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Originally published In Press as doi:10.1074/jbc.M409935200 on September 8, 2004

J. Biol. Chem., Vol. 279, Issue 46, 48389-48396, November 12, 2004
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A Lipid Peroxidation-derived Inflammatory Mediator

IDENTIFICATION OF 4-HYDROXY-2-NONENAL AS A POTENTIAL INDUCER OF CYCLOOXYGENASE-2 IN MACROPHAGES*{boxs}

Takeshi Kumagai{ddagger}, Nao Matsukawa{ddagger}, Yayoi Kaneko{ddagger}, Yoshiaki Kusumi§, Masako Mitsumata§, and Koji Uchida{ddagger}||

From the {ddagger}Graduate School of Bioagricultural Sciences and Institute for Advanced Research, Nagoya University, Nagoya 464–8601 and the §Department of Pathology, Nihon University School of Medicine, Tokyo 173–8610, Japan

Received for publication, August 30, 2004 , and in revised form, September 7, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid to eicosanoids, which mediate a variety of biological actions involved in vascular pathophysiology. In the present study, we investigated the role of lipid peroxidation products in the up-regulation of COX-2, an inducible isoform responsible for high levels of prostaglandin production during inflammation and immune responses. COX-2 was found to colocalize with 4-hydroxy-2-nonenal (HNE), a major lipid peroxidation-derived aldehyde, in foamy macrophages within human atheromatous lesions, suggesting that COX-2 expression may be associated with the accumulation of lipid peroxidation products within macrophages. To test the hypothesis that lipid peroxidation products might be involved in the regulation of prostanoid biosynthesis, we conducted a screen of oxidized fatty acid metabolites and found that, among the compounds tested, only HNE showed inducibility of the COX-2 protein in RAW264.7 macrophages. In addition, intraperitoneal administration of HNE resulted in an increase in cell numbers in the peritoneal cavity that was associated with significant increases in the peritoneal and tissue levels of COX-2 in mice. To understand the possible signaling mechanism underlying the inducing effect of HNE on COX-2 up-regulation, we examined the phosphorylation events that may lead to COX-2 induction and found that HNE did not stimulate the induction of nitric oxide synthase and activation of NF-{kappa}B but significantly activated p38 mitogen-activated protein kinase and its upstream kinase in RAW264.7 macrophages. Tyrosine kinases, such as the epidermal growth factor-like and Src family tyrosine kinases, appeared to mediate the stabilization of COX-2 mRNA via the p38 mitogen-activated protein kinase pathway. These findings suggest that HNE accumulated in macrophages/foam cells may represent an inflammatory mediator that plays a role in stimulation of the inflammatory response and contributes to the progression of atherogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Atherosclerosis is a disorder of 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, including cytokines, prothrombotic substances, and eicosanoids that cause the clinical manifestations and acute clinical complications of atherosclerosis. The eicosanoids derived from the metabolism of arachidonate have been extensively investigated because several studies have focused on their close relation to atherogenesis (1, 2). In macrophages, as well as in other cell types, arachidonate metabolites are synthesized by the cyclooxygenase enzyme. Presently, two isoforms of cyclooxygenase have been identified: cyclooxygenase-1 (COX-1),1 which is the constitutive form, and cyclooxygenase-2 (COX-2), which is the inducible form. COX-1, with an mRNA transcript of 2.8–3.0 kb, is present under normal conditions in most tissues and is responsible for housekeeping functions. On the other hand, COX-2, with an mRNA transcript of 4.0–4.5 kb, is not normally present under basal conditions or is present in very low amounts. However, COX-2 is rapidly induced by various stimuli, including proinflammatory cytokines such as interleukin-1{beta} and tumor necrosis factor-{alpha}, growth factors, and tumor promoters, to result in prostaglandin synthesis associated with inflammation and carcinogenesis (3). In macrophages, COX-2 expression appears to be mediated through both mitogen-activated protein kinase (MAPK) and nuclear factor-{kappa}B (NF-{kappa}B) signaling pathways (4).

Several lines of evidence indicate that the oxidative modification of protein and the subsequent accumulation of the modified proteins have been found in cells during aging, oxidative stress, and in various pathological states including premature diseases, muscular dystrophy, rheumatoid arthritis, and atherosclerosis (5, 6). The important agents that give rise to the modification of a protein may be represented by reactive aldehydic intermediates such as ketoaldehydes, 2-alkenals, and 4-hydroxy-2-alkenals (7). These reactive aldehydes are considered important mediators of cell damage because of their ability to covalently modify biomolecules that can disrupt important cellular functions and can cause mutations (8, 9). Furthermore, the adduction of aldehydes to apolipoprotein B in low density lipoproteins (LDL) has been strongly implicated in the mechanism by which LDL is converted to an atherogenic form that is taken up by macrophages, leading to the formation of foam cells (10, 11). 4-Hydroxy-2-nonenal (HNE), among the reactive aldehydes, is a major product of lipid peroxidation and is believed to be largely responsible for the cytopathological effects observed during oxidative stress (8, 9).

