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of July 28, 2017.
Apolipoprotein E and Peptide Mimetics
Modulate Inflammation by Binding the SET
Protein and Activating Protein Phosphatase
2A
Dale J. Christensen, Nobutaka Ohkubo, Jessica Oddo,
Michael J. Van Kanegan, Jessica Neil, Fengqiao Li, Carol A.
Colton and Michael P. Vitek
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2011 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2011; 186:2535-2542; ;
doi: 10.4049/jimmunol.1002847
http://www.jimmunol.org/content/186/4/2535
The Journal of Immunology
Apolipoprotein E and Peptide Mimetics Modulate
Inflammation by Binding the SET Protein and Activating
Protein Phosphatase 2A
Dale J. Christensen,*,† Nobutaka Ohkubo,‡ Jessica Oddo,* Michael J. Van Kanegan,*
Jessica Neil,* Fengqiao Li,* Carol A. Colton,‡ and Michael P. Vitek*,‡
T
he apolipoprotein E (APOE represents the gene; apoE
represents the protein) holoprotein is a multifunctional
299-amino-acid protein with a recognized role in cholesterol transport. Low density lipoprotein (LDL) particles containing the apoE holoprotein have been shown to bind to the
LDLR and the LDLR-related protein (LRP) to facilitate uptake of
the lipoprotein particles into cells (1–3). In addition to cholesterol transport, apoE has significant immunomodulatory properties,
including the capacity to suppress lymphocyte proliferation, Ig
synthesis, and neutrophil activation (4–7). These immunomodulatory properties appear to be biologically relevant, because apoEdeficient animals have impaired immunity after exposure to Listeria monocytogenes and are more susceptible to endotoxemia
after inoculation with LPS or Klebsiella pneumonia (8, 9). ApoEdeficient mice, compared with wild-type mice, show differences
in delayed-type hypersensitivity and IgM responses to Ag (10).
These observations suggest that apoE has important biological
functions independent of its role in cholesterol metabolism.
When apoE is added to mitogen-treated lymphocytes and
monocytes/macrophages, two readouts of Th cell responsiveness
*Cognosci, Inc., Research Triangle Park, NC 27709; †Division of Hematology, Department of Medicine, Duke University Medical Center, Durham, NC 27710; and
‡
Division of Neurology, Department of Medicine, Duke University Medical Center,
Durham, NC 27710
Received for publication August 23, 2010. Accepted for publication November 19,
2010.
This work was supported by the National Institutes of Health for the Small Business Innovation Research Grants R44 AG 020473 (to M.P.V.), R41 DK 075161 (to
M.P.V.), R44 NS 052920 (to F.L.), R43 AI 072864 (to D.J.C.), and R43 CA 137941
(to D.J.C.).
Address correspondence and reprint requests to Dr. Michael P. Vitek, Cognosci, Inc.,
P.O. Box 110606, Research Triangle Park, NC 27709. E-mail address: mikevitek@
cognosci.com
Abbreviations used in this article: apoE, apolipoprotein E protein; APOE, apolipoprotein E gene; IKK, I-kB kinase; I2PP2A, inhibitor-2 of PP2A; LDL, low-density
lipoprotein; LRP, LDLR-related protein; NP-40, Nonidet P-40; PP2A, protein phosphatase 2A; siRNA, small interfering RNA.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002847
are reduced: proliferation, and production of IL-2 (T cell growth
factor) (11). In addition, apoE suppresses human T lymphocyte
proliferation ex vivo, independent of the T cell mitogen used (12).
The immunosuppressive effect of apoE on Th cell responsiveness
was validated using lymphocytes and macrophages from mice
made deficient for apoE by targeted gene disruption (13). When
macrophages derived from apoE-deficient (APOE2/2) and apoEsufficient wild-type (APOE+/+) control mice were sensitized to
OVA, a 4-fold increase in the Ag-presenting ability of lymphocytes derived from Ag-sensitized APOE knockout over wild-type
mice was observed. The APOE2/2 APCs also facilitated a stronger Th1 response ex vivo by activating Th cells to produce more
IFN-g, compared with Ag-specific Th cells activated with
APOE+/+ macrophages (13). Furthermore, apoE-deficient macrophages and B cells express greater amounts of CD80, a molecule that is crucial for activation of Ag-specific lymphocytes (13,
14).
In an attempt to define the domains of the apoE holoprotein
responsible for its immunomodulatory activity, we and our collaborators created a peptide corresponding to the receptor binding
region and consisting of residues 133–149 of apoE. We found that
treatments with this apoE[133–149] peptide suppressed microglial
activation and secretion of inflammatory mediators (e.g., TNF-a
and IL-6 proteins) in primary microglial cultures and microglial
cell lines (15), in a manner that matches the anti-inflammatory
effect of the apoE holoprotein (16). In addition, apoE[133–149]
inhibited a macrophage signaling cascade and suppressed macrophage activation in the same fashion as the entire apoE protein
(17). Finally, the apoE[133–149] peptide significantly reduced serum TNF-a and IL-6 production at both the mRNA and protein
level in mice treated with LPS (18). This suppression of cytokine
production suggests that apoE[133–149] acts as an apoE-mimetic,
which is supported further by a report indicating that a similar
apoE[130–149] peptide composed of residues 130–149 was shown
to potently bind to the LRP1 (19).
Given the anti-inflammatory and immunomodulatory activities
described above, there is a paucity of knowledge about the mole-
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The molecular mechanism by which apolipoprotein E (apoE) suppresses inflammatory cytokine and NO production is unknown.
