Published online before print September 6, 2007, doi: 10.1161/CIRCRESAHA.107.154831
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© 2007 American Heart Association, Inc.
Molecular Medicine |
Smooth Muscle Cells and Myofibroblasts Use Distinct Transcriptional Mechanisms for Smooth Muscle 
-Actin Expression
From the Department of Molecular Physiology and Biological Physics (Q.G., T.Y., G.K.O.), University of Virginia, Charlottesville; and Division of Cardiology (J.L.), Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Mass.
Correspondence to Gary K. Owens, PhD, Department of Molecular Physiology and Biological Physics, University of Virginia, MR5 Room 1220, 415 Lane Rd, Charlottesville, VA 22908. E-mail gko{at}virginia.edu
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Abstract |
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There has been considerable controversy regarding the lineage relationship between smooth muscle cells (SMCs) and myofibroblasts, because they express a number of common cell-selective markers including smooth muscle (SM)
-actin. We have shown previously that MCAT elements within the SM -actin promoter confer differential activity in cultured SMCs versus myofibroblasts. In the present study, to determine the role of MCAT elements in vivo, we generated transgenic mice harboring an SM -actin promoter–enhancer–LacZ reporter gene containing MCAT element mutations and compared transgene expression patterns with wild-type SM -actin promoter–enhancer–LacZ transgenic mice. Results showed no differences in LacZ expression patterns in adult SMC-containing tissues. However, of interest, mutations of MCAT elements selectively abolished transgene expression in myofibroblasts within granulation tissue of skin wounds. In addition, mutations of MCAT elements caused a delay in the induction of transgene expression in SMCs, as well as loss of expression in cardiac and skeletal muscles during embryogenesis. Results of small interfering RNA–induced knockdown experiments showed that RTEF-1 regulated SM -actin transcription in myofibroblasts, but not in differentiated SMCs. Moreover, quantitative chromatin immunoprecipitation assays revealed that RTEF-1 bound to the MCAT element–containing region within the SM -actin promoter in myofibroblasts, whereas transcriptional enhancer factor (TEF)-1 was bound to the same region in differentiated SMCs. These results provide novel evidence that, although both SMCs and myofibroblasts express SM -actin, they use distinct transcriptional control mechanisms for regulating its expression. Results also indicate that the MCAT element-mutated SM -actin promoter–enhancer is a useful tool to direct gene expression selectively in differentiated SMCs.
Key Words: smooth muscle -actin • transgenic mouse • MCAT elements • transcriptional enhancer factor-1
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Smooth muscle cells (SMCs) play a key role in the maintenance of vascular homeostasis as well as the development of vascular diseases including atherosclerosis and restenosis.1 Myofibroblasts are induced de novo in multiple pathological states, such as the granulation tissue of contracting wounds and fibroproliferative diseases, and play a major role in the inflammatory response.2,3 In addition, both SMCs and myofibroblasts contribute to a wide range of human diseases including vein graft remodeling, tumor metastasis, and myocardial remodeling accompanying renovascular hypertension.1–4 Although the roles of SMCs and myofibroblasts in these diseases appear to be different, there has been considerable controversy regarding the identity and difference between these 2 cell types.1,5 The major reason for this controversy is based on the fact that these cell types share a number of cell-selective marker genes including smooth muscle (SM)
-actin and SM22.1,5 However, the distinction between these cell types and the mechanisms by which they regulate expression of their marker genes are of major importance, both because their contributions to human diseases are different and because the elucidation of pathways that distinguish these 2 cell types is likely to provide novel therapeutic strategies for selectively targeting these 2 cell types.
