Basic Fibroblast Growth Factor Antagonizes Transforming Growth Factor-β1–Induced Smooth Muscle Gene Expression Through Extracellular Signal–Regulated Kinase 1/2 Signaling Pathway Activation
Objective— Transforming growth factor-β1 (TGFβ1) and fibroblast growth factor (FGF) families play a pivotal role during vascular development and in the pathogenesis of vascular disease. However, the interaction of intracellular signaling evoked by each of these growth factors is not well understood. The present study was undertaken to examine the molecular mechanisms that mediate the effects of TGFβ1 and basic FGF (bFGF) on smooth muscle cell (SMC) gene expression.
Methods and Results— TGFβ1 induction of SMC gene expression, including smooth muscle protein 22-α (SM22α) and smooth muscle α-actin, was examined in the pluripotent 10T1/2 cells. Marked increase in these mRNA levels by TGFβ1 was inhibited by c-Src-tyrosine kinase inhibitors and protein synthesis inhibitor cycloheximide. Functional studies with deletion and site-directed mutation analysis of the SM22α promoter demonstrated that TGFβ1 activated the SM22α promoter through a CC(A/T-rich)6GG (CArG) box, which serves as a serum response factor (SRF)–binding site. TGFβ1 increased SRF expression through an increase in transcription of the SRF gene. In the presence of bFGF, TGFβ1 induction of SMC marker gene expression was significantly attenuated. Transient transfection assays showed that bFGF significantly suppressed induction of the SM22α promoter–driven luciferase activity by TGFβ1, whereas bFGF had no effects on the TGFβ1-mediated increase in SRF expression and SRF:DNA binding activity. Mitogen-activated protein kinase kinase-1 (MEK1) inhibitor PD98059 abrogated the bFGF-mediated suppression of TGFβ1-induced SMC gene expression.
Conclusion— Our data suggest that bFGF-induced MEK/extracellular signal-regulated kinase signaling plays an antagonistic role in TGFβ1-induced SMC gene expression through suppression of the SRF function. These data indicate that opposing effects of bFGF and TGFβ1 on SMC gene expression control the phenotypic plasticity of SMCs.
- basic fibroblast growth factor
- transforming growth factor-β1
- serum response factor
- smooth muscle cells
Phenotypic modulation of smooth muscle cells (SMCs) contributes to development of atherosclerotic and restenotic lesions. There is considerable interest in identifying the various extracellular signals that regulate SMC phenotype and the molecular mechanisms underlying such SMC plasticity. Transforming growth factor-β1 (TGFβ1) is one of the primary differentiation factors for SMCs.1 TGFβ1 upregulates several SMC differentiation markers, such as smooth muscle α-actin (SMα-actin), smooth muscle myosin heavy chain, SM22α, and h1 calponin in vitro. Moreover, TGFβ1 induces expression of these SMC differentiation maker genes in a variety of nonsmooth muscle precursor cell types in culture, including multipotent embryonic fibroblast (10T1/2 cells) and neural crest cells.2,3 These results suggest that TGFβ1 evokes an important signal that induces SMC differentiation.
In contrast, basic fibroblast growth factor (bFGF) is one of the most important mitogenic growth factors for SMCs1 and plays an important role in the onset and development of vascular disease. Several studies indicated that experimental reduction in bFGF expression inhibits SMC proliferation after intimal injury. Lindner et al suggested that in injured arteries, bFGF and FGF receptor-type 1 may be involved in the continued proliferative response of SMCs leading to neointima formation.4 SMCs respond to bFGF stimulation with proliferation, migration, and cytokine secretion occurring after vascular lesion.5
The promoter of almost all examined SMC-specific genes contain CArG (CC[A/T]6GG) box,6,7 which serves as a binding site for serum response factor (SRF)8. An increasing number of studies provide direct evidence that the CArG boxes are required for promoter activation of SMC genes in vitro and in vivo.9–11 The smooth muscle calponin gene contains CArG box within the first intron, which mediates SMC-specific enhancer activity.12 However, the signaling pathways that regulate SRF expression and function have been poorly understood.
