Download Expression of the LXR Protein in Human Atherosclerotic Lesions

Expression of the LXR␣ Protein in Human
Atherosclerotic Lesions
Yuichiro Watanabe, Shuying Jiang, Wakako Takabe, Riuko Ohashi, Toshiya Tanaka,
Yasutoshi Uchiyama, Keiko Katsumi, Hiroko Iwanari, Noriko Noguchi, Makoto Naito,
Takao Hamakubo, Tatsuhiko Kodama
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Objective—Liver X–activated receptor ␣ (LXR␣) regulates multiple genes controlling cholesterol metabolism and
transport. To clarify its role in atherogenesis, we established a monoclonal antibody recognizing native human LXR␣
protein and studied the expression pattern in human atherosclerotic lesions.
Methods and Results—A novel monoclonal antibody PPZ0412 was raised against the ligand-binding domain of LXR␣,
which can be used for immunostaining of human LXR␣ protein. LXR␣ protein was detected in the nucleus of
macrophages in the liver, spleen, or lung and also in hepatocytes and adipocytes. In atherosclerotic lesions, the LXR␣
protein was detected in macrophages positive for scavenger receptor class A and/or CD68.
Conclusions—In the human body, the LXR␣ protein is highly expressed in macrophage lineage cells and foam cells in
atherosclerotic lesions and is identified as a target for intervention in atherosclerotic disease. (Arterioscler Thromb Vasc
Biol. 2005;25:622-627.)
Key Words: LXR␣ 䡲 atherosclerosis 䡲 macrophages 䡲 monoclonal antibody
iver X–activated receptor ␣ (LXR␣; NR1H3) is a member of the nuclear receptor superfamily that forms a
functional heterodimer with retinoid X receptors (RXRs).1,2
LXR␣/RXRs heterodimers bind to DR-4 –type sequence elements known as the LXR response element in their target
genes. LXR␣ is activated by oxidized derivatives of cholesterol (oxysterols), such as 22(R)-hydroxycholesterol, 24(S)hydroxycholesterol, or 24(S), 25-epoxycholesterol.3,4 In experimental animals, LXR␣ mRNA is abundantly expressed in
tissues that participate in lipid metabolism, such as white
adipose tissue, liver, intestine, and also in macrophages.5–7 In
the liver, where LXR␣ mRNA is highly expressed, activation
of LXR induces de novo fatty acid biosynthesis, which has
led to the suggestion that LXR␣ is a sensor of the balance
between cholesterol and fatty acid metabolism.8,9 In macrophages, LXR␣ induces its target genes, such as ABCA1,
ABCG1, and apolipoprotein (apo) E, which are involved in
cholesterol efflux.10 –12
The identification of LXR␣ as a potential cholesterol
sensor that governs cholesterol metabolism and transport
opens up new possibilities for intervention in the treatment of
atherosclerosis. One potential problem with an LXR␣ agonist
is that it upregulates fatty acid synthesis, resulting in hypertriglyceridemia.6 On the other hand, recent studies have
L
demonstrated that LXR may play an atheroprotective role.
Systemic administration of an LXR agonist reduced atherosclerosis in LDLR⫺/⫺ and apoE⫺/⫺ mice.13 Further, bone
marrow transplantation from LXR␣/␤⫺/⫺ mice increased
lesion formation in these same models.14
These results suggested that LXR␣ expressed in macrophages in atherosclerotic lesions may play a critical role in
atherosclerotic disease. Given the complexity of the LXR␣
effects, it is of great interest to identify the LXR␣ protein
expression pattern in the human atherosclerotic lesion. Such
studies to date have been hampered by the lack of antibodies
capable of detecting the native LXR␣ protein.
Previously, we reported the establishment of a monoclonal
antibody against the N-terminal domain of human LXR␣, and
we also reported the induction of endogenous human LXR␣
protein K-8607 during differentiation from monocytes to
macrophages.15 Unfortunately, the reported anti–N-terminal
antibody did not possess sufficient specificity to detect
endogenous LXR␣ protein on immunohistochemical analysis. Therefore, tissue distribution and the subcellular localization of the endogenous LXR ␣ protein remain
unelucidated.
To identify the endogenous LXR␣ protein in human
tissues, we established a novel monoclonal antibody against
Original received August 11, 2004; final version accepted November 24, 2004.
