Liver X Receptor-dependent Repression of Matrix Metalloproteinase-9 Expression in Macrophages*

Antonio CastrilloDagger §, Sean B. Joseph§, Chaitra Marathe§, David J. Mangelsdorf||, and Peter TontonozDagger §**

From the Dagger  Howard Hughes Medical Institute and the § Department of Pathology and Laboratory Medicine, University of California, Los Angeles, California 90095 and the  Howard Hughes Medical Institute, Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9050

Received for publication, December 23, 2002, and in revised form, January 15, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Matrix metalloproteinases (MMPs) are zinc endopeptidases that degrade extracellular matrix (ECM) components during normal and pathogenic tissue remodeling. Inappropriate expression of these enzymes contributes to the development of vascular pathology, including atherosclerosis. MMP-9 is expressed in its active form in atherosclerotic lesions and is believed to play an important role in vascular remodeling, smooth muscle cell migration, and plaque instability. We demonstrate here that the liver X receptors (LXRs) LXRalpha and LXRbeta inhibit basal and cytokine-inducible expression of MMP-9. Treatment of murine peritoneal macrophages with the synthetic LXR agonists GW3965 or T1317 reduces MMP-9 mRNA expression and blunts its induction by pro-inflammatory stimuli including lipopolysaccharide, interleukin-1beta , and tumor necrosis factor alpha . In contrast, macrophage expression of MMP-12 and MMP-13 is not altered by LXR ligands. We further show that the ability of LXR ligands to regulate MMP-9 expression is strictly receptor-dependent and is not observed in macrophages obtained from LXRalpha beta null mice. Analysis of the 5'-flanking region of the MMP-9 gene indicates that LXR/RXR heterodimers do not bind directly to the MMP-9 promoter. Rather, activation of LXRs represses MMP-9 expression, at least in part through antagonism of the NFkappa B signaling pathway. These observations identify the regulation of macrophage MMP-9 expression as a mechanism whereby activation of LXRs may impact macrophage inflammatory responses.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Matrix metalloproteinases (MMPs)1 are a large family of Zn2+-containing endopeptidases that play a key role in degrading extracellular matrix (ECM) components. MMP activation has been implicated in physiological and pathological tissue remodeling in multiple contexts, including cancer invasion, inflammation, organ development, angiogenesis, and wound healing (1). The MMP family contains more than 20 members, including collagenases, gelatinases, matrilysins, stromelysins, and metalloelastases. Active MMPs trigger the degradation of ECM components such as collagens, elastin, gelatin, fibronectin, laminin, and proteoglycans. In addition, MMP activity induces the processing of several non-ECM proteins, including alpha 1-antitrypsin, alpha 2-macroglobulin, plasminogen, and serum amyloid protein A (2). The MMP activity is regulated at several levels, including gene transcription, cellular secretion, activation of proenzymes, and binding to tissue inhibitors of metalloproteinases (TIMPs). Increasing experimental evidence supports a direct link between elevated MMP activity and diseases that involve enhanced turnover of the ECM, such as atherosclerosis and rheumatoid arthritis (3).

Enhanced expression of several MMPs, including MMP-9 and MMP-1, has been documented in both murine and human atherosclerotic lesions. Macrophages are a primary source of MMP expression, and their elaboration of these enzymes is believed to contribute to smooth muscle cell migration, neointima formation, and plaque rupture (4). MMP-9 degrades vessel wall collagens, elastin, and vitronectin (5), and matrix degradation in atherosclerotic plaques can be localized to areas of macrophage accumulation (6). It has also been demonstrated that MMP-9 is transiently up-regulated in experimental models of vascular injury and that its activity contributes to the development of intimal lesions by promoting smooth muscle cell migration (7). Previous work has shown that macrophage MMP-9 expression is induced by pro-inflammatory stimuli including cytokines, chemokines, and bacterial wall components (8-11).

