Homeobox Protein Hex Induces SMemb/Nonmuscle Myosin Heavy Chain-B Gene Expression Through the cAMP-Responsive Element
Abstract—Recent studies have shown that the homeobox gene Hex plays an important role in inducing differentiation of vascular endothelial cells. In this study, we examined the expression of Hex in vascular smooth muscle cells (VSMCs) in vitro and in vivo. Immunohistochemistry showed a marked induction of Hex protein in neointimal VSMCs after balloon injury in rat aorta. Western and reverse transcriptase–polymerase chain reaction analyses demonstrated that Hex was abundantly expressed in cultured VSMCs, whereas it was undetectable in other cell types or in normal aorta. The expression pattern of Hex was similar to that of SMemb/NMHC-B, a nonmuscle isoform of myosin heavy chain that we have previously reported to be a molecular marker of dedifferentiated VSMCs. We next examined the role of Hex in SMemb gene transcription. Promoter analysis demonstrated that the sequence identical to consensus cAMP-responsive element (CRE) located at −481 of the SMemb promoter was critical for Hex responsiveness. Mutant Hex expression vector, which lacks the homeodomain, failed to stimulate SMemb gene transcription, suggesting the requirement of the homeodomain for its transactivation. Elecrophoretic mobility shift assay showed that Hex binds to a consensus binding sequence for homeobox proteins, but not to CRE. Cotransfection of protein kinase A expression vector increased the ability of Hex to stimulate SMemb promoter activity in a CRE-dependent manner. Overexpression of CRE binding protein (CREB), but not Mut-CREB which contains mutation at Ser133, strongly activated Hex-induced SMemb promoter activity. These results suggest that Hex mediates transcriptional induction of the SMemb/NMHC-B gene via its homeodomain, and Hex can function as a transcriptional modulator of CRE-dependent transcription in VSMCs.
Hex is a homeobox gene that is composed of a class of transcription factors that specify the body plan and regulate development of a wide variety of organisms.1 This transcription factor was originally isolated from hematopoietic tissues by polymerase chain reaction (PCR) using degenerate oligonucleotide primers corresponding to conserved homeodomain sequences.2 Expression of Hex transcripts is restricted to particular organs including hematopoietic tissue, lung, and liver.2 3 Whole-mount in situ hybridization demonstrated that Hex is transiently expressed in the nascent blood islands of the visceral yolk sac and later in embryonic angioblasts and endocardium4 5 and is generally downregulated during terminal cell differentiation.6 Furthermore, the overexpression of Hex using a retroviral vector resulted in the overgrowth of vascular endothelial cells.4 These results suggest that Hex plays a role in the development and growth of vascular endothelial cells. However, the expression of Hex in vascular smooth muscle cells (VSMCs) and its role in the transcription of VSMC genes remain unknown.
Proliferation and phenotypic modulation of VSMCs are a hallmark of vascular lesions.7 The concept of phenotypic modulation is well illustrated at the molecular level by changes in the regulation of the contractile proteins, such as smooth muscle (SM) α-actin,8 SM myosin heavy chain (MHC),9 10 11 myosin light chain,12 calponin,13 and SM22α.14 We and others have shown that VSMCs express at least 4 MHC variants including 2 nonmuscle variants of MHC.9 10 11 15 One non-muscle–specific isoform, NMHC-B/SMemb, is expressed during embryonic development of the aorta, declines in the neonate and adult, and is reinduced during atherogenesis. In contrast, SM1 and SM2, which are expressed in differentiated VSMCs, decline during atherogenesis.9 10 11 These changes in MHC expression suggest that an induced expression of the SMemb gene is associated with the development of vascular disease. In spite of extensive efforts, the molecular mechanisms underlying regulated expression of the vascular disease-relevant genes remain to be determined.
