Studies of the novel PDGFs, focusing on PDGF-D

Abstract: Thirty years ago the classical platelet-derived growth factors (PDG17s), PDGF-A and PDGF-B were discovered. For a long time they were thought to be the only PDG17 isoforms to exist, but recently two novel PDGF molecules were identified, namely PDGF-C and PDGF-D. This finding was unexpected and indicates that the PDGF signalling system is much more complex than was previously thought. The four PDG17 chains form five different di sulphide-1 inked homo- and heterodimers; PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD, which exert their biological effects by binding to, and signalling through two receptor tyrosine kinases, PDGFR-alpha and PDGFR-beta. The biological effects of PDGF-A and PDGF-B have been extensively studied. They are known to be major mitogens for several cell types of mesenchymal origin and have been implicated in several pathological conditions, such as atherosclerosis and fibrotic diseases. The discovery of two new members of the PDG17 family makes it important to further characterize the players in the PDGFR signalling system in order to understand their different biological functions. Part of this work describes the identification of the most recent found member of the PDGFfamily, PDGFD. In a computer-assisted search, a cDNA clone encoding PDGF-D was found. The full-length cDNA encoded a polypeptide of 370 amino acids. The secreted protein turned out to be a disulphide-linked homodimer, which displays a two-domain structure similar to that found in PDGF-C, with an N-terminal CUB domain and a C-terminal PDGF/VEGF homology domain. Secreted PDGF-DD was found to be a latent protein, which requires proteolytic removal of the CUB domains for activity. Proteolytically processed PDGF-DD preferentially binds to and signal through PDGFR-beta. Further, the human genes for PDGF-C and PDGF-D were found to be located on chromosomes 4q32, and 1 1q22.3 to 23.2, respectively. Characterization of the genes showed that the PDGFC gene contained 6 exons, while the PDGFD gene contained 7 exons. The biological role(s) and the in vivo responses of the novel PDG17s are less well known. Data presented herein suggest that PDGF-D might have a role in cardiac fibrosis. This was indicated when PDGF-D was overexpressed in the heart of transgenic mice. Overexpression of PDGF-D induced cardiac fibrosis and hypertrophy, which subsequently caused cardiac failure. In addition, vascular changes, with dilated microvessels, and proliferation of the smooth muscle cells leading to thickening of arterial walls, was seen. The protease uPA has recently been reported to activate PDGF-DD, and in a study where skeletal muscle injury was experimentally induced in uPA deficient mice, uPA was found to be required for efficient regeneration of damaged muscle. Data presented herein establish a role of PDGF-D in skeletal muscle development and regeneration. Using cultured myoblasts, PDGF-DD was found to stimulate proliferation and migration of the myoblasts in vitro, as well as inhibiting their differentiation. In summary, the findings presented in this thesis improve our understanding of the biological function of PDGF-D and enhance the knowledge of the complexity of the PDGF/PDGFR signalling system