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Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:1060-1067
Published online before print April 3, 2008, doi: 10.1161/ATVBAHA.108.164350
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:1060.)
© 2008 American Heart Association, Inc.

Brief Reviews

Regulation of Endogenous Apolipoprotein E Secretion by Macrophages

Maaike Kockx; Wendy Jessup; Leonard Kritharides

From the Macrophage Biology Group (M.K., W.J., L.K.), Centre for Vascular Research, University of New South Wales, Sydney; and the Department of Cardiology (L.K.), Concord Repatriation General Hospital, Sydney South Western Area Health Service, University of Sydney, Australia.

Correspondence to Leonard Kritharides, Macrophage Biology Group, Centre for Vascular Research, Room 405C Wallace Wurth Building, University of New South Wales, High Street, Kensington, Sydney, NSW 2050, Australia. E-mail l.kritharides{at}unsw.edu.au


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowBasic Biology of ApoE...
 down arrowRegulation of ApoE Secretion...
 down arrowFuture Directions
 down arrowReferences
 
Apolipoprotein E has critical roles in the protection against atherosclerosis and is understood to follow the classical constitutive secretion pathway. Recent studies have indicated that the secretion of apoE from macrophages is a regulated process of unexpected complexity. Cholesterol acceptors such as apolipoprotein A-I, high density lipoprotein, and phospholipid vesicles can stimulate apoE secretion. The ATP binding cassette transporter ABCA1 is involved in basal apoE secretion and in lipidating apoE-containing particles secreted by macrophages. However, the stimulation of apoE secretion by apoA-I is ABCA1-independent, indicating the existence of both ABCA1-dependent and -independent pathways of apoE secretion. The release of apoE under basal conditions is also regulated, requiring intact protein kinase A activity, intracellular calcium, and an intact microtubular network. Mathematical modeling of apoE turnover indicates that whereas some pools of apoE are committed to either secretion or degradation, other pools can be diverted from degradation toward secretion. Targeted inhibition or stimulation of specific apoE trafficking pathways will provide unique opportunities to regulate the biology of this important molecule.


Key Words: lipid and lipoprotein metabolism • atherosclerosis pathophysiology • mechanisms of atherosclerosis • cell biology/structural biology • cell signalling/signal transduction


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowBasic Biology of ApoE...
 down arrowRegulation of ApoE Secretion...
 down arrowFuture Directions
 down arrowReferences
 
Apolipoprotein E (apoE) is a 34-kDa protein that is produced and secreted by many cell types such as hepatocytes, smooth muscle cells, neuronal cells, and macrophages. As a constituent of plasma lipopoproteins, apoE directs movement of lipids from the periphery to the liver, where high affinity binding of apoE to the LDL receptor and other members of the LDL-receptor family facilitates uptake of the lipoprotein particles. A critical role for apoE in the protection against atherosclerosis was clearly established by the development of the apoE knockout mouse and subsequent studies which showed that macrophage-specific expression of apoE was antiatherogenic.1,2 The mechanisms by which macrophage apoE is antiatherogenic may include stimulating the removal of excess cholesterol from macrophage foam cells,3 as well as anti-inflammatory,4 antiproliferative,5 and immunomodulatory properties.6,7 The regulation of the secretion of apoE from macrophages is thus of great importance to our understanding of the atherosclerotic process.

The aim of this review is to describe the regulation of the secretion of apoE by macrophages, in light of recent observations indicating that its trafficking and secretion are under the control of signaling pathways. Although recycling of lipoprotein-derived apoE is referenced briefly for comparison, a detailed analysis of apoE recycling is beyond the scope of the present review and the interested reader is directed to recent excellent reviews on this topic.8–10


*    Basic Biology of ApoE in Macrophages
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 up arrowAbstract
 up arrowIntroduction
 *Basic Biology of ApoE...
down arrowRegulation of ApoE Secretion...
 down arrowFuture Directions
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Transcriptional Regulation of ApoE Synthesis
Transcription of macrophage apoE can be induced by differentiation, exposure to cytokines, hormones, and lipids such as cholesterol or oxysterols. In addition to proximal promoter elements, apoE transcription is modulated by 2 distal flanking sequences termed multienhancer 1 (ME.1) and multienhancer 2 (ME.2), which were shown to direct macrophage and adipose-specific expression in transgenic mice.11 Transcription factors involved in the direct transcriptional regulation of apoE in macrophages included: AP-1,12,13 NF-{kappa} B,12 LXR,14 and PPAR{gamma}.15 The LXR/RXR heterodimer regulates apoE transcription in response to lipids through interaction with conserved LXR response elements in the multienhancer regions. Studies in LXR-deficient mice indicate that LXRs mediate lipid-inducible rather than tissue-specific expression of apoE, and point to an important role of LXR in foam cell–related apoE expression in atherosclerosis.14 Regulation of apoE transcription by PPAR{gamma} is mediated via a PPRE element present in the intergenic region between the apoE and apoC1 genes, which is also well outside the proximal promoter.15

