From the Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana 70808
Received for publication, December 26, 2000, and in revised form, February 14, 2001
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ABSTRACT |
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Results from our previous studies
demonstrated that activation of Toll-like receptor 4 (Tlr4), the
lipopolysaccharide (LPS) receptor, is sufficient to induce
nuclear factor Cyclooxygenase (COX;1
prostaglandin endoperoxide (PGH2) synthase)
catalyzes the conversion of arachidonic acid to prostaglandin endoperoxide. This is the rate-limiting step in prostaglandin and thromboxane biosynthesis. Two isoforms of COX have been cloned from
various animal cells, constitutively expressed COX-1 (1-5) and
mitogen-inducible COX-2 (6-11). Numerous studies have demonstrated that the levels of prostaglandins in various tumors, or the tumor's biosynthetic capacity of prostaglandins, are greater when compared with
normal tissues (12-16). Recently, it has been shown that the inducible
form of COX is overexpressed in sites of inflammation and in many types
of tumor tissues (17-20). Overexpression of COX-2 in tumor tissues
occurs in both tumor cells and stromal cells including macrophages
(21). What causes the overexpression of COX-2 in such pathological
states is not clearly understood. COX-2 belongs to a family of
immediate early response genes that do not require precedent protein
synthesis for their expression (22). Therefore, elucidating the
signaling pathways leading to the expression of COX-2 is a key to
understanding why COX-2 is overexpressed in such pathological states
and can provide critical information for identifying potential targets
of modulation by pharmacological and dietary factors.
COX-2 expression is induced by various mitogenic stimuli in different
cell types (6, 9, 11, 23). The cis-acting NF The recent finding that murine Tlr4 is the LPS receptor (30) provided a
new impetus in elucidating LPS-induced signaling pathways and target
gene expression. Results from our previous studies indicated that
murine Tlr4 confers LPS responsiveness and that activation of Tlr4 is
sufficient to induce NF Activation of monocytes/macrophages is an important initial step in the
cascades of events leading to many inflammatory diseases including
endotoxemia (34). If activation of macrophages is modulated by types of
fatty acids through Tlr4, it can be inferred that risk for such
diseases may also be modified by different types of fatty acids.
Reagents--
All saturated and unsaturated fatty acids were
purchased from Nu-Chek (Eslyan, MN). Rumenic acid
(9(Z),11(E)-octadecadienoic acid; conjugated linoleic acid) was
purchased from Matreya (Pleasant Gap, PA). LPS was purchased
from Difco (Detroit, MI). Bovine serum albumin (fatty acid free and low
endotoxin, catalog number A8806; BSA) and human recombinant TNF Cell Culture--
RAW 264.7 cells (a murine macrophage-like cell
line, ATCC number TIB-71) or HT-29 cells (a human colon adenocarcinoma
cell line, ATCC number HTB-38) were cultured in Dulbecco's modified Eagle's medium containing 10% (v/v) heat-inactivated fetal bovine serum (Intergen) and 100 units/ml penicillin and 100 µg/ml
streptomycin (Life Technologies, Inc.) at 37 °C in a 5%
CO2/air environment. Cells (2 × 106) were
plated in 60-mm dishes (Falcon) and cultured for an additional 18 h to allow the number of cells to approximately double. Cells were
maintained in serum-poor (0.25% fetal bovine serum) medium for another
18 h prior to the treatment with indicated reagents.
Preparation of Fatty Acid-Albumin Complexes--
All fatty acids
were solubilized in ethanol. They were combined with fatty acid-free
and low endotoxin BSA at a molar ratio of 10:1 (fatty acid:albumin) in
serum-poor medium (0.25% fetal bovine serum). Fatty acid-albumin
complex solution was freshly prepared prior to each experiment.
