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Vascular smooth muscle cell activation and growth by 4-hydroxynonenal
Life Sciences 69 (2001) 689–697
Vascular smooth muscle cell activation and growth by 4-hydroxynonenal Hirobumi Kakishita, Yoshiyuki Hattori* Department of Endocrinology and Metabolism, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan Received 23 October 2000; accepted 11 December 2000
Abstract The present study examines the signal transduction mechanism that is involved in the growth of vascular smooth muscle cells exposed to 4-hydroxynonenal (HNE) in vitro. This aldehyde component of oxidized low-density lipoprotein has been identified in atherosclerotic lesion. Exposure to HNE caused ERK, JNK, and p38 MAP kinase activation as well as the induction of c-fos and c-jun gene expression. AP-1 activity was also significantly induced by HNE treatment. These intracellular activities appear to be the mechanism of HNE-caused mitogenesis. Indeed, HNE induced vascular smooth muscle cell proliferation as determened by Alamar-Blue assay and stimulated DNA synthesis as determined by bromodeoxyuridine incorporation. These observations are consistent with a role of lipid peroxidation products in vascular smooth muscle cell growth in atherogenesis. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Lipid peroxidation; Aldehyde; Atherosclerosis; Vasculare smooth muscle cell
Introduction The formation of atherosclerotic lesions is a complex process that is partly mediated by inflammatory and oxidative mechanisms including lipid peroxidation. Increasing evidence indicates that aldehydes generated endogenously during lipid peroxidation are causally involved in most of the pathophysiological effects associated with oxidative stress in cells and tissues [1,2]. Aldehydes derived from lipid peroxidation appear to be end products and remnants of lipid peroxidation processes that may also act as mediators for the primary free radicals that initiated lipid peroxidation. Among the aldehydes derived from lipid peroxidation, 4-hydroxy-2-nonenal (HNE) that can be produced from archidonic acid, linoleic acid, or their hydroperoxides in relatively large amounts in response to oxidative insult is believed to be largely responsible for the cytopathological effects associated with oxidative stress [1,2]. * Corresponding author. Tel.: 1 81 282 (87) 2150; fax: 1 81 282 (86) 4632. E-mail address: [email protected] (Y. Hattori) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 1 6 6 -3
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The extensive lipid peroxidation of oxidized low-density lipoprotein (LDL) generates HNE that is found in atherosclerotic lesions. Immunoreactive HNE is present at all stages of human atherosclerosis but not in normal human arteries [3] and has also been identified in the neointima of baboon arteries injured by balloon atheroplasty [4]. Lipid peroxidation products, and specifically HNE, stimulate chemotaxis and growth in other systems [5–7] and have been implicated in other pathological conditions thought to be related to oxidative stress [8–10]. Those observations support the view that HNE provides a link between oxidant generation, lipid peroxidation, and vascular smooth muscle cell (VSMC) proliferation in atherogenesis. The transcription factor activator prtein-1 (AP-1) consists of homo- or heterodimers of the proteins encoded by the fos and jun gene families and is believed to regulate genes involved in the control of cell growth and differentiation [11]. The transcriptonal activity of these binding factors is regulated by protein kinases related to the mitogen-activated protein (MAP) kinase superfamily. To date, at least three different subtypes of MAP kinases have been identified. These are in turn activated by distinct upstream dual specificty kinases, thus revealing the existence of protein kinase modules that can be indepedently and simultaneously activated. Whereas mitogens and growth factors lead to activation of protein kinase cascades resulting in activation of extracellular signal-regulated kinase (ERK) family MAP kinases, many forms of cellular stress preferentially triger two related signaling pathways [12–16]. These center on two MAP kinases or Jun N-terminal kinases (JNKs) and p38, also termed stress-activated protein kinase and reactivating kinase, respectively. The present study confirmed that HNE can stimulate VSMC growth [17]. We also obtained insight into the mechanisms involved by studying the induction of MAP kinases, c-fos and c-jun gene expression, and AP-1 activation.