Other modulators of macrophage apoE transcription have been described, including transforming growth factor (TGF)-beta which stimulates apoE expression by promoting binding of c-Jun to AP-1,13 and lipopolysaccharide (LPS), which represses apoE gene expression via both Tpl-2 and MEKK1 signaling pathways, leading to c-Jun and NF-{kappa}B action on distinct regions in the proximal promoter.12

Posttranslational Modifications to ApoE
ApoE is understood to follow the classical secretory pathway, whereby synthesis in the ER is followed by its movement to the Golgi and Trans Golgi network, and incorporation into vesicular structures, before being transported to the plasma membrane and secreted (Figure 1, pathway 1). Secreted apoE is typically released as phospholipid discs16 (Figure 1, pathway 6), however secreted apoE can vary in its density and association with lipid.17–19 As well as being released into the extracellular medium, a proportion of secreted apoE can be found bound to the cell surface (Figure 1, pathway 7), particularly in association with heparan sulfate proteoglycans.20,21 Cell surface pools may be reinternalized and subsequently degraded (Figure 1, pathways 3 and 4), or transported to the Golgi network for further modification22 (Figure 1, pathways 3 and 5), or released into the extracellular medium (Figure 1, pathway 3, 2, and 6).


Figure 1
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  		Figure 1. Candidate trafficking routes for constitutive secretion of apoE in macrophages. (1) Secretion via tubular and vesicular transport from the Golgi (G), after transport to the Golgi from the endoplasmic reticulum (ER); (2) Secretion via recycling endosomes (RE); (3) Recycling of secreted apoE into early endosomes (EE), and thereafter degraded in lysosomes or proteasomes (Lys/P), transported to Golgi, or secreted via RE; (4) Degradation of apoE in lysosomes or proteasomes (Lys/P), probably via late endosomes (LE); (5) Poorly glycosylated apoE on cell surface can reenter the Golgi compartment to be further glycosylated, presumably via EE (3) and then LE (5); (6) Secretion of apoE (or displacement of cell surface apoE), releases apoE into extracellular medium; (7) Binding of secreted apoE to the cell surface. Adapted from Heeren et al,9 Lucas et al,21 Zhao et al,22 Kockx et al,47 Duan et al,48 and Stow et al.78

ApoE is synthesized as a 38 500 Mr protein designated preapoE, containing a NH2-terminal 18-AA extension23 which mediates entry into the ER. After cleavage of the signal peptide in the ER, the protein is trafficked to the Golgi, where apoE is O-glycosylated on threonine 19424 and extensively sialylated.16 Glycosylation and sialylation can therefore act as useful markers of the intracellular trafficking of apoE. Glycosylation and sialylation are uniformly present in apoE freshly secreted from hepatocytes and macrophages.16,25 However, the majority of apoE found in the plasma is at least partially desialylated.16,23 These posttranslational modifications of apoE do not appear to affect receptor binding or lipoprotein clearance but may determine the preferential association with high density lipoprotein (HDL) particles.26 Studies using Chinese Hamster Ovary (CHO) cells defective in O-glycosylation demonstrated that glycosylation/sialylation is not obligatory for secretion of apoE.24,27 This is supported by other studies showing that oleic acid stimulates secretion of macrophage apoE while apparently reducing its sialylation.28 The possibility that glycosylation and sialylation may affect recycling of secreted apoE (see below) remains unexplored.

