Expression of the Smooth Muscle Myosin Heavy Chain Gene Is Regulated by a Negative-acting GC-rich Element Located between Two Positive-acting Serum Response Factor-binding Elements

Abstract

To identify cis-and trans-acting factors that regulate smooth muscle-specific gene expression, we studied the smooth muscle myosin heavy chain gene, a rigorous marker of differentiated smooth muscle. A comparison of smooth muscle myosin heavy chain promoter sequences from multiple species revealed the presence of a highly conserved 227-base pair domain (nucleotides ؊1321 to ؊1095 in rat). Results of a deletion analysis of a 4.3-kilobase pair segment of the rat promoter (nucleotides ؊4220 to ؉88) demonstrated that this domain was necessary for maximal transcriptional activity in smooth muscle cells. Gel-shift analysis and site-directed mutagenesis demonstrated that one true CArG and another CArG-like element contained within this domain were both recognized by the serum response factor and were both required for the positive activity attributable to this domain. Additional studies demonstrated that mutation of a GC-rich sequence within the 227-base pair conserved domain resulted in a nearly 100% increase in transcriptional activity. Gel-shift analysis showed that this GC-rich repressor element was recognized by both Sp1 and Sp3. These data demonstrate that transcriptional control of the smooth muscle myosin heavy chain gene is highly complex, involving both negative and positive regulatory elements, including CArG sequences found in the promoters of multiple smooth muscle differentiation marker genes.Intimal migration and proliferation of vascular smooth muscle cells (SMCs) 1 are known to play an integral role in development of atherosclerotic disease (1, 2), and numerous factors have been identified that have growth promoting and/or chemotactic activity for SMCs (2). An additional feature of SMCs within atherosclerotic lesions is that cells exhibit marked differences in morphology and protein expression patterns as compared with normal medial SMCs (3-6), a process referred to as "phenotypic modulation" (7). This is characterized by decreased expression of proteins that are characteristic of differentiated SMCs, including the SM isoforms of contractile proteins, as well as altered growth regulatory properties, lipid metabolism, matrix production, and decreased contractility (reviewed in Ref. 8). Of particular significance, many of these phenotypic alterations in intimal SMCs cannot simply be viewed as a consequence of atherosclerotic disease, but rather are likely to play a major role in its development and/or progression.Although the SMC exhibits a high degree of plasticity and, unlike skeletal and cardiac myocytes, does not undergo terminal differentiation, it is a very specialized cell, whose differentiated function is dependent upon the coordinate regulation of a large number of cell type-specific/selective products, including contractile proteins, receptors, signal transduction molecules, and ion channels (8). Contractile proteins that are highly abundant and specific to the SMC represent obvious candidates for studying molecular control of SMC differentiation. Consequently, it is not unexpected that the bes...

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