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.
Highlights
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
By comparing the 5Ј-flanking sequences of the rat, mouse, and rabbit smooth muscle myosin heavy chain (SM-MHC) genes, we identified two domains that were conserved among all three species
The most proximal domain encompassed the TATA box and contained two conserved CCTCCC elements. This region was previously studied in mouse, and the CCTCCC elements were shown to be necessary for the basal promoter activity ascribed to this domain [23]
Summary
Intimal migration and proliferation of vascular smooth muscle cells (SMCs) are known to play an integral role in development of atherosclerotic disease [1, 2], and numerous factors. 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 [3,4,5,6], 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). Immunohistochemical studies that discriminated between the SM-1 and SM-2 isoforms revealed that, in both rabbit and human aortae, SM-MHC protein was specific to the SMC and that SM-1 predominated early during embryonic development, with SM-2 appearing postnatally [18, 19] These studies indicate that, similar to smooth muscle ␣-actin, SM-MHC represents a highly rigorous SMC marker and a good candidate gene for discerning transcriptional mechanisms important for maintenance of the differentiated SMC phenotype
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