Expression of the LXR Protein in Human Atherosclerotic Lesions
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动脉硬化血栓血管生物学 2005年第3期
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 kodama@lsbm.org
Abstract
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.
We established the monoclonal antibody recognizing native human LXR protein PPZ0412 and studied the expression of LXR protein. In the human body, the LXR protein is highly expressed in macrophage lineage and atherosclerotic lesion foam cells and is identified as a target for intervention in atherosclerotic disease.
Key Words: LXR ? atherosclerosis ? macrophages ? monoclonal antibody
Introduction
Liver 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 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 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 LXRLBD and Monoclonal Antibody Generation
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-LXRLBD 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 LXRLBD 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 μmol/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 2.0x106 cells per dish for 16 hours before transfection. Transfections were performed with Effectene Transfection Reagent (QIAGEN) using 2 μg 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 μg/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 3x. The anti-FLAG mouse monoclonal antibody (Sigma; 20 μg/mL), control IgG (Sigma; 20 μg/mL), or PPZ0412 (20 μg/mL) and protein G Sepharose (Amersham) were added to the lysate and mixed by rotating the tubes at 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 μm 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 μg/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 LXRLBD
Figure 1A indicates the expression of GST-LXRLBD 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 (lane 3). The monoclonal antibody PPZ0412 was raised against this purified GST fusion protein.
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 GST-LXR-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 μg) were used. Lane 1, Mock; lane 2, human LXR; and lane 3, human LXR?. *Low-molecular-weight 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 significant effect of oxidation on the LXR protein amount in macrophages.
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 μg) and human monocytes (50 μg) and macrophages (50 μg) were used. Lane 1 shows LXR; lane 2, Monocyte; and lane 3, Macrophages. *Low-molecular-weight protein. B, Influence of oxidized LDL (100 μg/mL) addition on the LXR protein expression 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 FLAG-tagged 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.
Figure 3. Immunoprecipitation of human LXR using PPZ0412. Whole-cell extract from COS-7 cells transfected with FLAG-hLXR (250 μg) 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.
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.
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, x200; C and D, x400.
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 macrophage-like 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 positive in the nucleus of adipocytes, which were negative for CD68.
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, x200; E, F, G, and H, x400
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.
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, x100; C, D, and E, x200; F, G, and H, x400.
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 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 found during these experiments was the recognition of the lower-molecular-weight protein by PPZ0412. This lower-molecular-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 low-molecular-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.
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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 kodama@lsbm.org
Abstract
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.
We established the monoclonal antibody recognizing native human LXR protein PPZ0412 and studied the expression of LXR protein. In the human body, the LXR protein is highly expressed in macrophage lineage and atherosclerotic lesion foam cells and is identified as a target for intervention in atherosclerotic disease.
Key Words: LXR ? atherosclerosis ? macrophages ? monoclonal antibody
Introduction
Liver 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 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 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 LXRLBD and Monoclonal Antibody Generation
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-LXRLBD 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 LXRLBD 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 μmol/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 2.0x106 cells per dish for 16 hours before transfection. Transfections were performed with Effectene Transfection Reagent (QIAGEN) using 2 μg 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 μg/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 3x. The anti-FLAG mouse monoclonal antibody (Sigma; 20 μg/mL), control IgG (Sigma; 20 μg/mL), or PPZ0412 (20 μg/mL) and protein G Sepharose (Amersham) were added to the lysate and mixed by rotating the tubes at 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 μm 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 μg/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 LXRLBD
Figure 1A indicates the expression of GST-LXRLBD 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 (lane 3). The monoclonal antibody PPZ0412 was raised against this purified GST fusion protein.
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 GST-LXR-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 μg) were used. Lane 1, Mock; lane 2, human LXR; and lane 3, human LXR?. *Low-molecular-weight 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 significant effect of oxidation on the LXR protein amount in macrophages.
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 μg) and human monocytes (50 μg) and macrophages (50 μg) were used. Lane 1 shows LXR; lane 2, Monocyte; and lane 3, Macrophages. *Low-molecular-weight protein. B, Influence of oxidized LDL (100 μg/mL) addition on the LXR protein expression 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 FLAG-tagged 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.
Figure 3. Immunoprecipitation of human LXR using PPZ0412. Whole-cell extract from COS-7 cells transfected with FLAG-hLXR (250 μg) 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.
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.
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, x200; C and D, x400.
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 macrophage-like 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 positive in the nucleus of adipocytes, which were negative for CD68.
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, x200; E, F, G, and H, x400
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.
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, x100; C, D, and E, x200; F, G, and H, x400.
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 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 found during these experiments was the recognition of the lower-molecular-weight protein by PPZ0412. This lower-molecular-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 low-molecular-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.
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