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【摘要】
Objective— Acrolein, a known toxin in tobacco smoke, might be involved in atherogenesis. This study examined the effect of acrolein on expression of cyclooxygenase-2(COX-2) and prostaglandin (PG) production in endothelial cells.
Methods and Results— Cyclooxygenase (COX)-2 induction by acrolein and signal pathways were measured using Western blots, Northern blots, immunoflouresence, ELISA, gene silencing, and promoter assay. Colocalization of COX2 and acrolein-adduct was determined by immunohistochemistry. Here we report that the levels of COX-2 mRNA and protein are increased in human umbilical vein endothelial cells (HUVECs) after acrolein exposure. COX-2 was found to colocalize with acrolein-lysine adducts in human atherosclerotic lesions. Inhibition of p38 MAPK activity abolished the induction of COX-2 protein and PGE 2 accumulation by acrolein, while suppression of extracellular signal-regulated kinase (ERK) and JNK activity had no effect on the induction of COX-2 expression in experiments using inhibitors and siRNA. Furthermore, rottlerin, an inhibitor of protein kinase C (PKC ), abrogated the upregulation of COX-2 at both protein and mRNA levels.
Conclusion— These results provide that acrolein may play a role in progression of atherosclerosis and new information on the signaling pathways involved in COX-2 upregulation in response to acrolein and provide evidence that PKC and p38 MAPK are required for transcriptional activation of COX-2.
The present study demonstrates that acrolein, a known toxin in tobacco smoke, stimulates expression of COX-2 and enhances PG synthesis in endothelial cells through activation of PKC, p38 MAPK, and CREB pathway. Our finding suggests that acrolein may play a role in progression of atherosclerosis.
【关键词】 acrolein COX p MAPK atherosclerosis endothelial cells
Introduction
Activation of endothelial cells by proinflammatory stimuli has been established as an important link between risk factors and the pathologic mechanisms underlying atherosclerosis. 1 Thus, control of the inflammatory status of endothelial cells, which is achieved by a balance of pro- and antiinflammatory signals, is crucial to limiting the disease. Tobacco smoking induces inflammatory reactions 2 and promotes atherosclerosis 3; however, the mechanism that links cigarette smoking to an increased incidence of atherosclerosis is poorly understood.
Acrolein (CH 2 =CH-CHO), a major product of organic combustion, including tobacco smoking, is the most reactive, β-unsaturated aldehyde found widely in the environmol/Lent. Acrolein is highly reactive and is hazardous to human health. 4 Acrolein is produced by a wide variety of both natural and synthetic processes, including the incomplete combustion of organic materials. Acrolein also has been found to be formed from threonine by neutrophil myeloperoxidase at sites of inflammation 5 and has been identified as both a product and initiator of lipid peroxidation. 6 Recent studies have shown that acrolein levels are increased in many diseases such as atherosclerosis, Alzheimer disease, and diabetes, and is possibly related to pathogenesis in these conditions. 7–9 We and others have reported that acrolein elevates intracellular reactive oxygen species (ROS) levels, which leads to cell dysfunction. 8,10 ROS-mediated cell damage is an important etiologic factor in the pathogenesis of atherosclerosis. 11 ROS has been reported to induce the production of various atherogenic factors including inflammatory proteins. 12
Cyclooxygenase (COX) catalyzes the oxygenation of arachidonic acid to prostaglandin (PG) endoperoxides, which are converted enzymatically into PGs and thromboxane A2, both of which play physiological as well as pathologic roles in vascular function. Two distinct isoforms of COX have been identified in mammalian cells. COX-1 is constitutively expressed in a variety of cells such as vascular cells, fibroblasts, platelets, and epithelia, whereas COX-2 is absent from most normal tissues but is expressed in response to proliferative and inflammatory stimuli. 13 COX-2 is expressed in atherosclerotic lesions and is increased after vascular injury. Because chronic inflammation plays an important role in atherosclerosis, 1 COX-2 may participate in the genesis of atherosclerosis. 14,15
In view of the reports that inflammatory responses elicited by tobacco smoking are closely associated with atherogenesis, we hypothesized that acrolein, which is a main carbonyl component of tobacco smoke, might be involved in atherogenesis. In this report, we have explored the effect of acrolein on expression of COX-2 and PG production in endothelial cells. We showed (1) that acrolein induced COX-2 expression at both mRNA and protein levels, and (2) that this induction required the activation of PKC, p38 MAPK, and CREB. We also demonstrated (3) that treatment with p38 MAPK inhibitor reversed PGE 2 secretion by acrolein treatment, suggesting that the p38 MAPK pathway is important mechanistic component of this process.
