A Serum Response Factor-Dependent Transcriptional Regulatory Program Identifies Distinct Smooth Muscle Cell Sublineages

Abstract

The SM22␣ promoter has been used as a model system to define the molecular mechanisms that regulate smooth muscle cell (SMC) specific gene expression during mammalian development. The SM22␣ gene is expressed exclusively in vascular and visceral SMCs during postnatal development and is transiently expressed in the heart and somites during embryogenesis. Analysis of the SM22␣ promoter in transgenic mice revealed that 280 bp of 5 flanking sequence is sufficient to restrict expression of the lacZ reporter gene to arterial SMCs and the myotomal component of the somites. DNase I footprint and electrophoretic mobility shift analyses revealed that the SM22␣ promoter contains six nuclear protein binding sites (designated smooth muscle elements [SMEs] -1 to -6, respectively), two of which bind serum response factor (SRF) (SME-1 and SME-4). Mutational analyses demonstrated that a two-nucleotide substitution that selectively eliminates SRF binding to SME-4 decreases SM22␣ promoter activity in arterial SMCs by approximately 90%. Moreover, mutations that abolish binding of SRF to SME-1 and SME-4 or mutations that eliminate each SME-3 binding activity totally abolished SM22␣ promoter activity in the arterial SMCs and somites of transgenic mice. Finally, we have shown that a multimerized copy of SME-4 (bp ؊190 to ؊110) when linked to the minimal SM22␣ promoter (bp ؊90 to ؉41) is necessary and sufficient to direct high-level transcription in an SMC lineagerestricted fashion. Taken together, these data demonstrate that distinct transcriptional regulatory programs control SM22␣ gene expression in arterial versus visceral SMCs. Moreover, these data are consistent with a model in which combinatorial interactions between SRF and other transcription factors that bind to SME-4 (and that bind directly to SRF) activate transcription of the SM22␣ gene in arterial SMCs.Smooth muscle (SM) cells (SMCs) play important roles in organ systems throughout the body, subserving such diverse functions as modulation of arterial tone, controlling gastrointestinal motility, and regulating airway resistance. The unique functional capacities of this muscle cell lineage are determined by the expression of distinct sets of tissue-specific genes encoding contractile proteins, cell surface receptors, and intracellular enzymes (48). A feature that distinguishes the SMC lineage(s) from the striated muscle cell lineages is the capacity of SMCs to reversibly modulate their phenotype during postnatal development (13,39,49,62). For example, vascular SMCs located in the arterial tunica media are maintained in the resting, or G 0 /G 1 , phase of the cell cycle and express high levels of contractile protein isoforms (55). However, in response to vessel wall injury and the concomitant release of growth factors, these cells reenter the cell cycle, proliferate, and modulate their phenotype from primarily contractile to primarily synthetic (13,49,62). This phenotypic modulation has been implicated in the pathogenesis of cardiovascular diseases, including atherosclerosis ...

Transcriptional targeting of replication-defective adenovirus transgene expression to smooth muscle cells in vivo.

“…In all experiments, only third passage primary rat aortic SMCs were used. Immortalized rat vascular A7r5 SMCs, passage 4 human umbilical vein endothelial cells (HUVECs), and mouse NIH 3T3 fibroblasts were grown as described previously (31). Cells were placed in medium containing 2% FBS and infected with either 1, 10, or 100 PFU/cell of purified adenoviral stocks.…”

“…The 441-bp murine SM22 ␣ promoter has been shown previously to program arterial SMC-specific gene expression in transgenic mice (31,38,39). To test whether the SM22 ␣ promoter could be used to restrict the expression of a recombinant gene product encoded by an RDAd to SMCs, we constructed an RDAd (AdSM22-lacZ) containing the bacterial lacZ reporter gene under the transcriptional control of the murine SM22 ␣ promoter ( Fig.…”

“…The 441-bp murine SM22␣ promoter is active in embryonic skeletal muscle cells and the somites of transgenic mice (31). To determine whether AdSM22-lacZ programs transgene expression in adult skeletal muscle in vivo, 10 9 PFU of the AdSM22-lacZ virus was injected intramuscularly into the rat rectus abdominus and quadriceps muscles.…”

“…We have shown that the 441-bp SM22 ␣ promoter activates transcription exclusively in arterial SMCs during postnatal development in transgenic mice (31). To test whether the arterial SMC-specific SM22 ␣ promoter could be used to restrict the activity of an RDAd to SMCs, we generated AdSM22-lacZ, an RDAd containing the bacterial lacZ reporter gene under the transcriptional control of the 441-bp murine SM22 ␣ promoter.…”

Four isoforms of serum response factor that increase or inhibit smooth-muscle-specific promoter activity

“…These CArG boxes have been shown to be important in the control of gene expression in smooth-muscle cells in itro. Furthermore, promoter analysis in transgenic mice has shown that the CArG boxes in the smooth-muscle α-actin and SM22α promoters are required for the expression of reporter genes in vascular smooth-muscle cells (VSMCs) in the adult mouse [14][15][16]. The promoters of several cardiac-muscle genes also contain CArG boxes, implying a role for SRF in the control of cardiac-specific gene expression [17][18][19].…”