Expression and Functional Characterization of a Novel Putative Serine Carboxypeptidase in Vascular Smooth Muscle Cells

Abstract

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Pathology and Laboratory Medicine, 2008.A number of proteases have been shown to alter vascular smooth muscle cell proliferation and migration thus contributing to a variety of vascular disorders. We previously identified from cultured vascular smooth muscle cells (SMC) a novel gene designated retinoid-inducible serine carboxypeptidase (RISC). However, subsequent work indicated the induction of RISC by retinoids was incidental. As a result, we refer to RISC herein as serine carboxypeptidase 1 (SCPEP1), the Human Genome Organization (HUGO) approved gene name. SCPEP1 bears the signature catalytic triad found in all serine carboxypeptidases in nature, but nothing is known as to its function. In this thesis, I have characterized SCPEP1’s distribution and biological activities in vitro and in vivo with special emphasis on vascular SMC. We demonstrate that SCPEP1 is abundantly expressed in a variety of organ systems from the embryonic stage to adulthood. We further show that 55-kDa full-length SCPEP1 is cleaved into a 35-kDa fragment that accumulates inside the lysosomal compartment. Subsequently, we find SCPEP1 is highly expressed in the neointima after ligation injury where SMC continue to display cell cycle activity, migratory ability or remodeling. To evaluate the functional role of SCPEP1 in cultured vascular SMC, adenoviral-mediated overexpression of SCPEP1 is shown to promote vascular SMC proliferation and migration in vitro. Such effects are abolished with either a short hairpin RNA to Scpep1 or catalytic triad mutant (S167A) Scpep1. Silent mutations in SCPEP1 that evade Scpep1 RNA knockdown completely rescue the growth- and migration-inducing effects. In addition, I discovered a mutant SCPEP1 that doesn’t undergo cleavage, but still works in pro-growth and pro-migration effects, suggesting that cleavage of SCPEP1 is not necessary for mediating SMC growth and migration. To evaluate SCPEP1 function in vivo, I generated a knockout mouse. Scpep1-/- mice are viable and fertile with no obvious abnormalities. However, Scpep1-/- mice exhibit significantly decreased vascular remodeling. Collectively, my dissertation data suggest that SCPEP1 promotes vascular SMC proliferation and migration in a catalytic triad-dependent, cleavage-independent manner. SCPEP1 may therefore represent a new therapeutic target for the treatment of vascular occlusive diseases in the future