SDS-Polyacrylamide Gel Electrophoresis and
Immunoblotting--
These were performed as previously described (26,
35). Briefly, solubilized proteins were subjected to 8%
SDS-polyacrylamide gel electrophoresis for COX-2, iNOS, IL-1 Plasmids--
The luciferase reporter plasmids
(pGL2) containing the promoter region of the murine COX-2 gene
( Transient Transfection and Luciferase Assay--
These were
performed as described in our previous studies (27, 35). Briefly, RAW
264.7 cells were plated in 6-well plates (5 × 105
cells/well) and transfected with luciferase reporter plasmids and
HSP70- Statistical Analysis--
Data were analyzed by paired
t test.
Saturated Fatty Acids, but Not Unsaturated Fatty Acids, Induce
COX-2 Expression in RAW 264.7 Cells--
Saturated fatty acids induced
COX-2 expression as determined by both Western blot analysis (Fig.
1A) and luciferase reporter gene assay for COX-2 (Fig. 1B). Among the saturated fatty
acids (C8:0-C18:0) tested, lauric acid (C12:0) and palmitic acid
(C16:0) were most potent in inducing COX-2 expression (Fig.
1C). In addition to COX-2, the expression of other
inflammatory marker gene products such as iNOS and IL-1 Induction of COX-2 Expression by Saturated Fatty Acids Is Mediated
through the Activation of NF
Because naturally occurring fatty acids are known to bind and activate
PPARs (37-42), and some of PPAR activators induce COX-2 expression in
certain cell types (35, 43), we determined whether saturated fatty
acid-induced COX-2 expression is also mediated through the PPAR
signaling pathway. The 5'-flanking region of murine COX-2 contains PPAR
response element (PPRE)-like sequences (Fig.
3A). Thus, we determined
whether these sequences are required for saturated fatty acid-induced
COX-2 expression. The result showed that deletion of those sequences
did not affect the promoter activity of COX-2 reporter gene (Fig.
3A). Next, we determined whether a dominant-negative mutant
of PPAR Saturated Fatty Acid-induced COX-2 Expression Is Inhibited by a
Dominant-Negative Mutant of Tlr4--
Next, we attempted to identify
the upstream target in the NF Unsaturated Fatty Acids Inhibit Saturated Fatty Acid-induced COX-2
Expression, and This Inhibition Is Mediated through Suppression of
NF Unsaturated Fatty Acids Also Inhibit Constitutively Active Tlr4
( Unsaturated Fatty Acid Also Inhibits LPS-induced NF Most long-chain fatty acids are esterified in cellular lipids in
mammalian cells. Therefore, the concentrations of unesterified fatty
acids are believed to be low. However, fatty acids are rapidly released
by the action of various phospholipase A2 and
monoacylglycerol and diacylglycerol lipases in response to various
extracellular stimuli. In plasma the average concentration of free
fatty acid in postabsorptive state is <0.7 mM, and this
concentration may be much higher in absorptive phase after ingestion of
a fatty meal (45). Therefore, blood cells such as monocytes are
constantly exposed to relatively high concentrations of free fatty
acids. Fatty acids are known to regulate the expression of many genes involved in lipid metabolism (45) and modulate activity of signaling molecules such as phospholipase C and protein kinase C (46, 47). The
mechanism by which fatty acids can regulate gene expression is still
not well understood. However, some conceptual framework has been
proposed for the possible mechanism of actions.
Fatty acids and their oxidative metabolites are known to bind and
activate PPARs, the steroid-thyroid superfamily of nuclear receptors
(37-42). Two zinc finger motifs in the DNA binding domain of PPARs
bind PPREs located in the 5'-flanking region of PPAR responsive genes.
PPARs bind PPRE as a heterodimer with the retinoid X receptor.
Polyunsaturated fatty acids and other peroxisome proliferators induce
peroxisomal The inability of a dominant-negative mutant of PPAR It was shown that unsaturated fatty acids induce COX-2 expression in
mammary epithelial cells (43). Whether this induction is mediated
through PPARs has not been determined. However, to our surprise,
saturated fatty acids, but not unsaturated fatty acids, induce COX-2 in
RAW 264.7 cells (Fig. 1). Greater potency of lauric acid and palmitic
acid in inducing COX-2 expression among saturated fatty acids tested
(Fig. 1C) coincides with the abundance of these fatty acids
in the lipid A molecule (31). Lauric, myristic, and palmitic acids are
known to be major fatty acids acylated in the lipid A molecule (31).
