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J. Biol. Chem., Vol. 277, Issue 7, 4713-4721, February 15, 2002
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From the Department of Biochemistry, Faculty of Health Sciences,
McMaster University, Hamilton, Ontario L8N 3Z5, Canada
Received for publication, September 12, 2001, and in revised form, November 23, 2001
Liver X receptor Liver X receptors (LXR Recent work has shown that LXR Exposure of monocytes and other cell types to oxLDL and some of its
constituent oxysterols and oxidized lipids exert pleiotropic effects on
gene expression and elicit numerous cellular changes that are
associated with normal cell function and molecular processes linked to
pathological states (17). These include alteration in the expression of
genes that are not necessarily involved in regulating cellular lipid
content and homeostasis. For instance, studies have shown that the
expression of pro-inflammatory cytokines such as tumor necrosis
factor- Although oxLDL provides ligands for LXR Reagents--
22(R)-Hydroxycholesterol
(22(R)-HC) was obtained from Research Plus Ltd.;
22(S)-hydroxycholesterol (22(S)-HC),
25-hydroxycholesterol, 9-cis-retinoic acid (9cRA), and
Escherichia coli 011:B5 lipopolysaccharide (LPS) were
obtained from Sigma. Actinomycin D and cycloheximide were obtained from
Sigma. Oxidized low density lipoprotein (oxLDL) was obtained from
Intracel Perimmune (Rockville, MD).
Plasmids--
Human LXR Cell Culture--
THP-1 human monocytic cells (obtained from
American Type Tissue Collection) were cultured in RPMI 1640 media
supplemented with 10% v/v fetal bovine serum, 1% v/v
penicillin/streptomycin, and 1% v/v L-glutamine. Human
peripheral blood mononuclear cells were collected from healthy donors,
and monocytes were isolated from buffy coat preparations using a MACS
Monocyte Isolation kit (Milentyi Biotec) according to the
manufacturer's instructions. Cells were cultured in RPMI 1640 supplemented with 10% autologous serum, 1% v/v
penicillin/streptomycin, and 1% v/v L-glutamine. Experiments were initiated on the day blood was collected. COS-1 cells
(ATCC) were cultured in Dulbecco's minimum essential medium supplemented with 10% v/v fetal bovine serum, 1% v/v
penicillin/streptomycin, and 1% v/v L-glutamine.
Cytokine Assays--
THP-1 and human monocytes (106
cells/ml) were cultured as above and incubated in the presence of the
various compounds as indicated in the figure legends. Control cells
received the equivalent amount of vehicle (Me2SO,
ethanol, or water) as indicated. The levels of human TNF- RNA Analysis--
Total RNA from THP-1 cells (4 × 106 cells/sample) was isolated using the RNeasy Mini
isolation kit (Qiagen, Chatsworth, CA) and subjected to Northern
analysis under standard conditions using random-primed
32P-radiolabeled probes generated from cDNAs for human
TNF- Transfections and Luciferase Assay--
Transient transfection
of COS-1 cells was carried out using LipofectAMINE (Invitrogen, San
Diego, CA) according to the manufacturer's instructions. Briefly,
cells (3 × 105 cells/well in 6-well plates) were
transfected using 4 µl of LipofectAMINE along with 0.5 µg of a
luciferase reporter gene, 0.5 µg of pRC/CMV-LXR Metabolic Labeling and Immunoprecipitation--
THP-1 cells
(4 × 106 cells per sample) were cultured in
methionine- and cysteine-free RPMI containing 1% dialyzed fetal bovine serum and 100 µCi/ml [35S]methionine/cysteine
(PerkinElmer Life Sciences) for 12 h at 37 °C in the presence
of LXR and RXR ligands (10 µM) or vehicle, as described
in the figure legend. Cell extracts were prepared as described (30) and
precleared using goat IgG-agarose conjugate (Santa Cruz
Biotechnologies, Santa Cruz, CA). Equivalent amounts of radioactivity
from each sample were immunoprecipitated with polyclonal goat
anti-human TNF- Electrophoretic Mobility Shift Analysis--
Electrophoretic
mobility shift assays, using human LXR Oxysterols and 9cRA Cooperatively Induce the Production of TNF-
As we observed in cells treated with LPS (Fig. 1, A and
B), secretion of TNF-
Several reports have shown that oxLDL inhibits LPS-mediated TNF-
Several lines of evidence indicate that
22(R)-HC/9cRA-dependent induction of TNF- Oxysterols Induce TNF-
To determine if 22(R)-HC increased TNF-
TNF- LXR LXR
Examination of the
The foregoing establishes the human TNF- 9cRA Triggers Release of TNF-
To begin to unravel this multistep pathway, we carried out
order-of-addition experiments with oxysterols and 9cRA in the presence of various inhibitors. As outlined in Fig.
9, the basic approach involved
preincubating THP-1 cells with 22(R)-HC under various conditions for 12 h, a time interval that we previously determined to be sufficient to detect TNF-
Co-incubation of 22(R)-HC at t = 0 with the
protein synthesis inhibitor cycloheximide (which does not affect
TNF- The emergent role of LXR Transfection analysis and inhibitor studies demonstrated that the
TNF- TNF- The penetration of monocytes into the vascular intima and their
differentiation into lipid-loaded foam cells through influx of oxLDL by
scavenger receptors such as CD36 and SR-A is one of the earliest steps
in atherogenesis (17, 42). In this context, LXR In summary, we demonstrate that the human TNF- We thank J. Gauldie and M. Hitt (McMaster
University) for TNF- *
This work was supported in part by the Canadian Institutes
of Health Research.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.
§
To whom correspondence should be addressed: Tel.: 905-525-9140 (ext. 22184); Fax: 905-546-0800; E-mail: caponej@mcmaster.ca.