In the present work, we demonstrate that ligand activation of the nuclear receptors LXRalpha and LXRbeta inhibits the expression of MMP-9 in macrophages. We also show that LXR-dependent repression of MMP-9 is accomplished through inhibition of the nuclear factor-kappa B (NFkappa B) pathway rather than direct binding of LXR/RXR heterodimers to the MMP-9 promoter. These results define a novel function of LXRs in the control of macrophage gene expression and support the potential utility of LXR ligands in the modulation of macrophage inflammatory responses.

    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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Reagents and Plasmids-- GW3965 and T0901317 were provided by Tim Willson and Jon Collins (GlaxoSmithKline). Ligands were dissolved in dimethyl sulfoxide prior to use in cell culture. LPS from Salmonella typhimurium was purchased from Sigma. TNFalpha , IFN-gamma and IL-1beta were from PeproTech. pCMX expression plasmids for LXRalpha , LXRbeta and RXR have been described (12, 13). The MMP-9 promoter constructs (-670, -531, and -70) linked to the firefly luciferase gene have been also described (14).

Cell Culture and Transfections-- Peritoneal macrophages were obtained from thioglycolate-injected mice as described (15) and cultured in RPMI 1640 medium containing 10% fetal bovine serum. Bone marrow-derived macrophages (BMDMs) from age-matched WT and LXRalpha beta -/- mice were prepared as described (16). Briefly, bone marrow cells extracted from femurs were incubated for 24 h in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum. After 24 h, nonadherent cells were collected and grown for 6 days in complete DMEM supplemented with 30% of filtered supernatant from L929 cells expressing macrophage colony-stimulating factor (M-CSF). On day 6, adherent BMDMs were deprived of M-CSF for 24 h and cultured in DMEM containing 10% lipoprotein protein-deficient serum (LPDS) for another 24 h prior to any stimulation. RAW 264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum. For ligand treatments, cells were cultured in RPMI 1640 or DMEM medium supplemented with 5% LPDS (Intracel) and receptor ligands for 18 h prior to LPS or cytokine stimulation. Transient transfections of RAW 264.7 cells were performed in triplicate in 48-well plates. 2 × 105 cells were transfected for 6 h with reporter plasmid (100 ng/well), receptor plasmids (10-50 ng/well), pCMV-beta -galactosidase (50 ng/well), and pTKCIII (to a total of 200 ng/well) using OptiMem-1 media and Superfect reagent (Invitrogen). After transfection, cells were incubated in media containing 10% LPDS and the indicated ligands or vehicle for 12 h prior to stimulation with LPS for another 18 h. Luciferase activity was normalized to beta -galactosidase activity.

Western Blot Analysis-- Macrophages were cultured in 10-cm dishes at 90% confluence with 5 ml of RPMI containing 5% LPDS. After treatment, conditioned media were collected and briefly centrifuged at 500 × g. Supernatants were collected, and protein content was assayed using the Bio-Rad protein reagent. For intracellular proteins, cytosolic and nuclear extracts were prepared as described previously (17). Samples containing equal amounts of protein were boiled in 250 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 2% beta -mercaptoethanol, and then size separated in 8% SDS-PAGE. Proteins were transferred to nitrocellulose, and the membrane was blocked overnight by 0.01% Tween 20 phosphate-buffered saline containing 5% nonfat milk. MMP-9, Ikappa Balpha , IKK-1, and beta -actin proteins were detected following the enhanced chemoluminescence (ECL) technique (Amersham Biosciences). Rabbit anti-MMP-9 antibody was from Torrey Pines Biolabs (Houston, TX; catalog number TP-221; kindly provided by Lisardo Bosca, Spain). Rabbit anti-Ikappa Balpha , anti-IKK-1, and anti-beta -actin antibodies were from Santa Cruz Biotechnology (catalog numbers SC-371, SC-7183, and SC-7210, respectively). Different exposure times of the films were used to ensure that bands were not saturated.

