordingly, the reaction with free apolipoproteins caused significant net reduction of cellular cholesterol, predominantly in cholesteryl ester, in FB and MP, but not in SMC. While the efflux Km with apoA-I and A-II were 7.5 and 4.5 micrograms/ml for MP, those for SMC and FB were both 1 microgram/ml or lower, as low as 1/1500 and 1/500 of their plasma concentrations, respectively. The apparent efflux Km for HDL were, on the other hand, all in the range of 36 to 65 micrograms of protein/ml for SMC, FB, and MP, showing that the mode of cholesterol exchange of these cells with lipoprotein surface is not significantly different from each other. Thus, peripheral cells such as FB may provide a significant source of HDL by interacting with extracellular free apolipoproteins in interstitial fluid, reducing intracellularly accumulated cholesterol. However, SMC seem very resistant to this interaction, suggesting that atheromatous lesions predominantly consisting of SMC are resistant to regression.     

ABCA1 mediates concurrent cholesterol and phospholipid efflux to apolipoprotein A-I

Prior studies provide data supporting the notion that ATP binding cassette transporter A1 (ABCA1) promotes lipid efflux to extracellular acceptors in a two-step process: first, ABCA1 mediates phospholipid efflux to an apolipoprotein, and second, this apolipoprotein-phospholipid complex accepts free cholesterol in an ABCA1-independent manner. In the current study using RAW264.7 cells, ABCA1-mediated free cholesterol and phospholipid efflux to apolipoprotein A-I (apoA-I) were tightly coupled to each other both temporally and after treatment with ABCA1 inhibitors. The time course and temperature dependence of ABCA1-mediated lipid efflux to apoA-I support a role for endocytosis in this process. Cyclodextrin treatment of RAW264.7 cells partially inhibited 8Br-cAMP-induced efflux of free cholesterol and phospholipid to apoA-I. ABCA1-expressing cells are more sensitive to cell damage by high-dose cyclodextrin and vanadate, leading to increased lactate dehydrogenase leakage and phospholipid release even in the absence of the acceptor apoA-I. Finally, we could not reproduce a two-step effect on lipid efflux using conditioned medium from ABCA1-expressing cells pretreated with cyclodextrin.     

Cathepsins F and S block HDL3-induced cholesterol efflux from macrophage foam cells

In atherosclerosis, accumulation of cholesterol in macrophages may partially depend on its defective removal by high-density lipoproteins (HDL). We studied the proteolytic effect of cathepsins F, S, and K on HDL(3) and on lipid-free apoA-I, and its consequence on their function as inductors of cholesterol efflux from cholesterol-filled mouse peritoneal macrophages in vitro. Incubation of HDL(3) with cathepsin F or S, but not with cathepsin K, led to rapid loss of prebeta-HDL, and reduced cholesterol efflux by 50% in only 1min. Cathepsins F or K partially degraded lipid-free apoA-I and reduced its ability to induce cholesterol efflux, whereas cathepsin S totally degraded apoA-I, leading to complete loss of apoA-I cholesterol acceptor function. These results suggest that cathepsin-secreting cells induce rapid depletion of lipid-poor (prebeta-HDL) and lipid-free apoA-I and inhibit cellular cholesterol efflux, so tending to promote the formation and maintenance of foam cells in atherosclerotic lesions.     

Regulatory Insertion Removal Restores Maturation, Stability and Function of ΔF508 CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel is a large multidomain membrane protein that matures inefficiently during biosynthesis. Its assembly is further perturbed by the deletion of F508 from the first nucleotide-binding domain (NBD1) responsible for most cystic fibrosis. The mutant polypeptide is recognized by cellular quality control systems and is proteolyzed. CFTR NBD1 contains a 32-residue segment termed the regulatory insertion (RI) not present in other ATP-binding cassette transporters. We report here that RI deletion enabled F508 CFTR to mature and traffic to the cell surface where it mediated regulated anion efflux and exhibited robust single chloride channel activity. Long-term pulse-chase experiments showed that the mature ΔRI/ΔF508 had a T_1/2 of ∼ 14 h in cells, similar to the wild type. RI deletion restored ATP occlusion by NBD1 of ΔF508 CFTR and had a strong thermostabilizing influence on the channel with gating up to at least 40 °C. None of these effects of RI removal were achieved by deletion of only portions of RI. Discrete molecular dynamics simulations of NBD1 indicated that RI might indirectly influence the interaction of NBD1 with the rest of the protein by attenuating the coupling of the F508-containing loop with the F1-like ATP-binding core subdomain so that RI removal overcame the perturbations caused by F508 deletion. Restriction of RI to a particular conformational state may ameliorate the impact of the disease-causing mutation.     

