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HNE as an inducer of COX-2
Author’s Accepted Manuscript HNE as an inducer of COX-2 Koji Uchida
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PII: DOI: Reference:
S0891-5849(17)30064-3 http://dx.doi.org/10.1016/j.freeradbiomed.2017.02.004 FRB13197
To appear in: Free Radical Biology and Medicine Received date: 14 December 2016 Revised date: 31 January 2017 Accepted date: 1 February 2017 Cite this article as: Koji Uchida, HNE as an inducer of COX-2, Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2017.02.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
HNE as an inducer of COX-2
Koji Uchida* Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
*Corresponding author. Koji Uchida, Ph.D. Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan. Tel: 81-3-5841-5127, Fax: 81-3-5841-8026. E-mail: [email protected]
Abstract Cyclooxygenase-2 (COX-2), an inducible isoform responsible for high levels of prostaglandin (PG) production during inflammation and immune responses, mediate a variety of biological actions involved in vascular pathophysiology. COX-2 is induced by various stimuli, including proinflammatory cytokines, to result in PG synthesis associated with inflammation and carcinogenesis. 4-Hydroxy-2-nonenal (HNE) is one of a group of small molecules that can induce COX-2 expression. The mechanistic studies have revealed that the HNE-induced COX-2 expression results from the stabilization of COX-2 mRNA mediated by the p38 mitogen-activated protein kinase signaling pathway and uniquely requires a serum component, which is eventually identified to be modified low-density lipoproteins (LDLs), such as the oxidized form of LDLs. It has also been shown that HNE-induced COX-2 expression is mechanistically linked to the expression of transcription factor p53 and that the overexpression of COX-2 is associated with down-regulation of a proteasome subunit, leading to the enhanced accumulation of p53 and ubiquitinated proteins and to the enhanced sensitivity toward HNE. Thus, the overall mechanism and pathophysiological role of the COX-2 induction by HNE have become increasingly evident. 1
ABBREVIATIONS: COX, cyclooxygenase; HNE, 4-hydroxy-2-nonenal; LDLs, low-density lipoproteins; MAPK, mitogen-activated protein kinase; NF-kB, nuclear factor-kB
Keywords: 4-Hydroxy-2-nonenal, COX-2
Introduction Atherosclerosis is a disorder of the lipid metabolism as well as a chronic inflammatory disease. Monocyte-derived macrophages play a prominent role in the formation and progression of atherosclerotic plaque, particularly after their transformation into foam cells. When activated by inflammatory stimuli, the macrophages synthesize and secrete various mediators, which cause the clinical manifestations and acute clinical complications of atherosclerosis. The eicosanoids derived from the metabolism of arachidonate, among those mediators, have been extensively investigated because several studies have focused on their close relation to atherogenesis. Cyclooxygense (COX) is a key enzyme catalyzing the rate-limiting step that converts free arachidonic acid to prostaglandin (PG) H2 on the arachidonic cascade [1]. COX exists in two distinct isozymes (COX-1 and COX-2), one of which, COX-2, is primarily responsible for inflammation [2]. COX-2 is not normally present under the basal conditions or is present in very low amounts; however, it is rapidly induced in response to a wide variety of cytokines, growth factors, and ligands of G protein-coupled receptors. COX-2 is responsible for high levels of PG production during inflammation and immune responses and mediates a variety of biological actions involved in vascular pathophysiology. The induction of the COX-2 gene expression is regulated at both transcriptional (promoter-based) and post-transcriptional levels [3-5]. Both mitogen-activated protein kinase (MAPK) and nuclear factor-kB (NF-kB) signaling pathways have been shown to mediate the COX-2 gene expression [6]. 2
There is considerable evidence that low-density lipoproteins (LDLs) is oxidatively modified in vivo, and that this modification results in an increase in its proinflammatory and proatherogenic properties. Several oxidized fatty acids generated in the oxidized LDLs have been shown to play a role as important signaling molecules in the context of the atherosclerotic lesion. In this article, I will illustrate a comprehensive summary of our studies on the activation of pro-inflammatory signaling mechanisms by one of the most studied products of lipid peroxidation, 4-hydroxy-2-nonenal (HNE).
Identification of HNE as an inducer of COX-2 In view of the observation that liver injury associated with oxidative stress is accompanied by increased PG synthesis, it is hypothesized that lipid peroxidation products may be involved in the up-regulation of the PG biosynthesis. Indeed, in an alcohol-fed rat, a model of alcoholic liver disease, alcohol over-intake increases the formation of HNE-modified proteins and is associated with the COX-2 and proinflammatory cytokine TNF-a expression [7, 8]. In addition, the HNE-specific epitopes have been detected in foamy macrophages within human atheromatous lesions [9] where the pro-inflammatory responses, including COX-2 expression, are being accelerated. To determine if lipid peroxidation could be involved in the COX-2 expression, Kumagai et al. [10] conducted a screen of oxidized fatty acids on COX-2 induction in rat liver epithelial RL34 and mouse macrophage RAW264.7 cell lines and demonstrated that HNE could specifically stimulate the COX-2 expression (Fig. 1). They have also shown that the depletion of the GSH pools in the cells with L-buthionine-S,R-sulfoximine significantly reduced the HNE-induced expression of COX-2 whereas the N-acetylcysteine pretreatment reversely led to a dose-dependent enhancement of the COX-2 expression [10]. These findings suggest the intracellular GSH status may be strictly related to the HNE-induced COX-2 expression. Of interest, they also observed that the a,b-unsaturated aldehydes, such as acrolein, crotonaldehyde, and 2-nonenal, possessing an analogous functionality to HNE, were all inactive on the COX-2 3
induction. These studies represent a first demonstration of a link between COX-2 and HNE.
