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Molecular Endocrinology 20 (4): 786-794
Copyright © 2006 by The Endocrine Society

Regulation of Macrophage Inflammatory Gene Expression by the Orphan Nuclear Receptor Nur77

Liming Pei, Antonio Castrillo and Peter Tontonoz

Howard Hughes Medical Institute and Department of Pathology and Laboratory Medicine, University of California, Los Angeles, California 90095

Address all correspondence and requests for reprints to: Peter Tontonoz, M.D., Ph.D., Howard Hughes Medical Institute, University of California Los Angeles School of Medicine, Box 951662, Los Angeles, California 90095-1662. E-mail: ptontonoz{at}mednet.ucla.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Members of the nuclear hormone receptor superfamily have emerged as important regulators of macrophage gene expression in inflammation and disease. Previous studies have shown that the lipid-activated receptors peroxisomal proliferator-activated receptor and liver X receptor inhibit nuclear factor-{kappa}B (NF-{kappa}B) signaling and inflammatory gene expression. We recently identified the NR4A subfamily of orphan nuclear receptors (Nur77/NR4A1, Nurr1/NR4A2, and NOR1/NR4A3) as lipopolysaccharide- and NF-{kappa}B-responsive genes in macrophages. However, the role of these transcription factors in macrophage gene expression is unknown. We demonstrate here that, in contrast to peroxisomal proliferator-activated receptor and liver X receptor, the role of NR4A receptors in macrophages is proinflammatory. Retroviral expression of Nur77 in macrophages leads to the transcriptional activation of multiple genes involved in inflammation, apoptosis, and cell cycle control. One particularly interesting Nur77-responsive gene is the inducible kinase IKKi/IKK{epsilon}, an important component of the NF-{kappa}B signaling pathway. The IKKi promoter contains a functional NR4A binding site and is activated by all three NR4A receptors in transient transfection assays. Consistent with the activation of IKKi, expression of Nur77 in macrophages potentiates the induction of inflammatory gene expression in response to lipopolysaccharide. These results identify a new role for NR4A orphan nuclear receptors in the control of macrophage gene expression during inflammation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MEMBERS OF THE nuclear hormone receptor superfamily are now recognized to be key regulators of macrophage gene expression during inflammation and atherosclerosis. Both the peroxisomal proliferator-activated receptors (PPARs) and liver X receptors (LXRs) are established mediators of lipid metabolic and inflammatory gene expression in cells of the artery wall, including macrophages (1, 2). Lipid loading of macrophages leads to the transcriptional activation of PPARs and LXRs by providing the cell with oxidized fatty acid and oxysterol ligands, respectively (2, 3). The balance of evidence suggests that PPARs and LXRs are negative regulators of inflammatory gene expression in macrophages. Although the mechanisms involved remain to be defined, both PPAR and LXR have been reported to antagonize the nuclear factor-{kappa}B (NF-{kappa}B) signaling pathway and to inhibit induction of gene expression in response to lipopolysaccharide (LPS) (4, 5, 6). Consistent with this proposed antiinflammatory activity, genetic loss of either PPAR{gamma} or LXR expression from macrophages accelerates atherosclerosis in murine models (7, 8). In addition, a number of synthetic PPAR and LXR ligands have now been shown to inhibit lesion formation in mice (9, 10). A potential role for other nuclear receptors in promoting inflammatory gene expression in macrophages has not been adequately explored.

The orphan nuclear receptors Nur77 (NR4A1; also known as TR3 and NGFI-B), Nurr1 (NR4A2), and NOR1 (NR4A3) were initially characterized as growth factor-inducible genes (11, 12, 13). In contrast to receptors such as PPAR and LXR, structural studies suggest that these receptors do not bind small-molecule ligands (14, 15). Rather, the activity of NR4A receptors is controlled at the level of protein expression and posttranslational modification (16). Previous work has identified important functions for members of this subfamily in apoptosis in lymphocytes and other cell types (17, 18, 19) and the development of dopaminergic neurons (20). NR4A proteins have been reported to bind to the NBRE (NGFI-B response element) sequence (AAAGGTCA) as monomers (21) and to the palindromic NurRE (Nur77 response element) sequence (TGATATTTX6AAATGCCA) as homodimers (22). Nur77 and Nurr1, but not NOR1, can form heterodimers with retinoic acid receptor and may also have a role in retinoid signaling (23).