Using an affinity purification approach, we found that peptide mimetics of apoE, derived from its receptor binding domain residues
130–150, bound to the SET protein, which is a potent physiological inhibitor of protein phosphatase 2A (PP2A). Both holo-apoE
protein and apoE-mimetic peptides bound to the C-terminal region of SET, which is then associated with an increase in PP2Amediated phosphatase activity. As physiological substrates for PP2A, the LPS-induced phosphorylation status of signaling MAPK
and Akt kinase is reduced following treatment with apoE-mimetic peptides. On the basis of our previous report, in which apoEmimetic peptides reduced I-kB kinase and NF-kB activation, we also demonstrate a mechanism for reduced production of
inducible NO synthase protein and its NO product. These data provide evidence for a novel molecular mechanism by which
apoE and apoE-mimetic peptides antagonize SET, thereby enhancing endogenous PP2A phosphatase activity, which reduces levels
of phosphorylated kinases, signaling, and inflammatory response. The Journal of Immunology, 2011, 186: 2535–2542.
2536
APOLIPOPROTEIN E AS A MODULATOR OF INFLAMMATORY SIGNALING
Materials and Methods
Materials
All peptides were synthesized by PolyPeptide Laboratories (San Diego,
CA), using standard Fmoc-based chemistry and purified to .95% purity.
The sequences synthesized were apoE[133–149]: acetyl-LRVRLASHLRKLRKRLL-amide and COG1410: acetyl-AS(aib)LRKL(aib)KRLL-amide,
where (aib) signifies amino-iso-butyric acid. All buffers and reagents
were from Sigma unless otherwise specified.
Detection of LRP and LDLR by Western blotting
Primary mouse peritoneal macrophages were obtained by i.p. injection of
four 25- to 30-g male C57BL/6 mice with 1 ml 5 mM sodium periodate and
repeating this injection 72 h later. Peritoneal macrophages were harvested
by lavage with cold PBS containing 10 U/ml heparin sulfate, 24 h after the
second sodium periodate injection. Cells were collected by centrifugation
and washed with 1 ml chilled PBS and lysed in Nonidet P-40 (NP-40) buffer
(50 mM Tris, 0.2% NP-40, 150 mM NaCl). BV2 microglial cells were
grown to log phase in a T75 flask, stimulated with LPS (100 ng/ml), and
cells were lysed in NP-40 buffer. Protein concentration of the lysate was
determined using the BCA assay (Pierce) and adjusted to 5 mg total protein
per milliliter of solution. Laemmli protein sample buffer (43, 75 ml) was
added to the cell lysate (25 ml), and the solutions were heated to 90˚C for 5
min. Protein solutions were loaded onto duplicate gels and were separated
by SDS-PAGE, blotted onto nitrocellulose membranes (Bio-Rad). The
nitrocellulose membranes were blocked using 5% nonfat milk in TBST
(containing 0.1% Tween 20) for 3 h, then washed with TBST. One
membrane was incubated overnight at 4˚C in a rabbit anti-LDLR Ab
(Cayman Chemical). The other membrane was incubated in a mouse
monoclonal anti-LRP Ab (CalBiochem) and a rabbit anti–b-GAPDH Ab
(Santa Cruz). Membranes were washed with TBST for 1 h, with three
changes of the wash solution, and incubated with the appropriate IRDye
680 or IRDye 800 secondary Abs. Protein bands were visualized and
quantitated using an Odyssey Infrared scanner (LiCor). Following the first
read of the LDLR blot, a rabbit anti-cyclophilin B Ab (Abcam) was added,
allowed to bind, washed, and the blot incubated again with the secondary
Ab. The blot was again read to provide a loading control protein band of
the appropriate mass.
TNF-a production by peritoneal macrophages or BV2 cells
Primary peritoneal macrophages as obtained above were resuspended in
DMEM/10% FBS/1% pen-strep/1% HEPES/1% L-glutamine media (Life
Technologies). Cells were seeded at 2 3 105 cells per well into a 48-well
tissue culture plate and incubated in 5% CO2 at 37˚C overnight to adhere
to the plate. The culture media were removed the following morning and
replaced with 1% FBS/DMEM containing peptide treatments. The apoE
[133–149] peptide was added to a final concentration of either 5 or 10 mM,
and COG1410 was added at final concentrations of 2 or 4 mM. Media
containing the peptide or no peptide were incubated with the cells for 30
min before addition of 100 ng/ml LPS and 300 ng/ml mouse IFN-g each to
a third of the wells, and a vehicle control to the remaining wells. Treated
cells were incubated for 24 h at 37˚C in 5% CO2 to stimulate. BV2 cells
were grown overnight and seeded at 5 3 104 cells per well in a 48-well
plate. Cell treatments were performed as described for the peritoneal
macrophages, except LPS (200 ng/ml) was used without IFN-g to stimulate TNF-a production. After 24 h, media were removed and TNF-a
quantified using ELISA (Invitrogen). Cell numbers were determined with
the MTT assay (Sigma). Values for TNF-a were normalized to MTT values
prior to plotting.
BV2 uptake of biotin-labeled apoE[133–149]peptide and SET
immunofluorescence staining
BV2 cells were plated at a density of 8 3 103 cells per well onto poly-Dlysine–coated 12-mm glass coverslips and grown overnight in complete
media (DMEM/10% FBS/13 nonessential amino acids/13 sodium pyruvate; Life Technologies). Biotin-labeled apoE[133–149] was added at
a concentration of 1 mM in complete media and incubated at 37˚C for 2 h.
After treatment, the cells were washed with PBS and rinsed with 2 M
acetic acid (15 s) to remove extracellular bound ligand, and then cells were
washed thoroughly with PBS (3 3 500 ml) and fixed with 4% paraformaldehyde for 20 min. Cells were blocked and permeabilized by
treatment with 10% normal goat serum/1% BSA/0.1% Triton X-100 for
1 h, washed, and incubated with Alexa Fluor 555-labeled streptavidin
(Invitrogen) for 30 min at 37˚C. Next, cells were washed and incubated
with a rabbit polyclonal anti-SET primary Ab (Santa Cruz) diluted in
blocking buffer for 2 h at room temperature. Following a thorough wash,
cells were then labeled with an Alexa Fluor 488-labeled goat anti-rabbit
IgG secondary Ab (Invitrogen) and Hoescht-33342 nuclear stain (Pierce)
for 1 h at room temperature in PBS. The coverslips were then washed
thoroughly and mounted in Vectashield mounting media (Vector Labs) and
imaged with a Nikon Eclipse Ti-S fluorescence microscope. Images were
processed using Nikon NIS-Elements Basic Research software.