RNA Analysis-- Total RNA was extracted using Trizol reagent (Invitrogen). Northern analysis was performed as described (18). Blots were probed with specific radiolabeled probes for MMP-9, MMP-13, and MMP-12, and normalized using cDNA probe for 36B4. Probes were obtained by PCR using the following primers: MMP-9 Fwd, 5'-ttaccagcgccagccgacttttg-3'; MMP-9 Rev, 5'cgtcgtcgtcgaaatgggcatc-3'; MMP-13 Fwd, 5'-ggccagaactccccaaccat-3'; MMP-13 Rev, 5'-acccccaccccatacatctgaaagt-3', MMP-12 Fwd, 5'-ttctttgggctagaagcaactgggc-3'; and MMP12 Rev, 5'-gcagcttgaatagcagatgggatgc-3'. Real time quantitative PCR (TaqMan) analysis was performed using an Applied Biosystems 7700 sequence detector as described (19). Primer and probe sequences for MMP-9 were as follows: forward, 5'-tcaccttcacccgcgtgta3'; reverse, 5'-gtcctccgcgacaccaa-3'; and probe, 5'-/56-FAM-acccgaagcggacattgtcatccag/3BHQ-1/-3'. All assays were performed in duplicate, and cycle thresholds of individual genes were normalized to 36B4.

Electrophoretic Mobility Shift Assays-- The following oligonucleotides corresponding to consensus binding sites for NFkappa B and AP-1 were used (only one strand shown): NFkappa B, 5'-CCAACTGGGGACTCTCCCTTTGGGAACA-3'; and AP-1, 5'-TCGATTCCAAAGAGTCATCAG-3'. Oligonucleotides were labeled with Klenow enzyme (Promega). Electrophoretic mobility shift assays of nuclear extracts were performed by incubating DNA probe with 5 µg of nuclear protein for 30 min at 4 °C in a reaction mixture containing 1 µg/ml poly(dI·dC), 5% glycerol, 1 mM EDTA, 100 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, and 10 mM Tris-HCl, pH 7.8. Protein-DNA complexes were electrophoresed on a 6% native polyacrylamide gel and visualized by autoradiography. Competition with unlabeled oligonucleotides was performed using a 20-fold excess of double-stranded DNA in the binding reaction.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We reported previously that administration of a synthetic LXR ligand diminishes lipid accumulation and atherosclerotic lesion formation in murine models (19). In an effort to better understand the molecular basis of these effects, we used Affymetrix oligonucleotide arrays to identify novel LXR-regulated genes in macrophages (20, 21). These studies led to the identification of MMP-9 as a potential LXR-responsive gene. To validate these initial results, we examined basal and inducible expression of MMP-9 in cultured macrophages. Treatment of murine BMDMs with the synthetic LXR ligand GW3965 (22) significantly reduced basal MMP-9 expression as determined by Northern blotting and real time quantitative PCR assays (Fig. 1A, lanes 1-2). Because cytokines and bacterial endotoxins are potent inducers of MMP-9 production, we further investigated the effect of LXR ligand on MMP-9 response to these pro-inflammatory stimuli. BMDM were treated for 18 h with 2 µM GW3965, then stimulated for 24h with LPS or mouse recombinant TNFalpha or IL-1beta . MMP-9 expression was highly up-regulated in cells treated with all three stimuli (Fig. 1A, lanes 3-5). Preincubation of macrophages with the synthetic LXR agonist resulted in a substantial reduction of MMP-9 mRNA levels (Fig. 1A, lanes 6-8). The inhibitory effect of GW3965 on MMP-9 expression was dose-dependent over a concentration range of 100 nM to 2 µM (Fig. 1B). This dose response profile is similar to that observed for the induction of established LXR target genes such as ABCA1 and ABCG1 (13, 19, 22).