Published, JBC Papers in Press, December 10, 2001, DOI 10.1074/jbc.M108807200
The abbreviations used are:
LXR, liver X
receptor;
22(R)-HC, 22(R)-hydroxycholesterol;
22(S)-HC, 22(S)-hydroxycholesterol;
9cRA, 9-cis-retinoic acid;
LXRE, LXR-response element;
oxLDL, oxidized low density lipoprotein;
RXR, 9-cis-retinoic acid
receptor (retinoid X receptor);
TNF-
Oxysterol Activators of Liver X Receptor and
9-cis-Retinoic Acid Promote Sequential Steps in the
Synthesis and Secretion of Tumor Necrosis Factor-
from Human
Monocytes*
,
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
(LXR), is a
nuclear hormone receptor that is activated by oxysterols and plays a
crucial role in regulating cholesterol and lipid metabolism in liver
and cholesterol efflux from lipid-loaded macrophages. Here we show that
treatment of human peripheral blood monocytes or monocytic THP-1 cells
with the LXR ligand 22(R)-hydroxycholesterol
(22(R)-HC), in combination with 9-cis-retinoic
acid (9cRA), a ligand for the LXR heterodimerization partner retinoid X
receptor (RXR), results in the specific induction of the potent
pro-apoptotic and pro-inflammatory cytokine tumor necrosis factor-
(TNF-). Promoter analysis, inhibitor studies, and order-of-addition
experiments demonstrated that TNF- induction by 22(R)-HC
and 9cRA occurs by a novel two-step process. The initial step involves
22(R)-HC-dependent induction of TNF-
mRNA, and intracellular accumulation of TNF- protein, mediated
by binding of LXR/RXR to an LXR response element at position
879 of the TNF- promoter. Subsequent cell release of TNF-
protein occurs via a separable 9cRA-dependent,
LXR-independent step that requires de novo transcription
and protein synthesis. Our findings reveal a potentially new dimension
of the physiological role of LXR and identify a unique multistep
pathway of TNF- production that may be of consequence to the normal
function of LXR in monocyte/macrophages and in disease conditions such
as atherosclerosis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(NR1H3) and LXR
(NR1H2))1 are recently
described members of the nuclear hormone receptor superfamily of
ligand-activated transcription factors that are important in the
regulation of genes that govern cholesterol homeostasis in the liver
and cholesterol efflux from peripheral tissues. LXRs are bound by and
activated by specific cholesterol metabolites, including oxysterols,
and regulate the expression of target genes by binding to specific
promoter response elements (LXREs) in association with the obligate
heterodimerization partner, retinoid X receptor (RXR), the receptor for
9-cis-retinoic acid (9cRA) (1). Natural ligands for LXR
include 22(R)-hydroxycholesterol (22(R)-HC),
24(S),25-epoxycholesterol, and 25-hydroxycholesterol (2-4),
compounds that have been found free in serum and in association with
atherogenic oxidized low density lipoprotein (oxLDL) particles (5-7).
In the liver, LXR serves as a sterol sensor and regulates the
expression of genes that influence cholesterol metabolism and
homeostasis, including the genes encoding cholesterol 7-hydroxylase,
which controls the cholesterol/bile acid synthetic pathway, and sterol
regulatory element-binding protein-1c, a key transcription factor that
regulates expression of genes important in fatty acid biosynthesis (6, 8, 9-11). Studies using lxr null mice have shown that
LXR is essential for normal cholesterol homeostasis and secretion of
excess cholesterol in vivo (9).
plays a fundamental role in
macrophage biology by regulating cholesterol efflux from lipid-loaded cells. This is manifested by LXR-mediated induction of genes encoding the ATP-binding cassette proteins ABC-1 and ABCG1, which encode plasma membrane-associated reverse cholesterol transport proteins that mediate cholesteryl ester and free cholesterol efflux from monocytes and lipid-loaded macrophages (12-15). Effluxed
cholesterol is subsequently delivered to extracellular acceptors,
especially high density lipoprotein/ApoE, and transported back to the
liver where it is converted to bile acids and excreted (1). The key role of LXR as a master regulator in the overall process governing cholesterol efflux and reducing intracellular cholesterol levels is
underscored by the findings that the genes encoding ApoE, which is
necessary for high density lipoprotein formation is also a transcriptional target of LXR (16). The pivotal role of LXR in
regulating reverse cholesterol transport in macrophages is of
particular disease relevance, because lipid accumulation in these
cells, through the uptake of oxLDL, is of fundamental importance to the
etiology and pathogenesis of atherogenesis and atherosclerosis and
other chronic inflammatory diseases. oxLDL particles accumulate in
macrophages that have infiltrated the arterial intimal space, subsequently developing into lipid-loaded foam cells that comprise the
characteristic fatty streak of early atherosclerotic lesions (17). In
this context, LXR, by reducing intracellular cholesterol and lipid
accumulation, is considered to be anti-atherogenic. Consistent with
this, recent studies have shown that selective agonists of RXR, which
activate LXR/RXR heterodimers, significantly reduce lesion size and the
progression of atherosclerosis in apoE/ animals (18).
(TNF-), interleukin (IL)-1, IL-1, IL-6, IL-8, and
platelet-derived growth factor are differentially modulated in
macrophages in response to oxLDL or various oxysterols (19-22).
Similarly, other studies have demonstrated diverse effects of oxLDL and
oxysterols in vascular endothelial cells and smooth muscle cells
(23-25). The cytokines described above provoke local inflammatory
responses and can induce apoptosis of macrophages resident within the
arterial intimal space and smooth muscle cells in the medial layer of
the arterial wall (17). They can also promote T-cell infiltration and
tissue necrosis that contribute to the late stage necrotic core within
complex atherosclerotic lesions (26).
