Triglyceride-rich lipoprotein, remnant cholesterol, and apolipoproteins CII, CIII, and E in patients with schizophrenia.

Patients with schizophrenia show a disproportionally increased risk of cardiovascular disease. Hypertriglyceridemia is prevalent in this population; however, how this relates to levels of remnant cholesterol, triglyceride (TG)-rich lipoprotein (TRL) particle size and composition, TG turnover, and apolipoprotein (apo) and angiopoietin-like protein (ANGPTL) concentrations is unknown. Fasting levels of cholesterol (total [TC], LDL-C, HDL-C, non-HDL-C and remnant cholesterol) and TG were determined in 110 patients diagnosed with schizophrenia, and 46 healthy controls. TRL particle size, concentration and composition, and ß-hydroxybutyrate (TG turnover marker) were assessed by NMR. Levels of apoCII, apoCIII, apoE, ANGPTL3, ANGPTL4, and ANGPTL8 were measured by ELISA, and apoCII, apoCIII and apoE were further evaluated in HDL and non-HDL fractions. Patients with schizophrenia had significantly elevated TG, TG:apoB ratio, non-HDL-C, remnant cholesterol, non-HDL-apoCII and non-HDL-apoCIII, and HDL-apoE (all P < 0.05), lower HDL-C and apoA-I (all P < 0.001), and comparable apoB, TC, TC:apoB ratio, LDL-C, ß-hydroxybutyrate, ANGPTL3, ANGPTL4 and ANGPTL8 to healthy controls. Patients had a 12.0- and 2.5-fold increase in the concentration of large and medium TRL particles respectively, but similar cholesterol:TG ratio within each particle. Plasma TG, remnant cholesterol, and large and medium TRL particle concentrations correlated strongly with apoCII, apoCIII, and apoE in the non-HDL fraction, and with apoCIII and apoE in the HDL fraction in patients with schizophrenia. Differences in TG, HDL-C, TRL particle concentrations, apoCIII, and apoE persisted after adjustment for conventional risk factors. These results are consistent with impaired TRL lipolysis and clearance in patients with schizophrenia which may be responsive to targeting apoCIII.

ApoA-I Protects Pancreatic ß-Cells From Cholesterol-Induced Mitochondrial Damage and Restores Their Ability to Secrete Insulin.

BACKGROUND: High cholesterol levels in pancreatic ß-cells cause oxidative stress and decrease insulin secretion. ß-cells can internalize apo (apolipoprotein) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves ß-cell insulin secretion by reducing oxidative stress. METHODS: Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-ß-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mmol/L glucose was quantified by radioimmunoassay. Internalization of fluorescently labeled apoA-I by ß-cells was monitored by flow cytometry. The effects of apoA-I internalization on ß-cell gene expression were evaluated by RNA sequencing. ApoA-I-binding partners on the ß-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in ß-cells and isolated islets with MitoSOX and confocal microscopy. RESULTS: An F1-ATPase ß-subunit on the ß-cell surface was identified as the main apoA-I-binding partner. ß-cell internalization of apoA-I was time-, concentration-, temperature-, cholesterol-, and F1-ATPase ß-subunit-dependent. ß-cells with internalized apoA-I (apoA-I+ cells) had higher cholesterol and cell surface F1-ATPase ß-subunit levels than ß-cells without internalized apoA-I (apoA-I- cells). The internalized apoA-I colocalized with mitochondria and was associated with reduced oxidative stress and increased insulin secretion. The IF1 (ATPase inhibitory factor 1) attenuated apoA-I internalization and increased oxidative stress in Ins-1E ß-cells and isolated mouse islets. Differentially expressed genes in apoA-I+ and apoA-I- Ins-1E cells were related to protein synthesis, the unfolded protein response, insulin secretion, and mitochondrial function. CONCLUSIONS: These results establish that ß-cells are functionally heterogeneous, and apoA-I restores insulin secretion in ß-cells with elevated cholesterol levels by improving mitochondrial redox balance.

Identification of a Distinct Platelet Phenotype in the Elderly: ADP Hypersensitivity Coexists With Platelet PAR (Protease-Activated Receptor)-1 and PAR-4-Mediated Thrombin Resistance.

