Activation of Mitogen-Activated Protein Kinases (ERK/JNK) and AP-1 Transcription Factor in Rat Carotid Arteries After Balloon Injury
Abstract Smooth muscle cell proliferation is a key event in neointimal formation after balloon angioplasty. The molecular signals that mediate this process have yet to be identified. Mitogen-activated protein (MAP) kinases are thought to play a pivotal role in transmitting transmembrane signals required for cell proliferation in vitro. The present studies were designed to investigate whether the signal transduction pathways of MAP kinases were involved in the development of restenosis in the injured arteries. Rat carotid arteries were isolated at various time points after balloon injury, and activities of MAP kinases, including extracellular signal-regulated kinases (ERK), and stress activated protein kinases (SAPK)/c-Jun N-terminal protein kinases (JNK), were determined in protein extracts of the vasculature using protein kinase assay and Western blot analysis. After balloon angioplasty, ERK2 and JNK1 activities in the vessel wall increased rapidly, reached a high level in 5 minutes and maintained for 1 hour. A sustained increase in ERK2 kinase activity was observed over the next 7 days in the arterial wall and 14 days in neointima after injury. In contrast, opposite and uninjured arteries did not show significant changes in these kinase activities. Concomitantly, Western blot analysis confirmed that the ERK2 kinase in the injured vessels was indeed activated or phosphorylated, showing a slowly migrating species of a 42-kDa protein containing phosphorylated tyrosine. Kinase activation is followed by an increase in c-fos and c-jun gene expression and enhanced activator protein 1 (AP-1) DNA-binding activity. Thus, balloon injury rapidly activates the MAP kinases in rat carotid arteries. These kinase activations may be crucial in mediating smooth muscle cell proliferation in response to vascular angioplasty.
- mitogen-activated protein kinase
- signal transduction
- transcription factor AP-1
- vascular injury
- Received September 11, 1996.
- Accepted February 5, 1997.
Smooth muscle accumulation in the arterial intima is a key event in restenosis after angioplasty and bypass surgery and in the development of atherosclerotic lesions.1,2 The accumulation of arterial SMC during lesion formation is caused by a combination of proliferation and directed migration of arterial SMC from the media into the intima.1-4 Both of these activities can be induced by cytokines and growth factors, such as platelet-derived growth factors (PDGF), fibroblast growth factor (FGF), and transforming growth factor β1 (TGF-β1), which is believed to be produced within the arterial wall and/or circulating cells in response to the vascular injury.2 The precise signal transduction pathways that link to vascular injury and quantitative changes in such gene expression during the development of the neointima after balloon angioplasty are largely unknown.
Several studies have shown that arterial injury immediately induces early gene expressions such as c-fos, c-jun, and c-myc.5,6 In vitro studies have demonstrated that Fos and Jun proteins combine to form stable AP-1 heterodimers, which bind to AP-1 consensus sequences present in numerous genes associated with cell proliferative response and extracellular matrix production.7 These AP-1-regulated genes are believed to be involved in the development of neointima.8 The induction and activation of transcription factor AP-1 rely mainly on MAP kinases in cultured cells.
MAP kinases include ERK, SAPK/JNK, and p38/RK/CSBP kinases.9 ERK is activated in response to growth factors, cytokines and a variety of stress in cultured cells.10,11 This kinase is responsible for the activation and phosphorylation of a number of other regulatory proteins including S6 kinase, cPLA2, and transcription factors needed for the expression of genes involved in cell proliferation.12-14 In addition, its activation is also required for passing through certain checkpoints in the cell cycle, eg, G1/S and G2/M, in proliferating cells in vitro.15-17 SAPK (JNK), as the name implies, is highly activated in response to stresses, including UVC irradiation, heat shock, inflammatory cytokines, and inhibitors of protein synthesis.18-20 JNK (SAPK) was named for its ability to phosphorylate the c-Jun protein leading to its enhanced transcriptional activity.19,21 Recently, it has also been shown to be capable of phosphorylating the transcription factor ATF-2.22,23 Therefore, MAP kinases-mediated signal pathways may play a key role in initiating cell proliferation and differentiation.
Most of our knowledge about the activation and function of MAP kinases has come from studies on cultured cells. Little is known about their activation in vivo and their relevance to pathological conditions in animal models. So far, no systematic investigation examining the activation of the MAP kinases, as well as AP-1 activity, has been conducted in a well defined model of vascular injury. In this report, we examined the MAP kinase activation and AP-1 activity at various time intervals following vascular injury of rat carotid artery. We demonstrated that vascular injury leads to the rapid activation of MAP kinases and increased AP-1 activity in the arterial wall.
