Regulation of smooth muscle alpha-actin expression in vivo is dependent onCArG elements within the 5 ' and first intron promoter regions

The aims of the present studies were to define sufficient promoter sequence s required to drive endogenous expression of smooth muscle (SM) alpha-actin and to determine whether regulation of SM a-actin expression in vivo is de pendent on CArG (CC(A/T)(6)GG) cis elements. Promoter deletions and site di rected mutagenesis techniques were used to study gene regulation in transge nic mice as well as in smooth muscle cell (SMC) cultures. Results demonstra ted that a Lac Z transgene that contained 547 bp of the 5' rat SM alpha-act in promoter was sufficient to drive embryonic expression of SM cu-actin in the heart and in skeletal muscle but not in SMCs. Transient transfections i nto SMC cultures demonstrated that the conserved CArG element in the first intron had significant positive activity, and gel shift analyses demonstrat ed that the intronic CArG bound serum response factor. A transgene construc t from -2600 through the first intron (p2600Int/Lac Z) was expressed in emb ryos and adults in a pattern that closely mimicked endogenous SM alpha-acti n expression. Expression in adult mice was completely restricted to SMCs an d was detected in esophagus, stomach, intestine. lung, and nearly all blood vessels, including coronary, mesenteric, and renal vascular beds. Mutation of CArG B completely inhibited expression in all cell types, whereas mutat ion of the intronic CArG selectively abolished expression in SMCs, which su ggests that it may act as an SMC-specific enhancer-like element. Taken toge ther, these results provide the first in vivo evidence for the importance o f multiple CArG cis elements in the regulation of SM cu-actin expression.