SM -actin is among the most frequently used markers for both SMCs and myofibroblasts. SM -actin is first expressed in vascular SMCs during differentiation of the outflow tract and formation of aortic arch around embryonic day (E)9.5, and in adult animals, it is highly restricted to SMCs under normal circumstances.6 It is also transiently expressed in the cardiac and skeletal muscle during the embryonic development.6 In addition, it is also known to be expressed in activated myofibroblasts within granulation tissue, as part of the stromal response with neoplasia and during tissue fibrosis.2–4 We have shown previously that the promoter–enhancer region from –2560 bp through the first intron (+2784 bp) of the SM -actin gene is sufficient to drive LacZ transgene expression in vivo in transgenic mice in a manner that recapitulates expression of the endogenous gene throughout the development and in response to vascular injury.7,8 In addition, studies by Tomasek et al9 have shown that this SM -actin promoter–enhancer is sufficient to induce the transgene expression in myofibroblasts within the granulation tissue of skin wounds. This SM -actin promoter–enhancer contains multiple cis elements, including 3 highly conserved CArG elements, a transforming growth factor (TGF)ß1 control element (TCE), 2 E-boxes, and 2 MCAT elements.1,5 Mutational analyses in transgenic mice containing the SM -actin promoter–enhancer–LacZ (SM-LacZ) construct revealed that each of 3 CArG elements, the TCE, and the 2 E-boxes were independently required for proper expression of this gene in vivo.1,5,7 The preceding results provide evidence that a combination of multiple cis elements and cognate trans-binding factors coordinately regulates expression of the SM -actin gene in both SMCs and myofibroblasts. However, as yet, no cis elements or trans-binding factors, which play a unique role in regulating SM -actin expression within SMCs or myofibroblasts in vivo, have been reported.
MCAT elements (AGGAATG) have been shown to play a key role in the transcriptional activation of multiple muscle genes such as cardiac troponin T10, skeletal -actin,11 and ß-myosin heavy chain,11 by binding with transcriptional enhancer factor (TEF)-1 family members including TEF-1, DTEF-1, RTEF-1, and Tead2.12 Previous studies from our laboratory have shown that mutations of 2 highly conserved MCAT elements within the truncated rat SM -actin promoter (MCAT1 at –184 bp and MCAT2 at –320 bp) decrease the transcriptional activity in AKR-2B fibroblasts, L6 myoblasts, and aortic endothelial cells, whereas the same mutations increase the activity in cultured aortic SMCs.13 Strauch, Getz, and colleagues also have shown that mutation of MCAT1 element in mouse SM -actin promoter decreases the gene transcriptional activity in AKR-2B fibroblasts and that TEF-1 is able to bind to the MCAT1 element, as determined by electrophoretic mobility-shift assays.14,15 The authors also have shown that the flanking sequence of MCAT1 element is the binding site for 3 single-stranded DNA-binding proteins, Pur, Purß, and MSY1, and proposed possible mechanisms whereby these three proteins inhibit TEF-1–mediated transcription of the SM -actin gene.15 These results suggest that the function of MCAT elements within the SM -actin promoter is different between SMCs and myofibroblasts, although there are several limitations in the preceding studies.
First, these studies were performed entirely in cultured model systems, and the relevance to regulation in vivo remains unclear. Second, these studies used a truncated SM -actin promoter construct that is not sufficient to drive SM -actin expression in vivo.7 Third, it remains to be determined whether TEF-1 is the binding factor for MCAT elements in these cell types, because the DNA–protein binding, as determined by electrophoretic mobility-shift assay in vitro, provides no direct evidence of binding within intact chromatin in cells, and no loss-of-function experiments have been tested to determine whether TEF-1 family members regulate SM -actin expression. Therefore, the goals of the present studies were (1) to determine the role of MCAT elements within the full-length SM -actin promoter–enhancer on SM -actin transcription in vivo in SMCs and myofibroblasts based on analyses of transgenic mice containing mutations of MCAT elements within a SM -actin promoter–enhancer–LacZ construct and (2) to determine the molecular mechanisms by which MCAT elements and their binding factors regulate SM -actin transcription in SMCs and myofibroblasts using the loss-of-function approaches and the chromatin immunoprecipitation (ChIP) assays.
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Materials and Methods |
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Generation and Analysis of Transgenic Mice
Animal procedures were approved by the University of Virginia Animal Use and Care Committee. Transgenic mice harboring an SM
-actin promoter–enhancer–LacZ reporter gene containing MCAT element mutations (dMCAT-SM-LacZ mice) were generated using standard methods provided by University of Virginia Gene Targeting & Transgenic Facility. LacZ expression in embryos and tissues from adult mice was examined as described previously.7
Skin Wound Injury
Male 6- to 10-week-old transgenic mice were anesthetized with an intraperitoneal injection of ketamine and xylazine. Full-thickness skin wounds were made by a sterile blade on left and right sides of the dorsal skin and sutured with 6-0 silk. Seven or 14 days after the injury, the wounded skin was harvested for LacZ staining.