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Fig. 1.   The synthetic LXR agonist GW3965 inhibits MMP-9 expression in macrophages. A, BMDMs were pretreated for 18 h with vehicle or GW3965 (2 µM) in media containing 5% LPDS. Cells were then stimulated with LPS (200 ng/ml), IL-1-beta (20 ng/ml), or TNFalpha (20 ng/ml) for 24 h. RNA was isolated, and MMP-9 transcript levels were measured by Northern blotting and real time quantitative PCR (TaqMan). B, macrophages were treated for 24 h with the indicated concentrations of GW3965 and total RNA (10 µg per lane) was analyzed by Northern blotting and hybridized with 32P-labeled probes for MMP-9 and MMP-13. MMP-9 mRNA levels were also measured the by real time quantitative PCR. DMSO, Me2SO.

Next, we sought to determine whether the negative regulation observed for MMP-9 was unique or whether LXR activation similarly affected expression of other MMP family members in macrophages. We treated BMDM macrophages with LPS, TNFalpha , or IL-1beta in the absence or presence of two structurally unrelated LXR agonists, GW3965 and T1317 (23). As recent reports have shown that IFNs are potent inhibitors of MMP-9 expression in several cell types (14, 24), we also included IFN-gamma in our experiments as a positive control. Activation of LXR with either GW3965 or T1317 resulted in a significant inhibition of both basal and inducible MMP-9 expression (Fig. 2). As expected, IFN-gamma also inhibited MMP-9 expression in these cells in response to all three inflammatory stimuli. The pattern of expression of two other important metalloproteinases expressed by macrophages, MMP-12 and MMP-13 (25-27), was distinctly different from that of MMP-9. MMP-12 expression was repressed by LPS but was not affected by TNFalpha or IL-1beta . MMP-13 expression was induced by LPS but not by TNFalpha or IL-1beta . Surprisingly both MMP-12 and MMP-13 were repressed by IFN-gamma . Most importantly, in contrast to MMP-9, LXR agonists had no effect on the expression of MMP-12 and MMP-13.


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Fig. 2.   Differential regulation of macrophage MMP expression by LXR. BMDMs were cultured in DMEM containing 5% LPDS and incubated with LXR ligands (GW3965 (GW) or T1317 (T), 2 µM) or with IFNgamma (50 ng/ml) for 18 h. Macrophages were then challenged with LPS (200 ng/ml), IL-1-beta (20 ng/ml), or TNFalpha (20 ng/ml) for another 24 h as indicated. mRNA levels were measured by Northern blotting. DMSO, Me2SO.

To determine whether the inhibitory effect observed with GW3965 and T1317 is actually mediated by LXRs, we performed a series of experiments with macrophages derived from either WT mice or those with target disruptions of the LXRalpha and/or LXRbeta genes. Fig. 3A illustrates that the ability of GW3965 to inhibit both basal and LPS-induced expression of MMP-9 is completely abolished in peritoneal macrophages derived from LXRalpha beta -/- mice. Because GW3965 activates both LXRs, we also used knockout cells to determine whether the inhibitory effect of this ligand was mediated by LXRalpha , LXRbeta , or both. Peritoneal macrophages from mice carrying a single disruption of either LXRalpha or LXRbeta exhibited the same pattern of MMP-9 inhibition as WT macrophages after LXR agonist treatment (Fig. 3B). Thus, both LXRs are competent to mediate the inhibitory effect. We also confirmed these results with BMDM challenged with LPS for 6 and 18 h (Fig. 3C). GW3965 reduced expression of MMP-9 at both time points in WT cells, whereas no effect was observed in LXRalpha beta -/- cells. In contrast to MMP-9 expression, MMP-13 expression was not altered by LXR ligand and did not differ between LXR genotypes. To determine the effect of LXR activation on MMP-9 protein expression, we analyzed conditioned medium for secreted protein as described previously (14, 24, 28, 29). Inhibition of MMP-9 mRNA expression by an LXR agonist correlated with reduced levels of protein expression as revealed by Western blot analysis of conditioned media from WT BMDM treated for 24 h with LPS, TNFalpha , or IL-1beta (Fig. 3D). No change in MMP-9 protein expression was observed in LXRalpha beta -/- macrophages.