Methods
The Methods are provided as supplemental online material, available at http://atvb.ahajournals.org.
Results
Acrolein Induces COX-2 Expression and PGE 2 Production
To test the effects of acrolein on HUVECs, cells were incubated with medium containing acrolein at different concentrations, and COX-2 levels were analyzed by Western blot. COX-2 was strongly induced in HUVECs in dose- and time-dependent manner ( Figure 1A and 1 B). Northern blotting analysis showed that the COX-2 mRNA signal was more intense in the cells that had been treated with acrolein, compared with control cells. The induction was detected at 0.5 hour after the addition of acrolein and reached the maximal level after 1 hour ( Figure 1 C). Immunofluorescence analysis demonstrated that COX-2 levels were significantly increased in HUVECs incubated with acrolein for 16 hours ( Figure 1 D). We tested whether this COX-2 enzyme was responsible for acrolein-induced PGE 2 production in the culture media of cells stimulated with acrolein because COX-2 catalyzes biosynthesis of PGs. As shown in Figure 1 E, acrolein increased PGE 2 secretion by 8-fold at 16 hours, which was completely blocked by a selective COX-2 inhibitor, NS-398. The results indicate that acrolein can lead to COX-2 protein expression and subsequently PGE 2 biosynthesis in HUVECs.
Figure 1. Increase of COX-2 expression and PGE 2 production by acrolein in HUVECs, induction of COX-2 in mice administrated with acrolein, and colocalization of COX-2 with acrolein in human atherosclerotic lesions of blood vessels. After treatment of HUVECs with various concentrations of acrolein (A) at various times (B), the cells were washed twice with phosphate-buffered saline. The cell lysates were prepared and 20 µg samples of proteins were subjected to Western blotting using anti–COX-2 antibody and anti–β-actin antibody. C, HUVECs were treated with 10 µmol/L acrolein for indicated times. Total RNA was extracted, and 20 µg of the resulting RNA were analyzed by Northern blotting with 32 P-labeled human COX-2 probe. 28S and 18S rRNA were used as controls. D, Immunofluorescence staining analyses of COX-2 expression in HUVECs exposed to 10 µmol/L acrolein. Cells were treated and further processed as described in Methods. E, Cells were treated with 10 µmol/L acrolein in the presence or absence of NS-398 for 16 hours, and then release of PGE 2 was measured from supernatants as described in Methods. The values shown for PGE 2 production are the mean±SD of 3 independent experiments. * P <0.001 compared with untreated control cells. F, Immunoblot analysis of COX-2 in lung tissues of mice administrated acrolein (C: control, PBS only, A: acrolein 4 mg/ Kg). Human atherosclerotic blood vessel specimens were immunostained with anti-acrolein antibody (G) and anti–COX-2 (H) as described in Methods. Bar=50 µm.
Induction of COX-2 by Administration of Acrolein in Mice and COX-2 Colocalizes With Protein-Bound Acrolein in Atherosclerotic Lesions From Patients
To further study whether acrolein is capable of inducing COX-2 in vivo, mice administrated with acrolein (4 mg/kg) for 24 hours and we found COX-2 was induced by acrolein in lung tissues ( Figure 1 F). Next, we also examined the pathohistologic location of COX-2 and acrolein-bound protein in human atherosclerotic blood vessels samples to determine whether acrolein might be involved in COX-2 upregulation in vivo. Both COX-2 and protein-bound acrolein were rarely detected in nonatherosclerotic segments of these blood vessels (data not shown). In contrast, we found that both acrolein-lysine adducts ( Figure 1 G) and COX-2 ( Figure 1 H) colocalized in the blood vessel cells.