Cellular Localization in Macrophages
ApoE represents up to 8% to 10% of protein constitutively secreted from macrophages.29 Although a number of studies have investigated the localization of endogenous apoE in hepatocytes,30–32 detailed microscopic studies in macrophages are limited. Werb et al29 showed that apoE was distributed in the Golgi complex, ER, secretory vesicles, and coated vesicles of mouse peritoneal macrophages. Kruth et al33 demonstrated that cellular apoE can localize to invaginations of the plasma membrane containing cholesterol crystals or modified LDL, in so-called "surface-connected" compartments where it may facilitate cholesterol solubilization. In the studies of Werb, apoE was released from secretory storage granules in response to frustrated phagocytosis that occurred after contact with immobilized immune complexes.34 The same investigators also described reuptake and resecretion of secreted apoE.29 This observation of apparent "recycling" of macrophage-derived apoE has been confirmed and extended by several groups (see below). ApoE does not accumulate in lysosomes under normal circumstances, consistent with usually efficient lysosomal degradation, but may be observed when lysosomal activity is inhibited.35

The quantitative importance of cell surface–bound apoE, relative to internal vesicular apoE, as a source for secreted apoE varies between cell types. In hepatocytes (studies principally performed in HepG2 cells) up to 54% of total cellular apoE at steady-state is at the cell surface,36 existing in distinct heparinase- and lipid-releasable pools.20 In contrast, in primary macrophages,17,22,37 less than 10% of cell apoE is on the cell surface. This suggests that the binding of endogenous apoE to cell-surface proteoglycans varies between cell types and appears to be quantitatively more important in HepG2 cells than in macrophages. There is also variation between primary macrophages of different species, as primary murine macrophages have a larger cell surface pool of apoE (sensitive to pronase) than do primary human macrophages.38 In primary human macrophages heparinase has no effect on apoE secretion,37,38 suggesting that the cell-surface binding of apoE to proteoglycans may be a quantitatively minor regulator of the rate of apoE secretion under steady state conditions.

Recycling of Macrophage-Derived ApoE in Macrophages
Reuptake of endogenously synthesized and secreted apoE by macrophages was first observed by Werb et al.29 Recent studies have shown that apoE can undergo repeated cycles of internalisation and resecretion (Figure 1, pathways 3 and 2), with approximately 27% of internalized apoE recycled and resecreted within 4 hours.39 Endocytosis of exogenous lipoprotein-derived apoE was reported to follow a quite distinct intracellular route relative to endogenous apoE, the latter having a predominantly Golgi localization and the former having an endosomal/lysosomal localization.40 However, reendocytosed lipoprotein-derived apoE has also been identified in the Golgi network.41 Apparent inconsistencies in the literature may relate to the relative sizes of endogenous and recycled exogenous pools. The predominance of endogenous apoE in the Golgi and exogenous apoE in the endosomal compartment may still allow for some overlap of intracellular transport routes for exogenous and endogenous apoE.

Resecretion of reinternalized apoE is observed in macrophages, hepatocytes, fibroblasts, and apoE-transfected CHO cells. Recycling of 125I-apoE-VLDL delivered to CHO cells was enhanced by cyclodextrins but not by apoA-I,42 whereas apoA-I and HDL promoted recycling of exogenous lipoprotein-derived apoE in primary murine macrophages39 and hepatoma cells.43 Monensin44 and brefeldin A35 inhibit endogenous apoE secretion, but fail to affect recycling of 125I-apoE-VLDL delivered to CHO cells,42 supporting the existence of different secretory pathways for endogenous and recycled apoE. Recycling of lipoprotein-derived apoE has been associated with cholesterol removal from hepatoma cells,9 however in vivo hepatic recycling of macrophage-derived apoE is not associated with significant alterations to hepatocyte lipid metabolism.45 The quantitative importance of recycling to the stimulation of secretion of macrophage apoE by apoA-I, HDL, and other apolipoproteins (see below) requires clarification.

Intracellular Degradation
ApoE undergoes intracellular degradation in a post Golgi compartment in macrophages (Figure 1, pathway 4), HepG2 and CHO cells.35,46 Newly synthesized apoE has a half life of 22 minutes in macrophages47 and of 2.6 hours in HepG2 cells.46 Some studies have concluded degradation only occurs within lysosomes, whereas others have shown that degradation is sensitive to both lysosomal and proteasomal chemical inhibitors.35,46,48 Brefeldin A, which perturbs ER to Golgi transport, inhibits apoE degradation and causes intracellular accumulation of unglycosylated apoE with lower molecular weight.35 This suggests that entry of apoE into the Golgi network, and glycosylation of apoE, precede entry into the degradatory compartment.35 Degradation of apoE is inhibited by ALLN, an inhibitor of Ca2+ dependent proteases, in HepG2 cells, macrophages, and CHO cells expressing apoE.46,48 Furthermore, the degradation of apoE by HepG2 cells is inhibited by incubation in Ca2+-free medium. This has been interpreted as indicating that Ca2+-dependent proteases may contribute to intracellular degradation.46 However, interpretation is complicated by the effects of Ca2+ on intracellular signaling, and more recently, its direct role in apoE secretion.49