In the present study, we investigated the effects of TGFβ1 and bFGF on SMC gene expression in the pluripotent 10T1/2 cells and in vascular SMCs. The results demonstrated that TGFβ1 induces CArG-dependent SM22α gene expression via an increase in SRF expression, and Src family of tyrosine kinase is important for this effect. Furthermore, bFGF inhibits TGFβ1-induced SM22α gene expression via mitogen-activated protein kinase kinase-1 (MEK1) by repressing SRF function without affecting SRF expression. Thus, we suggest that TGFβ1 and bFGF act as antagonistic growth factors that regulate the CArG-dependent SMC marker gene expression by modulating SRF expression and function, respectively. These findings shed light on the role of TGFβ1 and bFGF for modulating SMC gene expression during development and in vascular disease, in which SMC phenotypic change plays a crucial role.
Materials and Methods
The Materials and Methods section can be found in an online supplement available at http://atvb.ahajournals.org.
TGFβ1 Induces Expression of SMC Marker Genes in 10T1/2 Cells
Northern blot analyses revealed that the levels of mRNAs for SM22α and SMα-actin were significantly induced by TGFβ1 in 10T1/2 cells. Embryonic type of smooth muscle myosine heavy chain (SMemb) and Krüppel-like zinc-finger transcription factor 5/basic transcription regulatory element binding protein 2 (KLF5/BTEB2), a transcription factor implicated in the regulation of SMemb and others, were also induced. Calponin mRNA was scarcely detected by Northern blot, but RT-PCR analysis revealed its significant induction by TGFβ1 in 10T1/2 cells. Analysis of temporal expression showed that SMα-actin and SM22α mRNA levels were induced by TGFβ1×2 hours and remained elevated for 12 hours after TGFβ1 stimulation. SRF mRNA levels were peaked at 6 hours after TGFβ1 stimulation (Figure IA through IC, available online at http://atvb.ahajournals.org).
As shown in Figure ID, cycloheximide (4 μg/mL, a nonspecific inhibitor for protein synthesis) alone modestly increased the expression of SM22α and SMα-actin mRNAs. TGFβ1-induced expression of SM22α and SMα-actin mRNAs was rather attenuated by cycloheximide. These results suggest that de novo protein synthesis is partly required for the TGFβ1-induced expression of SM22α and SMα-actin genes.
Src Family of Tyrosine Kinase Mediates TGFβ1-Induced Expression of SM22α and SMα-Actin Genes
As shown in Figure 1A, TGFβ1-induced SM22α mRNA expression in 10T1/2 cells was blocked by genistein (10 μmol/L; a tyrosine kinase inhibitor) but not by other protein kinase inhibitors, such as PD98059 (50 μmol/L; a specific inhibitor for MEK1, a mitogen-activated protein [MAP] kinase kinase for extracellular signal–regulated kinase [ERK]), SB203580 (10 μmol/L; a specific inhibitor for p38MAP kinase), calphostin C (1 μmol/L; protein kinase C inhibitor), and wortmannin (1 μmol/L; a PI3 kinase inhibitor). We then examined the effect of daidzein (10 μmol/L; an inactive analog of genistein) and other tyrosine kinase inhibitors, such as herbimycin A (1 μmol/L; a relatively specific inhibitor for Src tyrosine kinases), tyrphostin 23 (100 μmol/L; a broad inhibitor for tyrosine kinase), and protein phosphatase 1 (PP1; 4 μmol/L; a more specific inhibitor for Src tyrosine kinases). As shown in Figure 1B, tyrphostin 23 as well as genistein but not daidzein dramatically inhibited induction of SM22α by TGFβ1. Furthermore, herbimycin A and PP1 potently attenuated the TGFβ1-induced SM22α expression. To examine the effect of Src family of tyrosine kinase in cultured rat aortic SMCs (RASMCs), levels of SM22α and SMα-actin mRNA were measured by real-time RT-PCR in TGFβ1-treated RASMCs (Figure II, available online at http://atvb.ahajournals.org). TGFβ1-induced SM22α and SMα-actin mRNA expression in RASMCs was significantly blocked by herbimycin A. These results suggest that Src family of tyrosine kinase pathways mediate TGFβ1-induced SM22α gene expression.