Inhibition of p38 MAPK Abolishes Induction of COX-2 Protein Expression
Oxidative stress triggered by H 2 O 2 and treatment with lipid peroxidation end products have been found to activate MAP kinase pathways including ERK, JNK, and p38 MAPK. 16,17 To test whether acrolein activates MAP kinase pathways including ERK, JNK, and p38 MAPK in HUVECs, cells were exposed to acrolein in the culture medium. We found activation of these kinases by acrolein (supplemental Figure I). To determine whether the MAPK pathways were directly involved in the induction of COX-2 by acrolein, cells were pretreated with kinase inhibitors for 1 hour before adding acrolein. PD98059, which specifically inhibits the ERK, had no effect on the induction of COX-2 ( Figure 2 A), suggesting that the induction of COX-2 did not require the ERK pathways. SP600125, JNK specific inhibitor, 18 also did not prevent the induction of COX-2 protein by acrolein. In contrast, SB203580, a specific inhibitor for p38 MAPK, potently inhibited the induction of COX-2. Interestingly, we observed that acrolein did not affect COX-1 expression ( Figure 2 A). To determine whether the inhibition occurred at the level of transcription, Northern blot analysis was carried out. The levels of COX-2 mRNA were dramatically reduced by SB203580 but not by PD98059 and SP600125 ( Figure 2 B). We next investigated the effect of MAPK inhibitors on acrolein induced PGE 2 production. The p38 MAPK inhibitor, SB203580, dramatically suppressed acrolein-induced PGE 2 production ( Figure 2 C), suggesting that p38 MAPK signaling pathway is involved in the acrolein-induced PGE 2 biosynthesis. But PD98059 and SP600125 did not prevent the PGE 2 production by acrolein.
Figure 2. Effect of MAPK inhibitor on COX-2 induction and PGE 2 production in HUVECs. A, HUVECs were preincubated with the p38 MAPK inhibitor, SB (SB203580, 10 µmol/L), the MEK inhibitor, PD (PD98059, 40 µmol/L), and the JNK inhibitor, SP (SP600125, 20 µmol/L) for 30 minutes. The cells were then treated 10 µmol/L acrolein for 16 hours and 20 µg samples of whole cell lysates were subjected to the Western blotting using anti–COX-2 antibody and anti–COX-1 antibody. B, After preincubation with above inhibitors, the cells were treated with or without acrolein. After 1 hour, the cells were washed twice with phosphate-buffered saline. Total RNA was extracted, and 20 µg of the resulting RNA were analyzed by Northern blotting with 32 P-labeled human COX-2 probe. 28S and 18S rRNA were used as controls. C, HUVECs were preincubated with SB (10 µmol/L), PD (40 µmol/L), and SP (20 µmol/L) for 30 minutes. The cells were then treated 10 µmol/L acrolein for 16 hours and then release of PGE 2 was measured from supernatants as described in Methods. The values shown for PGE 2 production are the mean±SD of 3 independent experiments. * P <0.005 compared with untreated control cells.
To validate a role for p38 MAPK in the acrolein induced COX-2 upregulation, we inhibited p38 MAPK using siRNA transfection. Transfection of HUVECs with p38 MAPK siRNA duplex (250 nmol/Lol/L, 48 hours) abolished the induction of COX-2 by acrolein ( Figure 3 A). Figure 3 B shows that acrolein stimulated the transcription of COX-2 as well. When we performed transient transfections with a human COX-2 promoter-luciferase reporter construct (–1432/+59), 19 COX-2 reporter activity was increased to more than 100% by acrolein and decreased to basal levels by treatment of p38 MAPK inhibitor.