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Fig. 3.   LXR-dependent inhibition of MMP-9 in macrophages. A, thioglycolate-elicited peritoneal macrophages obtained from WT and LXRalpha beta -/- mice were incubated with 2 µM GW3965 (GW) for 18 h. Cells were treated with LPS (200 ng/ml) for 1.5, 3, and 6 h. mRNA levels of MMP-9 were measured by real time quantitative PCR. B, peritoneal macrophages from WT, LXRalpha -/-, LXRbeta -/-, or LXRalpha beta -/- mice were cultured with GW3965 or T1317 (2 µM) for 18 h and then stimulated with LPS (200 ng/ml) for 6 h. MMP-9 mRNA levels were quantitated by real time PCR. C, BMDM obtained from WT and LXRalpha beta -/- mice were pretreated with GW3965 (2µM) for 18 h and stimulated with LPS for 6 or 18 h. mRNA levels of MMP-9, MMP-13, and 36B4 were detected by Northern blotting. D, WT and LXRalpha beta -/- BMDMs were treated with GW3965 for 12 h and then challenged with LPS (200 ng/ml), IL-1beta (20 ng/ml), and TNFalpha (20 ng/ml) for 36 h. MMP-9 protein expression was analyzed by Western blotting. DMSO, Me2SO.

We also used WT and LXR null BMDM to characterize the effects of LXR agonists on TNFalpha -mediated induction of MMP-9 expression. Using TNFalpha as an inducer, we found that increasing concentrations of this cytokine (0.1-100 ng/ml) stimulated MMP-9 expression in both LXR WT and null cells. However, the genetic absence of LXR led to a significant increase in both basal and stimulus-dependent expression (Fig. 4A). This observation suggests that LXR null cells are more responsive than WT cells to TNFalpha stimulation, at least with respect to MMP-9 expression. It is also notable that the ability of LXR agonists to block MMP-9 expression was evident even in the presence of very high concentrations (100 ng/ml) of TNFalpha . We also performed a time course experiment to assess the persistence of the LXR inhibitory effects in the face of sustained inflammatory signals. Expression of MMP-9 mRNA in response to a moderate dose of TNFalpha (10 ng/ml) steadily increased over time (2-24 h, Fig. 4B). Longer periods of time were also evaluated, but no further increase was observed beyond 24 h (not shown). At all time points, the presence of GW3965 blunted the TNFalpha -dependent induction of MMP-9 in WT but not in LXRalpha beta -/- cells.


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Fig. 4.   Activation of LXR pages TNFalpha -induced MMP-9 expression. A, BMDMs obtained from WT and LXRalpha beta -/- mice were treated with vehicle or GW3965 (GW) (2 µM) for 18 h and stimulated for another 24 h with the indicated concentrations (ng/ml) of TNFalpha . B, BMDMs were cultured in 5% LPDS DMEM media containing GW3965 for 18 h. The cells were then activated for the indicated times with 10 ng/ml TNFalpha . MMP-9 transcript levels were analyzed by Northern blotting and/or real time quantitative PCR. DMSO, Me2SO.