CArG Box Is Required for TGFβ1-Mediated Increase in SM22α Promoter Activity
To determine the effects of TGFβ1 on SM22α promoter activity, a series of 5′-deletion constructs of SM22α promoter was transfected into 10T1/2 cells and cultured RASMCs (Figure IIIA and IIIB, available online at http://atvb.ahajournals.org). TGFβ1 induced promoter activity of −305Luc, −215Luc, and −158Luc, whereas −115Luc was unresponsive. Because the sequence between −158 and −115 contains 1 CArG box, (5′-CCAAATATGG-3′, located at −150), we then tested the role of CArG box in the induction of promoter activity in response to TGFβ1. We introduced nucleotide substitutions of 5 successive bases into the flanking or core sequence within the CArG box in the context of the −158Luc construct (Figure 2A and 2B). Luciferase activity in unstimulated cells transfected with −158m1Luc, −158m2Luc, and −158m3Luc was significantly lower than that of wild-type promoter construct −158Luc. More importantly, neither −158m2Luc nor −158m3Luc were responsive to TGFβ1. These results suggest that integrity of CArG box is essential for activation of the SM22α promoter by TGFβ1.
TGFβ1 Stimulates SRF Gene Expression at the Transcriptional Level Through Src Family of Tyrosine Kinase Pathway
To determine whether TGFβ1 increases SRF binding activity, we performed electrophoretic mobility shift assay (EMSA) by using the nuclear protein extracts from unstimulated or TGFβ1-stimulated 10T1/2 cells and the oligonucleotide-containing sequence between −158 and −133 of the SM22α promoter as probe. Shifted complex was increased in amounts in TGFβ1-treated 10T1/2 cells, and anti-SRF antibody completely supershifted the complex (Figure 3A and Figure IVA, available online at http://atvb.ahajournals.org). An increase in SRF binding by TGFβ1 was observed in the presence of PP1 because PP1 by itself increased SRF binding (Figure 3A). We next performed Northern blot analysis to test the effect of Src inhibition on TGFβ1-induced SRF mRNA expression in 10T1/2 cells. TGFβ1 induction of SRF was completely inhibited by PP1 or herbimycin, both of which are the Src family kinase inhibitors (Figure 3B). Western blot analysis showed the increase in SRF protein levels in response to TGFβ1 (Figure IVB). TGFβ1 effects on SRF promoter activity were evaluated by transfecting −2052SRF/Luc, a luciferase reporter gene containing SRF promoter region between −2052 and 114 into 10T1/2 cells and cultured RASMCs. As shown in Figure IVC, TGFβ1 stimulated luciferase activity of −2052SRF/Luc by 2.5-fold in 10T1/2 cells and by 1.7-fold in RASMCs. These results indicate that TGFβ1 stimulates SRF gene expression at least partly at the transcriptional level through Src family of tyrosine kinase pathway.
bFGF Inhibits TGFβ1-Induced SM22α and SMα-Actin Gene Via Activation of MEK1
To investigate the effects of mitogenic stimulation on TGFβ1-induced SM22α and SMα-actin gene expression, we used various growth factors, including platelet-derived growth factor (PDGF)-BB, vascular endothelial growth factor (VEGF), interleukin (IL)-6, epidermal growth factor (EGF) and bFGF in 10T1/2 cells (Figure VA, available online at http://atvb.ahajournals.org). Among these, bFGF markedly inhibited TGFβ1 induction of mRNA levels for SM22α, SMα-actin, and calponin. In contrast, dedifferentiated markers, such as SMemb and KLF5/BTEB2, were not affected (Figure 4A).
To investigate the signaling pathways involved in this repression, we tested the effects of inhibitors for protein kinases on TGFβ1-induced SM22α gene expression. As shown in Figure 4B, bFGF inhibited TGFβ1-induced SM22α expression by 55% and 17% in the absence and presence of PD98059 (MEK1 inhibitor), respectively, in 10T1/2 cells. In contrast, SB203580, genistein, and wortmannin had no effect on bFGF inhibition. To examine whether PD98059 inhibits bFGF effect in cultured RASMCs, levels of SM22α mRNA were measured by real-time RT-PCR in TGFβ1-treated or bFGF-treated RASMCs. As shown in supplemental Figure VB, bFGF did not attenuate TGFβ1-induced SM22α expression in the presence of PD98059 in RASMCs. These results indicated that bFGF represses TGFβ1-induced expression of the SM22α gene via activation of MEK1.
To examine whether bFGF affects TGFβ1-induced SM22α promoter activity, we performed luciferase assays. bFGF significantly attenuated TGFβ1 effects on the SM22α promoter, and PD98059 inhibited such an effect of bFGF (Figure VC).