An expanded Materials and Methods section can be found in the online data supplement at http://circres.ahajournals.org.
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Results |
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Mutations of MCAT Elements Abolished SM
-Actin Transcription in Myofibroblasts Within Granulation Tissue of Skin Wounds, Whereas They Had No Effect on SM -Actin Transcription in Adult SMCsTo determine the role of MCAT elements in SM
-actin transcription in vivo, we generated multiple independent transgenic mouse founder lines harboring mutations of MCAT elements within the context of a –2560- to +2784-bp SM -actin promoter–enhancer construct (dMCAT-SM-LacZ mice) (Figure 1A). The MCAT mutations used have been shown to completely disrupt the binding of TEF-1 family members, as determined by electrophoretic mobility-shift assay.13 We first determined the transgene expression patterns in multiple tissues from adult dMCAT-SM-LacZ mice relative to wild-type SM-LacZ mice. LacZ expression was seen in SMC-containing tissues, including the aorta, stomach, and intestine, but not in non-SM tissues such as the heart and skeletal muscle, which was comparable to wild-type SM-LacZ mice (Figure 1B and 1C). Similar LacZ staining patterns were obtained from 4 independent founder lines.
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Transgene expression was then examined in myofibroblasts within the granulation tissue of skin wounds in 2 independent founder lines of wild-type SM-LacZ and dMCAT-SM-LacZ mice. Full-thickness skin wounds were made, and the wounded skin containing the granulation tissue was harvested 7 and 14 days after injury. Myofibroblasts were identified by the positive staining of endogenous SM -actin expression in both wild-type SM-LacZ and dMCAT-SM-LacZ mice (Figure 2C, 2D, 2G, and 2H). However, of major significance, the LacZ transgene was activated in only myofibroblasts of wild-type SM-LacZ mice (Figure 2A and 2E), and no LacZ staining was seen in myofibroblasts of dMCAT-SM-LacZ mice (Figure 2B and 2F). Results suggest that MCAT elements are required for SM -actin transcription in myofibroblasts at early and late time points of granulation tissue formation within skin wounds, but they are dispensable for SM -actin transcription in adult differentiated SMCs.
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MCAT Elements Were Required for SM -Actin Transcription at the Initial Stages of Embryonic Development
Transgene expression patterns were also examined during embryonic development. Of major interest, results of LacZ staining in 4 independent founder lines showed no transgene expression in dMCAT-SM-LacZ mouse embryos at E10.5 and E12.5, whereas the transgene was readily detectable in smooth, cardiac, and skeletal muscles in the wild-type SM-LacZ transgenic counterpart (Figure 3A and 3B). At E13.5, the transgene was activated in SMCs in dMCAT-SM-LacZ embryos. However, in contrast to the wild-type SM-LacZ mouse embryos, which expressed the transgene at high levels in cardiac and skeletal muscles, transgene expression was completely restricted to the SMC-containing tissues. SMC-specific LacZ expression was more obvious in dMCAT-SM-LacZ embryos at E15.5 (Figure 3C). These results clearly indicate that MCAT elements are absolutely required for the initial activation of SM -actin transcription in SMCs, as well as in cardiac and skeletal myoblasts during embryonic development in vivo.