BACKGROUND: Thrombin (via PAR [protease-activated receptor]-1 and PAR-4) and ADP (via P2Y12 receptors) are potent endogenous platelet activators implicated in the development of cardiovascular disease. We aimed to assess whether platelet pathways alter with aging. METHODS: We characterized platelet activity in community-dwelling volunteers (n=174) in the following age groups: (1) 20 to 30 (young); (2) 40 to 55 (middle-aged); (3) ≥70 years (elderly). Platelet activity was assessed by aggregometry; flow cytometry (surface markers [P-selectin: alpha granule release, CD63: dense granule release, PAC-1: measure of conformationally active GPIIb/IIIa at the fibrinogen binding site]) measured under basal conditions and after agonist stimulation [ADP, thrombin, PAR-1 agonist or PAR-4 agonist]); receptor cleavage and quantification; fluorometry; calcium flux; ELISA. RESULTS: The elderly had higher basal platelet activation than the young, evidenced by increased expression of P-selectin, CD63, and PAC-1, which correlated with increasing inflammation (IL [interleukin]-1ß/IL-6). The elderly demonstrated higher P2Y12 receptor density, with greater ADP-induced platelet aggregation (P<0.05). However, elderly subjects were resistant to thrombin, achieving less activation in response to thrombin (higher EC50) and to selective stimulation of both PAR-1 and PAR-4, with higher basal PAR-1/PAR-4 cleavage and less inducible PAR-1/PAR-4 cleavage (all P<0.05). Thrombin resistance was attributable to a combination of reduced thrombin orienting receptor GPIbα (glycoprotein Ibα), reduced secondary ADP contribution to thrombin-mediated activation, and blunted calcium flux. D-Dimer, a marker of in situ thrombin generation, correlated with platelet activation in the circulation, ex vivo thrombin resistance, and circulating inflammatory mediators (TNF [tumor necrosis factor]-α/IL-6). CONCLUSIONS: Aging is associated with a distinctive platelet phenotype of increased basal activation, ADP hyperreactivity, and thrombin resistance. In situ thrombin generation associated with systemic inflammation may be novel target to prevent cardiovascular disease in the elderly.

Rapid Release of Interleukin-1ß from Human Platelets Is Independent of NLRP3 and Caspase.

OBJECTIVE: Platelets are critical in mediating both rapid responses to injury and the development and progression of coronary disease. Several studies have shown that, after prolonged exposure to agonists, they produce and release inflammatory mediators including interleukin-1ß (IL-1ß), via the classical pathway (NLRP3 inflammasome and caspase-1 cleavage to release active IL-1ß) as described for leukocytes. This study aimed to determine whether there is rapid release of IL-1ß in response to soluble platelet agonists and whether such rapid release is NLRP3- and caspase-1-dependent. METHODS AND RESULTS: Using flow cytometry to detect platelet activation (and release of α and dense granule contents) and the combination of Western blotting, enzyme-linked-immunosorbent assay, and immunogold labeling transmission electron and immunofluorescence microscopy, we identified that resting human platelets contain mature IL-1ß. Platelets release IL-1ß within minutes in response to adenosine diphosphate (ADP), collagen, and thrombin receptor agonists, but not in response to conventional NLRP3 inflammasome agonists-lipopolysaccharide and adenosine triphosphate. The rapid release of IL-1ß in response to ADP and thrombin receptor agonists was independent of caspases (including caspase-1) and NLRP3. Immature and mature IL-1ß were identified as low-abundance proteins on transmission electron microscopy of human platelets, and were localized to the platelet cytosol, open canalicular system, and the periphery of α granules. CONCLUSION: Unlike monocytes and neutrophils, human platelets are capable of rapid agonist- and time-dependent release of IL-1ß by a mechanism which is independent of caspase-1 and NLRP3.

Free Thiol ß2-GPI (ß-2-Glycoprotein-I) Provides a Link Between Inflammation and Oxidative Stress in Atherosclerotic Coronary Artery Disease.