Involvement of p38 MAPK pathway The NF-kB signal transduction cascade is a major stress response signaling pathway for the COX-2 gene expression. In mice and humans, the COX-2 promoter has binding sites for many transcription factors, including NF-kB in the 5' region of the COX-2 gene [11], and the requirement of the activation of NF-kB to induce the expression of COX-2 in the lipopolysaccharide-stimulated macrophages has been described [6]. Based on the discovery of HNE as a potential inducer of COX-2, several studies focusing on the HNE-induced signaling mechanisms for the COX-2 expression have been performed [12, 13]. Initially, it was anticipated that the NF-kB-dependent signaling pathway might mediate the HNE-stimulated COX-2 induction. However, no significant change in the IkB and NF-kB levels after treatment with HNE was observed. Kumagai et al. [12, 13] found that, instead of the NF-kB pathway, HNE elicited a rapid and significant phosphorylation of p38 mitogen-activated protein kinase (MAPK) and activate MAPK kinase (MKK)3/MKK6, a specific MAPKK of p38 MAPK. In addition, the relationship between COX-2 mRNA stability and HNE-activated p38 MAPK pathway was also revealed (Fig. 2). Involvement of a Src-dependent p38, ERK/c-Jun pathway was recently proposed as a major regulator of HNE-induced COX-2 expression in YPEN-1 cells [14]. Thus, the HNE-induced COX-2 gene expression is, at least in part, regulated at post-transcriptional levels via the p38 MAPK pathway.
Involvement of p53 and Sp1 To investigate transcriptional regulation of the COX-2 gene in response to HNE, Kumagai et al. [15] examined whether the HNE-induced COX-2 expression was mechanistically linked to the expression of p53, a transcription factor that regulates the response to a variety of stimuli, and found that the COX-2 levels were inversely 4
correlated with the p53 levels. In addition, the down-regulation of p53 with the antisense oligonucleotides against p53 significantly enhanced the expression of COX-2 mRNA and protein. These findings and the fact that COX-2 protein is undetectable in normal epithelial cells suggest that mutations of p53 may contribute to the increased expression of COX-2. Of interest, 4-oxo-2-nonenal, an analog of HNE, is unable to induce COX-2 while it activates a p53 signaling pathway [16]. On the other hand, it was hypothesized that Sp1, a general transcription factor that is involved in various inducible and constitutive gene expressions, might also be involved in the induction of COX-2 in response to HNE [15]. This speculation was based on the facts that (i) p53 suppresses various gene expressions through preventing Sp1 activity, (ii) The rat COX-2 promoter region has no putative p53-binding elements, and a minimal promoter region required for the basal transcription of the human COX-2 gene has been demonstrated to contain GC-rich proximal sequences that are specifically bound by Sp1, and (iii) p53 negatively regulates Sp1 through the formation of a p53-Sp1 heterocomplex. The immunoprecipitation experiments indeed showed that p53 bound to Sp1 in intact cells under normal conditions and HNE elicited the dissociation. It was also observed that the dissociation of p53-Sp1 complexes was accompanied by the nuclear translocation of Sp1. In addition, electrophoretic mobility shift assays using the oligonucleotide containing the Sp1 consensus element as a probe showed that HNE treatment resulted in a time-dependent increase in the Sp1 DNA binding activity. Although the regulatory mechanism of dissociation of p53-Sp1 complex remains unclear, the involvement of a phosphatidylinositol 3-kinase pathway, which induces p53 degradation through mdm2 phosphorylation, may not be unlikely. HNE indeed activates phosphatidylinositol 3-kinase/AKT pathway in vascular smooth muscle cells and wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, significantly inhibited the COX-2 expression [17]. Taken together, it has been hypothesized that down-regulation of p53 followed by the activation of a transcription factor Sp1 might be involved in the HNE-induced COX-2 gene expression (Fig. 2). 5
Outcome of COX-2 overexpression. Although a causal role for COX-2 has been proposed, mechanisms by which COX-2 function contributes to the pathogenesis of hyperplastic disease are not well defined. To examine if there is any correlation between COX-2 and p53 protein levels, Kumagai et al. [15] recently established the COX-2-overexpressing derivatives of RL34 cells by stable transfection with COX-2 cDNA. They investigated the COX-2-mediated change in gene expression by microarray analysis and observed significant up-regulation of acetylcholinesterase-associated collagen, isopentenyl diphosphate-dimethylallyl diphosphate isomerase, and p38 MAPK genes. In addition, the expression of genes involved in the phase II detoxification response, such as glutathione S-transferase Yb and Yc subunits, was also significantly up-regulated. They also observed significant down-regulation of proteasome subunits RC1 and RN3, transforming growth factor b-3, heat shock protein 27, apolipoprotein E, and prostacyclin synthase. Most notably, the proteasome RC1 subunit was dramatically down-regulated by ~26-fold in the COX-2 overexpressed cells. Consistent with the COX-2-mediated down-regulation of proteasome, a moderate reduction of the proteasome activities was observed. This proteasome dysfunction mediated by the COX-2 overproduction was associated with the enhanced accumulation of p53 and ubiquitinated proteins, leading to the enhanced sensitivity toward HNE. These results suggest the existence of a causal link between COX-2 and p53, which may represent a toxic mechanism of electrophilic lipid peroxidation products (Fig. 3).
