Liver X Receptor Agonists Inhibit Cytokine-Induced Osteopontin Expression in Macrophages Through Interference With Activator Protein-1 Signa
http://www.100md.com
循环研究杂志 2005年第4期
the Division of Endocrinology and Molecular Medicine (D.O., J.F.S., D.B.), University of Kentucky College of Medicine, Lexington, Ken
Division of Endocrinology (Y.T., F.B., V.H.C., R.E.L., W.A.H.), Diabetes and Hypertension, David Geffen School of Medicine, University of California, Los Angeles, Calif
the Division of Bone and Mineral Diseases (D.W.T.), Washington University Medical Center, St Louis, Mo.
Abstract
Osteopontin (OPN) is a proinflammatory cytokine and adhesion molecule implicated in the chemoattraction of monocytes and in cell-mediated immunity. We have recently reported that genetic OPN-deficiency attenuates the development of atherosclerosis in apoEeC/eC mice identifying OPN as potential target for pharmacological intervention in atherosclerosis. Synthetic agonists for the Liver X Receptor (LXR), members of the nuclear hormone receptor superfamily, prevent the development of atherosclerosis by regulating cholesterol homeostasis and suppressing inflammatory gene expression in macrophages. We demonstrate here that LXR ligands inhibit cytokine-induced OPN expression in macrophages. Two synthetic LXR ligands, T0901317 and GW3965, inhibited TNF-, IL-1, INF- and lipopolysaccharide induced OPN mRNA and protein expression in RAW 264.7 macrophages. Transient transfection experiments revealed that LXR ligands suppress cytokine-induced OPN promoter activity. Deletion analysis, heterologous promoter assays, and site-directed mutagenesis identified an activator protein-1 (AP-1) consensus site at -76 relative to the initiation site that supports OPN transcription in macrophages and mediates the effects of LXR ligands to inhibit OPN transcription. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that LXR agonists inhibit cytokine-induced c-Fos and phospho-c-Jun binding to this AP-1 site. Cytokine-induced c-Fos and phospho-c-Jun protein expression was inhibited by LXR ligands and overexpression of c-Fos and c-Jun reversed the inhibitory effect of LXR ligands on OPN promoter activity in transactivation assays. Finally, treatment of C57BL/6J mice with LXR ligands inhibited OPN expression in peritoneal macrophages indicating that the observed effects of LXR ligands to inhibit OPN expression are applicable in vivo. These observations identify the regulation of macrophage OPN expression as a mechanism whereby LXR ligands may impact macrophage inflammatory responses and atherosclerosis. The full text of this article is available online at http://circres.ahajournals.org.
Key Words: osteopontin liver X receptor nuclear receptor activator protein-1
Introduction
Osteopontin (OPN) is as an extracellular matrix protein that has recently been characterized as a proinflammatory cytokine with pleiotropic functions in inflammation.1,2 OPN promotes monocyte adhesion, migration, activation, and cytokine expression as well as macrophage survival.3eC6 Additional proinflammatory functions of OPN include the ability to stimulate the activation of matrix-metalloproteinases (MMPs) promoting the invasive behavior of monocytes at sites of inflammation.5,6 OPN further induces interleukin (IL)-12 and inhibits IL-10 expression in macrophages, suggesting critical regulatory roles in inflammation and early type-1 cytokine expression.4
OPN is highly expressed in atherosclerotic lesions where it is secreted by macrophages, vascular smooth muscle cells (VSMCs), and endothelial cells (ECs).7 We and other investigators have recently used genetic approaches to define the causal contribution of OPN to the development of atherosclerosis.6,8 Using OPN-deficient mice crossed into atherosclerosis-prone apoEeC/eC mice and infused with angiotensin II, we have provided evidence that OPN promotes the development of atherosclerosis and abdominal aortic aneurysms.6 In particular, OPN derived from hematopoietic cells contributes to atherosclerosis, indicating an important role of macrophage-derived OPN for the development of atherosclerosis.6 The mechanisms by which OPN contributes to atherosclerosis involve the regulation of monocyte recruitment into the arterial wall, the induction of vascular cytokine and MMP expression, and the regulation of macrophage survival in the arterial wall.6 Because OPN appears to specifically promote these early inflammatory mechanisms leading to atherosclerosis, OPN expression in macrophages may potentially provide a novel pharmacological target for the treatment of atherosclerosis.
The liver X receptor (LXR) is an important regulator of cholesterol, free fatty acid, and glucose metabolism.9 In addition to its importance in lipid and glucose metabolism, LXR activation has recently been demonstrated to regulate immune processes and to inhibit inflammatory gene expression in macrophages.10,11 Synthetic LXR agonists have been demonstrated to prevent atherosclerosis in murine models and to inhibit inflammation.10,12 In the present study, we demonstrate that LXR ligands inhibit cytokine-induced OPN expression in macrophages. This effect is mediated by an inhibition of c-Jun/c-Fos DNA binding activities to the proximal OPN promoter, which impairs AP-1eCdependent OPN transcription. These results define a novel mechanism by which LXR ligands may impact macrophage inflammatory responses and atherosclerosis.
Materials and Methods
Materials
T0901317 was purchased from Sigma-Aldrich. GW3965 was kindly provided by Dr Peter Tontonoz (University of California, Los Angeles, Calif). Primary antibodies were purchased from Santa Cruz Biotechnology: USF1 (sc-8983), USF2 (sc-861), c-Fos (sc-8047 or sc-253), FosB (sc-7203), Fra1 (sc-602), Fra2 (sc-604), jun B (sc-8051), c-Jun (sc-45), phospho-c-Jun (sc-822), jun D (sc-74), Oct1 (sc-232), and Oct-2 (sc-233). The OPN antibody (MPIIIB101) was obtained from the University of Iowa Hybridoma Bank.
Cell Culture
RAW 264.7 macrophages (American Type Culture Collection, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) as directed. THP-1 monocytes (ATCC) and HPBM (Cambrex Bio Science Walkersville) were cultured in RPMI1640 medium supplemented with 10% FBS according to the manufacturer’s instruction. Murine peritoneal macrophages were isolated and cultured as described.6 For ligand treatment, cells were serum-deprived by culture in 0.5% FBS and treated with the LXR ligand T0901317 or GW3965 for 24 hours before LPS or cytokine stimulation (R&D Systems). For all data shown, individual experiments were repeated at least three times with different lots or preparation of cells.
Northern Blotting
Northern Blotting was performed as previously described.6 Blots were cohybridized with cDNA encoding the constitutively expressed housekeeping gene Chinese hamster ovary gene B (CHOB) to assess equal loading of samples. Quantification was performed by densitometry and normalization to CHOB mRNA expression.
Western Blot Analysis
Cells were harvested at the indicated time points and sonicated in cell lysis buffer (CellSignaling). Nuclear extracts were isolated using the NE-PER Kit according to the manufacturer’s instructions (Pierce Biotechnology, Inc). Western blotting was performed as recently described.6
Plasmids and Transient Transfections
The OPN promoter constructs and c-Fos and c-Jun expression vectors were used as previously described.13 The AP-1 site at eC76 in the 2-kb OPN promoter construct was muted from eC80CCTCATGACeC71 to eC80CGGCGGGACeC71 using the QuickChange II siteeCdirected mutagenesis kit (Stratagene). The AP-1 luciferase reporter construct driven by seven AP-1 consensus sites was commercially obtained from Stratagene.
RAW 264.7 macrophages were transfected with 1 e DNA using LipofectAMINE 2000 (Invitrogen). Eight hours after transfection, cells were cultured in DMEM supplemented with 0.5% FBS in the presence of the LXR ligand T0901317 or GW3965 for 12 hours before cytokine stimulation. Luciferase activity was assayed 24 hours after stimulation using a Dual Luciferase Reporter Assay System (Promega). Transfection efficiency was adjusted by normalizing firefly luciferase activities to renilla luciferase activities generated by cotransfection with 10 ng pRL-CMV (Promega). All experiments were repeated at least 3 times with different cell preparations.
