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Vascular Biology |
From the Departments of Medicine and Biological Science (H.D., T.I., M.Y., H.K., H.S., K.K.-K., T.T., T.M, E.O., M.A., M.K.) and Ophthalmology (H.A.), Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan; the Institute for Genetic Medicine (L.K.), Department of Biochemistry and Molecular Biology, and Department of Medicine, Keck School of Medicine of the University of Southern California, Los Angeles, Calif.
Correspondence to Masahiko Kurabayashi, MD, PhD, Department of Medicine and Biological Science, Gunma University Graduate School of Medicine 3-39-15 Showa-machi, Maebashi, Gunma, 371-8511, Japan. E-mail mkuraba{at}med.gunma-u.ac.jp
| Abstract |
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Methods and Results Immunohistochemistry revealed that HERP1 and myocardin expression was localized to SMC in the neointima of balloon-injured rat aorta and in human coronary atherosclerotic lesions. Expression of both HERP1 and myocardin was elevated in cultured VSMCs compared with medial SMC. Overexpressed HERP1 inhibited the myocardin-induced SMC marker gene expression in 10T1/2 cells. HERP1 protein interfered with the SRF/CArGbox interaction in vivo and in vitro. Immunoprecipitation assays showed that HERP1 physically interacts with SRF.
Conclusions HERP1 expression was associated with the SMC proliferation and dedifferentiation in vitro and in vivo. HERP1 may play a role in promoting the phenotypic modulation of VSMCs during vascular injury and atherosclerotic process by interfering with SRF binding to CArG-box through physical association between HERP1 and SRF.
Myocardin is a potent SRF coactivator for VSMC differentiation. HERP1, a target gene of Notch, is a transcriptional repressor in vascular system. Both factors are coinduced in synthetic VSMCs. HERP1 inhibits myocardin-dependent SMC differentiation by preventing SRF from DNA binding through physical association with SRF.
Key Words: HERP1 myocardin serum response factor smooth muscle cells
| Introduction |
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, caldesmon, and calponin. Because the genes encoding these proteins are differentially expressed depending on the proliferative state of VSMCs, transcription factors regulated by numerous stimuli are responsible at least in part for the distinct pattern of gene expression seen in synthetic VSMCs.
There is mounting evidence that most SMC marker proteins such as SM-MHC and SM22
are controlled by serum response factor (SRF), which binds to a sequence known as a CArG box and recruits a potent coactivator, myocardin, for SMC differentiation.1 When myocardin is ectopically expressed in nonmuscle cells, it can induce SMC differentiation.2,3 Most importantly, mouse embryos deficient for myocardin show no evidence of vascular SMC, indicating myocardin as a necessary and sufficient factor for SMC differentiation in vivo.4 These observations, in conjunction with downregulation of SMC marker genes in synthetic VSMC, led us to speculate that myocardin expression might be downregulated in synthetic VSMCs. As yet, however, it is not clarified whether reduced expression of SMC marker genes in synthetic VSMC results from downregulation of myocardin expression.
Although Notch signaling is required during angiogenesis and in vascular homeostasis, the mechanisms by which Notch regulates vascular function remain to be elucidated. In vertebrates, receptors, ligands, and other components of Notch signaling are expressed in vasculature.5 Mutations of genes involved in Notch pathway in mice lead to abnormalities in many tissues including the vascular system.5 Human diseases such as Alagille syndrome and CADASIL, which show abnormalities in the cardiovascular system, are caused by mutations of the Notch ligand Jagged-1 and the receptor Notch-3, respectively.5 Such findings clearly demonstrate a crucial role of the Notch pathway in vascular development and homeostasis. Recently, several studies have reported with some controversial findings that expression level of several Notch components was significantly affected after vascular injury, suggesting that Notch pathway also plays a role in the pathogenesis of vascular diseases.6,7
We and others have recently identified HERP family (for HES-related repressor protein, also referred to as Hesr, Hey, HRT, CHF, and gridlock) that is predominantly expressed in cardiovascular system.813 Some of members of HERP family are proved to be direct downstream targets of Notch and acts as transcriptional repressor.1315 Among them, HERP1 and HERP2 play a crucial role in vascular development in vivo because double knockout of the HERP1 and HERP2 genes in mice resulted in embryonic death with a global lack of vascular remodeling.16 Mutant singly deficient for gridlock, HERP1 homologue of zebrafish, also showed disturbance of assembly of the aorta.12 Of particular note, Notch signaling including multiple target genes generally functions as negative regulator of differentiation in various cells.17 These findings, along with induction of Notch components in injured VSMCs, or synthetic VSMCs, strongly suggest that Notch target genes, HERP1 and HERP2, are also induced in synthetic VSMCs and play a critical role for development of vascular disease as negative regulator of VSMC differentiation.