Here we demonstrate that Hex is expressed in cultured VSMCs in vitro, and we show that its expression is induced in neointimal smooth muscle cells (SMCs) in rat aorta after balloon injury in vivo, whereas its expression is undetectable in normal aorta or other cell types. We also show that the expression pattern of Hex is similar to that of SMemb, which we have previously reported to be a molecular marker for dedifferentiated VSMCs. Our results indicate that Hex activates the SMemb gene expression through a cAMP-responsive element (CRE) motif16 and suggest that the role of Hex in modulating the transcription of the genes whose expression is regulated by the protein kinase A (PKA) signaling pathway.
Materials and Methods
SMCs and rat neonatal cardiac myocytes were prepared as described previously.17 18 C2/2 cells are an established cell line derived from rabbit aortic SMCs.17 NIH 3T3 fibroblasts, COS-7, and calf pulmonary artery endothelial (CPAE) cells were obtained from the American Type Cell Culture Collection. Cells were incubated in DMEM supplemented with 10% FBS and grown in a humidified incubator equilibrated with 5% CO2 at 37°C.
RNA Isolation and Reverse Transcriptase (RT)–PCR
Total RNA isolation, RT-PCR, and Southern blotting were performed as previously described.18 Sequences of primers for amplifying Hex and GAPDH cDNA are as follows: Hex sense, 5′-TTCCCGCGGACGGTGAACGAC-3′; Hex antisense, 5′-TCATCCAGCATTAAAGTAGCCTTT-3′; GAPDH sense, 5′-ACCACAGTCCATGCCATCAC-3′; and GAPDH antisense, 5′-TCCACCACCCTGTTGCTGTA-3′.
A short peptide specifying the carboxyl terminal end of rabbit Hex (N-ASQEDLESEISEDSDQEVDIEGDKGYFNAG-C) was synthesized and used for immunization. The synthetic short peptide was conjugated with BSA and injected subcutaneously into rabbits at biweekly intervals. Titer of antisera was determined by ELISA.
Immunohistochemistry and Western Blot Analysis
All procedures were performed according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). Immunohistochemistry of Hex was performed as described previously.19 Thoracic aorta of adult male Wister rats were injured with a 2F balloon embolectomy catheter as described previously.20 Two weeks after balloon injury, the animals were euthanized, and thoracic aortae were subjected to immunohistochemistry. Immunostaining with anti-Hex antibody was performed by using the Vectastain Elite ABC kit (Vector Laboratories, Inc). Western blot analysis was performed as described previously.19 In brief, tissue or cell extracts were separated on 12% SDS-PAGE. The gels were stained with Coomassie brilliant blue or the proteins were electrophoretically transblotted onto a nitrocellulose membrane. The membrane was immunologically stained with anti-Hex antibody or anti–α-actinin antibody (Sigma).
The promoter/luciferase fusion plasmids, SMemb-3.8k and SM1/2–3.4k, have been described.21 22 Serial deletion constructs, Del-950, Del-500, Del-481, Del-461, and Del-185 were prepared by using the Deletion Kit (Takara). For generation of Del-481μ, Del-481 was digested by AatII, blunted by Klenow enzyme, and then ligated by T4 ligase. Hex expression vector (Hex/cytomegalovirus [CMV]) was constructed by PCR. The coding region of murine Hex cDNA was amplified by PCR using upstream primers with a HindIII site; 5′-CCCAAGCTTATGCAGTTCCCGCACCCGGGG-3′, and the reverse primer with an XbaI site; 5′-CCCTCTAGATCATCCAGCATTAAAGTAGCC-3′. The PCR product was subcloned into pRc/CMV (Invitrogen). For generation of mutant Hex expression plasmid that lack its homeodomain (ΔHex/CMV), PCR products of Hex (1–400) and Hex (586–816) were ligated and inserted into pRc/CMV. 10×CRE-TK/luciferase vector was prepared as follows: 2 complementary oligonucleotides containing CRE were annealed and 10 copies of the annealed oligonucleotides were inserted into TK/pGL3, which contains the thymidine kinase (TK) promoter upstream of the luciferase gene, as follows: CRE top, 5′-TCGAGGATTGCCTGACGTCAGAGAGCC-3′, and CRE-bottom, 5′-TCGAGGCTCTCTGACGTCAG-GCAATCC-3′. The sequence responsible for CRE is underlined. CRE binding protein (CREB), Mut-CREB, and PKA expression vector and TK/pGL3 plasmid were generous gifts from Drs Mark Montminy (Salk Institute for Biological Studies) and Jean-Michel Gauthier (Laboratoire GlaxoWellcome), respectively.