OBJECTIVE: Atherosclerotic coronary artery disease is well recognised as an inflammatory disorder that is also influenced by oxidative stress. ß2-GPI (ß-2-glycoprotein-I) is a circulating plasma protein that undergoes post-translational modification and exists in free thiol as well as oxidized forms. The aim of this study was to assess the association between these 2 post-translational redox forms of ß2-GPI and atherosclerotic coronary artery disease. Approach and Results: Stable patients presenting for elective coronary angiography or CT coronary angiography were prospectively recruited. A separate group of patients after reperfused ST-segment-elevation myocardial infarction formed an acute coronary syndrome subgroup. All patients had collection of fasting serum and plasma for quantification of total and free thiol ß2-GPI. Coronary artery disease extent was quantified by the Syntax and Gensini scores. A total of 552 patients with stable disease and 44 with acute coronary syndrome were recruited. While total ß2-GPI was not associated with stable coronary artery disease, a higher free thiol ß2-GPI was associated with its presence and extent. This finding remained significant after correcting for confounding variables, and free thiol ß2-GPI was a better predictor of stable coronary artery disease than hs-CRP (high-sensitivity C-reactive protein). Paradoxically, there were lower levels of free thiol ß2-GPI after ST-segment-elevation myocardial infarction. CONCLUSIONS: Free thiol ß2-GPI is a predictor of coronary artery disease presence and extent in stable patients. Free thiol ß2-GPI was a better predictor than high-sensitivity C-reactive protein.

TRAIL-Expressing Monocyte/Macrophages Are Critical for Reducing Inflammation and Atherosclerosis.

Circulating tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) levels are reduced in patients with cardiovascular disease, and TRAIL gene deletion in mice exacerbates atherosclerosis and inflammation. How TRAIL protects against atherosclerosis and why levels are reduced in disease is unknown. Here, multiple strategies were used to identify the protective source of TRAIL and its mechanism(s) of action. Samples from patients with coronary artery disease and bone-marrow transplantation experiments in mice lacking TRAIL revealed monocytes/macrophages as the main protective source. Accordingly, deletion of TRAIL caused a more inflammatory macrophage with reduced migration, displaying impaired reverse cholesterol efflux and efferocytosis. Furthermore, interleukin (IL)-18, commonly increased in plasma of patients with cardiovascular disease, negatively regulated TRAIL transcription and gene expression, revealing an IL-18-TRAIL axis. These findings demonstrate that TRAIL is protective of atherosclerosis by modulating monocyte/macrophage phenotype and function. Manipulating TRAIL levels in these cells highlights a different therapeutic avenue in the treatment of cardiovascular disease.

Cell-specific production, secretion, and function of apolipoprotein E.

Apolipoprotein E (apoE) is a 34-kDa glycoprotein that is secreted from many cells throughout the body. ApoE is best known for its role in lipoprotein metabolism. Recent studies underline the association of circulating lipoprotein-associated apoE levels and the development for cardiovascular disease (CVD). Besides its well-established role in pathology of CVD, it is also implicated in neurodegenerative diseases and recent new data on adipose-produced apoE point to a novel metabolic role for apoE in obesity. The regulation of apoE production and secretion is remarkably cell and tissue specific. Here, we summarize recent insights into the differential regulation apoE production and secretion by hepatocytes, monocytes/macrophages, adipocytes, and the central nervous system and relevant variations in apoE biochemistry and function.

The E3 ubiquitin ligase, HECTD1, is involved in ABCA1-mediated cholesterol export from macrophages.

The ABC lipid transporters, ABCA1 and ABCG1, are essential for maintaining lipid homeostasis in cells such as macrophages by exporting excess cholesterol to extracellular acceptors. These transporters are highly regulated at the post-translational level, including protein ubiquitination. Our aim was to investigate the role of the E3 ubiquitin ligase HECTD1, recently identified as associated with ABCG1, on ABCG1 and ABCA1 protein levels and cholesterol export function. Here, we show that HECTD1 protein is widely expressed in a range of human and murine primary cells and cell lines, including macrophages, neuronal cells and insulin secreting ß-cells. siRNA knockdown of HECTD1 unexpectedly decreased overexpressed ABCG1 protein levels and cell growth, but increased native ABCA1 protein in CHO-K1 cells. Knockdown of HECTD1 in unloaded THP-1 macrophages did not affect ABCG1 but significantly increased ABCA1 protein levels, in wild-type as well as THP-1 cells that do not express ABCG1. Cholesterol export from macrophages to apoA-I over time was increased after knockdown of HECTD1, however these effects were not sustained in cholesterol-loaded cells. In conclusion, we have identified a new candidate, the E3 ubiquitin ligase HECTD1, that may be involved in the regulation of ABCA1-mediated cholesterol export from unloaded macrophages to apoA-I. The exact mechanism by which this ligase affects this pathway remains to be elucidated.