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cular mechanism by which apoE and its peptide mimetics are able
to suppress the immune response. Clues to the molecular mechanism can be derived from the reports of Kawamura et al. and Singh
et al. (20, 21) Kawamura and coworkers (20) reported that the apoE
holoprotein inhibits IL-1b signaling in vascular smooth muscle
cells, resulting in reduced production of inflammatory mediators,
including NO and PGE2. This effect was shown to be mediated
through decreased phosphorylation and reduction of nuclear localized NF-kB leading to reduced production of inducible NO synthase (22). Our collaborators, Singh et al. (21), also reported that
COG112, another apoE-mimetic peptide composed of the apoE
[133–149] peptide fused to the antennapedia protein transduction
domain peptide, also reduced activation of the NF-kB pathway. In
each case, it was noted that phosphorylation of the I-kB kinase
(IKK) was reduced by treatment with apoE or its mimetic peptide.
To understand the role of apoE and determine the mechanism by
which these apoE and apoE-mimetic peptides inhibit inflammatory
processes and reduce phosphorylation of NF-kB, we sought to
determine if the suppression of TNF-a production following LPS
stimulation was mediated by the known apoE receptors, LRP and
LDLR, and to identify other cellular proteins that bound to the
apoE[133–149] peptide. We found a significant dose-dependent
suppression of TNF-a production by two apoE-mimetic peptides, apoE[133–149] and COG1410, following LPS stimulation
of either primary mouse peritoneal macrophages or BV2 microglial cells. This is noteworthy because both LRP and LDLR are
expressed in peritoneal macrophages, but they are not expressed in
the BV2 cell line, suggesting that the anti-inflammatory activity
occurs by a receptor-independent mechanism. Beyond the mechanism of uptake, we now report that apoE and apoE-mimetic
peptides bind to the SET oncoprotein and that the apoE holoprotein interacts with SET through its C-terminal region from
amino acids 177–277. The protein SET is also known as inhibitor2 of protein phosphatase 2A (I2PP2A) and exists at elevated levels
in the brains of those with Alzheimer’s disease relative to normal
age-matched controls (23) and in leukemia cancer cells (24–26).
Binding of the apoE-mimetic peptide to SET results in activation of protein phosphatase 2A (PP2A), with a corresponding
dephosphorylation of PP2A targets. Signaling modules consisting
of various kinases and PP2A have been reported to increase the
fidelity of a signal transduction pathway by tightly regulating activation and deactivation processes. Evaluation of these modules
has identified numerous kinases that interact with PP2A, including
the MAPK proteins p38 kinase (27), JNK (28), and ERK (29, 30).
Furthermore, PP2A negatively regulates Akt and its target, IKK,
which normally participates in the activation of the NF-kB transcription factor (31, 32). NF-kB has been shown to positively
regulate production of inducible NO synthase (22) from the NOS2
gene, resulting in increased NO production. Accordingly, we
found reduced levels of phosphorylated p38 MAPK and the Akt
kinase that activates the NF-kB pathway. We also report that SET
is essential for normal inflammatory responses to LPS because
small interfering RNA (siRNA)-mediated reduction of the SET
protein level significantly inhibits the inflammatory response following LPS stimulation. Taken together, these results suggest a
novel mechanism by which apoE and apoE-mimetic peptides
modulate biological pathways that are critical for inflammatory
signaling by antagonism of SET, resulting in activation of PP2A.
The Journal of Immunology
Affinity purification of apoE[133–149] binding proteins
BV2 microglial cells were grown to log phase in a T75 flask, stimulated with
LPS (100 ng/ml), and biotin-labeled apoE[133-149] (10 mM) or biotin (10
mM) was added for 2 h; a lysate was prepared in NP-40 buffer (50 mM
Tris, 0.2% NP-40, 150 mM NaCl) by a single freeze–thaw cycle. Protein
concentration of the lysate was determined using the BCA assay (Pierce)
and adjusted to 5 mg total protein per milliliter of solution. Streptavidin–
agarose beads (1 ml) were washed with 10 ml NP-40 buffer, and 0.5 ml
beads were added to 1 ml extract. After incubation the beads were collected by filtration through a disposable minicolumn (Bio-Rad) and the
flow-through extract collected for analysis. Following washing with 100 ml
chilled NP-40 buffer, the beads were removed from the column and collected by centrifugation, and 75 ml 43 Laemmli buffer was added. Beads
were vortexed and heated to 90˚C for 10 min to ensure that all proteins
were released from the beads. Proteins were separated by SDS-PAGE,
visualized by Coomassie staining, and identified by mass spectrometry.
Affinity purification and Western blotting of mouse and human
brain lysate
Coimmunoprecipitation of apoE and SET
GST-SET fusion constructs were a kind gift from Dr. Ye (Emory University, Atlanta, GA). GST fusion proteins were expressed in E. coli bacterial
strain BL21 and purified using B-Per GST Fusion Protein Purification Kit
(Pierce) according to the manufacturer’s protocol. Purified protein was
eluted from the column, using 50 mM reduced glutathione, dialyzed in
SET buffer (50 mM Tris pH 7.5, 0.5 mM EDTA, 1 mM benzamadine, 0.5
mM PMSF, and 10% glycerol) and concentrated to 100 mg/ml, using
Centricon concentrators (Millipore). Purified human apoE (1 mg; Calbiochem) was incubated with purified GST-SET fusion protein (1 mg)
at 4˚C for 2 h in binding buffer (50 mM Tris pH 7.5, 0.5 mM EDTA,
1 mM benzamadine, 0.5 mM PMSF, 10% glycerol, and 0.25% NP-40).