Electrophoretic Mobility Shift Assay
Oligodeoxynucleotides were labeled with [-32P]-ATP and incubated with 4 e of nuclear extract for 20 minutes. DNA-protein complexes were analyzed by electrophoresis using a 6% nondenaturing acrylamide gel. For competitive oligonucleotide assays, 100-fold excess of unlabeled oligodeoxynucleotide was added to the nuclear extracts 20 minutes before the addition of the radiolabeled probe. Supershift experiments were performed by incubating 4 e of nuclear extract with 0.5 e of the indicated antibody for 20 minutes before addition of the radiolabeled probe. For the treatment studies, RAW 264.7 macrophages were cultured for 24 hours in the presence of 5 eol/L T0901317 (0.5% FBS) and stimulated with TNF- or IL-1 for either 1 or 6 hours as indicated.
Chromatin Immunoprecipitation Assays
ChIP assays were performed using a chromatin immunoprecipitation (ChIP) assay kit (Upstate) according to the manufacture’s instructions. Soluble chromatin was prepared from RAW 264.7 macrophages treated with 5 eol/L T0901317 for 24 hours followed by stimulation with TNF- (20 ng/mL) for 1 hour. Chromatin was immunoprecipitated with antibodies (2 e) directed against c-Fos (sc-8047) and phospho-c-Jun (sc-822). Final DNA extractions were PCR amplified using primer pairs that cover AP-1 consensus sequence at eC76 in the OPN promoter as follows: forward 5'-AACCAGAGGAGGAAGTGTAGGA-3'; antisense 5'-ACCAATCAGGAGGTGGAGTGATGT-3'.
Animal Studies
Seven-week-old male C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, Maine). T0901317 was dissolved at 50 mg/mL in DMSO and further diluted 1:5 in 0.9% PBS. Mice were treated with daily intraperitoneal injections of T0901317 at 50 mg/kg/d or vehicle for 7 days. Four days after beginning of the treatment 1.5 mL thioglycollate (4% wt/vol) was injected intraperitoneally. Three days later peritoneal macrophages were isolated and analyzed for OPN mRNA expression. All animal protocols were approved by the University of California, Los Angeles Animal Research Committee and complied with all federal, state, and industrial regulations.
Statistical Analysis
Analysis of variance (ANOVA) and paired or unpaired t test were performed for statistical analysis as appropriate. Probability values less than 0.05 were considered to be statistically significant. Results are expressed as mean±SEM.
Results
LXR Agonists Suppress OPN Expression in Macrophages
Based on our previous studies demonstrating that macrophage-derived OPN contributes to the development of accelerated atherosclerosis,6 we first determined the regulation of OPN expression in macrophages by different proinflammatory stimuli. As depicted in Figure 1A, OPN protein expression in RAW 264.7 macrophages was significantly upregulated by TNF, IL-1, INF-, and LPS. Northern blotting further revealed that these cytokines induced OPN mRNA, indicating that OPN regulation in macrophages occurs at the gene expression level (Figure 1B).
Because LXR agonists have recently been proposed as therapeutic approaches to treat cardiovascular disease and to suppress inflammatory gene expression in macrophages, we next investigated the effect of two different LXR ligands on macrophage OPN expression.9,14 Consistent with recent reports,15,16 treatment of macrophages with both LXR agonists increased their viability as assessed by annexin V-FITC FACS analysis (data not shown). Preincubation of macrophages with the synthetic LXR agonists resulted in a substantial suppression of cytokine-induced OPN protein and mRNA levels (Figure 1A and 1B), which was dose-dependent over a concentration range of 100 nmol/L to 5 eol/L (Figure 1C). Similarly, LXR agonists suppressed cytokine-induced OPN mRNA and protein expression in human peripheral blood monocytes and THP-1 monocytes (Figure 2A and 2B).
LXR Agonists Suppress Cytokine-Induced OPN Transcription
We next transiently transfected RAW 264.7 macrophages with a 2-kb OPN promoter fragment to determine whether cytokine-induced OPN promoter activity is suppressed by LXR agonists. Cytokine-induced OPN promoter activity was completely inhibited by T0901317 (Figure 3A). Both LXR agonists T0901317 and GW3965 inhibited TNF-eCinduced OPN promoter activity in a dose-dependent manner (Figure 3B). These results suggest that LXR ligands suppress cytokine-induced OPN expression by inhibiting transcription of the OPN gene.
The Proximal OPN Promoter Confers Transcriptional Regulation of OPN in Macrophages
The mechanism for the inhibition of cytokine-induced OPN transcription by LXR ligands is unlikely to be direct, as activation of LXR by its ligands has not been reported to function as a transcriptional repressor. Moreover, sequence analysis of the 5'-flanking region of the OPN gene did not reveal the presence of potential LXR response elements. To identify the promoter elements that regulate cytokine-induced OPN gene transcription and mediate the transcriptional suppression of OPN in macrophages by LXR agonists, we used a series of 5'-deletion constructs (Figure 4A). Whereas TNF-eCinduced transcriptional activity of the eC83OPN-Luc promoter construct was completely suppressed by the LXR agonist, a eC61OPN-Luc construct was not inducible by TNF- and exhibited a transcriptional activity only slightly above the background signal. Comparable results were obtained after stimulation with IL-1, INF-, and LPS (data not shown). These findings indicate that basal and TNF-eCinduced OPN promoter activity in macrophages is dependent on transcription factor binding sites located between eC83 to eC61 relative to the transcription initiation site.
We next performed heterologous promoter assays to confirm that the proximal eC83 to eC61 region of the OPN promoter confers the transcriptional regulation of OPN in macrophages. The basal transcriptional activity of the OPN(eC83/eC45)RSVLuc construct was approximately 30-fold greater than that of the minimal RSVLuc (Figure 4B). The OPN(eC83/eC45)RSVLuc conferred TNF- responsiveness compared to the unresponsive RSV minimal promoter. Moreover, TNF-eCinduced transcriptional activity of the OPN(eC83/eC45)RSVLuc was substantially inhibited by treatment with the LXR agonist. These results suggest that the proximal OPN promoter region eC83 to eC45 assembles transcriptional complexes that mediate both basal transcriptional activity and the suppression of cytokine-induced OPN promoter activity by LXR ligands.
LXR Agonists Suppress Cytokine-Induced OPN Promoter Activity by Negatively Interfering With AP-1 Transcription Activity
Based on previous studies demonstrating an important role of the AP-1 site located at eC76 for the transcriptional regulation of the OPN gene, a mutation in this AP-1 site was generated.13,17 Whereas treatment with TNF- resulted in an induction of the wild-type OPN promoter, the promoter containing a mutation of this AP-1 site exhibited low basal activity and was not induced by TNF- (Figure 5A). We next cotransfected macrophages with the eC83OPN-Luc reporter and eukaryotic expression vectors for c-Fos and c-Jun (Figure 5B). Overexpression of c-Fos and c-Jun resulted in a complete loss of the effect of LXR ligands to inhibit TNF- induced OPN transcriptional activation. Furthermore, TNF-eCinduced transcriptional activity of a heterologous promoter driven by multiple AP-1 response elements was dose-dependently inhibited by the LXR agonist (Figure 5C). In concert, these findings suggest that the suppression of cytokine-induced OPN promoter activity by LXR ligands is mediated through negative interference with c-Fos/c-Jun acting on the proximal OPN promoter.