Six-month-old male Wistar rats were maintained on a light/dark (12/12 hours) cycle at 24°C and received food and water ad libitum. All procedures were performed according to protocols approved by the Institution Committee for use and care of laboratory animals. Injury to the left common carotid artery was performed in the standard procedure as described previously.1 Briefly, rats underwent anesthesia with thiopental (40 mg/kg), and injury was performed with a 2-F Fogarty balloon catheter. The catheter was inserted through an incision made in the external carotid artery and advanced along the length of the carotid artery to the aortic arch. The balloon was then inflated and passed three times along the length of the carotid artery. The balloon catheter was then removed, and the external carotid artery was permanently ligated. The time necessary to pass the inflated balloon catheter back and forth into the carotid artery three times was 18 seconds. To keep the time of the injury constant, we maintained the time of balloon inflation constant at 18 seconds and used equal volume for balloon inflation (standard procedure) for the experiments. For a gentle injury, one-third of the volume of balloon inflation used for the standard procedure was employed, and the time of balloon inflation was 18 seconds. For experiments with mechanical stretch, carotid arteries were longitudinally prolonged 10 to 15% of the original length 3 times within 18 seconds. Such transient stretch did not cause morphologically detectable injury in the cells.24,25 At various time intervals following injury, animals were killed and carotids free of adventitia were collected and frozen immediately in liquid nitrogen.
For conventional histology, tissue fragments were fixed in 4% buffered (pH 7.2) formaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (HE).
The procedure used for protein extracts and kinase activity assay was similar to that described previously26,27 with a slight modification. Briefly, frozen carotid tissues were homogenized with polytron homogenizer on ice with buffer A containing 20 mmol/L Hepes (pH 7.4), 50 mmol/L β-glycerophosphate, 2 mmol/L EGTA, 1 mmol/L DTT, 1 mmol/L Na3VO4, 1% Triton X-100, 10% glycerol, 2 μmol/L leupeptin, 400 μmol/L PMSF, and 10 U/mL aprotinin. The homogenate was incubated on ice for 15 minutes. After centrifugation at 17,000g for 30 minutes, the supernatant was collected, and the protein concentration was measured with Bio-Rad protein assay reagent (Bio-Rad).
One-half milliliter of the supernatant containing 0.3 mg proteins was incubated with 5 μL of antibodies against mammalian ERK2 or JNK1 (Santa Cruz Biochem., Santa Cruz, Calif) for 2 hours at 4°C with rotation. These antibodies have been shown specifically to recognize mammalian ERK2p42 and JNK1p54.28,29 Subsequently, 40 μL of protein A-Sepharose 4B suspension was added, and rotating continued for 1 hour at 4°C. The immunocomplexes were precipitated by centrifugation and washed 2 times with the buffer A, B (500 mmol/L LiCl, 100 mmol/L Tris, 1 mmol/L DDT, 0.1% Triton X-100; pH7.6), and C (20 mmol/L Mops, 2 mmol/L EGTA, 10 mmol/L MgCl2, 1 mmol/L DDT, 0.1% Triton X-100; pH 7.2), respectively.
The activities of ERK2 in the immunocomplexes were measured as described previously.18,26,30,31 Briefly, the immunocomplexes were incubated with 35 μL of the buffer C supplemented with myelin basic protein (MBP; 6μg; Sigma), γ-32P-ATP (5 μCi), and MgCl2 (50 mmol/L) for 20 minutes at 37°C with vortexing every 5 minutes. To stop the reaction, 15 μL of 4× Laemmli buffer was added, and the mixture was boiled for 5 minutes. Proteins in the kinase reaction were resolved by SDS-PAGE (15% gel) and subjected to autoradiography.
