Liver X Receptor-dependent Repression of
Matrix Metalloproteinase-9 Expression in Macrophages*
Antonio
Castrillo
§,
Sean B.
Joseph§,
Chaitra
Marathe§,
David J.
Mangelsdorf¶
, and
Peter
Tontonoz
§**
From the
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 |
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)
LXR
and LXR
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-1
, and tumor necrosis factor
. 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 LXR
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 NF
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 |
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
1-antitrypsin,
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 LXR
and LXR
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-
B (NF
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 |
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. TNF
,
IFN-
and IL-1
were from PeproTech. pCMX expression plasmids for
LXR
, LXR
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
LXR

/
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-
-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
-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%
-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, I
B
, IKK-1, and
-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-I
B
, anti-IKK-1, and anti-
-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 NF
B
and AP-1 were used (only one strand shown): NF
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 |
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 TNF
or IL-1
. 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- (20 ng/ml), or TNF (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, TNF
, or IL-1
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-
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-
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
TNF
or IL-1
. MMP-13 expression was induced by LPS but not by
TNF
or IL-1
. Surprisingly both MMP-12 and MMP-13 were repressed
by IFN-
. 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 IFN (50 ng/ml) for 18 h.
Macrophages were then challenged with LPS (200 ng/ml), IL-1- (20 ng/ml), or TNF (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 LXR
and/or LXR
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 LXR

/
mice. Because GW3965 activates both LXRs,
we also used knockout cells to determine whether the inhibitory effect
of this ligand was mediated by LXR
, LXR
, or both. Peritoneal
macrophages from mice carrying a single disruption of either LXR
or
LXR
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 LXR

/
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, TNF
, or IL-1
(Fig. 3D). No change
in MMP-9 protein expression was observed in LXR

/
macrophages.

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Fig. 3.
LXR-dependent inhibition of MMP-9
in macrophages. A, thioglycolate-elicited peritoneal
macrophages obtained from WT and LXR  / 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, LXR / , LXR / ,
or LXR  / 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 LXR  / 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 LXR  / BMDMs were treated with GW3965 for 12 h and
then challenged with LPS (200 ng/ml), IL-1 (20 ng/ml), and TNF
(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 TNF
-mediated induction of MMP-9 expression. Using TNF
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
TNF
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 TNF
. 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 TNF
(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
TNF
-dependent induction of MMP-9 in WT but not in
LXR

/
cells.

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Fig. 4.
Activation of LXR pages
TNF -induced MMP-9 expression.
A, BMDMs obtained from WT and LXR  / 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 TNF . 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 TNF . 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 NF
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 NF
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 LXR
and RXR
. 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 LXR
/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 NF
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 LXR
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 NF
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-LXR , pCMX-RXR , and pCMV- -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 -galactosidase activity. DMSO,
Me2SO.
|
|
The results of Fig. 5 strongly suggest that LXR activation inhibits
MMP-9 expression through antagonism of the NF
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 I
B
protein were analyzed by Western blotting.
I
B
is degraded within the first 30 min of stimulation with LPS
and then rapidly re-synthesized to provide negative feedback control
and limit NF
B activation. Fig. 6
demonstrates that the upstream signaling pathway leading to I
B
degradation is not altered in response to LXR activation. I
B
is
degraded to the same extent with or without GW3965 after 30 min of LPS challenge in both WT and LXR

/
macrophages. We also analyzed the expression levels of IKK-1, one of the catalytic subunits responsible for I
B
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 I
B
phosphorylation-degradation.

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Fig. 6.
Activation of LXR does not inhibit
I B degradation.
Thioglycolate-elicited peritoneal macrophages obtained from WT and
LXR  / 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, I B , and -actin were
measured by Western blotting. DMSO, Me2SO.
|
|
To further investigate the mechanism of LXR effects on the NF
B
pathway, we examined NF
B DNA binding activity. WT or LXR

/
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 NF
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 NF
B proteins at 30 min in WT and
LXR

/
macrophages treated with increasing concentrations of
GW3965. Again, the LPS-induced NF
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 NF
B activation are not significantly impaired by LXR
and suggest that LXR agonists may exert their effects downstream of
NF
B binding to DNA.

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Fig. 7.
LXR activation does not prevent
NF 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 NF B or AP-1 and analyzed by electrophoretic mobility shift
assay. B, thioglycolate-elicited peritoneal macrophages
obtained from WT and LXR  / 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 NF B DNA sequences was analyzed by
electrophoretic mobility shift assay. DMSO,
Me2SO.
|
|
 |
DISCUSSION |
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, LXR
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 LXR
and LXR
are
negative regulators of MMP-9 expression in macrophages and
are consistent with our recent observations that LXR agonists repress the expression of multiple NF
B target genes (20). The LXR-selective agonists GW3965 and T1317 inhibit MMP-9 expression induced
by LPS, TNF
, and IL-1
, suggesting that they are likely to act on common downstream effectors of these signaling pathways. Previous studies have highlighted the central role of NF
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 NF
B and AP-1. Activated NF
B can be detected in atherosclerotic lesions, mainly within macrophages and smooth muscle cells, whereas little NF
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 NF
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 NF
B
activation is not affected by LXR. LPS-induced I
B degradation and
nuclear translocation of NF
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 NF
B
binding to DNA. More research will be needed to determine the mechanism underlying LXR suppressive effects.
Interference with NF
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 NF
B activity without affecting either I
B degradation or NF
B nuclear translocation (14, 24).
Interestingly, our results show that IFN-
also inhibits the
expression of MMP-12 and MMP-13, reinforcing the recently proposed
suppressive facet of IFN-
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 PPAR
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 PPAR
-dependent repression
remains to be elucidated, because suppressive effects with PPAR
ligands have also been observed in PPAR
/
cells (47).
In summary, our results identify LXR
and LXR
as negative
regulators of MMP-9 expression in macrophages. LXR
activation interferes with NF
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;
NF
B, nuclear factor
B;
RXR, retinoid X receptor;
LPS, lipopolysaccharide;
TNF
, tumor necrosis factor
;
IFN-
, interferon
;
IL-1
, interleukin 1
;
BMDM, bone marrow-derived
macrophage;
WT, wild type;
DMEM, Dulbecco's modified Eagle's medium;
LPDS, lipoprotein-deficient serum;
IKK-1, I
B kinase 1;
PPAR, peroxisome proliferator-activated receptor.
 |
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