This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Herring, B. P. Articles by Zhou, J. Search for Related Content PubMed PubMed Citation Articles by Herring, B. P. Articles by Zhou, J. Related Collections Related Article

(Circulation Research. 2007;101:856.)
© 2007 American Heart Association, Inc.

Editorials

mCAT Got YouR TEF?

B. Paul Herring, Jiliang Zhou

From the Department of Cellular and Integrative Physiology, Indiana University School of Medicine.

Correspondence to Prof Brian P. Herring, Indiana University School of Medicine, Department of Cellular and Integrative Physiology, 635 Barnhill Drive, MS350E, Indianapolis, IN 46202. E-mail pherring{at}iupui.edu

See related article, pages 883–892

Key Words: smooth muscle -actin • TGFß, transcriptional enhancer factor 1 • serum response factor • smooth muscle • myofibroblasts

	TEF and RTEF-1 Binding to the Smooth Muscle -Actin Gene Distinguishes Myofibroblasts and Adult Smooth Muscle Cells

Top TEF and RTEF-1 Binding... What Controls the Switch... The MCAT Mutated Smooth... References

 
Adult smooth muscle cells are highly plastic and can exhibit a range of phenotypes in response to different environmental and developmental cues. Their phenotypes can range from quiescent highly contractile cells with high levels of characteristic contractile protein isoforms, to highly proliferative cells that secrete large amounts of extracellular matrix and express only low levels of smooth muscle-specific isoforms of contractile proteins. These two states are often referred to as differentiated and dedifferentiated or phenotypically modulated states.¹ In reality the situation is more complex, with smooth muscle cells likely existing in a continuum of phenotypes between these two extremes. This smooth muscle cell plasticity often makes the unequivocal identification of smooth muscle cells challenging, particularly for the more dedifferentiated smooth muscle cells that are very similar to fibroblasts. The situation is further complicated by the existence of cell types with phenotypes part way between a fibroblast and a fully differentiated adult smooth muscle cell, namely myoepithelial and myofibroblast cells, and pericytes.^2–4 A major challenge to developmental biologists is determining how these cells relate to each other, determining whether they are derived from common or distinct precursors, and whether myofibroblasts or pericytes can become smooth muscle cells. One approach to begin to answer these questions is to determine the molecular mechanisms that control the phenotype of each of these cell types. A new study by Gan and colleagues,⁵ described in this issue of Circulation Research, provides definitive molecular evidence that distinguishes myofibroblasts from adult smooth muscle cells by the distinct transcriptional mechanisms that they use to direct expression of a shared molecular marker, smooth muscle -actin. In this elegant study, the authors demonstrate that the binding of RTEF-1 to MCAT elements within the smooth muscle -actin promoter is required for transcription in myofibroblasts but not in adult smooth muscle cells (Figure). These results reveal that although both adult smooth muscle cells and myofibroblasts express smooth muscle -actin, its expression in these cell types is regulated by distinct cis- and trans-acting factors. That myofibroblasts and adult smooth muscle cells use distinct cis-regulatory elements to control expression of a single gene should not, however, be too surprising given previous studies showing that a single gene often uses distinct elements for expression even among adult smooth muscle cells in different tissues. This phenomenon is perhaps best illustrated by earlier studies by the Owens group which showed that distinct regions of the smooth muscle myosin heavy chain gene direct expression in different vascular beds and among different visceral smooth muscle tissues.⁶ Perhaps the most intriguing finding of this study is that the MCAT elements required for expression of smooth muscle -actin in myofibroblasts are also required for expression in smooth muscle cells during early embryonic development. This provides unequivocal evidence that smooth muscle cells also use distinct regulatory elements to drive expression of a gene in embryonic and adult cells. This finding certainly begs the question as to what distinguishes an embryonic smooth muscle cell from a myofibroblast and raises the possibility that myofibroblasts could represent a resident population of embryonic smooth muscle cells that persist, undifferentiated, in an adult. Careful lineage mapping using temporally and spatially restricted lineage markers will be required to answer this important question.