The assay for JNK1 activity was performed similarly as described above. The substrate used was GST-c-Jun (the plasmid was provided by Dr. Woodgett), which was produced in Escherichia coli and isolated using glutathione Sepharose 4B RediPack Columns (Pharmacia Biotech Inc., Piscataway, NJ) according to the manufacturer’s protocol. Proteins in the kinase reaction were resolved by SDS-PAGE (12% gel) and subjected to autoradiography.18,26,30,31
Western Blot Analysis
Protein extracts (30 μg/lane) prepared from the arterial tissues as described above were separated by electrophoresis through a 10% SDS-polyacrylamide gel and transferred to an immobilon-p transfer membrane.32 The membranes were processed with the monoclonal antibody against ERK2, JNK1 or phosphotyrosine (Transduction Laboratory, Lexington, Ky). Specific antigen-antibody complexes were then detected with the ECL Western Blot Detection Kit (Amersham Co., Arlington Heights, Ill).
RNA Extraction and Northern Analysis
Freshly harvested tissues were homogenized and the RNA extracted using RNA Stat-60TM (Tel-Test “B”, Inc., Friends-wood, Tex). Total RNA (10 μg/lane) was fractionated by electrophoresis on formaldehyde-agarose gels and transferred to nylon membranes (Gene Screen Plus, DuPont, Boston, Mass). Hybridizations were performed using α-32P-labeled cDNA probes (ATCC, Rockville, Md) for specific mRNA species and standard procedures as described previously.33,34 Accuracy of loading and transfer, as well as the integrity of the RNA was verified by analysis of 18 and 28s RNA on the same blots.
Gel Mobility Shift Assays
For gel shift analysis, the procedure for protein extraction was similar to that described above except that the buffer contained 20 mmol/L Hepes (pH 7.5), 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.4 mol/L NaCl, 0.2 mmol/L DTT, 1 mmol/L Pefablock SC (Boehringer Mannheim, Mannheim, Germany) 20% glycerol, and 1 μg/m leupeptin. The supernatant was used for the assay after protein concentration was measured.
The procedure used was similar to that described previously.26 In short, 20 μg of aortic tissue protein extract was incubated with 0.5 ng of an oligonucleotide containing the AP-1 binding sequence (5′-CGCTTGATGACTCAGCCGGAA-3′) labeled with [γ-32P] ATP. For competition experiment, a mutant AP-1 oligonucleotide (5′-CGCTTGATGACTTGGCCGGAA-3′) was also used. Reaction buffer contained 10 mmol/L Tris, (pH 7.5), 1 mmol/L DTT, 1 mmol/L EDTA, 50 mmol/L NaCl, 5% glycerol, and 1 μg poly(dIdC) as a nonspecific competitor. Samples were electrophoresed through a 4% polyacrylamide gel and exposed to autoradiographic film. Super-shift assays were performed using antibodies against Fos, Jun, Fra-1, Fra-2, ATF2, JunB, or JunD (Santa Cruz Biochem). The antibodies were added to samples after the initial binding reactions between protein extracts, and oligonucleotides were allowed to take place.
Analysis of variance was performed for multiple comparisons. The Mann-Whitney U test was used for comparison between two groups. A P value less than .05 was considered statistically significant.
To verify the neointima formation after balloon injury, rat carotid arteries were injured and harvested at various time points. Figure 1⇓ shows results of a representative HE-stained sections of carotid arteries. The neointima was seen at days 21, 14, 7 and 5, whereas no significant morphological changes were observed between 10 minutes and 3 days after balloon injury.
Activation of ERK2 and JNK1
ERK2 activity of protein extracts derived from individual carotid artery was measured using MBP as a substrate. Figure 2A⇓ shows results of an experiment examining ERK2 activities in the vessel wall, and Fig 2B⇓ summarizes data from three independent experiments. Two control groups were employed in these experiments: (1) animals were killed immediately after removal from the housing facility and carotids free of adventitia were collected and (2) animals were killed at various time intervals following injury, and contralateral right carotids were collected as controls for possible effect of anesthesia and surgery. Because no significant difference in ERK2 and JNK activation was seen in tissues between the two control groups, either one of the two control groups was used in the present study.
ERK2 activities were at low levels in uninjured control vessels. Balloon injury resulted in a rapid activation of the ERK2 kinase, with eight to tenfold increase of activity between 5 minutes and 1 hour after the injury compared with those uninjured contralateral right carotids and uninjured control carotids. The ERK2 kinase activity declined by 6 hours post-injury but remained elevated. During the next 7 days, ERK2 activities were maintained at a higher level in the regenerating left carotid compared with uninjured arteries. By 14 days post-injury, ERK2 activities did not show any significant difference between the regenerating left carotid and uninjured contralateral right carotid. Because rats treated with restraint resulted in MAP kinase activation in blood vessels,26 in the present experiment, we minimized the time (<1 minute) to perform the anesthesia. Data show that ERK2 activity was low in the contralateral right carotids at various time points (control group 2; time points: 5 minutes to 21 days), which was not significantly different from control vessels (control group 1; time point: 0).