From the Department of Systems Biology and Medicine (Y.W., W.T., T.T., N.N., T.H., T.K.), Research Center for Advanced Science and Technology,
the University of Tokyo; the Immunology Research Department (Y.W.), Tokyo New Drug Research Laboratories II, Pharmaceutical Division, Kowa Co
Ltd; Perseus Proteomics Inc (S.J., Y.U., K.K., H.I.), Tokyo; and the Department of Cellular Function (S.J., R.O., M.N.), Division of Cellular and
Molecular Pathology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
Correspondence to Tatsuhiko Kodama, Laboratory for Systems Biology and Medicine at RCAST #34, The University of Tokyo, 4-6-1 Komaba,
Meguro, Tokyo 153-8904, Japan. E-mail [email protected]
© 2005 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
622
DOI: 10.1161/01.ATV.0000154489.53077.4e
Watanabe et al
LXR␣ Protein in Human Atherosclerotic Lesions
623
the human LXR␣ ligand-binding domain (LBD). This monoclonal antibody can specifically recognize endogenous human LXR␣ on immunoblot and immunohistochemical analyses. Using this antibody, we studied the expression of the
human LXR␣ protein both in normal human tissues and in
atherosclerotic lesions.
Methods
Expression of the LXR␣LBD and Monoclonal
Antibody Generation
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The LBD of the human LXR␣ (amino acids 164 to 447) was
expressed as a glutathione S-transferase (GST) fusion protein using
the expression vector pGEX4T-2 (Amersham Biosciences). Fusion
proteins were induced in BL-21 (Stratagene) and purified using
Glutathione Sepharose 4B (Amersham Biosciences). Recombinant
GST-LXR␣LBD was used for 3 cycles of immunization against
female BALB/c mice. Three days after the final administration, mice
were euthanized and lymphocytes from the spleen were isolated and
fused with NS-1 myeloma cells, as previously described.16 The fused
cells were cultured in HAT (0.1 mmol/L hypoxanthine, 0.1 mmol/L
aminopterin, and 0.16 mmol/L thymidine) selection medium for 10
to 14 days at 37°C to select for the surviving fusion clones.
Hybridomas were selected by ELISA against the purified recombinant GST-fused LXR␣LBD using tissue culture supernatant. Selected hybridoma clones were purified by limited dilution. For mass
production, 9 hybridoma clones were grown in mice ascites. Ascitic
fluids were collected and purified using ammonium sulfate.
Cell Culture and Oxidized Low-Density
Lipoprotein Preparation
Human primary monocytes/macrophages were obtained as previously described5 and maintained in RPMI 1640 medium supplemented with 10% FBS.
Human low-density lipoprotein (LDL) was isolated from the
plasma of healthy volunteers by the method of Goldstein et al.17,18
After dialysis with EDTA-free PBS, 1.7 mg/mL LDL were oxidized
with 100 ␮mol/L CuCl2 at 37°C for 18 hours.
Transient Transfection and Immunoblot Analysis
COS-7 cells were cultured in DMEM containing 10% FBS. Cells
were plated in a 100-mm dish at 2.0⫻106 cells per dish for 16 hours
before transfection. Transfections were performed with Effectene
Transfection Reagent (QIAGEN) using 2 ␮g of the pcDNA3-hLXR␣
expression vector.
Nuclear extracts were obtained as previously described.19 Aliquots
of each sample were resolved by SDS-PAGE (10%) and transferred
to polyvinylidene difluoride membranes (ProBlott, Applied Biosystems). After blocking the membranes with BlockAce (Dainippon
Pharmaceutical Co Ltd) for 3 hours at room temperature, immunoblotting was performed with an anti-LXR␣ antibody PPZ0412 (1
␮g/mL) as the primary antibody. Peroxidase-conjugated anti-mouse
IgG antibody (Sigma, St Louis, Mo) was used as the secondary
antibody, and SuperSignal West Dura Extended Duration Substrate
(Pierce) was used as the substrate for chemiluminescent detection.
As a control, we used a nuclear extract of transfected COS-7 cells
transfected with pcDNA3-LXR␣.
