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Cardiovascular Research 2001 51(1):151-159; doi:10.1016/S0008-6363(01)00274-7
© 2001 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Combined effect of retinoic acid and basic FGF on PAI-1 gene expression in vascular smooth muscle cells

Atai Watanabea, Masahiko Kurabayashia,*, Masashi Araia, Kenichi Sekiguchia and Ryozo Nagaib

aSecond Department of Internal Medicine, Gunma University School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511, Japan
bDepartment of Cardiovascular Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

mkuraba{at}med.gunma-u.ac.jp

* Corresponding author. Tel.: +81-27-220-8140; fax: +81-27-220-8150

Received 18 September 2000;
   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Aberrant regulation of the synthesis and degradation of the extracellular matrix (ECM) is associated with the pathophysiology of vascular disease. Plasminogen activator inhibitor-1 (PAI-1) plays a crucial role in regulating the quantity and composition of ECM. However, regulatory mechanisms underlying the expression of the PAI-1 gene remain unclear. We examined the effects of all-trans-retinoic acid (atRA), either alone or in combination with mitogenic growth factor, basic fibroblast growth factor (bFGF), on the PAI-1 expression in cultured vascular smooth muscle cells (SMCs). Methods: Cultures of the rabbit vascular smooth muscle cell line C2/2 were used to study the effects of atRA and bFGF separately or together. Results: Treatment of vascular SMCs with atRA in combination with bFGF resulted in an additional increase in PAI-1 expression both at the mRNA and protein levels. In contrast, tissue-type plasminogen activator, urokinase-type plasminogen activator and tissue factor mRNA levels were only minimally affected. The all-trans-RA- and bFGF-mediated increases in PAI-1 mRNA levels were markedly attenuated by the tyrosine kinase inhibitor genistein, but not by MEK1 or p38MAP kinase inhibitors. The rate of decrease in PAI-1 mRNA levels after actinomycin D treatment was not affected by atRA and bFGF. Transient transfection of the PAI-1 promoter-luciferase reporter gene, which contains 967 bp of the 5'-flanking region of the human PAI-1 gene, revealed that atRA and bFGF additionally increased transcription from this promoter. Progressive 5'-deletion revealed that the promoter region required for such an effect lies between –967 and –260, which contains no canonical sequence for the RA-response element. In agreement with the role of PAI-1 in the inhibition of fibrinolytic activity which stimulates ECM degradation, cell migration was inhibited by treatment with atRA and bFGF. Conclusions: These results indicate that atRA and bFGF can function in a combined fashion and induce PAI-1 synthesis in vascular SMCs, and suggest a role for these two compounds in regulating SMC migration.

KEYWORDS Growth factors; Protein kinases; Remodeling; Signal transduction; Smooth muscle


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Vascular smooth muscle cells (SMCs) play a critical role in the progression of neointima formation after vascular injury [1,2]. A wealth of data demonstrates that the migration of SMCs, which is essential for neointima formation, requires degradation of extracellular matrix (ECM) components [3–5]. The balance between fibrinolysis controlled by fibrinolytic enzymes, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), and coagulation regulated by the specific plasminogen activator inhibitor PAI-1 and the procoagulant protein tissue factor (TF) is important for degradation of ECM [6].

The fibrinolytic system comprises the inactive proenzyme plasminogen, which can be converted to the active enzyme plasmin, which induces the degradation of many extracellular proteins either directly or through the activation of latent matrix metalloproteinases (MMPs) [7,8]. The enzymatic action of tissue-type plasminogen activator (tPA) and of urokinase-type plasminogen activator (uPA), the two physiological plasminogen activators, is controlled by several plasminogen activator inhibitors. Of these, plasminogen activator inhibitor-1 (PAI-1) is believed to be of primary importance [9]. Since expression of the plasminogen system has also been demonstrated in SMCs and leukocytes in human atherosclerotic plaque [10], normal as well as atherosclerotic or restenotic vessels are able to express the components of the plasminogen system required for controlled plasmin proteolysis after vascular injury.

