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Circulation Research. 2007;101:607-616
Published online before print July 27, 2007, doi: 10.1161/CIRCRESAHA.107.157198
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(Circulation Research. 2007;101:607.)
© 2007 American Heart Association, Inc.

Cellular Biology

Secretion of Apolipoprotein E From Macrophages Occurs via a Protein Kinase A– and Calcium-Dependent Pathway Along the Microtubule Network

Maaike Kockx, Dongni Lily Guo, Thierry Huby, Philippe Lesnik, Jason Kay, Tharani Sabaretnam, Eve Jary, Michael Hill, Katharina Gaus, John Chapman, Jennifer L. Stow, Wendy Jessup, Leonard Kritharides

From the Macrophage Biology Group (M.K., D.L.G., K.G., W.J., L.K.), Centre for Vascular Research, School of Medical Sciences, University of New South Wales, Australia; INSERM U551 (T.H., P.L., J.C.), Dyslipoproteinemia and Atherosclerosis Research Unit, Paris, France; UMR S551 (T.H., P.L., J.C.), Université Pierre et Marie Curie-Paris6, Paris, France; Institute for Molecular Bioscience (J.K., J.L.S.), The University of Queensland, Brisbane, Australia; The Anzac Research Institute (T.S.), University of Sydney; Department of Physiology and Pharmacology (E.J., M.H.), University of New South Wales, Australia; and Department of Cardiology (L.K.), Concord Repatriation General Hospital, University of Sydney, Australia.

Correspondence to L. Kritharides, Department of Cardiology, Concord Hospital, Sydney, NSW 2139, Australia. E-mail l.kritharides{at}unsw.edu.au


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowConclusions
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Macrophage-specific expression of apolipoprotein (apo)E protects against atherosclerosis; however, the signaling and trafficking pathways regulating secretion of apoE are unknown. We investigated the roles of the actin skeleton, microtubules, protein kinase A (PKA) and calcium (Ca2+) in regulating apoE secretion from macrophages. Disrupting microtubules with vinblastine or colchicine inhibited basal secretion of apoE substantially, whereas disruption of the actin skeleton had no effect. Structurally distinct inhibitors of PKA (H89, KT5720, inhibitory peptide PKI14–22) all decreased basal secretion of apoE by between 50% to 80% (P<0.01). Pulse-chase experiments demonstrated that inhibition of PKA reduced the rate of apoE secretion without affecting its degradation. Confocal microscopy and live cell imaging of apoE–green fluorescent protein–transfected RAW macrophages identified apoE–green fluorescent protein in vesicles colocalized with the microtubular network, and inhibition of PKA markedly inhibited vesicular movement. 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 by 77.2% (P<0.01). Injection of c57Bl6 apoE+/+ bone marrow–derived macrophages into the peritoneum of apoE–/– C57Bl6 mice resulted in time-dependent secretion of apoE into plasma, which was significantly inhibited by transient exposure of macrophages to BAPTA-AM and colchicine and less effectively inhibited by H89. We conclude that macrophage secretion of apoE occurs via a PKA- and calcium-dependent pathway along the microtubule network.


Key Words: apolipoprotein E • atherosclerosis • macrophages • signaling pathways


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowConclusions
 down arrowReferences
 
Apolipoprotein (apo)E is a 34-kDa protein that has important roles in remnant lipoprotein clearance,1 Alzheimer’s disease, and lymphocyte activation.2 ApoE represents a large proportion of total protein constitutively secreted from macrophages.3 Vessel-specific and macrophage-specific secretion of apoE protects against atherosclerosis.4,5 ApoE expression is increased during macrophage differentiation and by cholesterol loading6,7 and is decreased by interleukin-1, granulocyte/macrophage-colony stimulating factor, lipopolysaccharide, and interferon-{gamma}.8–10