The present study describes a series of experiments that have explored the role of HERP1 in the phenotypic modulation of VSMC. Our in vitro analyses along with the immunohistochemical study showed that HERP1 plays an important role in modulating VSMC phenotypes, and this was caused by the ability of HERP1 to interfere with SRF binding to CArG box by physically associating with SRF. We propose that Notch-HERP pathway is one of the complex stimuli to modulate VSMC phenotypes, and that the stage of VSMC differentiation is determined by positive regulator myocardin and negative regulator HERP1.
| Materials and Methods |
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| Results |
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Induction of HERP1 and Myocardin Expression in Neointima After Balloon Injury
We examined whether expression of HERPs and myocardin are affected in neointima after balloon injury. Immunohistochemistry of 14-day balloon-injured rat aortas and control vessels revealed that whereas only a few cells were positive for HERP1 in medial layer in sham-operated aorta, HERP1 staining was colocalized with SM
-actinpositive cells in neointima (Figure 2A). HERP1-positive cells also present in the thin layer of media adjacent to adventitia. Because many ligands and receptors for Notch are induced in neointima after vascular injury,7 strong staining for HERP1 in neointima implicates that HERP1 is induced as a downstream target gene of Notch. In contrast to HERP1, HERP2 was barely stained in aorta from both sham-operated and balloon-injured rats, suggesting cell type-specific expression of HERP family members (data not shown). Unexpectedly, myocardin, a positive regulator for VSMC differentiation, was clearly detected in the neointima, which is characterized by synthetic phenotype of VSMC. These observations allowed us to speculate that function of myocardin is antagonized by certain factor(s) such as HERP1, a target gene of Notch, which functions in most cases as negative regulator for differentiation.
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Expression of HERP1 and Myocardin in Human Coronary Atherosclerotic Lesions
To examine the expression of HERP1 and myocardin in human atherosclerotic lesions, we next double-stained human coronary atherectomy tissues. We confirmed that the tissues contained SMC and endothelial cells, revealed by SM
-actin and von Willebrand factor expression, respectively (data not shown). As shown in Figure 2B, HERP1-positive cells almost colocalized with cells stained positive for SM
-actin and myocardin. These findings suggest that both HERP1 and myocardin are coexpressed in VSMCs, and play a role in the development of vascular disease.
Myocardin and HERP1 Transcripts Are Induced in Cultured Rat Aortic SMC
To determine the expression of the myocardin and HERP1 genes in vitro, we performed Northern blot analysis using total RNA from rat aorta, media of aorta, and cultured rat aortic SMC (RASMC). As shown in Figure 3, HERP1 gene transcripts were significantly increased in cultured RASMC when compared with that in aorta and in media of aorta, which also supports the notion that HERP1 may negatively regulate VSMC differentiation. HERP2 induction was not detected (data not shown). Of particular note, gene transcripts of myocardin were also increased in cultured RASMC. These data led us to postulate that stage of VSMC differentiation may be determined by balance of expression levels between myocardin and certain negative regulator(s) including HERP1.
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HERP1 Inhibits Induction of Myocardin-Dependent SMC Marker Genes
To examine whether HERP1 affects myocardin-induced VSMC differentiation, we compared expression of several SMC marker genes such as SM-MHC and SM22
in 10T1/2. As reported,2,3 myocardin strongly induced smooth muscle markers, SM-MHC and SM22
(Figure 4A, lane 3). When HERP1 was simultaneously expressed, however, induction of these markers by myocardin was dramatically decreased (Figure 4A, lane 4). Importantly, HERP1 did not affect the mRNA levels of SRF (Figure 4A) and protein expression of myocardin (Figure 4B). Immunostaining further revealed expression of myocardin in HERP1-positive cells (data not shown). These observations strongly suggest that HERP1 inhibits myocardin-dependent SMC differentiation by abrogating function of myocardin protein, not by myocardin expression. We also observed similar results using cultured RASMC. Additional HERP1 introduced by adenovirus markedly repressed expression of SM-MHC without affecting expression of SRF and myocardin (Figure 4C). Of interest, expression of SM22
, an early marker of SMC differentiation, was marginally altered, suggesting that the stage of SMC differentiation is determined by relative abundance between HERP1 and myocardin.