Retinoids play a pivotal role in the development and differentiation of diverse tissues [11,12]. Retinoids exert pleiotropic effects on a variety of cell types, including vascular SMC in vitro [13,14]. In addition, retinoids are able to reduce the intimal thickening induced by experimental injury in vivo [15].

A large number of growth factors, cytokines and vasoregulatory molecules participate in the formation of the atherosclerotic lesions resulting from the excessive inflammatory fibroproliferative response to various forms of insults to endothelial cells and SMC of the artery [16]. Among these is basic fibroblast growth factor (bFGF), a peptide growth factor which plays a key role in mediating cell proliferation, tissue differentiation, and tissue repair [17].

A recent study has suggested that the effect of retinoic acids on cell proliferation is dependent on the growth condition of the cells; retinoic acid stimulates DNA synthesis and cellular proliferation under conditions in which growth factors are absent in culture, but it inhibits growth in the presence of growth factors [18]. The molecular mechanisms behind these observations remain to be elucidated. The aim of the present study was to investigate the effects of atRA and bFGF, two distinct vasoactive substances, on the fibrinolytic system in vascular SMCs. To achieve this, we used cultured vascular SMCs and examined gene expression of PAI-1, tPA, uPA, and TF. We further determined the molecular mechanism underlying the combined effect of atRA and bFGF on PAI-1 gene expression.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Materials
All-trans-retinoic acid (atRA), calphostin C, genistein, and actinomycin D were purchased from Sigma. PD 98059 and tyrphostin A23 were obtained from BIOMOL Research Laboratories, and SB 203580, herbimycin A (RG-50810) were purchased from Calbiochem. All reagents were used at the recommended concentrations to avoid cell toxicity. Phenol red-free Dulbecco's modified Eagle's medium (DMEM), L-glutamine, fetal calf serum (FCS), streptomycin, and penicillin were obtained from Gibco. Basic fibroblast growth factor (bFGF) and a random primer kit were obtained from Boehringer Mannheim. Recombinant human platelet-derived growth factor (PDGF) was provided by Calbiochem. [{alpha}-32P]dCTP (3000 Ci/mmol) was from Amersham. The stock solution of atRA was prepared in ethanol, preserved at –30°C, and handled in reduced light.

2.2 Cell culture and treatments
C2/2 cells, an established cell line derived from rabbit aortic smooth muscle cell, were obtained from Life Science Center, Biochemical Research Lab, Asahi Chemical Industry [19]. Physiological and electromicroscopic studies have demonstrated that C2/2 cells conserve smooth muscle phenotype [20]. Cells were grown in phenol red-free DMEM supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mmol/l L-glutamine. Cells were incubated at 37°C and equilibrated with 5% CO2 in humidified air. At confluence, the culture medium was changed to serum-free DMEM. Twenty-four hours later, atRA and/or bFGF were added at the specified concentrations. Control cells received ethanol or dimethyl sulfoxide (DMSO) as a vehicle control, and the final concentration of ethanol or DMSO in the medium did not exceed 0.1%. When inhibitors were used, cells were pretreated for 1 h with varying doses of inhibitors before the addition of atRA and/or bFGF. At the indicated time, the cultures were lysed with Isogen (Nippon Gene) and stored at –80°C until isolation of RNA, or supernatants were harvested and stored at –80°C until assay of PAI-1 production. All our experiments were performed in serum-free medium to eliminate the possible modulatory factor(s) contained in sera.

2.3 Isolation of RNA and Northern blot analysis
Total RNA was extracted from the cells using Isogen according to the manufacturer's procedure. Each RNA (15 µg) was denatured, subjected to electrophoresis through a 1.2% agarose gel in the presence of formaldehyde and then transferred onto Biodyne nylon membranes (Pall BioSupport). The membranes were optimally cross-linked with UV light (Stratagene), and hybridized for 14 h at 42°C with cDNA probes for either human PAI-1, tPA, uPA or TF. After hybridization, the filters were washed under conditions of high stringency in 2xSSC containing 0.1% SDS. All filters were stripped and then sequentially hybridized with a cDNA encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which was used as a loading control. The cDNA probes were obtained by polymerase chain reaction (PCR) as previously described [21], and labeled with [{alpha}-32P]dCTP using a random primed DNA labeling kit. Autoradiography was performed using an intensifying screen and Kodak XAR5 (Eastman Kodak Company) film at –80°C for 3–7 days. The hybridization signal was quantitated by densitometry, normalized to that obtained with GAPDH.