ApoE degradation has been inhibited by up to 60% to 70% by the use of lysosomal chemical inhibitors.35 Concentrations of the lysosomal inhibitor chloroquine, which effectively inhibited apoE degradation (25 to 100 µmol/L), did not stimulate apoE secretion.35 In other studies, a cysteine protease inhibitor (ALLN) and lysosomal inhibitors did not stimulate apoE secretion despite effectively inhibiting degradation.21 The lack of stimulation of apoE secretion when degradation is inhibited suggests that some cellular pools of apoE are committed either to degradation or secretion.21 In this model, committed entry into the lysosomal or other degradatory compartment precludes reentry into the secretory compartment. However, as a number of studies have shown that stimulation of apoE secretion by HDL and apoA-I decreases net degradation of apoE,17,47,50 it is likely that there are other "uncommitted" pools of apoE otherwise destined for degradation which can be redirected to secretion.

We have recently modeled the secretion and degradation of apoE in human macrophages measured by [35S]-methionine labeling pulse-chase turnover studies and fitting the data to a first order equation (Figure 2A).17 The data best fit a model in which [35S]-apoE could exist within mobile (Em) or stable (Es) pools, where Em, but not Es, was available for secretion or degradation. In response to stimulation of apoE secretion with apoA-I (see below), the rate of degradation decreased, whereas Es was unchanged. These results support that some Em pools are "uncommitted" and accessible to either secretory or degradatory pathways.


Figure 2
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  		Figure 2. Modeling of apoE secretion in macrophages. A, Simple model of apoE trafficking and secretion [35S]-Metabolic labeling studies of apoE secretion can be fitted to a simple model with the first order equation {delta}Em/ {delta}t=–(k1+k2)xEm as described.17 Key components to the model are stable (Es) and mobile (Em) pools of apoE and the interconnection between degradation and secretion of Em. ApoA-I stimulates secretion and decreases degradation of Em without affecting Es.17 B, Proposed revised model incorporating 2 mobile pools Em1 and Em2. The PKA inhibitor H89 inhibits secretion of apoE without increasing degradation and increases the stable pool Es.49 This pool of apoE (Em2) is inaccessible to the degradatory compartment, unlike the pool of apoE (Em1) mobilized by apoA-I, suggesting the presence of 2 distinct pools, one committed to secretion (Em2), the other accessible to both secretory and degradatory compartments (Em1). As H89 is less effective at inhibiting apoA-I–induced apoE secretion than it is at inhibiting constitutive apoE secretion, a second, PKA-independent route of secretion of Em2 is also proposed.

Stimulated Secretion of ApoE
As distinct from factors that increase apoE secretion by stimulating its synthesis (eg, LXR/RXR ligands, cholesterol enrichment, cytokines,14,51,52 a number of agents have been shown to directly increase apoE secretion (independent of transcription). These include, LDL,48,53 HDL,47,50 apoA-I, A-II, A-IV, apoE,17–19,38 phospholipid vesicles (PLV),38,50 heparinase, oleic acid, and lactoferrin.20,21,28 Phospholipid discs containing apoA-I are more potent stimuli of apoE secretion than phospholipid vesicles or lipid-free apoA-I.17 Such stimulation presumably occurs at the level of the Golgi or post-Golgi compartments, as the secreted apoE is almost always to some extent glycosylated and sialylated. Most studies to date have not been designed to discriminate between stimulated de novo secretion of endogenous apoE and stimulated resecretion of reinternalized endogenous apoE.39

The observation that apoE can stimulate its own secretion from macrophages raises the potential of a positive feedback loop, which may be of particular importance in the microenvironment of the arterial wall. Physiological concentrations of apoE in the arterial wall have been demonstrated to be atheroprotective.54 The natural isoforms of apoE vary in their ability to exogenously stimulate secretion. Exogenous apoE4 is less effective at stimulating apoE3 secretion from macrophages than are apoE3 and apoE2,17 which may be relevant for the increased atherosclerotic risk associated with the apoE4 phenotype.55 This compares with studies on the secretion of endogenous apoE isoforms where apoE2 is secreted by macrophages less effectively than apoE3 or apoE456 and recycling studies of exogenous apoE4 which suggest that apoE4 recycles less efficiently than other isoforms.57

