A novel smooth muscle-specific enhancer regulates transcription of the smooth muscle myosin heavy chain gene in vascular smooth muscle cells

Transient DNA transfection analysis of 5' end deletion mutants of the rabbit smooth muscle myosin heavy chain (SMHC) gene promoter was performed in primary cultures of rabbit vascular smooth muscle cells (VSMC), A positive element located at position -1,332 upstream of the transcription start site consistently gave the high est relative chloramphenicol acetyltransferase (CAT) activity (6.3 +/- 1.5-fold over the minimal SMHC promoter), suggesting that inclusion of the extra 107-base pair (bp) DNA fragment between -1,332 and -1,225 could significantly enhance CAT activity in VSMC. Transfection of mutants into several muscle and nonmuscle cell lines did not show any significant CAT activity above control, showing that factors unique to smooth muscle cells were required for SMHC expression, Gel shift analysis indicated that multiple factors interacted with the 107-bp element, two of which appeared to show smooth muscle specificity. Tests of enhancer function in transfected VSMC indicated that the 107-bp fragment behaved as a classical enhancer, i.e. independently of position and orientation. These results indicate that a novel DNA element may regulate the tissue-restricted expression of the SMHC gene and provides the first example of a role for a smooth muscle-specific enhancer in VSMC.