View larger version (20K): [in this window] [in a new window]   Figure. Transcriptional regulation of smooth muscle -actin in myofibroblasts and adult smooth muscle cells. A number of cis-acting regulatory elements have been shown to be required for regulation of the smooth muscle -actin promoter in myofibroblasts (left panel) and in smooth muscle cells (right panel) in vivo (for simplicity only the proximal promoter region is shown). In myofibroblasts TGFß has been shown to increase expression of SRF, RGC-32, EF17,12,16; Gan and colleagues also show that TGFß promotes binding of RTEF-1 to the MCAT elements that are required for TGFß-mediated increases in smooth muscle -actin in myofibroblasts. Binding of TEF family members to the MCAT1 element can be antagonized by the single stranded DNA binding proteins Pur/ß.17 In addition, phosphorylation of SMAD3 facilitates its nuclear accumulation where it can interact with SRF or EF1.12,18 In contrast, in adult smooth muscle cells MCAT elements bind to TEF, which does not stimulate promoter activity. A number of signaling pathways have been shown to be important for expression of smooth muscle-specific proteins in adult smooth muscle cells including increases in intracellular calcium via voltage gated calcium channels, activation of RhoA and ROK, Gß subunits of G heterotrimeric proteins, and PKG.19–23 Together these molecules increase the activity of SRF complexes to activate transcription of the smooth muscle -actin gene. Complexes containing myocardin related transcription factors are shown, although other positive-acting SRF complexes likely play important roles in specific smooth muscle cell types.

	What Controls the Switch From MCAT-Dependent to MCAT-Independent Transcription Regulation During Development of Embryonic Smooth Muscle?

Top TEF and RTEF-1 Binding... What Controls the Switch... The MCAT Mutated Smooth... References

 
Mutation of the MCAT elements within the smooth muscle -actin promoter completely abolished expression of a transgene in early embryonic smooth muscle, before E13.5, but did not affect expression later in development or in adult tissues. The smooth muscle -actin promoter thus undergoes a dramatic switch between embryonic day 12.5 and day 13.5 from being dependent on MCAT elements and presumably RTEF-1 for activation, to a gene that is totally independent of these elements. Most surprising is the finding that this switch seems to occur in all smooth muscle tissues, both vascular and visceral. This raises the intriguing questions as to what happens at this stage of development to initiate this switch. Is this specific for smooth muscle -actin or are other genes coordinately regulated? Although we obviously do not yet have answers to these questions, it seems reasonable to postulate that this switch occurs in response to changes in the microenvironment, such as a change in hormone signaling. One potential mechanism accounting for this switch would be a change in dependence of the differentiating smooth muscle cells on TGFß signaling. Embryonic smooth muscle cells may switch from being dependent on TGFß signaling before E13.5 to being largely independent of TGFß after this time. This would be analogous to the switch that endothelial cells undergo from being dependent on VEGF during early development to becoming VEGF independent in the adult. In support of this proposal, RTEF-1 binding to MCAT elements is required for TGFß-mediated induction of -actin in myofibroblasts and for -actin expression in embryonic smooth muscle cells. TGFß stimulation of myofibroblasts has also been shown to increase expression of serum response factor (SRF),⁷ which is critically required both for smooth muscle -actin expression in smooth muscle cells and for TGFß-mediated activation of smooth muscle -actin in myofibroblasts.^8,9 In addition, TGFß is known to be important for induction of smooth muscle differentiation both in vitro and in vivo.^10,11 However, although SRF is required to maintain the differentiated phenotype of smooth muscle cells throughout development, it is not clear whether TGFß is required, under normal conditions, in adult smooth muscle cells. We speculate that the switch from MCAT-dependent transcription of the smooth muscle -actin gene to MCAT-independent transcription may also reflect a switch from TGFß-dependent SRF complexes, such as SRF-SMAD3-EF1, to TGFß-independent complexes, such as SRF-myocardin (Figure).^12,13 As discussed by Gan and colleagues, a switch to SRF-myocardin complexes likely does not simply reflect an induction of myocardin expression, as myocardin mRNA is readily detectable in the gut by E12.5.¹⁴ A caveat to this, however, is that studies on myocardin protein expression have not been performed because of the low specificity of most available antibodies, hence it is not known whether myocardin protein expression directly parallels its mRNA levels.