Materials and methods Cell culture and RNA extraction Rat VSMC were isolated by elastase and collagenase digestion of thoracic aortae from male Wistar as previously described [18]. Cells in passage 10–15 were used for experiments. Total RNA was extracted from confluent human VSMC using a modified guanidinium isothiocyanate method [19]. mRNA analysis of c-fos and c-jun Total cDNA was synthesized from the total RNA using avian myeloblastosis virus reverse transcriptase and random 9-mers as primers. The cDNA was amplified using PCR with primers derived from the published sequences of rat c-fos [20] and c-jun [21]. The PCR products for c-fos and c-jun were labelled with [a-32P]dCTP by random priming and used as probes. Blotting proceeded as described [22]. After probing for c-fos or c-jun mRNA expression, the filter was stripped and reprobed for the presence of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Radioactivity in the blots was quantified using a BAS2000 image analyzer (Fuji Photo Film Co., Tokyo, Japan).
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MAPK assay To create stable reporter cell lines with which to evaluate MAPK activation, we used transreporting systems with GAL4 fusion transactivators as pathway-specific sensors [23]. These systems use a fusion trans-activator plasmid consisting of the DNA binding domain of the yeast GAL4 (residues 1 to 147) protein and the activation domain of Elk-1, c-Jun, or Chop, respectively. We first transfected the pFR-Luc plasmid (that contains the luciferase gene which is controlled by a promoter that responds to GAL4 fusions; Stratagene) with a pSV40/ Zeo2 plasmid containing a Zeocin expression cassette (Invitrogen) into rat VSMC. Successive rounds of selection in antibiotic medium (Zeocin 300 mg/ml) isolated Zeocin-resistant clones. We also transfected Zeocin-resistant cell lines with the fusion trans-activator pFA2Elk1, pFA2-cJun, or pFA-Chop plasmids (Stratagene). Successive rounds of selection with G418 (500 mg/ml), generated clones that gave the best response when transfected with the positive control vectors for each reporting system. These clones were further analyzed. AP-1 activation To study AP-1 activation, the cells were stably transfected with a cis-reporter plasmid containing the luciferase reporter gene linked to seven repeats of AP-1 binding sites (pAP-1-Luc: Stratagene). The pAP-1-Luc plasmid was transfected together with a pSV2neo helper plasmid (Clontech, Palo Alto, CA, USA) into rat VSMC using FuGEN 6 transfection reagent (Boehringer Mannheim, Mannheim, Germany). The cells were cultured in the presence of G418 (Clontech) at a concentration of 500 mg/ml with medium replacement at 2 to 3 day intervals. Approximately 3 weeks later, G418-resistant clones were isolated using a cloning cylinder and the expression of luciferase activity was analyzed individually. Several clones were selected for analysis of AP-1 activation. Luciferase activity was measured using a Luciferase assay kit (Stratagene). Cell proliferation and DNA synthesis Cell proliferation was assessed using Alamar Blue assay. As a redox indicator, Alamar Blue (Serotec Ltd.) is reduced by reactions innate to cellular metabolism and thus provides an indirect measure of viable cell number. VSMC were growth-arrested in 96-well plates and treated with HNE for 48 hours. Then Alamar Blue (10w/v% in PBS) was added to the cells and 3hours later fluorescence was determined in a cytofluorometer at 544 nm excitation and 590 nm emission wavelength in a cytofluorometer (Fluoroskan Asent FL, Labsystems). The amount of DNA synthesis was assessed by determining BrdU incorporation by VSMC using an ELISA kit (Amersham). VSMC were growth arrested in 96-well plates, then HNE was added in the presence of BrdU for 24 h. After fixation and blocking, a peroxidase-labeled anti-BrdU antibody was added. The substrate was tetramethylbanzidine, and the color was read at 450 nm in a spectrophotometer. Statistical analysis Data are presented as mean 6 SEM. Multiple comparisons were evaluated by ANOVA followed by Fisher’s protected least significant difference test. Two experiments were compared using Student’s unpaired t test. A value of P,.05 was considered statistically significant.
