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(Circulation Research. 2003;92:840.)
© 2003 American Heart Association, Inc.

Molecular Medicine

Smooth Muscle -Actin Gene Requires Two E-Boxes for Proper Expression In Vivo and Is a Target of Class I Basic Helix-Loop-Helix Proteins

Meena S. Kumar, Jennifer A. Hendrix, A. Daniel Johnson, Gary K. Owens

From the Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville. Current affiliation for A.D.J. is Department of Biology, Wake Forest University, Winston-Salem, NC.

Correspondence to Gary K. Owens, PhD, Department of Molecular Physiology and Biological Physics, University of Virginia, 415 Lane Rd, MR5 Room 1220, PO Box 801394, Charlottesville, VA 22908. E-mail gko{at}virginia.edu

Changes in the differentiated state of smooth muscle cells (SMCs) play a key role in vascular diseases, yet the mechanisms controlling SMC differentiation are still largely undefined. We addressed the role of basic helix-loop-helix (bHLH) proteins in SMC differentiation by first determining the role of two E-box (CAnnTG) motifs, binding sites for bHLH proteins, in the transcriptional regulation of the SMC differentiation marker gene, smooth muscle -actin (SM -actin), in vivo. Mutation of one or both E-boxes significantly reduced the expression of a -2560- to 2784-bp SM -actin promoter/LacZ reporter gene in vivo in transgenic mice. We then determined the potential role of class I bHLH proteins, E12, E47, HEB, and E2-2, in SM -actin regulation. In cotransfection experiments, E12, HEB, and E2-2 activated the SM -actin promoter. Activation by HEB and E2-2 was synergistic with serum response factor. Additionally, the dominant-negative/inhibitory HLH proteins, Id2, Id3, and Twist, inhibited both the E12 and serum response factor–induced activations of the SM -actin promoter. Finally, we demonstrated that E2A proteins (E12/E47) specifically bound the E-box–containing region of the SM -actin promoter in vivo in the context of intact chromatin in SMCs. Taken together, these results provide the first evidence of E-box–dependent regulation of a SMC differentiation marker gene in vivo in transgenic mice. Moreover, they demonstrate a potential role for class I bHLH factors and their inhibitors, Id and Twist, in SM -actin regulation and suggest that these factors may play an important role in control of SMC differentiation and phenotypic modulation.

Key Words: smooth muscle -actin • transgenic mice • E-box • basic helix-loop-helix protein

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