The OPN Promoter Region eC80 to eC65 Assembles AP-1, Oct-1, and USF-1 Protein-DNA Complexes
In addition to the reported AP-1 site at eC76,13,17 the OPN promoter sequence between eC80 and eC65 contains a CCTCATGA motif resembling an atypical E-box as well as an Oct-1 (CTCATGA) cognate.13 As depicted in Figure 6A (lane 1 and 2), EMSA experiments revealed three specific complexes (labeled A, B, and C). Competition experiments confirmed AP-1eClike protein-DNA interactions (lane 3), an Oct-1 cognate (lane 4), and an USF-1 cognate (lane 5). A duplex oligonucleotide possessing a CCTT transition, which selectively disrupts the putative E-box (MUT1), can still compete for complex A and B, but not for complex C (lane 6). A mutant disrupting the AP-1, Oct-1, and E-box did not compete for any of the complexes formed at this promoter region (lane 7). A cold oligonucleotide MUT3 competed for the formation of all three complexes, indicating that only the intact cognate CCTCATGAC was sufficient to compete for assembly of complexes. In concert, these findings suggest that the CCTCATGAC motif between eC80 and eC71 relative to the transcription initiation site assembles three DNA/protein complexes consisting of AP-1, Oct-1, and USF-1 binding.
We next performed supershift experiments using nuclear extracts isolated from TNF-eCstimulated RAW 264.7 macrophages. Antibodies against c-Fos and Ser-63 phosphorylated c-Jun substantially diminished complex formation and supershifted complexes formed at eC80 to eC65 of the OPN promoter (Figure 6C). In addition, antibodies against Fra-1 and JunB partially supershifted the AP-1 complex, whereas an antibody directed against Fra-2 diminished the complex but did not result in a supershift of the complex. In contrast, antibodies to FosB, unphosphorylated c-Jun, and JunD had no effect on complex formation. Finally, antibodies directed against USF-1 and Oct-1 confirmed the presence of USF-1 and Oct-1 protein/DNA complexes. This data confirms that AP-1 (mainly c-Fos and phosphorylated c-Jun), Oct-1, and USF-1 transcription factors assemble protein/DNA complexes at the OPN promoter region eC80 to eC65.
AP-1 Binding to the eC80 to eC65 OPN Promoter Motif Is Suppressed by LXR Agonists
We next examined the effect of LXR ligands on the formation of protein/DNA complexes at the eC80 to eC65 OPN promoter sequence. Murine RAW 264.7 macrophages were serum-deprived for 24 hours and stimulated with TNF- or IL-1. TNF-eC and IL-1eCinduced AP-1 binding activities were completely inhibited by treatment with the LXR agonist (Figure 7A), whereas Oct-1 and USF-1 binding activities were not significantly regulated.
To confirm that c-Fos and phospho-c-Jun bind to the endogenous OPN promoter and that LXR agonists interfere with this binding, we next performed ChIP assays. PCR amplification using primer pairs that cover the AP-1 site at eC76 in the OPN promoter, demonstrated that this OPN promoter region coimmunoprecipitated with c-Fos and phospho-c-Jun after stimulation with TNF- (Figure 7B). This induction of c-Fos and phospho-c-Jun binding to the proximal OPN promoter was substantially inhibited by the LXR agonist. In concert, these findings indicate that TNF-eCinduced c-Fos and phospho-c-Jun binding to the AP-1 site at eC76 of the proximal OPN promoter is inhibited by LXR agonists.
TNF-eCInduced c-Fos and Phospho-c-Jun Protein Expression Is Inhibited by LXR Ligands
To further determine whether the inhibition of AP-1 binding by LXR ligands reflects changes in c-Fos or phospho-c-Jun expression levels, protein levels of both transcription factors were determined by Western blotting analysis. TNF-eCInduced c-Fos (Figure 8A) and phospho-c-Jun (Figure 8B) protein expression was significantly inhibited by treatment with the LXR agonist. Thus, suppression of TNF- and IL-1 induced binding to the AP-1 site at eC76 by LXR ligands reflects at least in part the regulation of c-Fos and phospho-c-Jun protein expression.
LXR Agonists Inhibit OPN Expression in Macrophages In Vivo
To finally determine whether the effects of LXR agonists to suppress OPN expression are applicable in vivo, T0901317 was administered to C57BL/6J mice for 7 days before isolation of peritoneal macrophages. The mRNA expression level of the LXR target gene ABCA1 was induced by 4.3-fold in peritoneal macrophages elicited from mice treated with T9091317, indicating sufficient availability of the LXR compound (data not shown). OPN protein (Figure 9A) and mRNA (Figure 9B) expression was substantially decreased in peritoneal macrophages from T0901317-treated mice compared with vehicle-treated mice (76±8% and 64±5% decrease in OPN mRNA and protein expression levels, respectively; n=4, P<0.005). Similarly, treatment of elicited murine peritoneal macrophages with the LXR agonist T0901317 dose-dependently suppressed TNF-eCinduced OPN protein expression (Figure 9C). Taken together, these results demonstrate that LXR ligands inhibit OPN expression in murine peritoneal macrophages and further support an important role for LXR ligands to suppress proinflammatory gene expression in vitro and in vivo.
Discussion
LXR signaling pathways have recently been proposed as potential target for therapeutic interventions in cardiovascular diseases.9,14 Whereas the antiatherogenetic effects of LXR agonists have been attributed to the modulation of cholesterol efflux from macrophages and induction of reverse cholesterol transport to the liver, recent evidence indicates that LXR ligands also modulate inflammatory gene expression.10,11 In the present study, we demonstrate that LXR agonists inhibit expression of OPN, a key inflammatory gene in macrophages. This inhibition is mediated through an inhibition of AP-1eCdependent transcriptional activation of the OPN promoter. These observations further support an important role for LXR agonists to suppress macrophage inflammatory responses.
We have recently demonstrated that OPN promotes atherogenesis by modulating leukocyte chemotaxis, cytokine and MMP expression, and macrophage survival.6 However, the molecular mechanisms that regulate OPN expression in macrophages during inflammatory processes are incompletely understood. OPN expression in macrophages was substantially induced after cytokine stimulation and this induction was transcriptionally suppressed by two LXR agonists. The OPN promoter is remarkably responsive to various stimuli, including cytokines, growth factors, and hormones.1 We identified a CCTCATGAC cognate in the OPN promoter located between eC80 and eC71 that supports basal and cytokine-induced transcriptional activity of the OPN promoter in macrophages. Consistent with previous reports in VSMCs, cold-competition and immunological supershift assays revealed AP-1 binding to this site.13,17 A variety of inflammatory genes are under the control of AP-1eCdependent signaling pathways; however, the transcriptional regulation of the OPN promoter by AP-1 in macrophages has not previously been investigated. The importance of this AP-1 site for the transcriptional activity of the OPN promoter was further demonstrated by a complete loss of basal and inducible OPN promoter activity after site-directed mutagenesis of this AP-1 site. Further characterization of the AP-1 complex binding to the OPN promoter revealed the presence of c-Fos and Ser-63 phosphorylated c-Jun binding activities. In addition to an induction of transcription from Fos and Jun genes, AP-1 activity is regulated through phosphorylation of c-Jun and phosphorylated c-Jun is the most potent transactivator of the AP-1 complex.18,19 Phosphorylation of c-Jun at Ser-63, located within its transactivation domain, potentiates its ability to activate transcription as either a homodimer or a heterodimer with c-Fos.19,20 Consistent with this, we observed that primarily Ser-63 phosphorylated c-Jun was present in the AP-1 complex bound to the CCTCATGAC cognate between eC80 and eC71 as opposed to unphosphorylated c-Jun.
The analysis of the OPN promoter did not reveal the presence of any putative LXRE response elements, indicating that the inhibition of cytokine-induced OPN transcription by LXR ligands likely involves an indirect mechanism through regulation of other transcription factors supporting OPN transcription. Because AP-1 binding activities appeared to be important for the transcriptional activity of the OPN promoter in macrophages, we next analyzed whether LXR agonists interfere with AP-1 binding to the proximal OPN promoter. EMSA and ChIP experiments indicated that LXR ligands suppress c-Fos and phospho-c-Jun binding to the AP-1 site at eC76 of the proximal OPN promoter. Results from transactivation studies further suggested that LXR ligands inhibit transactivation of a heterologous AP-1eCdriven promoter and that overexpression of c-Fos and c-Jun reverses the inhibition of OPN promoter activity by LXR ligands. Finally, LXR ligands inhibit cytokine-induced c-Fos and phospho-c-Jun protein expression, which may contribute at least in part to the observed inhibition of AP-1 binding to the proximal OPN promoter.