To evaluate TGFβ1 or bFGF pathway for ERK1/2 activation, 10T1/2 cells were treated with TGFβ1 or bFGF. Figure 4C and Figure VD show ERK phosphorylation was detected at 5 minutes and remained 60 minutes after bFGF stimulation. Although ERK phosphorylation was detected 5 minutes after TGFβ1 stimulation, this phosphorylation significantly attenuated thereafter.
Next, we performed Western blot analyses to test whether activation of MEK1 pathways by bFGF inhibits TGFβ1-induced c-Src activation. As shown in Figure VI (available online at http://atvb.ahajournals.org), bFGF as well as TGFβ1 induced c-Src activation, and simultaneous stimulation with TGFβ1 and bFGF exerts the additive effect on c-Src phosphorylation. PD98059 by itself induced c-Src phosphorylation, and thus an induction of c-Src phosphorylation by bFGF was not blocked by PD98059. However, these data imply that the inhibitory effects of bFGF on TGFβ1-induced SM22α expression are not mediated through inhibition of c-Src phosphorylation.
MEK1 Inhibits SRF Function Independent of Expression and DNA Binding Activity of SRF
To determine whether MEK1 activation inhibits SM22α expression, 10T1/2 cells and cultured RASMCs were transfected with expression vector for MEK1 or empty vector pcDNA3 along with the SM22α-luciferase reporter gene. Overexpression of MEK1 reduced TGFβ1-stimulated luciferase activity of −305Luc (Figure 5 and Figure VIIA, available online at http://atvb.ahajournals.org). Furthermore, induction of the SM22α promoter activity by SRF was prevented by MEK1 overexpression in 10T1/2 cells (Figure 5).
The observation that bFGF inhibited the TGFβ1-induced SM22α promoter activity led us to test whether bFGF inhibits SRF expression. Interestingly, bFGF had no measurable effects on SRF expression as assessed by Northern blot and Western blot analyses. Furthermore, EMSA showed that bFGF did not decrease and rather increased the binding of SRF to the CArG box (Figure VIIB through VIID). Collectively, these results suggest that bFGF inhibits SM22α expression not through inhibition of the SRF protein synthesis or inhibition of DNA binding but possibly through repression of SRF function (Figure 6).
The current study shows TGFβ1 actions on SMC gene expression in 10T1/2 cells and RASMCs. TGFβ1-directed SMC gene expression is mediated through Src-tyrosine kinase activation and is associated with the increases in SRF gene expression at the transcriptional level. We also demonstrated that TGFβ1 induction on SMC gene expression is significantly inhibited by bFGF. ERK1/2 activation mediates this effect of bFGF on TGFβ1 induction of SMC gene expression; in fact, inducible expression of SM22α was attenuated in the presence of PD98059. In addition, phosphorylation of ERK1/2 by transfecting MEK1 expression plasmid inhibited TGFβ1 action on SM22α promoter. Furthermore, bFGF did not inhibit TGFβ1-induced SRF expression and DNA binding activity. Finally, when several growth factors were tested for their capacity to inhibit TGFβ1 action on SMC gene expression, we observed that PDGF-BB, VEGF, PGI2, IL-6, and EGF did not possess the potent capacity of inhibiting TGFβ1-driven SM22α expression in 10T1/2 cells. Thus, our data highlight the critical role of bFGF–MEK1 pathway in inhibition of TGFβ1-driven SMC differentiation.
It is worth stressing that TGFβ1 signaling was inhibited by Src family of tyrosine kinase inhibitor. This finding is somewhat surprising because it has been described that TGFβ1 decreased Src kinase activity or induced degradation of activated Src kinase.13 Very few precedent reports showed the activation of Src tyrosine kinases by TGFβ1. Su et al have shown that in bovine articular chondrocyte, TGFβ1-mediated induction of tissue inhibitor of metalloproteinases-3 expression was inhibited by Src tyrosine kinase inhibitor as well as by serine/threonine protein kinase inhibitor.14 We confirmed the activation of Src family of tyrosine kinase by Western blot analysis using the anti-c-Src antibody. Although the precise mechanisms remain to be determined, it is possible that TGFβ1 signaling varies depending on cell types.
