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Saturated Fatty Acids, but Not Unsaturated Fatty Acids, Induce the Expression of Cyclooxygenase-2 Mediated through Toll-like Receptor 4*

  • Joo Y. Lee
    Footnotes
    Affiliations
    Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana 70808
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  • Kyung H. Sohn
    Footnotes
    Affiliations
    Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana 70808
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  • Sang H. Rhee
    Affiliations
    Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana 70808
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  • Daniel Hwang
    Correspondence
    To whom correspondence should be addressed:
    Affiliations
    Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana 70808
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  • Author 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.
Open AccessPublished:May 18, 2001DOI:https://doi.org/10.1074/jbc.M011695200
      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 (
      • DeWitt D.L.
      • Smith W.L.
      ,
      • Merlie J.P.
      • Fagan D.
      • Mudd J.
      • Needleman P.
      ,
      • Yokoyama C.
      • Takai T.
      • Tanabe T.
      ,
      • DeWitt D.L.
      • El-Harith E.A.
      • Kraemer S.A.
      • Andrews M.J.
      • Yao E.F.
      • Armstrong R.L.
      • Smith W.L.
      ,
      • Yokoyama C.
      • Tanabe T.
      ) and mitogen-inducible COX-2 (
      • Kujubu D.A.
      • Fletcher B.S.
      • Varnum B.C.
      • Lim R.W.
      • Herschman H.R.
      ,
      • Xie W.
      • Chipman J.G.
      • Robertson D.L.
      • Erikson R.L.
      • Simmons D.L.
      ,
      • O'Banion M.K.
      • Sadowski H.B.
      • Winn V.
      • Young D.A.
      ,
      • Hla T.
      • Neilson K.
      ,
      • Jones D.A.
      • Carlton D.P.
      • McIntyre T.M.
      • Zimmerman G.A.
      • Prescott S.M.
      ,
      • Feng L.
      • Sun W.
      • Xia Y.
      • Tang W.W.
      • Chanmugam P.
      • Soyoola E.
      • Wilson C.B.
      • Hwang D.
      ). 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 (
      • Bennett A.
      • Berstock D.A.
      • Carroll M.A.
      • Stamford I.F.
      • Wilson A.J.
      ,
      • Bennett A.
      • McDonald A.M.
      • Stamford I.F.
      • Charlier E.M.
      • Simpson J.J.
      • Zebro T.
      ,
      • Powles T.J.
      • Dowsett M.
      • Easty G.C.
      • Easty D.M.
      • Neville A.M.
      ,
      • Fulton A.
      • Rios A.
      • Loveless S.
      • Heppner G.
      ,
      • Levine L.
      ). Recently, it has been shown that the inducible form of COX is overexpressed in sites of inflammation and in many types of tumor tissues (
      • Kutchera W.
      • Jones D.A.
      • Matsunami N.
      • Groden J.
      • McIntyre T.M.
      • Zimmerman G.A.
      • White R.
      • Prescott S.M.
      ,
      • Eberhart C.E.
      • Coffey R.J.
      • Radhika A.
      • Giardiello F.M.
      • Ferrenbach S.
      • Dubois R.N.
      ,
      • Sano H.
      • Kawahito Y.
      • Wilder R.L.
      • Hashiramoto A.
      • Mukai S.
      • Asai K.
      • Kimura S.
      • Kato H.
      • Kondo M.
      • Hla T.
      ,
      • Kargmann S.L.
      • O'Neill G.P.
      • Vickers P.J.
      • Evans J.F.
      • Mancini J.A.
      • Jothy S.
      ). Overexpression of COX-2 in tumor tissues occurs in both tumor cells and stromal cells including macrophages (
      • Hwang D.
      • Scollard D.
      • Byrne J.
      • Levine E.
      ). 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 (
      • Herschman H.R.
      ). 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 (
      • Kujubu D.A.
      • Fletcher B.S.
      • Varnum B.C.
      • Lim R.W.
      • Herschman H.R.
      ,
      • Hla T.
      • Neilson K.
      ,
      • Feng L.
      • Sun W.
      • Xia Y.
      • Tang W.W.
      • Chanmugam P.
      • Soyoola E.
      • Wilson C.B.
      • Hwang D.
      ,
      • Lee S.H.
      • Soyoola E.
      • Chanmugam P.
      • Hart S.
      • Sun W.
      • Zhong H.
      • Liou S.
      • Simmons D.
      • Hwang D.
      ). The cis-acting NFκB element is present in the 5′-flanking regions of COX-2 genes of different species (
      • Fletcher B.S.
      • Kujubu D.A.
      • Perrin D.M.
      • Herschman H.R.
      ,
      • Yamamoto K.
      • Arakawa T.
      • Ueda N.
      • Yamamoto S.
      ). 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 (
      • Hwang D.
      • Jang B.C., Yu, G.
      • Boudreau M.
      ,
      • Rhee S.H.
      • Hwang D.
      ). Proinflammatory cytokines, such as TNFα and IL-1, also activate NFκB and induce COX-2 expression in many cell types (
      • Schwenger P.
      • Alpert D.
      • Skolnik E.Y.
      • Vilcek J.
      ,
      • Schwenger P.
      • Bellosta P.
      • Vietor I.
      • Basilico C.
      • Skolnik E.Y.
      • Vilcek J.
      ).
      The recent finding that murine Tlr4 is the LPS receptor (
      • Poltorak A.
      • He X.
      • Smirnova I.
      • Liu M.Y.
      • Huffel C.V.
      • Du X.
      • Birdwell D.
      • Alejos E.
      • Silva M.
      • Galanos C.
      • Freudenberg M.
      • Ricciardi-Castagnoli P.
      • Layton B.
      • Beutler B.
      ) 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 (
      • Rhee S.H.
      • Hwang D.
      ). The lipid A moiety possesses most of the biological activities of LPS (
      • Raetz C.R.
      ). 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 (
      • Raetz C.R.
      ). 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 (
      • Munford R.S.
      • Hall C.L.
      ,
      • Kitchens R.L.
      • Ulevitch R.J.
      • Munford R.S.
      ). 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 (
      • Poltorak A.
      • He X.
      • Smirnova I.
      • Liu M.Y.
      • Huffel C.V.
      • Du X.
      • Birdwell D.
      • Alejos E.
      • Silva M.
      • Galanos C.
      • Freudenberg M.
      • Ricciardi-Castagnoli P.
      • Layton B.
      • Beutler B.
      ), 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 (
      • Rietschel E.T.
      • Brade H.
      ). 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.