A link between lipoprotein modification and inflammatory response A unique finding related to the HNE-induced COX-2 gene expression is that the modified LDLs might be involved in the COX-2 induction. Kanayama et al. [18] found that HNE could induce COX-2 only in the presence of serum. They also identified the modified LDL, including oxLDLs, as a bona fide active component essential for the induction of 6
COX-2 by HNE. In addition, they characterized cellular events and established that the combination of HNE and oxLDLs cooperatively induced COX-2 gene expression through a novel mechanism, by which HNE up-regulates gene expression of the scavenger receptor CD36 and promotes the CD36-mediated COX-2 induction by the modified LDLs (Fig. 4). These findings represent a demonstration of a link between the oxidative modification of LDLs and the activation of the inflammatory potential of macrophages. However, an association of the CD36/oxidized LDLs pathway with the MAPK pathways and/or the transcription factors (p53 and Sp1) in the HNE-mediated induction of COX-2 expression still remain unclear.
Concluding remarks Mechanistic studies so far have revealed that (i) HNE-induced COX-2 expression resulted from the stabilization of COX-2 mRNA that is mediated by the p38 MAPK signaling pathway, (ii) the transcription factors p53 and Sp1 play a role in the HNE-induced COX-2 expression, and (iii) signal transduction mechanisms in the COX-2 expression by HNE require a serum component. These findings indicate that HNE among various lipid peroxidation products is very unique in the way of activating signal transduction pathways leading to the induction of COX-2. It has also been shown that COX-2 overproduction is associated with down-regulation of a proteasome subunit and the resultant proteasome dysfunction results in the enhanced accumulation of p53 and ubiquitinated proteins, leading to enhanced sensitivity toward electrophiles. Although the detailed mechanisms by which COX-2 overexpression down-regulates the proteasome subunit remain unclear, interaction of the COX-2 polypeptide with regulatory protein(s) on gene expression of the proteasome subunit may not be unlikely. Further studies are required to define at a molecular level this novel mechanism of COX-2 function and to assess its physiological relevance. The discovery of HNE as a COX-2 inducer also represents a further demonstration of a link between the oxidative modification of LDL and activation of the inflammatory potential of macrophages, since the observed effect 7
could be relevant in atheromata, where close contact between macrophages and oxidized lipids might ultimately result in the development of an inflammatory response, together with a cell failure to repair tissue damage. This phenomenon may thus represent an important contributing feature in an early step in the process of macrophage transformation into the foam cells composing the fatty streak, a primary histologic aspect of incipient atherosclerosis. Our future challenge is to identify a target molecule that triggers the signal transduction pathways leading to the COX-2 expression in early stages of atherosclerosis. Studies focusing on these biochemical steps would also extend our understanding of the regulation of signaling cascades stimulated by various reactive products.
REFERENCES [1] Smith, W. L.; Garavito, R.M.; DeWitt, D.L. Prostaglandin Endoperoxide H Synthases (Cyclooxygenases)-1 and -2. J. Biol. Chem. 271:33157-33160; 1996. [2] Smith, W. L.; Langenbach, R. Why there are two cyclooxygenase isozymes. J. Clin. Invest. 107:1491-1495; 2001. [3] Ryseck, R.P.; Raynoschek, C.; Macdonald-Bravo, H.; Dorfman, K.; Mattei, M.; Bravo, R. Identification of an immediate early gene, pghs-B, whose protein product has prostaglandin synthase/cyclooxygenase activity. Cell Growth Differ 3:443-450; 1992. [4] Yamagata, K.; Andreasson, K.I.; Kaufmann, W.E.; Barnes, C.A.; Worley, P.F. Expression of a mitogen-inducible cyclooxygenase in brain neurons: regulation by synaptic activity and glucocorticoids. Neuron 11:371-386; 1993. [5] Newton, R. J.; Seybold, J.; Kuitert, L. M.; Bergmann, M.; Barnes, P. J. Repression of cyclooxygenase-2 and prostaglandin E2 release by dexamethasone occurs by transcriptional and post-transcriptional mechanisms involving loss of polyadenylated mRNA. J. Biol. Chem. 273:32312-32321; 1998. [6] Huang, D.; Jang, B. C.; Yu, G.; Boudreau, M. Expression of mitogen-inducible 8