, and macrophages may
generate endogenous oxysterols that can activate LXR (6), a role for
LXR and LXR agonists in cytokine production in monocytes/macrophages has not been described. Here, we demonstrate that administration of the
LXR-specific ligand 22(R)-HC to peripheral blood-derived human monocytes and to monocytic THP-1 cells, in the presence of 9cRA,
results in the specific and selective production of bioactive TNF-,
a key pro-inflammatory and pro-apoptotic cytokine. We further show that
TNF- production occurs via a novel two-step process that involves an
initial oxysterol-dependent induction of TNF- mRNA
and intracellular protein accumulation mediated by LXR/RXR binding to an LXRE in the TNF- promoter, and subsequent cell release
of TNF- protein via a separable 9cRA-dependent,
LXR-independent step, which requires de novo transcription
and protein synthesis. These observations establish a novel role for
LXR and oxysterols in monocyte function and identify a unique pathway
of cytokine production in these cells.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and RXR mammalian expression
vectors pRC/CMV-LXR and pSG5-RXR have been previously described
(27, 28). Luciferase reporter plasmids used were
pXP1-TNF(1311)luc, which contains 1.3 kb of the human
TNF- promoter/regulatory region linked to the luciferase gene (29).
Plasmids used in promoter mapping experiments were constructed
from pXP1-TNF(1311)luc as follows; pTNF(914/359)luc was generated by a BstXI
collapse of pTNF(1311)luc, pTNF(971/762)luc
was generated by a MscI collapse of
pTNF(1311)luc, pTNF(641/493)luc was
generated by a StuI collapse of pTNF(1311)luc, and pTNF(987/105)luc was generated by a SstI
collapse of pTNF(1311)luc. pTNF(932/851)luc
was generated by cloning a single copy of a synthetic double-stranded
oligonucleotide corresponding to nucleotides 932 to 851 of the
TNF- promoter into the BamHI of the luciferase expression
vector pGL2luc (Promega). pTNF(894/866)luc
and pTNFmut(894/866)luc contained a single copy of a
synthetic double-stranded oligonucleotide corresponding to nucleotides
894 to 866 of the TNF- promoter, and a mutant variant thereof,
respectively (sequences provided below), into the BamHI site
of expression vector pGL2luc (Promega). All plasmid
constructions were verified by DNA sequence analysis.
, IL-1,
or IL-6 present in the culture media were measured, as specified, by
enzyme-linked immunosorbent assays (ELISA) using OptEIA kits (BD
PharMingen, San Diego, CA) according to the manufacturer's instructions.
, and glyceraldehyde-3-phosphate dehydrogenase as described
(30). Radioactive bands were quantified by phosphorimaging analysis of
the dried gel and normalized to the radioactivity present in the
glyceraldehyde-3-phosphate dehydrogenase signal, which was used as an
internal standard. RNA analysis was also performed by reverse
transcriptase-PCR using a commercially available kit (MBI Fermentas,
Hamilton, Ontario, Canada).
, and 0.5 µg
pSG5-RXR (28), as indicated. Transfections included 0.5 µg of
pCMVlacZ, which encodes the gene for -galactosidase, to
serve as an internal control for transfection efficiency. Total DNA and
promoter dosage were kept constant with pRC/CMV and pSG5 empty vectors
(Invitrogen), as appropriate. RXR and/or LXR ligands were dissolved in
ethanol or Me2SO and added to a final concentration of 10 µM each. Control cells received the equivalent amount of vehicle. Cell extracts were prepared 48 h post-transfection, and luciferase assays and -galactosidase assays were carried out as
previously described (28).
IgG (TNF- N-19, Santa Cruz Biotechnologies) followed by protein G-Sepharose (Boehringer-Ingelheim, Germany). Immune
complexes were resolved by 15% SDS-polyacrylamide gel electrophoresis, and radioactivity in dried gels was quantified by phosphorimaging analysis.
and RXR synthesized
in vitro by transcription of their corresponding cDNAs
and translation using commercially available systems (Promega), were
carried out as described previously (27, 28). Binding reactions were
carried out with a radiolabeled synthetic double-stranded oligonucleotide probe corresponding to nucleotides 932 to 851 of
the human TNF- promoter (see Fig. 6B) and probes
corresponding to putative LXREs between residues 932/903 (site 1, 5'-ACCTCTGGGGACATGTGACCACAGCAATGG-3') and
residues 894/866 (site 2, 5'-TGTCCAGGGCTATGGAAGTCGAGTATCG-3'), respectively (putative DR4 half-sites are underlined). A mutant variant
of the site 2 894/866 oligonucleotide was also used (5'-TGTCCAtttgatTGGAAGTCGAGTATCG-3', the
mutated residues are in lowercase). Competitor DNAs included the
oligonucleotide
5'-CTTGCGGTTCCCAGGGTTTAAATAAGTTCATCTA-3', corresponding to the LXRE from the MTV promoter (8) and
non-radiolabeled versions of the oligonucleotides described above.
Protein concentration in binding reactions was kept constant with
unprogrammed lysate as appropriate.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
from Unstimulated THP-1 Cells and Primary Human Monocytes--
To
determine if natural oxysterol activators of LXR can stimulate the
production of pro-inflammatory and pro-apoptotic cytokines from
unstimulated human monocytes, we used THP-1 cells, a human monocyte/macrophage cell line that expresses both LXR ( and isoforms) and its heterodimeric partner RXR (31, 32). Treatment of
THP-1 cells with either the LXR-specific ligand 22(R)-HC or the RXR-selective ligand 9cRA had no effect on the secretion of the
primary cytokines TNF-, IL-1, or IL-6, as measured by
enzyme-linked immunosorbent assays (ELISA) (Fig.
