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* 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.
Results from our previous studies demonstrated that activation of Toll-like receptor 4 (Tlr4), the lipopolysaccharide (LPS) receptor, is sufficient to induce nuclear factor κB 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 κB 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.
COX
cyclooxygenase
LPS
lipopolysaccharide
PPAR
peroxisome proliferator-activated receptors
PPRE
peroxisome proliferator response element
Tlr
Toll-like receptor
NIK
NFκB-inducing kinase
NFκB
nuclear factor κB
IκBα
inhibitor κBα
IL-1α
interleukin-1α
iNOS
inducible form of nitric-oxide synthase
GAPDH
glyceraldehyde-3-phosphate dehydrogenase
TNFα
tumor necrosis factor α
BSA
bovine serum albumin
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 (
). 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 (
). 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 (
). 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 (
). Results from our previous studies demonstrated that the activation of NFκB is required to induce maximal expression of COX-2 in the lipopolysaccharide (LPS)-stimulated macrophage cell line (
) 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 NFκB activation and expression of COX-2 in macrophages (
). Lipid A of Escherichia coli andSalmonella typhimurium is a β,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 (
). 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 (
). 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 (
), 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.
Activation of monocytes/macrophages is an important initial step in the cascades of events leading to many inflammatory diseases including endotoxemia (
). 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.
EXPERIMENTAL PROCEDURES
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 TNFα were purchased from Sigma. Polyclonal antibodies for COX-2 were prepared and characterized as described previously (
). Antibodies for iNOS and IL-1α 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 β-galactosidase enzyme system were purchased from Promega (Madison, WI). All other reagents were purchased from Sigma unless described otherwise.
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
). Briefly, solubilized proteins were subjected to 8% SDS-polyacrylamide gel electrophoresis for COX-2, iNOS, IL-1α, 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).
Plasmids
The luciferase reporter plasmids (pGL2) containing the promoter region of the murine COX-2 gene (−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 NFκB site, Kpn-COX2-Fmut and Hind-COX2-Rev were used as primers. Each polymerase chain reaction fragment was inserted into theKpnI 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 NFκB 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× NFκB-luciferase reporter construct was a gift from Frank Mercurio (Signal Pharmaceuticals, San Diego, CA). HSP70-β-galactosidase reporter plasmid was from Robert Modlin (University of California, Los Angeles, CA). The expression plasmids for a constitutively active form of Tlr4 (ΔTlr4) and a dominant-negative mutant, ΔTlr4(P712H), were prepared as described previously (
). The expression plasmid of the wild-type NFκB-inducing kinase (NIK), pRK-NIK(wt), was gift from Mike Rothe (Tularik, South San Francisco, CA). The dominant-negative mutant of inhibitor κB (pCMV4-IκBα(ΔN)) was provided by Dean Ballard (Vanderbilt University, Nashville, TN). The constitutively active form of MyD88 (FLAG-MyD88(ΔToll)) was kindly provided by Jurg Tschopp (University of Lausanne) (
). The dominant-negative mutant of mouse PPARγ (pCMX-PPARγ(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.
Transient Transfection and Luciferase Assay
These were performed as described in our previous studies (
). Briefly, RAW 264.7 cells were plated in 6-well plates (5 × 105 cells/well) and transfected with luciferase reporter plasmids and HSP70-β-galactosidase plasmid as an internal control using SuperFect transfection reagent (Quiagen) according to the manufacturer's instruction. Luciferase and β-galactosidase enzyme activities were determined using the luciferase assay system and β-galactosidase enzyme system (Promega, Madison, WI) according to the manufacturer's instruction. Luciferase activity was normalized by β-galactosidase activity.
Statistical Analysis
Data were analyzed by pairedt test.
RESULTS
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-1α 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).
Figure 1Saturated fatty acids, but not unsaturated fatty acids, induce the expression of COX-2, iNOS , and IL-1α.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-1α, 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-β-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 β-galactosidase enzyme activities were measured as described under “Experimental Procedures.” Relative luciferase activity was determined by normalization with β-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.
Induction of COX-2 Expression by Saturated Fatty Acids Is Mediated through the Activation of NFκB
In our previous studies it was demonstrated that activation of NFκB is sufficient and required to induce maximal expression of COX-2 in LPS-stimulated RAW 264.7 cells (
). Therefore, we determined whether saturated fatty acid-induced COX-2 expression is mediated through the activation of NFκB in RAW 264.7 cells. Lauric acid activated NFκB 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 IκBα plasmid (Fig. 2B). In addition, lauric acid-induced COX-2 expression was significantly reduced in the COX-2 promoter reporter gene containing the mutated NFκB site as compared with the one containing the wild-type NFκB site (Fig. 2C).