After translation, the protein is transported from the endoplasmic reticulum to the Golgi, where it undergoes O-linked glycosylation,11 after which apoE is either secreted or degraded.12 A proportion of secreted apoE binds to cell surface proteoglycans and can be released from the cell surface or internalized and then degraded or recycled to the cell surface.13 Intracellular apoE has a half-life of 22 minutes,14 and most of the apoE synthesized is rapidly degraded.12 Secretion redirects apoE away from intracellular degradation, thereby increasing the total pool in the arterial wall.14,15 Recycling of exogenous apoE derived from very-low-density lipoprotein has also been described,16 but there are important differences between the trafficking of exogenous and endogenous apoE.17 HDL,14 reconstituted A-I phospholipid particles, and apoA-I15,18,19 stimulate secretion of apoE. The ATP-binding cassette transporter ABCA1 is implicated in basal apoE secretion, but not that stimulated by apoA-I,15,20 and a recent report suggests involvement of ABCG1 in basal secretion of apoE.21 The signaling pathways regulating constitutive and stimulated traffic/secretion of apoE are unknown.

In general terms, constitutive and stimulus-regulated secretion of proteins involves transport of proteins within secretory vesicles from the endoplasmic reticulum via the Golgi to the plasma membrane.22 Vesicular traffic occurs along actin microfilaments and/or microtubules.23,24 Regulation involves interactions between signaling molecules and motor proteins associated with either actin or tubulin.23,25 Protein kinase (PK)A regulates traffic of many proteins at different steps along the constitutive secretory pathway, including transport from endoplasmic reticulum to Golgi, exit out of the Golgi, and transport from the trans Golgi network to the plasma membrane.26,27 Calcium is another important regulator and is required for the regulated secretion of neurotransmitters and hormones from endocrine and neuronal cells.28,29

We investigated the role of these pathways in the secretion of apoE from macrophages. ApoE secretion is demonstrated to be PKA- and calcium-dependent and to involve the microtubular network.


*    Materials and Methods
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 up arrowIntroduction
 *Materials and Methods
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 down arrowConclusions
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Reagents
Vinblastine, colchicine, cytochalasin D, H89, KT 5720, PKA inhibitory fragment 14 -22, and 8-bromoadenosine-3',5'-cyclic-monophosphate (8-bromo-cAMP), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate-acetoxymethyl ester (BAPTA-AM), and EGTA were obtained from Sigma. Latrunculin B, forskolin, 3'-isobutylmethylxanthine (IBMX), U73122, U73343, 2-aminoethoxydiphenylborate (2-APB), and thapsigargin were purchased from Calbiochem, and the St-Ht31 inhibitory and control peptides were from Promega. Molecular Probes supplied Fura-2-AM. Human apoA-I, LDL, acetylated LDL, and lipoprotein-deficient serum were prepared as described.30 An apoE–green fluorescent protein (GFP) construct was generated in house by removing the stop codon on the apoE cDNA and tagging the C terminus of the complete human apoE gene to the N terminus of pEGFP-N1 (BD Clontech) in frame (see the expanded Materials and Methods section in the online data supplement at http://circres.ahajournals.org).

Secretion of ApoE In Vivo
All animal procedures were performed at the Central Animal Facility of the Medical Faculty of La Pitié Hospital, with approval from the Direction Departementale des Services Vétérinaires, Paris, France, under strict compliance with European Community Regulations. Bone marrow–derived macrophages (BMDMs) were obtained from wild-type female C57BL/6 mice and seeded in corning dishes in L929 conditioned medium.31 Matured macrophages (4 days in culture) were cholesterol loaded by incubation in with 50 µg/mL acetylated LDL for 48 hours. Cells were detached from the plates using Accutase, washed, resuspended in RPMI medium 1640 containing 0.1% (wt/vol) BSA at 8x106 cells/mL and exposed to the indicated concentrations of H89, colchicine, and BAPTA-AM for 30 minutes at 37°C. After treatment, cells were washed, an aliquot was stored at –20°C, and 4x106 cells in 500 µL of PBS were injected intraperitoneally into fasted female apoE-deficient mice (n=6 for each condition; Charles River Laboratories, Wilmington, Mass). Blood was collected in heparin and EDTA before and 1, 2, and 3 hours after intraperitoneal injection by retroorbital puncture. At 3 hours, mice were euthanized, peritoneal lavage (PBS) was collected, and cells and supernatant were separated by ultracentrifugation and stored in RIPA buffer.