cyclooxygenase induced by lipopolysaccharide: mediation through both mitogen-activated protein kinase and NF-kB signaling pathways in macrophages. Biochem. Pharmacol. 54:87-96; 1997. [7] Nanji A.A.; Miao L.; Thomas P.; Rahemtulla A.; Khwaja S.; Zhao S.; Peters D.; Tahan S.R.; Dannenberg A.J. Enhanced cyclooxygenase-2 gene expression in alcoholic liver disease in the rat. Gastroenterology 112:943–951; 1997. [8] Li C.J.; Nanji A.A.; Siakotos A.N.; Lin R.C. Acetaldehyde-modified and 4-hydroxynonenal-modified proteins in the livers of rats with alcoholic liver disease. Hepatology 26:650–657; 1997. [9] Uchida, K.; Itakura, K.; Kawakishi, S.; Hiai, H.; Toyokuni, S.; Stadtman, E.R. Characterization of epitopes recognized by 4-hydroxy-2-nonenal specific antibodies. Arch. Biochem. Biophys. 324:241-248; 1995. [10] Kumagai, T.; Kawamoto, Y.; Nakamura, Y.; Hatayama, I.; Satoh, K.; Osawa, T.; Uchida, K. 4-Hydroxy-2-nonenal,the end product of lipid peroxidation, is a specific inducer of cyclooxygenase-2 gene expression. Biochem. Biophys. Res. Commun. 273:437-771; 2000. [11] Reddy, S. T.; Wadleigh D. J.; Herschman, H. R. Transcriptional regulation of the cyclooxygenase-2 gene in activated mast cells. J. Biol. Chem. 275:3107-3113; 2000. [12] Kumagai, T.; Nakamura, Y.; Osawa, T.; Uchida, K. Role of p38 mitogen-activated protein kinase in the 4-hydroxy-2-nonenal-induced cyclooxygenase-2 expression. Arch. Biochem. Biophys. 397:240-245; 2002. [13] Kumagai, T.; Matsukawa, N.; Kaneko, Y.; Kusumi, Y.; Mitsumata, M.; Uchida, K. A lipid peroxidation-derived inflammatory mediator: Identification of 4-hydroxy-2-nonenal as a potential inducer of cyclooxygenase-2 in macrophages. J. Biol. Chem. 279:48389-48396; 2004. [14] Jang, E. J.; Jeong, H. O.; Park, D.; Kim, D. H.; Choi, Y. J.; Chung, K. W.; Park, M. H.; Yu, B. P.; Chung, H. Y. Src tyrosine kinase activation by 4-hydroxynonenal upregulates p38, ERK/AP-1 signaling and COX-2 expression in YPEN-1 Cells. 9
PLoS ONE 10:e0129244; 2015. [15] Kumagai, T.; Usami, H.; Matsukawa, N.; Nakashima, F.; Chikazawa, M.; Shibata, T.; Noguchi, N.; Uchida, K. Functional interaction between cyclooxygenase-2 and p53 in response to an endogenous electrophile. Redox Biol. 4:74-86; 2015. [16] Shibata, T.; Iio, K.; Kawai, Y.; Shibata, N.; Kawaguchi, M.; Toi, S.; Kobayashi, M.; Kobayashi, S.; Yamamoto, K.; Uchida, K. Identification of a lipid peroxidation product as a potential trigger of the p53 pathway. J. Biol. Chem. 281:1196-1204; 2006. [17] Lee, S.J.; Seo, K.W.; Yun, M.R.; Bae, S.S.; Lee, W.S.; Hong, K.W.; Kim, C.D. 4-hydroxynonenal enhances MMP-2 production in vascular smooth muscle cells via mitochondrial ROS-mediated activation of the Akt/NF-kappaB signaling pathways. Free Radic. Biol.Med. 45:1487-1492; 2008. [18] Kanayama, M.; Yamaguchi, S.; Shibata, T.; Shibata, N.; Kobayashi, M.; Nagai, R.; Arai, H.; Takahashi, K.; Uchida, K. Identification of a serum component that regulates cyclooxygenase-2 gene expression in cooperation with 4-hydroxy-2-nonenal. J. Biol. Chem. 282:24166-24174; 2007.
10
Fig. 1.
Identification of HNE as the most active inducer of COX-2 in RAW264.7
macrophages (adapted from ref. 13). RAW264.7 macrophages were treated for 6 h with 50 mM of the indicated compounds and COX-2 induction was examined by an immunoblot analysis. Abbreviations: ACR, acrolein; CRA, crotonaldehyde; ONE, 4-oxo-2-nonenal; 9(R)-HODE, 9R-hydroxy-10E,12Z-octadecadienoic acid; (+)13-HODE, (+)13-hydroxy-9Z,11E,-octadecadienoic acid; 9(S)-HpODE, 9S-hydroperoxy-10E,12Z-octadecadienoic acid; 13(S)-HpODE, 13S-hydroperoxy-9Z,11E-octadecadienoic acid; 9-OxoODE, 9-oxo-10E,12Z-octadecadienoic acid; 13-OxoODE, 13-oxo-9Z,11E-octadecadienoic acid; 7KC, 7-ketocholesterol; (+)13-HODE cholesteryl ester, (+)13-hydroxy-9Z,11E-octadecadienoic acid cholesteryl ester; Leukotoxin, (+)9(10)epoxy-12Z-octadecenoic acid.
Fig. 2.
Model for mechanisms by which HNE up-regulates COX-2 (adapted from
ref. 14). HNE stabilizes COX-2 mRNA through the p38 MAPK signaling pathway, leading to the up-regulation of COX-2. On the other hand, the present work suggests an alternative mechanism, by which HNE induces COX-2 gene expression through down-regulation of p53 followed by the activation of Sp1.
Fig. 3.
Regulatory mechanism for COX-2 expression mediated by proteasome
dysfunction followed by p53 up-regulation (adapted from ref. 14).
Fig. 4.
A proposed mechanism for COX-2 expression by the combined stimulus of
HNE and oxidized LDLs (adapted from ref. 17).
HNE, generated during oxidative
stress and LDL oxidation, up-regulates gene expression of the scavenger receptor CD36. The up-regulation of CD36 is accompanied by the enhanced uptake of oxidized LDLs through CD36 and promotes the CD36-mediated COX-2 induction by oxidized LDLs. 11
Highlights
࣭HNE is an inducer of COX-2. ࣭HNE-mediated COX-2 induction involves p38 MAPK pathway. ࣭HNE-mediated COX-2 induction involves p53 and Sp1 ࣭COX-2 overproduction leads to the enhanced sensitivity toward HNE.