2.4 Promoter-luciferase vector
PAI-1-luciferase reporter gene vector containing an approximate 3.4-kb DNA insert was kindly provided by Dr. Douglas E. Vaughan (Vanderbilt University Medical Center, Nashville, TN, USA). Progressive deletions from the 5' end of the human PAI-1 gene promoter fragments were constructed by using PCR methods to create fragments of PAI-1 promoter into the pGL3-Basic luciferase vector (Promega) as previously described [21]. All fragments had an identical 3' end (i.e. the XhoI site at portion +82) and 5' end (i.e. the KpnI site at each portion). The identity of all new constructs was confirmed by sequencing.

2.5 Transient transfection and luciferase assay
For transient transfection experiments, C2/2 cells were cultured in 35-mm dishes using phenol red-free DMEM supplemented with 10% FCS. When cells reached 60% confluence, they were transfected using Tfx-50 (Promega), with 1 µg of the PAI-1 pGL3 constructs. After incubation for 12 h, the medium was replaced with serum starvation DMEM for 12 h and then cells were treated with atRA (0.32 µM) and/or bFGF (32 ng/ml). After 12 h, the cells were washed with PBS, and lysed in 120 µl of Cell Culture Lysis Reagent (Promega). The luciferase activity of each cell lysate was measured using a Berthold Lumat LB9501 luminometer, and was normalized to the cellular protein concentration. The protein concentration of each sample was measured using a Bio-Rad DC protein assay system (Bio-Rad Laboratories). Each transfection was repeated at least three times in duplicate.

2.6 ELISA for PAI-1
The concentrations of PAI-1 produced were measured using a commercially available ELISA kit (American Diagnostica) as stated in the figure legends. The culture supernatants were collected after stimulation for 24 h and the absorbance was measured at 450 nm. PAI-1 production was normalized to the volume of the medium and cell number.

2.7 Cell migration assay
The migration of C2/2 cells was assayed using a commercially available quantitative cell migration assay kit (Chemicon International). A cell suspension (2.5x105 cells) in quenching medium (serum-free DMEM containing 5% BSA) or media plus indicated reagents were loaded into the upper chambers, which were separated by a BSA or collagen I-coated membrane from the lower chambers containing the chemoattractant PDGF-BB (10 ng/ml). After 24 h incubation, non-migratory cells on the upper surface were removed with cotton swabs. The chamber membranes were stained with crystal violet staining solution. Stained migratory cells were solubilized with extraction buffer and the absorbance was measured at 570 nm. The number of migratory cells appearing on the underside of the chamber was expressed as absorbance.

2.8 Statistical analysis
Results are presented as the mean±standard error of the mean (S.E.M.) for at least three separate experiments. Analysis of variance (ANOVA) was used to analyze the significance of the results. P<0.05 was considered statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Regulation of PAI-1 mRNA by atRA and/or bFGF
To investigate the modulation of PAI-1 gene expression by atRA and/or bFGF, C2/2 cells were incubated in fresh serum-free medium 24 h before the addition of increasing concentrations of either atRA or bFGF. The effects of either atRA or bFGF on PAI-1 gene expression were dose-dependent with maximal stimulation occurring at concentrations of 0.32 µM and 32 ng/ml, respectively (Fig. 1A). Higher doses of atRA (0.64 µM) or bFGF (64 ng/ml) did not induce a further expression of PAI-1 mRNA. However, simultaneous stimulation with atRA and bFGF further potentiated PAI-1 mRNA levels even after each ligand alone induced a maximum expression of PAI-1 mRNA (Fig. 1A). In contrast, tPA, uPA and TF mRNA levels were not altered by treatment with atRA alone or in combination with bFGF (data not shown). As shown in Fig. 1B, the combined effect of atRA and bFGF was time-dependent. All-trans-RA action on PAI-1 mRNA levels was detected as early as 2 h after treatment. The enhancement reached a maximum after 4 h of treatment and was followed by a decrease to basal levels at 24 h. bFGF-induced enhancement of PAI-1 mRNA levels reached a peak after 2 h and declined thereafter.