1A). However,
co-administration of both compounds resulted in a potent and specific
increase in extracellular TNF- protein. Similar findings were
observed with the LXR activator 25-hydroxycholesterol when used in
combination with 9cRA (data not presented). TNF- induction was
observed at physiological concentrations of 22(R)-HC and at
levels shown to activate both the and isoforms of LXR
(EC50 5 µM and EC50 3 µM for LXR and LXR, respectively) (2-4). The
amount of extracellular TNF- produced by co-administering
22(R)-HC and 9cRA was ~50% of the levels observed from
cells stimulated with the potent endotoxin lipopolysaccharide (LPS). A
similar level of TNF- production was also observed in primary human
peripheral blood monocytes treated with 22(R)-HC and 9cRA
(Fig. 1B; 50 and 35 pg/ml for primary cells and THP-1 cells,
respectively), indicating that the response to these compounds is not
peculiar to THP-1 cells.
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Fig. 1.
22(R)-Hydroxycholesterol,
oxidized LDL (OxLDL), and
9-cis-retinoic acid (9cRA)
specifically induce production of TNF- from
THP-1 cells and primary human monocytes. A, THP-1 cells
or B, freshly prepared primary human monocytes were
incubated for 96 h in the presence of 22(R)-HC,
22(S)-HC, and/or 9cRA (each 10 µM), or LPS (10 ng/ml), as indicated, and the levels of TNF-, IL-1, and IL-6 were
quantified by ELISA. Data presented represent the average (±S.D.) of
three independent experiments. C, THP-1 cells were incubated
for 96 h in the presence of oxLDL (50 µg/ml) and 9cRA (10 µM), as indicated, and levels of TNF- were quantified
by ELISA. Data presented represent the average (±S.D.) of three
independent experiments.
by stimulated macrophages is
normally accompanied by the coordinate secretion of several other
primary pro-inflammatory cytokines, such as IL-1 and IL-6, in a
manner consistent with the acute phase response (33).
22(R)-HC/9cRA, however, had no effect on the production of
IL-1 or IL-6 from THP-1 cells or from primary monocytes (Fig. 1,
A and B) suggesting that 22(R)-HC/9cRA stimulates a pathway of induction of TNF- that is selective for this
cytokine and distinct from other pathways.
expression in activated macrophages, whereas other studies have shown
that oxLDL can induce TNF- in resting macrophages (19-23). However,
the findings have been inconsistent and can be dependent upon cell
type, species, and/or the concentration, composition, and degree of
modification of oxLDL. Given that 22(R)-HC is reportedly present in oxLDL (6), we tested whether oxLDL could also stimulate TNF- production in resting THP-1 cells. As shown in Fig.
1C, treatment of cells with oxLDL had minimal effect on
TNF- production as compared with control untreated cells. However,
co-administration of oxLDL with 9cRA led to an 8-fold induction. Thus,
under our experimental conditions, oxLDL cooperates with 9cRA in the
induction of TNF- protein expression in THP-1 cells.
is
specific and is mediated through endogenous LXR. For instance,
22(S)-hydroxycholesterol (22(S)-HC), a
stereoisomer of 22(R)-HC that binds to LXR with similar
affinity as does 22(R)-HC but does not activate the receptor
(4), failed to induce TNF- secretion from THP-1 cells or primary
monocytes, either alone or in combination with 9cRA (Fig. 1,
A and B). Importantly, 22(S)-HC inhibited 22(R)-HC/9cRA-mediated induction of TNF- in a
dose-dependent manner. As shown in Fig.
2, a 2-fold molar excess of
22(S)-HC vis à vis 22(R)-HC
almost completely eliminated extracellular TNF- induction. This
inhibition was not due to generalized cell toxicity, because final
concentrations of oxysterols up to 30 µM had no
deleterious effects on cell viability as determined by trypan blue
staining (data not presented). Finally, all-trans-retinoic acid, a natural activator of the retinoic acid receptor, could not
substitute for 9cRA and failed to induce TNF- when administered alone or with 22(R)-HC (data not presented). We also
examined kinetics of TNF- induction. As shown in Fig.
3, significant amounts of TNF- were
generated at the earliest time point examined (12 h) and continued to
accumulate over 96 h. The foregoing, along with further evidence
described below, is consistent with induction being directly mediated
by pre-existing, endogenous factors such as LXR and RXR.
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Fig. 2.
22(S)-HC inhibits
22(R)-HC/9cRA-mediated secretion of
TNF- . THP-1 cells were incubated with
22(R)-HC and 9cRA (10 µM each) for 96 h
in the presence of increasing concentrations of 22(S)-HC as
indicated. Supernatants were collected and assayed for TNF- as above
in Fig. 1. Cell viability remained at or above 95% over the course of
the experiment as monitored by trypan blue exclusion. The data
presented represent the average (±S.D.) from three separate
experiments.
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Fig. 3.
Time course of TNF-
production from THP-1 cells in the presence of
22(R)-HC and 9cRA. THP-1 cells were incubated in
the presence of 22(R)-HC and 9cRA (10 µM each)
for 96 h, and supernatants were assayed for TNF- by ELISA at
the times indicated. The values presented represent the average
(±S.D.) from three independent experiments.
mRNA and Protein Expression in THP-1
Cells but Subsequent Protein Secretion Requires 9cRA--
LXR/RXR
belongs to a subclass of nuclear receptor heterodimers that can be
activated, under certain circumstances, by ligands for either
heterodimer partner (1, 8, 34). The finding that co-administration of
22(R)-HC and 9cRA was necessary to detect extracellular
TNF- implies that both LXR and RXR need to be activated by their
cognate ligands to mediate TNF- induction. However, the simultaneous
requirement of both ligands for LXR/RXR to activate target gene
transcription is unusual. An alternative explanation is that
22(R)-HC and 9cRA participate in separate and distinct steps
in TNF- production, a hypothesis tested and discussed further below.
mRNA levels,
we performed Northern blot analysis on mRNA from THP-1 cells.