Short-term cooling increases serum triglycerides and small high-density lipoprotein levels in humans.

BACKGROUND: Cold exposure and ß3-adrenergic receptor agonism, which both activate brown adipose tissue, markedly influence lipoprotein metabolism by enhancing lipoprotein lipase-mediated catabolism of triglyceride-rich lipoproteins and increasing plasma high-density lipoprotein (HDL) levels and functionality in mice. However, the effect of short-term cooling on human lipid and lipoprotein metabolism remained largely elusive. OBJECTIVE: The objective was to assess the effect of short-term cooling on the serum lipoprotein profile and HDL functionality in men. METHODS: Body mass index-matched young, lean men were exposed to a personalized cooling protocol for 2 hours. Before and after cooling, serum samples were collected for analysis of lipids and lipoprotein composition by 1H-nuclear magnetic resonance. Adenosine triphosphate-binding cassette A1 (ABCA1)-mediated cholesterol efflux capacity of HDL was measured using [3H]cholesterol-loaded ABCA1-transfected Chinese hamster ovary cells. RESULTS: Short-term cooling increased serum levels of free fatty acids, triglycerides, and cholesterol. Cooling increased the concentration of large very low-density lipoprotein (VLDL) particles accompanied by increased mean size of VLDL particles. In addition, cooling enhanced the concentration of small LDL and small HDL particles as well as the cholesterol levels within these particles. The increase in small HDL was accompanied by increased ABCA1-dependent cholesterol efflux in vitro. CONCLUSIONS: Our data show that short-term cooling increases the concentration of large VLDL particles and increases the generation of small LDL and HDL particles. We interpret that cooling increases VLDL production and turnover, which results in formation of surface remnants that form small HDL particles that attract cellular cholesterol.

Measurement of Macrophage-Specific In Vivo Reverse Cholesterol Transport in Mice.

Reverse cholesterol transport (RCT) is one of the main processes that is thought to protect against cardiovascular disease. RCT constitutes the removal of cholesterol from peripheral sites, its transport through the plasma compartment for delivery to the liver for excretion. Here, we describe an in vivo RCT method that incorporates these steps, measuring movement of cholesterol from macrophages to the plasma, the liver, and finally to the feces in mice.

Human macrophage cathepsin B-mediated C-terminal cleavage of apolipoprotein A-I at Ser228 severely impairs antiatherogenic capacity.

Apolipoprotein A-I (apoA-I) is the major component of HDL and central to the ability of HDL to stimulate ATP-binding cassette transporter A1 (ABCA1)-dependent, antiatherogenic export of cholesterol from macrophage foam cells, a key player in the pathology of atherosclerosis. Cell-mediated modifications of apoA-I, such as chlorination, nitration, oxidation, and proteolysis, can impair its antiatherogenic function, although it is unknown whether macrophages themselves contribute to such modifications. To investigate this, human monocyte-derived macrophages (HMDMs) were incubated with human apoA-I under conditions used to induce cholesterol export. Two-dimensional gel electrophoresis and Western blot analysis identified that apoA-I is cleaved (∼20-80%) by HMDMs in a time-dependent manner, generating apoA-I of lower MW and isoelectric point. Mass spectrometry analysis identified a novel C-terminal cleavage site of apoA-I between Ser228-Phe229 Recombinant apoA-I truncated at Ser228 demonstrated profound loss of capacity to solubilize lipid and to promote ABCA1-dependent cholesterol efflux. Protease inhibitors, small interfering RNA knockdown in HMDMs, mass spectrometry analysis, and cathepsin B activity assays identified secreted cathepsin B as responsible for apoA-I cleavage at Ser228 Importantly, C-terminal cleavage of apoA-I was also detected in human carotid plaque. Cleavage at Ser228 is a novel, functionally important post-translational modification of apoA-I mediated by HMDMs that limits the antiatherogenic properties of apoA-I.-Dinnes, D. L. M., White, M. Y., Kockx, M., Traini, M., Hsieh, V., Kim, M.-J., Hou, L., Jessup, W., Rye, K.-A., Thaysen-Andersen, M., Cordwell, S. J., Kritharides, L. Human macrophage cathepsin B-mediated C-terminal cleavage of apolipoprotein A-I at Ser228 severely impairs antiatherogenic capacity.

