This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 25/11/2328    most recent 01.ATV.0000185829.47163.32v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Doi, H. Articles by Kurabayashi, M. Search for Related Content PubMed PubMed Citation Articles by Doi, H. Articles by Kurabayashi, M.

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:2328.)
© 2005 American Heart Association, Inc.

Vascular Biology

HERP1 Inhibits Myocardin-Induced Vascular Smooth Muscle Cell Differentiation by Interfering With SRF Binding to CArG Box

Hiroshi Doi; Tatsuya Iso; Miki Yamazaki; Hideo Akiyama; Hiroyoshi Kanai; Hiroko Sato; Keiko Kawai-Kowase; Toru Tanaka; Toshitaka Maeno; Ei-ichi Okamoto; Masashi Arai; Larry Kedes; Masahiko Kurabayashi

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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Objective— Myocardin is a coactivator of serum response factor (SRF) required for vascular smooth muscle cell (VSMC) differentiation. HERP1 is a transcriptional repressor, which is abundantly expressed in vascular system and is known to function as a target gene of Notch. However, the role of HERP1 in the pathogenesis of vascular lesions remains unknown. The present study characterizes the expression of HERP1 in normal and diseased vessels, and tests the hypothesis that HERP1 inhibits SRF/myocardin-dependent SMC gene expression.

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/CArG–box 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Phenotypic modulation of vascular smooth muscle cells (VSMCs) from contractile to synthetic forms plays a pivotal role in the pathogenesis of vascular diseases including atherosclerosis and restenosis after angioplasty.¹ It is well-established that VSMC phenotype is regulated by a complex array of local environmental cues including humoral factors, cell-cell and cell-matrix interactions, inflammatory stimuli, and mechanical stresses. Such complex stimuli downregulate a number of genes required for the contractile phenotype in synthetic VSMCs. These include smooth muscle myosin heavy chain (SM-MHC), SM22, 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.^8–13 Some of members of HERP family are proved to be direct downstream targets of Notch and acts as transcriptional repressor.^13–15 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
The Materials and Methods section can be found in an online supplement available at http://atvb.ahajournals.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Differential Expression Pattern of HERP1, HERP2, and Myocardin in Adult Tissues
Although HERP1 and HERP2 are abundantly expressed in developing cardiovascular system, their expression in adult tissue has not been clearly illustrated. Northern blot analysis showed that human HERP1 was abundantly expressed in heart and less strongly expressed in skeletal muscle, whereas human HERP2 was easily detected in other tissues, as well as in heart (Figure 1A). In the cardiovascular system, both HERP1 and HERP2 transcripts were abundantly expressed in aorta as well as heart (Figure 1B). In adult rats, HERP1 mRNA was expressed in several organs including heart, but most strongly in aorta. HERP2 expression in lung was much higher than that in other tissues such as heart and aorta (Figure 1C). Expression of myocardin was also tested in rat multiple organs (Figure 1C). Myocardin was markedly expressed in heart and gastrointestinal tract, but only weakly in aorta, which is in contrast to previous study reporting that in humans, myocardin expression was much stronger in aorta than in heart.¹⁸ These findings suggest that expression of HERP1, HERP2, and myocardin is differentially regulated in adult tissues.

View larger version (77K): [in this window] [in a new window]   Figure 1. Tissue distribution of HERP1, HERP2, and myocardin. Human adult multiple-tissue blot (A) and cardiovascular blot (B) were purchased form Clontech for Northern blot analysis (poly(A)+ RNA, 2 µg/lane). Positions of RNA size markers are shown on the right in kb. C, Total RNA was isolated from indicated organs of adult Wister rats and subjected to Northern blot analysis (total RNA, 15 µg/lane).

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-actin–positive 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.

View larger version (57K): [in this window] [in a new window]   Figure 2. HERP1 and myocardin are coexpressed in neointima after vascular injury and in -actin–positive cells of human coronary atherectomy tissues. A, Immunohistochemistry of tissues from sham-operated rat aorta (upper) and balloon-injured aorta (lower). Two weeks after operation, aortas were harvested and stained with hematoxylin-eosin (HE), or indicated antibodies. Note strong induction of HERP1 and myocardin in neointima after vascular injury. Original magnification x400. Scale bar=20 µm. B, Human coronary atherectomy tissues were double-stained with several combinations of indicated antibodies. Merged images are on the right. Yellow in the merged images indicates overlapping area. Original magnification x100. Scale bar=200 µm.