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Results To verify that the HNE treatment of the cells resulted in a functional increase in ERK, JNK, and p38 activities, activation of the transcription factor Elk-1, cJun, or Chop was respectively evaluated with trans-reporting systems using GAL4 fusion transactivators as pathway-specific sensors. When activated by phosphorylation, these fusion proteins binds to the promoter and induce luciferase expression. Therefore, luciferase activity in stable cell lines reflects the activation status of the fusion transactivator and, hence, the activation status of corresponding signal pathways. Fig. 1 shows that the activity of JNK increased along with HNE concentration whereas Elk and Chop activation peaked at 5 mM HNE. ERK induced by HNE (5 mM) was completely inhibited by PD98059 (30 mM), while Chop activation by HNE (5 mM) was also prevented by SB203580 (10 mM). We next investigated whether HNE modulates AP-1 activity as well as the expression of cfos and c-jun gene. We evaluated c-fos and c-jun mRNA levels by Northern blotting. While c-Fos mRNA levels were low in unstimulated cells, HNE increased the levels of c-Fos mRNA, to a peak at 2.5 mM, then rather decreased at higher concentrations (Fig. 2). On the other hand, c-jun mRNA is constitutively present and it further increased as a function of HNE concentration (Fig. 2). Since c-fos and c-jun are the major constituents of the transcriptional factor AP-1 and HNE increased the gene expression of these protooncogenes, we tested the effect of HNE on AP-1 activation in VSMC. We determined AP-1 activity by measuring AP-1-dependent transcription in rat VSMC stably transfected with a luciferase reporter constract. The activity of AP-1 was slightly increased by HNE at z1mM, significantly activated by higher concentrations of HNE, peaked at 2.5 mM, then declined (Fig. 3).
Fig. 1. Effect of HNE on activation of Elk-1, cJun, or Chop. Quiescent VSMC (transfected with pFR-Luc/pFA2Elk1:closed circles, pFR-Luc/pFA2-cJun: closed triangles, or pFR-Luc/pFA-Chop: closed squares) were incubated with various concentrations of HNE. After 3 h, cells were lysed, and luciferase activities were measured. Data are mean 6 S.E.M. of triplicate observations. * P,.05 and ** P,.01 compared with control (no HNE).
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Fig. 2. Effect of HNE on mRNA expression for c-fos and c-jun in VSMC. Cells were treated with various concentrations of HNE. After 1 h, total RNA was isolated and analyzed by Northern blot hybridization with specific probes for rat c-fos and c-jun.
To determine whether HNE stimulates VSMC growth, we examined the effect of 2.5 mM HNE on VSMC proliferation and DNA synthesis. This concentration of HNE is that at which AP-1 is most potentyl activated. After exposing of growth-arrested VSMC to HNE for 48 hours, cell proliferation was determined by Alamar-Blue assay. Fluorescent values as indicator of viable cell number were significantly increased after exposure to HNE (Fig. 4). We also measured the effect of HNE on DNA synthesis by determining incorporation of BrdU in
Fig. 3. Effect of HNE on AP-1 activation. Quiescent VSMC (transfected with pAP-1-Luc) were treated with different concentrations of HNE. After 3 h, cells were lysed, and luciferase activities were measured. Data are mean 6 S.E.M. of triplicate observations. * P,.05 and ** P,.01 compared with control (no HNE).
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Fig. 4. Effect of HNE on VSMC growth. (A) VSMC were growth-arrested in plates and treated with HNE (2.5 mM) for 48 h. Viable cell number was assessed using Alamar Blue assay. (B) VSMC were growth-arrested in plates and treated with HNE (2.5 mM) in the presence of BrdU for 24 h. Incorporation of BrdU in VSMC was determined using an ELISA kit. * P,.05 compared with control (no HNE).