HDL particle size is a critical determinant of ABCA1-mediated macrophage cellular cholesterol export.

RATIONALE: High-density lipoprotein (HDL) is a heterogeneous population of particles. Differences in the capacities of HDL subfractions to remove cellular cholesterol may explain variable correlations between HDL-cholesterol and cardiovascular risk and inform future targets for HDL-related therapies. The ATP binding cassette transporter A1 (ABCA1) facilitates cholesterol efflux to lipid-free apolipoprotein A-I, but the majority of apolipoprotein A-I in the circulation is transported in a lipidated state and ABCA1-dependent efflux to individual HDL subfractions has not been systematically studied. OBJECTIVE: Our aims were to determine which HDL particle subfractions are most efficient in mediating cellular cholesterol efflux from foam cell macrophages and to identify the cellular cholesterol transporters involved in this process. METHODS AND RESULTS: We used reconstituted HDL particles of defined size and composition, isolated subfractions of human plasma HDL, cell lines stably expressing ABCA1 or ABCG1, and both mouse and human macrophages in which ABCA1 or ABCG1 expression was deleted. We show that ABCA1 is the major mediator of macrophage cholesterol efflux to HDL, demonstrating most marked efficiency with small, dense HDL subfractions (HDL3b and HDL3c). ABCG1 has a lesser role in cholesterol efflux and a negligible role in efflux to HDL3b and HDL3c subfractions. CONCLUSIONS: Small, dense HDL subfractions are the most efficient mediators of cholesterol efflux, and ABCA1 mediates cholesterol efflux to small dense HDL and to lipid-free apolipoprotein A-I. HDL-directed therapies should target increasing the concentrations or the cholesterol efflux capacity of small, dense HDL species in vivo.

Sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) is a novel nucleotide phosphodiesterase regulated by cholesterol in human macrophages.

Cholesterol-loaded foam cell macrophages are prominent in atherosclerotic lesions and play complex roles in both inflammatory signaling and lipid metabolism, which are underpinned by large scale reprogramming of gene expression. We performed a microarray study of primary human macrophages that showed that transcription of the sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) gene is up-regulated after cholesterol loading. SMPDL3A protein expression in and secretion from primary macrophages are stimulated by cholesterol loading, liver X receptor ligands, and cyclic AMP, and N-glycosylated SMPDL3A protein is detectable in circulating blood. We demonstrate for the first time that SMPDL3A is a functional phosphodiesterase with an acidic pH optimum. We provide evidence that SMPDL3A is not an acid sphingomyelinase but unexpectedly is active against nucleotide diphosphate and triphosphate substrates at acidic and neutral pH. SMPDL3A is a major source of nucleotide phosphodiesterase activity secreted by liver X receptor-stimulated human macrophages. Extracellular nucleotides such as ATP may activate pro-inflammatory responses in immune cells. Increased expression and secretion of SMPDL3A by cholesterol-loaded macrophage foam cells in lesions may decrease local concentrations of pro-inflammatory nucleotides and potentially represent a novel anti-inflammatory axis linking lipid metabolism with purinergic signaling in atherosclerosis.

Pharmacological inhibition of dynamin II reduces constitutive protein secretion from primary human macrophages.