Signaling pathways may also induce covalent modification of TEF or RTEF-1 or they may cause changes in chromatin structure to facilitate binding of TEF as compared with RTEF-1 to the MACT elements in the smooth muscle -actin promoter. Signaling-induced changes in chromatin structure, regulated by either ATP-dependent remodeling complexes or by changes in HAT or HDAC activity, would have the advantage that these changes could simultaneously alter the binding of a number of transcription factors, thereby facilitating a coordinated switch between different transcription regulatory complexes. This may allow an embryonic smooth muscle cell to switch from a myofibroblast type mode of regulation of smooth muscle -actin to a more adult smooth muscle type of mode (Figure). An exciting challenge of future investigations will be to capitalize on these exciting findings to elucidate the mechanisms controlling the switch from MCAT-dependent to MCAT-independent transcription during smooth muscle development.

	The MCAT Mutated Smooth Muscle -Actin Promoter Provides a Valuable Tool for Smooth Muscle-Specific Gene Targeting

Top TEF and RTEF-1 Binding... What Controls the Switch... The MCAT Mutated Smooth... References

 
Gan and colleagues report that the MCAT mutated smooth muscle -actin promoter failed to be expressed in embryonic skeletal or cardiac muscle. Besides indicating the importance of this element for smooth muscle -actin expression in these tissues, mutation of the MCAT element, resulted in the restriction of the expression of the altered promoter to smooth muscle cells throughout development. This finding has important implications for the use of the mutant promoter to target expression of proteins, such as cre recombinase, to smooth muscle tissues. To date all of the currently available smooth muscle-restricted promoters, used to target gene expression in vivo, have ectopic expression in other tissues, either in the heart and skeletal muscle during embryonic development (eg, SM22 and wild-type smooth muscle -actin promoters) or in eggs and sperm of transgenic mice (smooth muscle myosin heavy chain promoter).¹⁵ The MCAT mutated smooth muscle -actin promoter will thus provide a unique and very valuable tool for targeting gene expression to adult smooth muscle tissues.

	Acknowledgments

 
Sources of Funding

This work was supported by NIH grants HL58571, DK61130, and DK65644. Dr Zhou was partially supported by a fellowship from American Heart Association.

Disclosures

None.

	Footnotes

 
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

	References

Top TEF and RTEF-1 Binding... What Controls the Switch... The MCAT Mutated Smooth... References

 

1. Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev. 2004; 84: 767–801.[Abstract/Free Full Text]
2. McAnulty RJ. Fibroblasts and myofibroblasts: their source, function and role in disease. Int J Biochem Cell Biol. 2007; 39: 666–671.[CrossRef][Medline] [Order article via Infotrieve]
3. Clarke C, Sandle J, Lakhani SR. Myoepithelial cells: pathology, cell separation and markers of myoepithelial differentiation. J Mammary Gland Biol Neoplasia. 2005; 10: 273–280.[CrossRef][Medline] [Order article via Infotrieve]
4. Yamagishi S, Imaizumi T. Pericyte biology and diseases. Int J Tissue React. 2005; 27: 125–135.[Medline] [Order article via Infotrieve]
5. Gan Q, Yoshida T, Li J, Owens GK. Smooth muscle cells and myofibroblasts use distinct transcriptional mechanisms for smooth muscle -actin expression. Circ Res. 2007; 101: 883–892.[Abstract/Free Full Text]
6. Manabe I, Owens GK. The smooth muscle myosin heavy chain gene exhibits smooth muscle subtype-selective modular regulation in vivo. J Biol Chem. 2001; 276: 39076–39087.[Abstract/Free Full Text]
7. Hirschi KK, Lai L, Belaguli NS, Dean DA, Schwartz RJ, Zimmer WE. Transforming growth factor-beta induction of smooth muscle cell phenotpye requires transcriptional and post-transcriptional control of serum response factor. J Biol Chem. 2002; 277: 6287–6295.[Abstract/Free Full Text]
8. Mack CP, Owens GK. Regulation of smooth muscle alpha-actin expression in vivo is dependent on CArG elements within the 5' and first intron promoter regions. Circ Res. 1999; 84: 852–861.[Abstract/Free Full Text]
9. Hautmann MB, Adam PJ, Owens GK. Similarities and differences in smooth muscle alpha-actin induction by TGF-beta in smooth muscle versus non-smooth muscle cells. Arterioscler Thromb Vasc Biol. 1999; 19: 2049–2058.[Abstract/Free Full Text]
10. Sinha S, Hoofnagle MH, Kingston PA, McCanna ME, Owens GK. Transforming growth factor-beta1 signaling contributes to development of smooth muscle cells from embryonic stem cells. Am J Physiol Cell Physiol. 2004; 287: C1560–1568.[Abstract/Free Full Text]
11. Goumans MJ, Mummery C. Functional analysis of the TGFbeta receptor/Smad pathway through gene ablation in mice. Int J Dev Biol. 2000; 44: 253–265.[Medline] [Order article via Infotrieve]
12. Nishimura G, Manabe I, Tsushima K, Fujiu K, Oishi Y, Imai Y, Maemura K, Miyagishi M, Higashi Y, Kondoh H, Nagai R. DeltaEF1 mediates TGF-beta signaling in vascular smooth muscle cell differentiation. Dev Cell. 2006; 11: 93–104.[CrossRef][Medline] [Order article via Infotrieve]
13. Pipes GC, Creemers EE, Olson EN. The myocardin family of transcriptional coactivators: versatile regulators of cell growth, migration, and myogenesis. Genes Dev. 2006; 20: 1545–1556.[Abstract/Free Full Text]
14. Du KL, Ip HS, Li J, Chen M, Dandre F, Yu W, Lu MM, Owens GK, Parmacek MS. Myocardin is a critical serum response factor cofactor in the transcriptional program regulating smooth muscle cell differentiation. Mol Cell Biol. 2003; 23: 2425–2437.[Abstract/Free Full Text]
15. Wamhoff BR, Sinha S, Owens GK. Conditional mouse models to study developmental and pathophysiological gene function in muscle. Handb Exp Pharmacol. 2007; 178: 441–468.[Medline] [Order article via Infotrieve]
16. Li F, Luo Z, Huang W, Lu Q, Wilcox CS, Jose PA, Chen S. Response gene to complement 32, a novel regulator for transforming growth factor-beta-induced smooth muscle differentiation of neural crest cells. J Biol Chem. 2007; 282: 10133–10137.[Abstract/Free Full Text]
17. Kelm RJ Jr, Cogan JG, Elder PK, Strauch AR, Getz MJ. Molecular interactions between single-stranded DNA-binding proteins associated with an essential MCAT element in the mouse smooth muscle alpha-actin promoter. J Biol Chem. 1999; 274: 14238–14245.[Abstract/Free Full Text]
18. Qiu P, Feng XH, Li L. Interaction of Smad3 and SRF-associated complex mediates TGF-beta1 signals to regulate SM22 transcription during myofibroblast differentiation. J Mol Cell Cardiol. 2003; 35: 1407–1420.[CrossRef][Medline] [Order article via Infotrieve]
19. Mack CP, Somlyo AV, Hautmann M, Somlyo AP, Owens GK. Smooth muscle differentiation marker gene expression is regulated by RhoA-mediated actin polymerization. J Biol Chem. 2001; 276: 341–347.[Abstract/Free Full Text]
20. Wei L, Zhou W, Croissant JD, Johansen FE, Prywes R, Balasubramanyam A, Schwartz RJ. RhoA signaling via serum response factor plays an obligatory role in myogenic differentiation. J Biol Chem. 1998; 273: 30287–30294.[Abstract/Free Full Text]
21. Wamhoff BR, Bowles DK, McDonald OG, Sinha S, Somlyo AP, Somlyo AV, Owens GK. L-type voltage-gated Ca²⁺ channels modulate expression of smooth muscle differentiation marker genes via a Rho kinase/myocardin/SRF-dependent mechanism. Circ Res. 2004; 95: 406–414.[Abstract/Free Full Text]
22. Reusch HP, Schaefer M, Plum C, Schultz G, Paul M. Gbeta gamma mediate differentiation of vascular smooth muscle cells. J Biol Chem. 2001; 276: 19540–19547.[Abstract/Free Full Text]
23. Boerth NJ, Dey NB, Cornwell TL, Lincoln TM. Cyclic GMP-dependent protein kinase regulates vascular smooth muscle cell phenotype. J Vasc Res. 1997; 34: 245–259.[Medline] [Order article via Infotrieve]

Related Article:

Smooth Muscle Cells and Myofibroblasts Use Distinct Transcriptional Mechanisms for Smooth Muscle -Actin Expression

Qiong Gan, Tadashi Yoshida, Jian Li, and Gary K. Owens
Circ. Res. 2007 101: 883-892. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Herring, B. P. Articles by Zhou, J. Search for Related Content PubMed PubMed Citation Articles by Herring, B. P. Articles by Zhou, J. Related Collections Related Article