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SM -Actin Transcription Was MCAT Element Independent in Cultured Aortic SMCs, But It Was Dependent on MCAT Elements in All-Trans-Retinoic Acid–Treated A404 Cells and TGFß1-Treated AKR-2B Cells
Results from dMCAT-SM-LacZ mice showed that SM -actin transcription was dependent on MCAT elements in early embryonic SMCs and myofibroblasts, whereas it was independent of MCAT elements in SMCs during late embryogenesis and in adult mice. To determine the molecular determinants of these differences in the MCAT element dependency of SM -actin transcription, we screened a number of SM -actin–expressing cultured cell lines, including rat aortic SMCs, all-trans-retinoic acid (RA)-treated A404 cells, and TGFß1-treated AKR-2B cells. Cultured rat aortic SMCs have been widely used as a model of differentiated SMCs and have been shown to express all known SMC differentiation markers and exhibit agonist-induced calcium transients.1,16,17 RA-treated A404 cells and TGFß1-treated AKR-2B cells have been proposed to be culture models for initial induction of SMC differentiation18 and activated myofibroblasts,14 respectively. As shown in Figure 4, transient transfection assays of the wild-type SM-LacZ plasmid and the dMCAT-SM-LacZ plasmid into these cell types revealed that transcriptional activity of the SM -actin gene was significantly decreased by mutations of MCAT elements in RA-treated A404 cells and TGFß1-treated AKR-2B cells. In contrast, mutations of MCAT elements did not decrease, but rather increased, the SM -actin promoter activity in rat aortic SMCs. We also tested effects of mutation of each of MCAT elements (MCAT1 and MCAT2) on the SM -actin promoter activity and found that both MCAT1-SM-LacZ and MCAT2-SM-LacZ constructs exhibited similar effects as the dMCAT-SM-LacZ construct. In addition, we excluded the possibility that the difference in MCAT element dependency was caused by the difference in species by using cultured SMCs derived from the mouse thoracic aorta. Consistent with rat aortic SMCs, mouse aortic SMCs exhibited MCAT element–independent SM -actin transcription (data not shown). Taken together, results indicate that rat aortic SMCs exhibit MCAT element–independent SM -actin transcription similar to that of adult differentiated SMCs in vivo in our transgenic mouse studies. In contrast, RA-treated A404 cells and TGFß1-treated AKR-2B cells, respectively, appear to model embryonic SMCs and skin wound myofibroblasts wherein SM -actin transcription is MCAT element dependent.
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Suppression of RTEF-1 Decreased SM -Actin Transcription in RA-Treated A404 Cells and TGFß1-Treated AKR-2B Cells, Whereas Suppression of Any of TEF-1 Family Members Had No Effect on SM -Actin Transcription in Aortic SMCs
Members of the TEF-1 family, including TEF-1, DTEF-1, and RTEF-1, have been shown to be trans-binding factors for MCAT elements and to be induced during mouse embryogenesis around E8.0 to E10.0,12 whereas Tead2 was expressed only in the hindbrain and barely detectable in any adult tissues.19 We hypothesized that the distinct availability of TEF-1 family members, including TEF-1, DTEF-1, and RTEF-1, may contribute to the differences in MCAT element dependency of SM -actin transcription in aortic SMCs versus RA-treated A404 cells and TGFß1-treated AKR-2B cells. To test this hypothesis, we first examined the expression and localization of TEF-1 family members in these cell types. Results of semiquantitative RT-PCR showed that mRNA expression of TEF-1, DTEF-1, and RTEF-1 was not different among aortic SMCs, RA-treated A404 cells, and TGFß1-treated AKR-2B cells (Figure 5A). Results also showed that protein expression levels of TEF-1, DTEF-1, and RTEF-1 were similar among all 3 cell types and that all members were localized in the nucleus, as determined by Western blotting of nuclear and cytoplasmic fractions (Figure 5B). These results suggest that expression and localization of TEF-1 family members are not responsible for the differences in MCAT element dependency in aortic SMCs versus RA-treated A404 cells and TGFß1-treated AKR-2B cells.