Glutathione-agarose or apoE Ab E6D7 (Calbiochem) with protein G
beads (Santa Cruz) was added and incubated for 45 min. Beads were
washed 43 with TBS + 0.1% NP-40 and extracted in SDS sample buffer
for 10 min at 90˚C. Samples were run on 12.5% SDS-PAGE gel, transferred to nitrocellulose, and immunoblotted with rabbit anti-GST (Cell
Signaling) and mouse anti-apoE (Calbiochem). IRDye-labeled secondary
anti-rabbit and anti-mouse Abs (LiCor) were used for imaging on a LiCor
Odyssey imaging system, as described above.
Competitions with COG1410 and apoE[133–149] peptides
Following the procedure above, competitions were performed using the
BV2 lysate. Non–biotin-labeled apoE[133–149] or COG1410 was added to
individual aliquots of lysate in increasing concentrations and incubated for
1 h prior to affinity purification and Western blotting of the SET, as described above.
PP2A activity in mouse brain
C57BL/6 mice were intravenously injected (5 ml/kg; n = 5 per group) with
either COG1410 (5 mg/kg in lactated Ringer’s solution) or lactated
Ringer’s solution. After 30 min the mice were anesthetized, sacrificed by
cervical dislocation, their brains removed, and flash frozen. The brain was
ground to a fine powder, as above, and lysed using Qiagen’s mammalian
proteome lysis kit. Protein was quantified using BCA (Pierce), and 500 mg
total protein was combined with 4 ml anti-PP2A Ab (1D6; Upstate) and 50
ml protein A–agarose beads in a total volume of 500 ml. The mixture was
shaken for 2 h at 4˚C, and then beads were collected by centrifugation.
Following four washes, 50 ml phosphatase assay buffer (Upstate) was
added to the beads, vortexed, and 50 ml bead slurry was added to one well
of a 96-well plate. A 10 mM stock of 6,8-difluoro-4-methylumbelliferyl
phosphate (Invitrogen) was diluted to 100 mM in assay buffer, and 50 ml
was added to each well of the plate; then the fluorescence intensity
of a fluorescent product produced by cleavage of phosphate from 6,8difluoro-4-methylumbiliferyl phosphate, a synthetic phosphatase substrate,
was measured using a plate reader. Fluorescence reads were performed
every 3 min, and shaking every 30 s, over a 30-min period.
Analysis of Akt and p38 phosphorylation following LPS
stimulation
BV2 cells were plated at 5 3 105 cells per well and were allowed to adhere
overnight in a six-well plate in DMEM/10% FBS (Life Technologies). Cells
were pretreated for 15 min with 5 mM apoE[133–149] peptide before adding
100 ng/ml LPS for 30 min. Protein extracts from cell cultures were lysed in
Mammalian Protein Extraction Reagent (Pierce) with protease inhibitors.
Equal amounts of cellular protein were added to 43 Laemmli buffer and
subjected to SDS-PAGE. After transferring to nitrocellulose membranes, the
blots were blocked for 30 min with Odyssey Blocking Buffer (LiCor) and
incubated with phospho-Akt (BioSource) or total Akt Abs (Cell Signaling)
in Super Block (ThermoSci). Proteins were visualized using LiCor secondary Abs on an Odyssey scanner, as described above. Similar blots were
incubated with phospho-p38 or total p38 Abs (Cell Signaling).
siRNA knockdown of SET
A confluent flask of BV2 cells was diluted 1:50 in normal growth media, and
500 ml per well was plated into 24-well plates. The cells were allowed
to adhere overnight before transfection. Once the cells adhered, 80 ml Dharmafect-1 was added to 1960 ml Optimem, and 350 ml of each siRNA (2 mM in
water) (I2PP2A from Santa Cruz, noncoding control from Dharmafect)
was added to 350 ml Optimem. These solutions were incubated at room
temperature for 5 min before addition of the Dharmafect mixture (625 ml)
to each siRNA mixture. After 20 min, 5 ml media (DMEM, penicillin/
streptomycin, nonessential amino acids, sodium pyruvate, and 1% FBS)
was added to each siRNA/Dharmafect mixture. Treatment of cells with the
siRNA preparations was accomplished by aspirating media from cells,
washing once with 250 ml PBS, and adding 500 ml siRNA/Dharmafect
preparation to the cells. After incubation for 24 h, media were removed,
cells were washed with 250 ml PBS, and fresh media (500 ml) containing
treatments (no treatment control, 5 mM COG1410, 100 ng/ml LPS, or
COG1410 plus LPS) were added. Following incubation (16 h) cells were
harvested by washing once with 500 ml PBS and lifting with Versene. Cells
were collected by centrifugation (5 min at 1000 3 g) and washed with PBS
to remove Versene. A Greiss assay (Promega) was performed on media
immediately following the cell harvest, according to the manufacturer’s
protocol. The effect of siRNA treatment on SET and PP2A was determined
by lysing the cell pellets in Qiagen’s mammalian proteome extraction
buffer. Proteins were quantified and diluted such that 20 mg total protein
was loaded per lane following addition of 43 Laemmli buffer and heating
for 10 min at 90˚C. Following transfer to nitrocellulose, Western blotting
was performed as above with anti-SET (Santa Cruz), anti–b-actin (Santa
Cruz), and anti-PP2Ac (Upstate) Abs.
Statistical analysis
All statistical analyses were performed using either one-way ANOVA or
Student t test, with statistical significance defined as p , 0.05 and values
expressed as mean 6 SEM. For comparison of three or more values,
ANOVA was used with the Neuman–Keuls post hoc test. When two groups
were compared, the Student t test was used.