The results presented above suggest that ligand activation of LXR inhibits transcription of the MMP-9 gene. The mechanism for this effect is unlikely to be direct, as LXR has not been reported to function as a ligand-dependent transcriptional repressor. Moreover, sequence analysis of the 5'-flanking region of the MMP-9 gene did not reveal the presence of potential LXR response elements (8, 30). However, the MMP-9 promoter does contain binding sites for both NFkappa B and AP-1 transcription factors, which are activated under pro-inflammatory conditions (10). We explored the possibility that LXR agonists may antagonize signaling pathways that up-regulate MMP-9 expression. We studied the effects of LXR on a luciferase reporter driven by the human MMP-9 promoter (-670 bp to +30 bp). This promoter fragment contains binding sites for NFkappa B and AP-1, Ets, and SP-1, and has been reported to contain the majority of the sequences responsible for MMP-9 activation under inflammatory conditions (14, 24) (Fig. 5). The MMP-9 promoter was transiently transfected into RAW 264.7 macrophages along with expression vectors for LXRalpha and RXRalpha . Following transfection, cells were treated with LPS and/or GW3965 as indicated in Fig. 5. As expected, a 10-fold increase in MMP-9 promoter activity was observed in response to LPS. Pretreatment with GW3965 led to a moderate reduction in luciferase activity in the presence of control vector, whereas a marked suppression was observed when LXRalpha /RXR expression vectors were co-transfected (Fig. 5A). To localize the regions important for mediating LXR-dependent repression, we analyzed truncated MMP-9 promoter constructs. Macrophages transfected with a construct containing sequences from -531 bp to + 30 bp of the promoter, which lacks the NFkappa B binding site, showed reduced luciferase activity after LPS treatment (Fig. 5B). However, the presence of the AP-1 site in this construct still permitted a 3-fold increase in luciferase activity. Remarkably, expression of this construct was not inhibited by GW3965 treatment even when LXRalpha was overexpressed. No significant luciferase activity was observed with a construct containing the minimal promoter of MMP-9 (-73 bp; Fig. 5C). These results indicate that the ability of LXR to inhibit the MMP-9 promoter requires the NFkappa B binding site.


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Fig. 5.   LXR activation inhibits MMP-9 promoter activity. RAW 264.7 cells were transiently transfected with MMP-9 promoter constructs (pGL3-670, pGL3-531, and pGL3-73) with or without pCMX, pCMX-LXRalpha , pCMX-RXRalpha , and pCMV-beta -galactosidase as described under "Materials and Methods." After transfection, macrophages were incubated with vehicle or GW3965 (GW) (2 µM) for 12 h in DMEM containing 10% LPDS. Cells were then stimulated with 100 ng/ml LPS for 18 h. Luciferase activity was normalized to beta -galactosidase activity. DMSO, Me2SO.

The results of Fig. 5 strongly suggest that LXR activation inhibits MMP-9 expression through antagonism of the NFkappa B signaling pathway. To investigate potential mechanisms for this effect, we treated WT peritoneal macrophages with GW3965 and then activated them with 200 ng/ml LPS. Cells were harvested at different time points, and soluble extracts were collected. Cytosolic proteins were size separated, and the levels of Ikappa Balpha protein were analyzed by Western blotting. Ikappa Balpha is degraded within the first 30 min of stimulation with LPS and then rapidly re-synthesized to provide negative feedback control and limit NFkappa B activation. Fig. 6 demonstrates that the upstream signaling pathway leading to Ikappa Balpha degradation is not altered in response to LXR activation. Ikappa Balpha is degraded to the same extent with or without GW3965 after 30 min of LPS challenge in both WT and LXRalpha beta -/- macrophages. We also analyzed the expression levels of IKK-1, one of the catalytic subunits responsible for Ikappa Balpha phosphorylation. Again, no significant changes were observed after GW3965 treatment in either genotype (Fig. 6). Taken together, these data indicate that LXR-mediated inhibition of MMP-9 expression is downstream of Ikappa Balpha phosphorylation-degradation.


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Fig. 6.   Activation of LXR does not inhibit Ikappa Balpha degradation. Thioglycolate-elicited peritoneal macrophages obtained from WT and LXRalpha beta -/- mice were pretreated with GW3965 (2 µM) for 18 h and challenged with 100 ng/ml LPS for 30 min. Cytosolic and nuclear extracts were obtained as described under "Materials and Methods." The protein levels of IKK-1, Ikappa Balpha , and beta -actin were measured by Western blotting. DMSO, Me2SO.