The fact that deacylation of these fatty acids from LPS results in loss
of endotoxic activity (32, 33) implies an important role of these fatty
acids in LPS-mediated signal transmission. NF The results presented in Figs. 4 and 6 suggest that activation of
NF Although the detail mechanism by which saturated and unsaturated fatty
acids interact with Tlr4 or its associated molecules is not known, the
results presented in this report represent a novel mechanism by which
fatty acids modulate signaling pathways and the expression of target
genes. Furthermore, the results imply the possibility that cellular
expression of COX-2 and other inflammatory markers in monocytes and
macrophages can be differentially regulated by different types of free
fatty acids that in turn can be altered by kinds of dietary fats
consumed. These results further raise important questions as to whether
activation of monocytes/macrophages and the propensity of endotoxemia
can be modulated by types of plasma fatty acids and whether unsaturated
fatty acids can provide prophylactic efficacy against endotoxemia.
Elucidating the mechanisms of the differential regulation of gene
expression and activation of macrophages by types of fatty acids will
help us to understand how different kinds of dietary fat modify risks
of many chronic and acute inflammatory diseases.
B activation and expression of inducible
cyclooxygenase (COX-2) in macrophages. Saturated fatty acids (SFAs)
acylated in lipid A moiety of LPS are essential for biological
activities of LPS. Thus, we determined whether these fatty acids
modulate LPS-induced signaling pathways and COX-2 expression in
monocyte/macrophage cells (RAW 264.7). Results show that SFAs,
but not unsaturated fatty acids (UFAs), induce nuclear factor
B
activation and expression of COX-2 and other inflammatory markers. This
induction is inhibited by a dominant-negative Tlr4. UFAs inhibit COX-2
expression induced by SFAs, constitutively active Tlr4, or LPS.
However, UFAs fail to inhibit COX-2 expression induced by activation of
signaling components downstream of Tlr4. Together, these results
suggest that both SFA-induced COX-2 expression and its inhibition by
UFAs are mediated through a common signaling pathway derived from Tlr4.
These results represent a novel mechanism by which fatty acids modulate
signaling pathways and target gene expression. Furthermore, these
results suggest a possibility that propensity of monocyte/macrophage
activation is modulated through Tlr4 by different types of free fatty
acids, which in turn can be altered by kinds of dietary fat consumed.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B element is
present in the 5'-flanking regions of COX-2 genes of different species
(24, 25). Results from our previous studies demonstrated that the
activation of NF
B is required to induce maximal expression of COX-2
in the lipopolysaccharide (LPS)-stimulated macrophage cell line (26,
27). Proinflammatory cytokines, such as TNF
and IL-1, also activate
NF
B and induce COX-2 expression in many cell types (28, 29).
B activation and expression of COX-2 in
macrophages (27). The lipid A moiety possesses most of the biological
activities of LPS (31). Lipid A of Escherichia coli and
Salmonella typhimurium is a
,1-6-linked disaccharide of
glucosamine, acylated with R-3-hydroxylaurate or myristate and
phosphorylated at positions 1 and 4'. The 3-hydroxyl groups of these
saturated fatty acids are further 3-O-acylated by lauric
acid, myristic acid, or palmitic acid (31). These acyl-linked saturated
fatty acids are subject to hydrolysis by acyloxyacyl hydrolase; the
deacylated lipid A loses its endotoxic properties and acts as an
antagonist against lipid A (32, 33). This implies that fatty acids
acylated in lipid A may play an important role in ligand recognition
and receptor activation for Tlr4. In light of the finding that murine
Tlr4 is the LPS receptor (30), it is important to determine whether
these fatty acids modulate Tlr4-mediated signaling pathways and the
expression of target gene products. If they do, this will represent a
new paradigm for the mechanism by which gene expression is regulated by
fatty acids.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
were
purchased from Sigma. Polyclonal antibodies for COX-2 were prepared and
characterized as described previously (21, 23). Antibodies for iNOS and
IL-1
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Donkey anti-rabbit IgG antibodies conjugated to horseradish
peroxidase were purchased from Amersham Pharmacia Biotech. ECL
Western blotting detection reagents were purchased from Amersham
Pharmacia Biotech. SuperFect transfection reagent was purchased from
Qiagen (Valencia, CA). A luciferase assay system and
-galactosidase
enzyme system were purchased from Promega (Madison, WI). All other
reagents were purchased from Sigma unless described otherwise.
