Abstract
Kallikrein-related peptidase 12 (KLK12) is a kallikrein family peptidase involved in angiogenesis – a complex biological process in which the sprouting, migration and stabilization of endothelial cells requires extracellular matrix remodeling. To characterize the molecular mechanisms associated with KLK12′s proangiogenic activity, we evaluated its ability to hydrolyze various matrix proteins. Our results show that KLK12 efficiently cleaved the human extracellular matrix proteins fibronectin and tenascin, both of which are involved in the regulation of endothelial cell adhesion and migration. For fibronectin, the major proteolytic product generated by KLK12 was a 29 kDa fragment containing the amino-terminal domain and the first five type I fibronectin-domains, which are essential for regulating fibronectin assembly. We also demonstrated that KLK12-mediated fibronectin proteolysis antagonizes fibronectin polymerization and fibronectin fibril formation by endothelial cells, leading to an increase in cell migration. Furthermore, a polyclonal antibody raised against KLK12′s proteolytic cleavage site on fibronectin prevented the KLK12-dependent inhibition of fibronectin polymerization and the KLK12-mediated pro-migratory effect on endothelial cells. Taken as a whole, our results indicate that KLK12′s proangiogenic effect is mediated through several molecular mechanisms.
Highlights
IntroductionBlood vessel formation (comprising vasculogenesis and angiogenesis) is a critical physiologic process in all human organs, and dysregulation of this process is often observed in human diseases (notably during tumorigenesis)[1,2,3]
Blood vessel formation is a critical physiologic process in all human organs, and dysregulation of this process is often observed in human diseases[1,2,3]
A time-dependent experiment showed that KLK12 rapidly released a proteolytic product with an apparent molecular mass of 29 kDa (E:S ratio: 1:50, Fig. 1B and D), which accumulated over time
Summary
Blood vessel formation (comprising vasculogenesis and angiogenesis) is a critical physiologic process in all human organs, and dysregulation of this process is often observed in human diseases (notably during tumorigenesis)[1,2,3]. Vasculogenesis involves the recruitment of progenitor cells (i.e. angioblasts) and their differentiation into endothelial cells (ECs), resulting in the de novo formation of blood vessels. Angiogenesis corresponds to the extension of pre-existing vessels through EC sprouting, migration and proliferation These cellular behaviors are tightly controlled in space and time by a complex balance between pro- and anti-angiogenic factors, cell-cell interactions, and cell-extracellular matrix (ECM) interactions[2,3]. The objective of the present study was to identify the molecular mechanisms involved in the regulation of angiogenesis by KLK12. To this end, we investigated KLK12′s ability to hydrolyze various matrix proteins with important roles in angiogenesis, and determined the associated functional consequences
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