Transfection and Luciferase Assay
All of the transfection and luciferase assays were performed using CPAE cells. Cationic liposome-mediated transfection and luciferase assays were performed according to the manufacturer’s specifications (Promega). Data are mean±SEM of at least 3 independent experiments in duplicate.
Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assays (EMSAs)
Preparation of nuclear extracts and EMSA were done as previously described.19 The sequences of the oligonucleotides used as a probe were as follows, with CRE motif underlined and mutations of the wild type in bold face: SMemb-481, 5′-GGGGGGTGACGTCAGGCCTCT-3′; CRE mut, 5′-GGGGGGTGCAACCAGGCCTCT-3′; HS for homeobox protein binding sequence, 5′-TGCCACTTAATCATT-AAGGGAGCC-3′; and HS mut, 5′-TGCCACTGCCTCAGCCA-GGGAGCC-3′.
Hex Is Expressed in Cultured VSMCs
RT-PCR analysis demonstrated that Hex mRNA is ubiquitously expressed in vivo, although little was present in aorta (Figure 1A⇓). In cultured cells, both primary cultures of rat aortic VSMCs and C2/2 cells, an established VSMC line derived from rabbit aorta, express Hex mRNA. Because most VSMCs in normal aorta show the contractile phenotype and primary cultured VSMCs exhibit the synthetic phenotype,23 these data suggest that expression of Hex is dependent on the differentiation state of VSMCs.
Western blot analysis of the various tissues showed that the measurable amount of Hex protein is detected only in liver. As shown in Figure 1B⇑, cultured VSMCs but not CPAE cells (endothelial cells), NIH 3T3 cells (fibroblasts), or cardiac myocytes expressed Hex protein as indicated by the single band with an approximate molecular mass of 35 kDa. These results indicate that expression of Hex protein in cultured VSMCs is cell type specific.
Hex Is Induced in Neointima After Balloon Injury
We next examined whether Hex is expressed in neointimal VSMCs in response to injury. As shown in Figure 2A⇓, immunohistochemistry of the cross section of rat aorta showed that Hex was significantly induced in neointima 1 week after balloon injury, whereas the medial layer showed little expression. Immunoreactivity to anti-Hex antibody appears to be specific because preimmune serum showed no reactivity. Expression of Hex reached its peak at 2 weeks after balloon injury and declined thereafter. Western blot analysis of the protein extracts from aorta showed that Hex was strongly induced in balloon-injured aorta as compared with normal aorta (Figure 2B⇓). As shown in Figure 2C⇓, comparison of the time course of the Hex expression with that of the SMemb expression revealed that both proteins were induced in a similar fashion in response to vascular injury. Taken together with the data indicating Hex expression in VSMCs (Figure 1B⇑), these results suggest that expression of Hex is induced in neointimal SMCs.
Hex Transactivates the SMemb Gene
To examine the transcriptional effects of Hex on the SMemb promoter, CPAE cells were cotransfected with the SMemb promoter-luciferase construct SMemb-3.8k that contains 3.8 kb of the promoter region along with the Hex expression vector (Hex/CMV). CPAE cells were utilized for reporter assays for the lack of endogenous Hex expression (see Figure 1B⇑). As shown in Figure 3A⇓, cotransfection of Hex/CMV significantly induced the luciferase activity of the SMemb promoter. The effect of Hex/CMV is apparently promoter specific, because the SM1/2 promoter-luciferase construct SM1/2-3.4k was not responsive to Hex.