, and
GAPDH immunoblot analyses. Following electrophoresis, the gel was
transferred to a polyvinylidene difluoride membrane, and the membrane
was blocked to prevent nonspecific binding of antibodies in TBS-T (20 mM Tris-HCl, 137 mM NaCl, 0.05% (v/v) Tween
20, pH 7.6) containing 5% nonfat dried milk (Carnation).
Immunoblotting was performed using respective polyclonal antibodies
followed by incubation with anti-rabbit IgG coupled to horseradish
peroxidase. The membrane was exposed on an x-ray film (Eastman Kodak
Co.) using ECL Western blot detection reagents (Amersham Pharmacia Biotech).
3201/+93 or
1017/+93) were provided by David Dewitt (Michigan
State University, East Lansing, MI). To prepare the wild-type COX-2
promoter fragment, polymerase chain reaction was performed with the
primers named Kpn-COX2-For and Hind-COX2-Rev using the murine COX-2
(
1017/+93) luciferase reporter plasmid as a template. To
prepare the mutant COX-2 promoter fragment-containing mutated NF
B
site, Kpn-COX2-Fmut and Hind-COX2-Rev were used as primers. Each
polymerase chain reaction fragment was inserted into the
KpnI and HindIII sites of pGL2 to generate the
wild-type or mutant COX-2 (
410/+86) luciferase reporter constructs, respectively. The sequence for the wild-type NF
B site, GGGATTCCC, was changed to GGCCTTCCC. All promoter sequences were confirmed by DNA sequencing. The primers used are as follows: Kpn-COX2-For, 5'-GACGGTACCGAGAGGTGAGGGGATTCCC-3'; Hind-COX2-Rev,
5'-CAGAAGCTTGGTGGAGCTGGCAGGATG-3'; Kpn-COX2- Fmut,
5'-GACGGTACCGAGAGGTGAGGGCCTTCCC-3'. 2× NF
B-luciferase reporter construct was a gift from Frank Mercurio (Signal
Pharmaceuticals, San Diego, CA). HSP70-
-galactosidase reporter
plasmid was from Robert Modlin (University of California, Los Angeles,
CA). The expression plasmids for a constitutively active form of Tlr4
(
Tlr4) and a dominant-negative mutant,
Tlr4(P712H), were prepared
as described previously (27). The expression plasmid of the wild-type NF
B-inducing kinase (NIK), pRK-NIK(wt), was gift from Mike Rothe (Tularik, South San Francisco, CA). The dominant-negative mutant of
inhibitor
B (pCMV4-I
B
(
N)) was provided by Dean Ballard (Vanderbilt University, Nashville, TN). The constitutively active form
of MyD88 (FLAG-MyD88(
Toll)) was kindly provided by Jurg Tschopp
(University of Lausanne) (36). The dominant-negative mutant of
mouse PPAR
(pCMX-PPAR
(L466A/L467A)) was from Ira Schulman (Ligand Pharmaceuticals, San Diego, CA). All DNA constructs were prepared in large scale using an EndoFree plasmid maxi kit
(Qiagen, Valencia, CA) for transfection.
-galactosidase plasmid as an internal control using SuperFect
transfection reagent (Quiagen) according to the manufacturer's instruction. Luciferase and
-galactosidase enzyme activities were
determined using the luciferase assay system and
-galactosidase enzyme system (Promega, Madison, WI) according to the manufacturer's instruction. Luciferase activity was normalized by
-galactosidase activity.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was also
induced by lauric acid in a dose-dependent manner (Fig.