We have recently shown that expression of all three NR4A receptors is highly inducible in macrophages by diverse inflammatory stimuli including oxidized low-density lipoprotein (LDL) (24). Treatment of macrophages with LPS or proinflammatory cytokines triggers a rapid transcriptional induction of Nur77, Nurr1, and NOR1 expression through activation of NF-{kappa}B signaling. We also found that Nur77 is expressed in macrophages within human atherosclerotic lesions, suggesting a potential role for NR4A receptors as transcriptional mediators of inflammatory signals during atherogenesis. At present, however, the transcriptional targets of NR4A receptors in macrophages are unknown. Here we demonstrate that retroviral expression of Nur77 in macrophages leads to the up-regulation of a battery of genes involved in inflammation, apoptosis, and cell cycle control. We further show that the gene encoding the inducible kinase IKKi is a direct target for Nur77 and that expression of Nur77 potentiates the induction of inflammatory gene expression by Toll-like receptor (TLR)3 and TLR4 ligands. We propose that NR4A orphan receptors are downstream effectors of inflammatory signaling pathways in activated macrophages.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Previous studies have shown that macrophage expression of Nur77, Nurr1, and NOR1 is highly inducible by LPS and inflammatory cytokines (24); however, the targets of NR4A receptors in this cell type are unknown. We used a gain of function approach to identify potential target genes for NR4A receptors in macrophages. Stable RAW cell lines were derived that constitutively expressed Nur77, Nurr1, or NOR1 from a retroviral vector (see Materials and Methods). These stable cell lines express individual NR4A receptors to a level comparable to that induced by LPS (data not shown). The use of the retroviral transduction allows for very large pools of stable transfectants to be analyzed, minimizing the impact of clone-to-clone variation. RNA from cell lines expressing LacZ control (RAW-LacZ), Nur77 (RAW-Nur77), Nurr1 (RAW-Nurr1), or NOR1 (RAW-NOR1) was subjected to transcriptional profiling using Affymetrix murine 430A cDNA microarrays. Selected differentially expressed genes are shown in Table 1Go. The greatest number of induced genes was observed in RAW-Nur77 cells. Interestingly, many genes were up-regulated by two or more NR4A family members, suggesting that these receptors have a number of common target genes. However, we also identified genes that appeared to be specifically responsive to one NR4A receptor. Among the significantly induced genes were those implicated in inflammatory signaling (MARCKS, NIK, IKKi/IKK{epsilon}), cell cycle control (cyclin D2), and apoptosis (cathepsin E) (Table 1Go). For example, the most highly induced gene (39-fold by Nur77) in our microarray analysis was MARCKS, an essential protein and protein kinase C substrate that is involved in diverse processes including migration and phagocytosis in a variety of cell types (25). Two important kinases involved in inflammatory signaling, IKKi and NIK, were also induced by NR4A expression and will be discussed in detail below.


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  		Table 1. Microarray Analysis of Gene Expression in RAW Macrophages Overexpressing NR4A Receptors

 
Next we used real-time PCR to verify differential expression of a number of these candidate NR4A responsive genes in RAW-Nur77 macrophages (Fig. 1Go). As shown in Fig. 1AGo, MARCKS, cyclin D2, NIK, IKKi, and cathepsin E were significantly induced in RAW cells expressing Nur77 compared with those expressing empty vector. In contrast, expression of a control gene, cyclin E2, was not altered in response to Nur77 expression. Importantly, two independently derived pools of RAW-Nur77 cells gave qualitatively similar results (nos. 1 and 2, Fig. 1Go). To exclude the possibility that the NR4A responsiveness of these genes was peculiar to RAW cells, we also used retroviral vectors to establish J774 macrophages that expressed either empty vector, control protein [green fluorescent protein (GFP)], or Nur77. As shown in Fig. 1BGo, MARCKS, cyclin D2, NIK, IKKi, and cathepsin E showed a similar pattern of induction in two independently derived populations of J774-Nur77 cells.


Figure 1
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  		Fig. 1. Induction of Candidate NR4A Target Genes in RAW and J774 Macrophages Stably Expressing Nur77

A, RNA was isolated from control or RAW264.7 cells expressing Nur77 from a retroviral vector. Relative RNA expression of target genes in vector control and two independent RAW-Nur77 cell lines is shown. B, RNA was isolated from J774 cells expressing retroviral vector, GFP, or Nur77. Relative RNA expression of target genes in vector, GFP, and two independent Nur77-expressing cell lines is shown. Gene expression was measured by real-time PCR. Vect, Vector.