Atherogenesis involves several aspects of chronic inflammation and wound healing. Indeed, the atheroma is considered a special case of tissue response to injury. Injurious stimuli may include lipoproteins trapped within lesions where protein and lipid moieties have undergone oxidative modifications. In a recent study, oxidized LDL components were shown to activate CD36, an important scavenger receptor, mediating the uptake of oxidized LDL (12, 13). These findings suggest that oxidized LDL contains a pro-atherogenic molecule that plays a role in foam cell formation and the pathogenesis of atherosclerosis. In view of the observation that increased eicosanoid production is closely associated with atherogenesis (1, 2), we hypothesized that an oxidized LDL component might be involved in the up-regulation of the prostaglandin biosynthesis. Even though human atherosclerotic lesions have been shown to contain a number of oxidized LDL components, we possess no information concerning their pro-inflammatory function (COX-2 inducibility) in macrophages.

In the present study, we have demonstrated that COX-2 colocalizes with protein-bound HNE in macrophage-derived foam cells within atheromatous lesions. We then evaluated the effect of the oxidized fatty acid metabolites on COX-2 induction in RAW264.7 macrophages and identified HNE as the potential inducer of COX-2 expression. Moreover, the involvement of HNE in COX-2 expression is also suggested by our demonstrations that COX-2 is up-regulated in mice administered with HNE. Finally, a signal transduction mechanism involved in the COX-2 expression stimulated by HNE is shown.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—HNE was purchased from Cayman Chemical Co. (Ann Arbor, MI). 4-Oxo-2-nonenal was synthesized by the oxidation of HNE-dimethyl acetal with pyridinium dichlorochromate followed by HCl hydrolysis (14). Anti-COX-1 monoclonal and anti-COX-2 polyclonal antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-p38 MAPK (Thr-180/Tyr-182) and anti-MKK3/MKK6 (Ser-189/207) phospho-specific antibodies were purchased from New England Labs, Inc. (Beverly, MA). The p38 MAPK-specific inhibitor SB203580 was from BIOMOL (Plymouth Meeting, PA). The tyrosine kinase inhibitors herbimycin, Na3VO4, PP2, and AG1478 were purchased from Calbiochem. First-strand cDNA synthesis kit was from Invitrogen. Horseradish peroxidase-linked anti-goat IgG was from DAKO Corp. Anti-rabbit IgG immunoglobulin-conjugated horseradish peroxidase, enhanced chemiluminescence (ECL) Western blotting detection reagents, and Hybond ECL nitrocellulose membranes were obtained from Amersham Biosciences. Protein concentration was measured using the BCA protein assay reagent obtained from Pierce.

Cell Culture—The murine macrophage cell line RAW264.7 was kindly given by Dr. Murakami (Kinki University, Japan). The cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 units/ml), streptomycin (100 µg/ml), L-glutamine (588 µg/ml), and 0.16% NaHCO3 at 37 °C in an atmosphere of 95% air and 5% CO2. 50–60% confluent cells were subjected to experiments in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum.

Immunoblot Analysis—The HNE-treated and untreated cells were washed twice with phosphate-buffered saline (pH 7.0) and lysed with lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride). After protein quantification, equal amounts of protein were (total protein, 20–50 µg) boiled with Laemmli sample buffer for 5 min at 100 °C (15). The samples were run on 10% SDS-polyacrylamide gels, transferred to a nitrocellulose membrane, incubated with 5% skim milk in TTBS (Tris-buffered saline containing 10% Tween 20) for blocking, washed, and treated with the primary antibodies. After washing with TTBS, blots were further incubated for 1 h at room temperature with IgG antibody coupled to horseradish peroxidase in TTBS. Blots were then washed three times in TTBS before visualization. An ECL kit was used for detection.

RNA Isolation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)—Total RNA was isolated with ISOGEN reagent (Nippon Gene Co., Tokyo, Japan). The RNA concentration was determined by measuring the absorbance at 260 nm. The RT reaction was performed with 5 µg of total RNA and an oligo(dT) primer using the First-strand cDNA synthesis kit. PCR reactions were carried out using 0.5 µl of cDNA in 25 µl of 10 mM Tris-HCl, pH 8.3, containing 50 mM KCl, 0.1% Triton X-100, 200 µM dNTPs, 1 µM of each forward and reverse primer, and 2 units of rTaq DNA polymerase (Toyobo Co., Osaka, Japan). The amplification conditions were: COX-2, 94 °C for 15 s (denaturation), 54 °C for 15 s (annealing), and 72 °C for 30 s (extension) for 26 cycles; GAPDH, 94 °C for 15 s, 56 °C for 15 s, and 72 °C for 30 s for 25 cycles, followed by 72 °C for 10 min. The following primers were used: COX-2, (F) 5'-CCCTGCTGGTGGAAAAGCCTGGTCC-3' and (R) 5'-TACTGTAGGGTTAATGTCATCTAG-3'; GAPDH, (F) 5'-AACCCATCACCATCTTCCAGGAGC-3' and (R) 5'-CACAGTCTTCTGAGTGGCAGTGAT-3'.