Results
ApoE-mimetic peptides suppress TNF-a production by an
LDLR/LRP-independent mechanism
To determine if suppression of inflammatory cytokine production
by apoE-mimetic peptides following LPS stimulation was due to
cell surface apoE receptors, the expression of LRP and LDLR was
evaluated by Western blotting on mouse peritoneal macrophages
and on the BV2 cell line. Expression of both LRP and LDLR was
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Frozen mouse and human brain samples were ground to a fine powder, using
a mortar and pestle cooled with liquid nitrogen. Extracts were prepared by
suspending 100 mg brain powder in NP-40 buffer and performing a freeze–
thaw cycle. Lysates were diluted to 1 mg/ml and 1-ml aliquots precleared
with streptavidin–agarose beads, as above. Biotin–apoE[133–149] (10 mg)
was added to 1 mg protein extract from BV2 cells, mouse brain, and human brain. As a negative control, biotin (0.1 mg) and non–biotin-labeled
apoE[133–149] peptide (10 mg) were added to another 1-mg sample of
total protein extract. After incubation for 2 h (4˚C), 50 ml fresh, washed
beads were added to the lysate/peptide mixtures, with incubation being
continued for an additional 2 h. Following four washes with 1 ml chilled
NP-40 buffer, 75 ml 43 Laemmli buffer was added and the beads were
heated to 90˚C for 10 min. Proteins were separated by SDS-PAGE and
blotted onto nitrocellulose membranes (Bio-Rad), and bound SET protein
was detected by Western blotting. Nitrocellulose membranes were blocked
using 5% nonfat milk in TBST containing 0.1% Tween 20 for 3 h, washed
with TBST, and incubated overnight at 4˚C in a rabbit anti-SET Ab (Santa
Cruz). Membranes were washed with TBST for 1 h with three changes of
the wash solution and incubated with a donkey anti-rabbit Ab labeled with
IRDye 680. Protein bands were visualized and quantitated using an Odyssey Infrared scanner (LiCor).
2537
2538
APOLIPOPROTEIN E AS A MODULATOR OF INFLAMMATORY SIGNALING
FIGURE 1. ApoE-mimetic peptides reduce TNF-a production by an
LRP/LDLR-independent mechanism. A, Cell lysates (40 mg per lane) from
mouse peritoneal macrophage (PM) and BV2 microglial cells were run
on duplicate PAGE gels and Western blotted with anti-LDLR and anticyclophilin B Abs or anti-LRP and anti-GAPDH Abs. No LDLR or LRP
was observed in the BV2 cell lysates. B, Elicited mouse peritoneal macrophages were plated overnight and treated with either vehicle or LPS (100
ng/ml) plus mouse IFN-g (300 ng/ml) in the presence or absence of the
indicated concentrations (mM) of the apoE[133–149] or COG1410 peptides (n = 6 wells per treatment). Media were removed and TNF-a
quantified by ELISA and normalized to a standard curve. C, Same as B,
except BV2 microglial cells were treated with LPS (200 ng/ml) in the
presence or absence of the indicated concentrations (mM) of the apoE
[133–149] or COG1410 peptides vehicle, LPS alone, apoE[133–149], and
LPS plus apoE[133–149].
peptides occurred through an LDLR/LRP-independent mechanism.
ApoE-mimetic peptides and apoE are transported into the
cytoplasm and bind to SET/I2PP2A
Because apoE[133–149] exerted anti-inflammatory effects independent of LRP/LDLR expression, we evaluated the ability of the
apoE[133–149] peptide to become internalized into cells. BV2
cells were treated with an apoE[133–149] peptide that had biotin
covalently attached to the N-terminal amine group of this peptide
for use as an affinity tag, and with a nonbiotinylated apoE[133–
149] peptide. After 2 h, the cells were washed with acid to strip
cell surface/receptor-bound peptide, and the cells were fixed and
subsequently permeabilized. After staining with Alexa Fluor 555labeled streptavidin, red fluorescence from Cy3 was found in the
cells treated with the biotin-labeled apoE[133–149] peptide, but
was not observed in the cells treated with apoE[133–149] peptide
lacking the biotin tag (Fig. 2A). The distribution of the red fluorescent signal indicated that apoE[133–149] peptide was transported into the cytoplasm of these cells.
To determine if the intracellular apoE[133–149] was binding
to an internal protein, we used the biotin-labeled apoE[133–149]
peptide to affinity purify cellular proteins that might bind to the
apoE portion of the peptide. The biotin-tagged apoE-peptide was
added to LPS-stimulated BV2 microglial cells. After incubation,
the cells were lysed, and proteins bound to the biotinylated peptide
were recovered as a complex by binding to agarose-conjugated
streptavidin. After extensive washing of the agarose beads, bound
proteins were eluted from the beads by heating in Laemmli buffer,
separated by SDS-PAGE, and visualized with Coomassie staining
(Fig. 2B). Two proteins of ∼39 and 25 kDa were readily observed
in lanes containing the biotin-tagged apoE-mimetic peptide (lanes
4 and 5), but were absent in the control lane (lane 6) containing
free biotin mixed with apoE[133–149] peptide lacking the covalently coupled biotin tag. Mass spectroscopy identified both
bands as the protein SET, also known as I2PP2A, which exists in
full-length and protease-cleaved forms (34, 35). To extend this
observation to tissues, similar affinity purifications were performed with extracts from BV2 cells, mouse brain, and human
brain. The binding of SET to the biotin-labeled apoE[133–149]
peptide was observed by affinity purification followed by Western
blotting in all three extracts (Fig. 2C). To confirm the localization
of the SET protein in BV2 cells and to verify that SET and apoE
[133–149] are found in the same intracellular compartment, we
stained the BV2 cells treated with biotin-labeled apoE[133–149]
with an anti-SET Ab. We detected the SET Ab with an Alexa Fluor
488-labeled secondary Ab and observed green fluorescence in the
cytoplasm of BV2 cells. We previously observed that this same
intracellular compartment contained the biotin-labeled apoE[133–
149] peptide, as detected by Alexa Fluor 555–streptavidin staining
(Fig. 2A).