Cross-talk between different transcription factors and a similar negative interference with AP-1 activity has been described for ligand-induced activation of other nuclear receptors, such as the retinoic acid receptor (RAR),21 the glucocorticoid receptor (GR),22 or the peroxisome proliferatoreCactivated receptor (PPAR).23 Although PPAR ligands inhibit AP-1eCdependent signaling pathways, agonists for this receptor have been demonstrated to inhibit PMA-induced OPN expression in macrophage through interference with homeobox-like A/T-rich sequences between eC990 and eC981 of the OPN promoter.24 These findings suggest that the mechanisms by which LXR and PPAR agonists suppress OPN expression are distinct and likely dependent on the mechanisms of OPN induction. However, additional studies are necessary to delineate in more detail the molecular mechanism involved in the negative regulation of c-Fos/c-Jun transactivation by LXR ligands.
In conclusion, data presented in this study demonstrate the observation that cytokine-induced OPN expression in macrophages depends on AP-1 binding to the proximal OPN promoter. LXR agonists inhibit cytokine-induced OPN expression and interfere negatively with AP-1 binding to the proximal OPN promoter by inhibiting c-Fos and phospho-c-Jun protein expression. The suppression of OPN expression by LXR agonists are applicable in vivo as OPN expression is inhibited in mouse peritoneal macrophages elicited after treatment with the LXR agonist. Because OPN is a key component for the inflammatory response and the recruitment of monocytes/macrophages into the atherosclerotic lesion, inhibition of cytokine-induced OPN expression by LXR agonists could provide a potential target for therapeutic intervention in human cardiovascular disease.
Acknowledgments
This study was supported by a Scientist Development Grant 0435239N from the American Heart Association to Dennis Bruemmer and by a National Institutes of Health Grant HL 58328 to Willa A. Hsueh. Florian Blaschke was supported by a research fellowship from Philip Morris USA Incorporated.
References
Denhardt DT, Giachelli CM, Rittling SR. Role of osteopontin in cellular signaling and toxicant injury. Annu Rev Pharmacol Toxicol. 2001; 41: 723eC749.
O’Regan A. The role of osteopontin in lung disease. Cytokine Growth Factor Rev. 2003; 14: 479eC488.
Giachelli CM, Lombardi D, Johnson RJ, Murry CE, Almeida M. Evidence for a role of osteopontin in macrophage infiltration in response to pathological stimuli in vivo. Am J Pathol. 1998; 152: 353eC358.
Ashkar S, Weber GF, Panoutsakopoulou V, Sanchirico ME, Jansson M, Zawaideh S, Rittling SR, Denhardt DT, Glimcher MJ, Cantor H. Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity. Science. 2000; 287: 860eC864.
Weber GF, Zawaideh S, Hikita S, Kumar VA, Cantor H, Ashkar S. Phosphorylation-dependent interaction of osteopontin with its receptors regulates macrophage migration and activation. J Leukoc Biol. 2002; 72: 752eC761.
Bruemmer D, Collins AR, Noh G, Wang W, Territo M, Arias-Magallona S, Fishbein MC, Blaschke F, Kintscher U, Graf K, Law RE, Hsueh WA. Angiotensin IIeCaccelerated atherosclerosis and aneurysm formation is attenuated in osteopontin-deficient mice. J Clin Invest. 2003; 112: 1318eC1331.
Giachelli CM, Bae N, Almeida M, Denhardt DT, Alpers CE, Schwartz SM. Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J Clin Invest. 1993; 92: 1686eC1696.
Matsui Y, Rittling SR, Okamoto H, Inobe M, Jia N, Shimizu T, Akino M, Sugawara T, Morimoto J, Kimura C, Kon S, Denhardt D, Kitabatake A, Uede T. Osteopontin deficiency attenuates atherosclerosis in female apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2003; 23: 1029eC1034.
Tontonoz P, Mangelsdorf DJ. Liver X receptor signaling pathways in cardiovascular disease. Mol Endocrinol. 2003; 17: 985eC993.
Joseph SB, Castrillo A, Laffitte BA, Mangelsdorf DJ, Tontonoz P. Reciprocal regulation of inflammation and lipid metabolism by liver X receptors. Nat Med. 2003; 9: 213eC219.
Castrillo A, Joseph SB, Marathe C, Mangelsdorf DJ, Tontonoz P. Liver X receptor-dependent repression of matrix metalloproteinase-9 expression in macrophages. J Biol Chem. 2003; 278: 10443eC10449.
Joseph SB, McKilligin E, Pei L, Watson MA, Collins AR, Laffitte BA, Chen M, Noh G, Goodman J, Hagger GN, Tran J, Tippin TK, Wang X, Lusis AJ, Hsueh WA, Law RE, Collins JL, Willson TM, Tontonoz P. Synthetic LXR ligand inhibits the development of atherosclerosis in mice. Proc Natl Acad Sci U S A. 2002; 99: 7604eC7609.
Bidder M, Shao J-S, Charlton-Kachigian N, Loewy AP, Semenkovich CF, Towler DA. Osteopontin transcription in aortic vascular smooth muscle cells is controlled by glucose-regulated upstream stimulatory factor and activator protein-1 activities. J Biol Chem. 2002; 277: 44485eC44496.
Lund EG, Menke JG, Sparrow CP. Liver X receptor agonists as potential therapeutic agents for dyslipidemia and atherosclerosis. Arterioscler Thromb Vasc Biol. 2003; 23: 1169eC1177.
Joseph SB, Bradley MN, Castrillo A, Bruhn KW, Mak PA, Pei L, Hogenesch J, O’Connell RM, Cheng G, Saez E. LXR-dependent gene expression is important for macrophage survival and the innate immune response. Cell. 2004; 119: 299eC309.
Valledor AF, Hsu L-C, Ogawa S, Sawka-Verhelle D, Karin M, Glass CK. Activation of liver X receptors and retinoid X receptors prevents bacterial-induced macrophage apoptosis. PNAS. 2004; 101: 17813eC17818.
Renault M-A, Jalvy S, Belloc I, Pasquet S, Sena S, Olive M, Desgranges C, Gadeau A-P. AP-1 is involved in UTP-induced osteopontin expression in arterial smooth muscle cells. Circ Res. 2003; 93: 674eC681.
Devary Y, Gottlieb RA, Smeal T, Karin M. The mammalian ultraviolet response is triggered by activation of Src tyrosine kinases. Cell. 1992; 71: 1081eC1091.
Smeal T, Binetruy B, Mercola DA, Birrer M, Karin M. Oncogenic and transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serines 63 and 73. Nature. 1991; 354: 494eC496.
Deng T, Karin M. c-Fos transcriptional activity stimulated by H-Ras-activated protein kinase distinct from JNK and ERK. Nature. 1994; 371: 171eC175.
Schule R, Rangarajan P, Yang N, Kliewer S, Ransone L, Bolado J, Verma I, Evans R. Retinoic acid is a negative regulator of AP-1-responsive genes. Proc Natl Acad Sci U S A. 1991; 88: 6092eC6096.
Jonat C, Rahmsdorf HJ, Park KK, Cato AC, Gebel S, Ponta H, Herrlich P. Antitumor promotion and antiinflammation: down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. Cell. 1990; 62: 1189eC1204.
Delerive P, Martin-Nizard F, Chinetti G, Trottein F, Fruchart J-C, Najib J, Duriez P, Staels B. Peroxisome proliferator-activated receptor activators inhibit thrombin-induced endothelin-1 production in human vascular endothelial cells by inhibiting the activator protein-1 signaling pathway. Circ Res. 1999; 85: 394eC402.