To determine if the injury was correlated with the activation of SAPK/JNK kinase in the vasculature, JNK1 activities were determined using GST-c-Jun as a substrate. As shown in Fig 3⇓, this treatment resulted in the rapid activation of JNK1, with maximum activity (>eightfold greater elevation compared with untreated control group 1) achieved 5 to 10 minutes after injury. The kinase activity declined 6 hours after injury.
Because the vessel wall mass increases several days after injury, it would be interesting to note whether ERK activity shows any difference between neointima and media. To address this issue, neointima and media of carotid arteries were carefully separated 2 weeks after injury and homogenized on ice. Protein extracts (0.3 mg), either from media or neointima, were used for ERK2 immunoprecipitation. ERK2 activity was calibrated based on the same amount of total proteins from either media or neointima. Data shown in Fig 4⇓ provide evidence that the magnitude of ERK2 activation in neointima is significantly higher than that in media. These results support the notion that activated MAP kinases were localized mainly in neointima 14 days after angioplasty.
To determine whether mechanical stress is involved in ERK activation in the early phase, carotid arteries injured gently (1/3 balloon volume of the standard procedure) or simply stretched (10 to 15% prolongation). Data shown in Fig 5⇓ provided evidence that stretching of the vessel wall resulted in significant activation of ERK2 kinases. Gentle injury to carotid arteries also activated ERK2 kinases although the magnitude of ERK2 activity was significantly lower compared with that obtained from standard injury (Fig 5⇓). We have also examined the situation of endothelial cells after gentle injury and found that no integrated endothelium was detectable (data not shown). These findings indicate that the magnitude of ERK2 activation correlated positively with the degree of balloon injury or the mechanical stress to the arterial wall.
Both ERK2 and JNK1 kinases are activated by dual phosphorylation of tyrosine and threonine residues in response to mitogenic or stress stimuli.10,11 Molecular weight of ERK2 shows a difference in both activated and inactivated forms, whereas JNK1 usually does not change. We performed a Western blot analysis using protein extracts from the arterial tissues and the antibody recognizing the ERK2 and JNK1. The activated (phosphorylated) form of p42 was identified based on its slower electrophoretic mobility compared with the unmodified (nonphosphorylated) form (Fig 6A⇓). Uninjured and contralateral vessels (control group 2) did not show any shift in the electrophoretic mobility of p42 protein seen on Western blots. These results demonstrated further that vascular injury causes the ERK activation or phosphorylation. No significant shift of JNK1 in the electrophoretic mobility were found (Fig. 6B⇓), which is consistent with observations by others using different types of cells.31
Protein Tyrosine Phosphorylation
It has been demonstrated that activation of the MAP kinases occurs through phosphorylation at tyrosine and threonine residues, and, therefore, we sought to obtain direct evidence for their tyrosine phosphorylation in the balloon-injured arteries. In addition, it would be interesting to see if other tyrosine-containing proteins were phosphorylated in the process of the vascular injury. Phosphotyrosine containing proteins in protein extracts from the arterial wall were visualized on Western blots using a monoclonal antiphosphotyrosine antibody PY20. As shown in Fig 7⇓, balloon injury resulted in rapid tyrosine phosphorylation or enhanced tyrosine phosphorylation of proteins with molecular masses of 42, 47, and 66 kDa, respectively. Reprobing of the same blots with antibodies against ERK2 verified that the 42-kDa band corresponded to ERK2 (data not shown). The JNK1 was not recognized by antiphosphotyrosine antibody because the tyrosine phosphorylated JNK1 lacks the epitope which is recognized by many antiphosphotyrosine antibodies.10 The identities of other tyrosine phosphorylated proteins p47 and p66 are not clear at present. They may represent other important components of the signaling pathways involved in vascular cell response to injury.
c-fos and jun Gene Expression
Both c-fos and c-jun gene expression have been shown to rely on MAP kinase-dependent phosphorylation of transcription factors, including p62TCF and Jun in cultured cells.35,36 Therefore, we examined whether injury-induced MAP kinase activation was associated with enhanced expression of these genes. As shown in Fig 8⇓, both c-fos and c-jun mRNA expression was induced in response to injury. The induction was rapid and transient, with maximal expression achieved 30 minutes after injury.