To determine if apoE[133–149] binding to SET was consistent with its function as a mimetic of the apoE holoprotein, we
investigated whether the apoE holoprotein bound to SET by
performing interaction studies with purified apoE and GST-SET
fusion proteins. Bacterially expressed GST-SET proteins were
purified and represented the 1–77 N-terminal portion of SET,
the 1–177 region that lacks the C-terminal fragment, and the
full-length 1–277 SET protein (36). The purified GST-SET fusion
proteins (and GST alone, as a control) were incubated with
purified human apoE prior to affinity purification using either glutathione-agarose, or anti-apoE Ab/protein G–agarose complexes. Western blotting was used to detect SET–apoE interactions. An interaction was observed between the full-length GST-
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observed in the peritoneal macrophage, but expression of both
receptors was notably absent in the BV2 cell line (Fig. 1A). With
the established difference between LRP and LDLR expression
in LPS-reactive cells of these two sources, we evaluated the ability of apoE[133–149] and the related COG1410 peptide to suppress TNF-a production following LPS stimulation. COG1410 is
shorter than apoE[133–149] and was engineered to have a stabilized a-helical structure through introduction of helix-stabilizing
aminoisobutyric acid residues, which provided for enhanced
in vitro and in vivo anti-inflammatory activity relative to the
natural apoE[133–149] peptide in other studies (33). Peritoneal
macrophages showed no notable production of TNF-a when
treated with no compound or with either of the apoE-mimetic
peptides alone. Significant levels of TNF-a were produced following stimulation with LPS and IFN-g, and the levels of TNF-a
production were reduced in a dose-dependent manner by treatment
with either apoE[133–149] or COG1410 (Fig. 1B). Treatment of
BV2 cells in a similar manner also demonstrated significant suppression of TNF-a production following LPS stimulation (Fig.
1C). These data from BV2 cells that lack LDL/LRP receptors
indicate that the anti-inflammatory activity of the apoE-mimetic
The Journal of Immunology
2539
SET fusion protein and human apoE (Fig. 3) in both GSTmediated affinity and anti–apoE-mediated affinity purifications.
No interaction was observed between the GST control protein and
apoE or for the GST-SET fusion proteins that lacked the Cterminal region from 177 to 277.
To confirm the specificity of the interaction, we used peptidemediated affinity purification and Western blotting techniques to
demonstrate that nonbiotinylated versions of the apoE[133–149]
and COG1410 could inhibit the interaction of the biotin–apoE
[133–149] peptide with SET. Equal amounts of BV2 lysates were
incubated with varying concentrations of nonbiotinylated peptides
prior to addition of the biotin-labeled apoE[133–149] peptide,
followed by affinity purification on streptavidin–agarose beads
and detection of bound SET by Western blotting. Both the apoE
[133–149] peptide and COG1410 were able to inhibit binding of
SET to the biotin-labeled apoE[133–149] peptide and reduce the
amount of SET detected by Western blotting (Fig. 4). The competition observed with each peptide suggests that the apoE[133–
149] peptide and the COG1410 peptide share a similar binding
site on the SET protein.
ApoE-mimetic peptide treatment activates PP2A in vivo
FIGURE 3. ApoE associates with the C-terminal region of SET. Purified
human apoE was incubated with recombinant GST or GST-SET 1–77, 1–
177, and 1–277 fusion proteins. Immunoprecipitation with glutathione–
agarose shows that apoE bound to the full-length GST-SET:1–277 fusion
protein (lane 9, upper blot), but not to truncated GST-SET fusion proteins,
when probed with an anti-apoE Ab. Conversely, immunoprecipitation using an anti-apoE Ab showed only full-length SET binding to apoE (lane
13, lower blot) when probed with an anti-SET Ab.
Previous reports indicate that SET is a potent physiological inhibitor of PP2A. Because apoE peptides and apoE bind to SET, this
suggests a novel mechanism whereby apoE/peptide increases PP2A
activity within a cell by binding and forming a complex with SET,
thereby reducing the amount of SET that is available to inhibit
PP2A. To test this hypothesis, mice were injected with COG1410
or a vehicle control, and brains were removed 1 h later and assayed
for PP2A activity. PP2A was selectively immunoprecipitated from
equal amounts of brain protein lysates from COG1410 and control
brain lysates using Ab 1D6 (Upstate), which recognizes the Cterminal region of its PP2Ac subunit. Immunoprecipitated PP2Amediated phosphatase activity was quantified, and the rate of
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FIGURE 2. ApoE-mimetic peptides enter cells and bind to SET. A, BV2 cells were plated, grown overnight, and treated with either apoE[133–149] (top
row) or biotin-labeled apoE[133–149] (bottom row) for 2 h before acid washing to remove surface-bound compounds. Cells were then fixed, permeabilized,
and stained with Alexa Fluor 555-labeled streptavidin (AF-SA) and rabbit anti-SET Ab. After washing, the cells were stained with an Alexa Fluor 488labeled anti-rabbit IgG and Hoechst-33342 dye. Images were acquired by fluorescence microscopy showing that biotin-labeled apoE[133–149] was localized in the cytoplasm (red staining in cells, as indicated by the white arrow) and that SET also localized in the cytoplasm (green in cells). Overlap of the
SET and apoE[133–149] is seen in the merged image by the yellow-orange color, as indicated by the white arrow. B, Lysates of BV2 microglial cultures
were incubated with biotin–apoE[133–149] (20 mM) or free biotin without the apoE[133–149] peptide, followed by incubation with streptavidin–agarose.