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Effects of knockdown of each TEF-1 family member on SM -actin transcription were then tested by using small interfering (si)RNA expression plasmids for TEF-1 family members and the SM-LacZ construct. Efficient and specific suppression of TEF-1 family members by corresponding siRNA expression plasmids was validated by cotransfecting each of siRNA expression plasmids (pMighty-TEF-1, pMight-DTEF-1, and pMighty-RTEF-1) and each of FLAG-tagged expression plasmids for TEF-1 family members (Figure 6A). Results showed that siRNA-induced suppression of TEF-1, DTEF-1, and RTEF-1 had no effect on transcriptional activity of the SM -actin gene in rat aortic SMCs (Figure 6B). In contrast, siRNA-induced knockdown of RTEF-1, but not TEF-1 or DTEF-1, selectively decreased the SM -actin promoter activity by 72% and 61% in RA-treated A404 cells (Figure 6C) and TGFß1-treated AKR-2B cells (Figure 6D), respectively. These results were confirmed by testing the effects of siRNA suppression of TEF-1 family members on endogenous SM -actin expression in these cell types (Figure II in the online data supplement). Results provide evidence (1) that RTEF-1 contributes to the transcription of the SM -actin gene in RA-treated A404 cells and TGFß1-treated AKR-2B cells, in which SM -actin transcription is MCAT element dependent, and (2) that the SM -actin transcriptional activity in rat aortic SMCs is not dependent on any of these TEF-1 family members, which is consistent with the observations that mutations of MCAT elements did not decrease the SM -actin promoter activity in these cells (Figure 4A).
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Association of TEF-1 Family Members With MCAT Elements in the SM -Actin Promoter Within Intact Chromatin Was Different Between MCAT Element–Independent Versus MCAT Element–Dependent Cell Types
Results thus far have shown that SM -actin transcription was dependent on RTEF-1 in RA-treated A404 cells and TGFß1-treated AKR-2B cells, but not in rat aortic SMCs, although all 3 TEF-1 family members were expressed and localized in a similar manner in these cells types. To further define molecular mechanisms responsible for differential MCAT element dependency among these cell types, the association of TEF-1 family members with the MCAT element–containing SM -actin promoter was examined using quantitative ChIP assays. Because of the proximity between MCAT elements and CArG elements within the SM -actin promoter, PCR amplification by our primer sets reflects the enrichment of DNA fragments from both elements (Figure 7A). As shown in Figure 7B, TEF-1 was associated with the MCAT element–containing SM -actin promoter in rat aortic SMCs to a similar extent as binding of serum response factor (SRF) to this promoter region. However, neither DTEF-1 nor RTEF-1 was associated with this promoter in rat aortic SMCs. In contrast, RTEF-1 was associated with the MCAT element–containing SM -actin promoter in RA-treated A404 cells and TGFß1-treated AKR-2B cells (Figure 7C and 7D). Indeed, TEF-1 was not associated with this promoter in these cells. None of the TEF-1 family members nor SRF was associated with the SM -actin promoter in undifferentiated A404 cells in which SM -actin expression was not activated (Figure 7E). Taken together, the results suggest that TEF-1 is the binding factor of MCAT elements in cells in which SM -actin transcription is MCAT element independent, whereas RTEF-1 was associated with MCAT elements within cells in which SM -actin transcription is MCAT element dependent.
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To determine whether differential binding of TEF-1 family members to MCAT elements observed in cultured cells also occurred in vivo, the in vivo ChIP assays were performed using adult mouse thoracic aorta and the granulation tissue of skin wounds. The results showed that TEF-1 was the binding factor of MCAT elements in mouse thoracic aorta, whereas RTEF-1 was associated with the MCAT element–containing SM -actin promoter in the granulation tissue of skin wounds (Figure 8). We also detected the enrichment of DTEF-1 within the SM -actin promoter in granulation tissues, which might reflect the heterogeneity of myofibroblasts. These results provide compelling evidence that differential binding of TEF-1 family members to the MCAT-containing SM -actin promoter within intact chromatin is among the molecular determinants of MCAT element dependency for SM -actin transcription in vivo.
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Discussion |
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SMCs and myofibroblasts are known to play a critical role in the pathophysiology of a wide range of major human diseases.1–5 However, the molecular identity of these cells has been obscured by the fact that they express a number of common cell-selective marker genes, including SM
-actin.5 Of major significance, results of the present studies show that, although both SMCs and myofibroblasts express SM -actin at high levels, the mechanisms whereby they activate expression of this gene can be distinguished at the molecular level, in that SM -actin expression in myofibroblasts but not in SMCs within adult mice was dependent on MCAT elements found at positions –184 and –320 bp of the SM -actin promoter in vivo. Moreover, although both cell types expressed TEF-1, DTEF-1, and RTEF-1, MCAT elements were associated with different TEF-1 family members within the context of intact chromatin in myofibroblasts versus SMCs both in cultured cell systems and in vivo. Taken together, results indicate (1) that identification of signaling pathways and molecules that activate MCAT elements will provide insight regarding mechanisms that contribute to myofibroblast activation in multiple disease states including skin wound healing and (2) that our MCAT element–mutated SM -actin promoter–enhancer can be used as a means to distinguish SMCs versus myofibroblasts and for purposes of driving SMC-specific expression in vivo.