To further investigate the mechanism of LXR effects on the NFkappa B pathway, we examined NFkappa B DNA binding activity. WT or LXRalpha beta -/- macrophages were preincubated with GW3965 and then activated with 200 ng/ml LPS. After 30 min of stimulation, cytoplasmic and nuclear extracts were collected. Electrophoretic mobility shift assays were performed by incubating these extracts with radiolabeled consensus binding sites for NFkappa B or AP-1. No significant changes were observed in the protein-DNA complexes in the presence of 1 µM GW3965 (Fig. 7A). We also analyzed the binding of NFkappa B proteins at 30 min in WT and LXRalpha beta -/- macrophages treated with increasing concentrations of GW3965. Again, the LPS-induced NFkappa B DNA binding was not altered by LXR ligand and was not different between genotypes (Fig. 7B). These observations indicate that the signaling pathways that lead to NFkappa B activation are not significantly impaired by LXR and suggest that LXR agonists may exert their effects downstream of NFkappa B binding to DNA.


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Fig. 7.   LXR activation does not prevent NFkappa B or AP-1 binding to DNA. A, thioglycolate-elicited peritoneal macrophages were preincubated with LXR ligand (2 µM) for 18 h and stimulated with LPS for another 30 min. Nuclear proteins were incubated with 32P-labeled oligonucleotides containing consensus binding sites for NFkappa B or AP-1 and analyzed by electrophoretic mobility shift assay. B, thioglycolate-elicited peritoneal macrophages obtained from WT and LXRalpha beta -/- mice were cultured with the indicated concentrations of GW3965 (GW) for 18 h. After 30 min of stimulation with LPS (100 ng/ml), nuclear extracts were collected. Binding to NFkappa B DNA sequences was analyzed by electrophoretic mobility shift assay. DMSO, Me2SO.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The liver X receptors are members of the nuclear receptor superfamily activated by oxysterols and play an important role in maintaining lipid and lipoprotein homeostasis. In macrophages wherein both LXRs are highly expressed, these receptors control the cholesterol efflux pathway through the regulation of target genes including ABCA1 and apolipoprotein E (apoE) (12, 31-33). The synthetic LXR ligand GW3965 reduces the development of atherosclerosis in low density lipoprotein receptor (LDLR) and apoE-deficient mice (19). Conversely, LXRalpha beta null mice exhibit foam cell accumulation in multiple tissues, including aorta, lung, spleen, and brain (19, 34, 35). Transplantation of LXR-deficient bone marrow into LDLR and apoE knockout mice results in increased atherosclerosis, demonstrating the relevance of macrophage LXR activity in the prevention of atherosclerosis (34). In an effort to better understand the molecular basis of these effects, we have endeavored to identify novel LXR target genes in macrophages. In the present study we have shown that ligand activation of LXRs in macrophages inhibits both basal and cytokine-inducible expression of the MMP-9 gene. These observations support a role for LXRs in the regulation of macrophage inflammatory responses.

The relationship between chronic inflammation and atherosclerosis is clearly evident. Components and mediators of the immune system, including macrophages, T cells, complement, and inflammatory cytokines are key players in the atherogenic process (36-38). MMP-9 is highly expressed by macrophages and smooth muscle cells in areas of atheroma formation and participates in the degradation of ECM components at the shoulder regions of atherosclerotic lesions (7, 26, 39). This proteolytic activity is proposed to lead to plaque instability and ultimately to plaque rupture and the resulting thrombus formation. Targeted disruption of the MMP-9 gene results in decreased smooth muscle cell migration in vitro and in vivo and reduces intimal hyperplasia (29). In addition, administration of broad MMP inhibitors in mouse models results in reduced progression of atherosclerotic lesions and neointima formation (26, 40). However, the availability of specific MMP inhibitors is still very limited. There is therefore considerable interest in identifying alternative mechanisms by which excessive MMP production can be limited.

