Homeobox Protein Hex Facilitates Serum Responsive Factor–Mediated Activation of the SM22α Gene Transcription in Embryonic Fibroblasts
Objective— Hex (hematopoietically expressed homeobox), a member of homeobox family of transcription factors, has been implicated in the vascular development because of its expression in hemangioblast, a hypothetical stem cell that gives rise to both angioblasts and hematopoietic lineages. In the present study, we examined the role of Hex in the differentiation of vascular smooth muscle cells.
Methods and Results— We constructed adenovirus expressing Hex, to which we refer to as AxCA/Hex, and transduced murine embryonic fibroblasts, 10T1/2 cells. Northern blot analyses showed that Hex increased the mRNA levels of smooth muscle α-actin and SM22α but not of calponin and smooth muscle myosin heavy chain. Transient transfection assays showed that Hex activates the transcription from the SM22α promoter in a CArG box-dependent manner. Electrophoretic mobility shift assays demonstrate that Hex is not able to bind to CArG box, but binding of serum responsive factor (SRF) to CArG box is enhanced in AxCA/Hex-transduced cells. Recombinant Hex protein produced by in vitro translation system augmented the binding activity of SRF to CArG box. Immunoprecipitation experiments revealed the physical association between Hex and SRF.
Conclusions— Hex induces transcription of the SM22α gene by facilitating the interaction between SRF and its cognate binding site in pluripotent embryonic fibroblasts.
Homeobox transcription factors control a range of cellular activity including induced expression of a set of lineage-specific genes.1 A family of vertebrate homeobox genes related to the Drosophila tinman (tin) gene plays a crucial role for cardiogenic differentiation. Nkx2.3, Nkx2.5, and Nkx2.7 are expressed in the developing heart tissues.2,3 In particular, Nkx2.5 is selectively expressed in cardiac cell progenitors before cardiogenic differentiation4,5 and activates cardiac muscle-specific genes, including cardiac α-actin,6 myosin heavy chain,5 and atrial natriuretic peptide genes.7 Ablation of Nkx2.5 gene function in the mouse leads to atypical cardiac development.8,9 In contrast, little is currently known about the transcription factors involved in formation of the embryonic vasculature, although many angiogenic peptides and their signal transduction cascades have been identified that regulate the process of vasculogenesis and angiogenesis.
The divergent homeobox gene Hex, also known as Prh, is expressed in early endothelial precursors and angioblasts, as well as in a range of multipotent hematopoietic progenitor cells.10,11 Hex is expressed in the nascent blood islands of the visceral yolk sac and later in embryonic angioblasts.11,12 Endothelial expression of Hex is transient and commences slightly after expression of the receptor tyrosine kinase gene, flk-1, which is known to be essential for vascular development.12 Once endothelial cell differentiation commences, Hex expression starts to decline.11 Such a downregulation of Hex expression appears to be required for terminal differentiation to occur. Overexpression of Xenopus laevis Hex (XHex) in the frog embryo causes disruption to developing vascular structure and an increase in the number of vascular endothelial cells.12 Because endothelial precursors are originated from blood islands, and because smooth muscle cells (SMC) arise from multiple types of progenitors including endothelial cells, we hypothesize that Hex, which is implicated in the endothelial and blood differentiation, is also involved in SMC differentiation.
Recently, we have identified that Hex expression is induced in neointima after balloon injury.13 Because Hex expression is associated with the expression of the embryonic/nonmuscle isoform of myosin heavy chain (SMemb/NMHC-B) gene in neointima, we have examined whether Hex regulates the expression of this gene. We found that Hex activates the promoter activity of the SMemb/NMHC-B gene through cAMP-responsive element binding protein (CREB) binding site in vitro. However, it is unlikely that interaction between Hex and CREB plays a role in regulating the SMC differentiation marker genes, including SM α-actin and SM22α,14 which are known to be expressed in neointima, because no previous studies have identified functional CRE within SMC marker gene promoters. Thus, the role of Hex in regulating the expression of SMC-specific genes remains to be determined.
In the present study, we demonstrated that Hex activates the endogenous smooth muscle α-actin (SM α-actin) and SM22α genes in non-SMC. In addition, we showed that such an induction is dependent on intact CArG (CC(A/T)6GG) box, which serves as the efficient binding site for serum responsive factor (SRF) and plays a crucial role in the expression of the SMC differentiation marker genes. By using electrophoretic mobility shift assays of in vitro-translated proteins of Hex and SRF, we then asked how this induction was mediated. Although Hex was not able to bind to CArG box, it enhanced the binding activity of SRF to CArG box. Immunoprecipitation assays showed that Hex interacts in vitro with SRF. On the basis of the results presented here, we propose that Hex can impart the SMC-specific function to the CArG box of SM22α by physically interacting with SRF.