Exposure of cells to 22(R)-HC led to an 11-fold induction in
the steady-state level of TNF- mRNA in comparison to untreated
cells (Fig. 4A). oxLDL also
resulted in induction of TNF- mRNA, albeit more modestly than
with 22(R)-HC. In contrast, 22(S)-HC did not
affect TNF- mRNA levels. Interestingly, the addition of 9cRA did
not further augment the 22(R)-HC-mediated increase; indeed,
TNF- mRNA expression was somewhat diminished (from 11- to
7-fold) under these circumstances. Similar results were obtained when
RNA was analyzed by reverse transcriptase-PCR (data not presented).
Fig. 4B shows a time course analysis of TNF- mRNA
induction in the presence of 22(R)-HC. As shown in the
figure, TNF- mRNA was detectable at the 12-h time point and
remained at this steady-state level. Thus, TNF- protein secretion
from THP-1 cells, as shown in Fig. 3, correlates with induction of
TNF- mRNA. The 22(R)-HC-mediated increase in TNF-
mRNA was not ablated in the presence of cycloheximide, consistent with induction being mediated by pre-existing, endogenous factors (Fig.
4C). Thus, 22(R)-HC increased the steady-state
levels of TNF- mRNA and did so independently of exogenously
added 9cRA.
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Fig. 4.
22(R)-HC induces
TNF- mRNA and protein expression in THP-1
cells. A, THP-1 cells were incubated as in Fig. 1 for
96 h with the indicated compounds, and total RNA was isolated from
treated cells and subjected to Northern blot analysis using a human
TNF- cDNA probe or a human glyceraldehyde-3-phosphate
dehydrogenase cDNA probe. B, time course of expression
of 22(R)-HC-mediated TNF- mRNA induction. THP-1 cells
were incubated with 22(R)-HC (10 µM) for the
times indicated and TNF- mRNA was subjected to Northern blot
analysis as described above. C, expression of
22(R)-HC-mediated TNF- mRNA does not require de
novo protein synthesis. THP-1 cells were incubated for 48 h
with 22(R)-HC (10 µM) in the presence or
absence of cycloheximide (16 mM). Total RNA was subjected
to Northern blot analysis as above. D, TNF- mRNA
induced by 22(R)-HC is translated. THP-1 cells were
metabolically labeled with [35S]methionine/cysteine in
the presence of the indicated compounds for 12 h (10 µM 22(R)-HC and 9cRA, 10 ng/ml LPS). Cell
extracts were immunoprecipitated with anti-human TNF- IgG, and
proteins were resolved by SDS-polyacrylamide gel electrophoresis. Data
are representative of two independent experiments. The 17-kDa band
represents the mature form of TNF-.
mRNA expression is induced by diverse factors in monocytes,
however, in the absence of cell activation, the mRNA is normally
translationally silent. Translation of TNF- mRNA is tightly
regulated, both by factors that mediate nucleocytoplasmic transport and
by mRNA silencing and stability factors that recognize determinants
in the 3'-untranslated region of TNF- mRNA (35-37). To
determine if TNF- mRNA made in response to 22(R)-HC
is translated, we metabolically labeled THP-1 cells with
[35S]methionine/cysteine and carried out
immunoprecipitation analysis using antibodies to human TNF-.
Cell-associated immunoreactive TNF-, with a mobility consistent with
the mature size of the protein, was detected in extracts prepared from
cells exposed to 22(R)-HC and at levels that were three to
four times higher than the control, untreated cells (Fig.
4D). As expected from the Northern analysis, treatment with
22(S)-HC or 22(S)-HC/9cRA had no effect on
TNF- protein levels. Also as expected, significant levels of
cell-associated TNF- protein were not observed in cells co-administered 22(R)-HC and 9cRA (Fig. 4C),
because TNF- protein is secreted under these circumstances (see Fig.
1, A and B). Similarly, very low levels of
cell-associated TNF- protein were detected in LPS-treated cells
(Fig. 4D), again, a consequence of the protein being rapidly
secreted following its synthesis (see Fig. 1, A and
B). Thus, TNF- mRNA induced by 22(R)-HC is
translated; however, the protein remains cell-associated.
/RXR Heterodimers Transactivate the TNF-
Promoter--
The above findings suggest that the human
TNF- gene is a target for LXR/RXR-mediated
transactivation. To determine this directly, we carried out transient
transfection assays in COS-1 cells using a luciferase reporter gene
linked to a 1.3-kb promoter/regulatory region of the human
TNF- gene (29). COS-1 cells express modest amounts of
endogenous RXR but very low levels of LXR, and, therefore, transactivation of LXR target genes in these cells is dependent upon
ectopic expression of LXR (27). As shown in Fig.
5, 22(R)-HC had no effect on
the activity of the TNF- reporter gene. Similar findings were
obtained with a control LXR target reporter gene pTK-DR4luc,
confirming that these cells express low levels of endogenous LXR (data
not presented). However, in the presence of ectopically expressed human
LXR, 22(R)-HC led to an 8- to10-fold induction in
reporter gene activity compared with untreated cells (Fig. 5).
Transfection of an expression vector for human RXR had no
significant effect on reporter gene activity, either in the absence or
presence of 9cRA. However, in the presence of expression vectors for
both LXR and RXR, 22(R)-HC led to a 20-fold increase in promoter activity, indicating that full induction required the
presence of sufficient amounts of both LXR and RXR. 9cRA did not
affect LXR/RXR activity, indicating that ligand occupancy of RXR
was insufficient to activate the LXR/RXR heterodimer in this
promoter context. Interestingly, however, 9cRA inhibited 22(R)-HC, LXR/RXR-mediated transactivation by ~50%,
consistent with findings from the Northern analysis of THP-1 cells (see
Fig. 3A). The reasons for this reduction are not clear at
present.
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Fig. 5.
The human TNF-
promoter is a target for transactivation by
LXR and RXR.
