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doi:10.1369/jhc.6A7120.2007
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Volume 55 (6): 641-649, 2007
Copyright ©The Histochemical Society, Inc.
Expression of Liver X Receptor 
in Rat Fetal Tissues at Different Developmental Stages
Division of Cellular and Molecular Pathology, Department of Cellular Function, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan (AS,TKawasaki,TKazawa,RO,SJ,MN); Perseus Proteomics, Inc., Tokyo, Japan (SJ,HI); and Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan (TM,TT,TH,JS,TKodama)
Correspondence to: Makoto Naito, MD, PhD, Division of Cellular and Molecular Pathology, Department of Cellular Function, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori 1-757, 951-8510, Niigata, Japan. E-mail: mnaito{at}med.niigata-u.ac.jp
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Summary |
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 Top Summary IntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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The liver X receptor (LXR) is a nuclear receptor that acts as a sterol sensor and metabolic regulator of cholesterol and lipid homeostasis. Using a novel LXR
-specific antibody for immunohistochemistry, we evaluated cellular expression of LXR in fetal rat tissues. In the fetal liver, LXR-positive macrophages appeared at 12 days and their number peaked at 18 days of gestation. In contrast, hepatocytes expressed LXR during the later stage of gestation, suggesting the functional development of the liver during ontogeny. Later, macrophages in spleen and thymus expressed LXR, and some mononuclear cells in the vascular lumen compatible to primitive/fetal macrophages in the fetal circulation were found to express LXR. In vitro, rat monocytes did not express LXR, but monocyte-derived macrophages cultured in the presence of macrophage-colony stimulating factor revealed the distinct expression of LXR in nucleoli. These findings suggest that LXR plays a role in the differentiation of fetal macrophages, particularly hepatic macrophages, in rat development. (J Histochem Cytochem 55:641–649, 2007)
Key Words: LXR • development • liver • macrophage • rat • nucleoli
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Introduction |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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LIVER X RECEPTORS (LXR) are nuclear receptors initially identified in the liver and that form a functional heterodimer with the retinoid X receptor (RXR) to recognize and bind to its hormone response elements (Apfel et al. 1994
; Willy et al. 1995). LXRs are activated by a number of oxidized derivatives of cholesterol (oxysterols) (Peet et al. 1998a; Edwards et al. 2002). LXR is expressed in the liver, intestine, adipose tissue, and in macrophages, whereas LXRß is expressed in most tissues (Kohro et al. 2000; Lu et al. 2001; Annicotte et al. 2004; Watanabe et al. 2005). LXR null mice display severe abnormalities in lipid and bile acid homeostasis (Peet et al. 1998b; Edwards et al. 2002). These studies imply that the role of LXR is as a sterol sensor and metabolic regulator of cholesterol and lipid homeostasis. Recent studies have established that reciprocal regulation of inflammation and lipid metabolism in macrophages through LXR plays a critical role in atherosclerotic lesions (Tangirala et al. 2002; Joseph et al. 2003). High levels of LXR mRNA have been shown in human monocyte-derived macrophages (Kohro et al. 2000; Whitney et al. 2001; Watanabe et al. 2005). LXR is also a direct target gene of LXR and LXRß in human macrophages and enhances its own expression (Whitney et al. 2001). These macrophage-specific expressions of LXR are suggested to prevent the transformation of macrophages into foam cells by amplifying the process of reverse cholesterol transport (Whitney et al. 2001; Tangirala et al. 2002; Joseph et al. 2003). However, little information is available about the expression of LXR in the development and differentiation of macrophages during rat development.
Using a newly produced antibody, we previously demonstrated that LXR protein was expressed in hepatocytes, adipocytes, and macrophages in human tissues as well as in foam cells in atherosclerotic lesions (Watanabe et al. 2005). In this study we examined the cellular expression of LXR in fetal rat tissues involved in lipid metabolism, such as a yolk sac and liver and tissue macrophages. We also investigated expression of LXR and nuclear localization in monocyte-derived macrophages in vitro.
