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

Circ Res. 2003 May 2;92(8):840-7. doi: 10.1161/01.RES.0000069031.55281.7C. Epub 2003 Mar 27.

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 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3T3 Cells
  • Actins / genetics*
  • Animals
  • Base Sequence
  • Basic Helix-Loop-Helix Transcription Factors
  • Binding Sites / genetics
  • Cells, Cultured
  • Chromatin / genetics
  • Chromatin / metabolism
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Gene Expression
  • Gene Expression Regulation
  • Helix-Loop-Helix Motifs / genetics
  • Humans
  • Inhibitor of Differentiation Protein 2
  • Lac Operon / genetics
  • Mice
  • Mice, Transgenic
  • Muscle, Smooth, Vascular / cytology
  • Muscle, Smooth, Vascular / metabolism*
  • Mutation
  • Nuclear Proteins*
  • Promoter Regions, Genetic / genetics*
  • Protein Binding
  • Rats
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Repressor Proteins*
  • Sequence Homology, Nucleic Acid
  • Serum Response Factor / genetics
  • Serum Response Factor / metabolism
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Transfection
  • Twist-Related Protein 1

Substances

  • Actins
  • Basic Helix-Loop-Helix Transcription Factors
  • Chromatin
  • DNA-Binding Proteins
  • ID2 protein, human
  • Id2 protein, rat
  • Idb2 protein, mouse
  • Inhibitor of Differentiation Protein 2
  • Nuclear Proteins
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • Serum Response Factor
  • TWIST1 protein, human
  • Transcription Factors
  • Twist-Related Protein 1