COS-1 cells were transfected with a human TNF-
promoter/luciferase reporter gene (TNF-(1311)luc) in
the absence or presence of expression vectors for human LXR and
human RXR along with 22(R)-HC (10 µM) and
9cRA (10 µM), as indicated, and luciferase activity was
measured. The values presented represent the average (±S.D.), relative
to untreated cells (taken as 1) from three independent transfections
carried out in triplicate, and normalized for protein and
-galactosidase expression levels.
/RXR Heterodimers Bind Directly to an Upstream LXRE in the
TNF- Promoter--
LXR/RXR heterodimers activate target gene
transcription by binding to LXR response elements (LXRE) that consist
of a hexanucleotide direct repeat element related to the consensus
half-site TGACCT separated by four nucleotides (DR4) (8). The human
TNF- promoter contains numerous transcription factor binding,
including multiple sites for NF-
B and AP-1, however, an obvious LXRE
is not apparent. To begin to identify promoter elements that mediate
LXR responsiveness, we generated a series of deletions in the TNF-
promoter and tested their activity in transfection assays in the
presence of co-expressed LXR/RXR. As demonstrated in Fig.
6A, the full-length promoter was induced 18-fold by 22(R)-HC in the presence of
co-expressed LXR/RXR, whereas deletion derivatives lacking
nucleotides spanning 987 to 105 or 914 to 359 (relative to the
transcription start site) were inactive. A derivative missing
nucleotides 971 to 762 was partially active, resulting in a 3-fold
induction, whereas a reporter gene derivative missing nucleotides 640
to 493 was induced 9-fold. These findings indicate that multiple
regions in the TNF- promoter participate in full responsiveness to
22(R)-HC and LXR/RXR and that sequences between 971
to 762 are of particular importance.
View larger version (16K):
[in a new window]
Fig. 6.
LXR /RXR
transactivates the human TNF- promoter
via a distal response element. A, COS-1 cells were
transfected as described above with pTNF(1311)luc or
various promoter derivatives, as indicated, along with human LXR and
human RXR expression vectors in the presence of 22(R)-HC
(10 µM). The values presented represent the average
(±S.D.) 22(R)-HC, LXR/RXR-mediated -fold induction
(relative to corresponding untreated cells, which were taken as 1) from
three independent transfections carried out in triplicate and
normalized for protein and -galactosidase expression levels.
B, relevant nucleotide sequence of the TNF- promoter
showing the position of two putative LXREs (Sites 1 and
2).
971 to 762 region revealed the presence of two
degenerate TGACCT motifs configured in a DR4 arrangement centered at
residues 918 (site 1) and 879 (site 2), respectively, that could
potentially serve as LXREs (see Fig. 6B). Consistent with
this, a synthetic subfragment spanning residues 932 to 851, which
contained these putative elements, was sufficient to confer responsiveness to 22(R)-HC and LXR/RXR (5- to 6-fold
induction) when appended to a heterologous promoter
(pTNF(932/851)luc, Fig. 6A). To determine if
LXR binds directly to this promoter region, a radiolabeled
oligonucleotide probe corresponding to this region was tested by
electrophoretic mobility shift assay using LXR and RXR proteins
synthesized in vitro. As shown in Fig.
7A, a protein-DNA complex was
observed only in the presence of both LXR and RXR (lane
d). This complex was specific, because it was competed by an
unlabeled bona fide LXRE oligonucleotide (MTV LXRE; compare
lanes d with lanes e and f) but not by
nonspecific DNA. To determine if LXR/RXR targets either of the
putative LXREs, we synthesized oligonucleotides spanning site 1 and
site 2 and used these in DNA-binding competition assays. As shown in Fig. 7B, the site 2 but not the site 1 oligonucleotide was
able to compete out binding of LXR/RXR to the 932/851
fragment (compare lanes d and e with lane
c), indicating that LXR/RXR targets site 2. Consistent with
this, radiolabeled site 2 probe (Fig. 7C, lane
f), but not site 1 probe (lane c), formed a specific protein-DNA complex with LXR/RXR (lanes g and
h). Finally, a derivative of site 2 in which the 5'-half
site was mutated did not generate a protein-DNA complex with
LXR/RXR, confirming that LXR/RXR targets the DR4 direct
repeat element (lane i). Unequivocal confirmation that site
2 constitutes a bona fide LXRE is shown in Fig.
8. Thus, the site 2 oligonucleotide, but
not the mutant derivative, conferred LXR/RXR responsiveness onto a heterologous promoter in vivo.
View larger version (33K):
[in a new window]
Fig. 7.
LXR /RXR
heterodimers bind directly and specifically to the site 2 distal
element in the TNF- promoter.
A, a radiolabeled DNA probe corresponding to nucleotides
932 to 851 of the TNF- promoter was incubated with in
vitro synthesized LXR and/or RXR, in the presence or absence
of excess unlabeled MTV LXRE probe, as indicated, and protein-DNA
complexes were resolved by gel electrophoresis. The first
lane represents probe incubated with unprogrammed reticulocyte
lysate. B, site 2 oligonucleotide inhibits protein-DNA
complex formation. Labeled 932/851 probe, was incubated as above
with LXR/RXR in the absence or the presence of 10- or 100-fold
molar excess of unlabeled oligonucleotide corresponding to site 1 and/or site 2, as indicated. C, LXR/RXR binds to site
2. Labeled probes corresponding to site 1, site 2, or the mutated site
2 oligonucleotide were incubated with LXR/RXR in the absence or
presence of excess unlabeled competitor probe, as indicated, and
protein-DNA complexes were resolved by gel electrophoresis.
View larger version (22K):
[in a new window]
Fig. 8.
The TNF- LXRE
confers LXR/RXR responsiveness to a heterologous promoter.