VSMC. Incubating VSMC with HNE over 24 hours increased the incorporation of BrdU compared with control (Fig. 4). Discussion The present study showed that HNE stimulates MAP kinases, induces c-fos and c-jun mRNA expression and increase AP-1 activity. Those effects were dependent on a narrow concentration range of HNE. Primarily activated in response to growth stimulation, ERK is implicated in cellular proliferation and differentiation [24–26], whereas JNK and p38 MAP
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kinases are charcterized by a powerful response to cellular stress such as UV light, osmotic stress, DNA damaging agents, and proinflammatory cytokines [14,27,28]. Activation of JNK has also been implicated in the induction of apoptosis in response to growth factor withdrawal and other envioronmental stimuli and in stimulating cell proliferation and transformation [29,30]. We demonstrated that HNE treatment activates ERK, which could lead to the induction of c-fos gene expression. We found that HNE also stimulated JNK and p38, which might be responsible for the induction of c-jun gene expression. Being the major components of the AP-1 transcription factor, c-fos and c-jun, once activated, can subsequently activate the transcription of several genes controlling cellular growth. Indeed, HNE induced VSMC growth, which is consistent with other observations [17]. Intracellular peroxide production by HNE has been demonstrated in rat liver epithelial cells [2]. It has been demonstrated that H2O2 significantly activated MAP kinases in VSMC [31] and we confirmed H2O2-induced activation of MAP kinases in our system. Accordingly, intracellular peroxide appears to play a crucial role in the HNE-induced activation of stress signaling pathways. HNE-induced peroxide, at least in part, seems to be responsible for activation of MAP kinases. One potentially significant consequence of oxidative stress is increased VSMC proliferation [32]. Regulation of the redox state of the cell may be a general mechanism by which growth signals are transduced and HNE is a potential source of intracellular pro-oxidants. Intracellular peroxide production by HNE has been demonstrated in rat liver epithelial cells [2]. Some levels of oxidant stress is required for cell growth. However, HNE is cytotoxic at higher concentrations [33–35]. Other oxidation products, such as H2O2 and oxidized LDL, over a narrow concentration range can also cause both proliferatiive and cytotoxic effects [36,37]. Thus, a narrow concentration range of HNE may activate cellular signal transduction leading to cell growth, whereas higher concentrations may have cytotoxic effects. This may be relevant in atherogenesis, where cell growth, apoptosis, and necrosis are thought to mutually contribute to lesion formation. In summary, HNE induces VSMC proliferation at the concentration of which causes the activation of MAP kinases, the induction of gene expression of c-fos and c-jun, and the increase in AP-1 activity. Acknowledgments This work was supported in part by a grant from Japan Private School Promotion Foundation and by Grants-in-Aids from the Ministry of Education, Science, and Culture. References 1. Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radical Biology & Medicine 1991;11(1):81–128. 2. Uchida K, Shiraishi M, Naito Y, Torii Y, Nakamura Y, Osawa T. Activation of stress signaling pathways by the end product of lipid peroxidation. 4-hydroxy-2-nonenal is a potential inducer of intracellular peroxide production. Journal of Biological Chemistry 1999;274(4):2234–42. 3. Jurgens G, Chen Q, Esterbauer H, Mair S, Ledinski G, Dinges HP. Immunostaining of human autopsy aortas
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Vascular smooth muscle cell activation and growth by 4-hydroxynonenal Hirobumi Kakishita, Yoshiyuki Hattori* Department of Endocrinology and Metabolism, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan Received 23 October 2000; accepted 11 December 2000
Abstract The present study examines the signal transduction mechanism that is involved in the growth of vascular smooth muscle cells exposed to 4-hydroxynonenal (HNE) in vitro. This aldehyde component of oxidized low-density lipoprotein has been identified in atherosclerotic lesion. Exposure to HNE caused ERK, JNK, and p38 MAP kinase activation as well as the induction of c-fos and c-jun gene expression. AP-1 activity was also significantly induced by HNE treatment. These intracellular activities appear to be the mechanism of HNE-caused mitogenesis. Indeed, HNE induced vascular smooth muscle cell proliferation as determened by Alamar-Blue assay and stimulated DNA synthesis as determined by bromodeoxyuridine incorporation. These observations are consistent with a role of lipid peroxidation products in vascular smooth muscle cell growth in atherogenesis. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Lipid peroxidation; Aldehyde; Atherosclerosis; Vasculare smooth muscle cell