Homeodomain Is Required for Hex-Induced Transactivation
To assess whether the homeodomain in Hex affects SMemb gene transcription, the mutant Hex expression vector (ΔHex/CMV), which lacks its homeodomain, was transfected into CPAE cells. First we tested whether both Hex and ΔHex are localized to the nuclei after transfection. Anti-Hex antibody clearly detected the expression of either Hex or ΔHex (Figure 3B⇑). We then examined the effects of Hex and ΔHex on the promoter activity of SMemb-3.8k. As shown in Figure 3C⇑, Hex/CMV transactivated the SMemb promoter activity ≈7-fold higher as compared with control vector, but ΔHex/CMV showed no effect on this promoter. These data suggest that the homeodomain is critical for the transactivation of Hex.
Hex Activates the SMemb Gene Expression Through CRE
To define the Hex response element within the SMemb promoter, a series of SMemb promoter deletion constructs was cotransfected with Hex/CMV in CPAE cells. The SMemb-luciferase constructs Del-950 and Del-481 as well as SMemb-3.8k were similarly activated by Hex. However, further deletion to −461 almost completely eliminated Hex responsiveness. Likewise, Del-185 and promoterless pGL3 constructs did not respond to Hex (Figure 4A⇓). These results suggest that the 5′-end of the Hex-response sequence is localized to the sequence between −481 and −461. Sequence analysis of this region revealed an sequence identical to CRE, 5′-TGACGTCA-3′, at −481. To test whether CRE is required for the activation by Hex, the mutant constructs SMemb-3.8kμ and Del-481μ (Figure 4B⇓) with disrupted CRE motifs in SMemb-3.8k and Del-481, respectively, were cotransfected with Hex/CMV. As shown in Figure 4C⇓, mutation of CRE clearly abolished the increase in promoter activity of SMemb-3.8k and Del−481 by Hex. These data suggest that CRE at −481 is critical for activation of the SMemb promoter by Hex.
Hex Stimulates CRE-Dependent Gene Transcription
To test whether the CRE motif at −481 is functional, a PKA expression vector was cotransfected with the SMemb promoter. As shown in Figure 5A⇓, PKA overexpression increased the promoter activity of SMemb-3.8k (2.5-fold). Cotransfection of Hex expression vector alone increased the SMemb promoter activity by 3-fold. We next examined the synergistic effects of PKA on Hex-induced SMemb promoter activity. Cotransfection of PKA expression vector significantly enhanced Hex-induced SMemb promoter activity (14-fold). To verify whether the transactivation of Hex is CRE dependent, we used 10×CRE-TK promoter, in which 10 copies of short fragments containing the consensus CRE motif were inserted into the TK-luciferase reporter vector. As shown in Figure 5B⇓, the 10×CRE-TK promoter was activated by Hex expression vector by 2.5-fold. PKA overexpression further augmented this activation. These data strongly suggest that the transactivating function of Hex is CRE dependent, and it is further potentiated by the PKA signaling pathway.
CREB Potentiates Hex-Induced SMemb Promoter Activity
Because CREB constitutes the major component of the PKA-mediated gene transcription and Hex activates the SMemb gene expression through CRE, we tested whether CREB plays a role in Hex-induced SMemb gene transcription. As shown in Figure 5C⇑, CREB by itself activated SMemb promoter activity by 4-fold, and more importantly, cotransfection of CREB with Hex expression vector strongly enhanced the activation of SMemb promoter by Hex (≈70-fold). Specificity of this effect was determined by using the construct Mut-CREB in which residue Ser133 is mutated. Mut-CREB has been reported not to activate the CRE-dependent transcription because Ser133 is essential for CREB function.24 Mut-CREB had no effects on SMemb promoter activity, and cotransfection with Hex only minimally increased Hex-mediated SMemb promoter activity. These results suggest that Hex and CREB synergistically activate SMemb gene and further support our finding that PKA signaling increases Hex-mediated SMemb expression.