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Materials and Methods |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Plasmid and Transient Transfection
Rat LXR
expression vector was constructed using cDNAs obtained from reverse transcription polymerase chain reaction (RT-PCR) of rat insulinoma INS-1E cells poly(A)+ RNA. Primers for full-length rLXR (nucleotides 22–1363, GenBank Accession Number NM031627) were as follows: 5'-GGGGTACCGAGATGTCCTTGTGGCTG-3' and 5'-GGGGATCCGTCATTCGTGGACATCCCAG-3'. PCR-amplified products were digested and inserted into the KpnI and BamHI site of pcDNA3 vector (Invitrogen; Carlsbad, CA). CHO cells were plated in 10 cm2 plates at 5 x 105 cells 18 hr prior to transfection. Transfections were performed with TransIT LT-1 transfection reagent (Mirus; Madison, WI) using 8 µg of pcDNA3-rLXR expression vector per plate. Nuclear extracts for immunoblotting were prepared as described previously (Sakai et al. 1996).
Animals
Wistar rats (260–300 g) were obtained from Charles River Inc. (Tokyo, Japan) and maintained under standard conditions at the Laboratory Animal Center of Niigata University School of Medicine. All animals were allowed free access to laboratory chow and tap water. Rats were mated overnight. The day when vaginal plug formation was detected was counted as day 1. Pregnant or newborn rats were killed the same day by ether anesthesia. Fetal tissues, including the liver and yolk sac from 11 days of gestation (E11) to E20 and rat tissues at 1, 14, and 56 days after birth, were obtained for immunohistochemical (IHC) staining. The tissue sample of liver for real-time PCR was obtained from rats at E14, 16, 18, and 56 days after birth. For immunoblotting, tissue samples of heart, lung, liver, spleen, and kidney were obtained from rats at 56 days after birth. For the culture of peripheral blood mononuclear cells (PBMCs), peripheral blood from rats at 56 days after birth was also collected from the aorta.
Cell Preparation and Culture
PBMC preparation and cell culture were previously described (Liu et al. 2005). Briefly, PBMCs were isolated by Lymphoprep (Axis Shild; Oslo, Norway). PBMCs were suspended at a concentration of 1 x 106 cells/ml in RPMI 1640 medium (Sigma-Aldrich; Irvine, UK) without serum and placed in Lab-Tek multiwell tissue culture chambers (Nagle Nunc International; Naperville, IL). After a 2-hr incubation at 37C, non-adherent cells were removed and RPMI 1640 medium with 10% fetal bovine serum (FBS; JRH Biosciences, Lenexa, KS) containing 1000 U/ml of macrophage-colony stimulating factor (kindly provided by Morinaga Milk Co.; Kanagawa, Japan) was added. For IHC staining, the 2-hr incubated cells and 7-day cultured cells were washed with PBS, fixed with cold acetone for 30 min, and prepared with 0.5% Triton X-100 for 15 min. May-Giemsa staining was performed for morphological examination of cultured cells. The 2-hr incubated cells and 7-day cultured cells were collected for real-time PCR and immunoblotting.
Antibodies
Mouse anti-human monoclonal antibody LXR (PPZ0412) was purchased from Perseus Proteomics Inc. (Tokyo, Japan) (Watanabe et al. 2005). A mouse anti-rat monocyte/macrophage and dendritic cell monoclonal antibody ED1 was purchased from BMA Biomedicals (Augst, Switzerland). Mouse monoclonal antibody for class A macrophage scavenger receptor (MSR-A) was kindly provided by Prof. M. Takeya (Second Department of Pathology, Kumamoto University School of Medicine). Chicken monoclonal antibody for actin (JLA20) was purchased from Oncogene Sciences (Uniondale, NY). Anti-LXR, anti-ED1, and anti-MSR-A antibodies were used for IHC at dilutions of 1:100, 1:500, and no dilution, respectively. Anti-LXR and anti-actin antibodies were used for immunoblotting at dilutions of 1:1000 and 1:2000, respectively.
IHC
Tissues were fixed with 10% formalin and embedded in paraffin. To retrieve antigens, specimens were deparaffinized and heated in sodium citrate buffer, pH 6.0, for 15 min at 120C. Endogenous peroxidase activity was quenched using 3% H2O2–methanol for 15 min, and then the specimens were blocked with 10% normal goat serum before overnight incubation with the primary antibodies at 4C. As the second step, peroxidase (PO)-conjugated goat anti-mouse immunoglobulin (F[ab']2; Nichirei Corporation, Tokyo, Japan) was reacted for 60 min. They were carefully washed three times with PBS in each step. To visualize PO activity, 3,3'-diaminobenzidine (DAB; Dojindo Laboratories, Kumamono, Japan) was used as the substrate in 0.05 M Tris–HCl buffer (pH 7.6) containing 0.01% H2O2. Nuclear staining was performed with hematoxylin. IHC staining for the culture cells was the same as above except for retrieving antigens. IHC double staining between LXR and ED1 was performed to investigate whether LXR-positive cells were tissue macrophages. Briefly, the first reaction was performed using anti-LXR antibody and the specimens were visualized as brown with DAB; then, the specimens were incubated at 4C overnight with ED1. They were visualized as blue with 4-chloro-1-naphthol. IHC staining patterns were examined using samples collected from three individual rats. The numbers of LXR-, ED1-, and MSR-A-positive cells with nuclei per mm2 were counted using a micrometer.