Regulation of adenosine 3':5'-monophosphate efflux from animal cells.

Cyclic AMP efflux was measured following hormonal stimulation of adenylate cyclase in a variety of animal cells including C-6 rat glioma cells, WI-38 human fibroblasts, and avian erythrocytes. Using a variety inhibitors of mitochondrial function and glycolysis, a correlation was noted between cellular ATP levels and the rate of cyclic AMP efflux in all cells examined. A relationship between the efflux rate and the magnitude of the membrane potential was not observed. Pharmacological agents which inhibited cyclic AMP egress in these cells without reducing ATP levels included several prostaglandins (A greater than B greater than E greater than F) and probenecid. The characteristics of the cyclic AMP efflux system resemble those of the organic anion transport system.     

A domain mimic increases DeltaF508 CFTR trafficking and restores cAMP-stimulated anion secretion in cystic fibrosis epithelia

The major disease-causing mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine 508 (F508), which adversely affects processing and plasma membrane targeting of CFTR. Under conditions predicted to stabilize protein folding, F508 CFTR is capable of trafficking to the plasma membrane and retains cAMP-regulated anion channel activity. Overexpression is one factor that increases CFTR trafficking; therefore, we hypothesized that expression of a domain mimic of the first nucleotide-binding fold (NBF1) of CFTR, i.e., the site of F508, may be sufficient to overwhelm the quality control process or otherwise stabilize F508 CFTR and thereby restore cAMP-stimulated anion secretion. In epithelial cells expressing recombinant F508 human (h)CFTR, expression of wild-type NBF1 increased the amount of both core-glycosylated and mature protein to a greater extent than expression of F508 NBF1. Expression of wild-type NBF1 in the F508 hCFTR cells increased whole cell Cl^– current density to 50% of that in cells expressing wild-type hCFTR. Expression of NBF1 in polarized epithelial monolayers from a F508/F508 cystic fibrosis mouse (MGEF) restored cAMP-stimulated transepithelial anion secretion but not in monolayers from a CFTR-null mouse (MGEN). Restoration of anion secretion was sustained in NBF1-expressing MGEF for >30 passages, whereas MGEN corrected with hCFTR progressively lost anion secretion capability. We conclude that expression of a NBF1 domain mimic may be useful for correction of the F508 CFTR protein trafficking defect in cystic fibrosis epithelia. protein processing; mouse; retrovirus; gene therapy     

Caveolin-1 and regulation of cellular cholesterol homeostasis

Caveolae are 50- to 100-nm cell surface plasma membrane invaginations present in terminally differentiated cells. They are characterized by the presence of caveolin-1, sphingolipids, and cholesterol. Caveolin-1 is thought to play an important role in the regulation of cellular cholesterol homeostasis, a process that needs to be properly controlled to limit and prevent cholesterol accumulation and eventually atherosclerosis. We have recently generated caveolin-1-deficient [Cav-1(-/-)] mice in which caveolae organelles are completely eliminated from all cell types, except cardiac and skeletal muscle. In the present study, we examined the metabolism of cholesterol in wild-type (WT) and Cav-1(-/-) mouse embryonic fibroblasts (MEFs) and mouse peritoneal macrophages (MPMs). We observed that Cav-1(-/-) MEFs are enriched in esterified cholesterol but depleted of free cholesterol compared with their wild-type counterparts. Similarly, Cav-1(-/-) MPMs also contained less free cholesterol and were enriched in esterified cholesterol on cholesterol loading. In agreement with this finding, caveolin-1 deficiency was associated with reduced free cholesterol synthesis but increased acyl-CoA:cholesterol acyl-transferase (ACAT) activity. In wild-type MPMs, we observed that caveolin-1 was markedly upregulated on cholesterol loading. Despite these differences, cellular cholesterol efflux from MEFs and MPMs to HDL was not affected in the Cav-1-deficient cells. Neither ATP-binding cassette transporter G1 (ABCG1)- nor scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux was affected. Cellular cholesterol efflux to apolipoprotein A-I was not significantly reduced in Cav-1(-/-) MPMs compared with wild-type MPMs. However, ABCA1-mediated cholesterol efflux was clearly more sensitive to the inhibitory effects of glyburide in Cav-1(-/-) MPMs versus WT MPMs. Taken together, these findings suggest that caveolin-1 plays an important role in the regulation of intracellular cholesterol homeostasis and can modulate the activity of other proteins that are involved in the regulation of intracellular cholesterol homeostasis.     

