The nucleotide receptor P2Y13 is a key regulator of hepatic High-Density Lipoprotein (HDL) endocytosis

Cell surface receptors for high-density lipoprotein (HDL) on hepatocytes are major partners in the regulation of cholesterol homeostasis. We recently identified a cell surface ATP synthase as a high-affinity receptor for HDL apolipoprotein A-I (apoA-I) on human hepatocytes. Stimulation of this ectopic ATP synthase by apoA-I triggered a low-affinity-receptor-dependent HDL endocytosis by a mechanism strictly related to the generation of ADP. This suggests that nucleotide G-protein- coupled receptors of the P2Y family are molecular components in this pathway. Only P2Y₁ and P2Y₁₃ are present on the membrane of hepatocytes. Using both a pharmacological approach and small interference RNA, we identified P2Y₁₃ as the main partner in hepatic HDL endocytosis, in cultured cells as well as in situ in perfused mouse livers. We also found a new important action of the antithrombotic agent AR-C69931MX as a strong activator of P2Y₁₃-mediated HDL endocytosis. Received 9 May 2005; received after revision 24 June 2005; accepted 1 September 2005     

Apolipoprotein M affecting lipid metabolism or just catching a ride with lipoproteins in the circulation?

Apolipoprotein M (apoM) is a novel apolipoprotein found mainly in high-density lipoproteins (HDL). Its function is yet to be defined. ApoM (25 kDa) has a typical lipocalin ?-barrel fold and a hydrophobic pocket. Retinoids bind apoM but with low affinity and may not be the natural ligands. ApoM retains its signal peptide, which serves as a hydrophobic anchor to the lipoproteins. This prevents apoM from being lost in the urine. Approximately 5% of HDL carries an apoM molecule. ApoM in plasma (1 μM) correlates strongly with both low-density lipoprotein (LDL) and HDL cholesterol, suggesting a link to cholesterol metabolism. However, in casecontrol studies, apoM levels in patients with coronary heart disease (CHD) and controls were similar, suggesting apoM levels not to affect the risk for CHD in humans. Experiments in transgenic mice suggested apoM to have antiatherogenic properties; possible mechanisms include increased formation of pre-? HDL, enhanced cholesterol mobilization from foam cells, and increased antioxidant properties. Received 28 November 2008; received after revision 15 December 2008; accepted 16 December 2008     

Cholesterol feeding to rats does not modulate the expression of binding sites for HDL on liver membranes

Summary The binding of HDL, Apo-E-free, was studied in rats fed a cholesterol rich diet for 2, 4 and 7 days. Plasma cholesterol increased up to 16-fold (from 55 to 900 mg/dl); liver cholesterol was also raised, from 0.5 to 16 mg/g of tissue. The HDL binding to membrane preparations was not affected while the binding of VLDL was reduced to about 50% of the controls. These data show, therefore, that liver binding sites for HDL are refractory to regulation by dietary cholesterol.     

The ATP synthase (F0−F1) complex in oxidative phosphorylation

The transmembrane electrochemical proton gradient generated by the redox systems of the respiratory chain in mitochondria and aerobic bacteria is utilized by proton translocating ATP synthases to catalyze the synthesis of ATP from ADP and P_i. The bacterial and mitochondrial H⁺-ATP synthases both consist of a membranous sector, F₀, which forms a H⁺-channel, and an extramembranous sector, F₁, which is responsible for catalysis. When detached from the membrane, the purified F₁ sector functions mainly as an ATPase. In chloroplasts, the synthesis of ATP is also driven by a proton motive force, and the enzyme complex responsible for this synthesis is similar to the mitochondrial and bacterial ATP synthases. The synthesis of ATP by H⁺-ATP synthases proceeds without the formation of a phosphorylated enzyme intermediate, and involves co-operative interactions between the catalytic subunits.     

Net transport of cholesterol from cells of the human EA.hy 926 endothelial cell line to high density lipoproteins

EA.hy 926 cells, a human endothelial cell line, show characteristics of differentiated endothelial cells. The cells express saturable binding of apo E-free¹²⁵I-high density lipoprotein₃ (HDL₃). B_max increased from 71 to 226 ng HDL₃ bound/mg cell protein after cholesterol loading of the confluent endothelial cells with cationized low density lipoprotein (LDL). The affinity did not change after cholesterol enrichment (K_d was 37 g HDL₃ protein/ml for control cells and 31 g/ml, for loaded cells). Incubation of cholesterol-loaded EA.hy 926 cells with native HDL and LDL had different effects on cellular cholesterol levels. Incubation with HDL decreased both esterified and unesterified cellular cholesteryl, but LDL did not change total cellular cholesterol However, LDL tended to increase cellular cholesteryl esters, with a concomitant decrease of unesterified, cellular cholesterol. Incubation of endothelial cells with both HDL and LDL also resulted in decreased total cellular cholesterol levels. These data show that cationized LDL-loaded human endothelial EA.hy 926 cells can be used to study the net transport of cellular cholesterol to HDL, the first step in reverse cholesterol transport.     