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.

View larger version (43K): [in this window] [in a new window]   Figure 3. Both HERP1 and myocardin transcripts are induced in cultured RASMC. Total RNA was isolated from rat aorta, media of aorta, or cultured RASMCs, and analyzed by Northern blot analysis for HERP1 and myocardin mRNAs. Media (noncultured RASMCs) was prepared from aortic tissue from several rats after removing intimal and adventitial layers by enzymatic digestion and mechanical sweeping with cotton swab. The number of passage times for cultured RASMCs is indicated.

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.

View larger version (22K): [in this window] [in a new window]   Figure 4. HERP1 inhibits induction of myocardin-dependent SMC marker genes. A, 10T1/2 cells were transfected with the indicated expression vectors. Three days after transfection, total RNA was isolated and gene expression was assayed by reverse-transcription polymerase chain reaction. GAPDH was measured as an internal control. B, 10T1/2 cells were transfected with the indicated expression vectors. Three days after transfection, total cellular lysates were prepared for Western blotting with either anti-FLAG or anti-HA antibodies. C, Cultured RASMC were infected with Ad-empty or Ad-HERP1 at day 0 and day 5 at a multiplicity of infection of 20. Ten days after infection, total RNA was extracted for reverse-transcription polymerase chain reaction.

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 HES–HERP 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 HES–HERP member may be able to inhibit myocardin-dependent gene expression in a similar fashion in various cells where one of the HES–HERP members and myocardin are simultaneously expressed.

View larger version (22K): [in this window] [in a new window]   Figure 5. HERP1 inhibits transactivation of SMC marker genes by myocardin. 10T1/2 cells were transiently transfected with luciferase reporter plasmids for SM-MHC or SM22 with indicated expression vectors. Three days after transfection, cells were lysed and assayed for luciferase activity. *P<0.05 relative to myocardin alone. Values represent the mean±SD.

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, HA–myocardin, 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.

View larger version (21K): [in this window] [in a new window]   Figure 6. HERP1 interferes with SRF binding to CArG box through physical association with SRF. A, EMSA was performed with in vitro-translated proteins for SRF and HERP1 and a radiolabeled probe containing the c-fos CArG element (upper). Loading amount of SRF was confirmed by Western blot analysis (lower). B, RASMC was infected with Ad-empty or Ad-HERP1. Four days later, cells were formaldehyde cross-linked, and sonicated chromatin was immunoprecipitated with anti-SRF antibody or normal IgG. Recovered DNA was subjected to polymerase chain reaction using primers encompassing the 5' CArG-box element within the SM–MHC promoter. C, Coimmunoprecipitation assay for HERP1 and SRF. 293T cells were transfected with combination of expression vectors for FLAG-HERP1 and/or SRF. Three days after transfection, immunoprecipitation was performed using normal mouse IgG or anti-FLAG antibody. Bound proteins were separated by SDS-PAGE, followed by Western blot analysis with anti-SRF antibody. Protein level from each expression vector in crude extracts was confirmed by Western blot analysis with anti-FLAG or anti-SRF antibodies.

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 GST–HERP1 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 SRF–myocardin-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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our results indicate that HERP1 and myocardin are colocalized in cultured VSMC, as well as in dedifferentiated VSMC in neointima and human coronary atherosclerotic lesions. These in vivo findings were extended to in vitro experiments whereby exogenous HERP1 markedly and specifically suppressed the myocardin-induced expression of SMC marker genes, such as SM-MHC and SM22 in 10T1/2 cells. This was because of the ability of HERP1 to interact with SRF, thereby interfering with SRF–CArG 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.^22–26 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 SRF–CArG box binding. There are several precedent reports that demonstrate the inhibition of SMC marker gene expression by interference with SRF–CArG interaction. HOP, an unusual homeodomain protein, bound to MADS box of SRF and weakly repressed SRF-dependent transcription by inhibiting SRF–DNA 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 SRF–DNA 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 SRF–DNA 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 SRF–CArG interaction.²⁵ Despite the very similar approach to detecting the interference with SRF–CArG 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.^30–32 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

 
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 (M.K.) and Japan Heart Foundation Grant for Research on Atherosclerosis Update (T.I.). We thank Yoshiko Nonaka for excellent technical help. We are grateful to T. Ueyama, S. Izumo, I. Manabe, and R. Nagai for critical reagents.