RNA Stability Assay—RAW264.7 cells were treated with HNE for 2 h to induce COX-2 mRNA expression. The medium was then changed to fresh medium containing actinomycin D (5 µg/ml) with or without an inhibitor reagent. At different time intervals, cells were collected for RNA preparation. RNA was examined by RT-PCR analysis. The levels of COX-2 mRNA were normalized for the intensity of the GAPDH signals.

Animal Experiments—Male C57BL/6J mice (Japan SLC Inc., Hamamatsu, Japan) were obtained at 7 weeks of age and individually housed in plastic cages (five/cage) at 23 °C with a 12-h light cycle. For 12 days they were fed unrestricted amounts of water and the control diet as follows: 20% casein, 3.5% mineral (93G-MX), 5.0% vitamin (93-VX), 0.2% choline chloride, 5.0% corn oil, 4.0% cellulose powder, 22.1% sucrose, and 44.2% starch. Animals received a single intraperitoneal injection of 10 µmol HNE (200 µl of PBS). Control animals received an equal volume of PBS. They were sacrificed by decapitation at 0, 6, and 24 h after the administration. Following sacrifice, the mice were injected with 4 ml of cold saline. The peritoneal cavity was gently massaged and the lavage fluid withdrawn and pooled. This procedure was repeated three times. Following washes with Hanks' balanced salt solutions, peritoneal cells were suspended in RPMI 1640 containing 10% fetal calf serum and incubated for 2 h at 37 °C, 5% CO2. After the incubation, nonadherent cells were removed by washing three times with Hank's Balanced Salt Solutions, and adherent cells were recovered with trypsin treatment and counted. For immunohistochemical analysis of COX-2, tissues from the control and HNE-treated animals were immediately removed, fixed in Bouin's solution, embedded in paraffin, cut to 3-µm thickness, and used for immunohistochemical analyses by an avidin-biotin complex method with alkaline phosphatase. Briefly, after deparaffinization with xylene and ethanol, anti-COX-2 polyclonal antibody (0.5 µg/ml), biotin-labeled anti-rabbit IgG (diluted 1:300; Vector Laboratories), and avidin-biotin complex (diluted 1:100; Vector) were sequentially used. Procedures using PBS or the IgG fraction (0.5 µg/ml) of normal rabbit serum instead of anti-COX-2 polyclonal antibody showed no or negligible positive responses.

Immunohistochemical Detection of COX-2 and Protein-bound HNE in Human Atherosclerotic Coronary Artery—Coronary artery specimens were obtained at routine autopsy procedures from patients with atherosclerosis and used for histopathological and immunohistochemical examinations. Each autopsy was performed at Nihon University Itabashi Hospital after family members granted informed consent in accordance with the Ethical Guidelines of Human Materials at Nihon University School of Medicine. Tissue samples from each case were fixed in 20% formalin, dehydrated, embedded in paraffin, and stored at room temperature. Serial 4-µm thick sections were cut from paraffin materials and used for hematoxylin-eosin staining or immunohistochemical staining. After deparaffinization and rehydration, the sections were quenched for 15 min with 3% hydrogen peroxide, rinsed in PBS, and boiled for 10 min in 100 mM citrate buffer, pH 6.0, for antigen retrieval. The tissues were pretreated with 5% cow powder milk in PBS for 30 min at 37 °C and incubated for 60 min at 37 °C with a mouse monoclonal anti-CD68 antibody (KP-1; DAKO) at a dilution of 1:200, goat polyclonal anti-COX-2 antibody (Santa Cruz Biotechnology) at a dilution of 1:200, or the mouse monoclonal anti-HNE-histidine antibody (RS17) at a dilution of 0.1 µg/ml. Antibody binding was visualized using the Histo-Fine SimpleStain kits (Nichirei Corp.), according to the manufacturer's instructions. 3,3'-Diaminobenzidine tetrahydrochloride was used as the chromogen, and hematoxylin was used as the counterstain. Sections from which the primary antibodies were omitted served as negative reaction controls. The localizations of HNE and COX-2 immunoreactivities were verified by consecutive sections stained with hematoxylineosin and immunostained for CD68.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Colocalization of COX-2 with Protein-bound HNE in Human Atherosclerotic Lesions—To obtain evidence for the involvement of lipid peroxidation products in COX-2 overproduction in vivo, we examined the pathohistologic location of COX-2 and a representative lipid peroxidation product, HNE, in human atherosclerotic coronary arterial samples. The non-atherosclerotic arterial wall had little or no COX-2 and HNE (data not shown), whereas in and around the advanced atherosclerotic core region both COX-2 and protein-bound HNE localized in the cytoplasm of foamy macrophages (Fig. 1, A and B). These macrophages were identified by histopathological and immunohistochemical features on hematoxylin-eosin-stained (data not shown) or CD68-immunostained (panel C) sections. It was also found that both COX-2 and HNE colocalized with other blood vessel cell types, but the brightest signals were shown in macrophage-derived foam cells. No immunoreaction product deposits were detected in sections with omission of the primary antibodies (panel D). These data suggest that COX-2 expression may be associated with the accumulation of lipid peroxidation products within macrophages.