CREB but Not Hex Binds to CRE
To pursue the molecular mechanism underlying the activation of the CRE-dependent transcription by Hex, we examined whether Hex is capable of binding directly to the CRE motif. The labeled oligonucleotide probe containing the consensus CRE motif, which we designated as SMemb-481, was incubated with nuclear extracts from VSMCs. As shown in Figure 6A⇓, a DNA:protein complex was formed with this probe in a sequence-specific manner; 100-fold molar excess of unlabeled probe, but not its mutated sequence, completely abolished the complex formation. Supershift experiments indicated that this shifted complex contains CREB but not Hex, because incubation of the nuclear extracts with anti-CREB antibody clearly shifted the complex, whereas anti-Hex antibody had no effect on the complex. The presence of Hex in the nuclear extracts was confirmed by incubating the nuclear extracts with the probe containing the consensus binding sequence for Hex, 5′-ATTAA-3′.2 A labeled probe containing this motif, designated as HS (homeobox protein binding sequence), formed a complex with nuclear extracts from cultured VSMCs. As shown in Figure 6B⇓, the single band corresponding to the DNA:protein complex was abolished by 100-fold molar excess of unlabeled HS probe but not by a mutated HS probe. Furthermore, a supershifted band was detected by addition of anti-Hex antibody to the reaction, indicating that Hex, which is expressed in cultured VSMCs, has the ability to bind to DNA under our experimental conditions. Consistent with the results of Western blot analysis (Figure 1B⇑), our EMSA data showed that the shifted complex was detected in the nuclear extracts from primary cultures of VSMCs and C2/2 cells, but not from CPAE cells, NIH 3T3 fibroblast cells, or cardiac myocytes (Figure 6B⇓). These results suggest that Hex is specifically expressed in VSMCs.
In the present study, we demonstrate that Hex mRNA is expressed in cultured VSMCs but not in other cell types in vitro and that its protein is induced in neointimal SMCs after balloon injury. In addition, we show that Hex specifically activates transcription driven by the SMemb promoter, which is known to be a molecular marker for dedifferentiated VSMCs, and the cis element required for the transactivation is identical to CRE. Furthermore, activation of the SMemb gene by Hex is potentiated by overexpression of PKA or CREB. From these results, we conclude that Hex activates SMemb gene transcription through a novel mechanism by which Hex activates the PKA-dependent transcriptional machinery in VSMCs.
Three lines of evidence support the hypothesis that Hex plays a role in dedifferentiation of VSMCs. Previous studies have suggested the role of Hex in cellular differentiation in vascular endothelial cells4 5 23 and leukemic cells.6 First, we observed that Hex expression vector transactivates the SMemb promoter but not the SM1/2 promoter. Because dedifferentiated VSMCs predominantly express the SMemb rather than the SM1/2 gene,9 10 11 these results favor the notion that Hex promotes dedifferentiation of VSMCs. Second, we showed that Hex protein and its transcripts are preferentially expressed in VSMCs of synthetic phenotype rather than in normal aorta. As shown in the immunohistochemistry of balloon-injured aorta, Hex was strongly induced in neointima in which most SMCs are of synthetic phenotype.9 10 11 Lastly, we showed that Hex and SMemb expression are colocalized within the neointima. These findings suggest that Hex may play a role in regulating gene expression in dedifferentiated VSMCs.
The role of Hex in the dedifferentiation of VSMCs is unexpected but not surprising, because previous studies have shown that Hex expression is lost on differentiation into mature monocytes, macrophages, and megakaryocytes6 and is downregulated once endothelial cell differentiation commences in the developing Xenopus4 and mouse.5 Furthermore, other homeobox genes have also been reported to be involved in the process of cellular dedifferentiation or proliferation. For example, Hox 11, the homeobox gene most related to Hex, disrupts a G2/M cell-cycle checkpoint.25 Another homeobox gene, Evx-1, transactivates the tenascin gene,26 the expression of which is developmentally regulated and is induced after balloon injury to the vessel wall.27 These reports support our assumption that Hex may play a role in the establishment of the synthetic phenotype as well as in the maintenance of this phenotype via transcriptional activation of dedifferentiated VSMC genes.
