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

Abstract

Key Words: Scpep1 Ⅲ smooth muscle Ⅲ neointima Ⅲ protease Ⅲ knockout S mooth muscle cells (SMCs) are critical for blood vessel homeostasis, but they also contribute to the pathogenesis of several vasculopathies. In response to arterial injury, SMCs shift from a quiescent, contractile phenotype to a proliferative, synthetic state that undermines normal arterial function leading to neointimal formation. 1,2 Myriad factors and cytokines, as well as proteases and their associated substrates, have been implicated in vascular pathology, 3-5 but additional proteases are likely involved in this process. For example, the serine carboxypeptidase cathepsin A (CTSA) cleaves a number of substrates (eg, endothelin-1) that effect pathological changes in the vessel wall. 6 -8 Recently, a mutant CTSA allele defective for enzyme activity was knocked into the wild-type (WT) locus of mice and shown to confer a decrease in the inactivation of endothelin-1, elevated arterial blood pressure, and altered elastogenesis. 9 Serine carboxypeptidases belong to the family of serine proteases and are most prevalent in the plant kingdom, where they function in numerous processes related to growth and development. 10 Three serine carboxypeptidases are found in mammals and each shares the same catalytic triad (serine, aspartic acid, and histidine) found in plant homologs. 10,11 We previously reported a novel serine carboxypeptidase from cultured SMCs in a screen for retinoid-induced genes. 12 We call this protease serine carboxypeptidase (SCPEP)1 because it contains several conserved domains common to all members of the serine carboxypeptidase family, including a substrate-binding domain and the catalytic triad. Northern blotting and in situ hybridization studies demonstrated Scpep1 mRNA in SMCs of the aorta and proximal convoluted tubular epithelium of the kidney. 12 More recently, we developed an antibody to SCPEP1 and showed its cleavage from a mature 55-kDa isoform to a 35-kDa isoform in all adult mouse tissues studied, including vascular SMCs and renal proximal convoluted tubular epithelium. 13 The biological substrates for SCPEP1, however, remain a mystery. We therefore consider SCPEP1 an orphan protease. Here, we have performed gain-and loss-of-function studies in vitro and in vivo to provide the first biological insight into SCPEP1 function. was extracted from cultured SMCs and tissues with TRIzol reagent (Invitrogen). RT-PCR was performed using the ProSTAR System (Stratagene). Short hairpin RNAs were generated as described. 14 SCPEP1 mutagenesis was performed using a QuikChange sitedirected mutagenesis kit (Stratagene). All constructs were then incorporated into adenovirus (Invitrogen). The animal protocol was approved by the Institutional Animal Care and Use Committee at the University of Rochester. Scpep1 knockout (KO) mice were generated through the University of Rochester Transgenic Core and back-crossed to C57BL/6 mice. Twelve-week-old Scpep1 KO mice or WT littermates were subjected to complete common carotid artery ligation as d...

Genome‐wide study on intramuscular fat in Italian Large White pig breed using the PorcineSNP60 BeadChip

“…SCPEP1 gene was originally identified in rat aortic smooth muscle cells by screening for retinoid-inducible genes (Chen et al 2001). Lee et al (2009) study demonstrates a role for SCPEP1 activity in modulating smooth muscle proliferation, migration and vascular remodelling. Nothing is known about SCPEP1 gene and function on skeletal muscle but further studies are needed to clarify the role of this serine carboxypeptidase in this specific tissue.…”

Serine Carboxypeptidase SCPEP1 and Cathepsin A Play Complementary Roles in Regulation of Vasoconstriction via Inactivation of Endothelin-1

“…Later study by Lee at al. [27] reported that the Scpep1 -null mice generated by replacing exons 1 and 2 of the Scpep1 gene with Neo cassette show a decrease in medial and intimal cell proliferation as well as in vessel remodelling following arterial injury. The same study also reported that a ∼50% knockdown of endogenous Scpep1 in mouse ASMC line showed dramatic decrease in serum-stimulated growth.…”

Vascular smooth muscle cell differentiation – 2010

“…Despite extensive surveying and testing, there are no known substrates for SCPEP1 making this protein an “orphan protease”. Genetic inactivation of SCPEP1 does not show an overt phenotype as mice survive and breed without any histological evidence of pathology[109],[110]. However, upon ligation injury of the carotid artery, SCPEP1 null mice show reduced neointimal load suggesting that SCPEP1 directs VSMC migration and proliferation.…”

“…However, upon ligation injury of the carotid artery, SCPEP1 null mice show reduced neointimal load suggesting that SCPEP1 directs VSMC migration and proliferation. Indeed, adenoviral delivery of SCPEP1 to VSMC causes accelerated growth and migration in a catalytic triad-dependent manner[110]. Interestingly, SCPEP1 is secreted from cells in a non cleaved manner though it is not clear as yet whether extracellular SCPEP1 exhibits biological activity.…”