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HERP1 Suppresses Myocardin-Dependent Transactivation of SM-MHC and SM22
Promoter
To test whether HERP1 is able to repress myocardin-dependent transactivation of smooth muscle marker genes, we next performed luciferase reporter gene assays with SM-MHC and SM22
promoters (Figure 5). Overexpressed myocardin strongly transactivated the promoters. However, when HERP1 was expressed at the same time, this induction was dramatically reduced in a dose-dependent manner. Overexpression of HERP1 marginally affected basal transcription of SM-MHC and SM22
promoters, suggesting that inhibition of myocardin-dependent transactivation by HERP1 was not through binding to promoter DNA. We next studied whether other HESHERP family members also possess the same function as HERP1 does. We observed essentially the same results when HERP2 and HES1 were used. These findings suggest that any HESHERP member may be able to inhibit myocardin-dependent gene expression in a similar fashion in various cells where one of the HESHERP members and myocardin are simultaneously expressed.
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HERP1 Interferes With SRF Binding to CArG Box Through Physical Association With SRF
It has been reported that myocardin, SRF, and DNA probe containing CArG box form a ternary complex in electrophoretic mobility shift assay (EMSA).19 Our data described (Figures 4 and 5
), along with the ability of myocardin to form the ternary complex, raise the question of whether HERP1 disrupts the ternary complex to inhibit myocardin-dependent SMC differentiation. To address this possibility, we performed EMSA with in vitro-translated SRF, HAmyocardin, and HERP1 proteins. As shown in Figure 6A, SRF alone showed a strong band (lane 2), which was supershifted by anti-SRF antibody (lane 5). Of interest, intensity of SRF-specific band was significantly reduced by additional HERP1 in a dose-dependent manner (lanes 3 and 4), which suggests that HERP1 disrupts interaction between SRF and the DNA probe. Because HERP1 per se did not show any specific band with the probe (lane 1), HERP1 seems to disrupt the interaction by associating with SRF directly rather than competing with SRF to bind the CArG box on the probe. When SRF and myocardin were simultaneously incubated, a new band appeared (Figure I lane 1, available online at http://atvb.ahajornals.org). This new band seems to be ternary complex band of SRF-myocardin-DNA probe because it was abolished by additional HA antibody (lane 3), but not by normal IgG antibody (lane 4). Most importantly, this band also disappeared by coincubation of HERP1 protein (lane 2), suggesting that HERP1 disrupts the formation of the ternary complex.
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To determine whether HERP1 disrupts interaction between SRF and the CArG elements in vivo, we performed chromatin immunoprecipitation assays. Results showed that HERP1 overexpression in cultured RASMCs caused marked reductions in SRF binding to CArG-containing regions of the SM-MHC promoter within intact chromatin (Figure 6B, lanes 5 and 6). To more rigorously test for physical interactions between HERP1 and SRF or myocardin, we further studied in vivo interaction between HERP1 and SRF by immunoprecipitation assay. When both FLAG-HERP1 and SRF were simultaneously expressed in cells, strong interaction was observed (Figure 6C, lane 2). The association was specific because it was not observed when FLAG-HERP1 was absent (lane 1) or when control IgG was used for immunoprecipitation (lane 3). GST-pulldown experiment confirmed the direct interaction between HERP1 and SRF in vitro (Figure IV, lane 3; available online at http://atvb.ahajornals.org). We next examined whether HERP1 also represses SRF binding to myocardin. Coimmunoprecipitation assays with myocardin, SRF, and HERP1 expression vectors showed that HERP1 did not affect the binding between SRF and myocardin (Figure II, available online at http://atvb.ahajornals.org). Our data suggest that HERP1 is likely to inhibit myocardin-dependent SMC differentiation by physical association with SRF, followed by interfering with SRF binding to CArG box.
The Basic Helix-Loop-Helix Domain of HERP1 Is Required for Physical Interaction With SRF and Repression of Myocardin-Induced SMC Gene Transcription
To determine the domains of HERP1 that mediate the interaction with SRF and repress myocardin-induced SMC gene expression, we first performed GST-pulldown assay using various truncated mutants of mouse GSTHERP1 fusion proteins. Among 3 mutants, only the basic helix-loop-helix domain of HERP1 as well as full-length HERP1 directly associated with SRF (Figure IV). Next, we performed luciferase assay using various HERP1 truncated mutants. To allow all the truncated mutants of HERP1 to translocate into nuclei, we used GAL-fusion proteins. As expected, the basic helix-loop-helix domain suppressed myocardin-dependent SMC gene transactivation to the same degree as full-length HERP1 did. However, the OCY region in HERP1 also suppressed myocardin-dependent SMC gene transactivation (Figure V, available online at http://atvb.ahajornals.org). Our data suggest that HERP1 represses SRFmyocardin-dependent SMC differentiation through physical interaction between basic helix-loop-helix domain of HERP1 and SRF, and some mechanisms other than physical interaction may be involved in OCY region-mediated repression of SRF function.