Immunoblotting
Immunoblotting was performed as described previously (Saito et al. 2005). Briefly, samples were lysed in a solution containing 150 mM NaCl, 50 mM Tris–HCl (pH 8.0), 5 mM EDTA, 1% Triton X-100 and 1 mM PMSF, 1 mM leupeptin, 1 mM aprotinin for 30 min on ice and centrifuged at 12,000 x g at 4C for 30 min. The extracted protein was resolved by SDS-PAGE. After electrophoresis, the protein was transferred to PVDF membrane (Amersham; Aylesbury, UK). Blots were pretreated with 5% skim milk overnight and then incubated with anti-LXR and anti-actin for 1 hr. They were then incubated with secondary anti-mouse IgG horseradish peroxidase-conjugated antibody (Amersham) diluted 1:2000 for 30 min, washed three times with PBS between each step of the procedure, and visualized with the ECL detection system (Amersham) according to the manufacturer's instruction.
Isolation of RNA and Analysis of mRNA
Total RNA was isolated from the liver tissues and cultured cells using the method of acid guanidinium–phenol–chloroform. Of the total RNA, 1 µg was mixed with 1X RT buffer (Invitrogen), 0.01 M of dithiothreitol (Invitrogen), 20 U of RNase inhibitor (Promega), 500 µM of deoxynucleoside triphoshates (dNTPs; Pharmacia, Uppsala, Sweden), 1 µg of random primer (Promega), 100 U of reverse transcriptase (Invitrogen), and distilled water to bring the final volume to 20 µl. The mixture was incubated at 42C for 60 min and then boiled at 95C for 3 min. Real-time PCR was performed using a chromo-4 four-color real-time PCR system (Bio-Rad; Hercules, CA) with SYBR Green technology, and the threshold cycle numbers were calculated using Opticon Monitor ver 3.1 (Bio-Rad). The reaction mixture consisted of 1 µl of sample cDNA, 1X iQ SYBR Green Supermix (Bio-Rad), 200 nM of each of the primers, and distilled water to bring to the final volume of 20 µl. PCR was carried out with the following primers described elsewhere: LXR (Hoekstra et al. 2003), forward: 5'-TCAGCATCTTCTCTGCAGACCGG-3', reverse: 5'-TCATTAGCATCCGTGGGAACA-3'; ß-actin (Watanabe et al. 2003), forward: 5'-TGGAATCCTGTGGCATCCATGAAAC-3', reverse: 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'. The protocol was 40 cycles of denaturation at 95C for 10 sec, annealing at 55C for 30 sec, and elongation at 70C for 30 sec. Reactions were performed in duplicate and threshold cycle numbers were averaged. ß-actin was used for normalizing expression data.
Immunofluorescence
Double immunofluorescence was described previously (Iguchi et al. 2005). Briefly, cells cultured for 7 days were washed with PBS, fixed with 4% paraformaldehyde for 5 min at 4C, prepared with 0.5% Triton X-100 for 5 min at 4C, and blocked with 10% goat serum for 30 min at room temperature. They were stained with an anti-nucleophosmin (Zymed; South San Francisco, CA) and an anti-LXR labeled with Zenon Alexa Fluor 488 labeling kit (Molecular Probes; Eugene, OR) at dilutions of 1:100 and 1:50, respectively. For nucleophosmin detection, Alexa Fluor 594 goat anti-mouse IgG (Molecular Probes) was used as a secondary antibody at a dilution of 1:2000. Control experiments were carried out by omitting the primary antibody. Specimens were counterstained with DAPI (Vector Laboratories; Burlingame, CA). Immunofluorescence was captured with a confocal laser-scanning microscope (LSM510META; Carl Zeiss, Jena, Germany).
Statistics
Three rats were examined for each procedure. Real-time PCR was carried out in duplicate. Data were expressed as the mean ± SD. Statistical analysis was carried out with SPSS version 11.0 (SPSS, Inc.; Chicago, IL). Correlation test between continuous quantitative and qualitative variables (t-test) was used to determine significances; p<0.05 was considered statistically significant.