1A). Unlike saturated fatty acids, all unsaturated fatty
acids (C18:1n-9, C18:2n-6, C20:4n-6,
C20:5n-3, and C22:6n-3) and conjugated linoleic
acid (cLA) tested were unable to induce COX-2 expression in RAW 264.7 cells (Fig. 1D).
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Fig. 1.
Saturated fatty acids, but not unsaturated
fatty acids, induce the expression of COX-2, iNOS, and
IL-1 . A, RAW 264.7 cells
maintained in serum-poor (0.25%) medium were treated with indicated
concentrations of lauric acid (C12:0) solubilized with BSA at a molar
ratio of 10:1 (fatty acid:BSA). After 11 h, cell lysates were
analyzed by COX-2, iNOS, IL-1
, or GAPDH immunoblot. Lane
1, cells treated in medium alone; lanes 2-5, cells
treated with lauric acid in medium with BSA; and lane 6,
cells treated in medium with 10 µM BSA without fatty
acid. B, cells were transfected with a luciferase reporter
plasmid for COX-2 promoter and HSP70-
-galactosidase reporter plasmid
as an internal control and treated with various concentrations of
lauric acid (C12:0) or (C) 75 µM of each
saturated fatty acid for 24 h. The luciferase and
-galactosidase enzyme activities were measured as described under
"Experimental Procedures." Relative luciferase activity was
determined by normalization with
-galactosidase activity.
D, cells were treated with 75 µM of each fatty
acid for 11 h. Cell lysates were analyzed by COX-2 or GAPDH
immunoblot. The panels contain representative data from more
than three different experiments. Values are mean ± S.E.
(n = 3). *, significantly different from the vehicle
control; p < 0.05. RLA, relative luciferase
activity.
B--
In our previous studies it was
demonstrated that activation of NF
B is sufficient and required to
induce maximal expression of COX-2 in LPS-stimulated RAW 264.7 cells
(27). Therefore, we determined whether saturated fatty acid-induced
COX-2 expression is mediated through the activation of NF
B in RAW
264.7 cells. Lauric acid activated NF
B in a
dose-dependent manner (Fig.
2A). The expression of COX-2
induced by lauric acid was inhibited by co-transfection of a
dominant-negative mutant of I
B
plasmid (Fig. 2B). In
addition, lauric acid-induced COX-2 expression was significantly
reduced in the COX-2 promoter reporter gene containing the mutated
NF
B site as compared with the one containing the wild-type NF
B
site (Fig. 2C).
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Fig. 2.
Lauric acid (C12:0)-induced expression of
COX-2 is inhibited by a dominant-negative mutant of
I B
or by mutation in
the NF
B site in COX-2 promoter.
A, RAW 264.7 cells were transfected with a luciferase
(Luc) reporter plasmid for NF
B response element and
treated with indicated concentrations of lauric acid (C12:0) for
24 h. B, cells were co-transfected with a luciferase
reporter plasmid for COX-2 promoter and the expression plasmid
containing a dominant-negative mutant of I
B
(I
B
(
N)) and then
treated with 75 µM lauric acid (C12:0) for 24 h.
C, cells were transfected with a luciferase reporter plasmid
for COX-2 promoter containing the wild-type NF
B site or the mutated
NF
B site. Relative luciferase activity (RLA) was
determined as described in Fig. 1. The panels contain
representative data from more than three different experiments. Values
are mean ± S.E. (n = 3). *, significantly
different from the vehicle control (A), the control
(C12:0+vector) (B), or the data obtained using COX-2
promoter with the wild-type NF
B site (C);
p < 0.05.