One of the major findings in the present study is that Hex activates SMemb gene transcription via CRE but not AT-rich sequences to which Hex is known to bind.2 Cotransfection of various SMemb promoter/luciferase constructs with Hex expression vector demonstrated that a CRE at −481 of the SMemb promoter was critical for the activation by Hex, and mutation of the CRE motif completely abolished Hex-induced increase in reporter activity. Hex also stimulated a 10×CRE-TK promoter but not TK promoter alone, confirming that the transactivating function of Hex is CRE dependent. These findings are not consistent with the previous reports indicating that most homeobox proteins are known to regulate gene transcription through AT-rich sequences.1 However, there are some precedent reports in which homeobox proteins transactivate gene expression through a sequence distinct from AT-rich sequences. For example, Drosophila eve homeobox protein binds to both AT-rich and GC-rich sites with a similar affinity,28 whereas zen protein is capable of binding to only GC-rich sites.29 A report on Pbx may be relevant to this study because Pbx regulates the CYP17 promoter, which encodes 17α-hydroxylase/17,20-lyase cytochrome P-450, through direct binding to CRE.30 Thus, it is becoming clear that the transactivation by homeobox proteins is not solely dependent on the authentic AT-rich sequence, which is considered to be a core of the homeobox binding sequence.
Our data in this study raised the possibility that Hex induces gene expression by interacting with CREB. The results from EMSA showed that Hex did not bind directly to CRE, whereas CREB constitutes a DNA-protein complex formed with CRE as expected. Reporter analysis of the SMemb promoter revealed that cotransfection of both Hex and CREB expression vectors synergistically stimulate CRE-dependent gene transcription. Interaction between homeobox proteins and CREB has been recently described. Lorentz et al31 reported that intestinal homeobox protein, Cdx2, interacts with CREB and increases the ability of Cdx2 to transactivate target genes through AT-rich sequences. Therefore, Hex might interact with DNA binding protein to CRE, such as CREB, and stimulate gene transcription. Results of our cotransfection assays using mutant Hex expression vector suggest that the interaction between Hex and CREB takes place through the homeodomain. We attempted to determine the physical interaction between Hex and CREB by using the following 2 different approaches: 2-hybrid luciferase assay and immunoprecipitation experiments. However, we did not obtain the data suggesting the physical interaction between Hex and CREB by both approaches. Although we cannot entirely exclude such a possibility, these results rather suggest that synergistic activation of the SMemb promoter by Hex and CREB may not be due to the direct interaction between these 2 factors. The precise molecular mechanism, including participation of the coactivators such as CBP/p300, will deserve further study.
Our findings that PKA and CREB activate Hex-mediated transcription of the SMemb gene suggest the role of cAMP-PKA-CREB pathway in cellular proliferation because SMemb expression is associated with cytokinesis of VSMCs. Although a number of studies have demonstrated that the cAMP-PKA pathway inhibits cellular proliferation, accumulating evidence indicates that cAMP also mediates mitogenesis of the cells, including VSMCs. Owen32 reported that prostaglandin E1 via cAMP induces DNA synthesis in the quiescent A10 rat embryo VSMC line. In addition, CREB, an effector of the cAMP-dependent pathway, forms heterodimers with c-Fos to transactivate the cyclin A promoter through CRE in cultured VSMCs.33 These observations support our assumption that effects of the cAMP-PKA signaling pathway on stimulating dedifferentiation may be partly mediated through the activation of the genes lying downstream of Hex.
In summary, we demonstrate that the homeobox protein Hex, which is induced in neointimal SMCs after balloon injury, activates the SMemb gene transcription through its homeodomain. Furthermore, our data indicate that Hex activates transcription in a CRE-dependent manner in corroboration with the PKA or CREB expression vectors. Taken together, these data suggest that Hex may play a role in the phenotypic modulation of VSMCs, and interaction between PKA- or CREB-dependent transcriptional machinery and homeobox proteins may provide novel insight into the molecular mechanisms underlying the development of vascular disease in which proliferation and phenotypic change of VSMCs play a major role.
This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan and a grant from the Japanese Cardiovascular Foundation (to M.K. and R.N.).
Original received June 1, 2000; resubmission received October 9, 2000; revised resubmission received November 6, 2000; accepted November 6, 2000.
- © 2001 American Heart Association, Inc.
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