Glycosphingolipid accumulation inhibits cholesterol efflux via the ABCA1/apolipoprotein A-I pathway: 1-phenyl-2-decanoylamino-3-morpholino-1-propanol is a novel cholesterol efflux accelerator

Cellular glycosphingolipid (GSL) storage is known to promote cholesterol accumulation. Although physical interactions between GSLs and cholesterol are thought to cause intracellular cholesterol "trapping," it is not known whether cholesterol homeostatic mechanisms are also impaired under these conditions. ApoA-I-mediated cholesterol efflux via ABCA1 (ATP-binding cassette transporter A1) is a key regulator of cellular cholesterol balance. Here, we show that apoA-I-mediated cholesterol efflux was inhibited (by up to 53% over 8 h) when fibroblasts were treated with lactosylceramide or the glucocerebrosidase inhibitor conduritol B epoxide. Furthermore, apoA-I-mediated cholesterol efflux from fibroblasts derived from patients with genetic GSL storage diseases (Fabry disease, Sandhoff disease, and GM1 gangliosidosis) was impaired compared with control cells. Conversely, apoA-I-mediated cholesterol efflux from fibroblasts and cholesterol-loaded macrophage foam cells was dose-dependently stimulated (by up to 6-fold over 8 h) by the GSL synthesis inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). Unexpectedly, a structurally unrelated GSL synthesis inhibitor, N-butyldeoxynojirimycin, was unable to stimulate apoA-I-mediated cholesterol efflux despite achieving similar GSL depletion. PDMP was found to up-regulate ABCA1 mRNA and protein expression, thereby identifying a contributing mechanism for the observed acceleration of cholesterol efflux to apoA-I. This study reveals a novel defect in cellular cholesterol homeostasis induced by GSL storage and identifies PDMP as a new agent for enhancing cholesterol efflux via the ABCA1/apoA-I pathway.     

VIP-dependent increase in F508del-CFTR membrane localization is mediated by PKCε

The most common cystic fibrosis causing mutation F508del induces early degradation and reduced trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels to the apical membrane of epithelial cells. In the human nasal epithelial cells JME/CF15, we previously reported that vasoactive intestinal peptide (VIP) exposure corrects trafficking and membrane insertion of functional F508del-CFTR channels at 37°C. Correction of trafficking was PKA dependent, whereas enhanced membrane localization involved PKC. In the present study, we have identified PKCε as the isoform involved in VIP-dependent F508del-CFTR membrane insertion. Iodide effluxes were used to monitor the presence of VIP-rescued functional F508del-CFTR channels at the surface of JME/CF15 cells maintained at 37°C. Iodide efflux peaks measured in response to stimulation with forskolin were insensitive to PKC α, β, γ, δ, ζ inhibitors. In contrast, efflux peaks were completely inhibited by pretreatment with the PKCε inhibitor peptide EAVSLKPT with an IC(50) of 4.9 μM or by PKCε small interfering RNA (siRNA). Immunostaining and confocal microscopy confirmed that membrane localization of F508del-CFTR induced by VIP was abolished in the presence of EAVSLKPT but not with other isoform inhibitors. In recombinant baby hamster kidney cells, endogenously expressing PKCε but no VIP receptor, wild-type, and F508del-CFTR sensitivity to cpt-cAMP stimulation was increased by PMA treatment. Biotinylation assays and immunoblots confirmed that PMA (0.5-2 h) induced a greater than threefold increase in membrane CFTR, whereas forskolin had no effect. The PMA effect was abolished by specifically inhibiting PKCε (EAVSLKPT IC(50) = 5.7 μM) but not other PKC isoforms. Taken together, these results indicate that stimulating PKCε by VIP or PMA increases membrane insertion and activity of WT- and F508del-CFTR.     

Rab11b Regulates the Apical Recycling of the Cystic Fibrosis Transmembrane Conductance Regulator in Polarized Intestinal Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, ΔF508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells.     