Received January 31, 2005; accepted June 12, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease Physiol Rev. 2004; 84: 767–801.[Abstract/Free Full Text]

2. Yoshida T, Sinha S, Dandre F, Wamhoff BR, Hoofnagle MH, Kremer BE, Wang DZ, Olson EN, Owens GK. Myocardin is a key regulator of CArG-dependent transcription of multiple smooth muscle marker genes. Circ Res. 2003; 92: 856–864.[Abstract/Free Full Text]

3. Wang Z, Wang DZ, Pipes GC, Olson EN. Myocardin is a master regulator of smooth muscle gene expression. Proc Natl Acad Sci U S A. 2003; 100: 7129–7134.[Abstract/Free Full Text]

4. Li S, Wang DZ, Wang Z, Richardson JA, Olson EN. The serum response factor coactivator myocardin is required for vascular smooth muscle development. Proc Natl Acad Sci U S A. 2003; 100: 9366–9370.[Abstract/Free Full Text]

5. Iso T, Hamamori Y, Kedes L. Notch signaling in vascular development. Arterioscler Thromb Vasc Biol. 2003; 23: 543–553.[Abstract/Free Full Text]

6. Wang W, Campos AH, Prince CZ, Mou Y, Pollman MJ. Coordinate Notch3-hairy-related transcription factor pathway regulation in response to arterial injury. Mediator role of platelet-derived growth factor and ERK. J Biol Chem. 2002; 277: 23165–23171.[Abstract/Free Full Text]

7. Lindner V, Booth C, Prudovsky I, Small D, Maciag T, Liaw L. Members of the Jagged/Notch gene families are expressed in injured arteries and regulate cell phenotype via alterations in cell matrix and cell-cell interaction. Am J Pathol. 2001; 159: 875–883.[Abstract/Free Full Text]

8. Kokubo H, Lun Y, Johnson RL. Identification and expression of a novel family of bHLH cDNAs related to Drosophila hairy and enhancer of split. Biochem Biophys Res Commun. 1999; 260: 459–465.[CrossRef][Medline] [Order article via Infotrieve]

9. Leimeister C, Externbrink A, Klamt B, Gessler M. Hey genes: a novel subfamily of hairy- and Enhancer of split related genes specifically expressed during mouse embryogenesis. Mech Dev. 1999; 85: 173–177.[CrossRef][Medline] [Order article via Infotrieve]

10. Nakagawa O, Nakagawa M, Richardson JA, Olson EN, Srivastava D. HRT1, HRT2, and HRT3: a new subclass of bHLH transcription factors marking specific cardiac, somitic, and pharyngeal arch segments. Dev Biol. 1999; 216: 72–84.[CrossRef][Medline] [Order article via Infotrieve]

11. Chin MT, Maemura K, Fukumoto S, Jain MK, Layne MD, Watanabe M, Hsieh CM, Lee ME. Cardiovascular basic helix loop helix factor 1, a novel transcriptional repressor expressed preferentially in the developing and adult cardiovascular system. J Biol Chem. 2000; 275: 6381–6387.[Abstract/Free Full Text]

12. Zhong TP, Rosenberg M, Mohideen MA, Weinstein B, Fishman MC. gridlock, an HLH gene required for assembly of the aorta in zebrafish. Science. 2000; 287: 1820–1824.[Abstract/Free Full Text]

13. Iso T, Sartorelli V, Chung G, Shichinohe T, Kedes L, Hamamori Y. HERP, a new primary target of Notch regulated by ligand binding. Mol Cell Biol. 2001; 21: 6071–6079.[Abstract/Free Full Text]

14. Nakagawa O, McFadden DG, Nakagawa M, Yanagisawa H, Hu T, Srivastava D, Olson EN. Members of the HRT family of basic helix-loop-helix proteins act as transcriptional repressors downstream of Notch signaling. Proc Natl Acad Sci U S A. 2000; 97: 13655–13660.[Abstract/Free Full Text]

15. Iso T, Sartorelli V, Poizat C, Iezzi S, Wu HY, Chung G, Kedes L, Hamamori Y. HERP, a novel heterodimer partner of HES/E(spl) in Notch signaling. Mol Cell Biol. 2001; 21: 6080–6089.[Abstract/Free Full Text]

16. Fischer A, Schumacher N, Maier M, Sendtner M, Gessler M. The Notch target genes Hey1 and Hey2 are required for embryonic vascular development. Genes Dev. 2004; 18: 901–911.[Abstract/Free Full Text]

17. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in development. Science. 1999; 284: 770–776.[Abstract/Free Full Text]

18. Du KL, Ip HS, Li J, Chen M, Dandre F, Yu W, Lu MM, Owens GK, Parmacek MS. Myocardin is a critical serum response factor cofactor in the transcriptional program regulating smooth muscle cell differentiation. Mol Cell Biol. 2003; 23: 2425–2437.[Abstract/Free Full Text]

19. Wang D, Chang PS, Wang Z, Sutherland L, Richardson JA, Small E, Krieg PA, Olson EN. Activation of cardiac gene expression by myocardin, a transcriptional cofactor for serum response factor. Cell. 2001; 105: 851–862.[CrossRef][Medline] [Order article via Infotrieve]

20. Sakata Y, Xiang F, Chen Z, Kiriyama Y, Kamei CN, Simon DI, Chin MT. Transcription factor CHF1/Hey2 regulates neointimal formation in vivo and vascular smooth muscle proliferation and migration in vitro. Arterioscler Thromb Vasc Biol. 2004; 24: 2069–2074.[Abstract/Free Full Text]

21. Wang W, Prince CZ, Hu X, Pollman MJ. HRT1 modulates vascular smooth muscle cell proliferation and apoptosis. Biochem Biophys Res Commun. 2003; 308: 596–601.[CrossRef][Medline] [Order article via Infotrieve]

22. Shin CH, Liu ZP, Passier R, Zhang CL, Wang DZ, Harris TM, Yamagishi H, Richardson JA, Childs G, Olson EN. Modulation of cardiac growth and development by HOP, an unusual homeodomain protein. Cell. 2002; 110: 725–735.[CrossRef][Medline] [Order article via Infotrieve]

23. Wang Z, Wang DZ, Hockemeyer D, McAnally J, Nordheim A, Olson EN. Myocardin and ternary complex factors compete for SRF to control smooth muscle gene expression. Nature. 2004; 428: 185–189.[CrossRef][Medline] [Order article via Infotrieve]

24. Liu Y, Sinha S, McDonald OG, Shang Y, Hoofnagle MH, Owens GK. Kruppel-like factor 4 abrogates myocardin-induced activation of smooth muscle gene expression. J Biol Chem. 2004.

25. Proweller A, Pear WS, Parmacek MS. Notch signaling represses myocardin-induced smooth muscle cell differentiation. J Biol Chem. 2005; 280: 8994–9004.[Abstract/Free Full Text]

26. Cao D, Wang Z, Zhang CL, Oh J, Xing W, Li S, Richardson JA, Wang DZ, Olson EN. Modulation of smooth muscle gene expression by association of histone acetyltransferases and deacetylases with myocardin. Mol Cell Biol. 2005; 25: 364–376.[Abstract/Free Full Text]

27. Chen F, Kook H, Milewski R, Gitler AD, Lu MM, Li J, Nazarian R, Schnepp R, Jen K, Biben C, Runke G, Mackay JP, Novotny J, Schwartz RJ, Harvey RP, Mullins MC, Epstein JA. Hop is an unusual homeobox gene that modulates cardiac development. Cell. 2002; 110: 713–723.[CrossRef][Medline] [Order article via Infotrieve]

28. Miano JM. Serum response factor: toggling between disparate programs of gene expression. J Mol Cell Cardiol. 2003; 35: 577–593.[CrossRef][Medline] [Order article via Infotrieve]

29. Iso T, Chung G, Hamamori Y, Kedes L. HERP1 is a cell type-specific primary target of Notch. J Biol Chem. 2002; 277: 6598–6607.[Abstract/Free Full Text]

30. Dahlqvist C, Blokzijl A, Chapman G, Falk A, Dannaeus K, Ibanez CF, Lendahl U. Functional Notch signaling is required for BMP4-induced inhibition of myogenic differentiation. Development. 2003; 130: 6089–6099.[Abstract/Free Full Text]

31. Itoh F, Itoh S, Goumans MJ, Valdimarsdottir G, Iso T, Dotto GP, Hamamori Y, Kedes L, Kato M, Dijke Pt P. Synergy and antagonism between Notch and BMP receptor signaling pathways in endothelial cells. Embo J. 2004; 23: 541–551.[CrossRef][Medline] [Order article via Infotrieve]

32. Elagib KE, Xiao M, Hussaini IM, Delehanty LL, Palmer LA, Racke FK, Birrer MJ, Shanmugasundaram G, McDevitt MA, Goldfarb AN. Jun blockade of erythropoiesis: role for repression of GATA-1 by HERP2. Mol Cell Biol. 2004; 24: 7779–7794.[Abstract/Free Full Text]

33. Majesky MW, Lindner V, Twardzik DR, Schwartz SM, Reidy MA. Production of transforming growth factor beta 1 during repair of arterial injury. J Clin Invest. 1991; 88: 904–910.