Figure 1
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  		Fig. 1 Effects of atRA in combination with bFGF on PAI-1 mRNA expression. (A) Northern blot analysis. Confluent C2/2 cells were incubated in serum-free medium for 24 h before testing. Cells were stimulated with increasing concentrations of atRA or bFGF for 3 h as described. At the maximum doses of atRA or bFGF, cells were also stimulated with increasing amounts of bFGF or atRA, respectively. Then total RNA (15 µg) was extracted and analyzed by Northern blots. The membranes were successively hybridized with the PAI-1 and GAPDH cDNA probes. (B) Northern blot analysis showing the time course of atRA- and/or bFGF-induced expression of PAI-1 mRNA. C2/2 cells were cultured with atRA (0.32 µM) and/or bFGF (32 ng/ml) for the indicated times, then total RNA was extracted and analyzed by Northern blots for PAI-1 and GAPDH mRNAs. (C) Densitometric analysis of the Northern blots. PAI-1 mRNA levels were normalized against the levels of GAPDH mRNA; the results are arbitrarily indicated as values relative to PAI-1 mRNA levels in the control cells and are the mean±S.E.M. of three separate experiments in duplicate. *P<0.05 compared with control cells (n=6).

 
3.2 Combined effect of atRA and bFGF on PAI-1 mRNA expression was mediated through a tyrosine kinase-dependent pathway
To investigate the intracellular mechanisms involved in the combined induction of the PAI-1 gene, C2/2 cells were treated for 1 h with each of the various inhibitors of intracellular signaling pathways before the addition of atRA and/or bFGF. First, we investigated whether MAP kinases are important mediators for the effects of atRA and bFGF on the PAI-1 gene expression in C2/2 cells. Fig. 2A shows that inhibition of ERKs and p38 MAPK signaling with PD98059 and SB203580, respectively, had no effects on the atRA- and bFGF-induced PAI-1 mRNA expression. Calphostin C, a specific inhibitor of PKC, had no effects either. In contrast, genistein (50 µM), which competes at the ATP-binding site and thus has a broad range of action [22], blocked atRA/bFGF-stimulated PAI-1 mRNA expression, whereas herbimycin A, which binds to Src-homology motifs, which confer a narrower range of action [23], had no effect on this induction. Tyrphostin A23, which potently inhibits the epidermal growth factor (EGF) receptor tyrosine kinases [24], increased basal levels of PAI-1 mRNA. Our data support the involvement of genistein-sensitive tyrosine kinases in atRA/bFGF-induced PAI-1 expression. We also observed that genistein blocked the inducible expression of the PAI-1 gene by either atRA or bFGF alone (Fig. 3). To assess whether the observed reduction in PAI-1 mRNA levels was due to a non-specific inhibition of mRNA or unequal RNA application, the blots were also probed with a GAPDH cDNA probe. GAPDH mRNA levels did not change under any experimental condition.


Figure 2
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  		Fig. 2 Effects of protein kinase inhibitors on the atRA- and bFGF-induced increase in PAI-1 mRNA levels. (A) Northern blot analysis. C2/2 cells were cultured with or without each inhibitor for 1 h, then atRA and bFGF were added simultaneously. The incubations were continued for 3 h, then total RNA was extracted and analyzed by Northern blots for PAI-1 and GAPDH mRNAs. (B) Densitometric analysis of the Northern blots. PAI-1 mRNA levels were normalized to the GAPDH signal. The results are arbitrarily indicated as values relative to PAI-1 mRNA levels in the control cells and are the mean±S.E.M. of three separate experiments in duplicate. *P<0.05 compared with control cells (n=6). PD, PD98059 (50 µM); SB, SB203580 (10 µM); Calph, calphostin C (100 nM); Gen, genistein (50 µM); Tyr, tyrphostin A23 (100 µM); Herb, herbimycin A (1 µM).

