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

Abstract

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 α-actin (SM α-actin), in vivo. Mutation of one or both E-boxes significantly reduced the expression of a −2560- to 2784-bp SM α-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 α-actin regulation. In cotransfection experiments, E12, HEB, and E2-2 activated the SM α-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 α-actin promoter. Finally, we demonstrated that E2A proteins (E12/E47) specifically bound the E-box–containing region of the SM α-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 α-actin regulation and suggest that these factors may play an important role in control of SMC differentiation and phenotypic modulation.