Immunoprecipitation
Immunoprecipitation study was performed as follows. COS-7 cells
transiently transfected with the FLAG-tag fusion human LXR␣
expression vector were used as source. Cells were scraped in
immunoprecipitation buffer (50 mmol/L Tris-HCl, pH 7.5,
10 mmol/L EDTA, 150 mmol/L NaCl, 10% glycerol, and 0.1%
NP-40) supplemented with protease inhibitors. The scraped cells
were lysed by freezing and thawing 3⫻. The anti-FLAG mouse
monoclonal antibody (Sigma; 20 ␮g/mL), control IgG (Sigma; 20
␮g/mL), or PPZ0412 (20 ␮g/mL) and protein G Sepharose (Amersham) were added to the lysate and mixed by rotating the tubes at
Figure 1. Establishment of monoclonal antibody against human
LXR␣ LBD. A, Purification of immunogen. Whole-cell lysates
from E coli transformed with GST-LXR␣-DBD or purified GSTLXR␣-DBD proteins were used. Lane 1 shows E coli after IPTG
induction; lane 2, E coli before IPTG induction; and lane 3, purified immunogen. B, Specificity of monoclonal antibody
PPZ0412. Whole-cell extract obtained from COS-7 cells transfected with human LXR␣ and LXR␤ (20 ␮g) were used. Lane 1,
Mock; lane 2, human LXR␣; and lane 3, human LXR␤. *Lowmolecular-weight protein.
4°C. The antibody-protein G Sepharose conjugate was collected by
centrifugation. After washing twice, the conjugates were resolved by
SDS-PAGE. To detect the FLAG-tagged fusion human LXR␣,
anti-FLAG antibody conjugated with horseradish peroxidase
(Sigma) was used.
Immunohistochemistry
Immunohistochemical analysis was performed as described previously.20 Human tissues were fixed for 1 day at room temperature in
10% formalin. The samples were sequentially dehydrated with an
alcohol series and embedded in paraffin. Antigen retrieval was
performed by heating the sections in an autoclave at 121°C for 15
minutes. During heating, the sections were immersed in 0.1 N citrate
buffer solution (pH 6.5). The paraffin sections (4 ␮m thick) were
then treated with normal horse serum to minimize nonspecific
staining. These tissues were incubated with a monoclonal antibody
against LXR␣ (PPZ0412) dissolved in 1% BSA/PBS at a final
concentration of 10 ␮g/mL for 2 hours at 25°C. After several washes
with PBS, the sections were stained with a secondary antibody
(Simple Stain MAX-PO, Nichirei, Tokyo, Japan) for 1 hour. To
prevent endogenous peroxidase reactions, the samples were pretreated with 0.3% H2O2 in cold methanol for 30 minutes. Finally, 0.1
mg/mL of 3, 3⬘-diaminobenzidine tetrahydrochloride was applied to
sections for 10 minutes. The sections were counterstained with
hematoxylin. Cultured cells were fixed in 4% paraformaldehyde and
immunostained by the same method as above.
In separate sets of experiments, sections of the human aorta were
double-stained with the anti-CD68 or anti–scavenger receptor class
A (SR-A) antibodies. In brief, the sections were immunostained
using the first primary monoclonal antibody and diaminobenzidine
as described above. After a wash with glycine/HCl buffer for 1 hour,
the sections were incubated with a second primary antibody and the
Vectastain ABC-PO substrate kit (Vector).21
Results
Generation of Monoclonal Antibody
Against LXR␣LBD
Figure 1A indicates the expression of GST-LXR␣LBD in
Escherichia coli (E coli). The expression of this fusion
protein was induced in E coli by isopropyl ␤ - D thiogalactoside (IPTG; lanes 1 and 2). The induced GST
fusion protein was purified using Glutathione Sepharose 4B
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Figure 2. Detection of endogenous human LXR␣ protein
expressed in the monocyte-derived macrophage. A, Whole-cell
extracts from COS-7 cells transfected with human LXR␣ (5 ␮g)
and human monocytes (50 ␮g) and macrophages (50 ␮g) were
used. Lane 1 shows LXR␣; lane 2, Monocyte; and lane 3, Macrophages. *Low-molecular-weight protein. B, Influence of oxidized LDL (100 ␮g/mL) addition on the LXR␣ protein expression
in macrophages.
(lane 3). The monoclonal antibody PPZ0412 was raised
against this purified GST fusion protein.
Figure 1B indicates the result of immunoblot study with
PPZ0412 using protein obtained from COS-7 cells transfected with human LXR␣ or LXR␤ expression vectors. The
antibody bound specifically to proteins expressed in COS-7
cells transfected with the LXR␣ expression vector. PPZ0412
recognized a protein of apparent molecular weight (Mr) of 47
kDa. Additional proteins with an apparent Mr of 40 kDa were
also detected only in cells transfected with the LXR␣ expression vector. This monoclonal antibody did not bind to the
LXR␤ protein, which has an amino acid sequence highly
similar to LXR␣.