| Discussion |
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in 10T1/2 cells. This was because of the ability of HERP1 to interact with SRF, thereby interfering with SRFCArG complex formation. The observation that HERP1 accumulated in the cells of neointima of the injured artery is consistent with the previous study that CHF1/HERP1-null mice showed decreased neointima formation after wire injury and that proliferative and migratory activity of VSMC lacking CHF1 was decreased.20 Another study reported that HRT1/HERP2 facilitates VSMC growth through suppression of the cyclin kinase inhibitor p21 and promotes cell survival by increased expression of the anti-apoptotic kinase Akt in stable transformant of HRT1-overexpressed cells.21 Because biochemical characteristics of HERP2 are very similar to that of HERP1,14,15 HERP1 may be involved in cell growth and anti-apoptosis through those mechanisms during neointima formation.
It has been generally accepted that transition from contractile phenotype to synthetic phenotype is associated with upregulation of growth-promoting factors such as egr-1, Id, and c-jun, which are directly or indirectly inhibit the function of differentiation factors.1 Contrary to this concept, it is noteworthy that expression of myocardin, a differentiation-promoting factor, is induced in primary cultured RASMC, in neointima, and in atherosclerotic lesions, all of which are characterized by synthetic VSMC. It is intriguing to speculate that HERP1 may play a role in inhibiting the function of abundant myocardin, which allows the VSMC to proliferate. This assumption is currently undergoing investigation.
Recent studies revealed that coactivator function of myocardin is attenuated or abolished by several molecules by different mechanisms.2226 In the present study, we clearly showed that HERP1 inhibited myocardin-induced transactivation of SMC-marker genes by physical interaction with SRF, then interfering with SRFCArG box binding. There are several precedent reports that demonstrate the inhibition of SMC marker gene expression by interference with SRFCArG interaction. HOP, an unusual homeodomain protein, bound to MADS box of SRF and weakly repressed SRF-dependent transcription by inhibiting SRFDNA binding.22,27 KLF4, Krüppel-like transcription factor, repressed the expression of SMC marker genes by both downregulating myocardin expression and preventing SRF from associating with SMC gene promoters.24 Although the authors did not find the effect of KLF4 on SRFDNA binding in EMSA, they observed that overexpression of KLF4 was associated with reduction in SRF binding to CArG containing regions of SM
-actin promoter. Because MADS box is responsible for DNA binding,28 both HOP and KLF4, as well as HERP1, are likely to inhibit SRFDNA interaction through the interface of DNA binding, or MADS box. In contrast to our results, Proweller et al have recently shown that HERP1 inhibits the ability of myocardin to stimulate SRF-mediated SMC gene expression independent of the inhibition of SRFCArG interaction.25 Despite the very similar approach to detecting the interference with SRFCArG interaction including the in vitro-translated proteins, they did not find the inhibitory effects of HERP1 on SRF-CArG interaction in EMSA and chromatin immunoprecipitation assay. The precise reasons for such discrepant results deserve further experiments.
What are the upstream molecules of HERP1 induction in neointima? It is most likely that Notch signaling is the one because of following reasons: (1) HERP1 is a direct target gene of Notch in A10 cells derived from aortic SMC;29 (2) many ligands and receptors for Notch are strongly induced in injured SMC;7 and (3) Notch1-null mutant mice showed vascular remodeling defect with remarkable reduction in expression of both HERP1 and HERP2 in vascular system.16 However, HERP1 induction observed in cultured RASMCs was Notch-independent (data not shown). Recent studies revealed that in several cell lines, HERP/HES expression was also induced by other factors such as transforming growth factor (TGF)-ß super family and transcription factor c-Jun.3032 Because TGF-ß levels and c-fos expression were increased in balloon injury model,33,34 HERP1 induction in neointima may be caused by those factors. Interestingly, several members of TGF-ß super family have been reported to cross-talk with Notch signaling and amplify Notch stimulation.30,31 Given that both Notch and TGF-ß seem to be active in neointima, they may synergistically elevate HERP1 expression in neointima. Further studies will be needed to elucidate those signaling upstream of HERP1 induction in neointima.
In summary, we demonstrated that 2 transcription factors, HERP1 and myocardin, which have been shown to independently play critical roles in cardiovascular development, antagonistically affect SRF-dependent SMC gene expression. In addition, we presented that both factors are coinduced in synthetic VSMCs. These findings provide novel insight into the molecular mechanisms of phenotypic modulation of VSMCs that are closely associated with vascular disease and vascular development.
| Acknowledgments |
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Received January 31, 2005; accepted June 12, 2005.
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