Nuclear proteins bind a cis-acting element in the smooth muscle alpha-actin promoter

Abstract

Identification of the regulators of smooth muscle cell (SMC) gene expression is critical to understanding SMC differentiation and alterations in SMC phenotype in vascular disease. Previous studies revealed positive transcriptional activity within the chicken smooth muscle (SM) alpha-actin promoter region from -209 to -257. In the present study, transient transfections of wild-type and mutant chicken SM alpha-actin promoter/reporter gene constructs into rat aortic SMC demonstrated that the positive transcriptional activity of this region was abolished with a two base pair mutation in a conserved sequence motif at -225 to -233 (TGTTTATC to TACTTATC). Electrophoretic mobility shift assays revealed that nuclear factors bound promoter fragments containing this sequence and that specific mutations in the TGTTTATC motif abolished nuclear factor binding. Studies thus provide evidence for binding of a nuclear factor to a positive cis-acting element within the SM alpha-actin promoter. Further characterization of this factor may contribute to a better understanding of the molecular mechanisms that regulate differentiation of SMC in vascular disease.

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

“…20 Mutation of the single E-box in this upstream region had no effect on the activity of the chicken SM ␣-actin promoter in rat aortic SMC, but the positive activity associated with this region was completely abolished by a 2-bp mutation in a conserved TGTTTATC sequence. 21 Of key importance, results from these studies using a heterologous system are in direct contrast with previous results with this same fragment in a homologous system 20 (chicken SM ␣-actin promoter in chicken SMC) and with a similar fragment of the rat SM ␣-actin promoter in rat SMC. 16 Unlike in the heterologous system, results in the homologous system defined a negative regulatory activity to this promoter region.…”

“…Previous studies have shown that mutation of essential nucleotides in the E-box flanking the TGTTTATC element at position Ϫ215 in the chicken SM ␣-actin promoter had no effect on promoter activity. 21 Additionally, in separate studies, we have shown that mutation of either 1 of the 2 E-boxes in the rat SM ␣-actin promoter alone did not affect promoter activity. However, specific mutations in both E-boxes modestly reduced SM ␣-actin promoter activity when transfected into rat aortic SMC.…”

“…Although we cannot rule out the fact that MSSP may be involved in the regulation of the rat SM ␣-actin promoter, these data suggest that it is not mediated by the TGTTTATC-CCCA element. MSSP may be an important factor accounting for the differences in our data when we tested the chicken SM ␣-actin promoter in rat SMC 21 versus the rat SM ␣-actin promoter in rat SMC (Figure 4). Taken together, these data provide evidence that the cis-and trans-acting factors that regulate the negative activity of the SM ␣-actin promoter are tissue type or species specific.…”

“…However, despite attempts using homology based library screening and the yeast 2-hybrid system, no SMC specific HLH factor has as yet been identified (Owens and McNamara, unpublished data, 1999). A number of ubiquitously expressed HLH factors are present in cultured SMC including Id, and the immunoglobulin transcription factor-1 protein, 21 upstream stimulatory factor-1 (USF-1) and USF-2. 23 Additionally, SMC within neointimal lesions formed after experimental vascular injury in animals and within human atherosclerotic plaques have also been shown to express Id.…”

Design of a muscle cell-specific expression vector utilising human vascular smooth muscle α-actin regulatory elements

“…20 Although the gene is transiently expressed in developing cardiac and skeletal muscle, and in myofibroblasts within tumours and healing wounds, 21,22 in adults it is constitutively expressed solely in VSMCs and smooth musclerelated cells. 23 A number of elements controlling the transcription of VSMC ␣-actin have been defined in the chicken, 24,25 rat, 26,27 mouse [28][29][30] and human [31][32][33] (schematic in Figure 1a). The sequences immediately flanking the VSMC ␣-actin TATA box are highly conserved in human (89% Vs rat), mouse (98% Vs rat), rat and chicken (73% Vs rat) between approximately −255 and +12.…”