Structural studies of discoidal lipoprotein A-I

Apolipoprotein A-I (apoA-I) is a major exchangeable apolipoprotein of high-density lipoproteins (HDLs), and plays an important role in reverse cholesterol transport. This process involves transport of cholesterol from peripheral tissues to the liver for processing, thereby eliminating excess cholesterol from the body. The function of apoA-I and its interaction with other components of HDL, including lecithin-cholesterol acyltransferase, seems to be closely linked to its structural plasticity. ApoA-I is likely to undergo changes in its structure and orientation between the various HDL subclasses and, therefore, knowledge of the precise structure of apoA-I is essential for understanding its role in the antiatherogenic properties of HDL. This review focuses on the role of apoA-I in reverse cholesterol transport and the work done by various groups to determine the structure of apoA-I in discoidal HDL particles.     

Cell surface adenylate kinase activity regulates the F1-ATPase/P2Y13-mediated HDL endocytosis pathway on human hepatocytes

We have previously demonstrated on human hepatocytes that apolipoprotein A-I binding to an ecto-F₁-ATPase stimulates the production of extracellular ADP that activates a P2Y₁₃-mediated high-density lipoprotein (HDL) endocytosis pathway. Therefore, we investigated the mechanisms controlling the extracellular ATP/ADP level in hepatic cell lines and primary cultures to determine their impact on HDL endocytosis. Here we show that addition of ADP to the cell culture medium induced extracellular ATP production that was due to adenylate kinase and nucleoside diphosphokinase activities, but not to ATP synthase activity. We further observed that in vitro modulation of both ecto-NDPK and AK activities could regulate the ADP-dependent HDL endocytosis. But interestingly, only AK appeared to naturally participate in the pathway by consuming the ADP generated by the ecto-F₁-ATPase. Thus controlling the extracellular ADP level is a potential target for reverse cholesterol transport regulation. Received 13 July 2006; received after revision 29 August 2006; accepted 19 September 2006     

Interaction of lipid transfer protein with plasma lipoproteins and cell membranes

Summary The hydrophobic lipid components of lipoproteins, cholesteryl ester and triglyceride, are transferred between all lipoproteins by a specific plasma glycoprotein, termed lipid transfer protein (LTP). LTP facilitates lipid transfer by an exchange process in which cholesteryl ester and triglyceride compete for transfer. Thus, LTP promotes remodeling of the lipoprotein structure, and plays an important role in the intravascular metabolism of these particles and in the lipoprotein-dependent pathways of cholesterol clearance from cells. The properties of LTP, its mechanisms of action, its roles in lipoprotein metabolism, and its modes of regulation are reviewed along with recent data that suggest a possible role for this protein in directly modifying cellular lipid composition.     

Role of the ubiquitin–proteasome system in the regulation of P2Y13 receptor expression: impact on hepatic HDL uptake

The protective effect of high density lipoproteins (HDL) against atherosclerosis is mainly attributed to their capacity to transport excess cholesterol from peripheral tissues back to the liver for further elimination into the bile, a process called reverse cholesterol transport (RCT). Recently, the importance of the P2Y₁₃ receptor (P2Y₁₃-R) was highlighted in HDL metabolism since HDL uptake by the liver was decreased in P2Y₁₃-R deficient mice, which translated into impaired RCT. Here, we investigated for the first time the molecular mechanisms regulating cell surface expression of P2Y₁₃-R. When transiently expressed, P2Y₁₃-R was mainly detected in the endoplasmic reticulum (ER) and strongly subjected to proteasome degradation while its homologous P2Y₁₂ receptor (P2Y₁₂-R) was efficiently targeted to the plasma membrane. We observed an inverse correlation between cell surface expression and ubiquitination level of P2Y₁₃-R in the ER, suggesting a close link between ubiquitination of P2Y₁₃-R and its efficient targeting to the plasma membrane. The C-terminus tail exchange between P2Y₁₃-R and P2Y₁₂-R strongly restored plasma membrane expression of P2Y₁₃-R, suggesting the involvement of the intra-cytoplasmic tail of P2Y₁₃-R in expression defect. Accordingly, proteasomal inhibition increased plasma membrane expression of functionally active P2Y₁₃-R in hepatocytes, and consequently stimulated P2Y₁₃-R-mediated HDL endocytosis. Importantly, proteasomal inhibition strongly potentiated HDL hepatic uptake (>200 %) in wild-type but not in P2Y₁₃-R-deficient mice, thus reinforcing the role of P2Y₁₃-R expression in regulating HDL metabolism. Therefore, specific inhibition of the ubiquitin–proteasome system might be a novel powerful HDL therapy to enhance P2Y₁₃-R expression and consequently promote the overall RCT.     