12
Figure
COX-2
COX-2
COX-2
COX-2
Fig. 1
HNE Ubiquitinated p53 P38 MAPK pathway
Sp1 p53
p53 Proteasome
RNA binding factors
Sp1 Cox-2 gene
mRNA stabilization Cox-2 mRNA
Cox-2
Fig. 2
HNE p38 p53/Sp1
Cox-2
Proteasome
p53
Fig. 3
HNE EGFR
Oxidized LDL
CD36
CD36 expression
COX-2 expression
Fig. 4
www.elsevier.com
PII: DOI: Reference:
S0891-5849(17)30064-3 http://dx.doi.org/10.1016/j.freeradbiomed.2017.02.004 FRB13197
To appear in: Free Radical Biology and Medicine Received date: 14 December 2016 Revised date: 31 January 2017 Accepted date: 1 February 2017 Cite this article as: Koji Uchida, HNE as an inducer of COX-2, Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2017.02.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
HNE as an inducer of COX-2
Koji Uchida* Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
*Corresponding author. Koji Uchida, Ph.D. Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan. Tel: 81-3-5841-5127, Fax: 81-3-5841-8026. E-mail: [email protected]
Abstract Cyclooxygenase-2 (COX-2), an inducible isoform responsible for high levels of prostaglandin (PG) production during inflammation and immune responses, mediate a variety of biological actions involved in vascular pathophysiology. COX-2 is induced by various stimuli, including proinflammatory cytokines, to result in PG synthesis associated with inflammation and carcinogenesis. 4-Hydroxy-2-nonenal (HNE) is one of a group of small molecules that can induce COX-2 expression. The mechanistic studies have revealed that the HNE-induced COX-2 expression results from the stabilization of COX-2 mRNA mediated by the p38 mitogen-activated protein kinase signaling pathway and uniquely requires a serum component, which is eventually identified to be modified low-density lipoproteins (LDLs), such as the oxidized form of LDLs. It has also been shown that HNE-induced COX-2 expression is mechanistically linked to the expression of transcription factor p53 and that the overexpression of COX-2 is associated with down-regulation of a proteasome subunit, leading to the enhanced accumulation of p53 and ubiquitinated proteins and to the enhanced sensitivity toward HNE. Thus, the overall mechanism and pathophysiological role of the COX-2 induction by HNE have become increasingly evident. 1
ABBREVIATIONS: COX, cyclooxygenase; HNE, 4-hydroxy-2-nonenal; LDLs, low-density lipoproteins; MAPK, mitogen-activated protein kinase; NF-kB, nuclear factor-kB
Keywords: 4-Hydroxy-2-nonenal, COX-2
Introduction Atherosclerosis is a disorder of the lipid metabolism as well as a chronic inflammatory disease. Monocyte-derived macrophages play a prominent role in the formation and progression of atherosclerotic plaque, particularly after their transformation into foam cells. When activated by inflammatory stimuli, the macrophages synthesize and secrete various mediators, which cause the clinical manifestations and acute clinical complications of atherosclerosis. The eicosanoids derived from the metabolism of arachidonate, among those mediators, have been extensively investigated because several studies have focused on their close relation to atherogenesis. Cyclooxygense (COX) is a key enzyme catalyzing the rate-limiting step that converts free arachidonic acid to prostaglandin (PG) H2 on the arachidonic cascade [1]. COX exists in two distinct isozymes (COX-1 and COX-2), one of which, COX-2, is primarily responsible for inflammation [2]. COX-2 is not normally present under the basal conditions or is present in very low amounts; however, it is rapidly induced in response to a wide variety of cytokines, growth factors, and ligands of G protein-coupled receptors. COX-2 is responsible for high levels of PG production during inflammation and immune responses and mediates a variety of biological actions involved in vascular pathophysiology. The induction of the COX-2 gene expression is regulated at both transcriptional (promoter-based) and post-transcriptional levels [3-5]. Both mitogen-activated protein kinase (MAPK) and nuclear factor-kB (NF-kB) signaling pathways have been shown to mediate the COX-2 gene expression [6]. 2
There is considerable evidence that low-density lipoproteins (LDLs) is oxidatively modified in vivo, and that this modification results in an increase in its proinflammatory and proatherogenic properties. Several oxidized fatty acids generated in the oxidized LDLs have been shown to play a role as important signaling molecules in the context of the atherosclerotic lesion. In this article, I will illustrate a comprehensive summary of our studies on the activation of pro-inflammatory signaling mechanisms by one of the most studied products of lipid peroxidation, 4-hydroxy-2-nonenal (HNE).
Identification of HNE as an inducer of COX-2 In view of the observation that liver injury associated with oxidative stress is accompanied by increased PG synthesis, it is hypothesized that lipid peroxidation products may be involved in the up-regulation of the PG biosynthesis. Indeed, in an alcohol-fed rat, a model of alcoholic liver disease, alcohol over-intake increases the formation of HNE-modified proteins and is associated with the COX-2 and proinflammatory cytokine TNF-a expression [7, 8]. In addition, the HNE-specific epitopes have been detected in foamy macrophages within human atheromatous lesions [9] where the pro-inflammatory responses, including COX-2 expression, are being accelerated. To determine if lipid peroxidation could be involved in the COX-2 expression, Kumagai et al. [10] conducted a screen of oxidized fatty acids on COX-2 induction in rat liver epithelial RL34 and mouse macrophage RAW264.7 cell lines and demonstrated that HNE could specifically stimulate the COX-2 expression (Fig. 1). They have also shown that the depletion of the GSH pools in the cells with L-buthionine-S,R-sulfoximine significantly reduced the HNE-induced expression of COX-2 whereas the N-acetylcysteine pretreatment reversely led to a dose-dependent enhancement of the COX-2 expression [10]. These findings suggest the intracellular GSH status may be strictly related to the HNE-induced COX-2 expression. Of interest, they also observed that the a,b-unsaturated aldehydes, such as acrolein, crotonaldehyde, and 2-nonenal, possessing an analogous functionality to HNE, were all inactive on the COX-2 3
induction. These studies represent a first demonstration of a link between COX-2 and HNE.
