Proteolytic activation and biological functions of the novel PDGFs

Abstract: The platelet-derived growth factors (PDGFs) and their cell surface receptors regulate growth and migration of connective tissue cells of both the vascular network and other organ systems. They are known to be important for normal development and processes, such as wound healing and angiogenesis, but have also been implicated in many pathological conditions, such as malignancies and fibrotic disease. Therefore, it is a major challenge to thoroughly characterize this signalling system in order to develop novel therapeutic strategies for the treatment of these diseases. Today the PDGF family consists of four PDGF members, the classical PDGF-A and PDGF-B polypeptide chains, and the novel PDGF-C and PDGF-D chains. The novel PDGFs differ from the classical chains as they possess an additional, so called CUB domain, in front of the common growth factor domain (GFD). The PDGF chains are secreted from the producing cells as five disulphide-linked dimeric factors, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGFDD, that after proteolytic activation exert their effects on cells through differential binding to two tyrosine kinase receptors, PDGF-alpha and PDGF-beta. All four PDGF chains are synthesized as proproteins, but unlike the classical PDGF members, which are proteolytically activated intracellularly in the trans-Golgi network, the novel PDGFs are activated extracellularly by proteolytic removal of the CUB domains. This thesis describes the identification of the trypsin-like serine protease, tissueplasminogen activator (tPA), as a potent activator of PDGF-CC. tPA is best known for its role in vascular fibrinolysis, where it converts plasminogen into plasmin, which in turn degrades the fibrin network in blood clots. tPA was shown to activate PDGF-CC in the absence of plasminogen, which might explain some of the plasminogen-independent effects reported for tPA. Further, tPA was found to cleave PDGF-CC, but not the highly homologous PDGF-DD, due to specific interactions of the second kringle domain in tPA with both the CUB domain, and the GM, in PDGF-C. The interaction between tPA and PDGF-CC probably positions the protease domain of tPA and the cleavage site in PDGF-C in close proximity to each other. These findings greatly facilitate our understanding of the in vivo functions of PDGF-CC, as well as tPA, both in normal and pathological conditions. Similar to the classical PDGFs, data presented here establishes a role of the novel PDGFs in tumourigenesis, characterized by their ability to induce cellular transformation of NIH/3T3 cells and, subsequently to induce tumour growth in nude mice. Proteolytic removal of the CUB domains from the novel PDGFs was shown to expose a cryptic retention motif in the activated factors, enabling these processed forms to bind to extracellular structures. This affected the ability of these activated factors to induce tumour growth in vivo, emphasizing the importance of understanding the mechanism underlying activation of the PDGFs.