Production of active and passive anaphylactic shock in the WBB6F1 mouse,a mast cell-deficient strain

Summary The role of mast cells in active and passive anaphylactic shock was examined using the WBB6F₁ mouse, a genetically mast cell-deficient strain. Lethal anaphylactic shock occurred at high incidence rates in mice actively sensitized to bovine serum albumin (BSA). The reaction was specific to BSA since the shock could not be elicited by human or guinea pig serum albumin in these animals. Lethal shock could be prevented by CV-3988 but not by cyproheptadine, which suggests that the shock is mediated by PAF but not by histamine and serotonin. Similarly, lethal shock was provoked by homologous antigens in mice which had been passively sensitized with allogeneic anti-benzylpenicilloyl (BPO) IgG₁ monoclonal antibody or with allogeneic or xenogeneic anti-BSA antiserum, but not in those sensitized with allogeneic anti-BPO IgE monoclonal antibody. These findings suggest that mast cells are not necessarily required for anaphylactic shock in the mouse.     

Tangier disease: still more questions than answers

High-density lipoproteins (HDLs) play a central role in transporting cholesterol from peripheral tissues to the liver for elimination from the body. Impairment of HDL-mediated cholesterol transport favors cholesterol deposition in the arterial wall and promotes development of arteriosclerosis. Tangier disease is a severe HDL deficiency syndrome characterized by the accumulation of cholesterol in tissue macrophages and prevalent atherosclerosis. A three-decade search for a culprit in Tangier disease led to the identification of mutations in a cell membrane protein called ABCA1, which mediates the secretion of excess cholesterol from cells into the HDL metabolic pathway. Because of its ability to deplete cells of cholesterol and to raise plasma HDL levels, ABCA1 has become a promising therapeutic target for preventing cardiovascular disease.     

Is there a critical tissue oxygen tension for bioenergetic status and cellular pH regulation in solid tumors?

Bioenergetic and metabolic status have been correlated with tissue oxygenation in murine fibrosarcomas (FSaII) of varying sizes (44–600 mm³). Ratios of -nucleoside triphosphates to inorganic phosphate (NTP/P_i) and phosphocreatine to inorganic phosphate (PCr/P_i) ratios derived from³¹P nuclear magnetic resonance spectroscopy (NMR) were positively correlated to median tissue O₂ tension (pO₂) values using O₂-sensitive needle electrodes. pH declined during growth with intracellular acidosis being evident in tumors >350 mm³. Whereas lactic acid formation greatly contributed to this decline in small and medium-sized tumors, adenosine triphosphate (ATP) hydrolysis and slowing down of the activities of pumps involved in cellular pH regulation seem to be major factors responsible for intracellular acidification in bulky tumors. PCr levels decreased at an early growth stage, whilst ATP concentrations dropped in bulky malignancies only, coinciding with a decrease in adenylate energy charge and a substantial rise in the levels of total P_i On average, median pO₂ values of ca. 10 mmHg represent a critical threshold for energy metabolism. At higher median O₂ tensions, levels of ATP, phosphomonoester (PME) and total P_i were relatively constant. This coincided with intracellular alkalosis or neutrality and stable adenylate ratios. On average, median pO₂ values <10 mmHg coincided with intracellular acidosis, ATP depletion, a drop in energy charge and rising P_i levels.     

Cholesterol feeding to rats does not modulate the expression of binding sites for HDL on liver membranes

The binding of HDL, Apo-E-free, was studied in rats fed a cholesterol rich diet for 2, 4 and 7 days. Plasma cholesterol increased up to 16-fold (from 55 to 900 mg/dl); liver cholesterol was also raised, from 0.5 to 16 mg/g of tissue. The HDL binding to membrane preparations was not affected while the binding of beta VLDL was reduced to about 50% of the controls. These data show, therefore, that liver binding sites for HDL are refractory to regulation by dietary cholesterol.     