AP-1 Binding Activation
Members of the Fos and Jun protein family dimerize to form AP-1 transcription factor complexes that regulate the expression of other genes.37,38 To determine if AP-1 binding activity was increased in balloon-injured arteries, gel mobility shift assays using an oligonucleotide containing an AP-1 binding site were performed. Figure 9A⇓ shows a time course for AP-1 activation in rat arteries in response to injury. Consistent with our previous studies,26 two regions of binding activity were apparent. The broad slower migrating region designated with an arrow was found to represent specific binding because it was completed successfully by cold oligonucleotides corresponding to the AP-1 binding site but not by a mutated AP-1 oligonucleotide that has been shown to disrupt binding of the transcription factor (Fig 9B⇓). The faster migrating bands, designated NS, are presumed to represent nonspecific interaction as this binding activity was present in similar levels in arterial extracts from both treated and untreated animals and was not affected by the addition of cold AP-1 oligonucleotides. Increased AP-1- binding activity was evident within 30 minutes after injury, but maximum DNA-binding was not achieved until 3 hours after injury.
Figure 9C⇑ shows the results of gel mobility shift assays performed in the presence of antibodies specific to c-Fos, c-Jun, Fra-1, Fra-2, ATF2, JunB, or JunD proteins. Addition of either c-Fos or c-Jun antibody to the binding reaction resulted in a shift of the binding complexes to a slow migrating species, indicating the presence of both Fos and Jun proteins in the DNA-binding complexes.
The development of the neointima after balloon angioplasty is attributable mainly to vascular SMC proliferation and extracellular matrix accumulation.1-2 It has been demonstrated that the activation of MAP kinase cascades is one of the major pathways for the regulation of proliferation and cell growth in various cultured cells.10,11 In the present studies, we have demonstrated the in vivo activation of the MAP kinases in the regenerating carotids following balloon injury. This kinase activation may play a significant role in regulation of medial and intimal SMC proliferation in response to injury.
The precise signal responsible for the activation of these MAP kinases in the vasculature following injury remains to be clarified. The MAP kinase activation following injury may be mediated by mechanical stretching (balloon inflation pressure), between 5 minutes and 1 hour in the early phase. In the present study, we found that stretching of the vessel wall resulted in significant activation of ERK2 kinases and that the magnitude of ERK2 activation correlated positively with the degree of balloon injury to the arterial wall. Other reports support this notion that the stretching of cultured myocytes and isolated carotid arteries can activate MAP kinases.24,25 Fluid shear stress results in the ERK2 MAP kinase activation in endothelial cells.39 In addition, acute hypertension can also activate MAP kinases in the arterial wall.26 Our data demonstrated that maximum activity of both ERK and JNK kinases was achieved as early as 5 minutes after injury. These findings suggest that the kinase activation in the early phase after injury may be caused by mechanical stimulation of the vessel wall.
Accumulating evidence indicates that mechanical stress enhances growth or proliferation of vascular SMCs.40-42 For instance, a single transient mechanical stimulus of human vascular SMCs induces DNA synthesis.41 In carotid arteries, SMC proliferation is proportional to the degree of balloon injury in a rat model of angioplasty.42 Our findings indicated that mechanical stress, such as stretching and balloon inflation pressure, resulted in ERK activation as early as 5 minutes after injury in the arterial wall, but this did not apply to other factors, including artery ligation for 18 seconds (data not shown) and anesthesia. Thus, MAP kinases could be important signal transducers between mechanical stress and SMC proliferation in response to hemodynamic stress and angioplasty.
On the other hand, injury also results in the induction of many growth factors and their receptors such as PDGF, IGF-1 and TGF-β1, and PDGF receptors in the arterial wall.2,4 Numerous studies have suggested that these factors induce the migration and proliferation of SMC following injury.1-4 These factors are also recognized as activators of ERK in many types of cells in vitro.10,11 In the present study, we have demonstrated that the activity of the ERK remained elevating during 7 days post-injury and 14 days in neointima. This later phase kinase activation may be mediated by the growth factors induced locally by injury. Thus our finding suggests that the kinase activation is a key component in tranducing signals, eg, mechanical stress and cytokines, to the gene expression required for SMC proliferation in response to balloon injury.