Beads were washed repeatedly, heated in Laemmli sample buffer, and released proteins were run on an SDS-PAGE gel, as visualized with Coomassie
staining. Lane 1, m.w. markers; lanes 2 and 3, total BV2 lysate; lanes 4 and 5, proteins affinity purified with biotin–apoE[133–149]; lane 6, proteins affinity
purified with biotin alone. The arrowheads point to the 39-kDa SET protein and a 25-kDa fragment of SET. C, Biotin-labeled apoE[133–149] was incubated
with 1 mg of BV2 microglial cell lysate, mouse brain lysate, or human brain lysate followed by incubation with streptavidin–agarose beads, which were
then washed and processed as above. Affinity purified proteins were subjected to Western blotting with an anti-SET Ab. For each blot, lane 1, m.w. markers;
lane 2, 25 mg of total lysate; lane 3, pull-down with biotin only; lane 4, pull-down with biotin–apoE[133–149]. The major immunoreactive protein band
is SET.
2540
APOLIPOPROTEIN E AS A MODULATOR OF INFLAMMATORY SIGNALING
phosphate cleavage was found to be ∼55% higher (p , 0.05) in
brain extracts prepared from animals treated with COG1410 relative to vehicle-treated animals (Fig. 5).
ApoE-mimetic peptides modulate PP2A-regulated signaling
pathways
On the basis of data showing that apoE-mimetic peptides bind and
antagonize SET, resulting in activation of PP2A, we hypothesized
that activation of PP2A by SET antagonists (i.e., apoE-mimetic
peptides) would modulate PP2A-mediated signaling pathways.
Therefore, we evaluated the effect of apoE-mimetic peptides on
p38 MAPK and Akt phosphorylation following LPS stimulation
of BV2 microglial cells. Following LPS stimulation, reduced
phospho-p38 and phopho-Akt levels were observed in samples
treated with apoE[133–149] relative to samples treated with only
LPS (Fig. 6). These findings are also consistent with previously
reported reductions of activation of the NF-kB pathway by reducing IKK phosphorylation with COG112, an apoE-mimetic
peptide composed of the apoE[133–149] peptide fused to the
FIGURE 5. Subcutaneous administration of an apoE-mimetic peptide
increases PP2A-mediated phosphatase activity in brain. C57BL/6 mice
were injected with COG1410 (▼) or a vehicle control (d), and the activity
of PP2A in brain extracts in a no extract control reaction (n) was measured
using an immunoprecipitation/fluorometric assay, as described in Materials and Methods. The rate of phosphate cleavage in COG1410-treated
brain extracts was significantly increased over the vehicle control or no
extract control. p , 0.05 by ANOVA.
FIGURE 6. ApoE-mimetic peptides downregulate LPS-mediated phosphorylation of p38-MAP kinase and Akt kinase. A, BV2 microglial cells
were treated with vehicle, LPS alone, apoE[133–149], and LPS plus apoE
[133–149]. Total cell lysates were Western blotted, probed with anti-p38
MAP kinase or anti–phospho-p38 MAP kinase Abs, and proteins were
quantified by fluorescence intensity (LiCor Odyssey scanner). The ratio of
phospho-p38 MAP kinase divided by the total-p38 MAP kinase is plotted.
Phospho-p38 MAP kinase levels are significantly higher in the LPS
treatment than in the LPS plus apoE[133–149] treatment. B, The same
lysates prepared for A were used in Western blots probed with anti-Akt or
anti–phospho-Akt Abs, and proteins were quantified by fluorescence intensity (LiCor Odyssey scanner). The ratio of phospho-Akt divided by the
total Akt is plotted. Phospho-Akt kinase levels are significantly higher in
the LPS treatment than in the LPS plus apoE[133–149] treatment **p ,
0.01.
antennapedia protein transduction domain peptide (21). These
data provide mechanistic support for suppressed production of
inflammatory cytokines, whose transcription is regulated by the
MAPK and NF-kB pathways.
Finally, to validate that knockdown of SET produced a similar
response to antagonism with apoE-mimetic peptides in modulating the inflammatory signaling process, production of the SET protein was silenced using siRNA techniques. Transfection of BV2
microglial cells with a noncoding, control siRNA did not reduce
levels of LPS-induced NO production or the ability of COG1410 to
significantly suppress NO production (Fig. 7A). However, transfection with SET-specific siRNAs resulted in significantly reduced
production of NO relative to noncoding siRNA control samples
upon LPS stimulation and was not further changed by the addition
of COG1410. Although the amount of SET in the SET siRNAtransfected cells was greatly reduced, levels of the catalytic PP2Ac
subunit of PP2A were unchanged (Fig. 7B). Reduced NO production is consistent with increased PP2A functional activity
without increased expression of the catalytic PP2Ac subunit in the
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FIGURE 4. ApoE peptides compete with biotin–apoE[133–149] for
binding to SET. A, BV2 microglial cell lysates were incubated with no
competitor (–), apoE[133–149] (:), or COG1410 (▼) at the indicated
concentrations, followed by addition of biotin–apoE[133–149] and streptavidin–agarose beads. Beads were washed and heated, and released proteins were Western blotted and probed with anti-SET Abs. SET protein
bands were quantified on a LiCor Odyssey fluorescence scanner and are
plotted as arbitrary fluorescence units. Both the apoE[133–149] and the
COG1410 peptides significantly competed with biotin–apoE[133–149]
binding to SET in cell lysates. B, Western blots used for quantitation in A.
The Journal of Immunology
cells. In addition, reduction of SET levels also blocked the ability
of COG1410 to further repress NO production, indicating that
SET is the functional target of apoE and the apoE-mimetic peptides. Taken together, these data indicate that SET is an essential
component of the inflammatory signal transduction process and
that apoE and apoE-mimetic peptides modulate the function of
SET.
Discussion
Innate immune responses to pathogens and tissue damage are
largely driven by signaling through the TLR protein family. As an
example of one pathway, LPS-mediated activation of the TLR-4
receptor is associated with sequential phosphorylation of signaling kinases in a transient manner that typically results in the activation of transcription factors, including NF-kB. These transcription factors initiate expression of genes encoding inflammatory mediators such as cytokines and inducible NO synthase.
