SM-20 is a novel growth factor-induced, 40kD protein originally identified in vascular smooth muscle cells (VSMC). In the normal vessel wall, SM-20 is found only in medial VSMC; it is induced in intimal VSMC after balloon injury. SM-20 is also expressed in cardiac and skeletal muscle and in certain epithelial and nerve cells. In cultured VSMC, SM-20 immunostaining demonstrates a striking association with cytoplasmic filaments in a pattern distinct from alpha-actin staining. The current proposal is designed to determine the general function of SM20, the identity of the SM-20-associated filaments, and the specific biologic role of SM-20 in VSMC. The applicant has recently isolated cDNA and genomic clones of the Drosophila SM-20 homologue.
Aim 1 will utilize Drosophila genetics to examine the general function of SM-20 in an intact organism. To test the hypothesis that SM-20 is required in the development of normal Drosophila tissues, a reverse genetics approach will be employed to isolate mutant flies which do not express SM-20. The gene will be mapped to its chromosomal location and flies carrying nearby mutations, small deficiencies or P-element insertions will be used to generate SM-20 deficient flies. The phenotype of these flies will be examined and SM-20 will be reintroduced into transgenic flies to demonstrate that SM-20 expression rescues the mutant phenotype.
Aim 2 will focus on SM-20's biologic role in VSMC. Based upon the association of SM-20 with cytoplasmic filaments and its induction by growth factors and vessel injury, the applicant hypothesizes that SM-20 is a cytoskeletal protein likely to be involved in migration and growth or in maintaining the structure and shape of VSMC.
This aim has two parts. The first is directed at characterizing the SM-20-associated filaments. It will determine whether SM-20 is a component of a known filament or comprises a new VSMC filament. If a component of a known filament, the aim will characterize its interaction with other components of the filament. If novel, it will determine the characteristics of the new filament. The second part will employ antisense strategies to examine the biologic role of SM-20 in cultured VSMC. These antisense techniques will also be used to examine the role of SM-20 in filament assembly. These studies should provide substantial information on a novel molecule that may play an important role in the development and maintenance of the vasculature and may provide a new VSMC-specific target within the vessel wall.