Despite the fact that bFGF did not decrease but rather increased SRF mRNA and protein levels, bFGF significantly attenuated induction of TGFβ1-mediated SMC-specific genes. Then how can bFGF inhibit the effect of TGFβ1? We examined whether bFGF inhibits the binding of SRF to CArG box and found that bFGF had essentially no effect on the binding activity of SRF to the CArG box. These data led us to speculate that bFGF represses SRF function through: (1) post-transcriptional SRF modification; (2) an induction of repressors of SRF including SRFδ5,15 Id2,16 and SMART;17 (3) a repression of SRF coactivators such as ternary complex factor, p300/CBP, SRC-1, and myocardin;18,19 and (4) an alteration of chromatin structure regulated by histone acetylase or histone deacetylase activity.20,21 Studies to examine these possibilities are currently in progress.
Our finding that MEK1 inhibitor PD98059 attenuated bFGF-mediated repression of TGFβ1-induced SMα-actin and SM22α expression deserves further attention. Previous studies have provided ample evidence indicating that MEK1 activation leads to the increase in SRF-dependent transcription through activation of accessory factors that bind to the SRF–CArG box complex, including members of the Ets family of transcription factors, Elk-1, SAP-1a, b, SAP-2, and ERP-1.22 Thus, a critical question is how SRF function could selectively be upregulated or downregulated by MEK1. This issue appears to be analogous to the question regarding the differential effects of mitogenic stimulation on the expression of SMC-specific genes and c-fos gene, both of which contain functional CArG box.23 The most plausible explanation is that modulation of SRF function by MEK1 signaling is dependent on sequence flanking CArG box. However, our data indicate that PDGF and EGF, both of which are known to activate MEK1 signaling, are less potent than bFGF in inhibiting TGFβ1-induced SM22α expression in 10T1/2 cells. Thus, it is tempting to speculate that bFGF-specific events other than MEK1 activation are necessary to inhibit TGFβ1-induced SMC gene expression.
It has been shown that the nuclear factors involved in the TGFβ1 control element–mediated transcription belong to the zinc finger family of KLFs, such as GKLF and KLF5/BTEB2.24 MacLellan et al have described previously that TGFβ1-induced activation of skeletal α-actin promoter required cooperation of SRF, YY1, and transcriptional enhancer factor-1.25 However, our data suggest that GKLF, BTEB2/IKLF, or YY1 do not play a major role in repressing the SM22α promoter by bFGF. The reasons for such an assumption are: (1) mRNA for BTEB2/IKLF, which positively regulates SM22α promoter, was induced by bFGF; (2) mRNA for GKLF, which functions as a negative regulator of SM22α expression, was not changed by bFGF; and (3) anti-YY1 antibody did not affect the DNA:protein complex as assessed by EMSA (data not shown).
What is the in vivo relevance of our observation? It has been shown previously that in early, simple, and advanced atherosclerotic lesions, both TGFβ1 and bFGF were expressed in intimal SMCs. Although TGFβ1 acts as a bifunctional regulator for SMC differentiation depending on growth status and the presence of other growth factors, an increased expression of bFGF was associated consistently with SMC proliferation of the atheromatous lesions.26 However, the role of bFGF in SMC differentiation has been described poorly. Our data indicate that SMC or embryonic fibroblasts exposed simultaneously to TGFβ1 and bFGF express SMC marker genes significantly less than in response to TGFβ1 alone. This suggests that SMCs in atherosclerotic lesions that contain abundant bFGF are less differentiated than those expressing predominant TGFβ1. Given that undifferentiated SMCs that highly express the genes for proteases that degrade matrix proteins and bFGF may potentially be involved in plaque neovascularization, bFGF-driven events may contribute to formation of unstable plaques and to life-threatening complications of atheroma and provide new options for therapeutic intervention.
In summary, we demonstrated that 2 growth factors (TGFβ1 and bFGF), which have been shown independently to play critical roles in regulation of smooth muscle development, antagonistically affect SRF-dependent SMC gene expression. In addition, we demonstrate that bFGF-induced MEK1 signaling attenuates SRF function but not its DNA binding activity and expression. These findings provide novel insight into SRF function regulation during SMC differentiation, which is influenced profoundly by growth factors.
This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sport, and Culture of Japan and a grant from the Japan Cardiovascular Foundation. We thank Miki Yamazaki and Akemi Yoguchi for excellent technical help.
- Received December 26, 2003.
- Accepted May 24, 2004.
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