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Results |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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LXR
-specific Antibody for Rat TissuesFigure 1A indicates the results of immunoblotting with PPZ0412 using protein obtained from CHO cells transfected with the rat LXR
expression vector. The antibody bound specifically to the rat LXR protein with an apparent molecular mass of 47 kDa. Amino acid sequences of human LXR protein are highly similar to rat LXR, which has higher homology than human LXRß. The antibody recognized the human LXR ligand-binding domain (LBD), and the amino acid sequence is almost the same. The antibody did not cross-react with the rat LXRß protein (data not shown). These findings suggested that the antibody is specific for rat LXR protein.
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Immunoblotting revealed a distinct band corresponding to 47 kDa in the tissue samples of lung, liver, and spleen taken from adult rats. No distinct band was detected in the heart and kidney (Figure 1B). Immunohistochemically, expression of LXR
in adult rat tissue was clearly found in the nuclei of hepatocytes (Figure 2A
), Kupffer cells, alveolar macrophages, and macrophages in the spleen (Table 1
). Macrophages were confirmed by the IHC double staining using anti-LXR and anti-ED1 antibodies (Figure 2B). LXR staining pattern was homogeneous in the nuclei. Kupffer cells showed higher intensity of LXR expression than hepatocytes (Figure 2A). LXR expression pattern of adult rat tissues was the same as that of human tissues, indicating that this antibody is available for IHC staining of rat tissues.
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Expression of LXR
in Fetal Macrophages and Other CellsLXR
expression was first detected in the endodermal layer of the yolk sac at E11, and LXR expression in the yolk sac was observed throughout the fetal period (Table 1). However, no LXR-positive cells were present in the stroma or in the vascular lumen of the yolk sac.
LXR-positive macrophages first appeared in the fetal liver at E12 (Figure 2C) (Table 1). LXR-positive hepatic macrophages increased with gestational days (Figure 2D). We compared expression of LXR, ED1, and MSR-A in hepatic macrophages (Figure 3A
). LXR- and ED1-positive cells showed a peak at E18 and E16, respectively. MSR-A-positive cells increased with time and were highest in the adult liver.
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Hepatocytes were negative in LXR
immunostaining until E16. LXR-positive hepatocytes appeared at E18 (Figure 2D) (Table 1). Nuclear staining of hepatocytes became prominent after birth, but the intensity of the nuclear staining of hepatocytes was much weaker than that of macrophages (Figures 2A and 2D). LXR mRNA level of the fetal liver was highest at E18 (Figure 3B). However, LXR mRNA level of the adult liver was much higher than that of the fetal liver because of the increased expression of LXR mRNA in hepatocytes.
Interestingly, LXR-positive mononuclear cells were found in the vascular lumen at E16 and E18 (Figure 4A
). In contrast, no LXR-positive mononuclear cells were detected in the vascular lumen after birth (Figure 4B). We have reported that primitive/fetal macrophages circulate in the peripheral blood in the early stage of the fetal period, whereas monocytes first appear in blood at late gestational days and increase with each fetal day (Takahashi et al. 1996). These findings suggested that the LXR-positive cells may be fetal macrophages in the fetal circulation.
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At 16 or 18 days of gestation, LXR
-positive macrophages were detected in the spleen and thymus (Table 1). Weakly positive expression of LXR was observed in the brown adipose tissue at 18 days of gestation (data not shown). After birth, LXR-positive macrophages were present in several tissues (Table 1) as reported previously (Watanabe et al. 2005).
Expression of LXR in Monocyte-derived Macrophages
To investigate the relationship between expression of LXR and macrophage differentiation, we cultured monocytes obtained from adult rats. Adherent PBMC monocytes incubated for 2 hr were small, round in shape, and with scant cytoplasm (Figure 5A
). They showed negative expression for LXR (Figure 5B) and MSR-A (Figure 5C). In contrast, cells cultured for 7 days were large and with abundant cytoplasm (Figure 5D). Most of them distinctly expressed both LXR (Figures 5E and 6A
) and MSR-A (Figure 5F). LXR expression was observed in nuclei as dots and MSR-A expression was predominantly observed on the cell membrane in these cells. LXR mRNA and protein levels were also paralleled with LXR expression in these cells (Figures 6B and 6C).
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Interestingly, immunofluorescence double staining showed a colocalization of LXR
and nucleophosmin (Figure 7
). Nucleophosmin, also known as numatrin, No38 and B23, is characterized as a nucleolar protein functioning in the processing and transport of rRNA (Borer et al. 1989). These findings suggested that LXR is localized in nucleoli.