 
We further examined the time course of induction of these candidate target genes in macrophages using the TetOff inducible system. Stable J774 cell lines were derived that expressed GFP control or Nur77 under the control of a tetracycline (Tet)-dependent promoter (see Materials and Methods). Basal expression of Nur77 in J774-TetOff-Nur77 macrophages was approximately 10-fold higher than in GFP control cells, presumably due to incomplete repression by the TetOff system (Fig. 2Go). However, Nur77 mRNA expression was further induced after Tet removal from the cells. By contrast, expression of a control nuclear receptor, LXRß, did not change significantly in response to Tet removal. Remarkably, each of the candidate target genes was also induced after Tet removal, although the time course of induction was delayed compared with Nur77, consistent with a secondary response. Nur77 expression increased between 9 and 24 h after Tet removal, whereas MARCKS, cyclin D2, NIK, IKKi, and cathepsin E mRNA began to accumulate after 24 h. None of these genes were induced in control J774-TetOff-GFP cells, whereas the control gene GFP was abundantly expressed at 24 h, as monitored by fluorescence microscopy (Fig. 2Go and data not shown).


Figure 2
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  		Fig. 2. Time Course of Target Gene Induction in Macrophages Expressing Nur77 from an Inducible Promoter

J774-TetOff-GFP and J774-TetOff-Nur77 cells (see Materials and Methods) were cultured in 20 µg/ml Tet. To induce Nur77 expression, cells were washed thoroughly with PBS to remove Tet. Gene expression was analyzed by real-time PCR at the indicated time after Tet removal. dox, Doxycycline.

 
Next we investigated whether any of the candidate Nur77-responsive genes were direct targets for this transcription factor. Many Nur77-responsive genes revealed by our microarray analysis (Table 1Go) are known downstream of IKKi-IRF3-mediated antiviral pathways, including CXCL10/IP10, IFIT1, and others (26, 27, 28). This prompted us to focus on IKKi. DNA sequence analysis identified a potential NBRE at position –420 bp in the proximal promoter of the mouse IKKi gene (Fig. 3AGo). The ability of NR4A receptors to bind this sequence was tested in EMSAs. As shown in Fig. 3BGo, radiolabeled IKKi NBRE oligonucleotide was bound efficiently by in vitro translated Nur77, Nurr1, and NOR1 protein. This binding was sequence specific, as indicated by the ability of an excess of unlabeled oligonucleotide to compete for complex formation. By contrast, a mutated version of this NBRE was unable to bind NR4A proteins.


Figure 3
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  		Fig. 3. The IKKi Promoter Is a Direct Target for Nur77

A, Sequences of the NBRE and its flanking sequence (wild-type and mutated) at position –420 of the mouse IKKi promoter. B, Gel shift analysis showing in vitro translated Nur77, Nurr1, and NOR1 protein binding to the IKKi NBRE. Mut, Mutant; Comp, competitor; Ctrl, control.

 
We further tested the relevance of the NBRE sequence for regulation of the IKKi promoter in transient transfection assays. A luciferase reporter construct containing sequences from –1107 bp to +22 bp of the IKKi promoter was cotransfected into RAW cells along with expression vectors for Nur77, Nurr1, or NOR1. As shown in Fig. 4Go, all three NR4A family members transactivated the IKKi promoter. Furthermore, the NBRE sequence plays a critical role in inducible expression of the IKKi promoter, because introduction of a specific mutation in this sequence severely compromised the response to both NR4A receptors and LPS (Fig. 4Go). Together, the results of Figs. 3Go and 4Go establish the IKKi promoter as a direct target for NR4A transcription factors.


Figure 4
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  		Fig. 4. Transactivation of the IKKi Promoter by NR4A Receptors

RAW264.7 cells were transiently transfected with mouse IKKi promoter constructs, pCMV-ß-galactosidase, and either pCMV or pCMV-rNGFI-b or pCMV-mNurr1 or pCMV-mNOR1 expression vectors as described in Materials and Methods. Cells were treated, 24 h after transfection, with either PBS or LPS (500 ng/ml) for 18 h. Luciferase activity was normalized to ß-galactosidase activity. Data are means of triplicate wells ± SEM. mut, Mutant.