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  		FIG. 1.
Colocalization of COX-2 with a lipid peroxidation product in advanced atheromatous lesions of arterial tissue. Coronary arterial tissue specimen with atherosclerosis was immunostained with COX-2 (A), anti-HNE antibody (RS17) (B), and anti-CD68 antibody (C). No immunoreaction is visible on a section from which either primary antibody was omitted (D) as negative reaction controls.

 
Identification of HNE as a Specific COX-2 Inducer—To test whether lipid peroxidation products might be involved in regulation of prostaglandin biosynthesis, we conducted a screen of oxidized fatty acid metabolites. RAW264.7 macrophages were treated for 6 h with 50 µM of the indicated compounds, and COX-2 induction was examined by an immunoblot analysis. Remarkably, among the oxidized lipids tested only HNE showed inducibility of the COX-2 protein (Fig. 2). Of interest, 4-oxo-2-nonenal, an analog of HNE, did not show any effect on COX-2 induction. These results and the observation that COX-2 colocalized with HNE in human atherosclerotic lesions (Fig. 1) suggest that HNE might be involved in COX-2 induction in macrophages.



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  		FIG. 2.
Identification of HNE as the most active inducer of COX-2 in RAW264.7 macrophages. RAW264.7 macrophages were treated for 6 h with 50 µM of the indicated compounds, and COX-2 induction was examined by an immunoblot analysis. ACR, acrolein; CRA, crotonaldehyde; (±)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.

 
HNE Induces COX-2 Expression in RAW264.7 Macrophages—We then examined HNE (Fig. 3A) for an effect on COX-2 expression. When the cells were treated with 50 µM HNE, almost maximal increases in COX-2 protein levels in response to HNE treatment were observed by 6 h, whereas the amount of the COX-1 was essentially unchanged (Fig. 3B). Dose-dependent effects of HNE were also analyzed at the time of maximum induction (6 h), and the COX-2 protein was induced in the concentration range of 5–75 µM (Fig. 3C). Transcriptional regulation of COX activities was further demonstrated using RT-PCR. As illustrated in Fig. 3D, the effect of HNE on the COX-2 protein was correlated with increasing levels of COX-2 mRNA. The increase in the COX-2 message, therefore, coincided with a substantial rise in the COX-2 protein level. It has also been shown that oxidized LDL components, including HNE, could activate CD36, an important scavenger receptor, mediating the uptake of oxidized LDL (13). Consistent with this finding, HNE-induced COX-2 expression was functionally associated with the expression of other inflammation-related regulatory molecules, such as CD36 (Supplementary Fig. S1). These data suggest that HNE may represent an inflammatory mediator that is capable of directly inducing several pro-atherogenic molecules in macrophages.



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  		FIG. 3.
Induction of COX-2 expression by HNE in RAW264.7 macrophages. A, chemical structure of HNE. B, time-dependent induction of COX-1 (lower) and COX-2 (upper) proteins in the cells treated with 50 µM HNE. C, dose-dependent induction of COX-2 protein in the cells treated with HNE for 4 h. D, RT-PCR analysis of COX-1 (lower) and COX-2 (upper) in RAW264.7 macrophages treated with 50 µM HNE for different time intervals.

 
COX-2 Up-regulation in Mice Administered with HNE—To further examine whether HNE is capable of inducing COX-2 expression in vivo, mice were challenged intraperitoneal with 10 µmol HNE for 48 h, and changes in cell numbers and COX-2 levels in the peritoneal cavity were measured. As shown in Fig. 4A, left panel, the HNE-treated mice showed a 2.7-fold increase in cell numbers in the peritoneal cavity. This increase was because of HNE, as saline challenge (Control) did not induce a cellular infiltrate. This HNE-induced cellular infiltrate was associated with the increased expression of COX-2 in peritoneal macrophages (Fig. 4A, right panel). Moreover, we examined COX-2 levels in the tissues (liver, lung, kidney, and spleen) from mice challenged with HNE and found that COX-2 was significantly induced by HNE treatment in these tissues (Fig. 4B). An immunohistochemistry experiment also verified the induction of COX-2 in the tissues from mice treated with HNE (Fig. 4C).



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  		FIG. 4.
Induction of COX-2 in mice administered with HNE. Mice were challenged intraperitoneal with 10 µmol HNE for 6, 24, or 48 h, and changes in cell numbers and COX-2 levels in the peritoneal cavity and tissues were measured. A, the number of total inflammatory cells (left) and the COX-2 protein levels in the peritoneal cavity of mice administered with HNE for 48 h. The means of duplicate determinations are shown with S.D. bars. B, immunoblot analysis of COX-2 in the tissues of mice administered with HNE. C, immunohistochemical detection of COX-2 in the liver of mice administered with HNE.