Figure IA (available online at http://atvb.ahajournals.org)
indicates the result of immunoblot study with PPZ0412 using
protein obtained from COS-7 cells transfected with human
LXR␣ or FLAG-tagged human LXR␣ expression vectors.
PPZ0412 recognized a protein of apparent Mr of 47 kDa or 50
kDa. Additional proteins were detected in cells transfected
with the human LXR␣ expression vector or FLAG-tagged
human LXR␣ expression vector. Figure IB indicates the
result of immunoblot study with anti-FLAG antibody using
same protein. The protein of apparent Mr of 50 kDa was
detected using anti-FLAG antibody. There were no additional
bands.
Detection of Native Human LXR␣ Protein in
Human Monocyte-Derived Macrophages by the
Monoclonal Antibody PPZ0412
Figure 2A indicates the results of immunoblotting using
whole-cell extracts obtained from human monocytes and
macrophages. The monoclonal antibody PPZ0412 bound to
the 47-kDa protein in human monocyte-derived macrophages. The apparent molecular weight of this protein is
equal to that of the human LXR␣ protein expressed in COS-7
cells. An additional minor band of ⬇40 kDa was also
detected. As can be seen in Figure 2B, we could not detect
Figure 3. Immunoprecipitation of human LXR␣ using PPZ0412.
Whole-cell extract from COS-7 cells transfected with FLAGhLXR␣ (250 ␮g) was used for immunoprecipitation analysis. For
SDS-PAGE electrophoresis, 5% of the input fraction or the
supernatant fraction was used. After resolution with SDS-PAGE,
anti-FLAG antibody conjugated with horseradish peroxidase
was used for detection.
significant effect of oxidation on the LXR␣ protein amount in
macrophages.
Immunoprecipitation of the Human LXR␣ Protein
Figure 3 indicates the result of immunoprecipitation studies
using the LXR␣ protein tagged for the FLAG epitope at the
N-terminal domain expressed in COS-7 cells. This FLAGtagged protein was recognized by anti-FLAG antibody and
was immunoprecipitated. PPZ0412 also bound to this protein,
and the tagged LXR␣ protein was precipitated efficiently.
Control IgG was unable to bind to this protein. This indicates
the anti-FLAG antibody and PPZ0412 are able to specifically
recognize the FLAG-tag fusion human LXR␣.
Immunohistochemical Study of the Human Liver
We initially studied the localization of the endogenous LXR␣
protein in the human liver, because earlier studies by Northern blotting and RT-PCR have reported relatively abundant
expression of LXR␣ mRNA here. PPZ0412 recognizes the
protein in the nucleus of Kupffer cells and hepatocytes. As
can be seen in Figure 4C, the staining in the nucleus of
Kupffer cells (arrow heads) is more prominent than that in
hepatocytes. Control IgG did not exhibit significant binding.
This result is consistent with the reported expression pattern
of LXR␣ mRNA in the liver.
Expression of the LXR␣ Protein in Human Organs
Figure 5 depicts LXR␣ protein expression in human lung,
spleen, thymus, and adipose tissue. In the lung, the LXR␣
protein was detected in the nucleus of alveolar macrophages.
In the spleen and thymus, it was also detected in macrophagelike cells. These LXR␣-positive cells were also positive for
CD68, a marker for macrophage lineage cells (data not
shown). However, the expression of LXR␣ was not limited to
cells of macrophage lineage. In adipose tissue, LXR␣ was
Watanabe et al
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Figure 4. Immunohistochemistry of human liver using PPZ0412.
Paraffin sections were stained with PPZ0412. A positive reaction
(brown) is seen in the nucleus of Kupffer cells and hepatocytes
(A and C). No positive reaction is observable when normal
serum was used as the first antibody (B and D). Arrow heads
indicate nucleus of Kupffer cells. Original magnification: A and
B, ⫻200; C and D, ⫻400.
positive in the nucleus of adipocytes, which were negative for
CD68.