 

Figure 3
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  		Fig. 3 Effects of genistein on PAI-1 mRNA levels after treatment with either atRA or bFGF alone. (A) Northern blot analysis. C2/2 cells were cultured with or without genistein for 1 h, then atRA or bFGF was added. The incubations were continued for 3 h, then total RNA was extracted and analyzed by Northern blots for PAI-1 and GAPDH mRNAs. (B) Densitometric analysis. PAI-1 mRNA levels were normalized to the GAPDH signal. The results are arbitrarily indicated as values relative to PAI-1 mRNA levels in the control cells and are the mean±S.E.M. of three separate experiments in duplicate. *P<0.05 compared with control cells (n=6). Gen, genistein (50 µM).

 
3.3 All-trans-RA- and bFGF-induced PAI-1 mRNA expression at the transcriptional levels
To determine whether simultaneous stimulation with atRA and bFGF increases steady-state levels of PAI-1 mRNA at the transcriptional levels or at post-transcriptional levels, the measurement of the PAI-1 mRNA half-life was performed in the presence or absence of atRA and bFGF. C2/2 cells were stimulated for 4 h with a combination of 0.32 µM atRA and 32 ng/ml bFGF, then each medium was changed and actinomycin D (5 µg/ml) was added with or without atRA/bFGF. The incubations were continued for the next 6 h. The rate of decrease in PAI-1 mRNA levels after actinomycin D treatment was not affected by atRA/bFGF (Fig. 4). Thus, simultaneous stimulation with atRA and bFGF has no effect on the stability of PAI-1 mRNA, suggesting that the observed increase in PAI-1 mRNA levels by atRA/bFGF was due to an increase in transcription from the PAI-1 promoter. The half-life of the PAI-1 mRNA was not affected by either atRA or bFGF alone (data not shown).


Figure 4
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  		Fig. 4 Effect of actinomycin D on the atRA- and bFGF-induced increase in PAI-1 mRNA levels. (A) Northern blot analysis. C2/2 cells were pretreated with atRA (0.32 µM) and bFGF (32 ng/ml) for 3 h, then each medium was changed and actinomycin D (AD, 5 µg/ml) was added with or without atRA/bFGF. The incubations were continued for the next 6 h, then total RNA was extracted and analyzed by Northern blots for PAI-1 and GAPDH mRNAs. (B) Densitometric analysis of the Northern blots. PAI-1 mRNA levels were normalized against the levels of GAPDH mRNA. The data are expressed as the percentage of hybridization signals present at each time following AD addition, compared with the signal present at 0 h, and are the mean±S.E.M. of three separate experiments in duplicate. *P<0.05 compared with cells at 0 h (n=6).

 
3.4 All-trans-RA and bFGF increased PAI-1 promoter activity
Next, we performed a transient transfection assay to determine the promoter region mediating the effects of the atRA and bFGF on PAI-1 transcription. C2/2 cells were transiently transfected with –967Luc, and the luciferase activity was measured 12 h after stimulation with atRA and bFGF individually or together. As shown in Fig. 5, the increase in the luciferase activity of –967Luc was comparable between stimulation of atRA and bFGF (two-fold increase). Co-treatment with atRA and bFGF resulted in a higher luciferase activity (four-fold). Deletion of the 5'-flanking region to –260Luc eliminated such a response. These results indicate that the major sequence determinants of combined responsiveness in the PAI-1 promoter reside between –967 and –260, where no RA-response element (RARE)-like sequence was present.


Figure 5
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  		Fig. 5 Induction of luciferase activity with atRA and/or bFGF in C2/2 cells transfected with PAI-1 promoter-luciferase reporter gene constructs. C2/2 cells were transfected with the indicated reporter genes. At 12 h post-transfection, the medium was replaced with serum starvation medium for 12 h. Then atRA (0.32 µM) and/or bFGF (32 ng/ml) was added and incubations were continued for the next 12 h as described in Methods. Cell lysates were prepared and analyzed for luciferase activity. Bars represent the relative induction of luciferase activity after treatment with atRA and/or bFGF as compared with untreated control cells. Results represent the mean±S.E.M. of three separate experiments in duplicate. *P<0.05 compared with control cells (n=6). **P<0.05 compared with values in atRA and bFGF-treated cells (n=6).