Luciferase reporter genes containing a single copy of the wild type
site 2 oligonucleotide (pTNF(894/866)luc) or of a mutant
derivative (pTNFmut(894/866)luc) were transfected into
COS-1 cells in the presence or absence of 22(R)-HC along
with expression vectors for LXR and RXR as indicated, and
luciferase activity was measured. The values presented represent the
average (±S.D.), relative to untreated cells (taken as 1) from two
independent transfections carried out in triplicate, and normalized for
protein and -galactosidase expression levels. The sequence of the
wild type and mutant oligonucleotides are indicated.
gene as a novel
and direct target for LXR/RXR binding and transactivation and suggests that the increase in TNF- mRNA observed in monocytes treated with 22(R)-HC occurs, at least in part, at the level
of transcription.
from Cells via a Separate
LXR-independent Pathway That Requires de Novo Transcription and Protein
Synthesis--
As shown above, although 22(R)-HC increased
TNF- mRNA and protein levels in THP-1 cells, soluble TNF- was
only detected in the presence of co-administered 9cRA. This suggests
that 9cRA, either alone or in conjunction with 22(R)-HC,
participates in a distinct, post-translation step that triggers TNF-
release from cells. The block to TNF- secretion is apparently not
related to pro-TNF- protein processing, because the immunoreactive
TNF- protein synthesized in 22(R)-HC-treated cells
migrated with an apparent molecular mass of 17 kDa, consistent
with the mature form of the protein (see Fig. 4D). Indeed,
the 26-kDa pro-TNF- precursor was not detected.
mRNA and protein after
22(R)-HC treatment, and extracellular TNF- after
co-administration of 22(R)-HC/9cRA (see Figs. 3 and 4).
9cRA, and additional treatments as indicated in Fig. 9, was
administered after this 12-h interval, and cells were incubated for a
further 12 h, after which extracellular TNF- was measured by
ELISA. As shown in Fig. 9, lane d, addition of
22(R)-HC at t = 0 followed by 9cRA at
t = 12 h led to a 40-fold induction of
extracellular TNF- protein in comparison to untreated cells or cells
treated with 22(R)-HC alone (lanes a and
b). Thus, significant production of TNF- was observed
over a 24-h time period even when the addition of 22(R)-HC
and 9cRA was separated by a period of 12 h. As expected, TNF-
was not detected when 22(S)-HC was substituted in place of
22(R)-HC prior to addition of 9cRA (Fig. 9, lane
i).
View larger version (10K):
[in a new window]
Fig. 9.
9cRA triggers release of
TNF- protein in a process that requires
de novo transcription and protein synthesis but is
independent of LXR. THP-1 cells were sequentially incubated at
t = 0 and t = 12 h with the
indicated compounds. Cells were incubated for a further 12 h, and
culture supernatants were assayed for TNF- by ELISA. Final
concentrations were as follows; 22(R)-HC,
22(S)-HC, and 9cRA (10 µM); cycloheximide
(cyclo, 16 mM); and actinomycin D (act
D, 5 µg/ml). The values presented represent the average (±S.D.)
from three independent experiments carried out in triplicate.
mRNA induction as shown previously in Fig. 4C)
or 22(S)-HC (which inhibits 22(R)-HC induction as
shown in Fig. 2) followed by 9cRA at 12 h ablated soluble TNF-
production as expected, because TNF- mRNA and/or protein is not
induced under these conditions (lanes c and h). In contrast, inclusion of 22(S)-HC with 9cRA at 12 h
had no effect on extracellular TNF- production (Fig. 9, compare
lanes g and h, respectively). These results
suggest that the secretion step triggered by 9cRA is independent of
LXR. However, inclusion of cycloheximide (lane f) or the
transcription inhibitor actinomycin D (lane e) along with
9cRA at 12 h completely abolished TNF- secretion. Thus, 9cRA
appears to be required for the de novo synthesis of one or
more factors that, whereas not necessary for
22(R)-HC-mediated induction of TNF- mRNA and protein,
are required at a post-translational step for release of
cell-associated TNF-. In this context it is noteworthy that
extracellular TNF- was not detected when cells were first
preincubated with 9cRA followed by 22(R)-HC (Fig. 9, compare
lane d with lane j). This could simply be due to
degradation or loss of 9cRA activity during the 12-h preincubation time
interval prior to addition of 22(R)-HC. However, the finding
is also consistent with the possibility that a putative 9cRA-induced
factor needed for TNF- release is labile and/or only transiently
available soon after 9cRA treatment. This scenario could also provide
an explanation of our findings that sequential addition of
22(R)-HC followed by 9cRA led to a reproducibly more robust
production of TNF- over a 24-h interval as compared with when these
compounds were co-administered (40- versus 10-fold,
respectively; compare Fig. 9, lane d, with the 24-h time
point shown in Fig. 3). Thus, the former conditions would be expected
to lead to the accumulation of intracellular TNF- protein prior to
secretion mediated by the subsequent addition of 9cRA. In contrast,
simultaneous addition of 22R-HC and 9cRA might be expected
to result in the presence of less extracellular TNF- at the 24-h
time point because of the prior requirement for TNF- mRNA
synthesis and translation, and the postulated labile nature of a
9cRA-induced release factor.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in interconnected pathways that
control rates of lipid accumulation and efflux and in coordinating cellular responses to lipid-loading in monocytes and macrophages, and
its linkage to pathological conditions such as atherosclerosis has
placed this transcription factor under intense scrutiny (18, 31, 32,
38). The findings reported here identify a new and potentially
significant role for LXR and oxysterols in monocytes and macrophages
by demonstrating that LXR and its oxysterol ligands specifically
stimulate TNF- mRNA synthesis. Moreover, we show that cell
release of TNF- protein made in response to LXR activation is
mediated via an independent and separable mechanism that is stimulated
by the retinoid 9cRA but that does not require LXR. Our findings are
unusual in that secretion of TNF- was specific to this cytokine, and
the typical co-expression of other pro-inflammatory cytokines such as
IL-1 and IL-6 associated with the inflammatory response, and seen in
cells exposed to endotoxins such as LPS, was not observed. The
foregoing suggests that oxysterol activators of LXR and 9cRA cooperate
in a novel and selective pathway of cytokine induction that may be
specific to TNF-.