Immunohistochemical Study of Human
Atherosclerotic Lesions
To clarify the distribution of the LXR␣ protein in human
atherosclerotic lesions, we examined the lesioned aorta of
human subjects. As can be seen in Figure 6A and 6D, in
human plaque lesions the LXR␣ protein was mainly detected
in the nucleus of mononuclear cells and foam cells. LXR␣positive cells were not detected in normal aorta (Figure 6C
and 6F), suggesting that these LXR␣-positive mononuclear
cells were infiltrating during lesion formation. As can be seen
in Figure 6B and 6E, in advanced lesions the number of
LXR␣ protein–positive cells was decreased because of the
decrease of cellularity.
To identify the cell type of LXR␣ protein–positive cell, we
also stained the lesion with anti–SR-A and anti-CD68 antibodies. Figure 6G indicates that the LXR␣ and SR-A proteins
were detected in the same cells. The SR-A protein (purple)
was mainly detected in association with the cell membrane,
and the LXR␣ protein (brown) was detected mainly in the
nucleus. Figure 6H indicates the results of immunostaining
LXR␣ Protein in Human Atherosclerotic Lesions
625
Figure 6. Immunohistochemistry of human atherosclerotic
lesion. A through F, Paraffin sections were stained with
PPZ0412. A positive reaction (brown) is seen in the nucleus of
infiltrated cells (A and D). In advanced lesions, the number of
LXR␣ protein–positive cells decreased (B and E). No positive
reaction was seen in normal arteries (C and F). G, Sections were
stained with PPZ0412 (brown) and anti–SR-A (purple). The LXR␣
and SR-A proteins were detected in the same cells. H, Sections
were stained with anti-CD68 (dark purple) and PPZ0412 (brown).
Original magnification: A and B, ⫻100; C, D, and E, ⫻200; F, G,
and H, ⫻400.
for another macrophage lineage marker CD68 (dark purple)
and the LXR␣ protein (brown). CD68 was detected mainly in
the cytoplasm, and the LXR␣ protein was detected in the
nucleus. These results indicate that the LXR␣ protein is
expressed in macrophage lineage cells in various stages of
atherosclerotic lesions.
Discussion
In this study, we established a novel anti-LXR␣ monoclonal
antibody PPZ0412. The apparent molecular weight of the
protein detected by PPZ0412 (47 kDa) is in good agreement
with the expected molecular weight deduced from its reported
cDNA sequence (447 amino acids). This antibody can be
used for immunoblotting, immunoprecipitation, and also
immunohistochemistry for endogenous or overexpressed human LXR␣ protein in various cells.
PPZ0412 did not cross-react with the LXR␤ protein,
although the amino acid sequence of the LXR␤ protein is
closely similar to that of LXR␣. One of the problems we
Figure 5. Immunohistochemical localization of human LXR␣ protein in tissues.
Paraffin sections were stained with
PPZ0412. A positive reaction (brown) is
seen in the nucleus of macrophages in
lung (A and E), spleen (B and F), and
thymus (C and G). A weak positive reaction is also observable in adipocytes in
adipose tissue (D and H). Original magnification: A, B, C, and D, ⫻200; E, F, G,
and H, ⫻400
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found during these experiments was the recognition of the
lower-molecular-weight protein by PPZ0412. This lowermolecular-weight protein (* in Figure 1B, * in Figure IA, and
* in Figure 2A, lane 3) was detected only in cells expressing
the LXR␣ protein, and it was originally considered to be a
degradation product. The result of immunoblot study using
FLAG-tagged human LXR␣ (Figure I) supports the hypothesis, because an anti-FLAG antibody did not recognize the
lower-molecular-weight protein. The FLAG epitope was
fused to the LXR␣ protein at N-terminal domain, and if
degradation products were generated by N-terminal truncation, the FLAG antibody could not recognize the lowmolecular-weight degradation product. Recently, the presence of a splicing variant of LXR␣ mRNA was also reported.
It is an open question whether this protein was a degraded
LXR␣ protein or a splicing variant or characterized by
nonspecific binding. Further analysis will be needed to
characterize this additional immunoreactive protein. It was
found that PPZ0412 is able to specifically recognize the
endogenous human LXR␣ protein.
The expression pattern of the LXR␣ protein in human
tissue is consistent with the previously reported profile of
mRNA expression in both human tissue and experimental
animals.5–7 Using PPZ0412, LXR␣ protein was detected in
the Kupffer cells of the liver, alveolar macrophages in the
lung, and in other macrophages resident in the thymus and
spleen. LXR␣-positive cells were also positive for CD68, a
well-studied marker for macrophage lineage cells. This result
clearly indicates that major cell types expressing the LXR␣
protein in the human body are macrophage lineage cells.