Results of the present studies demonstrate that, in addition to myofibroblasts, embryonic SMCs at the initial stages of development also use MCAT elements for SM -actin transcription. LacZ staining of dMCAT-SM-LacZ mouse embryos at E10.5 and E12.5 clearly showed that SM -actin transcription at these early time points was dependent on MCAT elements, whereas these elements were dispensable for transcription after E13.5. The results provide novel evidence indicating that transcriptional control mechanisms for initial induction of SMC marker genes are different from the mechanisms for the maintenance of these genes. Although the precise mechanisms and factors responsible for the switch from MCAT element–dependent to MCAT element–independent SM -actin transcription during embryonic development are currently unclear, it is worth noting that the onset of expression of myocardin, a potent inducer of SMC marker genes, appears to be delayed in vascular SMCs, as compared with initial expression of multiple SMC marker genes including SM -actin and SM22.5 Indeed, Du et al20 showed that myocardin mRNA was not detectable in the descending aorta until E12.5, as determined by in situ hybridization, whereas it was expressed in the heart as early as E7.5. In addition, although myocardin has been shown to be a key regulator of CArG-containing SMC marker genes in cultured adult differentiated SMCs by loss-of-function experiments,16,20 there is controversy regarding the requirement of myocardin for the initial induction of SMC differentiation. That is, although Li et al21 showed that myocardin knockout mice died by E10.5 and mice exhibited no evidence of SMC differentiation, several investigators, including those workers, suggested that the failure to form SMC tissues may have been secondary to defects in differentiation of cardiomyocytes and/or to defective formation of the extraembryonic circulation.5 In support of this, recent studies by Pipes et al22 showed that myocardin was not cell-autonomously required for the induction of SMC lineage based on the observations that myocardin-null embryonic stem cells were able to differentiate into SMCs in the setting of chimeric knockout mice generated by injection of myocardin-null embryonic stem cells into the wild-type blastocysts. As such, it is interesting to hypothesize that the initial induction of SMC marker genes is dependent on MCAT elements and their binding factors but not myocardin and that MCAT elements are dispensable when myocardin expression reaches some critical level. However, it should be noted that myocardin was easily detectable in visceral SMCs such as the muscularis mucosa layer of the stomach and intestine and the urogenital ridge at E12.5,20 even though SM -actin transcription was MCAT element dependent in these tissues at this time point. Thus, the switch from MCAT element–dependent SM -actin transcription to MCAT element–independent SM -actin transcription is likely to be more complex than the simple presence or absence of a single factor such as myocardin, and further studies are needed to fully resolve these issues.