Headgroup-specific exposure of phospholipids in ABCA1-expressing cells

ABCA1 has been established to be required for the efflux of cholesterol and phospholipids to apolipoproteins such as apoA-I. At present, it is unclear whether ABCA1-mediated lipid exposure is specific with regard to lipid headgroups and whether it requires calcium activation and the presence of a lipid acceptor. In the present work, we found exofacial exposure of endogenous phosphatidylserine in the absence of apoA-I to be enhanced in ABCA1-GFP expressing MDCKII and HeLa cells compared with control cells. By using C6-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-labeled phospholipid analogues, we observed elevated redistribution of phosphatidylserine and phosphatidylethanolamine but not of phosphatidylcholine analogues from the cytoplasmic to the exoplasmic leaflet of the plasma membrane of ABCA1-GFP expressing cells. Whereas glyburide affected neither the level of exofacial endogenous PS nor the outward movement of the amino phospholipid analogues, the latter was sensitive to intracellular Ca2+ in ABCA1-GFP expressing cells, further enhancing outward analogue redistribution with respect to control cells. Both receptor-mediated endocytosis and fluidphase endocytosis were reduced in MDCKII cells expressing ABCA1-GFP. Glyburide raised the level of receptor-mediated endocytosis in the ABCA1-GFP expressing cell to the level of control cells in the absence of glyburide. In control cells, however, fluid-phase endocytosis but not receptor-mediated endocytosis was significantly reduced upon glyburide treatment.     

Mechanisms and consequences of cellular cholesterol exchange and transfer

It is apparent from consideration of the reactions involved in cellular cholesterol homeostasis that passive transfer of unesterified cholesterol molecules plays a role in cholesterol transport in vivo. Studies in model systems have established that free cholesterol molecules can transfer between membranes by diffusion through the intervening aqueous layer. Desorption of free cholesterol molecules from the donor lipid-water interface is rate-limiting for the overall transfer process and the rate of this step is influenced by interactions of free cholesterol molecules with neighboring phospholipid molecules. The influence of phospholipid unsaturation and sphingomyelin content on the rate of free cholesterol exchange are known in pure phospholipid bilayers and similar effects probably occur in cell membranes. The rate of free cholesterol clearance from cells is determined by the structure of the plasma membrane. It follows that the physical state of free cholesterol in the plasma membrane is important for the kinetics of cholesterol clearance and cell cholesterol homeostasis, as well as the structure of the plasma membrane. Bidirectional flux of free cholesterol between cells and lipoproteins occurs and rate constants characteristic of influx and efflux can be measured. The direction of any net transfer of free cholesterol is determined by the relative free cholesterol/phospholipid molar ratios of the donor and acceptor particles. Cholesterol diffuses down its gradient of chemical potential generally partitioning to the phospholipid-rich particle. Such a surface transfer process can lead to delivery of cholesterol to cells. This mechanism operates independently of any lipoprotein internalization by receptor-mediated endocytosis. The influence of enzymes such as lecithin-cholesterol acyltransferase and hepatic lipase on the direction of net transfer of free cholesterol between lipoproteins and cells can be understood in terms of their effects on the pool sizes and the rate constants for influx and efflux. Excess accumulation of free cholesterol in cells stimulates the rate of cholesteryl ester formation and induces deposition of cholesteryl ester inclusions in the cytoplasm similar to the situation in the 'foam' cells of atherosclerotic plaque. Clearance of cellular cholesteryl ester requires initial hydrolysis to free cholesterol followed by efflux of this free cholesterol. The rate of clearance of cholesteryl ester from cytoplasmic droplets is influenced by the physical state of the cholesteryl ester; liquid-crystalline cholesteryl ester is removed more slowly than cholesteryl ester in a liquid state.(ABSTRACT TRUNCATED AT 400 WORDS)     

Accumulation of cardiolipin and lysocardiolipin in fibroblasts from Tangier disease subjects

Tangier disease (TD) is an inherited disorder of lipid metabolism characterized by very low high density lipoprotein (HDL) plasma levels, cellular cholesteryl ester accumulation and reduced cholesterol excretion in response to HDL apolipoproteins. Molecular defects in the ATP binding cassette transporter 1 (ABCA1) have recently been identified as the cause of TD. ABCA1 plays a key role in the translocation of cholesterol across the plasma membrane, and defective ABCA1 causes cholesterol storage in TD cells. Not only cholesterol efflux, but also phospholipid efflux was shown to be impaired in TD cells. By use of thin layer chromatography, high performance liquid chromatography and time-of-flight secondary ion mass spectrometry, we characterized the cellular phospholipid content in fibroblasts from three homozygous TD patients. The cellular content of the major phospholipids was not found to be significantly altered in TD fibroblasts. However, the two phospholipids cardiolipin and lysocardiolipin, which make up minute amounts in normal cells, were at least 3–5-fold enriched in fibroblasts from TD subjects. A structurally closely related phospholipid (lysobisphosphatidic acid) has recently been shown to be enriched in Niemann–Pick type C, another lipid storage disorder. Altogether these data may indicate that the role of these phospholipids is a regulatory one rather than that of a bulk mediator of cholesterol solubilization in sterol trafficking and efflux.