Figure 3. Involvement of p38 MAPK in COX-2 induction by acrolein in HUVECs. A, Effect of p38 MAPK siRNA transfection on COX-2 induction by acrolein. HUVECs were transfected with siRNA against 250 nmol/L p38 MAPK. At 48 hours after transfection, cells were stimulated with 10 µmol/L acrolein for 30 minutes. Cells were lysed and Western blot analysis was performed using an antibody against COX-2. The stripped membrane was subjected to Western blotting with anti-p38 MAPK antibody and β-actin antibody, used as to an expression of the protein and internal control, respectively. B, COX-2 promoter activity. HUVECs were transfected with COX-2 promoter construct (–1432 to +59) for 48 hours. After transfection, cells were preincubated with p38 MAPK inhibitor, SB203580 (10 µmol/L), for 30 minutes before exposure to 10 µmol/L acrolein for 6 hours. Luciferase activity represents data that have been normalized to cotransfected β-galactosidase activity. Data are presented as means±SD of triplicate experiments. * P <0.005 compared with untreated control cells.
PKC Is Required for COX-2 Activation
Because our previous data indicated that acrolein activates PKC, 16 we examined the role of PKC in COX-2 induction by acrolein. Figure 4 A shows that acrolein rapidly activates PKC. Within 10 minutes, PKC activation reached the maximal level, and the kinase activity gradually decreased at 30 minutes after stimulation. To determine whether PKC plays a role in the signaling pathways controlling the induction of COX-2 by acrolein, we used an inhibitor specific for PKC, rottlerin. HUVECs were pretreated with 5 µmol/L concentrations of rottlerin for 1 hour, and then 10 µmol/L acrolein was added. After 16 hours, cells were collected, and the COX-2 protein levels were determined by Western blot analysis. Figure 4 B shows that 5 µmol/L rottlerin completely blocked the induction of COX-2 protein. Northern blot analysis ( Figure 4 C) shows that the inhibition occurred at the level of mRNA, suggesting that the activation of a PKC kinase activity is necessary for the upregulation of the COX-2 mRNA in response to acrolein treatment. Taken together, these data support the conclusion that the activation of PKC is required for COX-2 induction.
Figure 4. Involvement of PKC in COX-2 induction by acrolein in HUVECs. A, HUVECs were preincubated with PKC inhibitor, rottlerin (5 µmol/L), for 30 minutes. The cells were treated with 10 µmol/L acrolein for 16 hours and subjected to the Western blot for COX-2. B, After preincubation with rottlerin (5 µmol/L) for 30 minutes, the cells were treated with or without acrolein. After 1 hour, the cells were washed twice with phosphate-buffered saline. Total RNA was extracted, and 20 µg of the resulting RNA were analyzed by Northern blotting with 32 P-labeled human COX-2 probe. 28S and 18S rRNA were used as controls. C, HUVECs were stimulated by treatment with 10 µmol/L acrolein for the indicated times. Lysates prepared and 40 µg samples of proteins were analyzed by Western blotting using anti–phospho-PKC and PKC antibody as indicated. D, Effect of PKC on acrolein-induced p38 MAPK activation. HUVECs were preincubated with rotterin for 30 minutes after by stimulation with acrolein for 30 minutes. Cell lysates (30 µg) were prepared and analyzed for phosphorylation of p38 by Western blot analysis, using a specific antibody.
Activation of p38 MAPK Is Affected by Rottlerin
To determine whether the p38 MAPK activation is followed by PKC activation in response to acrolein treatment, HUVECs were pretreated with rottlerin, then were incubated with 10 µmol/L acrolein. The activation of p38 MAPK was determined by Western blot analysis ( Figure 4 D). We found that a significant decrease of p38 MAPK in the presence of rottlerin, suggesting that PKC is required for the activation of p38 MAPK by acrolein treatment.