Involvement of p38 MAPK pathway The NF-kB signal transduction cascade is a major stress response signaling pathway for the COX-2 gene expression. In mice and humans, the COX-2 promoter has binding sites for many transcription factors, including NF-kB in the 5' region of the COX-2 gene [11], and the requirement of the activation of NF-kB to induce the expression of COX-2 in the lipopolysaccharide-stimulated macrophages has been described [6]. Based on the discovery of HNE as a potential inducer of COX-2, several studies focusing on the HNE-induced signaling mechanisms for the COX-2 expression have been performed [12, 13]. Initially, it was anticipated that the NF-kB-dependent signaling pathway might mediate the HNE-stimulated COX-2 induction. However, no significant change in the IkB and NF-kB levels after treatment with HNE was observed. Kumagai et al. [12, 13] found that, instead of the NF-kB pathway, HNE elicited a rapid and significant phosphorylation of p38 mitogen-activated protein kinase (MAPK) and activate MAPK kinase (MKK)3/MKK6, a specific MAPKK of p38 MAPK. In addition, the relationship between COX-2 mRNA stability and HNE-activated p38 MAPK pathway was also revealed (Fig. 2). Involvement of a Src-dependent p38, ERK/c-Jun pathway was recently proposed as a major regulator of HNE-induced COX-2 expression in YPEN-1 cells [14]. Thus, the HNE-induced COX-2 gene expression is, at least in part, regulated at post-transcriptional levels via the p38 MAPK pathway.
Involvement of p53 and Sp1 To investigate transcriptional regulation of the COX-2 gene in response to HNE, Kumagai et al. [15] examined whether the HNE-induced COX-2 expression was mechanistically linked to the expression of p53, a transcription factor that regulates the response to a variety of stimuli, and found that the COX-2 levels were inversely 4
correlated with the p53 levels. In addition, the down-regulation of p53 with the antisense oligonucleotides against p53 significantly enhanced the expression of COX-2 mRNA and protein. These findings and the fact that COX-2 protein is undetectable in normal epithelial cells suggest that mutations of p53 may contribute to the increased expression of COX-2. Of interest, 4-oxo-2-nonenal, an analog of HNE, is unable to induce COX-2 while it activates a p53 signaling pathway [16]. On the other hand, it was hypothesized that Sp1, a general transcription factor that is involved in various inducible and constitutive gene expressions, might also be involved in the induction of COX-2 in response to HNE [15]. This speculation was based on the facts that (i) p53 suppresses various gene expressions through preventing Sp1 activity, (ii) The rat COX-2 promoter region has no putative p53-binding elements, and a minimal promoter region required for the basal transcription of the human COX-2 gene has been demonstrated to contain GC-rich proximal sequences that are specifically bound by Sp1, and (iii) p53 negatively regulates Sp1 through the formation of a p53-Sp1 heterocomplex. The immunoprecipitation experiments indeed showed that p53 bound to Sp1 in intact cells under normal conditions and HNE elicited the dissociation. It was also observed that the dissociation of p53-Sp1 complexes was accompanied by the nuclear translocation of Sp1. In addition, electrophoretic mobility shift assays using the oligonucleotide containing the Sp1 consensus element as a probe showed that HNE treatment resulted in a time-dependent increase in the Sp1 DNA binding activity. Although the regulatory mechanism of dissociation of p53-Sp1 complex remains unclear, the involvement of a phosphatidylinositol 3-kinase pathway, which induces p53 degradation through mdm2 phosphorylation, may not be unlikely. HNE indeed activates phosphatidylinositol 3-kinase/AKT pathway in vascular smooth muscle cells and wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, significantly inhibited the COX-2 expression [17]. Taken together, it has been hypothesized that down-regulation of p53 followed by the activation of a transcription factor Sp1 might be involved in the HNE-induced COX-2 gene expression (Fig. 2). 5
Outcome of COX-2 overexpression. Although a causal role for COX-2 has been proposed, mechanisms by which COX-2 function contributes to the pathogenesis of hyperplastic disease are not well defined. To examine if there is any correlation between COX-2 and p53 protein levels, Kumagai et al. [15] recently established the COX-2-overexpressing derivatives of RL34 cells by stable transfection with COX-2 cDNA. They investigated the COX-2-mediated change in gene expression by microarray analysis and observed significant up-regulation of acetylcholinesterase-associated collagen, isopentenyl diphosphate-dimethylallyl diphosphate isomerase, and p38 MAPK genes. In addition, the expression of genes involved in the phase II detoxification response, such as glutathione S-transferase Yb and Yc subunits, was also significantly up-regulated. They also observed significant down-regulation of proteasome subunits RC1 and RN3, transforming growth factor b-3, heat shock protein 27, apolipoprotein E, and prostacyclin synthase. Most notably, the proteasome RC1 subunit was dramatically down-regulated by ~26-fold in the COX-2 overexpressed cells. Consistent with the COX-2-mediated down-regulation of proteasome, a moderate reduction of the proteasome activities was observed. This proteasome dysfunction mediated by the COX-2 overproduction was associated with the enhanced accumulation of p53 and ubiquitinated proteins, leading to the enhanced sensitivity toward HNE. These results suggest the existence of a causal link between COX-2 and p53, which may represent a toxic mechanism of electrophilic lipid peroxidation products (Fig. 3).