 
The ability of Nur77 to induce IKKi and NIK expression points to an unexpected role for NR4A nuclear receptors in macrophage-inflammatory responses. Each of these genes has previously been shown to play important roles in inflammatory signaling. IKKi is induced by LPS (29, 30) and has been shown to phosphorylate IRF3 and to mediate antiviral responses (26, 27). NIK was identified as a component of the NF-{kappa}B complex and has been reported to phosphorylate IKK proteins, thereby transducing signals from upstream stimuli (31, 32, 33). The ability of Nur77 to regulate these two genes suggests that induction of Nur77 may potentiate gene activation in response to inflammatory stimuli. To test this hypothesis, we compared the response of RAW-vector and RAW-Nur77 cells to both the TLR4 ligand LPS and the TLR3 ligand polydeoxyinosinic deoxycytidylic acid (polyI:C). Note that although individual stable clones can show significant variation in inflammatory responses, the use of large pools of retrovirally transduced cells minimizes the impact of such variation in our system. As shown in Fig. 5Go, A and B, induction of cyclin D2 and MARCKS in response to LPS or poly I:C was enhanced in RAW-Nur77 cells compared with RAW-vector cells. Moreover, expression of Nur77 also potentiated the induction of inflammatory genes such as Mx1, TNF{alpha}, and IP10 in response to LPS and/or polyI:C. These observations suggest that Nur77 can function as a proinflammatory mediator in activated macrophages.


Figure 5
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  		Fig. 5. Expression of Nur77 in Macrophages Potentiates the Response to TLR Ligand Stimulation

RAW-vector and RAW-Nur77 cells were treated with either TLR3 ligand poly I:C (poly IC) (1 µg/ml) or TLR4 ligand LPS (10 ng/ml) for the indicated time in media containing 1% FBS. Gene expression was analyzed by real-time PCR. IP-10, Interferon-inducible protein 10.

 
Finally, we examined expression of our putative NR4A target genes in macrophages derived from mice lacking Nur77 expression. As shown in supplemental Fig. 1Go (published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org), comparable levels of MARCKS, cyclin D2, and IKKi were observed in wild-type and Nur77 null macrophages after LPS stimulation. However, given that NOR1 and Nurr1 are highly induced in Nur77 null cells, and given the fact that all three NR4A family members induce expression of these target genes in vitro (Table 1Go), it is likely that Nurr1 and NOR1 are able to compensate for loss of Nur77.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The NR4A nuclear receptors are among the earliest nuclear receptors to appear in evolution. Three closely related receptors, Nur77, Nurr1, and NOR1, are present in mammals, whereas a single homolog, DHR38, is found in Drosophila. Crystallography studies suggest that the ligand binding domain of these receptors does not contain a viable ligand-binding pocket (14, 15). These receptors appear to have evolved before the acquisition of the ability to bind ligand. Thus, NR4A receptors are transcription factors the activity of which is controlled primarily at the level of protein expression and/or posttranslational modification (16). The first member of this subfamily to be identified, Nur77 or NGFI-B, was cloned based on its rapid induction in response to NGF (hence the name NGFI-B) (11). Subsequent studies have documented induction of Nur77, Nurr1, and NOR1 in response to a variety of stimuli in a range of cells types. In this regard, the expression pattern of NR4A receptors is reminiscent of immediate early-response genes such as c-fos and c-jun. The diversity of signals that induce NR4A receptor expression suggests that the function of these receptors is likely to be highly dependent on cell type and context. Earlier studies of Nur77 and NOR1 focused primarily on their roles in T cell apoptosis (17) and endocrine functions (22, 34). We demonstrated previously that expression of all three NR4A receptors is highly inducible in macrophages by inflammatory cytokines and oxidized lipid such as oxLDL (24). In the present work, we have shown that retroviral expression of Nur77 in macrophages leads to the induction of genes involved in inflammation, apoptosis, and cell cycle control. We further showed that the IKKi gene is a direct target for Nur77 and that expression of Nur77 potentiates the induction of inflammatory gene expression by TLR3/4 ligands. This work highlights NR4A receptors as potentially important contributors to macrophage-inflammatory responses.