Isolation and Culture of Human Monocyte-Derived Macrophages
Human monocytes were isolated from white cell concentrates of healthy donors of the New South Wales Red Cross blood transfusion service, Sydney, Australia, using density gradient centrifugation after layering on Ficoll–Paque Plus (Amersham).15 After differentiation for 6 days, the cells were enriched with cholesterol by incubation in RPMI medium 1640 containing 10% lipoprotein-deficient serum (vol/vol) and acetylated LDL (50 µg/mL) for 2 days.15

Inhibitor Treatments
Cells were washed twice and incubated with or without various treatments in RPMI medium 1640 containing 0.1% BSA. After the indicated times, media were transferred to Eppendorf tubes, mixed with Complete protease inhibitor (Roche Applied Science), and 0.02 trypsin inhibitory units of aprotinin (Sigma), and spun 5 minutes at 1300g to remove any detached cells. The cultures were washed twice with PBS and then scraped and lysed in 0.1% Triton containing Complete protease inhibitor and 0.02 thrombin inhibitory units of aprotinin.

Measurement of ApoE Secretion
ApoE secreted into the medium was measured by ELISA and confirmed by Western blot analysis.15 Cellular apoE levels were determined by Western blot analysis.15 For murine in vivo studies, apoE in cells, peritoneal lavage supernatant and infranatant, and plasma were determined by Western blot analysis using a rabbit anti-mouse antibody (Biodesign) and using mouse plasma as a standard.

Analysis of ApoE mRNA by Real-Time PCR
Total RNA was isolated, and apoE mRNA levels were analyzed by quantitative RT-PCR.15

Pulse-Chase Experiments and Metabolic Labeling of Cell Proteins With [35S]-Methionine/Cysteine
Pulse-chase studies were performed as described previously.15 Secretion of total [35S]-labeled protein was measured by trichloroacetic acid precipitation, whereas [35S]-labeled apoE in cell lysates and medium was immunoprecipitated using a goat antibody to human apoE and protein A–Sepharose (Amersham Biosciences). Secreted lysozyme was determined by Western blotting using a rabbit anti-human lysozyme antibody (Dako). Immunoprecipitated [35S]-labeled apoE from cell lysates was separated by SDS-PAGE. The 34-kDa band was quantified by phosphorimaging (Photostimulated Luminescence, Fujix BAS-1000) and expressed as arbitrary units of [35S]-apoE per milligram of cell protein.15

Immunofluorescence Microscopy
Immunofluorescence was performed as described.32 Transiently transfected Raw 264.7 cells expressing apoE-GFP were fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 in blocking buffer (PBS containing 0.5% BSA). Primary antibodies to mouse anti–{alpha}-tubulin (Molecular Probes), Alexa 488 phalloidin (Molecular Probes), mouse anti-GM130 (BD Biosciences), and cy3-conjugated secondary antibodies (The Jackson Laboratory) were used in blocking buffer. Epifluorescence microscopy was performed using an Olympus Provis BX51 microscope equipped with a x100 oil objective and a DP70 camera. Confocal microscopy was performed using an LSM 510 META (Carl Zeiss Microscope Systems).

Cell Viability
Cell viability (always between 85% and 100%) was routinely assessed by light microscopic morphology, by estimation of cell protein using the BCA method30 and by measuring leakage of lactate dehydrogenase (LDH assay) into the medium.15

Data Analysis
The degradation and secretion of cellular apoE from pulse-chase experiments were simultaneously fitted to a first-order rate equation, with k1 and k2 describing the rate constants for secretion and degradation, respectively. equation


Formula 1

The first-order rate equation (Equation), was fitted to the experimental secretion and degradation data using a nonlinear least-squares fitting program (Solver, Microsoft Excel). The quality of the fit was evaluated by an error function as described.15,33 Cellular apoE was previously shown to exist in stable (Es) and mobile pools (Em).15

Data presented are means±SD of triplicate cultures from single experiments representative of at least 2 to 3 independent experiments. A significant difference between control and multiple treatment groups was assessed by ANOVA using Dunnett post hoc test for multiple comparisons. Comparisons of two groups were performed by unpaired Student’s t-test or Mann Whitney-U test as appropriate. IC50 values were determined by nonlinear regression using Graphpad Prism. Differences were considered significant at P<0.05. Mouse studies are presented as the means±SEM of n=6 mice for each treatment group.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowConclusions
 down arrowReferences
 
