Activation of the novel PDGFS by plasminogen activators in tumorigenesis

Sammanfattning: Members of the platelet-derived growth factor (PDGF) family are mitogens for cells of a mesenchymal origin and have important functions both during development and in the adult. Their role in developmental processes has been extensively investigated by methodical targeted gene inactivations, revealing critical involvement in microvascular pericyte recruitment, neural crest development and alveogenesis. In the adult, an active participation in tissue homeostasis, wound healing and human diseases such as oncogenesis, atherosclerosis and fibrosis has been substantiated. Cellular effects are exerted by five homo- or heterodimeric ligands (PDGF-AA, -BB, -AB, -CC and -DD) with individual binding affinities for the two structurally related receptor tyrosine kinases, PDGFRalpha and PDGFRbeta. Binding induces receptor dimerization and transphosphorylation, thereby initiating intracellular signaling events. Both PDGF-AA and PDGF-CC signal via PDGFRalpha, while PDGF-BB and PDGF-DD are believed to act preferentially via PDGFRbeta in vivo. In contrast to the two classical PDGFs, the novel and less characterized members, PDGF-CC and PDGF-DD, contain N-terminal complement subcomponents C1r/C1s, urchin EGF-like protein, bone morphogenic protein-1 (CUB) domains within the fulllength protein. So far, the functional role of the CUB domains is not fully understood, but they have to be proteolytically removed before the factors can bind to and activate their cognate receptors. In this thesis, we used different approaches to investigate how proteolytic activation of the novel PDGFs is regulated and what role this process may have in vivo. The first two studies describe the characterization of the biochemical interactions between the novel PDGFs and their specific proteolytic enzymes referred to as plasminogen activators. Experimental evidence also suggests that proteolytic activation of PDGF-DD reveals a retention motif mediating interactions with pericellular components. In the third study, we elucidate the role of the novel PDGFs in rhabdomyosarcoma and unravel regulatory mechanisms controlling the expression of plasminogen activators. Our data suggest that both PDGF-CC and PDGF-DD are promising therapeutic targets in this disease. Therefore, in the fourth study, we summarize data from an ongoing project where we have initiated an extensive production of monoclonal antibodies targeting PDGF-DD. Generated antibodies recognize full-length PDGF-DD and possess neutralizing activity. Future studies will enlighten their efficacy in vivo. In summary, our findings presented in this thesis show that plasminogen activators can directly interact with, and proteolytically process, latent PDGF-CC and PDGF-DD. Weshow that the proteolytic activation not only generates an active growth factor, but alsocontrols its bioavailability. Furthermore, we present evidence for an active role of the novel PDGFs in rhabdomyosarcoma and suggest that monoclonal antibodies can be a useful molecular tool for neutralization of PDGF activity both in vitro and in vivo.