promoter is a direct target for transactivation by LXR/RXR
heterodimers. This manifests principally through direct binding of
LXR/RXR to a bona fide LXRE present at position 879 of the TNF- promoter. In addition to this LXRE, there appear to be
other downstream regions of the promoter that are required for full LXR
responsiveness. Thus, a promoter derivative that was missing the
upstream LXRE (pTNF(
971-762)) was still activated by LXR/RXR,
albeit to a much lesser degree than derivatives that retained the
identified LXRE. This suggests that full activity requires the presence
of a second response element and/or cooperativity with other
transcription factors. Ongoing studies to further delineate LXR
protein-DNA and protein-protein interactions, and interplay with
other transcription factors will provide insights into the mechanisms
of transcriptional activation and potential convergence with other
signaling pathways that are known to be important for TNF- gene expression.
plays a predominant and pleiotropic role in inflammation and
host defense and, if left unregulated, can cause chronic inflammation
and septic shock. Consequently, production of bioactive TNF- is
stringently regulated by multiple transcriptional and post-transcriptional mechanisms that serve to modulate expression, abundance, modification, processing, stability, subcellular
localization, and secretion of TNF- mRNA and/or protein (35, 36,
39). Our findings identify a novel post-translational pathway of
control that is stimulated by 9cRA. Inhibitor studies and
order-of-addition experiments indicated that 9cRA acts as a step
subsequent to 22(R)-HC/LXR-mediated induction of TNF-
mRNA and protein synthesis. This post-translational step is
independent of LXR, because 9cRA-mediated TNF- release was not
ablated in the presence of the LXR antagonist 22(S)-HC. This
step, however, requires de novo transcription and protein synthesis because it was sensitive to inhibition by actinomycin D and
cycloheximide, respectively. The foregoing suggests that 9cRA,
potentially through activation of endogenous RXR, stimulates the
synthesis of one or more cellular factors required for cellular release
of mature TNF- protein. Although there is no precedent for such a
mechanism in macrophages, a previous report suggests that 9cRA and RXR
can promote the secretion of insulin from glucose-stimulated pancreatic
islet cells (40). It has been reported that biologically active, mature
TNF- in activated macrophages is retained in the Golgi complex and
is subsequently translocated from this intracellular pool by various
stimulants (39), perhaps mediated by mitogen-activated protein kinase
signaling cascades (41). It will be of interest to unravel the
mechanisms and pathways involved in 9cRA-mediated release of TNF-
and whether this process is specific to TNF- made in response to
22(R)-HC/LXR and involves other nuclear hormone receptors
such as RXR.
, by promoting
reverse cholesterol transport through stimulation of ABC-1 and ApoE1
expression, is considered to be anti-atherogenic. Recent findings that
LXR and RXR agonists reduce lesion size and the progression of
atherosclerosis in apoE/ animals (18) are consistent with this
view. In contrast, co-expression of pro-inflammatory cytokines,
including TNF-, are considered pro-atherogenic, because these agents
can promote inflammation, apoptosis, and necrosis in atheromatic
lesions, leading to late-stage lesion calcification and clinical
syndrome (17). Indeed, several reports have shown that certain
oxysterols induce apoptosis (43) and that atherosclerotic lesions
contain significant numbers of apoptotic cells as well as
immunoreactive TNF- (44, 45). However, the selective expression of
TNF- by oxysterols/LXR in the absence of the other primary cytokines
normally associated with inflammation suggests that LXR activation does
not elicit a generalized pro-inflammatory response. In this context,
specific oxysterol/LXR-dependent stimulation of TNF-
expression in monocytes/macrophages resident within the intima, coupled
with LXR-mediated cholesterol efflux, could lead to diminishment of
lesion size by inducing apoptosis of proliferating smooth muscle cells,
foam cells, and/or infiltrating T cells. However, a deleterious effect
of TNF- expression cannot be excluded a priori, because
TNF- could also serve to worsen the existing state by stimulating
inflammatory cytokine production from infiltrating T cells. TNF-
elicits pleiotropic and complex pathophysiological effects, and the
biological relevance of our findings may bear on diverse cellular
functions and physiological circumstances that are unrelated to any of
the scenarios discussed above. In this context, it is noteworthy that
any pathophysiological consequences of oxysterol-mediated TNF-
induction from monocytes/macrophages would presumably be evident only
under specific physiological circumstances where a second signal,
mediated by compounds such as 9cRA, is available to stimulate cell
release of TNF-.
gene is a
direct target for LXR transactivation in monocytes and identify a
unique and multistep pathway of TNF- production in these cells. Our
findings reveal a potentially important new dimension to the physiological role of LXR. Further investigations will help to elucidate the biological consequence of these findings to normal cellular function and pathological states.
ACKNOWLEDGEMENTS
and glyceraldehyde-3-phosphate dehydrogenase
cDNAs, respectively, J. Economou (University of California, Los
Angeles) for pXP1-TNF(1311) luc, and D. Mangelsdorf
(University of Texas Southwestern) for pTK-DR4luc. Special
thanks go to Ryan P. Sheahan for his help with some of the transfection
studies. We also thank K. Rosenthal and R. Woolstencroft (McMaster
University) for reagents and helpful advice.
FOOTNOTES
Holds an Ontario Graduate Scholarship from the Province of Ontario.
ABBREVIATIONS
, tumor necrosis
factor-;
IL, interleukin;
LPS, lipopolysaccharide;
CMV, cytomegalovirus;
ELISA, enzyme-linked immunosorbent assay;
ApoE, apolipoprotein E;
MTV, murine mammary tumor virus.
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