Leiomodin1, a novel SRF/MYOCD target gene preferentially expressed in vascular smooth muscle cells

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pharmacology and Physiology, 2014.Smooth muscle cell (SMC) differentiation is characterized by the expression of serum response factor (SRF)/myocardin (MYOCD) regulated contractile and cytoskeletal genes. Changes in the expression of these genes force SMCs to switch from displaying a characteristic contractile phenotype to a proliferative phenotype. Several studies have linked this phenotypic adaptation to various pathological diseases including vascular occlusive disorders, hypertension, heart failure and Alzheimer’s disease. SRF binds to >1200 permutations of a 10 base pair DNA sequence element known as a CArG box. This lab initiated the CArGome Project in 2004 to define all functional SRF-binding CArG boxes in the genome. One of the genes found to contain CArG boxes was Leiomodin1 (Lmod1). Lmod1 is one of three independently transcribed paralogous genes whose expression, regulation and function form the basis of this dissertation. Conventional and quantitative RT-PCR as well as Western blotting and immunohistochemistry indicate Lmod1 mRNA and protein expression is restricted to SMC-enriched tissues of the mouse. Using a bioinformatics approach, two conserved CArG boxes were found in the human LMOD1 promoter region. Luciferase assays reveal LMOD1 displays CArG-dependent SRF/MYOCD-induced activation. Consistent with these findings gel shift assays demonstrate binding between SRF and the two CArG boxes in human LMOD1. Transgenic mouse studies establish LMOD1 as a bonafide SRFCArG dependent promoter. In vivo ligation injury studies revealing attenuated Lmod1 mRNA and protein expression following injury offers novel evidence on LMOD1’s ability in facilitating a SMC contractile phenotype. However, in vitro studies demonstrating impairment in SMC proliferation and migration following endogenous LMOD1 knock down reveal otherwise. This contrast in findings demonstrates the need of performing more studies to fully determine whether or not LMOD1 is involved in facilitating a contractile SMC phenotype. Finally, immunofluorescence microscopy reveals endogenous LMOD1 knockdown compromises SMC actin filament assembly, a finding similar to other SMC marker proteins involved in contributing towards a SMC contractile phenotype. Taken together, these data demonstrate that Lmod1 is a novel SMC-restricted SRF/MYOCD target gene that appears to be required for facilitating a contractile SMC phenotype. Future studies should be directed towards the function of LMOD1 in normal and pathological processes associated with SMCs, particularly in the cardiovascular system