(44) alters the saturated fatty acid-induced COX-2
expression. The result showed that lauric acid-induced COX-2 expression
in cells co-transfected with a dominant-negative mutant of PPAR
plasmid was not altered as compared with control cells regardless of
whether the COX-2 reporter gene construct contains the PPRE-like
sequences or not (Fig. 3B). Together, these results suggest
that saturated fatty acid-induced COX-2 expression is not directly
mediated through the PPRE-like sequences in COX-2 gene.
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Fig. 3.
Lauric acid (C12:0)-induced COX-2 expression
is not affected by deletion of PPRE-like sequences in murine COX-2
promoter or by a dominant-negative mutant of
PPAR . A, RAW 264.7 cells were
transfected with a luciferase (Luc) reporter plasmid for
COX-2 promoter with or without PPRE-like sequences. B, cells
were co-transfected with a reporter plasmid for COX-2 promoter with or
without a dominant-negative mutant of PPAR
and then treated with
lauric acid (75 µM) for 24 h. Relative luciferase
activity (RLA) was determined as described in Fig. 1. The
panels contain representative data from more than three
different experiments. Values are mean ± S.E. (n = 3).
B signaling pathways through which the
saturated fatty acids activate NF
B and induce COX-2 expression.
Activation of Tlr4 is sufficient and necessary to activate NF
B and
to induce COX-2 expression in RAW 264.7 cells. Because of the
implication that lauric acid, myristic acid, or palmitic acid acylated
in the lipid A molecule may play an important role in transmitting the
LPS-mediated signal, we determined whether saturated fatty acid-induced
activation of NF
B and COX-2 expression are mediated through the
murine LPS receptor (Tlr4). If saturated fatty acid-induced COX-2
expression is mediated through Tlr4, co-transfection of cells with a
dominant-negative mutant of Tlr4 should lead to inhibition of COX-2
expression. The results show that the dominant-negative mutant of Tlr4
(
Tlr4(P712H)) inhibits both saturated fatty acid-induced NF
B
activation and COX-2 expression (Fig. 4,
A and B). These results suggest that the upstream
target in the signaling pathways through which saturated fatty acids
mediate NF
B activation and COX-2 expression is Tlr4 or its
associated molecules. However, these results do not allow us to
conclude whether saturated fatty acids directly interact with Tlr4.
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Fig. 4.
Lauric acid (12:0)-induced activation of
NF B and COX-2 expression are inhibited by a
dominant-negative mutant of Tlr4. RAW 264.7 cells were
co-transfected with a luciferase (Luc) reporter plasmid for
NF
B response element (A) or COX-2 promoter (B)
and the expression plasmid for a dominant-negative mutant of Tlr4
(
Tlr4(P712H)) and then treated with lauric
acid (75 µM) for 24 h. Relative luciferase activity
(RLA) was determined as described in Fig. 1. The
panels contain representative data from more than three
different experiments. Values are mean ± S.E. (n = 3). *, significantly different from the control (C12:0+vector);
p < 0.05.
B--
Unlike saturated fatty acids, unsaturated fatty acids were
unable to induce COX-2 expression (Fig. 1D). Furthermore,
they inhibited saturated fatty acid-induced NF
B activation (Fig.
5A) and COX-2 expression (Fig.
5B). These results indicate that inhibition of saturated
fatty acid-induced COX-2 expression by unsaturated fatty acids is
mediated through suppression of the NF
B signaling pathway. Together,
these results suggest that both the induction of COX-2 by saturated
fatty acids and its inhibition by unsaturated fatty acids are mediated
through the NF
B signaling pathway.
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Fig. 5.
Unsaturated fatty acids inhibit lauric acid
(C12:0)-induced activation of NF B and COX-2
expression. RAW 264.7 cells were transfected with a luciferase
(Luc) reporter plasmid for NF
B response element
(A) or COX-2 promoter (B) and pre-treated with 5 µM of each unsaturated fatty acid for 3 h and then
treated with lauric acid (75 µM) for an additional
21 h. Relative luciferase activity (RLA) was determined
as described in Fig. 1, and data are expressed as a percentage of the
control (C12:0). The panels contain representative data from
more than three different experiments. Values are mean ± S.E.
