Abstract
Smooth muscle cell (SMC) differentiation is defined largely by a number of cell-restricted genes governed directly by the serum response factor (SRF)/myocardin (MYOCD) transcriptional switch. Here, we describe a new SRF/MYOCD-dependent, SMC-restricted gene known as Leiomodin 1 (Lmod1). Conventional and quantitative RT-PCRs indicate that Lmod1 mRNA expression is enriched in SMC-containing tissues of the mouse, whereas its two paralogs, Lmod2 and Lmod3, exhibit abundant expression in skeletal and cardiac muscle with very low levels in SMC-containing tissues. Western blotting and immunostaining of various adult and embryonic mouse tissues further confirm SMC-specific expression of the LMOD1 protein. Comparative genomic analysis of the human LMOD1 and LMOD2 genes with their respective mouse and rat orthologs shows high conservation between the three exons and several noncoding sequences, including the immediate 5' promoter region. Two conserved CArG boxes are present in both the LMOD1 and LMOD2 promoter regions, although LMOD1 displays much higher promoter activity and is more responsive to SRF/MYOCD stimulation. Gel shift assays demonstrate clear binding between SRF and the two CArG boxes in human LMOD1. Although the CArG boxes in LMOD1 and LMOD2 are similar, only LMOD1 displays SRF or MYOCD-dependent activation. Transgenic mouse studies reveal wild type LMOD1 promoter activity in cardiac and vascular SMC. Such activity is abolished upon mutation of both CArG boxes. Collectively, these data demonstrate that Lmod1 is a new SMC-restricted SRF/MYOCD target gene.
Highlights
Smooth muscle cell (SMC) types are characterized by a growing number of cyto-contractile genes
To determine whether Leiomodin 1 (LMOD1) expression is modulated under conditions promoting SMC differentiation, we examined steady-state LMOD1 protein levels in Human coronary artery smooth muscle cells (HCASM) in growth or differentiation-inducing medium
Similar attenuated LMOD1 promoter activity is noted following transfection with MYOCD_v3, MRTF-A and MRTF-B (Fig. 5, C and D). These findings show that serum response factor (SRF) and members of the MYOCD family mediate LMOD1 promoter activity in a CArG-dependent fashion
Summary
Smooth muscle cell (SMC) types are characterized by a growing number of cyto-contractile genes. Smooth muscle cell (SMC) differentiation is defined largely by a number of cell-restricted genes governed directly by the serum response factor (SRF)/myocardin (MYOCD) transcriptional switch. Such activity is abolished upon mutation of both CArG boxes These data demonstrate that Lmod is a new SMC-restricted SRF/MYOCD target gene. Vascular and visceral SMCs are defined by the expression of a number of cell-restricted ion channels as well as signaling, cyto-contractile, and matrix-associated genes whose encoded proteins facilitate the major functions associated with the differentiated SMC phenotype [1]. Positioned CArG elements around the LMOD2 gene appear nonfunctional Together, these data demonstrate LMOD1 as a new, SMC-restricted member of the mammalian CArGome and highlight the importance of validating putative CArG-containing genes with wet lab assays
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