Basal Secretion of ApoE Is Dependent on an Intact Microtubule Network
We investigated whether secretion of apoE from human monocyte–derived macrophages (HMDMs) was dependent on an intact microtubule network and/or an intact actin skeleton by using 2 mechanistically distinct disruptors of the microtubule network (colchicine and vinblastine) and of the actin skeleton (cytochalasin D and Latrunculin B). Secretion of apoE was markedly inhibited by both colchicine and vinblastine but was unaffected by disruption of the actin skeleton (Table 1). The inhibition of apoE secretion was dose-dependent (Table 1) and time-dependent (data not shown), decreasing secretion of apoE by 83.2±8.2% and 72.3±1.1% for colchicine and vinblastine, respectively, after 1 hour. Cellular apoE levels were not affected by either treatment (data not shown).


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  		Table 1. Effect of Microtubule and Actin Skeleton Disruption on ApoE Secretion

Inhibition of PKA Decreases Basal ApoE Secretion
H89, an inhibitor of PKA, markedly and dose-dependently (0 to 40 µmol/L) inhibited secretion of apoE, reaching maximal inhibition of 74.9±4.6% (Figure 1A) without cytotoxicity (data not shown). Inhibition of apoE secretion by H89 was also time dependent (data not shown).


Figure 1
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  		Figure 1. Inhibition of PKA decreases apoE secretion. HMDMs were incubated with increasing concentrations of H89 (A) or 20 µmol/L H89, KT5720, or PKI14–22 (PKI) (B) or peptide inhibitors of AKAP-PKA-binding Ht31i and Ht31c (C). After 40 minutes, secreted apoE was measured. *P<0.05 relative to control.

Although it is possible PKA inhibition may decrease apoE transcription via activator protein-2,34 under the present experimental conditions, H89 had no effect on total macrophage apoE mRNA or protein levels (data not shown). The effect of H89 was attributable to its inhibition of PKA, and not off-target effects, as two other PKA inhibitors (KT5720 and PKI14–22) also decreased apoE secretion (Figure 1B). Spatial and temporal regulation of PKA is mediated by its binding to the A-kinase anchoring proteins (AKAPs).35 Figure 1C shows that disruption of PKA-AKAP anchoring using St-Ht31 inhibitory peptide (Ht31i) inhibited apoE secretion by 66.6±4.6% (100 µmol/L), whereas the inactive St-Ht31 control peptide had no effect, providing independent confirmation of PKA involvement. Cellular apoE levels were not affected by St-Ht31i (data not shown).

To determine whether PKA directly controls apoE secretion, or acts indirectly through control of its proteolytic turnover, macrophage proteins were pulse-labeled with [35S]-methionine/cysteine, and the kinetics of [35S]-apoE secretion and degradation were determined. H89 markedly decreased the secretion of [35S]-apoE in 1 hour from 19.1±3.3% to 5.7±2.5% of total (Figure 2). Under the same conditions, cellular [35S]-apoE decreased by 74.5±5.0% in control cells and by 66.8±5.0% in H89-treated cells. Net degradation of [35S]-apoE, calculated by subtracting secreted and cellular [35S]-apoE from total [35S]-apoE at time 0, was unchanged by H89.


Figure 2
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  		Figure 2. H89 inhibits secretion of a preformed pool of apoE, whereas apoE degradation is not affected. HMDMs were labeled with 250 µCi/mL [35S]methionine/cysteine for 3 hours and then washed and chased in medium containing unlabeled methionine/cysteine, without ({circ}) or with (bullet) 40 µmol/L H89. Secreted [35S]-apoE (A); cell-associated [35S]-apoE (B); net degradation of [35S]-apoE (C), calculated by subtracting residual [35S]-apoE in cell and media from [35S]-apoE in cells at time 0 (T0); model for [35S]-apoE turnover (D). All data are percentages of total [35S]-apoE at T0; *P<0.001 for [35S]-apoE secreted at 60 minutes, H89 vs control.

