Role of intron 1 in smooth muscle α-actin transcriptional regulation in activated mesangial cells in vivo

Background. The activation of glomerular mesangial cells is one of the early, important features of progressive glomerular disease. Smooth muscle alpha-actin (SM alpha A) is an excellent marker of activated mesangial cells. However, the mechanisms of SM alpha A regulation are only available from in vitro investigation. Methods. We examined in vivo promoter analysis of the SM alpha A gene-utilizing transgenic mice harboring different promoter regions of the SM alpha A gene fused to chloramphenicol acetyl transferase (CAT). CAT activities were tested in primary cultured mesangial cells and in glomerular legions of Habu venom glomerulonephritis. Results. The DNA sequence -891 to +3828, which contains exon 1, intron 1, and the first 14 bp of exon 2 in addition to the 5'-flanking sequence of the SM alpha A gene, induced high levels of transcription in activated mesangial cells in in vivo habu venom glomerulonephritis and in cultured mesangial cells derived from transgenic mice. The DNA region -891 to -124 was a positive element in mesangial cells derived from transgenic mice. Deletions (3316 or 137 bp) in intron 1 reduced transcription to undetectable levels. The 137 bp sequence is highly conserved among several species, containing one CArG box element, which is one of the key motifs for transcriptional activation of contractile-related proteins. In vitro transfection analysis failed to demonstrate these positive effects of intron 1 and region -891 to -124. Conclusions. In vivo promoter analysis of the SM alpha A gene provided new information about the transcriptional regulation of SMaA in activated mesangial cells. The DNA region -891 to -124 has a positive effect on SM alpha A transcription in cultured mesangial cells. The intron 1 region (+1088 to +1224) plays a pivotal role in SM alpha A transcription in activated mesangial cells in vivo. Further analysis of this conserved region in intron 1, including the CArG motif, will be of great value in understanding the molecular mechanisms of mesangial activation.