Characterization of an E-box–Dependent cis Element in the Smooth Muscle α-Actin Promoter

Abstract

Abstract-Identification of the regulators of smooth muscle specific gene expression is critical for understanding smooth muscle cell (SMC) differentiation and the alterations in SMC phenotype seen in vascular diseases. Previous studies have identified that a 2-bp mutation in a conserved cis-acting element (TGTTTATC) in the promoter of the chicken smooth muscle (SM) ␣-actin gene abolished nuclear factor binding and decreased transcriptional activity of a 271-bp SM ␣-actin promoter fragment when transfected into rat aortic SMC. However, the promoter region containing this conserved sequence has negative cis regulatory activity when studied in homologous systems. The goal of the present studies was to further characterize the transcriptional activity of the rat SM ␣-actin promoter region between -224 and -236 that is conserved across mammals. DNAse I analysis and electrophoretic mobility shift assays demonstrated that SMC nuclear proteins bound an extended sequence (TGTTTATCCCCATAA). Transient transfection experiments of wild-type and mutant rat SM ␣-actin promoter-luciferase constructs into rat aortic SMC revealed that promoter activity was enhanced by mutations of specific nucleotides in the TGTTTATCCCCA region. Interestingly, the TGTTTATCCCCA element in the rat SM ␣-actin promoter is centered between 2 canonical E-boxes. Mutations of the flanking E-boxes abolished the enhancement in promoter activity seen with mutation of the TGTTTATCCCCA element alone. Thus studies provide evidence for a regulatory cassette in the rat SM ␣-actin promoter that regulates gene expression via combinatorial interactions between 2 E-boxes and a newly described TGTTTATCCCCA element.

Id3 Is a Novel Atheroprotective Factor Containing a Functionally Significant Single-Nucleotide Polymorphism Associated With Intima–Media Thickness in Humans

“…As a readout of transcriptional activity, the smooth muscle alpha actin (SMaA) promoter, which has previously been shown to be activated by E12, in an E-box dependent manner, was used9, 23. As anticipated, transfection of E12 resulted in activation of the SMaA promoter, an effect which was inhibited by Id3105A in a concentration-dependent manner.…”

Krüppel-like Factor 4 (KLF4/GKLF) Is a Target of Bone Morphogenetic Proteins and Transforming Growth Factor β1 in the Regulation of Vascular Smooth Muscle Cell Phenotype

“…Four independent experiments were carried out. Experiments were normalized to total protein as because we and others have observed that viral promoters interfere with SM differentiation-marker promoter responsiveness due to interactions with CArG boxes present in these promoters (37). Data were analyzed using Student's t test, and differences were considered significant for p Ͻ 0.05.…”

MKL1 mediates TGF-β1-induced α-smooth muscle actin expression in human renal epithelial cells

“…Also, the ␣-SMA promoter of different species contains a number of highly conserved regions with consensus binding motifs for transcription factors. The 5Ј-flanking sequence upstream of the TATA box contains several cis-acting elements including two different CC(A/T-rich) 6 GG (CArG) boxes (named A and B), a MCAT motif, an E box, a TGF-␤1 control element, a TGF-␤1 hypersensitivity region, and a conserved TGTTTATC sequence (5,17,19,25,28,35). Recent studies indicate that different regulatory mechanisms induce ␣-SMA promoter activity in renal epithelial cells that convert to myofibroblasts during EMT (12,54,62).…”