Leiomodin 1, a new serum response factor-dependent target gene expressed preferentially in differentiated smooth muscle cells

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.

FIGURE 1.

Leiomodin 1 mRNA and protein expression analysis. A, RT-PCR analysis of Lmod1, Lmod2, and Lmod3 in the indicated adult mouse tissues. Note the similar mRNA expression pattern between Lmod1 and Cnn1, a well known SMC-restricted gene (43). B, quantitative RT-PCR of Lmod1 mRNA in the same tissues as A. Expression levels are displayed as normalized fold increases over brain (set to 1). C, Western blot of LMOD1 protein expression in mouse tissues and in 3T3 cells transfected either with an empty control plasmid or an expression plasmid carrying Lmod1 (top panel). LMOD1 protein expression following preabsorption of the LMOD1 antibody with LMOD1 antigen (middle panel). D, endogenous LMOD1 protein expression in PAC1 SMCs following siRNA knockdown of LMOD1. E, immunohistochemistry of LMOD1 protein in aorta (panels a and e), bladder (panels b and f), brain (panels c and g), and skeletal muscle (panels d and h) using either the LMOD1 antibody (panels a–d) or a nonimmune, isotype-matched IgG control (panels e–h). The bar in panel h is 30 μm for all panels. The data shown in all of the panels are representative of three independent experiments. Arrows in panels c and d indicate LMOD1 positive blood vessels.

FIGURE 2.

LMOD1 protein expression in developing mouse embryos. Immunohistochemistry of LMOD1 protein expression (red stain) in sagittal sections of E9.5 (A), E13.5 (C and D), and E15.5 (E and F) mouse embryos. An isotype-matched IgG control antibody shows no staining of an E9.5 embryo (B). Similar lack of staining was seen with the IgG control applied to E13.5 and E15.5 embryo sections (not shown). ao, aorta; br, bronchiole; bv, blood vessel; he, heart; in, intestine; li, liver; lu, lung; st, stomach. The bars in A and B represent 100 μm, and the bars in C–F represent 500 μm.

FIGURE 3.

Functional analysis of human LMOD1 and LMOD2 promoters. A, VISTA plot indicating nucleotide sequence homology between human (Hsa), mouse (Mmu) and rat (Rno) LMOD1 over a 50-kb genomic interval. The x axis represents the human base sequence, and the y axis indicates the percentage of homology between Hsa versus Mmu (top plot) and Hsa versus Rno (bottom plot). The bent arrow at the top represents the inferred transcription start site. Light teal peaks represent untranslated regions; dark blue peaks represent protein-coding exons; and pink peaks are the conserved nonprotein coding sequences. Sequence logos of two CArG-like elements near LMOD1 are shown below the VISTA plots and illustrate the conservation of each CArG box across six vertebrate species. B and C, LMOD1 and LMOD2 promoter activity in PAC1 SMCs transfected either with SRF-VP16 (B) or MYOCD_v3 (C). The promoter activity reported here and below is a ratio between luciferase and Renilla normalized to that of the pGL3 basic plasmid set to 1. Similar SRF/MYOCD-mediated promoter activity was observed in at least three independent studies.

FIGURE 4.

SRF binding to LMOD1 CArG elements. ³²P-Labeled double-stranded oligonucleotides containing CArG elements from CNN1 and LMOD1 promoter CArG Far and CArG Near were incubated with the in vitro translated (IVT) SRF in the presence of SRF or MEF2A antibody or a 100-fold molar excess of unlabeled wild type (WT) or mutant (Mut) oligonucleotide. SRF-CArG nucleoprotein complex (SRF) and supershift (SS) are indicated with arrows. Exposure times for each EMSA were 11 h (for CNN1) or 64 h (for LMOD1).

FIGURE 5.

Functional analysis of LMOD1 CArG elements in vitro. A, schematic of the wild type (WT) LMOD1 promoter and various CArG mutants. B and C, each indicated LMOD1 promoter construct was transfected into PAC-1 SMCs in the presence of SRF-VP16 (B) or MYOCD_v3 (C), and luciferase activity was measured. D, COS-7 cells were similarly transfected with WT or double CArG mutant LMOD1 promoter and either MYOCD_v3, MRTF-A, or MRTF-B. The results shown were reproduced in one independent experiment. *, p < 0.001; **, p < 0.01.

FIGURE 6.

LMOD1 expression in SRF-deficient cells and tissues. RT-PCR analysis of Lmod1 and Srf mRNA either in PAC-1 SMC transduced with shEGFP or shSRF for 72 h (A) or indicated tissues from adult mice carrying homofloxed Srf alleles and the tamoxifen (Tmx)-inducible Myh11-Cre driver (34) (B). Note the reduction in both Srf and Lmod1 mRNA in tissues derived from mice treated with Tmx (to activate Cre recombinase and effect excision of the Srf gene) versus the vehicle control (Oil). The latter results were extended by comparing protein expression of SRF (C, panels a and b) versus LMOD1 (C, panels c and d) in similar mice treated either with Tmx (C, panels b and d) or sunflower oil (C, panels a and c). The bar in panel d of C is 30 μm for all panels. The images in A and B were inverted in Adobe Photoshop so as to better indicate the bands in each gel.

FIGURE 7.

LMOD1 mRNA and protein expression in human cells overexpressing MYOCD. A, the indicated human cell lines were transduced with equal amounts of adenovirus carrying MYOCD or LacZ and endogenous LMOD1 and LMOD2 mRNA expression assessed by RT-PCR. B, same experiment as in A only LMOD1 and MYOCD protein expression were determined using Western blotting. This result was reproduced in an independent experiment.

FIGURE 8.

LMOD1 promoter activity in transgenic mice. Sagittal E12.5 day mouse embryos stained either with β-galactosidase to assess LMOD1 promoter activity (A–C and E–G) or an antibody to LMOD1 (D) or control IgG (H). Shown are three WT LMOD1 promoter mice (A–C) and three double CArG mutant LMOD1 promoter mice (E–G). The thick arrows indicate the heart, and the thinner arrows point to aorta and vessels of head. The bar in H is 1 mm for all panels. Eight of 25 wild type founders displayed promoter activity in cardiac muscle, and five of 25 showed promoter activity in vascular tissue. In contrast none of the 10 CArG mutant founders showed LMOD1 promoter activity in cardiac muscle or vascular tissue.