(n = 3). *, significantly different from the C12:0
alone; p < 0.05.
Tlr4)-induced COX-2 Expression, but They Do Not Inhibit COX-2
Expression Induced by Constitutively Active MyD88 or NIK, Which Lies
Downstream of Tlr4--
If saturated fatty acid-induced COX-2
expression is mediated through Tlr4, it is logical to determine whether
the inhibition of saturated fatty acid-induced COX-2 expression by
unsaturated fatty acids is also mediated through Tlr4. The results
showed that docosahexaenoic acid (C22:6n-3) partially
inhibits constitutively active Tlr4 (
Tlr4)-induced COX-2 expression
(Fig. 6A). MyD88 is the
immediate downstream adaptor protein that interacts directly with the
cytoplasmic domain of Tlr4. Activation of MyD88 leads to activation of
NF
B and COX-2 expression in RAW 264.7 cells (27). Therefore, if the
inhibition of saturated fatty acid-induced COX-2 expression by
unsaturated fatty acids is mediated through Tlr4, COX-2 expression
induced by the activation of signaling steps downstream of Tlr4 should
not be inhibited by unsaturated fatty acids. The results indeed show
that docosahexaenoic acid (C22:6n-3) is unable to inhibit
COX-2 expression induced by constitutively active MyD88 or NIK (Fig. 6,
B and C). These results suggest that both
induction of COX-2 expression by saturated fatty acids and its
inhibition by unsaturated fatty acids are mediated through Tlr4 or
molecules associated with Tlr4.
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Fig. 6.
Docosahexaenoic acid
(C22:6n-3) inhibits constitutively active Tlr4
( Tlr4)-induced, but not constitutively active
MyD88- or NIK-induced, COX-2 expression. RAW 264.7 cells were
co-transfected with a luciferase (Luc) reporter plasmid for
COX-2 promoter and the expression plasmid for a constitutively active
Tlr4 (
Tlr4) (A), a constitutively
active MyD88(
Toll) (B), or NIK (C) and then
treated with 20 µM docosahexaenoic acid
(C22:6n-3) for 11 h. Relative luciferase activity
(RLA) was determined as described in Fig. 1. The
panels contain representative data from more than three
different experiments. Values are mean ± S.E. (n = 3). *, significantly different from the control (
Tlr4 without
C22:6n-3); p < 0.05.
B Activation
and Expression of COX-2, iNOS, and IL-1
--
If the inhibition of
saturated fatty acid-induced COX-2 expression by unsaturated fatty
acids is mediated through Tlr4 or its associated molecules, unsaturated
fatty acids should also inhibit LPS-induced COX-2 expression. The
results indeed show that docosahexaenoic acid (C22:6n-3)
inhibits the LPS-induced expression of COX-2, iNOS, and IL-1
(Fig.
7A). Other unsaturated fatty
acids tested (Fig. 1D) also inhibit LPS-induced COX-2
expression (data not shown). Inhibition of LPS-induced NF
B
activation by docosahexaenoic acid (C22:6n-3) is
demonstrated by inhibition of LPS-induced degradation of I
B
protein (Fig. 7B). Furthermore, docosahexaenoic acid
(C22:6n-3) fails to inhibit TNF
-induced COX-2 expression
in a colon tumor cell line (HT-29) (Fig. 7C) reinforcing the
possibility that the inhibitory effect of unsaturated fatty acid on
saturated fatty acid- or LPS-induced expression of COX-2 is
specifically mediated through Tlr4 or its associated molecules.
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Fig. 7.