 
3.5 Regulation of PAI-1 production by atRA and/or bFGF
To assess whether the observed increase represents up-regulation in PAI-1 production, specific ELISA was performed. Confluent cultures of C2/2 cells were serum starved for 24 h and then incubated with fresh serum-free medium containing atRA (0.32 µM) and bFGF (32 ng/ml) individually or together for 24 h. Culture medium was harvested and the PAI-1 production was assayed. As shown in Fig. 6A, PAI-1 production was significantly stimulated by bFGF alone. All-trans-RA alone had a small effect on PAI-1 production. Consistent with the effects observed at the level of PAI-1 mRNA, a combined increase in PAI-1 production was observed when atRA was applied in combination with bFGF.


Figure 6
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  		Fig. 6 Effects of atRA and/or bFGF on PAI-1 production in the culture medium of C2/2 cells and effects on C2/2 migration. (A) PAI-1 production in C2/2 cells. Confluent cells were serum starved for 24 h and then incubated with fresh serum-free medium containing atRA (0.32 µM) and/or bFGF (32 ng/ml) for 24 h. Culture medium was harvested and the protein of PAI-1 was assayed by specific ELISA. Standard curves were constructed from dilutions of purified PAI-1. Values are the mean±S.E.M. of three separate experiments. *P<0.05 compared with values in untreated control cells (n=6). **P<0.05 compared with values in atRA- and bFGF-treated cells (n=6). (B) Effects of atRA and/or bFGF on C2/2 migration. C2/2 cells were stimulated with 10 ng/ml PDGF-BB, and the effects of atRA (0.32 µM) and/or bFGF (32 ng/ml) on cell migration were determined by measuring the absorbance, reflecting the number of cells that had migrated through a membrane coated with BSA or collagen I at 570 nm as described in Methods. The data are expressed as percentage absorbance for the cells treated with the reagents indicated, compared with the absorbance for PDGF-BB-treated control cells and are the mean±S.E.M. of three separate experiments. *P<0.05 compared with values in PDGF-BB-stimulated control cells with the corresponding type of coated membrane (n=3). BSA+collagen I indicates the average value of the results obtained from the BSA and collagen I-coated membrane.

 
3.6 Effects of atRA and/or bFGF on C2/2 migration
To investigate the effect of atRA and/or bFGF on cell migration, we performed a migration assay. The number of C2/2 migrating across the membrane was dependent on the presence of a chemoattractant, PDGF-BB. As shown in Fig. 6B, treatment with atRA and/or bFGF reduced the cell migration elicited by PDGF-BB regardless of the membrane coating (percent inhibition in the average of the results using the BSA and collagen I-coated membrane: 74, 70 and 63% by atRA, bFGF and the combination, respectively).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In this study, we report the novel observation that atRA alone or in combination with bFGF stimulates the production of PAI-1 from vascular SMCs at both the protein and mRNA levels. Furthermore, atRA and bFGF significantly decreased SMC migration. Our data suggest that atRA and bFGF induced PAI-1 mRNA in a gene-specific manner, since these ligands did not alter tPA nor uPA mRNA levels. In addition, the induction of PAI-1 was inhibited by the tyrosine kinase inhibitor genistein, thus suggesting that the tyrosine kinase-dependent step is involved in the regulation of PAI-1 gene expression by atRA and bFGF. Although numerous studies have documented that tyrosine kinases play a crucial role in mediating the effect of bFGF [25], few reports have described the involvement of these kinases in RA-mediated gene expression. Thus, the findings of combined activation of the PAI-1 gene by atRA and bFGF offer a novel insight into the mechanisms of the genetic response of vascular SMC to two distinct vasoactive substances, atRA and bFGF.

There are several previous reports suggesting that tyrosine kinase is involved in the regulation of gene expression by atRA [26,27]. Shang et al. demonstrated that atRA exerts IFN-like actions by phosphorylating the signal transducer and activator of transcription 1 (STAT1) in breast carcinoma cell line MCF-7 [28]. Thus it is intriguing to speculate that atRA increases PAI-1 promoter activity through inducing phosphorylation of STAT1. However, this seems unlikely because our Western blot analysis revealed that atRA treatment did not induce phosphorylation of STAT1 (data not shown). Investigation of the tyrosine kinases responsible for the increased transcription by atRA is currently in progress.