 
HNE Stimulated COX-2 Induction through an NF-{kappa}B-independent Mechanism—It has been shown that nitric oxide (NO) may increase prostaglandin synthesis by activation of the COX-2 protein (16–20). NO is known to react with iron-containing enzymes, resulting in their activation; in this respect, the heme-containing COX-2 enzyme is a potential target for the direct action of NO. Conversely, production of prostaglandin has been reported to increase or decrease NO production in a biphasic manner in murine macrophages and mesangial cells (21, 22). As shown in Fig. 5A, LPS significantly induced both COX-2 and iNOS in RAW264.7 cells, whereas HNE-induced COX-2 expression was not associated with iNOS induction. The observation that HNE-induced up-regulation of COX-2 was not associated with iNOS induction contradicts previous findings that NF-{kappa}B, a ubiquitous transcription factor, regulates gene expression of both COX-2 and iNOS (23–25). Hence, to ascertain whether NF-{kappa}B is involved in HNE-induced COX-2 expression, we examined NF-{kappa}B activation in RAW264.7 macrophages treated with HNE. As shown in Fig. 5, B–E, LPS induced transient reduction of the inhibitor-{kappa}B (I{kappa}B) level (panel B) and significantly enhanced the nuclear translocation of NF-{kappa}B (panel C), whereas no significant change in I{kappa}B and NF-{kappa}B levels was observed in the HNE-stimulated cells (D and E). Thus, it appears that HNE up-regulates COX-2 expression through an NF-{kappa}B-independent mechanism.



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  		FIG. 5.
Effect of LPS or HNE on iNOS induction and activation of NF-{kappa}B pathway in RAW264.7 macrophages. A, induction of iNOS. The cells were treated with 100 ng/ml of LPS in the presence of 100 units/ml of interferon-{gamma} or 50 µM of the indicated compound (HNE, ACR, or 13-oxoODE) for 6 h. Expression of iNOS and COX-2 proteins was detected by immunoblot analysis. ACR, acrolein; 13-oxoODE, 13-oxo-9Z,11E-octadecadienoic acid. B, LPS-induced transient reduction of I{kappa}B. C, LPS-induced nuclear translocation of NF-{kappa}B. D, changes in cytosolic I{kappa}B levels in cells treated with HNE. E, changes in cytosolic and nuclear NF-{kappa}B levels in cells treated with HNE. B–D, I{kappa}B and NF-{kappa}B proteins detected by immunoblot analysis.

 
Involvement of Tyrosine Kinases in HNE-induced COX-2 Ex pression—To understand the possible signaling mechanism underlying the inducing effect of HNE on COX-2 up-regulation, we examined the phosphorylation events that may lead to COX-2 induction. Involvement of tyrosine kinases was suggested by the observations that a tyrosine kinase inhibitor, herbimycin, significantly inhibited COX-2 induction (Fig. 6A), whereas sodium orthovanadate, a general inhibitor of protein tyrosine phosphatase, conversely potentiated it (Fig. 6B).



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  		FIG. 6.
Effect of pharmacological inhibitors of tyrosine kinase and protein tyrosine phosphatase on HNE-induced COX-2 expression in RAW264.7 macrophages. A, effect of a tyrosine kinase inhibitor. The cells were treated with 1 or 10 µM herbimycin for 1 h prior to incubation with 50 µM HNE for 4 h. B, effect of a protein tyrosine phosphatase inhibitor. The cells were treated with 1 or 10 µM sodium orthovanadate for 1 h prior to incubation with 50 µM HNE for 4 h.

 
To further investigate whether HNE can activate cellular protein kinases, the cells incubated with 50 µM HNE and the tyrosine phosphorylation of cellular proteins were determined by Western blot analysis with anti-phosphotyrosine antibody. As shown in Fig. 7A, several proteins with apparent molecular masses of 60, 80, and 120 kDa were the dominant proteins phosphorylated after incubation with HNE. The increase in tyrosine phosphorylation was detectable within 5 min during HNE treatment and up to at least 2 h after the treatment. In addition, HNE-induced tyrosine phosphorylation was significantly inhibited by PP2 and AG1478, the pharmacological inhibitors of the Src family and EGFR-like tyrosine kinases, respectively (Fig. 7, B and C). Therefore, the early increase in tyrosine phosphorylation led us to hypothesize that the activation of protein tyrosine kinases is probably one of the upstream signals of HNE-stimulated COX-2 induction in cells. In agreement with this hypothesis, the inhibitors of the Src family and EGFR-like tyrosine kinases significantly suppressed HNE-induced COX-2 expression (Fig. 7D). Thus, it is likely that multiple protein kinases may be involved in the induction of COX-2 by HNE.