Dynamins are fission proteins that mediate endocytic and exocytic membrane events and are pharmacological therapeutic targets. These studies investigate whether dynamin II regulates constitutive protein secretion and show for the first time that pharmacological inhibition of dynamin decreases secretion of apolipoprotein E (apoE) and several other proteins constitutively secreted from primary human macrophages. Inhibitors that target recruitment of dynamin to membranes (MiTMABs) or directly target the GTPase domain (Dyngo or Dynole series), dose- and time- dependently reduced the secretion of apoE. SiRNA oligo's targeting all isoforms of dynamin II confirmed the involvement of dynamin II in apoE secretion. Inhibition of secretion was not mediated via effects on mRNA or protein synthesis. 2D-gel electrophoresis showed that inhibition occurred after apoE was processed and glycosylated in the Golgi and live cell imaging showed that inhibited secretion was associated with reduced post-Golgi movement of apoE-GFP-containing vesicles. The effect was not restricted to macrophages, and was not mediated by the effects of the inhibitors on microtubules. Inhibition of dynamin also altered the constitutive secretion of other proteins, decreasing the secretion of fibronectin, matrix metalloproteinase 9, Chitinase-3-like protein 1 and lysozyme but unexpectedly increasing the secretion of the inflammatory mediator cyclophilin A. We conclude that pharmacological inhibitors of dynamin II modulate the constitutive secretion of macrophage apoE as a class effect, and that their capacity to modulate protein secretion may affect a range of biological processes.

Cellular cholesterol regulates ubiquitination and degradation of the cholesterol export proteins ABCA1 and ABCG1.

The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes.

Protein kinase C controls vesicular transport and secretion of apolipoprotein E from primary human macrophages.

Macrophage-specific apolipoprotein E (apoE) secretion plays an important protective role in atherosclerosis. However, the precise signaling mechanisms regulating apoE secretion from primary human monocyte-derived macrophages (HMDMs) remain unclear. Here we investigate the role of protein kinase C (PKC) in regulating basal and stimulated apoE secretion from HMDMs. Treatment of HMDMs with structurally distinct pan-PKC inhibitors (calphostin C, Ro-31-8220, Go6976) and a PKC inhibitory peptide all significantly decreased apoE secretion without significantly affecting apoE mRNA or apoE protein levels. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated apoE secretion, and both PMA-induced and apoAI-induced apoE secretion were inhibited by PKC inhibitors. PKC regulation of apoE secretion was found to be independent of the ATP binding cassette transporter ABCA1. Live cell imaging demonstrated that PKC inhibitors inhibited vesicular transport of apoE to the plasma membrane. Pharmacological or peptide inhibitor and knockdown studies indicate that classical isoforms PKCα/ß and not PKCδ, -ε, -θ, or -ι/ζ isoforms regulate apoE secretion from HMDMs. The activity of myristoylated alanine-rich protein kinase C substrate (MARCKS) correlated with modulation of PKC activity in these cells, and direct peptide inhibition of MARCKS inhibited apoE secretion, implicating MARCKS as a downstream effector of PKC in apoE secretion. Comparison with other secreted proteins indicated that PKC similarly regulated secretion of matrix metalloproteinase 9 and chitinase-3-like-1 protein but differentially affected the secretion of other proteins. In conclusion, PKC regulates the secretion of apoE from primary human macrophages.

Protein kinase A modulates the activity of a major human isoform of ABCG1.

ABCG1 is an ABC half-transporter that exports cholesterol from cells to HDL. This study set out to investigate differences in posttranslational processing of two human ABCG1 protein isoforms, termed ABCG1(+12) and ABCG1(-12), that differ by the presence or absence of a 12 amino acid peptide. ABCG1(+12) is expressed in human cells and tissues, but not in mice. We identified two protein kinase A (PKA) consensus sites in ABCG1(+12), absent from ABCG1(-12). Inhibition of PKA with either of two structurally unrelated inhibitors resulted in a dose-dependent increase in cholesterol export from cells expressing ABCG1(+12), whereas ABCG1(-12)-expressing cells were unaffected. This was associated with stabilization of the ABCG1(+12) protein, and ABCG1(+12)-S389 was necessary to mediate these effects. Mutation of this serine to aspartic acid, simulating a constitutively phosphorylated state, resulted in accelerated degradation of ABCG1(+12) and reduced cholesterol export. Engineering an equivalent PKA site into ABCG1(-12) rendered this isoform responsive to PKA inhibition, confirming the relevance of this sequence. Together, these results demonstrate an additional level of complexity to the posttranslational control of this human ABCG1 isoform that is absent from ABCG1(-12) and the murine ABCG1 homolog.

IL-1beta signals through the EGF receptor and activates Egr-1 through MMP-ADAM.