34. Miano JM, Vlasic N, Tota RR, Stemerman MB. Localization of Fos and Jun proteins in rat aortic smooth muscle cells after vascular injury. Am J Pathol. 1993; 142: 715–724.[Abstract]

This article has been cited by other articles:

				Q. Xiao, Z. Luo, A. E. Pepe, A. Margariti, L. Zeng, and Q. Xu Embryonic stem cell differentiation into smooth muscle cells is mediated by Nox4-produced H2O2 Am J Physiol Cell Physiol, April 1, 2009; 296(4): C711 - C723. [Abstract] [Full Text] [PDF]

				R. A. Deaton, Q. Gan, and G. K. Owens Sp1-dependent activation of KLF4 is required for PDGF-BB-induced phenotypic modulation of smooth muscle Am J Physiol Heart Circ Physiol, April 1, 2009; 296(4): H1027 - H1037. [Abstract] [Full Text] [PDF]

				J. N. Regan and M. W. Majesky Building a Vessel Wall With Notch Signaling Circ. Res., February 27, 2009; 104(4): 419 - 421. [Full Text] [PDF]

				M. Shin, H. Nagai, and G. Sheng Notch mediates Wnt and BMP signals in the early separation of smooth muscle progenitors and blood/endothelial common progenitors Development, February 15, 2009; 136(4): 595 - 603. [Abstract] [Full Text] [PDF]

				J. Zhou, E. K. Blue, G. Hu, and B. P. Herring Thymine DNA Glycosylase Represses Myocardin-induced Smooth Muscle Cell Differentiation by Competing with Serum Response Factor for Myocardin Binding J. Biol. Chem., December 19, 2008; 283(51): 35383 - 35392. [Abstract] [Full Text] [PDF]

				D. Morrow, S. Guha, C. Sweeney, Y. Birney, T. Walshe, C. O'Brien, D. Walls, E. M. Redmond, and P. A. Cahill Notch and Vascular Smooth Muscle Cell Phenotype Circ. Res., December 5, 2008; 103(12): 1370 - 1382. [Abstract] [Full Text] [PDF]

				D.L. Tharp, B.R. Wamhoff, H. Wulff, G. Raman, A. Cheong, and D.K. Bowles Local Delivery of the KCa3.1 Blocker, TRAM-34, Prevents Acute Angioplasty-Induced Coronary Smooth Muscle Phenotypic Modulation and Limits Stenosis Arterioscler. Thromb. Vasc. Biol., June 1, 2008; 28(6): 1084 - 1089. [Abstract] [Full Text] [PDF]

				K. Niessen and A. Karsan Notch Signaling in Cardiac Development Circ. Res., May 23, 2008; 102(10): 1169 - 1181. [Abstract] [Full Text] [PDF]

				Y. Tang, S. Urs, and L. Liaw Hairy-Related Transcription Factors Inhibit Notch-Induced Smooth Muscle {alpha}-Actin Expression by Interfering With Notch Intracellular Domain/CBF-1 Complex Interaction With the CBF-1-Binding Site Circ. Res., March 28, 2008; 102(6): 661 - 668. [Abstract] [Full Text] [PDF]

				F. A. High, M. M. Lu, W. S. Pear, K. M. Loomes, K. H. Kaestner, and J. A. Epstein Endothelial expression of the Notch ligand Jagged1 is required for vascular smooth muscle development PNAS, February 12, 2008; 105(6): 1955 - 1959. [Abstract] [Full Text] [PDF]

				T. Tanaka, H. Sato, H. Doi, C. A. Yoshida, T. Shimizu, H. Matsui, M. Yamazaki, H. Akiyama, K. Kawai-Kowase, T. Iso, et al. Runx2 Represses Myocardin-Mediated Differentiation and Facilitates Osteogenic Conversion of Vascular Smooth Muscle Cells Mol. Cell. Biol., February 1, 2008; 28(3): 1147 - 1160. [Abstract] [Full Text] [PDF]