By using a combination of siRNA-induced knockdown experiments and the in vitro and in vivo quantitative ChIP assays, the present studies demonstrated that TEF-1 was associated with MCAT elements within the SM -actin promoter in cells in which SM -actin transcription was independent of MCAT elements (ie, differentiated SMCs), whereas RTEF-1 was the major binding factor of MCAT elements in cells in which SM -actin transcription was MCAT element dependent (ie, myofibroblasts and embryonic SMCs). The results suggest that the differential binding of TEF-1 family members to MCAT elements is a critical determinant for MCAT element–dependent SM -actin transcription, although the underlying mechanisms are currently unknown. However, there are several possible mechanisms to explain these observations, which are not mutually exclusive. First, it is possible that cell type–specific cofactors for TEF-1 family members may mediate the selective binding of TEF-1 family members to MCAT elements. For example, results from several laboratories have shown that Vestigial-like 2 (Vgl-2), a skeletal muscle-specific cofactor of TEF-1 family members, was increased during the skeletal muscle differentiation in C2C12 cells and that the association of Vgl-2 with TEF-1 lowered the ability of TEF-1 to bind to the MCAT element within the skeletal -actin promoter.23–25 They also showed that, although the binding of TEF-1 to the MCAT element was decreased, the binding of RTEF-1 to the same MCAT element was increased during the skeletal muscle differentiation.25 These results suggest that Vgl-2 plays a role in the selection of TEF-1 family members to bind to the skeletal -actin MCAT element during the skeletal muscle differentiation. At present, however, no similar tissue-restricted member of the Vgl family has been identified in SMCs. Second, different posttranslational modifications of TEF-1 family members may contribute to their distinct association with MCAT elements within the SM -actin promoter. Indeed, protein kinase A–induced phosphorylation of TEF-1 has been reported to repress its binding activity to the MCAT element within the cardiac -myosin heavy chain gene in cardiac myocytes.26 Moreover, results of previous studies by Stewart and colleagues showed that, although TEF-1, DTEF-1, and RTEF-1 were expressed in cardiac myocytes and TEF-1 accounted for 85% of MCAT binding activity on the skeletal -actin promoter as determined by electrophoretic mobility-shift assay, DTEF-1 selectively mediated the skeletal -actin activation by 1-adrenergic signaling.27 By testing the phosphorylation status of TEF-1 family members, the authors found that the unique phosphorylation site in DTEF-1 was required for 1-adrenergic response and that the phosphorylation status of DTEF-1 altered its binding affinity to the MCAT element.27 It is possible that the phosphorylation status of each of TEF-1 family members is different among different cell types and alters the binding affinity of TEF-1 family members with MCAT elements within the SM -actin promoter. Third, the combinatorial interactions of MCAT elements and TEF-1 family members with other regulatory cis elements and trans-binding factors within the SM -actin promoter–enhancer might regulate the affinity of TEF-1 family members to MCAT elements. Of interest, SRF, the trans-binding factor for CArG elements, has been shown to interact with TEF-1 to regulate the skeletal -actin gene transcription in cardiomyocytes.28 In addition, myocardin might regulate the association of TEF-1 family members with MCAT elements because the induction of myocardin expression in vascular SMCs during the embryonic development appears to be related to the MCAT element–dependent versus MCAT element–independent SM -actin transcription, as described above. Identification of the mechanisms for distinct association of TEF-1 family members to MCAT elements will enhance our understanding of pathophysiology of multiple diseases in which myofibroblasts and SMCs are involved.
In summary, the results of the present studies provide novel evidence showing that expression of the SM -actin gene uses distinct transcriptional control mechanisms in different cell types and at different developmental stages. Our results also provide evidence that there are distinct binding patterns of TEF-1 family members to MCAT elements that may provide the molecular basis for differential MCAT element dependency between these cell types. Finally, the SM -actin promoter–enhancer containing mutations of MCAT elements may have utility for SMC-specific gene delivery or targeting because, unlike the wild-type SM -actin promoter, it is inactive in myofibroblasts as well as in cardiac and skeletal myoblasts.
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Acknowledgments |
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Sources of Funding
This work was supported by NIH grants R01HL38854, P01HL19242, and R37HL57353 (to G.K.O.) and by American Heart Association National Scientist Development Grant 0635253N (to T.Y).
Disclosures
None.
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Footnotes |
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Original received April 25, 2007; revision received July 31, 2007; accepted August 24, 2007.
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References |
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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.
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7. Mack CP, Owens GK. Regulation of smooth muscle -actin expression in vivo is dependent on CArG elements within the 5' and first intron promoter regions. Circ Res. 1999; 84: 852–861.
8. Regan CP, Adam PJ, Madsen CS, Owens GK. Molecular mechanisms of decreased smooth muscle differentiation marker expression after vascular injury. J Clin Invest. 2000; 106: 1139–1147.[Medline] [Order article via Infotrieve]
9. Tomasek JJ, McRae J, Owens GK, Haaksma CJ. Regulation of -smooth muscle actin expression in granulation tissue myofibroblasts is dependent on the intronic CArG element and the transforming growth factor-ß1 control element. Am J Pathol. 2005; 166: 1343–1351.
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