The 10T1/2 cells (murine embryonic fibroblast cells) have been previously described.15 Cells were grown at 37°C in a 5% CO2 atmosphere in Dulbecco modified Eagle’s medium (DMEM) supplement with 10% fetal bovine serum and 1% penicillin/streptomycin. Rat aorta-derived SMCs have been previously described.16
Recombinant Adenovirus Expression Constructs
The recombinant adenovirus vectors were generated as previously described.17 AxCA/LacZ and AxCA/Hex were prepared by inserting the β-galactosidase or Hex cDNAs, respectively, into the Ad E1-deleted region under the control the CAG promoter.
Northern Blot Analysis
Total RNA from 10T1/2 cells transduced with either AxCA/LacZ or AxCA/Hex was isolated using the ISOGEN reagent (Nippongene) in accordance with the manufacturer’s instructions. Northern blot analysis was performed as previously described.18
Western Blot Analysis
Cytosolic extracts from 10T1/2 cells infected with either AxCA/LacZ or AxCA/Hex were electrophoresed as previously described18 and blotted with anti-Hex antibody. Anti-Hex antibody was synthesized as previously described.13
The mouse SM22α promoter/luciferase fusion plasmid (SM22α/pGL2) was kindly provided by Dr K. Hasegawa (Kyowahakko Co Ltd, Tokyo, Japan). For generation of serial deletion construct, SM22α/pGL2 was digested with SacI and HindIII (SM22α-1309Luc), PstI and HindIII (SM22α-445Luc), SmaI and HindIII (SM22α-115Luc), gel-purified and subcloned into pGL3 (Promega). SM22α-158Luc and SM22α-171Luc were prepared by polymerase chain reaction (PCR) reaction with SM22α/pGL2 as a template. The forward and reverse primers used for generation of SM22α-171Luc were 5′-CCCGAGCTCGCTCCAACTTGGTG-TCTTTCC-3′ and 5′-GGGAAGCTT-GAAGGAGAGTAGCTTCGGTGT-3′ with SacI and HindIII sites (italic), respectively. The forward and reverse primers used for generation of SM22α-158Luc were 5′-CCCGAGCTCTTTC-CCCAAATATGGAGCCTG -3′ and 5′-GGGAAGCTTGAAGGAGAGTAGCTT-CGGTGT-3′. The PCR products were gel-purified, digested, and subcloned into pGL3.
For generation of mutants, the following forward and reverse primers were used in a PCR reaction with SM22α-158Luc as a template. The primers (mutations of wild-type sequence appear in bold) for SM22α-158m1Luc, SM22α-158m2Luc, and SM22α-158m3Luc were as follows: SM22α-158m1Luc, 5′-CCCGAGCTCGGGAACCAAATATGGAGCCTG-3′, SM22α-158m2Luc, 5′-CCCGAGCTCTTTCCAACCCTATGGAGCCTG-3′, and for SM22α-158m3Luc was 5′-CCCGAGCTCTTTCCCCAAAGCGTTAGCCTG -3′.
Hex expression plasmid (Hex/cytomegalovirus [CMV]) has been previously described.13 The expression plasmid SRF/pME18S, the coding region of mouse SRF, (kindly provided by Dr T. Miwa) has been described.19
Transfection and Luciferase Assays
The 10T1/2 cells were transfected with a series of the SM22α reporter construct (1 μg), along with 1 μg of expression plasmid by a modified calcium phosphate precipitation method as previously described.18 Cells were harvested in cell lysis buffer at 48 hours after transfection. Luciferase activities were assayed according to the manufacturer’s specifications (Promega).