Previously, Kohro et al reported that LXR␣ mRNA is the
most highly induced transcriptional regulator during differentiation from human primary culture monocytes to macrophages with M-colony–stimulating factor or granulocyte/
macrophage colony–stimulating factor.5 LXR␣ mRNA in
human macrophage lineage cells was far higher than that
found in the liver or other organs. These results provide
evidence that macrophage lineage cells in various human
organs are positive for the LXR␣ protein under normal
physiological condition. Previously, induction of LXR␣
mRNA by addition of oxidized LDL was reported in THP-1
cells or mouse macrophages,12 and we have reported that
LXR␣ gene is activated during macrophage differentiation
without further stimulation in human monocyte-derived macrophages.5,15 To examine the effect of oxidized LDL, we
investigated LXR␣ protein expression in macrophages. As
depicted in Figure 2B, we could not detect the significant
increase of LXR␣ protein by addition of oxidized LDL.
In addition to its detected presence in macrophage lineage
cells, LXR␣ protein was also detected in the hepatocytes and
adipocytes. Both of these cell types are actively involved in
the metabolism, transport, and storage of lipids. The intensity
of immunostaining was weaker in both hepatocytes and
adipocytes than in macrophage lineage cells. Recently, Seo et
al reported that treatment with LXR agonist enhanced adipocyte differentiation from primary human stromal vascular
cells obtained from subcutaneous adipose tissue. Treatment
of these cells with a synthetic LXR agonist resulted in
markedly enhanced adipocyte differentiation.22 LXR␣ plays a
role in the execution of adipocyte differentiation by regulation of both lipogenesis and adipocyte-specific gene
expression.
The result of immunohistochemical study indicated that
LXR␣ is expressed in macrophages present in atherosclerotic
lesions. Cells in the lesion expressing LXR␣ were also
positive for SR-A, indicating that they are active for the
uptake of modified lipoprotein. The LXR signaling pathway
in atherosclerosis has an established role in atherosclerosis.13,14,23 Joseph et al have shown that treatment with a
synthetic LXR agonist GW3965 can reduce atherosclerotic
lesion development in 2 mouse models (ie, LDLR⫺/⫺ and
apoE⫺/⫺ mice).13 Terasaka et al have reported the effectiveness of another synthetic agonist T-0901317.23 Ligands for
RXR, an LXR-heterodimer partner, were also efficacious in
reducing atherosclerosis.24 Furthermore, bone marrow transplantation from LXR␣/␤⫺/⫺ mice increases lesion formation
in these same models.14 The abundant expression of LXR␣
protein in infiltrating macrophages supports the hypothesis
that LXR␣ agonists have a beneficial effect against development of atherosclerosis in the arterial wall. If LXR␣ proteins
were mainly located in the foam cells of atherosclerotic
lesions, the activation of LXR␣ might activate the expression
of ABC transporters and help eliminate accumulated cholesterol from the foam cells. Recently, Joseph et al and Fowler
et al reported the reciprocal regulation of inflammation and
lipid metabolism by LXR.25,26 These studies reported that
LXR agonists can inhibit macrophage inflammatory gene
expression. The presence of LXR␣ protein in infiltrating
macrophages indicates that they are primed to respond LXR␣
agonists. Highly expressed LXR␣ proteins in various human
tissues can respond to LXR␣ agonists, and may suppress the
progression of inflammatory reactions under a variety of
conditions. Further studies will be necessary to assess the
effectiveness of treatment specifically targeted to LXR␣
activation. The monoclonal anti-human LXR␣ antibody described in this study will be a powerful tool to help analyze
the precise expression, localization, and function of LXR␣ in
human physiology and pathology and will greatly facilitate
progress toward realizing the therapeutic potential suggested
by the ongoing work in this field.
Acknowledgments
This work is supported by a grant from joint research & development
projects with academic institutes and private companies. The authors
acknowledge C. Nagao, A. Kikuchi, and A. Izumi for their excellent
technical assistance and Dr Kevin Boru of Pacific Edit for review of
the manuscript. We thank Dr David J. Mangelsdorf of the Howard
Hughes Medical Institute at the University of Texas Southwestern
Medical Center for providing the human LXR␣ expression vector for
positive control.
References
1. Apfel R, Benbrook D, Lernhardt E, Ortiz MA, Salbert G, Pfahl M. A
novel orphan receptor specific for a subset of thyroid hormone-responsive
elements and its interaction with the retinoid/thyroid hormone receptor
subfamily. Mol Cell Biol. 1994;14:7025–7035.