Introduction The formation of atherosclerotic lesions is a complex process that is partly mediated by inflammatory and oxidative mechanisms including lipid peroxidation. Increasing evidence indicates that aldehydes generated endogenously during lipid peroxidation are causally involved in most of the pathophysiological effects associated with oxidative stress in cells and tissues [1,2]. Aldehydes derived from lipid peroxidation appear to be end products and remnants of lipid peroxidation processes that may also act as mediators for the primary free radicals that initiated lipid peroxidation. Among the aldehydes derived from lipid peroxidation, 4-hydroxy-2-nonenal (HNE) that can be produced from archidonic acid, linoleic acid, or their hydroperoxides in relatively large amounts in response to oxidative insult is believed to be largely responsible for the cytopathological effects associated with oxidative stress [1,2]. * Corresponding author. Tel.: 1 81 282 (87) 2150; fax: 1 81 282 (86) 4632. E-mail address: [email protected] (Y. Hattori) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 1 6 6 -3
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The extensive lipid peroxidation of oxidized low-density lipoprotein (LDL) generates HNE that is found in atherosclerotic lesions. Immunoreactive HNE is present at all stages of human atherosclerosis but not in normal human arteries [3] and has also been identified in the neointima of baboon arteries injured by balloon atheroplasty [4]. Lipid peroxidation products, and specifically HNE, stimulate chemotaxis and growth in other systems [5–7] and have been implicated in other pathological conditions thought to be related to oxidative stress [8–10]. Those observations support the view that HNE provides a link between oxidant generation, lipid peroxidation, and vascular smooth muscle cell (VSMC) proliferation in atherogenesis. The transcription factor activator prtein-1 (AP-1) consists of homo- or heterodimers of the proteins encoded by the fos and jun gene families and is believed to regulate genes involved in the control of cell growth and differentiation [11]. The transcriptonal activity of these binding factors is regulated by protein kinases related to the mitogen-activated protein (MAP) kinase superfamily. To date, at least three different subtypes of MAP kinases have been identified. These are in turn activated by distinct upstream dual specificty kinases, thus revealing the existence of protein kinase modules that can be indepedently and simultaneously activated. Whereas mitogens and growth factors lead to activation of protein kinase cascades resulting in activation of extracellular signal-regulated kinase (ERK) family MAP kinases, many forms of cellular stress preferentially triger two related signaling pathways [12–16]. These center on two MAP kinases or Jun N-terminal kinases (JNKs) and p38, also termed stress-activated protein kinase and reactivating kinase, respectively. The present study confirmed that HNE can stimulate VSMC growth [17]. We also obtained insight into the mechanisms involved by studying the induction of MAP kinases, c-fos and c-jun gene expression, and AP-1 activation.
Materials and methods Cell culture and RNA extraction Rat VSMC were isolated by elastase and collagenase digestion of thoracic aortae from male Wistar as previously described [18]. Cells in passage 10–15 were used for experiments. Total RNA was extracted from confluent human VSMC using a modified guanidinium isothiocyanate method [19]. mRNA analysis of c-fos and c-jun Total cDNA was synthesized from the total RNA using avian myeloblastosis virus reverse transcriptase and random 9-mers as primers. The cDNA was amplified using PCR with primers derived from the published sequences of rat c-fos [20] and c-jun [21]. The PCR products for c-fos and c-jun were labelled with [a-32P]dCTP by random priming and used as probes. Blotting proceeded as described [22]. After probing for c-fos or c-jun mRNA expression, the filter was stripped and reprobed for the presence of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Radioactivity in the blots was quantified using a BAS2000 image analyzer (Fuji Photo Film Co., Tokyo, Japan).
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MAPK assay To create stable reporter cell lines with which to evaluate MAPK activation, we used transreporting systems with GAL4 fusion transactivators as pathway-specific sensors [23]. These systems use a fusion trans-activator plasmid consisting of the DNA binding domain of the yeast GAL4 (residues 1 to 147) protein and the activation domain of Elk-1, c-Jun, or Chop, respectively. We first transfected the pFR-Luc plasmid (that contains the luciferase gene which is controlled by a promoter that responds to GAL4 fusions; Stratagene) with a pSV40/ Zeo2 plasmid containing a Zeocin expression cassette (Invitrogen) into rat VSMC. Successive rounds of selection in antibiotic medium (Zeocin 300 mg/ml) isolated Zeocin-resistant clones. We also transfected Zeocin-resistant