The amount of apoE bound to, and released from, the cell surface can be expected to affect the stimulation achieved by various agents. Increased secretion when proteoglycan synthesis is inhibited suggests that cell surface binding can sequester apoE and inhibit its secretion.21 Cell surface apoE can also be bound to lipoprotein receptors, such that antiserum to the LDL receptor (LDLr)58 and suramin (which blocks ligand binding to the LDL-receptor related protein LRP) can reduce sequestration on the macrophage surface and increase apoE secretion.59 Simple displacement of apoE from the macrophage surface is unlikely to fully explain apoA-I–stimulated apoE secretion.17

As cell surface pools are relatively small, rapidly internalized, and degraded,21,37 vesicular traffic and exocytosis of apoE probably continuously replenish cell surface pools of apoE, in which case cell surface and intracellular apoE transport mechanisms may need to be considered as closely connected. Recent studies from our own group indicate that inhibition of intracellular vesicular trafficking results in very rapid inhibition of apoE secretion,49 implying that residence time on the cell surface is either relatively brief in primary human macrophages or contributes relatively little to apoE secretion.

ATP-Binding Cassette Transporters and ApoE Secretion
ABCA1, Constitutive Macrophage ApoE Secretion and Cholesterol Efflux
The ATP binding cassette transporter ABCA1 has been identified as the key regulator of cholesterol efflux to apoA-I and the protein which is defective in Tangier disease.60–62 ABCA1 activity contributes to basal constitutive secretion of apoE, as macrophages from patients with Tangier disease17,63 and normal macrophages in which ABCA1 activity or expression is inhibited show reduced apoE secretion.63 Similar observations were made in the brain, where murine ABCA1 deficiency was associated with lower tissue apoE levels and a markedly reduced secretion of apoE from astrocytes and microglial cells.64,65 The mechanism by which ABCA1 regulates apoE secretion is unclear. Cells from patients with Tangier disease and mice with ABCA1 deficiency have a morphologically expanded Golgi complex,66 in parallel with impaired intracellular transport, likely attributable to perturbed vesicular budding between the Golgi and the plasma membrane,67 and these characteristics would be expected to disturb apoE trafficking. The ABCA1 transporter has also been reported to modulate late endocytic trafficking of cholesterol and the Niemann Pick Type 1 (NPC1) protein,68 raising the additional possibility that late endosomal dysfunction could perturb apoE secretion in Tangier disease.

Although the pathways stimulating apoE secretion and cholesterol efflux are distinct,17,44 apoE, whether added to cells or during its secretion from cells, can itself mediate cholesterol efflux. ABCA1 primarily, but not exclusively, mediates cholesterol efflux to delipidated or lipid-poor apolipoproteins. There is evidence to suggest that exogenous apoE removes cholesterol via an ABCA1-mediated pathway, whereas secreted apoE may also remove cholesterol in an ABCA1-independent manner.69 Increasing lipidation of cholesterol acceptors decreases the contribution of ABCA1 to cholesterol efflux relative to that of ATP-binding cassette transporter G1 (ABCG1).70,71 This raises the possibility that ABCG1 may contribute to cholesterol efflux mediated by phospholipid-containing secreted apoE particles.

ABCG1 and ApoE Secretion
ABCG1 is a major mediator of cholesterol efflux to phospholipid-containing acceptor particles such as HDL, contributes to reverse cholesterol transport of macrophage cholesterol (reviewed in71), and may have a role in apoE expression or secretion. Deletion of macrophage ABCG1 has recently been shown to be associated with increased expression of ABCA1, increased cellular apoE levels, and increased secretion of apoE.72 Transplantation of these ABCG1-deficient macrophages into LDLr–/– mice led to an increase in plasma apoE concentrations.72 However, in another study, total body ABCG1-deletion had no effect on plasma levels of apoE.73 The discrepancy between the 2 studies may arise from differences between macrophage-specific and total body ABCG1-deletion, differences between LDLr-deficient and LDLr-replete animal models, or from differences in the severity of hypercholesterolemia achieved in the 2 studies. Assuming that there are circumstances in which ABCG1-deletion does modulate apoE secretion, it remains to be established whether this is secondary to increased apoE synthesis, to indirect effects on ABCA1 activity or cellular cholesterol status, or a direct effect of ABCG1 on apoE trafficking.