Electrophoretic Mobility Shift Assays
Nuclear extracts from 10T1/2 cells were prepared as previously described.20 The sequences for sense strand of double-stranded oligonucleotides used as probes or competitors in electrophoretic mobility shift assays (EMSA) were as follows (with a consensus motif in italics and mutations of wild-type sequence in bold): SM22α (-158/-133), 5′-GTCTTTCCCCAAATATGGAGCCTGTG-3′; mut-1, 5′- GTCGGGAACCAAAT-ATGGAGCCTGTG -3′; mut-2, 5′- GTCTTTCCAACCCTATGGAGCCTGTG-3′; mut-3, 5′- GTCTTTCCCCAAAGCGTTAGCCTGTG-3′’; mut-4, 5′- GTCTTTCCCCAAATATGGCTAAGGTG-3′; Homeobox protein, 5′-TGCCAC-TTAATCATTAAGGGAGCC-3′; CArG, 5′-TCGAGCCCCAAATATGGAGC-CCCAAATATGGAGC-3′; and Sp1, 5′-ATTCGATCGGGGCGGGGCGAGC-3′. Binding reactions were performed as previously described.21
In Vitro Synthesis of Hex and SRF
In vitro synthesis of Hex and SRF were performed using TNT T7 Quick Coupled Translation System (Promega) with Hex/pcDNA3 and SRF/pGEM2 as templates. For generation of Hex/pcDNA3, mouse Hex cDNA was inserted into pcDNA3.1 (Invitrogen). SRF/pGEM2, the coding region of human SRF, was kindly provided by Dr I. Manabe (University of Tokyo, Japan).
The 10T1/2 cells were transfected with SRF/pME18S and Hex/pcDNA3.1 (HA epitope tagged). At 24 hours after transfection, the protein lysis solution was immunoprecipitated with normal rabbit IgG, anti-SRF, and anti-HA probe antibodies (Santa Cruz Biotechnology, Inc). Immunoprecipitated protein lysis solution was electrophoresed on 15% SDS-polyacrylamide gels and then transferred onto nitrocellulose membrane (Schleicher and Schuell, Inc).
Hex Induces SMC-Specific Marker Gene Expression
To determine the role of Hex in the process of SMC differentiation, we first constructed AxCA/Hex, which contains mouse Hex cDNA. Figure 1A and 1B show the mRNA and protein expression of Hex in the 10T1/2 cells transduced with AxCA/Hex. Figure 1C shows that Hex expression is localized in the nuclei of 10T1/2 cells.
We next transduced 10T1/2 cells with AxCA/Hex and performed Northern blot analyses. As shown in Figure 2, the mRNA levels of SMC-specific genes, SM α-actin and SM22α, were significantly increased in AxCA/Hex-transduced 10T1/2 cells as compared with AxCA/LacZ-transduced 10T1/2 cells. In contrast, Hex did not affect the mRNA levels of these SMC-specific genes in rat aortic SMCs in which the SM α-actin and SM22α genes are highly expressed under basal condition.
Mapping of the Hex Responsive Region Within the SM22α Promoter
To investigate whether Hex regulates the SM22α expression at the transcriptional level, we performed transient transfection assays. We cotransfected 10T1/2 cells with SM22α (−1309/+41) Luc/reporter gene, designated as SM22α-1309Luc and Hex expression vector (Hex/CMV). As shown in Figure 3A, Hex/CMV increased luciferase activity of SM22α-1309Luc by ≈3-fold.
To delineate the sequence responsible for the activation of SM22α promoter by Hex, progressive deletion of 5′-flanking region was performed. The SM22α-promoter constructs SM22α-445Luc and SM22α-171Luc, in which the 5′ ends correspond to −445 and −171, respectively, were activated by Hex, whereas SM22α-115Luc and promoterless construct pGL3 were unresponsive. These data indicate that Hex-responsive sequence is localized between −171 and −115. Sequence analysis of this region revealed a sequence identical to CArG box, 5′-CCAAATATGG-3′, at −150. To test whether CArG box is necessary for the activation of the SM22α promoter by Hex, we made constructs in which mutation was introduced into CArG box in the context of SM22α-158Luc (Figure 3B and 3C). The construct SM22α-158m1Luc, which contains mutation outside CArG box, exhibited the same activation in promoter activity as the wild-type promoter construct SM22α-158Luc in response to Hex/CMV, whereas the constructs SM22α-158m2Luc and SM22α-158m3Luc both contain mutation within the CArG motif and did not respond to Hex/CMV.