The activation of this pathway, however, is limited with respect
to time and location, so that the response may be acute but does
not last for an extended period. In contrast, the strength with
which this pathway is activated does vary, resulting in variable
levels of phosphorylated signaling kinases and their associated
activities.
Phosphorylation of signaling kinases is associated with enhanced
kinase activity toward the next kinase in the pathway to propagate
the signal. Negative regulation of these pathways is accomplished
by removal of the phosphate from the signaling kinases to restore
the prestimulated, noninflammatory, or healthy state. In general, the
phosphorylation status of a protein kinase depends upon the balance between the activation of kinases that promote phosphorylation and the activity of phosphatase enzymes that remove the
phosphate groups from the protein. Given the simultaneous presence of these opposing forces, the regulation of inflammatory
pathways can largely be viewed as a balance between the activating kinases and the deactivating phosphatases. In the case of the
MAPK and NF-kB pathways, PP2A has been shown to mediate
the deactivating dephosphorylation events. Therefore, inhibition
of PP2A activity would be expected to result in an increased inflammatory response, whereas activation of PP2A would be
expected to inhibit the inflammatory response.
Like the PP2A phosphatase complex, the SET protein, MAPK,
and Akt kinases, we have demonstrated that the apoE-mimetic
peptides are localized in the cytoplasm of BV2 cells. Similarly,
exogenously applied biotin-labeled apoE holoprotein can be found
in the cytoplasm and nuclei of Chinese hamster ovary cells (37).
Furthermore, apoE protein has also been detected in the cytoplasm
of neurons, astrocytes, and glial cells in human brain samples (38).
In the case of the apoE holoprotein, reports suggested that apoE
binds to LDLR and LRP and is taken up into cells by receptormediated endocytosis (2, 3). The apoE holoprotein is known to
bind to LRP and LDLR, and the regions of the apoE protein ligand
responsible for this finding are contained in the region from amino
acids 130–150 (10). Similarly, an apoE[130–149] mimetic peptide
that is 3 aa longer than the apoE[133–149] peptide used in these
studies, binds to the LRP receptor with high affinity (19) through
localized interactions between the complement repeat-17 region
of LRP and the apoE[130–149] ligand, which were recently defined at the molecular structure level (39). Taken together, these
data suggest that apoE and the apoE-mimetic peptides bind to
LRP or LDLR and become internalized through receptor-mediated
endocytosis (40). Although beyond the scope of the current work,
the observation that apoE[133–149] gains entry into BV2 cells,
which do not express either LDLR or LRP, suggests that other
related receptors (40) may also allow for the apoE-mimetic peptides to gain entry into cells through endocytosis.
In this paper, we report that apoE and apoE-mimetic peptides
bind to the SET protein, a potent physiological inhibitor of PP2A.
Following activation of inflammatory signaling pathways via
TLR-4 pathway stimulation with LPS, as the MAPK and NF-kB
pathways become activated, binding of apoE and apoE-mimetic
peptides to SET would be associated with an increased level of
PP2A-mediated phosphatase activity, as we have reported. This
increase in PP2A activity would account for the reduced levels of
phosphorylated p38-MAP kinase and Akt kinase, as well as reduced NO release. We also observed that knockdown of SET using
siRNA resulted in a decrease in NO production that was similar to
the effect of antagonizing SET with the apoE-mimetic peptide.
These combined data suggest a novel molecular mechanism by
which apoE and apoE-mimetic peptides positively modulate PP2A
activity through antagonism of SET binding, with a concomitant
reduction in inflammatory signaling due to decreased levels of
activating phosphorylated signaling kinases. This mechanism is
also consistent with previous reports that 1) both the apoE[130–
149] peptide and the apoE holoprotein bind to LRP (3, 19); 2)
apoE[133–149] can be detected inside cells (this paper), and the
apoE holoprotein has also been found inside cells (37); 3) both
apoE-mimetic peptides and apoE holoprotein inhibit production of
NO, TNF-a, and other cytokines (15, 16, 18); and 4) both apoEmimetic peptides and apoE holoprotein reduce NF-kB activation
through inhibition of IKK phosphorylation (20, 21). These data,
when coupled with the current demonstration that both the apoEmimetic peptides and the apoE holoprotein bind to SET, suggest
that the peptides are true mimetics of the apoE protein in receptor binding and SET binding activities. Furthermore, the molecular mechanism newly described in this paper, whereby apoE and
apoE-mimetic peptides antagonize SET, increase cellular PP2A
phosphatase activity, and decrease activation of inflammatory sig-
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FIGURE 7. SET silencing or ApoE-mimetic peptides downregulate
LPS-mediated NO release. A, siRNA-induced knockdown of SET was
accomplished by adding a pool of three siRNA sequences for SET or
a noncoding control siRNA. Following transfection of the siRNA, cells
were allowed to recover and were stimulated with LPS (100 ng/ml) in the
presence or absence of 5 mM COG1410, as indicated (n = 3). The production of NO was quantitated using the Greiss assay and plotted. *p ,
0.05. ns, not significant. B, Cells from A were lysed, pooled by treatment,
and the extracts were analyzed by Western blotting to quantify b-actin (as
a loading control), SET, and the catalytic C subunit of PP2A.
2541
2542
APOLIPOPROTEIN E AS A MODULATOR OF INFLAMMATORY SIGNALING
nal transduction pathways, may ultimately be a key for elucidation of the apoE genotype effects in human disease.
Disclosures
M.P.V., D.J.C., J.O., J.N., and F.L. are employees of Cognosci. M.P.V. owns
shares of and serves on the Board of Directors of Cognosci. C.A.C., D.J.C.,
and M.P.V. have been reviewed and approved by the Duke Conflict of Interest Committee.
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