Oyama Y, Akuzawa N, Nagai R, Kurabayashi M. PPAR{gamma} ligand inhibits osteopontin gene expression through interference with binding of nuclear factors to A/T-rich sequence in THP-1 cells. Circ Res. 2002; 90: 348eC355.(Daisuke Ogawa, Jeffrey F.)
Division of Endocrinology (Y.T., F.B., V.H.C., R.E.L., W.A.H.), Diabetes and Hypertension, David Geffen School of Medicine, University of California, Los Angeles, Calif
the Division of Bone and Mineral Diseases (D.W.T.), Washington University Medical Center, St Louis, Mo.
Abstract
Osteopontin (OPN) is a proinflammatory cytokine and adhesion molecule implicated in the chemoattraction of monocytes and in cell-mediated immunity. We have recently reported that genetic OPN-deficiency attenuates the development of atherosclerosis in apoEeC/eC mice identifying OPN as potential target for pharmacological intervention in atherosclerosis. Synthetic agonists for the Liver X Receptor (LXR), members of the nuclear hormone receptor superfamily, prevent the development of atherosclerosis by regulating cholesterol homeostasis and suppressing inflammatory gene expression in macrophages. We demonstrate here that LXR ligands inhibit cytokine-induced OPN expression in macrophages. Two synthetic LXR ligands, T0901317 and GW3965, inhibited TNF-, IL-1, INF- and lipopolysaccharide induced OPN mRNA and protein expression in RAW 264.7 macrophages. Transient transfection experiments revealed that LXR ligands suppress cytokine-induced OPN promoter activity. Deletion analysis, heterologous promoter assays, and site-directed mutagenesis identified an activator protein-1 (AP-1) consensus site at -76 relative to the initiation site that supports OPN transcription in macrophages and mediates the effects of LXR ligands to inhibit OPN transcription. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that LXR agonists inhibit cytokine-induced c-Fos and phospho-c-Jun binding to this AP-1 site. Cytokine-induced c-Fos and phospho-c-Jun protein expression was inhibited by LXR ligands and overexpression of c-Fos and c-Jun reversed the inhibitory effect of LXR ligands on OPN promoter activity in transactivation assays. Finally, treatment of C57BL/6J mice with LXR ligands inhibited OPN expression in peritoneal macrophages indicating that the observed effects of LXR ligands to inhibit OPN expression are applicable in vivo. These observations identify the regulation of macrophage OPN expression as a mechanism whereby LXR ligands may impact macrophage inflammatory responses and atherosclerosis. The full text of this article is available online at http://circres.ahajournals.org.
Key Words: osteopontin liver X receptor nuclear receptor activator protein-1
Introduction
Osteopontin (OPN) is as an extracellular matrix protein that has recently been characterized as a proinflammatory cytokine with pleiotropic functions in inflammation.1,2 OPN promotes monocyte adhesion, migration, activation, and cytokine expression as well as macrophage survival.3eC6 Additional proinflammatory functions of OPN include the ability to stimulate the activation of matrix-metalloproteinases (MMPs) promoting the invasive behavior of monocytes at sites of inflammation.5,6 OPN further induces interleukin (IL)-12 and inhibits IL-10 expression in macrophages, suggesting critical regulatory roles in inflammation and early type-1 cytokine expression.4
OPN is highly expressed in atherosclerotic lesions where it is secreted by macrophages, vascular smooth muscle cells (VSMCs), and endothelial cells (ECs).7 We and other investigators have recently used genetic approaches to define the causal contribution of OPN to the development of atherosclerosis.6,8 Using OPN-deficient mice crossed into atherosclerosis-prone apoEeC/eC mice and infused with angiotensin II, we have provided evidence that OPN promotes the development of atherosclerosis and abdominal aortic aneurysms.6 In particular, OPN derived from hematopoietic cells contributes to atherosclerosis, indicating an important role of macrophage-derived OPN for the development of atherosclerosis.6 The mechanisms by which OPN contributes to atherosclerosis involve the regulation of monocyte recruitment into the arterial wall, the induction of vascular cytokine and MMP expression, and the regulation of macrophage survival in the arterial wall.6 Because OPN appears to specifically promote these early inflammatory mechanisms leading to atherosclerosis, OPN expression in macrophages may potentially provide a novel pharmacological target for the treatment of atherosclerosis.
The liver X receptor (LXR) is an important regulator of cholesterol, free fatty acid, and glucose metabolism.9 In addition to its importance in lipid and glucose metabolism, LXR activation has recently been demonstrated to regulate immune processes and to inhibit inflammatory gene expression in macrophages.10,11 Synthetic LXR agonists have been demonstrated to prevent atherosclerosis in murine models and to inhibit inflammation.10,12 In the present study, we demonstrate that LXR ligands inhibit cytokine-induced OPN expression in macrophages. This effect is mediated by an inhibition of c-Jun/c-Fos DNA binding activities to the proximal OPN promoter, which impairs AP-1eCdependent OPN transcription. These results define a novel mechanism by which LXR ligands may impact macrophage inflammatory responses and atherosclerosis.
Materials and Methods
Materials
T0901317 was purchased from Sigma-Aldrich. GW3965 was kindly provided by Dr Peter Tontonoz (University of California, Los Angeles, Calif). Primary antibodies were purchased from Santa Cruz Biotechnology: USF1 (sc-8983), USF2 (sc-861), c-Fos (sc-8047 or sc-253), FosB (sc-7203), Fra1 (sc-602), Fra2 (sc-604), jun B (sc-8051), c-Jun (sc-45), phospho-c-Jun (sc-822), jun D (sc-74), Oct1 (sc-232), and Oct-2 (sc-233). The OPN antibody (MPIIIB101) was obtained from the University of Iowa Hybridoma Bank.
Cell Culture
RAW 264.7 macrophages (American Type Culture Collection, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) as directed. THP-1 monocytes (ATCC) and HPBM (Cambrex Bio Science Walkersville) were cultured in RPMI1640 medium supplemented with 10% FBS according to the manufacturer’s instruction. Murine peritoneal macrophages were isolated and cultured as described.6 For ligand treatment, cells were serum-deprived by culture in 0.5% FBS and treated with the LXR ligand T0901317 or GW3965 for 24 hours before LPS or cytokine stimulation (R&D Systems). For all data shown, individual experiments were repeated at least three times with different lots or preparation of cells.
Northern Blotting
Northern Blotting was performed as previously described.6 Blots were cohybridized with cDNA encoding the constitutively expressed housekeeping gene Chinese hamster ovary gene B (CHOB) to assess equal loading of samples. Quantification was performed by densitometry and normalization to CHOB mRNA expression.
Western Blot Analysis
Cells were harvested at the indicated time points and sonicated in cell lysis buffer (CellSignaling). Nuclear extracts were isolated using the NE-PER Kit according to the manufacturer’s instructions (Pierce Biotechnology, Inc). Western blotting was performed as recently described.6
Plasmids and Transient Transfections
The OPN promoter constructs and c-Fos and c-Jun expression vectors were used as previously described.13 The AP-1 site at eC76 in the 2-kb OPN promoter construct was muted from eC80CCTCATGACeC71 to eC80CGGCGGGACeC71 using the QuickChange II siteeCdirected mutagenesis kit (Stratagene). The AP-1 luciferase reporter construct driven by seven AP-1 consensus sites was commercially obtained from Stratagene.
RAW 264.7 macrophages were transfected with 1 e DNA using LipofectAMINE 2000 (Invitrogen). Eight hours after transfection, cells were cultured in DMEM supplemented with 0.5% FBS in the presence of the LXR ligand T0901317 or GW3965 for 12 hours before cytokine stimulation. Luciferase activity was assayed 24 hours after stimulation using a Dual Luciferase Reporter Assay System (Promega). Transfection efficiency was adjusted by normalizing firefly luciferase activities to renilla luciferase activities generated by cotransfection with 10 ng pRL-CMV (Promega). All experiments were repeated at least 3 times with different cell preparations.
