doi: 10.1161/01.RES.0000015402.38996.61
© 2002 American Heart Association, Inc.
Editorials |
Smooth Muscle-Specific Transcription Without a CArG Box Element
From the Departments of Pathology and Molecular and Cellular Biology, Center for Cardiovascular Development, Baylor College of Medicine, Houston, Tex.
Correspondence to Dr Mark W. Majesky, Dept of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. E-mail mmajesky{at}bcm.tmc.edu
Key Words: smooth muscle • serum response factor • promoter • arterial injury
Studies of the transcriptional control of vascular smooth muscle-specific gene expression have highlighted the importance of a conserved DNA recognition element known as a CArG box. This element has the core sequence CC[AT]6GG and is the binding site for the central MADS box domain of the serum response factor (SRF). All smooth muscle cell (SMC)-specific promoter/enhancer elements characterized to date exhibit a strong dependence upon SRF binding to CArG elements located either in the 5' promoter region, the first intron, or both. It is therefore of considerable interest that a study by Layne et al1 found in this issue of Circulation Research reports the identification of regulatory elements from the mouse aortic carboxypeptidase-like protein (ACLP) promoter that drive reporter gene expression in transgenic mice in a vascular smooth muscle-specific pattern in the absence of a recognizable CArG element.
Mouse Aortic Carboxypeptidase-Like Protein
Mouse ACLP was discovered in a screen for human aortic proteins that interact with the basic helix-loop-helix (bHLH) domain of E47.2 E47, and its related family member E12, are heterodimeric binding partners for myogenic factors (MyoD and myogenin). Heterodimer formation is required for transcriptional activation by myogenic bHLH factors. One of the clones recovered in this screen was a truncated form of human ACLP, which was used to isolate a full-length clone. Sequence analysis of the full-length cDNA revealed that ACLP is a secreted protein that contains a discoidin I-like domain and a catalytically-inactive metallocarboxypeptidase domain. Proteins encoded by the truncated ACLP cDNA bound to E47, whereas full-length ACLP did not. ACLP protein distribution in adult mice is highly restricted to vascular smooth muscle, with no detectable expression in cardiac or skeletal muscle.2 Consistent with sequence analysis, ACLP protein was found in apparent association with components of the extracellular matrix (ECM).
Immunostaining of E15.5 mouse embryos showed expression of ACLP in the medial layer of large blood vessels, cartilage, airway basement membranes, and dermal fibroblasts.3 ACLP-/- mice were generated and the null mutation was found to be lethal at or around the time of birth.3 ACLP-deficient embryos had severe anterior abdominal wall defects resulting in extrusion of the abdominal viscera through the ventral body wall (gastroschisis). A low percentage of ACLP-/- mice survived to adulthood and exhibited defects in wound healing. Histological analysis suggested that delayed wound healing was secondary to lack of ACLP in the ECM produced by dermal fibroblasts. Taken together, these findings suggest that ACLP is a secreted protein associated with the ECM that is produced by arterial SMCs and other connective tissue cells and functions both in embryonic development and adult tissue repair.
ACLP Promoter
To better understand ACLP gene regulation in vascular smooth muscle, Layne et al cloned a 2.5-kb segment of the 5' flanking region of the ACLP promoter and examined its activity in transgenic mice.1 The 2.5-kb promoter drove expression of a nuclear-targeted LacZ reporter gene in venous and arterial SMCs of both large and small vessels. Endothelial cells were negative, as were cardiac, skeletal, and visceral smooth muscle tissues. As with ACLP immunostaining,3 dermal fibroblasts and cartilaginous elements of the skeleton were also positive for reporter gene expression. After arterial injury, ACLP promoter activity was upregulated and strongly expressed in the developing neointima. This pattern differs from that of other SMC marker genes including SM22
, SM-actin, and SM-MHC, which are downregulated after arterial injury. Sequence analysis of the 2.5-kb ACLP promoter was notable for the absence of a recognizable CArG box element. To date, all vascular SMC-specific genes whose transcriptional regulation has been examined have been found to contain one or more CArG elements in the 5'-promoter and/or first intron.
To identify positive regulatory elements for ACLP expression in vascular SMCs, 5' deletion constructs of the 2.5-kb ACLP promoter were examined in rat aortic SMCs (RASMCs) in vitro. Nearly full activity was retained after deletion of sequences upstream of -156 bp, whereas activity was lost with additional deletions to -122 bp. The ACLP promoter region between -156 and -122 bp is rich in GC content and was shown to bind Sp1 and Sp3 transcription factors present in RASMC nuclear extracts. Mutational analysis indicated that Sp1 and Sp3 binding sites were located within the same region identified by deletion analysis to contain most of the promoter activity in RASMCs. When transfected into the Drosophila cell line D.Mel.2 (which lacks endogenous Sp1), both Sp1 and Sp3 expression constructs strongly transactivated the -156-bp ACLP promoter. These findings argue that high levels of ACLP promoter activity in RASMCs are conferred by Sp1 and Sp3 nuclear factors binding to the GC-rich elements in the ACLP proximal promoter. Whether these ubiquitously expressed factors are also responsible for vascular smooth muscle-restricted expression of the 2.5-kb construct in transgenic mice is a critical question that remains to be answered.