Although the NR4A receptors were among the first orphan nuclear receptors cloned, our knowledge of their biological function is limited. In particular, very few direct target genes for these transcription factors have been identified. The apoptotic effects of Nur77 in T cells has been proposed to involve the target genes FasL and CD30 (35, 36). In the brain, the tyrosine hydroxylase and the proopiomelanocortin genes are established NR4A targets (34, 37). Using transcriptional profiling, we have expanded the list of potential NR4A target genes to include genes involved in multiple biological processes. Interestingly, some of the genes identified in our microarray study are also implicated in apoptosis, including caspase4, FAS, and FLICE-inhibitory protein. Whether these genes are critical mediators of NR4A proapoptotic effects remains to be tested.

Despite the longstanding observation that expression of Nur77 and other subfamily members is induced by growth factors, little data have emerged to link these receptors with the control of cell growth. NOR1 is known to form fusion proteins with several distinct partners in different types of human skeletal myxoid chondrosarcoma (38, 39, 40). Furthermore, Nur77/TR3 is one of 17 signature genes associated with metastasis of primary solid tumors (41). In the present study we also identified cyclin D2 as a target of Nur77. Cyclin D2 is one of the main gatekeepers in G1->S phase transition. Although the role of cyclin D2 in monocytic cells is not yet clear, the ability of Nur77 to control expression of this gene may provide a molecular mechanism for the recent observation that Nur77 has mitogenic activity in lung cancer cells (42).

The most unexpected functional class of genes to emerge from our microarray studies was those linked to inflammatory pathways. Previous studies have reported that expression of Nurr1 is elevated in rheumatoid arthritis synovial tissue; however, the functional consequence of receptor induction in this context has not been explored (43). In our study, genes induced by overexpression of Nur77 included the important signaling kinases IKKi and NIK. At least one of these genes, IKKi, is a direct target for NR4A receptors. Mutation of the NBRE in the proximal IKKi promoter compromised induction by both Nur77 and LPS, suggesting that Nur77 binding is essential for maximal response of this promoter to inflammatory stimuli. IKKi was first identified as an LPS- and phorbol 12-myristate 13-acetate-inducible kinase related to I{kappa}B kinases (29, 30). It has recently been shown to phosphorylate IRF3 and to mediate antiviral responses (26, 27). Consistent with the ability of Nur77 to induce IKKi, we observed that Mx1, a critical gene involved in innate antiviral responses and a downstream target of the IRF3 pathway, is more highly inducible in macrophages expressing Nur77. Multiple other IRF3 pathway antiviral genes, such as IFIT1 and IFI203, were also up-regulated in Nur77-expressing cells in our microarray analysis (Table 1Go). Expression of Nur77 also promoted the induction of TNF{alpha} and IP-10 by TLR3/4 ligands. The question of whether these inflammatory genes are induced as a primary effect of Nur77 or are induced secondary to the effect of up-regulated kinases such as IKKi will require further investigation. Together with the induction of NR4As by inflammatory stimuli, the control of multiple inflammatory signaling molecules by Nur77 points to a previously unappreciated role for Nur77/Nurr1/NOR1 in the innate immune response.

Like other nuclear receptors, Nur77, NOR1, and Nurr1 are transcription factors that regulate gene expression through direct binding to specific promoter elements in their downstream target genes. However, recent work has also suggested that in certain cell lines Nur77 can translocate from the nucleus to mitochondria to induce cytochrome c release and apoptosis through interaction with Bcl-2 (18, 44). We have previously shown that Nur77 protein accumulates rapidly in the nucleus in response to inflammatory stimuli (24). Furthermore, in the present work we show that Nur77, Nurr1, and NOR1 regulate expression of the IKKi gene through direct binding to a response element in the promoter. Thus, the NR4A-dependent changes in gene expression observed in macrophages in our study are likely to result from nuclear, rather than mitochondrial, effects.