2. Willy PJ, Umesono K, Ong ES, Evans RM, Heyman RA, Mangelsdorf
DJ. LXR, a nuclear receptor that defines a distinct retinoid response
pathway. Genes Dev. 1995;9:1033–1045.
3. Repa JJ, Mangelsdorf DJ. The liver X receptor gene team: potential new
players in atherosclerosis. Nat Med. 2002;8:1243–1248.
Watanabe et al
Downloaded from http://atvb.ahajournals.org/ by guest on August 3, 2017
4. Tontonoz P, Mangelsdorf DJ. Liver X receptor signaling pathways in
cardiovascular disease. Mol Endocrinol. 2003;17:985–993.
5. Kohro T, Nakajima T, Wada Y, Sugiyama A, Ishii M, Tsutsumi S,
Aburatani H, Imoto I, Inazawa J, Hamakubo T, Kodama T, Emi M. J
Atheroscler Thromb. Genomic structure and mapping of human orphan
receptor LXR alpha: upregulation of LXRa mRNA during monocyte to
macrophage differentiation. J Atheroscler Thromb. 2000;7:145–151.
6. Lu TT, Repa JJ, Mangelsdorf DJ. Orphan nuclear receptors as eLiXiRs
and FiXeRs of sterol metabolism. J Biol Chem. 2001;276:37735–37738.
7. Annicotte JS, Schoonjans K, Auwerx J. Expression of the liver X receptor
alpha and beta in embryonic and adult mice. Anat Rec A Discov Mol Cell
Evol Biol. 2004;277A:312–316.
8. Peet DJ, Turley SD, Ma W, Janowski BA, Lobaccaro JM, Hammer RE,
Mangelsdorf DJ. Cholesterol and bile acid metabolism are impaired in
mice lacking the nuclear oxysterol receptor LXR alpha. Cell. 1998;93:
693–704.
9. Repa JJ, Liang G, Ou J, Bashmakov Y, Lobaccaro JM, Shimomura I,
Shan B, Brown MS, Goldstein JL, Mangelsdorf DJ. Regulation of mouse
sterol regulatory element-binding protein-1c gene (SREBP-1c) by
oxysterol receptors, LXRalpha and LXRbeta. Genes Dev. 2000;14:
2819 –2830.
10. Repa JJ, Turley SD, Lobaccaro JA, Medina J, Li L, Lustig K, Shan B,
Heyman RA, Dietschy JM, Mangelsdorf DJ. Regulation of absorption and
ABC1-mediated efflux of cholesterol by RXR heterodimers. Science.
2000;289:1524 –1529.
11. Laffitte BA, Repa JJ, Joseph SB, Wilpitz DC, Kast HR, Mangelsdorf DJ,
Tontonoz P. LXRs control lipid-inducible expression of the apolipoprotein E gene in macrophages and adipocytes. Proc Natl Acad Sci
U S A. 2001;98:507–512.
12. Chawla A, Boisvert WA, Lee CH, Laffitte BA, Barak Y, Joseph SB, Liao
D, Nagy L, Edwards PA, Curtiss LK, Evans RM, Tontonoz P. A PPAR
gamma-LXR-ABCA1 pathway in macrophages is involved in cholesterol
efflux and atherogenesis. Mol Cell. 2001;7:161–171.
13. Joseph SB, McKilligin E, Pei L, Watson MA, Collins AR, Laffitte BA,
Chen M, Noh G, Goodman J, Hagger GN, Tran J, Tippin TK, Wang X,
Lusis AJ, Hsueh WA, Law RE, Collins JL, Willson TM, Tontonoz P.
Synthetic LXR ligand inhibits the development of atherosclerosis in mice.
Proc Natl Acad Sci U S A. 2002;99:7604 –7609.
14. Tangirala RK, Bischoff ED, Joseph SB, Wagner BL, Walczak R, Laffitte
BA, Daige CL, Thomas D, Heyman RA, Mangelsdorf DJ, Wang X, Lusis
AJ, Tontonoz P, Schulman IG. Identification of macrophage liver X
receptors as inhibitors of atherosclerosis. Proc Natl Acad Sci U S A.
2002;99:11896 –11901.
