Control of smooth muscle development by the myocardin family of transcriptional coactivators

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Differentiation of smooth muscle cells (SMCs) is accompanied by the transcriptional activation of an array of muscle-specific genes that confer the unique contractile and physiologic properties of this muscle cell type. The majority of smooth muscle genes are controlled by serum response factor (SRF), a widely expressed transcription factor that also regulates genes involved in cell proliferation. Myocardin and myocardin-related transcription factors (MRTFs) interact with SRF and potently stimulate SRF-dependent transcription. Gain- and loss-of-function experiments have shown myocardin to be sufficient and necessary for SMC differentiation. SMCs are highly plastic and can switch between differentiated and proliferative states in response to extracellular cues. Suppression of SMC differentiation by growth factor signaling is mediated, at least in part, by the displacement of myocardin from SRF by growth factor-dependent ternary complex factors. The association of SRF with myocardin and MRTFs provides a molecular basis for the activation of SMC genes by SRF and the responsiveness of the smooth muscle differentiation program to growth factor signaling.

Introduction

Smooth muscle cells (SMCs) are required for the formation and function of the cardiovascular, respiratory, genitourinary, and gastrointestinal systems. In contrast to cardiac and skeletal muscle cells, which undergo terminal differentiation, SMCs are highly plastic and can modulate their phenotypes between proliferative and differentiated states in response to extracellular cues [1]. Abnormalities in vascular SMC differentiation are responsible for a variety of disorders, including atherosclerosis, asthma, vascular restenosis following angioplasty, and hypertension.

Serum response factor (SRF) is a widely expressed transcription factor required for smooth muscle (SM) gene expression and differentiation [2]. Paradoxically, SRF also regulates genes involved in cell proliferation, which opposes the SM differentiation program. The recent discovery of myocardin and myocardin-related transcription factors (MRTFs), which act as SRF coactivators 3., 4.•, has revealed a mechanism for the activation of SMC genes and a molecular basis for suppression of SMC differentiation by growth factor signals. In this review, we discuss the roles of the myocardin family in the control of SM differentiation and signal-dependent gene expression.

Section snippets

Diversity of smooth muscle cells

SMCs express a set of contractile proteins that are distinct from those expressed in skeletal and cardiac muscles [1]. Genes that are activated during SM differentiation include those encoding SM-myosin heavy chain, SM α-actin, SM22, SM-myosin light chain kinase, and calponin. Certain of these genes are expressed transiently in developing cardiac and skeletal muscle cell lineages during embryogenesis, whereas others are specific to the SMC.

SMCs are highly heterogeneous and arise throughout the

Regulation of smooth muscle cell gene expression by SRF

Nearly every SMC gene analyzed to date has been found to be controlled by SRF (reviewed in 1., 2.), a widely expressed MADS (MCM1, Agamous, Deficiens, SRF) box transcription factor first identified as a serum-responsive activator of the c-fos promoter [8]. SRF binds as a homodimer to a consensus sequence CC(A/T)6GG, known as a CArG box or serum response element. Whereas the c-fos promoter contains a single CArG box, most SM genes contain two or more CArG boxes, which act cooperatively to

SRF cofactors

SRF, like other MADS box proteins, has a propensity to associate with other transcription factors, providing for combinatorial control of SRF target genes [17]. Ternary complex factors (TCFs) of the ETS-domain family, including Elk-1, SAP-1a and NET/SAP-2/Erp, were among the first SRF cofactors identified [18]. These TCFs interact with the MADS box of SRF through a short hydrophobic stretch known as a B-box. Stable association of TCFs with SRF also requires an ETS binding site (GGAA/T)

The myocardin family of transcriptional coactivators

Myocardin was discovered in an in silico screen for novel genes expressed specifically in the heart [3]. Myocardin stimulates SRF activity by forming a ternary complex with SRF on DNA and providing its strong transcriptional activation domain (TAD) to SRF, which otherwise is a very weak activator of transcription. Myocardin preferentially activates promoters containing two or more CArG boxes, an observation consistent with the known cooperativity of CArG boxes associated with muscle genes.