Docosahexaenoic acid
(C22:6n-3) inhibits LPS-induced expression of COX-2,
iNOS, and IL-1 and degradation of
I
B
in RAW 264.7 cells, but it fails to inhibit TNF
-induced
COX-2 expression in HT-29 cells. A, RAW 264.7 cells
were pretreated with indicated concentrations of docosahexaenoic acid
(C22:6n-3) for 3 h and then stimulated with LPS (100 ng/ml) for 8 h and analyzed by COX-2, iNOS, IL-1
, or GAPDH
immunoblot or (B) for 30 min and analyzed by I
B
immunoblot. C, colon cancer cells (HT-29) were pretreated
with various concentrations of docosahexaenoic acid
(C22:6n-3) for 3 h and then treated with TNF
(20 ng/ml) for 8 h. Cell lysates were analyzed by COX-2 and GAPDH
immunoblot. The panels contain representative data from more
than three different experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-oxidation and the expression of certain peroxisomal enzymes (45). Using PPAR
null mice, it was demonstrated that PPAR
is required for the induction of acyl-CoA oxidase by n-3 polyunsaturated fatty acids but not for the suppression of lipogenic enzymes by n-3 polyunsaturated fatty acids (48). These results indicate
that regulation of gene expression by fatty acids can be mediated
through signaling pathways other than PPARs.
to inhibit
saturated fatty acid-induced COX-2 expression suggests that the
induction was not mediated through activation of PPAR
. However, the
possibility that the induction of COX-2 expression by saturated fatty
acid is in part mediated through PPAR
or PPAR
cannot be ruled
out. Murine COX-2 gene contains PPRE-like sequences at positions
2354
to
2342 in the 5'-flanking region. Deletion of these sequences did
not affect the promoter activity of COX-2 reporter gene (Fig. 3)
suggesting that the PPRE-like sequences do not appear to be required
for saturated fatty acid-induced COX-2 expression. However, the
possibility that the saturated fatty acids in part stimulate or inhibit
other PPAR-responsive gene products, which in turn cause the induction
of COX-2 expression, cannot be ruled out.
B is one of the major
downstream signaling pathways derived from activation of the LPS
receptor, Tlr4 in RAW 264.7 cells (27). The results demonstrating that induction of COX-2 by lauric acid is mediated through activation of
NF
B (Fig. 2) and that this activation is inhibited by a
dominant-negative mutant of Tlr4 (Fig. 4A), suggest that the
most upstream signaling components affected by saturated fatty acids
include Tlr4 or molecules associated with Tlr4. Whether saturated fatty
acids can directly interact with Tlr4 or they interact with molecules
associated with either extracellular or intracellular domains of Tlr4
remains to be determined.
B and COX-2 expression induced by saturated fatty acids and
inhibition of this induction by unsaturated fatty acids are mediated
through a common signaling pathway derived from Tlr4. The possibility
that saturated fatty acids may act as a physiologically relevant
endogenous ligand for Tlr4 and that unsaturated fatty acids interfere
with saturated fatty acids in interacting with Tlr4 or molecules
associated with Tlr4 remains to be determined.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Walter A. Deutsch for reading the manuscript and Wei Fan for technical assistance.
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FOOTNOTES |
---|
* This work was supported in part by grants from the National Institutes of Health (DK-41868 and CA-75613), United States Department of Agriculture (9700918), and American Institute for Cancer Research (98A0978).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.
Contributed equally to this work.
§ Supported in part by a fellowship from the Korea Research Foundation.
¶ To whom correspondence should be addressed: Pennington Biomedical Research Center, 6400 Perkins Rd., Louisiana State University, Baton Rouge, LA 70808. Tel.: 225-763-2518; Fax: 225-763-3030; E-mail: hwangdh@pbrc.edu.
Published, JBC Papers in Press, March 2, 2001, DOI 10.1074/jbc.M011695200
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ABBREVIATIONS |
---|
The abbreviations used are:
COX, cyclooxygenase;
LPS, lipopolysaccharide;
PPAR, peroxisome proliferator-activated
receptors;
PPRE, peroxisome proliferator response element;
Tlr, Toll-like receptor;
NIK, NFB-inducing kinase;
NF
B, nuclear
factor
B;
I
B
, inhibitor
B
;
IL-1
, interleukin-1
;
iNOS, inducible form of nitric-oxide synthase;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
TNF
, tumor necrosis factor
;
BSA, bovine serum albumin.
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