Inhibition of lung carcinoma cell growth by high density lipoprotein-associated α-tocopheryl-succinate

-Tocopheryl-succinate (TS) is a synthetic, anti-neoplastic derivative of -tocopherol. Here we studied the effects of free and high-density lipoprotein subclass 3 (HDL₃)-associated TS on the growth of human (A549) and mouse Lewis (LL2) lung carcinoma cells. Both free and HDL3-associated TS inhibited A549 growth in a time- and concentration-dependent manner. Treatment of A549 cells with TS-enriched HDL3 led to DNA fragmentation and a time-dependent decrease in immunoreactivity of poly(ADP-ribose)polymerase. Uptake experiments revealed a high capacity for selective TS uptake in excess of holoparticle endocytosis. Overexpression of scavenger receptor class B, type I (SR-BI), the prime receptor mediating selective lipid uptake, in A549 cells resulted in significantly increased selective TS uptake, a finding associated with complete cellular growth arrest. The present in vitro findings were verified in an in vivo model: tumor inoculation in C57BL6 was performed with either wild-type, -galactosidase- or SR-BI-overexpressing LL2 cells. After tumor inoculation, the animals received six consecutive intravenous injections of TS. This experimental setup resulted in significantly reduced tumor burden in animals that were inoculated with SR-BI-overexpressing LL2 cells but not in animals inoculated with wild-type or -galactocidase-transfected cells. Based on our in vitro and in vivo findings, we propose that SR-BI could provide a novel route for HDL₃-mediated drug delivery of anti-neoplastic drugs.Received 8 March 2004; received after revision 7 April 2004; accepted 26 April 2004     

Digoxin and ouabain increase the synthesis of cholesterol in human liver cells

Digoxin and ouabain are steroid drugs that inhibit the Na⁺/K⁺-ATPase, and are widely used in the treatment of heart diseases. They may also have additional effects, such as on metabolism of steroid hormones, although until now no evidence has been provided about the effects of these cardioactive glycosides on the synthesis of cholesterol. Here we report that digoxin and ouabain increased the synthesis of cholesterol in human liver HepG2 cells, enhancing the activity and the expression of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), the rate-limiting enzyme of the cholesterol synthesis. This effect was mediated by the binding of the sterol regulatory element binding protein-2 (SREBP-2) to the HMGCR promoter, and was lost in cells silenced for SREBP-2 or loaded with increasing amounts of cholesterol. Digoxin and ouabain competed with cholesterol for binding to the SREBP-cleavage-activating protein, and are critical regulators of cholesterol synthesis in human liver cells. Received 10 January 2009; received after revision 11 February 2009; accepted 6 March 2009     

Mylip makes an Idol turn into regulation of LDL receptor

High blood low-density-lipoprotein (LDL) cholesterol is a serious health problem among an increased number of patients in the Western world. Statins and other cholesterol lowering drugs have proven to be beneficial as therapy but are not optimal and show adverse effects in some patients. The LDL receptor is a crucial determinant of cholesterol metabolism in the body and amenable for drug interventions. Novel insights into the physiology of this receptor come from studies on the ubiquitination and degradation of LDL receptor by the ubiquitin ligase Mylip/Idol that is induced in cells by the nuclear receptor, LXR. This may open up new possibilities in the future to influence LDL receptor levels and cholesterol metabolism pharmacologically in various diseases.     

Molecular mechanisms of phagocytic uptake in mammalian cells

Phagocytosis is a highly conserved, complex process that has evolved to counter the constant threat posed by pathogens, effete cells and debris. Classically defined as a mechanism for internalising and destroying particles greater than 0.5 mum in size, it is a receptor-mediated, actin-driven process. The best-studied phagocytic receptors are the opsono-receptors, FcgammaR and CR3. Phagocytic uptake involves actin dynamics including polymerisation, bundling, contraction, severing and depolymerisation of actin filaments. Recent evidence points to the importance of membrane remodelling during phagocytosis, both in terms of changes in lipid composition and delivery of new membrane to the sites of particle binding. Here we review the molecular mechanisms of phagocytic uptake and some of the strategies developed by microbial pathogens to manipulate this process.     

The role of P-glycoprotein/cellular prion protein interaction in multidrug-resistant breast cancer cells treated with paclitaxel

We previously reported that treatment with P-glycoprotein (P-gp) substrates promotes in vitro invasion in multidrug-resistant (MDR) breast cancer cells. This effect is initiated by the P-gp pump function and mediated by interaction of P-gp with some unknown component(s). However, the underlying mechanism(s) remains poorly understood. Here we confirm a novel physical interaction between P-gp and cellular prion protein (PrP^c). Blocking P-gp activity or depletion of PrP^c inhibited paclitaxel (P-gp substrate)- induced invasion. Paclitaxel further facilitated the formation of P-gp/PrP^c clusters residing in caveolar domains and promoted the association of P-gp with caveolin-1. Both caveolin-1 and the integrity of caveolae were required for the drug-induced invasion. In addition, the P-gp/PrP^c complex also played an important role in anti-apoptotic activity of MCF7/Adr cells.These data provide new insights into the mode by which MDR breast cancers evade cytotoxic attacks from P-gp substrates and also suggest a role for P-gp/ PrP^c interaction in this process. Received 4 September 2008; received after revision 16 November 2008; accepted 18 November 2008