The immediate-early gene Egr-1 controls the inducible expression of many genes implicated in the pathogenesis of a range of vascular disorders, yet our understanding of the mechanisms controlling the rapid expression of this prototypic zinc finger transcription factor is poor. Here we show that Egr-1 expression induced by IL-1beta is dependent on metalloproteinases (MMP) and a disintegrin and a metalloproteinase (ADAM). Pharmacologic MMP/ADAM inhibitors and siRNA knockdown prevent IL-1beta induction of Egr-1. Further, IL-1beta activates Egr-1 via the epidermal growth factor receptor (EGFR). This is blocked by EGFR tyrosine kinase inhibition and EGFR knockdown. IL-1beta induction of Egr-1 expression is reduced in murine embryonic fibroblasts (mEFs) deficient in ADAM17 despite unbiased expression of EGFR and IL-1RI in ADAM17-deficient and wild-type mEFs. Finally, we show that IL-1beta-inducible wound repair after mechanical injury requires both EGFR and MMP/ADAM. This study reports for the first time that Egr-1 induction by IL-1beta involves EGFR and MMP/ADAM-dependent EGFR phosphorylation.

Cholesterol accumulation inhibits ER to Golgi transport and protein secretion: studies of apolipoprotein E and VSVGt.

Cholesterol excess is typical of various diseases including atherosclerosis. We have investigated whether cholesterol accumulation in the ER (endoplasmic reticulum) can inhibit exit of vesicular cargo and secretion of proteins by studying apoE (apolipoprotein E), a significant glycoprotein in human health and disease. CHO (Chinese hamster ovary) cells expressing human apoE under a cholesterol-independent promoter incubated with cholesterol-cyclodextrin complexes showed increased levels of cellular free and esterified cholesterol, inhibition of SREBP-2 (sterol-regulatory-element-binding protein 2) processing, and a mild induction of ER stress, indicating significant accumulation of cholesterol in the ER. Secretion of apoE was markedly inhibited by cholesterol accumulation, and similar effects were observed in cells enriched with lipoprotein-derived cholesterol and in primary human macrophages. Removal of excess cholesterol by a cyclodextrin vehicle restored apoE secretion, indicating that the transport defect was reversible. That cholesterol impaired protein trafficking was supported by the cellular accumulation of less sialylated apoE glycoforms, and by direct visualization of altered ER to Golgi transport of thermo-reversible VSVG (vesicular stomatitis virus glycoprotein) linked to GFP (green fluorescent protein). We conclude that intracellular accumulation of cholesterol in the ER reversibly inhibits protein transport and secretion. Strategies to correct ER cholesterol may restore homoeostatic processes and intracellular protein transport in conditions characterized by cholesterol excess.

Glycosylation and sialylation of macrophage-derived human apolipoprotein E analyzed by SDS-PAGE and mass spectrometry: evidence for a novel site of glycosylation on Ser290.

Apolipoprotein E (apoE) is a 34-kDa glycoprotein secreted from various cells including hepatocytes and macrophages and plays an important role in remnant lipoprotein clearance, immune responses, Alzheimer disease, and atherosclerosis. Cellular apoE and plasma apoE exist as multiple glycosylated and sialylated glycoforms with plasma apoE being less glycosylated/sialylated than cell-derived apoE. Some of the glycan structures on plasma apoE are characterized; however, the more complicated structures on plasma and cellular/secreted apoE remain unidentified. We investigated glycosylation and sialylation of cellular and secreted apoE from primary human macrophages by one- and two-dimensional gel electrophoresis and mass spectrometry. Our results identify eight different glycoforms with (HexNAc)(2)-Hex(2)-(NeuAc)(2) being the most complex glycan detected on Thr(194) in both cellular and secreted apoE. Four additional glycans were identified on apoE(283-299), and using beta-elimination/alkylation by methylamine in vitro, we identified Ser(290) as a novel site of glycan attachment. Comparison of plasma and cellular/secreted apoE from the same donor confirmed that cell-derived apoE is more extensively sialylated than plasma apoE. Given the importance of the C terminus of apoE in regulating apoE solubility, stability, and lipid binding, these results may have important implications for our understanding of apoE biochemistry.