      DISCUSSION

      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 (
      • Jump D.B.
      • Clarke S.D.
      ). 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 (
      • Jump D.B.
      • Clarke S.D.
      ) and modulate activity of signaling molecules such as phospholipase C and protein kinase C (
      • Hwang S.C.
      • Jhon D.Y.
      • Bae Y.S.
      • Kim J.H.
      • Rhee S.G.
      ,
      • Nishizuka Y.
      ). 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 (
      • Forman B.M.
      • Chen J.
      • Evans R.M.
      ,
      • Gottlicher M.
      • Widmark E.
      • Li Q.
      • Gustafsson J.A.
      ,
      • Kliewer S.A.
      • Sundseth S.S.
      • Jones S.A.
      • Brown P.J.
      • Wisely G.B.
      • Koble C.S.
      • Devchand P.
      • Wahli W.
      • Willson T.M.
      • Lenhard J.M.
      • Lehmann J.M.
      ,
      • Krey G.
      • Braissant O.
      • L'Horset F.
      • Kalkhoven E.
      • Perroud M.
      • Parker M.G.
      • Wahli W.
      ,
      • Yu K.
      • Bayona W.
      • Kallen C.B.
      • Harding H.P.
      • Ravera C.P.
      • McMahon G.
      • Brown M.
      • Lazar M.A.
      ,
      • Xu H.E.
      • Lambert M.H.
      • Montana V.G.
      • Parks D.J.
      • Blanchard S.G.
      • Brown P.J.
      • Sternbach D.D.
      • Lehmann J.M.
      • Wisely G.B.
      • Willson T.M.
      • Kliewer S.A.
      • Milburn M.V.
      ). 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 (
      • Jump D.B.
      • Clarke S.D.
      ). 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 (
      • Ren B.
      • Thelen A.P.
      • Peters J.M.
      • Gonzalez F.J.
      • Jump D.B.
      ). 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 (
      • Meade E.A.
      • McIntyre T.M.
      • Zimmerman G.A.
      • Prescott S.M.
      ). 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. 1 C) coincides with the abundance of these fatty acids in the lipid A molecule (
      • Raetz C.R.
      ). Lauric, myristic, and palmitic acids are known to be major fatty acids acylated in the lipid A molecule (
      • Raetz C.R.
      ). The fact that deacylation of these fatty acids from LPS results in loss of endotoxic activity (
      • Munford R.S.
      • Hall C.L.
      ,
      • Kitchens R.L.
      • Ulevitch R.J.
      • Munford R.S.
      ) 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 (
      • Rhee S.H.
      • Hwang D.
      ). 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. 4 A), 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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