cell lines with the fusion trans-activator pFA2Elk1, pFA2-cJun, or pFA-Chop plasmids (Stratagene). Successive rounds of selection with G418 (500 mg/ml), generated clones that gave the best response when transfected with the positive control vectors for each reporting system. These clones were further analyzed. AP-1 activation To study AP-1 activation, the cells were stably transfected with a cis-reporter plasmid containing the luciferase reporter gene linked to seven repeats of AP-1 binding sites (pAP-1-Luc: Stratagene). The pAP-1-Luc plasmid was transfected together with a pSV2neo helper plasmid (Clontech, Palo Alto, CA, USA) into rat VSMC using FuGEN 6 transfection reagent (Boehringer Mannheim, Mannheim, Germany). The cells were cultured in the presence of G418 (Clontech) at a concentration of 500 mg/ml with medium replacement at 2 to 3 day intervals. Approximately 3 weeks later, G418-resistant clones were isolated using a cloning cylinder and the expression of luciferase activity was analyzed individually. Several clones were selected for analysis of AP-1 activation. Luciferase activity was measured using a Luciferase assay kit (Stratagene). Cell proliferation and DNA synthesis Cell proliferation was assessed using Alamar Blue assay. As a redox indicator, Alamar Blue (Serotec Ltd.) is reduced by reactions innate to cellular metabolism and thus provides an indirect measure of viable cell number. VSMC were growth-arrested in 96-well plates and treated with HNE for 48 hours. Then Alamar Blue (10w/v% in PBS) was added to the cells and 3hours later fluorescence was determined in a cytofluorometer at 544 nm excitation and 590 nm emission wavelength in a cytofluorometer (Fluoroskan Asent FL, Labsystems). The amount of DNA synthesis was assessed by determining BrdU incorporation by VSMC using an ELISA kit (Amersham). VSMC were growth arrested in 96-well plates, then HNE was added in the presence of BrdU for 24 h. After fixation and blocking, a peroxidase-labeled anti-BrdU antibody was added. The substrate was tetramethylbanzidine, and the color was read at 450 nm in a spectrophotometer. Statistical analysis Data are presented as mean 6 SEM. Multiple comparisons were evaluated by ANOVA followed by Fisher’s protected least significant difference test. Two experiments were compared using Student’s unpaired t test. A value of P,.05 was considered statistically significant.
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Results To verify that the HNE treatment of the cells resulted in a functional increase in ERK, JNK, and p38 activities, activation of the transcription factor Elk-1, cJun, or Chop was respectively evaluated with trans-reporting systems using GAL4 fusion transactivators as pathway-specific sensors. When activated by phosphorylation, these fusion proteins binds to the promoter and induce luciferase expression. Therefore, luciferase activity in stable cell lines reflects the activation status of the fusion transactivator and, hence, the activation status of corresponding signal pathways. Fig. 1 shows that the activity of JNK increased along with HNE concentration whereas Elk and Chop activation peaked at 5 mM HNE. ERK induced by HNE (5 mM) was completely inhibited by PD98059 (30 mM), while Chop activation by HNE (5 mM) was also prevented by SB203580 (10 mM). We next investigated whether HNE modulates AP-1 activity as well as the expression of cfos and c-jun gene. We evaluated c-fos and c-jun mRNA levels by Northern blotting. While c-Fos mRNA levels were low in unstimulated cells, HNE increased the levels of c-Fos mRNA, to a peak at 2.5 mM, then rather decreased at higher concentrations (Fig. 2). On the other hand, c-jun mRNA is constitutively present and it further increased as a function of HNE concentration (Fig. 2). Since c-fos and c-jun are the major constituents of the transcriptional factor AP-1 and HNE increased the gene expression of these protooncogenes, we tested the effect of HNE on AP-1 activation in VSMC. We determined AP-1 activity by measuring AP-1-dependent transcription in rat VSMC stably transfected with a luciferase reporter constract. The activity of AP-1 was slightly increased by HNE at z1mM, significantly activated by higher concentrations of HNE, peaked at 2.5 mM, then declined (Fig. 3).
Fig. 1. Effect of HNE on activation of Elk-1, cJun, or Chop. Quiescent VSMC (transfected with pFR-Luc/pFA2Elk1:closed circles, pFR-Luc/pFA2-cJun: closed triangles, or pFR-Luc/pFA-Chop: closed squares) were incubated with various concentrations of HNE. After 3 h, cells were lysed, and luciferase activities were measured. Data are mean 6 S.E.M. of triplicate observations. * P,.05 and ** P,.01 compared with control (no HNE).
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Fig. 2. Effect of HNE on mRNA expression for c-fos and c-jun in VSMC. Cells were treated with various concentrations of HNE. After 1 h, total RNA was isolated and analyzed by Northern blot hybridization with specific probes for rat c-fos and c-jun.