In previous studies, modeling apoE turnover and secretion, we identified that macrophage apoE exists in relatively "mobile" (Em) and "stable" (Es) pools (Figure 2D), the secretion (k1) and degradation (k2) of which can be described with a first-order rate equation (Equation).15 Fitting the experimental data in Figure 2 to the same first-order rate equation showed that k1 was 3.6-fold higher under control conditions than when PKA was inhibited but that inhibition of PKA did not affect k2 (Table 2). Concomitantly, H89 treatment increased the stable pool of cellular apoE (Es). Modeling data without an increase in Es, ie, assuming Es remains unaltered by H89, was incompatible with prespecified error functions of the model,15 confirming increased Es was an important consequence of PKA inhibition. Thus, PKA directs apoE secretion but not apoE degradation.


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  		Table 2. Summary of Modeling Parameters for ApoE Secretion

Visualization of ApoE Trafficking
To visualize the effect of PKA inhibition, RAW macrophages were transiently transfected with apoE-GFP. Preliminary studies established that apoE and apoE-GFP stably transfected into CHO-K1 cells were secreted with similar efficiency and were equally inhibited by H89 and stimulated by apoA-I, thereby indicating that apoE-GFP was a valid surrogate for apoE (Figure I in the online data supplement). ApoE-GFP demonstrated a typical perinuclear Golgi distribution, colocalizing with the Golgi marker GM130 (Figure 3), as well as being present in large vesicular structures distributed throughout the cell. In cells costained to depict F-actin, there was no apparent association of apoE-GFP vesicles with microfilaments. In cells with labeled microtubules, apoE-GFP vesicles were seen associated with microtubules in the cell periphery. H89 did not appreciably alter the overall distribution of apoE in live (see supplemental Movies 1 and 2) or in fixed cells (data not shown). Live cell imaging demonstrated apoE-GFP–containing vesicles moving out of the Golgi toward the cell surface and other vesicles with multidirectional trajectories (supplemental Movie 1). Total movement of apoE-containing vesicles was dramatically inhibited by H89 (P<0.0001; supplemental Movie 2), indicating PKA regulates the traffic of post-Golgi apoE-containing vesicles to and from the plasma membrane.


Figure 3
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  		Figure 3. PKA regulates post-Golgi traffic of apoE-containing vesicles. Raw264.7 cells expressing apoE-GFP (green) and immunostained for GM130 (blue) (A), allexa-488 phalloidin (red) (A), or anti-{alpha}-tubulin (red) (B) to stain cis/medial Golgi structures, actin skeleton, or microtubules, respectively. A, Some apoE-GFP is present in the Golgi, whereas bright green vesicles represent apoE-GFP scattered through the cytoplasm. B, ApoE-GFP vesicular structures scattered through the cell, and in multiple instances, these are closely aligned with microtubules (arrows). C, Graph describing movement of 50 apoE-GFP–containing vesicles during live cell imaging; horizontal lines represent median movement for each treatment group. *P<0.001 relative to control (Mann–Whitney U test). Vesicle movements are demonstrated in an example shown in a sequence of still images from supplemental Movie 1 (D). ApoE-GFP is seen as a single large vesicle that moves out of the Golgi toward the periphery. Scale bar=5 µm (A and B).

PKA Is Required for Stimulation of ApoE Secretion by ApoA-I
Whereas basal apoE secretion is ABCA1-dependent,15,21 apoE secretion stimulated by apoA-I is ABCA1-independent, suggesting dissociation of basal and stimulated secretion.15 ApoA-I–induced apoE secretion was dose-dependently inhibited by H89 (Figure 4A) but was less effectively inhibited than was basal apoE secretion (eg, 74.9±4.6% inhibition of basal versus 64.1±4.5% inhibition of A-I–stimulated apoE secretion). This difference was investigated in more detail by deriving IC50 values for basal and "apoA-I–specific" secretion. H89-dependent IC50 values were 2-fold lower for basal than for apoA-I–specific apoE secretion (12.2 and 28.4 µmol/L, respectively).