A link between lipoprotein modification and inflammatory response A unique finding related to the HNE-induced COX-2 gene expression is that the modified LDLs might be involved in the COX-2 induction. Kanayama et al. [18] found that HNE could induce COX-2 only in the presence of serum. They also identified the modified LDL, including oxLDLs, as a bona fide active component essential for the induction of 6
COX-2 by HNE. In addition, they characterized cellular events and established that the combination of HNE and oxLDLs cooperatively induced COX-2 gene expression through a novel mechanism, by which HNE up-regulates gene expression of the scavenger receptor CD36 and promotes the CD36-mediated COX-2 induction by the modified LDLs (Fig. 4). These findings represent a demonstration of a link between the oxidative modification of LDLs and the activation of the inflammatory potential of macrophages. However, an association of the CD36/oxidized LDLs pathway with the MAPK pathways and/or the transcription factors (p53 and Sp1) in the HNE-mediated induction of COX-2 expression still remain unclear.
Concluding remarks Mechanistic studies so far have revealed that (i) HNE-induced COX-2 expression resulted from the stabilization of COX-2 mRNA that is mediated by the p38 MAPK signaling pathway, (ii) the transcription factors p53 and Sp1 play a role in the HNE-induced COX-2 expression, and (iii) signal transduction mechanisms in the COX-2 expression by HNE require a serum component. These findings indicate that HNE among various lipid peroxidation products is very unique in the way of activating signal transduction pathways leading to the induction of COX-2. It has also been shown that COX-2 overproduction is associated with down-regulation of a proteasome subunit and the resultant proteasome dysfunction results in the enhanced accumulation of p53 and ubiquitinated proteins, leading to enhanced sensitivity toward electrophiles. Although the detailed mechanisms by which COX-2 overexpression down-regulates the proteasome subunit remain unclear, interaction of the COX-2 polypeptide with regulatory protein(s) on gene expression of the proteasome subunit may not be unlikely. Further studies are required to define at a molecular level this novel mechanism of COX-2 function and to assess its physiological relevance. The discovery of HNE as a COX-2 inducer also represents a further demonstration of a link between the oxidative modification of LDL and activation of the inflammatory potential of macrophages, since the observed effect 7
could be relevant in atheromata, where close contact between macrophages and oxidized lipids might ultimately result in the development of an inflammatory response, together with a cell failure to repair tissue damage. This phenomenon may thus represent an important contributing feature in an early step in the process of macrophage transformation into the foam cells composing the fatty streak, a primary histologic aspect of incipient atherosclerosis. Our future challenge is to identify a target molecule that triggers the signal transduction pathways leading to the COX-2 expression in early stages of atherosclerosis. Studies focusing on these biochemical steps would also extend our understanding of the regulation of signaling cascades stimulated by various reactive products.
REFERENCES [1] Smith, W. L.; Garavito, R.M.; DeWitt, D.L. Prostaglandin Endoperoxide H Synthases (Cyclooxygenases)-1 and -2. J. Biol. Chem. 271:33157-33160; 1996. [2] Smith, W. L.; Langenbach, R. Why there are two cyclooxygenase isozymes. J. Clin. Invest. 107:1491-1495; 2001. [3] Ryseck, R.P.; Raynoschek, C.; Macdonald-Bravo, H.; Dorfman, K.; Mattei, M.; Bravo, R. Identification of an immediate early gene, pghs-B, whose protein product has prostaglandin synthase/cyclooxygenase activity. Cell Growth Differ 3:443-450; 1992. [4] Yamagata, K.; Andreasson, K.I.; Kaufmann, W.E.; Barnes, C.A.; Worley, P.F. Expression of a mitogen-inducible cyclooxygenase in brain neurons: regulation by synaptic activity and glucocorticoids. Neuron 11:371-386; 1993. [5] Newton, R. J.; Seybold, J.; Kuitert, L. M.; Bergmann, M.; Barnes, P. J. Repression of cyclooxygenase-2 and prostaglandin E2 release by dexamethasone occurs by transcriptional and post-transcriptional mechanisms involving loss of polyadenylated mRNA. J. Biol. Chem. 273:32312-32321; 1998. [6] Huang, D.; Jang, B. C.; Yu, G.; Boudreau, M. Expression of mitogen-inducible 8