Identification of Nuclear Factor Binding Sites in the SM22α Promoter
To investigate the molecular mechanisms underlying the activation of the CArG-dependent transcription by Hex, we performed EMSA to determine whether Hex is capable of binding directly to CArG box. We used double-stranded oligonucleotide containing sequence from −158 to −133 of the SM22α gene as a probe (shown in Figure 4A) and incubated with nuclear extracts from 10T1/2 cells transduced with AxCA/Hex. As shown in Figure 4B, prominent shifted complex C1 was observed. Formation of C1 was sequence-specific because although addition of 100 molar excess of unlabeled probe or m1, m4, and CArG sequence almost completely abolished the complex formations, m2, m3, and Hex binding sequence had no effects on complex formation. Based on the sequence mutated in m2 and m3, these results indicate that sequence involved in the complex C1 formation is localized to CArG box between −150 and −141. We next performed supershift assay. Incubation of the nuclear extracts with anti-SRF antibodies completely supershifted complex C1. In contrast, anti-Hex and anti-Sp1 antibody had no effect on complex C1 formation (Figure 4C). These results suggest that C1 complex is constituted by SRF alone and Hex is not a measurable component of C1 complex.
Overexpression of Hex Enhances Binding of SRF to CArG Box but Does not Increase SRF Expression
To explore the mechanisms whereby Hex enhanced the promoter activity in CArG box-dependent manner, we first performed Western blot analysis and examined whether Hex had an effect on SRF protein levels. As shown in Figure 4D, Western blot analysis of 10T1/2 cells, which were transduced with either AxCA/LacZ or AxCA/Hex showed no discernable increase in Hex protein levels in Hex-transduced cells compared with LacZ-transduced cells. We then tested whether Hex increases the DNA binding of SRF. As shown in Figure 5A, the intensity of complex C1, which is constituted by SRF, was clearly increased in the nuclear extracts prepared from the cells transduced with AxCA/Hex. Intensity of Sp1 binding was equivalent between samples used in the EMSA, indicating that observed change in intensity of C1 complex is not caused by the difference or variation in quality and quantity of the nuclear extracts used (Figure 5B).
Recombinant Hex Enhances Binding of SRF to CArG Box
To further examine the interaction between Hex and SRF, we next performed EMSA using recombinant Hex (rHex) and SRF (rSRF) produced in a reticulocyte lysate system. As shown in Figure 5C, radioactive complex was barely detected when incubated with 1 μL of rSRF and 32P-labeled probe SM22α (−158/−133), which contains CArG box. Incubation of 2 μL of rSRF with the probe gave rise to a weakly shifted complex. Addition of rHex markedly enhanced SRF binding to CArG box in a dose-dependent manner. No retarded bands were observed when rHex or unprogrammed lysates were incubated with probe. Addition of unprogrammed lysate did not change SRF binding. SDS-PAGE of rSRF and rHex was performed for control (Figure 5D).
Hex Directly Binds to SRF
We asked whether the enhancement of SRF binding to CArG box in the presence of Hex was mediated through protein–protein association. We performed immunoprecipitation assay to investigate the interaction of SRF and Hex. The 10T1/2 cells were transfected with SRF/pME18S and Hex expression vector expressing the HA epitope-tagged Hex protein. Normal rabbit IgG, anti-HA probe antibody, and anti-SRF antibody were used for immunoprecipitation. The resulting immunoprecipitates were resolved by SDS-PAGE, transferred onto nitrocellulose membrane, and subjected to immunoblot analysis. As shown in Figure 6, the anti-SRF antibody coimmunoprecipitated HA epitope-tagged Hex and the anti-HA antibody coimmunoprecipitated SRF. Our results indicated that Hex and SRF form complexes in 10T1/2 cells.
In the present study, we showed that Hex activates endogenous SM α-actin and SM22α expression in 10T1/2 fibroblasts. In addition, we found that Hex induces SM22α promoter activity via proximal CArG element. With EMSA, using the nuclear extracts from AxCA/Hex-transduced cells and in vitro-translated Hex and SRF proteins, we demonstrated that Hex does not bind to CArG element of the SM22α promoter but rather increases the binding of SRF to this element. Furthermore, the immunoprecipitation assay showed that Hex physically interacts with SRF. Given that SRF plays a central role in the activation of the SMC marker genes, our data suggest that Hex may contribute to SMC differentiation by serving as positively acting cofactor for SRF.