Figure 4
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  		Figure 4. ApoA-I–stimulated apoE secretion is inhibited by H89. HMDMs were pretreated with H89 for 10 minutes, washed, and incubated for 40 minutes with H89 with or without 25 µg/mL apoA-I (A). ApoA-I–specific apoE secretion was calculated by subtracting basal apoE secretion from total apoE secreted to apoA-I. B, H89-mediated IC50 values for basal ({circ}) (12.2±0.26) and apoA-I–specific (bullet) (28.4±0.28) apoE secretion were determined by nonlinear regression. *P<0.05 relative to basal control (no H89); #P<0.05 relative to apoA-I control (no H89).

Stimulation of PKA Does Not Stimulate ApoE Secretion From HMDMs
We next investigated whether apoA-I stimulated PKA activity and whether stimulation of PKA activity increased apoE secretion. PKA activity in HMDMs was measured under basal conditions and after exposure to apoA-I or a combination of adenyl cyclase stimulation and phosphodiesterase inhibition (forskolin/IBMX). Forskolin/IBMX significantly increased PKA activity (supplemental Figure II). However, as no increase in PKA activity was induced by apoA-I, and forskolin/IBMX did not stimulate apoE secretion, stimulation of PKA activity is insufficient to increase apoE secretion.

Intracellular Calcium Signaling Is Required for Macrophage ApoE Secretion
Calcium signaling can be stimulated by PKA and in turn can regulate PKA activity.28,36 We therefore investigated whether calcium is involved in apoE secretion from HMDMs. Chelation of [Ca2+]i with BAPTA-AM decreased secretion of apoE in a concentration-dependent manner (Figure 5A), whereas chelation of extracellular Ca2+ using EGTA had no effect. Similar results were obtained when cells were treated with BAPTA-AM or EGTA in calcium-free medium, supporting the finding that apoE secretion is not dependent on an extracellular Ca2+ pool.


Figure 5
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  		Figure 5. Intracellular Ca2+ generated via PLC and IP3 are involved in apoE secretion. HMDMs were incubated with indicated concentrations of BAPTA-AM (A), EGTA (B), U73122, and U73343 (C) or 2-APB (D) for 40 minutes. *P<0.05 relative to control.

To determine whether [Ca2+]i was mobilized through activation of Phospholipase C (PLC) and subsequent inositol 3-phosphate (IP3) generation, cells were treated with the PLC inhibitor U73122, its inactive structural analog U73343, and an IP3 receptor (IP3R) antagonist, 2-APB. U73122 (10 µmol/L) inhibited basal apoE secretion by 53.0±11.4% and inhibited apoA-I–stimulated apoE secretion to a lesser extent (by 24±3%; data not shown), whereas the inactive analogue had no effect (Figure 5). 2-APB also dose-dependently inhibited apoE secretion. These data support a role for PLC and IP3 in the mobilization of [Ca2+]i during apoE secretion.

BAPTA-AM inhibited basal and apoA-I–stimulated apoE secretion similarly (77.2±2.8% and 78.3±3.6% inhibition, respectively). The possibility that apoA-I stimulates apoE secretion by triggering release of [Ca2+]i was investigated with fluorescence microscopy using Fura-2-AM. ApoA-I did not trigger release of [Ca2+]i (supplemental Figure III). Although positive controls ATP, thapsigargin, and the calcium ionophore A23187 all achieved marked increases in [Ca2+]i, none of these compounds stimulated apoE secretion.

Generalized Inhibition of the Constitutive Secretory Pathway in HMDMs
The effect of microtubule disruption, PKA inhibition, and chelation of [Ca2+]i on other proteins constitutively secreted by HMDMs was investigated. Colchicine, H89, and BAPTA-AM inhibited total protein secretion by 22.7%, 57.1%, and 64.8%, respectively. Secretion of total protein, [35S]-apoE, and lysozyme were concurrently inhibited (supplemental Table I). None of the inhibitors affected total cell-associated trichloroacetic acid–precipitable material (not shown). Thus the pathway inhibitors can be concluded to affect HMDM constitutive protein secretion in general.