Figure 2Lauric acid (C12:0)-induced expression of COX-2 is inhibited by a dominant-negative mutant of IκBα or by mutation in the NFκB site in COX-2 promoter.A, RAW 264.7 cells were transfected with a luciferase (Luc) reporter plasmid for NFκB 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 IκBα (IκBα(ΔN)) 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 NFκB site or the mutated NFκB 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 NFκB site (C);p < 0.05.
), 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 PPARγ (
) 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 PPARγ 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.
Figure 3Lauric 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 γ.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 PPARγ and then treated with lauric acid (75 μm) for 24 h. Relative luciferase activity (RLA) was determined as described in Fig. 1. Thepanels contain representative data from more than three different experiments. Values are mean ± S.E. (n= 3).
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 NFκB signaling pathways through which the saturated fatty acids activate NFκB and induce COX-2 expression. Activation of Tlr4 is sufficient and necessary to activate NFκB 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 NFκB 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 (ΔTlr4(P712H)) inhibits both saturated fatty acid-induced NFκB 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 NFκB 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.
Figure 4Lauric acid (12:0)-induced activation of NFκB 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 NFκB response element (A) or COX-2 promoter (B) and the expression plasmid for a dominant-negative mutant of Tlr4 (ΔTlr4(P712H)) and then treated with lauric acid (75 μm) for 24 h. Relative luciferase activity (RLA) was determined as described in Fig. 1. Thepanels 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.
Unsaturated Fatty Acids Inhibit Saturated Fatty Acid-induced COX-2 Expression, and This Inhibition Is Mediated through Suppression of NFκB
Unlike saturated fatty acids, unsaturated fatty acids were unable to induce COX-2 expression (Fig. 1D). Furthermore, they inhibited saturated fatty acid-induced NFκB 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 NFκB 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 NFκB signaling pathway.
Figure 5Unsaturated fatty acids inhibit lauric acid (C12:0)-induced activation of NFκB and COX-2 expression. RAW 264.7 cells were transfected with a luciferase (Luc) reporter plasmid for NFκB 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.
Unsaturated Fatty Acids Also Inhibit Constitutively Active Tlr4 (ΔTlr4)-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 (ΔTlr4)-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 NFκB and COX-2 expression in RAW 264.7 cells (
). 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.
Figure 6Docosahexaenoic acid (C22:6n-3) inhibits constitutively active Tlr4 (ΔTlr4)-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 (ΔTlr4) (A), a constitutively active MyD88(ΔToll) (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. Thepanels contain representative data from more than three different experiments. Values are mean ± S.E. (n= 3). *, significantly different from the control (ΔTlr4 without C22:6n-3); p < 0.05.
Unsaturated Fatty Acid Also Inhibits LPS-induced NFκB Activation and Expression of COX-2, iNOS, and IL-1α
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-1α (Fig.7A). Other unsaturated fatty acids tested (Fig. 1D) also inhibit LPS-induced COX-2 expression (data not shown). Inhibition of LPS-induced NFκB activation by docosahexaenoic acid (C22:6n-3) is demonstrated by inhibition of LPS-induced degradation of IκBα protein (Fig. 7B). Furthermore, docosahexaenoic acid (C22:6n-3) fails to inhibit TNFα-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.
Figure 7Docosahexaenoic acid (C22:6n-3) inhibits LPS-induced expression of COX-2, iNOS , and IL-1α and degradation of IκBα in RAW 264.7 cells, but it fails to inhibit TNFα-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-1α, or GAPDH immunoblot or (B) for 30 min and analyzed by IκBα 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 TNFα (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.
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 (
). 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 (
). 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 (
). 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 β-oxidation and the expression of certain peroxisomal enzymes (
). Using PPARα null mice, it was demonstrated that PPARα 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 (
). These results indicate that regulation of gene expression by fatty acids can be mediated through signaling pathways other than PPARs.
The inability of a dominant-negative mutant of PPARγ to inhibit saturated fatty acid-induced COX-2 expression suggests that the induction was not mediated through activation of PPARγ. However, the possibility that the induction of COX-2 expression by saturated fatty acid is in part mediated through PPARα or PPARδ 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.
It was shown that unsaturated fatty acids induce COX-2 expression in mammary epithelial cells (
). 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 (
) implies an important role of these fatty acids in LPS-mediated signal transmission. NFκB is one of the major downstream signaling pathways derived from activation of the LPS receptor, Tlr4 in RAW 264.7 cells (
). The results demonstrating that induction of COX-2 by lauric acid is mediated through activation of NFκB (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.
The results presented in Figs. 4 and 6 suggest that activation of NFκB 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.
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.
Acknowledgments
We thank Dr. Walter A. Deutsch for reading the manuscript and Wei Fan for technical assistance.
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