Electrophoretic Mobility Shift Assay
Oligodeoxynucleotides were labeled with [-32P]-ATP and incubated with 4 e of nuclear extract for 20 minutes. DNA-protein complexes were analyzed by electrophoresis using a 6% nondenaturing acrylamide gel. For competitive oligonucleotide assays, 100-fold excess of unlabeled oligodeoxynucleotide was added to the nuclear extracts 20 minutes before the addition of the radiolabeled probe. Supershift experiments were performed by incubating 4 e of nuclear extract with 0.5 e of the indicated antibody for 20 minutes before addition of the radiolabeled probe. For the treatment studies, RAW 264.7 macrophages were cultured for 24 hours in the presence of 5 eol/L T0901317 (0.5% FBS) and stimulated with TNF- or IL-1 for either 1 or 6 hours as indicated.
Chromatin Immunoprecipitation Assays
ChIP assays were performed using a chromatin immunoprecipitation (ChIP) assay kit (Upstate) according to the manufacture’s instructions. Soluble chromatin was prepared from RAW 264.7 macrophages treated with 5 eol/L T0901317 for 24 hours followed by stimulation with TNF- (20 ng/mL) for 1 hour. Chromatin was immunoprecipitated with antibodies (2 e) directed against c-Fos (sc-8047) and phospho-c-Jun (sc-822). Final DNA extractions were PCR amplified using primer pairs that cover AP-1 consensus sequence at eC76 in the OPN promoter as follows: forward 5'-AACCAGAGGAGGAAGTGTAGGA-3'; antisense 5'-ACCAATCAGGAGGTGGAGTGATGT-3'.
Animal Studies
Seven-week-old male C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, Maine). T0901317 was dissolved at 50 mg/mL in DMSO and further diluted 1:5 in 0.9% PBS. Mice were treated with daily intraperitoneal injections of T0901317 at 50 mg/kg/d or vehicle for 7 days. Four days after beginning of the treatment 1.5 mL thioglycollate (4% wt/vol) was injected intraperitoneally. Three days later peritoneal macrophages were isolated and analyzed for OPN mRNA expression. All animal protocols were approved by the University of California, Los Angeles Animal Research Committee and complied with all federal, state, and industrial regulations.
Statistical Analysis
Analysis of variance (ANOVA) and paired or unpaired t test were performed for statistical analysis as appropriate. Probability values less than 0.05 were considered to be statistically significant. Results are expressed as mean±SEM.
Results
LXR Agonists Suppress OPN Expression in Macrophages
Based on our previous studies demonstrating that macrophage-derived OPN contributes to the development of accelerated atherosclerosis,6 we first determined the regulation of OPN expression in macrophages by different proinflammatory stimuli. As depicted in Figure 1A, OPN protein expression in RAW 264.7 macrophages was significantly upregulated by TNF, IL-1, INF-, and LPS. Northern blotting further revealed that these cytokines induced OPN mRNA, indicating that OPN regulation in macrophages occurs at the gene expression level (Figure 1B).
Because LXR agonists have recently been proposed as therapeutic approaches to treat cardiovascular disease and to suppress inflammatory gene expression in macrophages, we next investigated the effect of two different LXR ligands on macrophage OPN expression.9,14 Consistent with recent reports,15,16 treatment of macrophages with both LXR agonists increased their viability as assessed by annexin V-FITC FACS analysis (data not shown). Preincubation of macrophages with the synthetic LXR agonists resulted in a substantial suppression of cytokine-induced OPN protein and mRNA levels (Figure 1A and 1B), which was dose-dependent over a concentration range of 100 nmol/L to 5 eol/L (Figure 1C). Similarly, LXR agonists suppressed cytokine-induced OPN mRNA and protein expression in human peripheral blood monocytes and THP-1 monocytes (Figure 2A and 2B).
LXR Agonists Suppress Cytokine-Induced OPN Transcription
We next transiently transfected RAW 264.7 macrophages with a 2-kb OPN promoter fragment to determine whether cytokine-induced OPN promoter activity is suppressed by LXR agonists. Cytokine-induced OPN promoter activity was completely inhibited by T0901317 (Figure 3A). Both LXR agonists T0901317 and GW3965 inhibited TNF-eCinduced OPN promoter activity in a dose-dependent manner (Figure 3B). These results suggest that LXR ligands suppress cytokine-induced OPN expression by inhibiting transcription of the OPN gene.
The Proximal OPN Promoter Confers Transcriptional Regulation of OPN in Macrophages
The mechanism for the inhibition of cytokine-induced OPN transcription by LXR ligands is unlikely to be direct, as activation of LXR by its ligands has not been reported to function as a transcriptional repressor. Moreover, sequence analysis of the 5'-flanking region of the OPN gene did not reveal the presence of potential LXR response elements. To identify the promoter elements that regulate cytokine-induced OPN gene transcription and mediate the transcriptional suppression of OPN in macrophages by LXR agonists, we used a series of 5'-deletion constructs (Figure 4A). Whereas TNF-eCinduced transcriptional activity of the eC83OPN-Luc promoter construct was completely suppressed by the LXR agonist, a eC61OPN-Luc construct was not inducible by TNF- and exhibited a transcriptional activity only slightly above the background signal. Comparable results were obtained after stimulation with IL-1, INF-, and LPS (data not shown). These findings indicate that basal and TNF-eCinduced OPN promoter activity in macrophages is dependent on transcription factor binding sites located between eC83 to eC61 relative to the transcription initiation site.
We next performed heterologous promoter assays to confirm that the proximal eC83 to eC61 region of the OPN promoter confers the transcriptional regulation of OPN in macrophages. The basal transcriptional activity of the OPN(eC83/eC45)RSVLuc construct was approximately 30-fold greater than that of the minimal RSVLuc (Figure 4B). The OPN(eC83/eC45)RSVLuc conferred TNF- responsiveness compared to the unresponsive RSV minimal promoter. Moreover, TNF-eCinduced transcriptional activity of the OPN(eC83/eC45)RSVLuc was substantially inhibited by treatment with the LXR agonist. These results suggest that the proximal OPN promoter region eC83 to eC45 assembles transcriptional complexes that mediate both basal transcriptional activity and the suppression of cytokine-induced OPN promoter activity by LXR ligands.
LXR Agonists Suppress Cytokine-Induced OPN Promoter Activity by Negatively Interfering With AP-1 Transcription Activity
Based on previous studies demonstrating an important role of the AP-1 site located at eC76 for the transcriptional regulation of the OPN gene, a mutation in this AP-1 site was generated.13,17 Whereas treatment with TNF- resulted in an induction of the wild-type OPN promoter, the promoter containing a mutation of this AP-1 site exhibited low basal activity and was not induced by TNF- (Figure 5A). We next cotransfected macrophages with the eC83OPN-Luc reporter and eukaryotic expression vectors for c-Fos and c-Jun (Figure 5B). Overexpression of c-Fos and c-Jun resulted in a complete loss of the effect of LXR ligands to inhibit TNF- induced OPN transcriptional activation. Furthermore, TNF-eCinduced transcriptional activity of a heterologous promoter driven by multiple AP-1 response elements was dose-dependently inhibited by the LXR agonist (Figure 5C). In concert, these findings suggest that the suppression of cytokine-induced OPN promoter activity by LXR ligands is mediated through negative interference with c-Fos/c-Jun acting on the proximal OPN promoter.
The OPN Promoter Region eC80 to eC65 Assembles AP-1, Oct-1, and USF-1 Protein-DNA Complexes
In addition to the reported AP-1 site at eC76,13,17 the OPN promoter sequence between eC80 and eC65 contains a CCTCATGA motif resembling an atypical E-box as well as an Oct-1 (CTCATGA) cognate.13 As depicted in Figure 6A (lane 1 and 2), EMSA experiments revealed three specific complexes (labeled A, B, and C). Competition experiments confirmed AP-1eClike protein-DNA interactions (lane 3), an Oct-1 cognate (lane 4), and an USF-1 cognate (lane 5). A duplex oligonucleotide possessing a CCTT transition, which selectively disrupts the putative E-box (MUT1), can still compete for complex A and B, but not for complex C (lane 6). A mutant disrupting the AP-1, Oct-1, and E-box did not compete for any of the complexes formed at this promoter region (lane 7). A cold oligonucleotide MUT3 competed for the formation of all three complexes, indicating that only the intact cognate CCTCATGAC was sufficient to compete for assembly of complexes. In concert, these findings suggest that the CCTCATGAC motif between eC80 and eC71 relative to the transcription initiation site assembles three DNA/protein complexes consisting of AP-1, Oct-1, and USF-1 binding.