Structural features of myocardin proteins

Myocardin belongs to the SAP (SAF-A/B, Acinus, PIAS) family of proteins, which play diverse roles in chromatin remodeling, transcriptional control, and fragmentation of DNA during apoptosis [31]. The 35-amino acid SAP domain has been predicted to adopt a helix-linker-helix structure with the potential to bind to DNA. The SAP domain of SAF-A binds to A/T rich genomic DNA associated with the nuclear matrix, the so-called nuclear matrix attachment region. Similarly, the SAP domain in myocardin is

Expression patterns of myocardin and MRTFs

During mouse embryogenesis, myocardin expression is first detected in cardiac precursor cells within the cardiac crescent at ~E7.5 and, thereafter, is maintained in cardiac myocytes in the atrial and ventricular chambers of the heart until adulthood [3]. In addition, myocardin is expressed in a subset of vascular and visceral smooth muscle cell types. Expression is especially robust in visceral SMCs of the stomach, bladder, and intestine, where myocardin expression precedes expression of smooth

Myocardin is necessary and sufficient for smooth muscle cell differentiation

Cardiac gene expression can be blocked in Xenopus embryos by injection of mRNA encoding a dominant negative mouse myocardin mutant into a ventral blastomere fated to form heart [3]. Similarly, expression of dominant negative myocardin in the P19CL6 teratocarcinoma cell line inhibits cardiogenesis [52]. While such studies suggest an essential role for myocardin in cardiogenesis, it is conceivable that a dominant negative myocardin mutant could simply perturb SRF activity and thereby block

Signaling pathways leading to SRF

Serum and purified growth factors stimulate SRF activity via two independent pathways, one dependent on phosphorylation of TCFs by a MAP kinase cascade, the other dependent on Rho signaling and actin dynamics [47] (Figure 3). Dominant negative MRTF mutants or inhibition of MRTF-A/B expression with RNA interference has been shown to specifically inhibit SRF-dependent activation of the c-fos promoter in response to serum and RhoA, whereas TCF-mediated activation was unaffected by the loss of MRTF

Antagonists of myocardin/MRTF activity

In principle, repressors of SRF activity should antagonize the activity of myocardin and MRTFs. There are multiple mechanisms through which such antagonism can occur. The small homeodomain-only protein HOP has been shown to suppress SRF activity by interfering with the ability of SRF to bind DNA. Accordingly, HOP can suppress myocardin-dependent activation of SRF 23., 24.. Similarly, GATA transcription factors act as powerful repressors of SRF activity by multiple mechanisms [49].

Switch between differentiation and proliferation of smooth muscle cells

How SRF selects between muscle-specific and growth-regulated genes is not fully understood. Any proposed mechanism for the activation of SM gene transcription must account for the ability of growth factor signals to reversibly suppress the differentiation program. In this regard, the reversible association of myocardin with SRF appears to provide a mechanism for plasticity of SM phenotypes [50].

Many, but not all, SM genes contain TCF binding sites near adjacent CArG boxes. In SMCs, myocardin

Conclusions and questions for the future

The discovery of myocardin and MRTFs has provided new models and mechanisms to account for the seemingly paradoxical roles of SRF in the control of cell proliferation and myogenesis, and has raised many intriguing questions for the future. For example, how is the expression of myocardin controlled within the earliest progenitors of the cardiac and SM lineages? What are the functions of MRTFs in vivo and to what extent are they redundant with myocardin? How do myocardin and MRTFs contribute to

Update

Following submission of this review, the following publications 53.•, 54.•, 55.• provided further insight into the functions of the myocardin family.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We thank Alisha Tizenor for assistance with graphics and Jennifer Page for editorial assistance. Work in the laboratory of EN Olson is supported by grants from the National Institutes of Health, The DW Reynolds Center for Clinical Cardiovascular Research, and The Robert A. Welch Foundation. D-Z Wang is a Basil O’Connor Scholar of The March of Dimes Birth Defects Foundation and is supported by the National Institutes of Health, Muscular Dystrophy Association and American Heart Association.

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