Phospholylation of CREB and Activation of CRE Transcription Factor by Acrolein
We next investigated acrolein-associated phosphorylation of cAMP-responsive element-binding protein (CREB), which is known as a regulator of COX-2 expression in several cells. 20,21 Acrolein strongly increased the phospholylation of CREB in HUVECs ( Figure 5 A), and the activation was completely abolished by p38 MAPK inhibitor but not by ERK and JNK inhibitor ( Figure 5 A). The COX-2 promoter contains multiple potential cis-activating regulator elements such as cAMP-responsive elements (CRE), NF- B, NF-IL6 (C/EBPβ), and E-box transcriptional elements, which have been identified as being involved in receptor-mediated COX-2 expression. 19,22–24 The identities of the cis-elements regulated by acrolein signal pathways are unknown. Electrophoretic mobility shift assay, using an end-labeled oligoprobe containing the CRE consensus, showed an increase in CRE binding activity by the treatment of acrolein ( Figure 5 B). The acrolein-stimulated increases in CRE binding activity were detected from 30 minutes, reached a maximal level at 1 hour, and decreased thereafter.
Figure 5. CREB activation and binding to COX-2 CRE on acrolein-stimulated HUVECs. A, CREB activation. HUVECs were preincubated with the p38 MAPK inhibitor, SB (SB203580, 10 µmol/L), the MEK inhibitor, PD (PD98059, 40 µmol/L), and the JNK inhibitor, SP (SP600125, 20 µmol/L) for 30 minutes. The cells were then treated 10 µmol/L acrolein, for CREB activation, for 30 minutes and 30 µg samples of whole cell lysates were subjected to Western blotting using anti–phospho-CREB antibody and anti-CREB antibody. B, CRE COX-2 binding. Cells were stimulated with 10 µmol/L acrolein indicated times under serum free conditions. Cells were taken out to prepare nuclear extract, and nuclear proteins were analyzed by EMSA to determine the DNA binding activity of CRE as described under Methods.
Discussion
Cigarette smoking is the leading risk factor in the etiology of atherosclerotic vascular disease. 3 Cigarette smoke can damage a number of organ systems; however, ECs are particularly vulnerable. Although it is unclear which smoke constituent is responsible for the deleterious effects, there are over 4000 different chemicals in cigarette smoke, and it is quite likely that a combined insult from several chemical constituents is responsible for the injury. 25 In this study, we show that acrolein, a main toxic component of cigarette smoke, 26 may contribute to the development of vascular disease through COX-2 induction.
The findings presented herein show that acrolein increases COX-2 mRNA, protein, and PG synthesis in HUVECs, time- and dose-dependently. Transient transfections demonstrated that acrolein treatment in HUVECs increases the rate of COX-2 transcription. The increased PG synthesis in HUVECs after treatment with acrolein reflects an increase in functional COX-2 protein, because NS398, a specific inhibitor of COX-2 enzyme activity, effectively blocked PG synthesis in the acrolein-treated cells. To our knowledge, this is the first report that acrolein increases COX-2 expression and PGE 2 production in any cell system. The signal transduction cascade that mediates activation of PKC, p38 MAPK, and CREB, leading to subsequent COX-2 induction and PGE 2 secretion, is diagrammed in supplemental Figure II. Acrolein is present at a level of 238 to 468 µg/cigarette, 26 and total aldehyde including acrolein generated by smoking one cigarette, if completely dissolved in the lung lining fluid, could be present at 2 to 3 mmol/L. 27 Therefore the concentration of acrolein in this study is considered to be physiological level.
