An A/T-rich cis-element is essential for rat angiotensin II type 1A receptor transcription in vascular smooth muscle...

Transcriptional mechanisms regulating the expression of the rat angiotensin II type 1A receptor (rAT1AR) gene were investigated in cultured rat vascular smooth muscle cells (VSMC). Transcriptional analyses of various 5'-deletion mutants of the rAT1AR promoter region, fused upstream from the firefly luciferase gene, demonstrated that a 71 base pair (bp) region (-557 to -486 bp, with respect to transcription initiation) was necessary for expression of this gene in VSMC. Electrophoretic mobility shift assays demonstrated that specific protein-DNA complexes were formed with the -516 to -486 bp region of the rAT1AR promoter when incubated with VSMC extract. Computer analysis of this region indicated the presence of an A/T-rich sequence (i.e., TTTAAAAATAAA) which is similar to a myocyte enhancer binding factor 2 (MEF2) cis-regulatory element (i.e., CTTAAAAATAAC). Site-directed mutagenesis of this A/T-rich sequence inhibited rAT1AR promoter activity in VSMC, suggesting that this region was necessary for expression of this gene in these cells. Immuno-gel shift experiments suggest that MEF2 heterodimers may interact with the A/T-rich sequence in the rAT1AR promoter. Additionally, it was demonstrated that a transcription factor non-homologous to MEF2 can also interact with this A/T-rich site in the rAT1AR promoter. Taken together, our results suggest that MEF2 heterodimers, and/or transcription factors non-homologous to MEF2, are required to regulate the expression of the rAT1AR gene in VSMC.