We next performed supershift experiments using nuclear extracts isolated from TNF-eCstimulated RAW 264.7 macrophages. Antibodies against c-Fos and Ser-63 phosphorylated c-Jun substantially diminished complex formation and supershifted complexes formed at eC80 to eC65 of the OPN promoter (Figure 6C). In addition, antibodies against Fra-1 and JunB partially supershifted the AP-1 complex, whereas an antibody directed against Fra-2 diminished the complex but did not result in a supershift of the complex. In contrast, antibodies to FosB, unphosphorylated c-Jun, and JunD had no effect on complex formation. Finally, antibodies directed against USF-1 and Oct-1 confirmed the presence of USF-1 and Oct-1 protein/DNA complexes. This data confirms that AP-1 (mainly c-Fos and phosphorylated c-Jun), Oct-1, and USF-1 transcription factors assemble protein/DNA complexes at the OPN promoter region eC80 to eC65.
AP-1 Binding to the eC80 to eC65 OPN Promoter Motif Is Suppressed by LXR Agonists
We next examined the effect of LXR ligands on the formation of protein/DNA complexes at the eC80 to eC65 OPN promoter sequence. Murine RAW 264.7 macrophages were serum-deprived for 24 hours and stimulated with TNF- or IL-1. TNF-eC and IL-1eCinduced AP-1 binding activities were completely inhibited by treatment with the LXR agonist (Figure 7A), whereas Oct-1 and USF-1 binding activities were not significantly regulated.
To confirm that c-Fos and phospho-c-Jun bind to the endogenous OPN promoter and that LXR agonists interfere with this binding, we next performed ChIP assays. PCR amplification using primer pairs that cover the AP-1 site at eC76 in the OPN promoter, demonstrated that this OPN promoter region coimmunoprecipitated with c-Fos and phospho-c-Jun after stimulation with TNF- (Figure 7B). This induction of c-Fos and phospho-c-Jun binding to the proximal OPN promoter was substantially inhibited by the LXR agonist. In concert, these findings indicate that TNF-eCinduced c-Fos and phospho-c-Jun binding to the AP-1 site at eC76 of the proximal OPN promoter is inhibited by LXR agonists.
TNF-eCInduced c-Fos and Phospho-c-Jun Protein Expression Is Inhibited by LXR Ligands
To further determine whether the inhibition of AP-1 binding by LXR ligands reflects changes in c-Fos or phospho-c-Jun expression levels, protein levels of both transcription factors were determined by Western blotting analysis. TNF-eCInduced c-Fos (Figure 8A) and phospho-c-Jun (Figure 8B) protein expression was significantly inhibited by treatment with the LXR agonist. Thus, suppression of TNF- and IL-1 induced binding to the AP-1 site at eC76 by LXR ligands reflects at least in part the regulation of c-Fos and phospho-c-Jun protein expression.
LXR Agonists Inhibit OPN Expression in Macrophages In Vivo
To finally determine whether the effects of LXR agonists to suppress OPN expression are applicable in vivo, T0901317 was administered to C57BL/6J mice for 7 days before isolation of peritoneal macrophages. The mRNA expression level of the LXR target gene ABCA1 was induced by 4.3-fold in peritoneal macrophages elicited from mice treated with T9091317, indicating sufficient availability of the LXR compound (data not shown). OPN protein (Figure 9A) and mRNA (Figure 9B) expression was substantially decreased in peritoneal macrophages from T0901317-treated mice compared with vehicle-treated mice (76±8% and 64±5% decrease in OPN mRNA and protein expression levels, respectively; n=4, P<0.005). Similarly, treatment of elicited murine peritoneal macrophages with the LXR agonist T0901317 dose-dependently suppressed TNF-eCinduced OPN protein expression (Figure 9C). Taken together, these results demonstrate that LXR ligands inhibit OPN expression in murine peritoneal macrophages and further support an important role for LXR ligands to suppress proinflammatory gene expression in vitro and in vivo.
Discussion
LXR signaling pathways have recently been proposed as potential target for therapeutic interventions in cardiovascular diseases.9,14 Whereas the antiatherogenetic effects of LXR agonists have been attributed to the modulation of cholesterol efflux from macrophages and induction of reverse cholesterol transport to the liver, recent evidence indicates that LXR ligands also modulate inflammatory gene expression.10,11 In the present study, we demonstrate that LXR agonists inhibit expression of OPN, a key inflammatory gene in macrophages. This inhibition is mediated through an inhibition of AP-1eCdependent transcriptional activation of the OPN promoter. These observations further support an important role for LXR agonists to suppress macrophage inflammatory responses.
We have recently demonstrated that OPN promotes atherogenesis by modulating leukocyte chemotaxis, cytokine and MMP expression, and macrophage survival.6 However, the molecular mechanisms that regulate OPN expression in macrophages during inflammatory processes are incompletely understood. OPN expression in macrophages was substantially induced after cytokine stimulation and this induction was transcriptionally suppressed by two LXR agonists. The OPN promoter is remarkably responsive to various stimuli, including cytokines, growth factors, and hormones.1 We identified a CCTCATGAC cognate in the OPN promoter located between eC80 and eC71 that supports basal and cytokine-induced transcriptional activity of the OPN promoter in macrophages. Consistent with previous reports in VSMCs, cold-competition and immunological supershift assays revealed AP-1 binding to this site.13,17 A variety of inflammatory genes are under the control of AP-1eCdependent signaling pathways; however, the transcriptional regulation of the OPN promoter by AP-1 in macrophages has not previously been investigated. The importance of this AP-1 site for the transcriptional activity of the OPN promoter was further demonstrated by a complete loss of basal and inducible OPN promoter activity after site-directed mutagenesis of this AP-1 site. Further characterization of the AP-1 complex binding to the OPN promoter revealed the presence of c-Fos and Ser-63 phosphorylated c-Jun binding activities. In addition to an induction of transcription from Fos and Jun genes, AP-1 activity is regulated through phosphorylation of c-Jun and phosphorylated c-Jun is the most potent transactivator of the AP-1 complex.18,19 Phosphorylation of c-Jun at Ser-63, located within its transactivation domain, potentiates its ability to activate transcription as either a homodimer or a heterodimer with c-Fos.19,20 Consistent with this, we observed that primarily Ser-63 phosphorylated c-Jun was present in the AP-1 complex bound to the CCTCATGAC cognate between eC80 and eC71 as opposed to unphosphorylated c-Jun.
The analysis of the OPN promoter did not reveal the presence of any putative LXRE response elements, indicating that the inhibition of cytokine-induced OPN transcription by LXR ligands likely involves an indirect mechanism through regulation of other transcription factors supporting OPN transcription. Because AP-1 binding activities appeared to be important for the transcriptional activity of the OPN promoter in macrophages, we next analyzed whether LXR agonists interfere with AP-1 binding to the proximal OPN promoter. EMSA and ChIP experiments indicated that LXR ligands suppress c-Fos and phospho-c-Jun binding to the AP-1 site at eC76 of the proximal OPN promoter. Results from transactivation studies further suggested that LXR ligands inhibit transactivation of a heterologous AP-1eCdriven promoter and that overexpression of c-Fos and c-Jun reverses the inhibition of OPN promoter activity by LXR ligands. Finally, LXR ligands inhibit cytokine-induced c-Fos and phospho-c-Jun protein expression, which may contribute at least in part to the observed inhibition of AP-1 binding to the proximal OPN promoter.