PKA, Microtubules, and Intracellular Ca2+ Play a Role in ApoE Secretion From Macrophages In Vivo
To study macrophage-mediated apoE secretion in vivo, BMDMs obtained from wild-type C57Bl/6 mice were treated with H89, colchicine, and BAPTA-AM in vitro for 30 minutes, washed, and then injected into the peritoneal cavity of apoE–/– mice. Blood was collected from the apoE–/– mice over 3 hours, and plasma was analyzed for the appearance of macrophage-derived apoE. ApoE was secreted in vivo, appearing in plasma time dependently (Figure 6), the peak rate of rise occurring between 1 and 2 hours, reaching a plateau between 2 and 3 hours. In vitro and in vivo rates of secretion of apoE (combining peritoneal lavage supernatant and total plasma apoE at 3 hours) from murine macrophages were comparable (0.32±0.01 versus 0.78±0.01 arbitrary units of apoE per milligram of cell protein per hour, respectively). Pretreatment of BMDMs with H89, colchicine, and BAPTA-AM decreased apoE secretion in vivo, with BAPTA-AM being almost completely inhibitory. Colchicine significantly inhibited apoE secretion at 1 hour by 29.3±14.5% but was not significantly inhibitory by 2 hours. H89 decreased apoE levels by 22.3±3.2% at 1 hour; however, this did not reach statistical significance (P=0.17).


Figure 6
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  		Figure 6. Secretion of apoE from macrophages in vivo. BMDMs from wild-type C57Bl/6 mice were treated with 40 µmol/L H89, 80 µmol/L colchicine, or 100 µmol/L BAPTA-AM for 30 minutes, harvested, washed, and injected into the peritoneal cavity of apoE–/– mice. Blood was collected at 0, 1, 2, and 3 hours. A, BMDM cell lysates before peritoneal injection showing no effect on cellular apoE levels by treatment. B, Time-dependent release of apoE into plasma (the Western blot represents 1 mouse; the graph represents means±SEM of 6 control mice at each time point). C, Effect of H89 (P=0.17), colchicine (P=0.04), and BAPTA-AM (P=0.0001) on secretion into plasma of macrophage apoE at 1 hour (all probability values vs control [Ctrl]). D, Effect of H89 (P=0.99), colchicine (P=0.23), and BAPTA-AM (P=0.0001) on secretion into plasma of macrophage apoE at 2 hours (all probability values vs control). Values represent means±SEM of 6 mice per treatment group.

As in vivo inhibition diminished over time, we investigated whether differences between in vivo and in vitro efficacy may be explained by reversible inhibition following cell washing (supplemental Table II). In vitro inhibition was indeed reversible after removal of H89 and colchicine but not after removal of BAPTA-AM.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowConclusions
 down arrowReferences
 
The present study is the first to characterize the major signaling pathways required for apoE secretion from macrophages. We have identified that apoE is secreted along the microtubular network and involves PKA and intracellular calcium signaling.

Secretion of apoE is important in the pathogenesis of atherosclerosis and other disease states. Macrophage-specific secretion of apoE increases cholesterol clearance and protects against the development of atherosclerosis.37,38 Removal of excess macrophage cholesterol and the antiinflammatory, antiproliferative, and immune-modulating properties of apoE are understood to contribute.2

ApoE is constitutively secreted from macrophages.3 Constitutive secretion has been understood as a continuous movement of vesicles to the plasma membrane followed by unregulated release of vesicular contents. Regulated macrophage protein secretion, for example after stimulation by cytokines, is distinct from constitutive secretion. For example, whereas tumor necrosis factor-{alpha} release is stimulated by lipopolysaccharide exposure,39 apoE synthesis and secretion are inhibited by lipopolysaccharide, interferon-{gamma}, and tumor necrosis factor-{alpha}, and apoE travels via different intracellular vesicular routes.39 Our data using trichloroacetic acid precipitation and quantification of lysozyme secretion indicate that other constitutively released proteins are PKA-, microtubule-, and calcium-dependent. Thus apoE secretion can be considered a prototype for the understanding of the secretion of other macrophage proteins. Recent studies have demonstrated that constitutive secretion is regulated,40,41 involving kinases, phosphatases, receptors, and, typically, the microtubule network.42

Microtubules mediate long-range transport in vesicle traffic via kinesin and dynein motor proteins.25 Most likely, cytoplasmic vesicle-bound PKA complexes with dynein and kinesin, as has been reported for the intracellular transport of pigment granules.43 In our study, interfering with the actin skeleton had no effect on apoE secretion, suggesting a distinction between actin-mediated phagocytosis or migration44,45 and macrophage-mediated protein secretion.