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  		FIG. 7.
Involvement of Src family and EGFR-like tyrosine kinases in HNE-induced COX-2 expression in RAW264.7 macrophages. A, HNE-induced tyrosine phosphorylation. RAW264.7 macrophages were treated with 50 µM HNE for different time intervals as indicated. B, inhibition of HNE-induced tyrosine phosphorylation by the Src family tyrosine kinase inhibitor PP2. C, inhibition of HNE-induced tyrosine phosphorylation by the EGFR-like tyrosine kinase inhibitor AG1478. D, inhibition of the HNE-induced COX-2 expression by the Src family and EGFR-like tyrosine kinase inhibitors. B–D, cells were treated with the inhibitors for 30 min prior to incubation with 50 µM HNE for 4 h.

 
Involvement of Tyrosine Kinases in p38 MAPK-mediated Stabilization of COX-2 mRNA—It has been shown that p38 MAPK plays a pivotal role in COX-2 induction (4). In addition, we have previously shown that HNE stimulates the p38 MAPK pathway in rat liver epithelial RL34 cells (26). Hence, we examined the involvement of tyrosine kinases in HNE-induced COX-2 expression through the p38 MAPK pathway. As shown in Fig. 8A, after stimulation with 50 µM HNE, p38 MAPK was rapidly and strongly phosphorylated within 5 min, and this phosphorylation was maintained after 60 min. MKK3/MKK6, which is an upstream kinase of p38 MAPK, was also phosphorylated by HNE treatment, and this phosphorylation was correlated with the p38 MAPK phosphorylation. HNE also activated c-Jun NH2-terminal kinase; however, a MAPK pathway, consisting of extracellular signal-regulated kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase kinases 1/2, was not phosphorylated by the HNE treatment (data not shown). The involvement of a p38 MAPK pathway in the HNE-induced COX-2 expression was suggested by the observation that SB203580, a selective p38 MAPK inhibitor, significantly suppressed HNE-induced COX-2 expression (Fig. 8B). To establish the possible connection between tyrosine kinases and MAPK, the effect of pharmacological inhibitors of the Src family and EGFR-like tyrosine kinases upon HNE-induced phosphorylation of p38 MAPK was examined. As shown in Fig. 8C, both tyrosine kinase inhibitors PP2 and AG1478 significantly suppressed the phosphorylation of p38 MAPK. The result suggests that HNE activates the p38 MAPK cascades via the Src family and EGFR-like tyrosine kinases.



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  		FIG. 8.
Involvement of Src family and EGFR-like tyrosine kinases in HNE-induced activation of p38 MAPK in RAW264.7 macrophages. A, activation of p38 MAPK and its upstream kinase MKK3 by HNE in RAW264.7 macrophages. The cells were treated with 50 µM HNE for different time intervals as indicated. Whole cell lysates were subjected to immunoblot analysis for detection of phospho-p38 MAPK and phospho-MKK3/MKK6. B, effect of a p38 MAPK inhibitor, SB203580, on HNE-induced COX-2 expression. The cells were treated with the inhibitor for 30 h prior to incubation with 50 µM HNE for 4 h. C, inhibition of HNE-induced p38 MAPK activation by the Src family and EGFR-like tyrosine kinase inhibitors (PP2 and AG1478). The cells were treated with the inhibitors for 30 min prior to incubation with 50 µM HNE for 4 h.

 
Because p38 MAPK modulates COX-2 mRNA levels by a change in mRNA stability (27–29), it was hypothesized that HNE induced COX-2 gene expression by raising the stability of COX-2 mRNA via the p38 MAPK pathway. To examine the involvement of tyrosine kinases in COX-2 mRNA stability, we analyzed COX-2 mRNA levels by means of an actinomycin D chase experiment (29, 30). In this experiment, because of the inhibition of transcription by the RNA synthesis inhibitor actinomycin D, subsequent mRNA levels depend on RNA stability without the transcriptional effect. If the increase in COX-2 mRNA is due to stabilization of mRNA and is mediated by the p38 MAPK pathway, COX-2 mRNA levels would decay more rapidly with p38 inhibitor treatment. As shown in Fig. 9A, in the absence of the p38 inhibitor, the COX-2 mRNA decayed at a slow rate (about 20% by 90 min). In contrast, the presence of the p38 MAPK inhibitor strongly enhanced COX-2 mRNA degradation (about 75% by 90 min), suggesting that HNE-induced COX-2 expression resulted from the stabilization of COX-2 mRNA that is mediated by the p38 MAPK signaling pathway. Furthermore, the Src family and EGFR-like tyrosine kinase inhibitors that suppressed p38 phosphorylation (Fig. 8) significantly promoted the COX-2 mRNA decay (Fig. 9, B and C). Thus, the tyrosine kinases, such as the EGFR-like and Src family tyrosine kinases, may mediate the stabilization of COX-2 mRNA via the p38 MAPK pathway.