Both inflammatory cytokines and oxidized lipids have been linked to the pathogenesis of atherosclerosis. The induction of NR4A receptors by these stimuli in macrophages and the ability of NR4As to induce certain genes involved inflammatory signaling raise the question of whether NR4A-dependent gene expression may affect atherogenesis. Previous work has addressed the function of NR4A receptors in other vessel wall cell types such as smooth muscle cells and endothelial cells. Martinez-Gonzalez and Badimon (45) have reported that NOR1 expression is induced in smooth muscle cells by LDL, and Gruber et al. (46) have reported that Nur77 mediates induction of plasminogen activator inhibitor 1 (PAI-1) expression in endothelial cells in response to TNF{alpha}. Other studies using a carotid artery ligation model showed reduced neointimal formation in transgenic mice expressing Nur77 specifically in smooth muscle cells (47). In the future, it will be of interest to determine the impact of the macrophage NR4A signaling pathway for the development of atherosclerosis in murine models. Given the redundancy we have observed in the regulation of target genes in macrophages, however, one might predict that loss of one or even two receptors may not be sufficient to abolish target gene regulation. Thus, it may be necessary to employ conditional knockout strategies to knock down multiple NR4A family members.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture, Reagents, and Transfections
RAW264.7 and J774 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA) and were cultured in DMEM containing 10% FBS. LPS and poly I:C were from Sigma Chemical Co. (St. Louis, MO). The mouse IKKi promoter (–1107 to +22 bp) was amplified by PCR and cloned into the NheI/XhoI site of pGL3 basic luciferase reporter vector (Promega Corp., Madison, WI). Two nucleotides of the NBRE site in IKKi promoter were mutated using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). Transient transfections of the IKKi promoter into RAW264.7 cells was performed in triplicate as described previously (48) using Lipofectamine 2000 (Invitrogen, San Diego, CA) according to manufacturer’s directions. Luciferase activity was normalized to internal ß-galactosidase control. Transfections were carried out in triplicate wells, and each experiment was repeated at least three times. Thioglycolate-elicited murine peritoneal macrophages were isolated from C57Bl/6 mice or Nur77–/– mice on a C57Bl/6 background and cultured as described elsewhere (24).

Construction of Stable Cell Lines
A murine stem cell virus-based retroviral vector and the pRevTRE-TetOn/Off system (CLONTECH Laboratories, Inc., Palo Alto, CA) were used to create cell lines stably expressing NR4A proteins. Retroviral infection of RAW264.7 and J774 cells was performed according to the manufacturer’s directions. Cells stably expressing mouse coding sequences of Nur77, Nurr1, NOR1, or control vectors (Mock, LacZ, luciferase, or GFP) were selected with either 1–4 µg/ml puromycin or 50–200 µg/ml hygromycin over a period of 1–2 wk. All constructs were verified by DNA sequencing.

RNA and Protein Analysis
Total RNA was extracted using Trizol reagent (Invitrogen). Real-time quantitative PCR for RNA analysis and Western blotting for protein analysis have been described previously (48). Primer sequences for real-time PCR are available on request. EMSA was performed as described (48). The sequence of oligos is shown in Fig. 3AGo. NR4A proteins were in vitro transcribed/translated using TnT quick coupled transcription/translation system (Promega) from pCR2.1-mNur77 (Invitrogen) and CMX-mNurr1 or CMX-mNOR1 (both kindly provided by Dr. B. Forman). For microarray analysis, RNA from RAW cells expressing Nur77, Nurr1, and NOR1 was used to probe Affymetrix murine 430A microarrays at the University of California Los Angeles microarray core facility. Microarray data were analyzed using Genespring software (Agilent, Palo Alto, CA).


   ACKNOWLEDGMENTS
 
We thank Dr. J. Milbrandt and P. Cohen for Nur77 null animals, Dr. S. Tetradis for pCI-mNur77 vector, and Dr. B. Forman for CMX-mNurr1 and CMX-mNOR1 plasmids. We thank Drs. O. Conneely, S. Mullican, and A. Hoffmann for discussions. We also thank V. Ramirez-Carrozzi for the help with TetOff system and members of the Tontonoz laboratory for helpful suggestions.


   FOOTNOTES
 
This work was supported by Grant HL 30568 from National Institutes of Health and a Bristol-Myers-Squibb Freedom-to-Discover Award in Cardiovascular Research.

P.T. is an investigator of the Howard Hughes Medical Institute at the University of California, Los Angeles.

Disclosure Statement: L.P. and A.C. have nothing to declare. P.T. consults for Asahi Kasei and Sumitomo and received lecture fees from Bristol-Myers Squibb.

First Published Online December 8, 2005

Abbreviations: GFP, Green fluorescent protein; IKKi, inducible kinase Ki; LDL, low-density lipoprotein; LPS, lipopolysaccharide; LXR, liver X receptor; NBRE, NGFI-B response element; NF-{kappa}B, nuclear factor-{kappa}B; polyI:C, polydeoxyinosinic deoxycytidylic acid; PPAR, peroxisomal proliferator-activated receptor; Tet, tetracycline; TLR, Toll-like receptor.

Received for publication August 15, 2005. Accepted for publication November 30, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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