Cross-talk between different transcription factors and a similar negative interference with AP-1 activity has been described for ligand-induced activation of other nuclear receptors, such as the retinoic acid receptor (RAR),21 the glucocorticoid receptor (GR),22 or the peroxisome proliferatoreCactivated receptor (PPAR).23 Although PPAR ligands inhibit AP-1eCdependent signaling pathways, agonists for this receptor have been demonstrated to inhibit PMA-induced OPN expression in macrophage through interference with homeobox-like A/T-rich sequences between eC990 and eC981 of the OPN promoter.24 These findings suggest that the mechanisms by which LXR and PPAR agonists suppress OPN expression are distinct and likely dependent on the mechanisms of OPN induction. However, additional studies are necessary to delineate in more detail the molecular mechanism involved in the negative regulation of c-Fos/c-Jun transactivation by LXR ligands.
In conclusion, data presented in this study demonstrate the observation that cytokine-induced OPN expression in macrophages depends on AP-1 binding to the proximal OPN promoter. LXR agonists inhibit cytokine-induced OPN expression and interfere negatively with AP-1 binding to the proximal OPN promoter by inhibiting c-Fos and phospho-c-Jun protein expression. The suppression of OPN expression by LXR agonists are applicable in vivo as OPN expression is inhibited in mouse peritoneal macrophages elicited after treatment with the LXR agonist. Because OPN is a key component for the inflammatory response and the recruitment of monocytes/macrophages into the atherosclerotic lesion, inhibition of cytokine-induced OPN expression by LXR agonists could provide a potential target for therapeutic intervention in human cardiovascular disease.
Acknowledgments
This study was supported by a Scientist Development Grant 0435239N from the American Heart Association to Dennis Bruemmer and by a National Institutes of Health Grant HL 58328 to Willa A. Hsueh. Florian Blaschke was supported by a research fellowship from Philip Morris USA Incorporated.
References
Denhardt DT, Giachelli CM, Rittling SR. Role of osteopontin in cellular signaling and toxicant injury. Annu Rev Pharmacol Toxicol. 2001; 41: 723eC749.
O’Regan A. The role of osteopontin in lung disease. Cytokine Growth Factor Rev. 2003; 14: 479eC488.
Giachelli CM, Lombardi D, Johnson RJ, Murry CE, Almeida M. Evidence for a role of osteopontin in macrophage infiltration in response to pathological stimuli in vivo. Am J Pathol. 1998; 152: 353eC358.
Ashkar S, Weber GF, Panoutsakopoulou V, Sanchirico ME, Jansson M, Zawaideh S, Rittling SR, Denhardt DT, Glimcher MJ, Cantor H. Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity. Science. 2000; 287: 860eC864.
Weber GF, Zawaideh S, Hikita S, Kumar VA, Cantor H, Ashkar S. Phosphorylation-dependent interaction of osteopontin with its receptors regulates macrophage migration and activation. J Leukoc Biol. 2002; 72: 752eC761.
Bruemmer D, Collins AR, Noh G, Wang W, Territo M, Arias-Magallona S, Fishbein MC, Blaschke F, Kintscher U, Graf K, Law RE, Hsueh WA. Angiotensin IIeCaccelerated atherosclerosis and aneurysm formation is attenuated in osteopontin-deficient mice. J Clin Invest. 2003; 112: 1318eC1331.
Giachelli CM, Bae N, Almeida M, Denhardt DT, Alpers CE, Schwartz SM. Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J Clin Invest. 1993; 92: 1686eC1696.
Matsui Y, Rittling SR, Okamoto H, Inobe M, Jia N, Shimizu T, Akino M, Sugawara T, Morimoto J, Kimura C, Kon S, Denhardt D, Kitabatake A, Uede T. Osteopontin deficiency attenuates atherosclerosis in female apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2003; 23: 1029eC1034.
Tontonoz P, Mangelsdorf DJ. Liver X receptor signaling pathways in cardiovascular disease. Mol Endocrinol. 2003; 17: 985eC993.
Joseph SB, Castrillo A, Laffitte BA, Mangelsdorf DJ, Tontonoz P. Reciprocal regulation of inflammation and lipid metabolism by liver X receptors. Nat Med. 2003; 9: 213eC219.
Castrillo A, Joseph SB, Marathe C, Mangelsdorf DJ, Tontonoz P. Liver X receptor-dependent repression of matrix metalloproteinase-9 expression in macrophages. J Biol Chem. 2003; 278: 10443eC10449.
Joseph SB, McKilligin E, Pei L, Watson MA, Collins AR, Laffitte BA, Chen M, Noh G, Goodman J, Hagger GN, Tran J, Tippin TK, Wang X, Lusis AJ, Hsueh WA, Law RE, Collins JL, Willson TM, Tontonoz P. Synthetic LXR ligand inhibits the development of atherosclerosis in mice. Proc Natl Acad Sci U S A. 2002; 99: 7604eC7609.
Bidder M, Shao J-S, Charlton-Kachigian N, Loewy AP, Semenkovich CF, Towler DA. Osteopontin transcription in aortic vascular smooth muscle cells is controlled by glucose-regulated upstream stimulatory factor and activator protein-1 activities. J Biol Chem. 2002; 277: 44485eC44496.
Lund EG, Menke JG, Sparrow CP. Liver X receptor agonists as potential therapeutic agents for dyslipidemia and atherosclerosis. Arterioscler Thromb Vasc Biol. 2003; 23: 1169eC1177.
Joseph SB, Bradley MN, Castrillo A, Bruhn KW, Mak PA, Pei L, Hogenesch J, O’Connell RM, Cheng G, Saez E. LXR-dependent gene expression is important for macrophage survival and the innate immune response. Cell. 2004; 119: 299eC309.
Valledor AF, Hsu L-C, Ogawa S, Sawka-Verhelle D, Karin M, Glass CK. Activation of liver X receptors and retinoid X receptors prevents bacterial-induced macrophage apoptosis. PNAS. 2004; 101: 17813eC17818.
Renault M-A, Jalvy S, Belloc I, Pasquet S, Sena S, Olive M, Desgranges C, Gadeau A-P. AP-1 is involved in UTP-induced osteopontin expression in arterial smooth muscle cells. Circ Res. 2003; 93: 674eC681.
Devary Y, Gottlieb RA, Smeal T, Karin M. The mammalian ultraviolet response is triggered by activation of Src tyrosine kinases. Cell. 1992; 71: 1081eC1091.
Smeal T, Binetruy B, Mercola DA, Birrer M, Karin M. Oncogenic and transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serines 63 and 73. Nature. 1991; 354: 494eC496.
Deng T, Karin M. c-Fos transcriptional activity stimulated by H-Ras-activated protein kinase distinct from JNK and ERK. Nature. 1994; 371: 171eC175.
Schule R, Rangarajan P, Yang N, Kliewer S, Ransone L, Bolado J, Verma I, Evans R. Retinoic acid is a negative regulator of AP-1-responsive genes. Proc Natl Acad Sci U S A. 1991; 88: 6092eC6096.
Jonat C, Rahmsdorf HJ, Park KK, Cato AC, Gebel S, Ponta H, Herrlich P. Antitumor promotion and antiinflammation: down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. Cell. 1990; 62: 1189eC1204.
Delerive P, Martin-Nizard F, Chinetti G, Trottein F, Fruchart J-C, Najib J, Duriez P, Staels B. Peroxisome proliferator-activated receptor activators inhibit thrombin-induced endothelin-1 production in human vascular endothelial cells by inhibiting the activator protein-1 signaling pathway. Circ Res. 1999; 85: 394eC402.
Oyama Y, Akuzawa N, Nagai R, Kurabayashi M. PPAR{gamma} ligand inhibits osteopontin gene expression through interference with binding of nuclear factors to A/T-rich sequence in THP-1 cells. Circ Res. 2002; 90: 348eC355.(Daisuke Ogawa, Jeffrey F.)