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  		FIG. 9.
Involvement of Src family and EGFR-like tyrosine kinases and p38 MAPK in stabilization of COX-2 mRNA. RAW264.7 macrophages were either left untreated or treated with HNE for 2 h, and the medium was then changed to fresh medium containing actinomycin D (Act. D) (5 µg/ml) with or without inhibitor. Total RNA was isolated at the times indicated and subjected to RT-PCR analysis for COX-2 mRNA and GAPDH mRNA. The data shown are representative of three independent experiments. A, effect of the p38 MAPK inhibitor on HNE-induced COX-2 mRNA stability. B, effect of the Src family tyrosine kinase inhibitor on HNE-induced COX-2 mRNA stability. C, effect of the EGFR-like tyrosine kinase inhibitor on HNE-induced COX-2 mRNA stability.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The presence of protein-bound lipid peroxidation products has been reported in many chronic human diseases, including atherosclerosis and neurodegenerative disorders (9). The immunohistochemical analysis of atherosclerotic lesions from the human aorta indeed demonstrated that intense HNE immuno-reactivity was associated with cells, primarily macrophages (Fig. 1). It is known from in vitro studies that all of the major cell types within the atherosclerotic lesions are capable of promoting the oxidation of LDL (31–35); therefore, the cell-associated staining patterns we observed are likely attributable to the cellular oxidation of LDL by endothelial cells, macrophages, and smooth muscle cells. The resulting oxidized LDL may be taken up by cells and may be the ultimate source of the lipids that accumulate in atherosclerotic lesions. In addition, intracellular granular staining observed in atherosclerotic lesions represents the presence of protein-bound lipid peroxidation products that had already been taken up by macrophages and are present within the cell in cytoplasmic organelles (36). This leads to the speculation that the lipid adduction renders proteins relatively resistant to intracellular proteolytic degradation, resulting in the marked accumulation of epitopes in macrophages. On the other hand, augmented COX-2 expression or prostaglandin overproduction in atherosclerotic lesions has also been reported. Schonbeck et al. (37) show that human atheromatous lesions contain COX-2, colocalizing mainly with macrophages of the shoulder region and lipid core periphery. The involvement of lipid peroxidation products in COX-2 over-expression was suggested by our demonstrations that, within atheromatous lesions, COX-2 appeared to colocalize with protein-bound HNE in macrophage-derived foam cells (Fig. 1).

To test the hypothesis that lipid peroxidation products may be involved in the up-regulation of prostaglandin biosynthesis, we evaluated the effect of oxidized fatty acid metabolites on COX-2 induction in RAW264.7 macrophages and identified HNE as an inducer of COX-2 expression (Fig. 2). HNE is a major electrophilic product of lipid peroxidation caused by oxidative stress that is formed by radical-initiated degradation of {omega}6-polyunsaturated fatty acids such as linoleic and arachidonic acids, two relatively abundant fatty acids in human cells (8). It has been shown that high concentrations of HNE can be generated during in vitro oxidation of LDL (8, 38, 39). Steady-state levels of HNE have also been determined in many tissues and body fluids. Under physiological conditions, the cellular concentration of HNE ranges from 0.1 to 3 µM (8, 9). The concentration of this endogenously generated aldehyde in cells is relatively high compared with the concentrations of exogenous cytotoxic agents that cells may encounter under different environmental conditions. However, under oxidative stress conditions, HNE can accumulate at even higher concentrations of 10 µM to 5 mM both in vivo and in vitro (8, 9, 40) despite an active cellular metabolism, because of its high lipid/water compartmentalization coefficient (8). For example, in rats exposed to CCl4 and in Long Evans Cinnamon rats, the level of HNE can reach up to 100 µM in hepatocytes (41–44). Similar concentrations of HNE have been reported to accumulate in cells when neurons are exposed to agents that induce membrane lipid peroxidation (45). Thus, the amounts of HNE generated in vivo may be sufficient for it to play a role in stimulating most of the responses that have been shown to induce at µM concentrations in vitro.

It is of interest to note that other lipid peroxidation products, possessing an analogous functionality to that of HNE, were all inactive on COX-2 induction. The inability of 4-oxo-2-nonenal to induce COX-2 is particularly interesting because this HNE analog has been recognized to be more reactive than HNE toward the DNA bases 2'-deoxyguanosine, 2'-deoxyadenosine, and 2'-deoxycytidine, as well as selected amino acids and proteins (46). This invites the speculation that there may be a specific cellular target of HNE that may directly or indirectly lead to COX-2 induction. Thus, our challenge is to define the target molecule that triggers signal transduction pathways leading to COX-2 induction.