cyclooxygenase induced by lipopolysaccharide: mediation through both mitogen-activated protein kinase and NF-kB signaling pathways in macrophages. Biochem. Pharmacol. 54:87-96; 1997. [7] Nanji A.A.; Miao L.; Thomas P.; Rahemtulla A.; Khwaja S.; Zhao S.; Peters D.; Tahan S.R.; Dannenberg A.J. Enhanced cyclooxygenase-2 gene expression in alcoholic liver disease in the rat. Gastroenterology 112:943–951; 1997. [8] Li C.J.; Nanji A.A.; Siakotos A.N.; Lin R.C. Acetaldehyde-modified and 4-hydroxynonenal-modified proteins in the livers of rats with alcoholic liver disease. Hepatology 26:650–657; 1997. [9] Uchida, K.; Itakura, K.; Kawakishi, S.; Hiai, H.; Toyokuni, S.; Stadtman, E.R. Characterization of epitopes recognized by 4-hydroxy-2-nonenal specific antibodies. Arch. Biochem. Biophys. 324:241-248; 1995. [10] Kumagai, T.; Kawamoto, Y.; Nakamura, Y.; Hatayama, I.; Satoh, K.; Osawa, T.; Uchida, K. 4-Hydroxy-2-nonenal,the end product of lipid peroxidation, is a specific inducer of cyclooxygenase-2 gene expression. Biochem. Biophys. Res. Commun. 273:437-771; 2000. [11] Reddy, S. T.; Wadleigh D. J.; Herschman, H. R. Transcriptional regulation of the cyclooxygenase-2 gene in activated mast cells. J. Biol. Chem. 275:3107-3113; 2000. [12] Kumagai, T.; Nakamura, Y.; Osawa, T.; Uchida, K. Role of p38 mitogen-activated protein kinase in the 4-hydroxy-2-nonenal-induced cyclooxygenase-2 expression. Arch. Biochem. Biophys. 397:240-245; 2002. [13] Kumagai, T.; Matsukawa, N.; Kaneko, Y.; Kusumi, Y.; Mitsumata, M.; Uchida, K. A lipid peroxidation-derived inflammatory mediator: Identification of 4-hydroxy-2-nonenal as a potential inducer of cyclooxygenase-2 in macrophages. J. Biol. Chem. 279:48389-48396; 2004. [14] Jang, E. J.; Jeong, H. O.; Park, D.; Kim, D. H.; Choi, Y. J.; Chung, K. W.; Park, M. H.; Yu, B. P.; Chung, H. Y. Src tyrosine kinase activation by 4-hydroxynonenal upregulates p38, ERK/AP-1 signaling and COX-2 expression in YPEN-1 Cells. 9
PLoS ONE 10:e0129244; 2015. [15] Kumagai, T.; Usami, H.; Matsukawa, N.; Nakashima, F.; Chikazawa, M.; Shibata, T.; Noguchi, N.; Uchida, K. Functional interaction between cyclooxygenase-2 and p53 in response to an endogenous electrophile. Redox Biol. 4:74-86; 2015. [16] Shibata, T.; Iio, K.; Kawai, Y.; Shibata, N.; Kawaguchi, M.; Toi, S.; Kobayashi, M.; Kobayashi, S.; Yamamoto, K.; Uchida, K. Identification of a lipid peroxidation product as a potential trigger of the p53 pathway. J. Biol. Chem. 281:1196-1204; 2006. [17] Lee, S.J.; Seo, K.W.; Yun, M.R.; Bae, S.S.; Lee, W.S.; Hong, K.W.; Kim, C.D. 4-hydroxynonenal enhances MMP-2 production in vascular smooth muscle cells via mitochondrial ROS-mediated activation of the Akt/NF-kappaB signaling pathways. Free Radic. Biol.Med. 45:1487-1492; 2008. [18] Kanayama, M.; Yamaguchi, S.; Shibata, T.; Shibata, N.; Kobayashi, M.; Nagai, R.; Arai, H.; Takahashi, K.; Uchida, K. Identification of a serum component that regulates cyclooxygenase-2 gene expression in cooperation with 4-hydroxy-2-nonenal. J. Biol. Chem. 282:24166-24174; 2007.
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Fig. 1.
Identification of HNE as the most active inducer of COX-2 in RAW264.7
macrophages (adapted from ref. 13). RAW264.7 macrophages were treated for 6 h with 50 mM of the indicated compounds and COX-2 induction was examined by an immunoblot analysis. Abbreviations: ACR, acrolein; CRA, crotonaldehyde; ONE, 4-oxo-2-nonenal; 9(R)-HODE, 9R-hydroxy-10E,12Z-octadecadienoic acid; (+)13-HODE, (+)13-hydroxy-9Z,11E,-octadecadienoic acid; 9(S)-HpODE, 9S-hydroperoxy-10E,12Z-octadecadienoic acid; 13(S)-HpODE, 13S-hydroperoxy-9Z,11E-octadecadienoic acid; 9-OxoODE, 9-oxo-10E,12Z-octadecadienoic acid; 13-OxoODE, 13-oxo-9Z,11E-octadecadienoic acid; 7KC, 7-ketocholesterol; (+)13-HODE cholesteryl ester, (+)13-hydroxy-9Z,11E-octadecadienoic acid cholesteryl ester; Leukotoxin, (+)9(10)epoxy-12Z-octadecenoic acid.
Fig. 2.
Model for mechanisms by which HNE up-regulates COX-2 (adapted from
ref. 14). HNE stabilizes COX-2 mRNA through the p38 MAPK signaling pathway, leading to the up-regulation of COX-2. On the other hand, the present work suggests an alternative mechanism, by which HNE induces COX-2 gene expression through down-regulation of p53 followed by the activation of Sp1.
Fig. 3.
Regulatory mechanism for COX-2 expression mediated by proteasome
dysfunction followed by p53 up-regulation (adapted from ref. 14).
Fig. 4.
A proposed mechanism for COX-2 expression by the combined stimulus of
HNE and oxidized LDLs (adapted from ref. 17).
HNE, generated during oxidative
stress and LDL oxidation, up-regulates gene expression of the scavenger receptor CD36. The up-regulation of CD36 is accompanied by the enhanced uptake of oxidized LDLs through CD36 and promotes the CD36-mediated COX-2 induction by oxidized LDLs. 11
Highlights
࣭HNE is an inducer of COX-2. ࣭HNE-mediated COX-2 induction involves p38 MAPK pathway. ࣭HNE-mediated COX-2 induction involves p53 and Sp1 ࣭COX-2 overproduction leads to the enhanced sensitivity toward HNE.
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Figure
COX-2
COX-2
COX-2
COX-2
Fig. 1
HNE Ubiquitinated p53 P38 MAPK pathway
Sp1 p53
p53 Proteasome
RNA binding factors
Sp1 Cox-2 gene
mRNA stabilization Cox-2 mRNA
Cox-2
Fig. 2
HNE p38 p53/Sp1
Cox-2
Proteasome
p53
Fig. 3
HNE EGFR
Oxidized LDL
CD36
CD36 expression
COX-2 expression
Fig. 4