Mouse ACLP Promoter Activity in Cultured SMCs Is Independent of SRF
The absence of a recognizable CArG box in the -2.5-kb mouse ACLP promoter together with the evidence that promoter activity in cultured SMCs depends on nuclear factors Sp1 and Sp3 suggested that ACLP promoter activity in SMCs is independent of SRF. However, the possibility remained that Sp1 or Sp3 may bind to DNA and recruit SRF through protein-protein interactions. Activation of the cardiac -actin promoter in skeletal muscle cells was shown to depend upon a muscle-specific multiprotein complex containing myogenin, Sp1, and SRF.4 To address the role of SRF in ACLP promoter activity, Layne et al1 used a truncated form of SRF (amino acids 1 to 269) that contains the DNA binding domain but lacks the C-terminal transactivation domain. This form of SRF has been shown to function as a dominant-negative (DN-SRF) for CArG-dependent transcription.5 Expression of DN-SRF had no effect on - 2.5-kb ACLP promoter activity in RAMSCs while strongly inhibiting the activity of a -441-bp SM22 promoter, a known target for SRF-dependent transactivation. Taken together, these data suggest that vascular smooth muscle-restricted expression of the 2.5-kb ACLP promoter in transgenic mice is independent of SRF/CArG interactions.
CArG-Dependent Versus CArG-Independent SMC-Specific Transcription
The finding that ACLP promoter sequences can drive SMC-specific transcription in an SRF-independent manner contrasts sharply with reports from a number of laboratories showing that SMC-specific expression requires intact SRF-binding CArG elements.6– 10 It is important to point out, however, that the number of smooth muscle-specific genes examined in transgenic mice to date is still quite small. Moreover, the genes that have been examined in detail are all either cytoskeletal or contractile proteins. This raises the interesting question as to whether transcriptional controls for SMC-selective expression of cytoskeletal/contractile protein genes will turn out to be different from those for secreted proteins that function in the extracellular environment of the vessel wall. There is evidence to suggest that this may be true. For example, SRF-dependent transcription is controlled by cytoskeletal actin treadmilling in NIH3T3 cells11 and requires rhoGTPase-mediated signaling in SMCs and skeletal myoblasts.12–14 The rhoGTPases (rho, rac, and cdc42) are control points for many processes involving actin filament dynamics and cytoskeletal remodeling. In muscle cells, rhoA-GTPase participates in formation of a muscle cytoskeleton via SRF-dependent activation of contractile and cytoskeletal genes that reinforce the muscle phenotype in a positive-feedback manner. SRF in turn regulates the transcription of actin and other cytoskeletal proteins. Moreover, SRF- /- embryonic stem cells exhibit defects in cell spreading, adhesion, and migration that are correlated with downregulation of the focal adhesion proteins FAK, ß1-integrin, talin, zyxin, vinculin, and actin.15 Transfection of SRF-/- embryonic stem cells with a constitutively active form of SRF (SRF-VP16) restored expression of focal adhesion components and F-actin synthesis, leading to a dramatic reorganization of actin filaments, lamellipodia extension, and normal cell adhesion. Thus, evidence suggests that SRF may be the central element of an autoregulatory feedback loop controlling actin polymerization, focal adhesion formation, and contraction in muscle cells. In this respect, it is intriguing that ACLP, a secreted protein localized in the ECM, appears not to require SRF-CArG box interactions for cell-specific expression in vivo. Moreover, the absence of a consensus CArG box in the tropoelastin gene, which also encodes an ECM protein in the vessel wall, and the regulation of tropoelastin transcription by Sp116 further support this possibility.
Summary and Implications
In addition to the potential utility of the ACLP promoter to direct gene expression to SMCs in injured or diseased arteries, the studies by Layne et al1 raise new questions about transcriptional controls for vascular SMC differentiation. Characterization of the mouse ACLP promoter suggests that SMC-restricted transcription does not necessarily require SRF/CArG interactions and is not always downregulated after arterial injury. Resolving how widely expressed factors such as Sp1, Sp3, and SRF contribute to vascular SMC-specific transcription will require greater understanding of the combinatorial interactions of nuclear factors and transcriptional coregulators that assemble on SMC marker genes and adaptation of advances in protein sequencing methods to identification of multiprotein complexes that govern SMC-specific transcription.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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