ApoE in the Formation of Nascent HDL Particles
ApoE secreted from macrophages is lipid-poor.74 However, on cholesterol enrichment of macrophages, apoE is secreted as discoidal particles with a density of <1.21g/mL.16–18,74 In these cholesterol-enriched conditions, secretion of apoE can promote cholesterol efflux in the absence of added cholesterol acceptors.74 Secreted apoE can also associate with exogenous HDL particles.44 After exposure of foam cell macrophages to apoA-I, most apoA-I–cholesterol–phospholipid particles formed are free of apoE, and secreted apoE is found on both apoE-only and apoE/apoA-I–containing particles.19 Interestingly, although apoA-I–stimulated apoE secretion (see below) is not directly dependent on ABCA1, its lipidation does require expression of ABCA1, as apoE secreted from ABCA1-deficient macrophages is only partially lipidated and floats at a higher density (>1.21 g/mL) than from normal macrophages.18 This suggests that lipidation of apoE may be dependent on efflux of phospholipid or cholesterol mediated by ABCA1.

ApoA-I–Mediated ApoE Secretion Is ABCA1-Independent
Whereas apoA-I–mediated cholesterol efflux is critically dependent on the expression and activity of ABCA1, we and others have demonstrated that apoA-I–stimulated apoE secretion is ABCA1-independent.17,18 Studies using recombinant apoA-I with selected deletions and point mutations established that there are different structural requirements for apoA-I–induced apoE secretion and apoA-I–induced cholesterol efflux.17 For example, deletion of the C-terminal domain (AA190–243) reduced apoA-I–induced cholesterol efflux, but promoted apoA-I–induced apoE secretion, and deletion of the central region (AA123-166) did not significantly affect cholesterol efflux but did inhibit apoE secretion. Additionally, a range of synthetic {alpha}-helical peptides which stimulated apoE secretion to an extent similar to intact apoA-I were incapable of stimulating cholesterol efflux.17 Stimulation of apoE secretion is a general property of {alpha}-helix containing molecules including apoA-II, A-IV, and apoE.17 Most importantly, macrophages from patients with Tangier disease, while secreting far less apoE under basal conditions, secreted normal amounts of apoE when exposed to apoA-I.17 These results indicate that apoA-I induces apoE secretion via an ABCA1-independent pathway, and that this is distinct from basal apoE secretion, which is ABCA1-dependent.


*    Regulation of ApoE Secretion From Macrophages
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 up arrowBasic Biology of ApoE...
 *Regulation of ApoE Secretion...
down arrowFuture Directions
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General Aspects of Regulated Protein Secretion From Macrophages
In the classical constitutive pathway implicated in apoE secretion, proteins travel from the ER to the Golgi, and thence through the Golgi apparatus to the plasma membrane in tubulo-vesicular carriers.75,76 In the secretion of other proteins, pathways regulating exocytic and endocytic vesicle traffic can interact at different levels,77,78 while using the same intermediate endo/lysosomal vesicles. Recent studies have shown that in macrophages the recycling endosome can act as a sorting station for interleukin (IL)-6 and tumor necrosis factor (TNF){alpha}.77 The role of "sorting stations" for apoE secretion requires investigation.

Regulated secretion, as observed in mast cells and platelets,79,80 is usually distinguished from classical constitutive secretion by the involvement of secretory granules containing accumulated secretory products that are released on cellular recognition of appropriate stimuli.75 A third, lysosomal secretory pathway, is used by cathepsins and involves a subset of specialized lysosomes with unique morphology and content that release their constituents in response to external stimuli.81 Finally, a nonclassical or ER/Golgi-independent secretory pathway is described.82 This is relevant to proteins which do not possess a signal sequence necessary for translocation to the ER, and this pathway is not inhibited by brefeldin A or monensin. IL-1 and high mobility group Box-1 (HMBG1) protein are secreted from macrophages via this route.82 Although constitutive secretion and regulated exocytosis of proteins have traditionally been considered quite distinct, apoE appears to combine constitutive secretion with acute regulation.