It is well known that COX-2 expression has been linked with activation of MAPK pathways and that the particular signaling pathway involved is dependent on the type of stimuli. In the present study, we demonstrated that acrolein induces COX-2 expression by activating p38 MAPK. In contrast, ERK and JNK did not contribute to acrolein-mediated COX-2 induction. Using siRNA-mediated suppression of p38 MAPK in HUVECs, we were able to gather additional evidence demonstrating role of p38 MAPK in the COX-2 induction by acrolein ( Figure 3 A).
p38 MAPK plays an important role in the expression of proinflammatory molecules and the regulation of cellular responses during infection and has been widely investigated for an effect on COX-2 at translational and transcriptional levels. 28 Interleukin (IL)-1β–induced transcription of COX-2 in a human microvascular endothelial cell line has been shown to require the combinatorial action of transcription factors, such as activated protein-2 (AP2), nuclear factor-IL-6 (NF-IL-6), and cAMP-responsive elements (CRE). 29 The CRE element in the COX-2 promoter is necessary for the induction of COX-2 transcription mediated by nitric oxide, proteasome inhibitors, and lipopolysaccharide (LPS). 30–32 Our gel shift assay using CRE probe from the COX-2 promoter indicates that CREB binds to a COX-2 CRE ( Figure 5 B). The involvement of a CRE may be of particular relevance to acrolein-induced COX-2 transcriptional activation.
The activation of PKC induces COX-2 expression in many cell types, such as astrocytic and endothelial cells. 33,34 Activation of PKC has been suggested to be a key event in the signal pathway leading to COX-2 expression. In the previous study, we showed that acrolein induce activation of PKC in HUVECs. 16 In present study we found that COX-2 expression was reduced by a PKC inhibitor, and the PKC activation leads to p38 MAPK activation and COX-2 expression ( Figure 4 D). Similarly, Kim et al report that epigallocatechin-3 gallate (EGCG)-induced COX-2 expression requires activation of p38 MAPK via PKC pathway in astrocyte and immortalized astroglial cells. 33
Acrolein levels are increased in patients with atherosclerosis as well as in cigarette smokers, 8,9 which is strongly associated with an increased risk of vascular disease in clinic. Thus increased acrolein levels might be involved in pathogenesis of atherosclerosis. On the other hand, augmented COX-2 expression or PG overproduction in atherosclerotic lesions has also been reported. Human atheromatous lesions contain COX-2, colocalizing mainly with macrophages of the shoulder region and lipid core periphery. COX-2 expression was also detected in smooth muscle cells and in endothelial cell in atherosclerosis. 35,36 Our results show that COX-2 appeared to colocalize with protein-bound acrolein in atherosclerotic blood vessels ( Figure 1G and 1 H). In addition, very recently, Shao et al reported that acrolein may interfere with normal high density lipoprotein (HDL), which protects from atherosclerosis, and with cholesterol transport by modifying specific sites in apoI, resulting in atherogenesis. 37 Taken together, the possibility that increased levels of acrolein may promote development of atherosclerosis must be seriously considered.
In summary, the present study demonstrates that acrolein, a known toxin in tobacco smoke, stimulates expression of COX-2 and enhances PG synthesis in HUVECs through activation of PKC, p38 MAPK, and CREB pathways. Our finding suggests that acrolein may play an important role in progression of atherosclerosis via an inflammatory response involving COX-2 expression.
Acknowledgments
We thank Drs D.Y. Park and S.H. Rhee for the gift of human blood vessel specimens sample and COX-2 promoter construct.
Sources of Funding
This study was supported, in part, by Grants-in Aid for 21 century Center of Excellence from the Ministry of Education, Science, Sports and Culture, Japan. Y.S. Park is the recipient of research fellowships from the Japan Society for the Promotion of Science for Young Scientists and the Kanae foundation. J. Kim is the recipient of an American Foundation for Urologic Disease fellowship.
Disclosures
None.
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作者单位：Yong Seek Park; Jayoung Kim; Yoshiko Misonou; Rina Takamiya; Motoko Takahashi; Michael R. Freeman; Naoyuki TaniguchiFrom the Department of Biochemistry (Y.S.P., Y.M., R.T., M.T., N.T.) and the Department of Disease Glycomics, Research Institute for Microbial Diseases (N.T.), Osaka University, Japan;