The results presented here demonstrate that LXRalpha and LXRbeta are negative regulators of MMP-9 expression in macrophages and are consistent with our recent observations that LXR agonists repress the expression of multiple NFkappa B target genes (20). The LXR-selective agonists GW3965 and T1317 inhibit MMP-9 expression induced by LPS, TNFalpha , and IL-1beta , suggesting that they are likely to act on common downstream effectors of these signaling pathways. Previous studies have highlighted the central role of NFkappa B and AP-1 transcription factors in the regulation of MMP-9 gene expression. These factors are activated by common stimuli, and their activity is crucial for the rapid response to cellular injury (41, 42). Several inflammatory molecules that are released in the atherosclerotic environment are potent inducers of NFkappa B and AP-1. Activated NFkappa B can be detected in atherosclerotic lesions, mainly within macrophages and smooth muscle cells, whereas little NFkappa B activation is detected in healthy vessels (43). Thus, the enhanced expression of inflammatory genes such as MMP-9 during the development of atherosclerosis can be ascribed to the activation of these transcription factors. Our results suggest that LXR activation leads to an impairment of NFkappa B action on the MMP-9 promoter. Unfortunately, the mechanism underlying this effect is not yet clear. We found that the upstream signaling pathway controlling NFkappa B activation is not affected by LXR. LPS-induced Ikappa B degradation and nuclear translocation of NFkappa B were not altered by LXR ligands and were not different in WT and LXR null macrophages. These observations suggest that LXR agonists may exert their effects downstream of NFkappa B binding to DNA. More research will be needed to determine the mechanism underlying LXR suppressive effects.

Interference with NFkappa B signaling has been proposed by other authors (14, 24) to explain the inhibitory effects of IFNs on MMP-9 expression in several cancer cell types. IFNs inhibit MMP-9 expression through activation of interferon regulatory factor 1 (IRF-1), which blocks NFkappa B activity without affecting either Ikappa B degradation or NFkappa B nuclear translocation (14, 24). Interestingly, our results show that IFN-gamma also inhibits the expression of MMP-12 and MMP-13, reinforcing the recently proposed suppressive facet of IFN-gamma in macrophages (44). Interestingly, other members of the nuclear receptor family, including PPAR and the glucocorticoid receptor (GR), have also been shown to inhibit MMP-9 expression. Administration of synthetic PPARgamma ligands to apoE-/- mice decreases the expression of MMP-9 in aortic tissue (45). In vitro, high concentrations of rosiglitazone inhibit macrophage activation, including MMP-9 expression (46). However the mechanism of PPARgamma -dependent repression remains to be elucidated, because suppressive effects with PPARgamma ligands have also been observed in PPARgamma -/- cells (47).

In summary, our results identify LXRalpha and LXRbeta as negative regulators of MMP-9 expression in macrophages. LXR activation interferes with NFkappa B function on the MMP-9 promoter under inflammatory situations without affecting its DNA binding capacity. Elucidation of the mechanism behind these effects may offer new strategies for limiting excessive MMP-9 expression in lesion macrophages and providing stability to the atherosclerotic plaque.

    ACKNOWLEDGEMENTS

We thank Tim Willson and Jon Collins for GW3965 and T1317, D. Boyd and M. Seiki for MMP-9 promoter constructs, and Lisardo Bosca and Carlos Lopez-Otin for reagents and comments.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| Investigator of the Howard Hughes Medical Institute at the University of Texas Southwestern Medical Center.

** Assistant Investigator of the Howard Hughes Medical Institute at the University of California, Los Angeles, and to whom correspondence should be addressed: Howard Hughes Medical Inst., UCLA School of Medicine, Box 951662, Los Angeles, CA 90095-1662. Tel.: 310-206-4546; Fax: 310-267-0382; E-mail: ptontonoz@mednet.ucla.edu.

Published, JBC Papers in Press, January 16, 2003, DOI 10.1074/jbc.M213071200

    ABBREVIATIONS

The abbreviations used are: MMP, matrix metalloproteinase; ECM, extracellular matrix; LXR, liver X receptor; NFkappa B, nuclear factor kappa B; RXR, retinoid X receptor; LPS, lipopolysaccharide; TNFalpha , tumor necrosis factor alpha ; IFN-gamma , interferon gamma ; IL-1beta , interleukin 1beta ; BMDM, bone marrow-derived macrophage; WT, wild type; DMEM, Dulbecco's modified Eagle's medium; LPDS, lipoprotein-deficient serum; IKK-1, Ikappa B kinase 1; PPAR, peroxisome proliferator-activated receptor.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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