Macrophage ApoE Secretion Involves Protein Kinase A, Intracellular Calcium, and the Microtubular Network
Recent studies in other systems have demonstrated that constitutive protein secretion is regulated, and involves kinases, phosphatases, receptors, and typically, the microtubule network.76,83,84 In view of previous studies indicating that apoE secretion could be stimulated by apoA-I and by HDL,17,19,38,47 we hypothesized that even basal apoE secretion must be under some form of regulation. We showed that disruption of microtubules inhibited basal apoE secretion, whereas disruption of the actin skeleton had no effect. Microscopy studies supported this by showing alignment of apoE-containing vesicles along the microtubules but not the actin cytoskeleton in RAW macrophages.49

Protein kinase A (PKA) regulates traffic of several proteins at different steps in the constitutive secretory pathway,85 and a range of PKA inhibitors decreased secretion of apoE.49 Live cell imaging of apoE-GFP–transfected RAW macrophages indicated that PKA is required for the movement of apoE-containing vesicles. Pulse-chase apoE turnover studies showed that inhibition of PKA specifically reduces the rate of apoE secretion, expanding the size of the intracellular apoE pool, but does not affect its degradation.49 This suggests that apoE-containing secretory vesicles accumulate within the cell in response to PKA inhibition, at a site inaccessible to lysosomal degradation (Figure 2). Importantly, an extracellular PKA agonist did not stimulate apoE secretion, indicating constitutive PKA activity is sufficient.

Another important regulator of secretion is the second messenger calcium, which is also required for the regulated secretion of neurotransmitters and hormones from endocrine and neuronal cells.86–88 Chelation of intracellular calcium ([Ca2+]i) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate-acetoxymethyl ester (BAPTA-AM) inhibited apoE secretion, but chelation of extracellular calcium with EGTA had no effect. As inhibitors of phospholipase C (PLC) and the inositol triphosphate (IP3) receptor (IP3R) also decreased secretion of apoE, a role for PLC and IP3R in the mobilization of [Ca2+]i during secretion of apoE is most likely.49

Although PKA and [Ca2+]i are required for apoE secretion, neither stimulation of PKA activity nor increased [Ca2+]i stimulated basal apoE secretion from macrophages; conversely apoA-1-stimulated apoE secretion was not accompanied by an increase in cellular PKA activity or [Ca2+]i. Thus [Ca2+]i and PKA can be considered permissive but do not explain how apoE secretion is stimulated. The IC50 for inhibiting basal apoE secretion with the PKA inhibitor H89 is half that for its inhibition of apoA-I–stimulated apoE secretion.49 Therefore it is likely that ABCA1-mediated basal apoE secretion occurs predominantly via a PKA-sensitive pathway, and that apoA-I–stimulated secretion additionally involves a contribution from an as yet undefined PKA-insensitive pathway (Figure 2B). Whether PKA and calcium interact during apoE secretion as described elsewhere,86,89,90 or act at different steps in the secretory pathway, requires further investigation.

As constitutive release of lysozyme and other proteins from macrophages were also found to be PKA-, microtubule-, and calcium-dependent,49 the regulation of apoE secretion can be considered a prototype for understanding the processes regulating the secretion of other macrophage proteins.


*    Future Directions
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The regulation of apoE secretion from macrophages provides an important avenue for rapidly regulating the concentration of apoE in local cellular environments such as the arterial wall, and potentially in the plasma. However there are quite major limitations to our current understanding of how secretion of apoE is regulated. The intracellular routes for recycling of macrophage-secreted apoE require further careful mapping and comparison made with those required for recycling of lipoprotein-derived apoE. Once these routes are defined, the relative quantitative contribution of resecretion of macrophage-derived apoE and secretion of Golgi-network-derived de novo apoE under basal and apoA-I–induced conditions can be understood. The signaling and vesicular pathways regulating apoE secretion in macrophages clearly need deeper exploration and will require identifying the different pathways involved and how they interact, including the signaling partners and subcellular sites of action for PKA. The relevance of these pathways to cells other than macrophages cannot be automatically assumed and will require in vitro evaluation before targeted manipulation of apoE secretion in defined tissues can be undertaken. Targeted inhibition or stimulation of specific apoE trafficking pathways in vivo will provide unique opportunities to test directly the role of secreted macrophage-derived apoE in regulating atherosclerosis.


*    Acknowledgments
 
The contributions of Kat Gaus to modelling studies and Jenny Stow and Jay Kay to trafficking studies performed in our laboratory are gratefully acknowledged.

Disclosures

Supported by National Health and Medical Research Council of Australia (grants 455252 and 482800).


*    Footnotes
 
Original received February 4, 2008; final version accepted March 21, 2008.


*    References
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 up arrowAbstract
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 *References
 

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