				S. Kennard, H. Liu, and B. Lilly Transforming Growth Factor- (TGF- 1) Down-regulates Notch3 in Fibroblasts to Promote Smooth Muscle Gene Expression J. Biol. Chem., January 18, 2008; 283(3): 1324 - 1333. [Abstract] [Full Text] [PDF]

				J. B.-D. Ponio, C. Wright-Crosnier, M.-T. Groyer-Picard, C. Driancourt, I. Beau, M. Hadchouel, and M. Meunier-Rotival Biological function of mutant forms of JAGGED1 proteins in Alagille syndrome: inhibitory effect on Notch signaling Hum. Mol. Genet., November 15, 2007; 16(22): 2683 - 2692. [Abstract] [Full Text] [PDF]

				N. Clement, M. Gueguen, M. Glorian, R. Blaise, M. Andreani, C. Brou, P. Bausero, and I. Limon Notch3 and IL-1beta exert opposing effects on a vascular smooth muscle cell inflammatory pathway in which NF-{kappa}B drives crosstalk J. Cell Sci., October 1, 2007; 120(19): 3352 - 3361. [Abstract] [Full Text] [PDF]

				T. Gridley Notch signaling in vascular development and physiology Development, August 1, 2007; 134(15): 2709 - 2718. [Abstract] [Full Text] [PDF]

				A. Fischer and M. Gessler Delta Notch and then? Protein interactions and proposed modes of repression by Hes and Hey bHLH factors Nucleic Acids Res., July 14, 2007; 35(14): 4583 - 4596. [Abstract] [Full Text] [PDF]

				S. M. Shirvani, L. Mookanamparambil, M. F. Ramoni, and M. T. Chin Transcription factor CHF1/Hey2 regulates the global transcriptional response to platelet-derived growth factor in vascular smooth muscle cells Physiol Genomics, June 19, 2007; 30(1): 61 - 68. [Abstract] [Full Text] [PDF]

				O. G. McDonald and G. K. Owens Programming Smooth Muscle Plasticity With Chromatin Dynamics Circ. Res., May 25, 2007; 100(10): 1428 - 1441. [Abstract] [Full Text] [PDF]

				M. S. Parmacek Myocardin-Related Transcription Factors: Critical Coactivators Regulating Cardiovascular Development and Adaptation Circ. Res., March 16, 2007; 100(5): 633 - 644. [Abstract] [Full Text] [PDF]

				H. Kokubo, S. Tomita-Miyagawa, Y. Hamada, and Y. Saga Hesr1 and Hesr2 regulate atrioventricular boundary formation in the developing heart through the repression of Tbx2 Development, February 15, 2007; 134(4): 747 - 755. [Abstract] [Full Text] [PDF]

				K. Kawai-Kowase and G. K. Owens Multiple repressor pathways contribute to phenotypic switching of vascular smooth muscle cells Am J Physiol Cell Physiol, January 1, 2007; 292(1): C59 - C69. [Abstract] [Full Text] [PDF]

				K. Hayashi, S. Nakamura, W. Nishida, and K. Sobue Bone Morphogenetic Protein-Induced Msx1 and Msx2 Inhibit Myocardin-Dependent Smooth Muscle Gene Transcription Mol. Cell. Biol., December 15, 2006; 26(24): 9456 - 9470. [Abstract] [Full Text] [PDF]

				H. Doi, T. Iso, H. Sato, M. Yamazaki, H. Matsui, T. Tanaka, I. Manabe, M. Arai, R. Nagai, and M. Kurabayashi Jagged1-selective Notch Signaling Induces Smooth Muscle Differentiation via a RBP-J{kappa}-dependent Pathway J. Biol. Chem., September 29, 2006; 281(39): 28555 - 28564. [Abstract] [Full Text] [PDF]

				G.C. T. Pipes, E. E. Creemers, and E. N. Olson The myocardin family of transcriptional coactivators: versatile regulators of cell growth, migration, and myogenesis. Genes & Dev., June 15, 2006; 20(12): 1545 - 1556. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 25/11/2328    most recent 01.ATV.0000185829.47163.32v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Doi, H. Articles by Kurabayashi, M. Search for Related Content PubMed PubMed Citation Articles by Doi, H. Articles by Kurabayashi, M.