Smooth muscle alpha-actin gene requires two E-boxes for proper expression in vivo and is a target of class I basic helix-loop-helix proteins.
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 alpha-actin (SM alpha-actin), in vivo. Mutation of one or both E-boxes significantly reduced the expression of a -2560- to 2784-bp SM alpha-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 alpha-actin regulation. In cotransfection experiments, E12, HEB, and E2-2 activated the SM alpha-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 alpha-actin promoter. Finally, we demonstrated that E2A proteins (E12/E47) specifically bound the E-box-containing region of the SM alpha-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 alpha-actin regulation and suggest that these factors may play an important role in control of SMC differentiation and phenotypic modulation.
Kumar
Meena S
MS
Department of Molecular Physiology and Biological Physics, University of Virginia, 415 Lane Rd, MR5 Room 1220, PO Box 801394, Charlottesville, Va 22908, USA.
Hendrix
Jennifer A
JA
Johnson
A Daniel
AD
Owens
Gary K
GK
D016475:3T3 Cells; D000199:Actins / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005786:Gene Expression Regulation; D018257:Helix-Loop-Helix Motifs / Q000235:genetics; D006801:Humans; D051797:Inhibitor of Differentiation Protein 2; D007763:Lac Operon / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D009154:Mutation; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D012689:Sequence Homology, Nucleic Acid; D026362:Serum Response Factor / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D051879:Twist-Related Protein 1
D000199:Actins; D051792:Basic Helix-Loop-Helix Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D051797:Inhibitor of Differentiation Protein 2; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D026362:Serum Response Factor; D014157:Transcription Factors; D051879:Twist-Related Protein 1; C497450:ID2 protein, human; C497451:Id2 protein, rat; C497452:Idb2 protein, mouse; C103173:TWIST1 protein, human