With regard to the cellular targets of HNE, the results of this study suggested that the Src family and EGFR-like tyrosine kinases might be essential for HNE-induced p38 MAPK activation (Figs. 6, 7, 8). The previous findings that (i) HNE triggers autophosphorylation of EGFR (47, 48), (ii) HNE induces cell growth inhibition and apoptotic cell death by targeting EGFR (49), and (iii) HNE causes reduction of the kinase activity of v-Src (50) suggest that HNE, originating from oxidized LDL or generated inside the cells during oxidative stress, may react directly with tyrosine kinases. Indeed, oxidized LDL (but not native LDL) have been shown to induce the formation of HNEEGFR and -PDGFR adducts, as evidenced by the presence in immunopurified EGFR and PDGFR of HNE protein epitopes reacting with anti-HNE protein antibodies and by the loss of free NH2 groups (determined by the radiolabeled specific probe, [3H] succinimidyl propionate) (47, 51). This oxidized LDL-induced derivatization of EGFR and PDGFR by HNE is associated with activation of the intrinsic tyrosine kinase and with autophosphorylation of the receptors and the subsequent recruitment of SH2-containing proteins. These effects are mimicked by the addition of free HNE to cell culture medium, which triggers both autophosphorylation and activation of the receptors. On the other hand, many studies have provided evidence linking the activation of tyrosine kinases to p38 MAPK (52). However, there is no clear mechanism as to how the Src family and EGFR-like tyrosine kinases may modulate the activity of p38 MAPK. The previous finding that activation of p38 MAPK in NIH 3T3 cells stably transformed by v-Src requires Ras and Rac suggests that the Src family and EGFR-like tyrosine kinases may regulate the activity of p38 MAPK through intermediate signaling pathways.

There is considerable evidence that p38 MAPK is important for regulating both COX-2 transcription and mRNA stability. Our previous study has shown that the p38 MAPK pathway plays a key role in the mechanism of HNE-induced COX-2 expression in hepatocytes (25). We indeed observed that the p38 MAPK pathway activated by HNE exerts its function by increasing the stability of COX-2 mRNA in RAW264.7 macrophages (Fig. 9). Using a classical RT-PCR approach, we found that, when transcription was blocked by the addition of actinomycin D, the stability of HNE-induced COX-2 mRNAs rapidly declined when the p38 MAPK-dependent signal was inhibited by SB203580 (Fig. 9A). Although the contributions of other possible p38 MAPK-regulated transcription factors that bind to the COX-2 promoter were not explored in this study, our results unambiguously show that p38 MAPK is required to extend the half-life of the COX-2 transcript in cells exposed to HNE.

On the other hand, the NF-{kappa}B signal transduction cascade is another major stress response signaling pathway. In mice and humans, the COX-2 promoter has many transcription factors, including NF-{kappa}B in the 5' region of the cox-2 gene (53–55), and the requirement of the activation of NF-{kappa}B to induce the expression of COX-2 in LPS-stimulated macrophages has been described (4). The NF-{kappa}B pathway has also been implicated in the expression of COX-2 stimulated by tumor necrosis factor-{alpha}, hypoxia, endothelin, and interleukin-1{beta} in endothelial cells (56) and hepatocytes (57). The present study showed that LPS induced transient reduction of the I{kappa}B level (Fig. 5B) and significantly enhanced the nuclear translocation of NF-{kappa}B (Fig. 5C). In contrast to the LPS-induced COX-2 expression, however, no significant change in I{kappa}B and NF-{kappa}B levels was observed in the HNE-stimulated macrophages (Fig. 5, D and E). Moreover, in agreement with this finding, HNE-induced COX-2 expression was not associated with the induction of the NF-{kappa}B-dependent gene product, iNOS (Fig. 5A). Thus, our finding that HNE up-regulates COX-2 without NF-{kappa}B activation is in striking contrast to the fact that NF-{kappa}B activation is necessary for LPS-induced COX-2 induction in RAW 264.7 macrophages (23–25).

In conclusion, we showed that HNE strongly induced COX-2 expression in macrophages. This finding represents a further demonstration of a link between the oxidative modification of LDL and activation of the inflammatory potential of macrophages. The observed effect 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.


   FOOTNOTES
 
* This work was supported by a research grant from the Ministry of Education, Culture, Sports, Science, and Technology, by the Center of Excellence (COE) Program in the 21st Century in Japan, and by research fellowships from the Japan Society for the Promotion of Science (to T. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure. Back

|| To whom correspondence should be addressed: Laboratory of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464–8601, Japan. Tel.: 81-52-789-4127; Fax: 81-52-789-5741; E-mail: uchidak{at}agr.nagoya-u.ac.jp.

1 The abbreviations used are: COX, cyclooxygenase; MAPK, mitogen-activated protein kinase; NF-{kappa}B, nuclear factor-{kappa}B; I{kappa}B, inhibitor-{kappa}B; LDL, low density lipoprotein; HNE, 4-hydroxy-2-nonenal; PBS, phosphate-buffered saline; LPS, lipopolysaccharide; EGFR, EGFR, epidermal growth factor receptor; PDGFR, platelet-derived growth factor receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MKK, mitogen-activated protein kinase kinase; iNOS, inducible nitric oxide synthase. Back



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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