To determine whether HNE stimulates VSMC growth, we examined the effect of 2.5 mM HNE on VSMC proliferation and DNA synthesis. This concentration of HNE is that at which AP-1 is most potentyl activated. After exposing of growth-arrested VSMC to HNE for 48 hours, cell proliferation was determined by Alamar-Blue assay. Fluorescent values as indicator of viable cell number were significantly increased after exposure to HNE (Fig. 4). We also measured the effect of HNE on DNA synthesis by determining incorporation of BrdU in
Fig. 3. Effect of HNE on AP-1 activation. Quiescent VSMC (transfected with pAP-1-Luc) were treated with different concentrations of HNE. After 3 h, cells were lysed, and luciferase activities were measured. Data are mean 6 S.E.M. of triplicate observations. * P,.05 and ** P,.01 compared with control (no HNE).
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Fig. 4. Effect of HNE on VSMC growth. (A) VSMC were growth-arrested in plates and treated with HNE (2.5 mM) for 48 h. Viable cell number was assessed using Alamar Blue assay. (B) VSMC were growth-arrested in plates and treated with HNE (2.5 mM) in the presence of BrdU for 24 h. Incorporation of BrdU in VSMC was determined using an ELISA kit. * P,.05 compared with control (no HNE).
VSMC. Incubating VSMC with HNE over 24 hours increased the incorporation of BrdU compared with control (Fig. 4). Discussion The present study showed that HNE stimulates MAP kinases, induces c-fos and c-jun mRNA expression and increase AP-1 activity. Those effects were dependent on a narrow concentration range of HNE. Primarily activated in response to growth stimulation, ERK is implicated in cellular proliferation and differentiation [24–26], whereas JNK and p38 MAP
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kinases are charcterized by a powerful response to cellular stress such as UV light, osmotic stress, DNA damaging agents, and proinflammatory cytokines [14,27,28]. Activation of JNK has also been implicated in the induction of apoptosis in response to growth factor withdrawal and other envioronmental stimuli and in stimulating cell proliferation and transformation [29,30]. We demonstrated that HNE treatment activates ERK, which could lead to the induction of c-fos gene expression. We found that HNE also stimulated JNK and p38, which might be responsible for the induction of c-jun gene expression. Being the major components of the AP-1 transcription factor, c-fos and c-jun, once activated, can subsequently activate the transcription of several genes controlling cellular growth. Indeed, HNE induced VSMC growth, which is consistent with other observations [17]. Intracellular peroxide production by HNE has been demonstrated in rat liver epithelial cells [2]. It has been demonstrated that H2O2 significantly activated MAP kinases in VSMC [31] and we confirmed H2O2-induced activation of MAP kinases in our system. Accordingly, intracellular peroxide appears to play a crucial role in the HNE-induced activation of stress signaling pathways. HNE-induced peroxide, at least in part, seems to be responsible for activation of MAP kinases. One potentially significant consequence of oxidative stress is increased VSMC proliferation [32]. Regulation of the redox state of the cell may be a general mechanism by which growth signals are transduced and HNE is a potential source of intracellular pro-oxidants. Intracellular peroxide production by HNE has been demonstrated in rat liver epithelial cells [2]. Some levels of oxidant stress is required for cell growth. However, HNE is cytotoxic at higher concentrations [33–35]. Other oxidation products, such as H2O2 and oxidized LDL, over a narrow concentration range can also cause both proliferatiive and cytotoxic effects [36,37]. Thus, a narrow concentration range of HNE may activate cellular signal transduction leading to cell growth, whereas higher concentrations may have cytotoxic effects. This may be relevant in atherogenesis, where cell growth, apoptosis, and necrosis are thought to mutually contribute to lesion formation. In summary, HNE induces VSMC proliferation at the concentration of which causes the activation of MAP kinases, the induction of gene expression of c-fos and c-jun, and the increase in AP-1 activity. Acknowledgments This work was supported in part by a grant from Japan Private School Promotion Foundation and by Grants-in-Aids from the Ministry of Education, Science, and Culture. References 1. Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radical Biology & Medicine 1991;11(1):81–128. 2. Uchida K, Shiraishi M, Naito Y, Torii Y, Nakamura Y, Osawa T. Activation of stress signaling pathways by the end product of lipid peroxidation. 4-hydroxy-2-nonenal is a potential inducer of intracellular peroxide production. Journal of Biological Chemistry 1999;274(4):2234–42. 3. Jurgens G, Chen Q, Esterbauer H, Mair S, Ledinski G, Dinges HP. Immunostaining of human autopsy aortas
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