Various stress stimuli that control cell growth use kinase cascades to transmit signals from the cell surface to the nucleus. Activation of the JNK/SAPK is believed to be critical for the cell’s response to environmental insults. Evidence obtained from study of cultured cells indicates that JNK/SAPK is involved in mediating cell growth or hypertrophy via activation of transcription factor AP-1.7,43 JNK/SAPK kinase can also phosphorylate or activate several transcription factors, including c-Jun, ATF2, Elk.23,44 ATF2 can dimerize not only with c-Jun but also with itself and some other members of the ATF family, including ATF3 CREB, and Elk-1, together with serum response factor, controls transcription from the serum response element. These transcription factors regulate gene expression, including matrix metalloproteinase, adhesion molecule E-selectin, NO synthase, IL-8, and proliferating cell nuclear antigen.43 These genes have been demonstrated to play a key role in the neointima formation. Our findings demonstrated that balloon injury induces JNK/SAPK activation in the arterial wall, suggesting a role of these protein kinases in response to injury. Thus, rapid JNK/SAPK activation in injured arteries could be important for mediating these gene expression, which are essential for proliferation of vascular smooth muscle cells.
The transcription factor AP-1 binds to AP-1 consensus sequences present in numerous genes associated with cell proliferative response and extracellular matrix production, such as FGF, endothelin-1, c-myc, collagenase and TGF-β1.7,8 Indeed, balloon injury to rat carotid artery induces TGF-β1 gene expression, and the levels of TGF-β1 transcripts in the regenerating left carotid remain elevated above those of the uninjured artery from 6 hours to 2 weeks after injury.45,46 The time course of the MAP kinase activation of an injured artery reported here was similar to that observed for TGF-β1 gene expression in the rat carotid arteries of balloon-injured models. Furthermore, the kinetics of the MAP kinase activation is also correlated with the time course of SMC proliferation following balloon injury of rat carotid arteries.47 It could be interesting to further study how AP-1 controls the gene expression in the process of SMC migration and growth in vivo.
Recent studies have focused on the signaling events in cultured cells from cardiovascular tissue, including myocytes and SMC. It has been shown that MAP kinase activation can induce a hypertrophic response in cardiac myocytes.24 Depletion of MAP kinase using an antisense oligodeoxynucleotide downregulates the phenylephrine-induced hypertrophic response in rat cardiac myocytes.48 Accumulating evidence indicates that MAP kinase phosphotase (MKP-1) specifically inhibits mitogen-induced activation of MAP kinases in cell lines.31,49 Recently, Lai et al50 reported a reduction of MKP-1 expression in carotid arteries in response to balloon injury that may be responsible for sustained activation of ERK2 in the injured arterial wall. In the present study we have demonstrated significant activation of MAP kinases in balloon injured arteries (up to 7 or 14 days). Inhibition of the MAP kinase activation by novel drugs or gene-therapeutic approaches may be useful for treatment of the vessel wall after angioplasty. It is an interesting issue and important for therapy in the future. At the present time, however, it is impossible to see the effect of ERK antagonist on neointima formation in vivo for several reasons. First, there is no effective way to deliver the antagonist to local SMCs. When the antagonist is introduced systematically, animals will die as a result of inhibition of the ERK function required for cell differentiation and proliferation in other tissues. When the antagonist is introduced locally, it will soon be discharged. Second, specificity of the antagonist is very low. Data derived from in vitro experiments show that ERK antagonists inhibit not only functions of MAP kinases but other protein kinases as well. Nevertheless, the therapeutic effect of ERK antagonist or inhibitor on neointima formation should be addressed when the specific antagonist or inhibitor is available. Thus, understanding of the mechanisms serving to regulate MAP kinase activities could lead to strategies for prevention or therapeutic intervention for vascular disorders.
Selected Abbreviations and Acronyms
|ERK||=||extracellular signal-regulated kinases|
|JNK||=||c-Jun N-terminal protein kinase|
|MBP||=||myelin basic protein|
|SAPK||=||stress-activated protein kinase|
|SMC||=||smooth muscle cell|
This work was supported by grant P11615-MED (to Q.X.) from the Austrian Science Foundation and a Hans- und Blaca-Moser-Stiftung (Q.X.). We thank Dr. J. Woodgett, Ontario Cancer Institute, Toronto, Canada, for providing the GST-c-Jun expression vector, Dr. G. Wick for critical reading of the manuscript, A. Jenenwein and G. Strum for excellent technical assistance, and T. Öttl for the preparation of photographs.
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