The observed interaction between atRA and bFGF may be explained by the possible phosphorylation of RAR by bFGF-mediated signaling because phosphorylation of RAR has been documented to potentiate the transactivating function of RAR [29]. This assumption is supported by a previous report in which an estrogen receptor, phosphorylated by MAPK, results in activation of the estrogen receptor as a transcription factor [30]. Alternatively, it is also possible that atRA-mediated signaling increases the activity of the bFGF receptor. This mechanism is analogous to that in a recent study by Yen et al. [31]. They demonstrated that RA induces myeloid differentiation of HL-60 cells by activating the signal transduction cascade originating from c-FMS, a transmembrane tyrosine kinase receptor of the PDGF subfamily. In addition, we cannot rule out the possibility that atRA- and bFGF-mediated signalings interact with each other at post-receptor levels.

Results of the transient transfection assays and standard decay assays using actinomycin D indicate that such a combined effect between atRA and bFGF is mainly regulated at the transcriptional levels. Searching for the putative regulatory sequence mediating bFGF-induced PAI-1 promoter activity revealed that AP1- and SP1-binding sites are in the promoter region between –967 and –260. We observed that PAI-1 gene expression is induced by RAR{alpha} overexpression (unpublished data) as well as by atRA, suggesting that atRA-induced PAI-1 gene transcription is an RAR-mediated effect. Despite the report that RA-induced signaling is mainly mediated through the binding of RAR receptor to its cognate binding sequence in the promoter region [32], no canonical RARE is found in the RAR{alpha}-responsive region. These results suggest that RAR{alpha} exerts its role by modulating DNA-binding transcription factors through direct or indirect protein–protein interaction. Further studies will obviously be required to understand the precise mechanism responsible for the atRA-induced transcription of the PAI-1 gene.

The finding that bFGF inhibits the migration of C2/2 cells in our migration assay system was surprising because a number of studies demonstrated that bFGF stimulates SMC migration [33,34]. However, given that the activation of fibrinolytic enzymes is a prerequisite for SMC migration [3–5], our results would be reconciled by the fact that, in C2/2 cells, bFGF did not induce expression of the tPA and uPA genes, which may play a critical role in activating the metalloproteinase system and in degrading ECM. The ability of cells to express tPA and uPA in response to bFGF may be different among cellular types, because Miralles et al. have shown that the uPA gene is regulated by bFGF differently in myoblasts from myotubes [35]. Thus, our results suggest that the action of bFGF on migration largely depends upon the cellular properties which determine the pattern of fibrinolysis-relevant genes expressed in response to bFGF.

In summary, the present study illustrates that atRA and bFGF induce PAI-1 gene expression, the principal inhibitory factors of the fibrinolytic system, without affecting tPA or uPA expression in vascular SMCs. Furthermore, we demonstrate that tyrosine kinase(s) is involved in the induction of PAI-1 triggered by atRA and bFGF. Although the functional sequelae of the augmented expression of the PAI-1 gene by atRA and bFGF in vascular SMCs in relation to neointima formation appear to be quite complex, SMC migration is actually inhibited by treatment with atRA and bFGF in our system. The secretion of PAI-1 could have the effects of both down-regulating plasmin-mediated ECM degradation and down-regulating MMPs activation [36], and may contribute substantially to the decrease in vascular SMC migration. At the site of the neointima, a growth factor such as bFGF is produced abundantly by vascular SMCs [37] and induces PAI-1 production. All-trans-RA treatment may augment this anti-proteolytic state of vascular SMCs by increasing the synthesis and secretion of PAI-1, thereby further decreasing the migration rate. The fact that atRA interacts with bFGF may provide the basis for the pleiotropic effects of atRA and a rationale for the use of retinoids as therapeutic agents to induce PAI-1 in the vessel wall.

Time for primary review 30 days.


   Acknowledgements
 
This work was supported, in part, by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Japan Cardiovascular Foundation.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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