Although cAMP/PKA can modulate apoE transcription,34 our data demonstrate PKA modulates apoE secretion under conditions in which mRNA or protein are unaltered. We therefore conclude that secretion is more sensitively and rapidly inhibited by inhibition of PKA than are apoE transcription and synthesis. This distinction may be important in vivo, as PKA-dependent signaling pathways can be rapidly regulated.

Pulse-chase studies confirmed that PKA affects secretion of preformed [35S]-apoE. As H89 decreased the rate constant for apoE secretion without diminishing the rate constant for degradation, this indicates PKA selectively modulates secretory trafficking of apoE without affecting degradation. The markedly reduced movement of vesicles containing apoE-GFP after H89 suggests that relatively immobile vesicles contribute to the increased stable pool of apoE during PKA inhibition.

ApoE secretion was inhibited by chelation of [Ca2+]i. As extracellular Ca2+ was not required for apoE secretion, triggered internalization of extracellular Ca2+ is unlikely to be involved in this process. Roles for both PKA and Ca2+ have been observed for many other secretory processes, and many levels of crosstalk between the 2 pathways exist. PKA potentiates the Ca2+ response by controlling PLC activity through its phosphorylation.46 PKA can also alter the properties of IP3Rs by phosphorylation and phosphorylates Ca2+ channels, regulating Ca2+ entry directly.47 Ca2+ can also affect the cAMP/PKA response, and several adenyl cyclases are Ca2+-dependent.48

Although macrophage apoE secretion appears to inhibit atheroma formation, a recent study suggests plasma apoE concentrations, which are principally liver-derived, correlate with cardiovascular risk.49 Thus comparative in vitro and in vivo studies must account for cellular specificity. We adapted a recently validated model of in vivo macrophage cholesterol efflux to establish an in vivo model of macrophage apoE secretion.50 In this model, the peritoneal space represents the interstitial space of the arterial wall, and all apoE appearing in plasma of apoE-deficient mice derives from intraperitoneally injected apoE+/+ macrophages. Time-dependent secretion of apoE into plasma was most sensitive to BAPTA-AM and less sensitive to colchicine and H89. Although the effects of H89 and colchicine were limited by transient exposure and reversible inhibition, the data do provide support for microtubule-, calcium-, and probably PKA-dependent macrophage apoE secretion in vivo. Definitive confirmation will require stable, sustained, and nontoxic in vivo inhibition of each of these pathways.

The difference in IC50 for H89-mediated inhibition of basal and apoA-I–induced apoE secretion may be explained by the presence of 2 pathways: one that is in common to basal secretion and apoA-I and another that is exclusive to apoA-I–induced apoE secretion. Stimulation of PKA activity, addition of 8-Br-cAMP or dibutyryl-cAMP (M.K., L.K. unpublished observations, 2007) and increased [Ca2+]i all failed to stimulate apoE secretion. Thus [Ca2+]i and PKA can be considered permissive but are, in themselves, insufficient to stimulate apoE secretion and do not explain apoA-I–specific apoE secretion.


*    Conclusions
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 up arrowIntroduction
 up arrowMaterials and Methods
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 *Conclusions
down arrowReferences
 
Macrophage secretion of apoE occurs via a PKA and calcium-dependent pathway along the microtubule network.


*    Acknowledgments
 
Sources of Funding

This work was supported by Project grant 455251 and Program grant 222722 from the National Health and Medical Research Council, Australia.

Disclosures

None.


*    Footnotes
 
Original received August 24, 2006; resubmission received June 4, 2007; accepted July 17, 2007.


*    References
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 up arrowIntroduction
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 *References
 

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M. Kockx, W. Jessup, and L. Kritharides
Regulation of Endogenous Apolipoprotein E Secretion by Macrophages
Arterioscler. Thromb. Vasc. Biol., June 1, 2008; 28(6): 1060 - 1067.
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