15. Watanabe Y, Tanaka T, Uchiyama Y, Takeno T, Izumi A, Yamashita H,
Kumakura J, Iwanari H, Shu-Ying J, Naito M, Mangelsdorf DJ,
Hamakubo T, Kodama T. Establishment of a monoclonal antibody for
LXR␣ Protein in Human Atherosclerotic Lesions
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
627
human LXRalpha: Detection of LXRalpha protein expression in human
macrophages. Nucl Recept. 2003;1:1.
Watanabe A, Hippo Y, Taniguchi H, Iwanari H, Yashiro M, Hirakawa K,
Kodama T, Aburatani H. An opposing view on WWOX protein function
as a tumor suppressor. Cancer Res. 2003;63:8629 – 8633.
Goldstein JL, Basu SK, Brown MS. Receptor-mediated endocytosis of
low-density lipoprotein in cultured cells. Methods Enzymol. 1983;98:
241–260.
Takabe W, Kanai Y, Chairoungdua A, Shibata N, Toi S, Kobayashi M,
Kodama T, Noguchi N. Lysophosphatidylcholine enhances cytokine production of endothelial cells via induction of L-type amino acid transporter
1 and cell surface antigen 4F2. Arterioscler Thromb Vasc Biol. 2004;24:
1640 –1645.
Caruccio L, Banerjee R. An efficient method for simultaneous isolation of
biologically active transcription factors and DNA. J Immunol Methods.
1999;230:1–10.
Jiang S, Tanaka T, Iwanari H, Hotta H, Yamashita H, Kumakura J,
Watanabe Y, Uchiyama Y, Aburatani H, Hamakubo T, Kodama T, Naito
M. Expression and localization of P1 promoter-driven hepatocyte nuclear
factor-4alpha (HNF4alpha) isoforms in human and rats. Nucl Recept.
2003;1:5.
Yamamoto T, Naito M, Moriyama H, Umezu H, Matsuo H, Kiwada H,
Arakawa M. Repopulation of murine Kupffer cells after intravenous
administration of liposome-encapsulated dichloromethylene diphosphonate. Am J Pathol. 1996;149:1271–1286.
Seo JB, Moon HM, Kim WS, Lee YS, Jeong HW, Yoo EJ, Ham J, Kang
H, Park MG, Steffensen KR, Stulnig TM, Gustafsson JA, Park SD, Kim
JB. Activated liver X receptors stimulate adipocyte differentiation
through induction of peroxisome proliferator-activated receptor gamma
expression. Mol Cell Biol. 2004;24:3430 –3444.
Terasaka N, Hiroshima A, Koieyama T, Ubukata N, Morikawa Y, Nakai
D, Inaba T. T-0901317, a synthetic liver X receptor ligand, inhibits
development of atherosclerosis in LDL receptor-deficient mice. FEBS
Lett. 2003;536:6 –11.
Claudel T, Leibowitz MD, Fievet C, Tailleux A, Wagner B, Repa JJ,
Torpier G, Lobaccaro JM, Paterniti JR, Mangelsdorf DJ, Heyman RA,
Auwerx J. Reduction of atherosclerosis in apolipoprotein E knockout
mice by activation of the retinoid X receptor. Proc Natl Acad Sci U S A.
2001;98:2610 –2615.
Joseph SB, Castrillo A, Laffitte BA, Mangelsdorf DJ, Tontonoz P.
Reciprocal regulation of inflammation and lipid metabolism by liver X
receptors. Nat Med. 2003;9:213–219.
Fowler AJ, Sheu MY, Schmuth M, Kao J, Fluhr JW, Rhein L, Collins JL,
Willson TM, Mangelsdorf DJ, Elias PM, Feingold KR. Liver X receptor
activators display anti-inflammatory activity in irritant and allergic
contact dermatitis models: liver-X-receptor-specific inhibition of inflammation and primary cytokine production. J Invest Dermatol. 2003;120:
246 –255.
Downloaded from http://atvb.ahajournals.org/ by guest on August 3, 2017
Expression of the LXRα Protein in Human Atherosclerotic Lesions
Yuichiro Watanabe, Shuying Jiang, Wakako Takabe, Riuko Ohashi, Toshiya Tanaka, Yasutoshi
Uchiyama, Keiko Katsumi, Hiroko Iwanari, Noriko Noguchi, Makoto Naito, Takao Hamakubo
and Tatsuhiko Kodama
Arterioscler Thromb Vasc Biol. 2005;25:622-627; originally published online December 29,
2004;
doi: 10.1161/01.ATV.0000154489.53077.4e
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