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Discussion |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Using a newly produced antibody, the present study demonstrated that LXR
protein was detected in macrophage lineage cells as well as in endodermal cells of the yolk sac, hepatocytes, and adipose tissue during rat development.
Among the macrophage lineage cells, LXR protein expression was first confirmed in hepatic macrophages during rat development, followed by appearance of LXR-positive macrophages in other tissues. The number of LXR-positive hepatic macrophages showed a peak just before birth, and LXR mRNA levels in fetal liver were highest just before birth. Macrophages are abundant in fetal liver and actively phagocytize various blood cells (Naito et al. 1997), suggesting that hepatic macrophages may be most active in lipid metabolism or other functions among fetal tissue macrophages.
Kohro et al. (2000) reported that LXR mRNA is the most highly induced transcriptional regulator during differentiation from human primary culture monocytes to macrophages. We have shown that mRNA and protein expression of LXR in rat macrophages was much higher than those in monocytes in vitro. These results provide evidence that LXR expression is regulated by the differentiation and functional status of macrophages. Rat monocytes did not express LXR by IHC, whereas we have found LXR-expressing mononuclear cells in the vascular lumen of rat fetuses. Immature macrophages, called primitive macrophages, first appear in the yolk sac and differentiate into fetal macrophages (Takahashi et al. 1996; Naito et al. 1997; Watanabe et al. 2003). Although there were no LXR-positive cells in the yolk sac, LXR-positive cells were found in the vascular lumen of rat fetuses at E16 and E18. IHC staining also demonstrated not only LXR-positive cells, but also ED1-positive cells in the vessels (data not shown). These findings suggest that LXR-positive cells in the fetal circulation are not monocytes, but fetal macrophages.
The distribution of LXR in the nucleus was reported in the LXR-overexpressing cells (Morello et al. 2005). To define the intranuclear localization of LXR more precisely, we examined rat monocyte-derived macrophages by immunofluorescence microscopy. Although the nuclear staining pattern of LXR was homogeneous in tissue sections (Watanabe et al. 2005), a dot-like staining pattern was observed in cultured macrophages. Immunofluorescence double staining revealed the colocalization of LXR and nucleophosmin, the latter of which is identified as a nucleolar protein (Borer et al. 1989). It is known that nucleolar size affects the rapid cell proliferation in cancer cells (Derenzini et al. 2000). Lipopolysaccharide-stimulated macrophages revealed upregulation of nucleophosmin and prevented apoptosis (Zhang et al. 2006). Regulation of the PPAR-
gene through LXR is recognized to control cellular differentiation and apoptosis in a human promyelocytic cell line, HL-60 (Kaul and Anand 2003). These findings suggest that the intranucleolar localization of LXR is transcriptionally significant in macrophage differentiation.
The liver plays a significant role in lipid metabolism, and hepatocytes in adult animals are the major cell type expressing LXR. However, expresssion of LXR in hepatic macrophages preceeded that in hepatocytes in the fetal liver. It is reported that liver acyl-CoA cholesterol acyltransferase activity is low in the early stage of gestation (Ding and Lilburn 2000). LXR is involved in the regulation of genes contributing to hepatobiliary cholesterol and bile acid homeostasis, but nuclear receptors involved in bile formation are poorly developed during the fetal stage (Balasubramaniyan et al. 2005). This biochemical evidence coincides with LXR expression in hepatocytes in the late stage of the rat fetal period. LXR expression was first detected in endodermal cells of the yolk sac at E11. Because the yolk sac mediates the transfer of lipid from the yolk to the circulation (Ding and Lilburn 2000), LXR expression in the yolk sac may play a crucial role in lipid transport before the development of liver function.
In summary, using IHC and immunoblotting techniques we clarified LXR expression in fetal macrophages. In addition to lipid metabolism, LXR may play an important role in the differentiation of fetal macrophages, particularly hepatic macrophages.
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Acknowledgments |
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This study was supported by the Program of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, the Focus 21 project of the New Energy and Industrial Technology Development Organization, and the Special Coordination Fund for Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology (Japan).
We thank S. Momozaki, K. Ohyachi, and T. Aoyama (Division of Cellular and Molecular Pathology, Department of Cellular Function, Niigata University Graduate School of Medical and Dental Science) for their excellent technical assistance.
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Footnotes |
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Received for publication October 19, 2006; accepted February 7, 2007
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