Accumulating evidence indicates that homeodomain factors, which contain distinct DNA-binding domains, achieve their transactivating action not only through DNA–protein interaction but also through protein–protein interaction. However, the fact that Hex is capable of increasing the binding activity of SRF to CArG box deserves particular attention because there are only a few precedent reports that describe similar observation. Grueneberg et al22 have shown that paired homeobox protein Phox enhances the DNA binding activity of SRF to the c-fos SRE. By EMSA, using recombinant SRF and GST-Phox proteins, they suggested that the ternary Phox-SRF-SRE complex was transient or unstable under EMSA conditions, because formation and dissociation of this complex were rapid in the presence of Phox. Likewise, Hautmann et al23 showed that Mhox, which is the murine homologue of Phox, increases SRF binding to the CArG box in SM α-actin promoter. In our study, although we did not determine the formation of the ternary Hex-SRF-CArG complex in our EMSA condition, we did observe that in vitro-translated Hex increased the binding of SRF to CArG box. Thus, Hex-SRF-CArG complex may be either transient or unstable. Other factors or post-transcriptional modification of Hex and SRF do not appear to be required to stabilize this complex.
The molecular mechanisms of how Hex increases the binding of SRF to CArG box of SM22α promoter remain unanswered. At least 3 possibilities are envisioned, which are not mutually exclusive. First, Hex could accelerate the exchange of a new SRF molecule onto the SRE. Second, Hex could act by eliciting a conformational change in SRF. Third, SRF and Hex could form stable cooperative complexes on DNA. We favor the third model because we detected the Hex/SRF complex by immunoprecipitation assays. Chen and Schwartz have described the possibility that coassociation of Nkx2.5 and SRF in the CArG box might be stabilized through a short peptide encompassing the amino terminus and helix 1, and perhaps helix 2 of Nkx2.5.24 Consistent with this view, overexpression of plasmid encoding mutated Hex, which lacks homeodomain, was not able to activate SM22α promoter (data not shown). Further studies to define the interactive regions shared by Hex and SRF are needed.
We have previously shown that Hex expression is induced in neointima after balloon injury in rats.13 Phenotypic modulation of vascular SMC, which occurs in response to vascular injury, has long been known to be associated with the downregulation of virtually every SMC differentiation marker and upregulation of dedifferentiated SMC markers, including the SMemb/NMHC-B.14,25 Among SMC differentiation markers, SM α-actin and SM22α, but not calponin and smooth muscle myosin heavy chain 1 and 2 (SM1 and SM2), are expressed in neointima, although their expression levels are reduced in neointima as compared with those in media. Interestingly, our Northern blot analysis showed that Hex induces SM α-actin and SM22α genes, but it has no effects on calponin and SM1/2 gene expression (data not shown). Thus, it is intriguing to speculate that Hex plays a role in inducing or maintaining the proliferating phenotype characterizing SMC in neointima. Our data suggest that Hex functions in a cell type-specific manner, because Hex overexpression does not induce SMC marker gene expression in bovine aortic endothelial cells. This result conforms to our hypothesis that Hex acts as a cofactor of SRF, which plays a crucial role in SMC gene expression. Furthermore, it is likely that Hex plays a dispensable role in maintaining the undifferentiated phenotype, because overexpression of ΔHex, which acts as an inhibitor of Hex, has no effects on SM α-actin and SM22α gene expression in vascular SMC (data not shown). Then, what is the molecular mechanism by which Hex is capable of inducing limited set of SMC marker gene expression, even though all of the SMC marker genes contain functional CArG box within their promoters? Taking into consideration that the SM α-actin and SM22α genes are induced at early stage of SMC differentiation, and that the calponin and SM1/2 genes are induced at later stage of differentiation,14 one of the most plausible explanations for differential expression of the SMC marker genes by Hex is that SRF levels may be only rate-limiting in activation of early SMC differentiation marker, and Hex is sufficient to drive transcription of these genes through increasing SRF binding activity. However, combinatorial interactions of SRF with many transcription factors such as GATA6, MEF2, and SRF cofactors may be required for the later stage of SMC differentiation markers to be expressed and, if so, Hex overexpression may not be sufficient for activating these genes. We are currently testing this hypothesis.
In conclusion, the present study showed that the SMC marker genes were induced by Hex through the interaction between Hex and SRF on CArG box. Regulatory role of Hex in SRF-dependent genes through protein–protein interaction supports the concept that function of SRF is modulated by a variety of SRF cofactors, including myocardin26 and C-reactive protein.27 Further investigation of the molecular basis for enhancement of the binding of SRF to CArG box by Hex will provide the clue for the understanding of the context-dependent and signal-responsive regulation of phenotypic modulation of vascular SMC.
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 by a grant from the Japan Cardiovascular Foundation (to R.N. and M.K.).
- Received May 10, 2004.
- Accepted June 21, 2004.
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