Structural Requirements for Activation of Latent Pdgf-cc by Tissue Plasminogen Activator

Platelet-derived growth factor C (PDGF-C) is one of four members in the PDGF family of growth factors, which are known mitogens and survival factors for cells of mesenchymal origin. PDGF-C has a unique two domain structure consisting of an N-terminal CUB and a conserved C-terminal growth factor domain, that are separated by a hinge region. PDGF-C is secreted as a latent dimeric factor (PDGFCC) which undergoes extracellular removal of the CUB domains to become a PDGF receptor a agonist. Recently, the multi-domain serine protease tissue plasminogen activator (tPA), a thrombolytic agent used for treatment of acute ischemic stroke, was shown to cleave and activate PDGF-CC. In this study we determine the molecular mechanism of tPA-mediated activation of PDGF-CC. Using various PDGFCC and tPA mutants, we were able to demonstrate that both the CUB and the growth factor domain of PDGF-C, as well as the kringle-2 domain of tPA, are required for the interaction and cleavage to occur. We also show that arginine 231 in PDGF-C is essential for tPA-mediated proteolysis and that the released “free” CUB domain of PDGF-C can act as a competitive inhibitor of the cleavage reaction. Further, we studied how the PDGF-C/tPA axis is regulated in primary fibroblasts and found that PDGF-C expression is down-regulated by hypoxia but induced by TGF-b 1 treatment. Elucidating the regulation and the mechanism of tPA-mediated activation of PDGF-CC will advance our knowledge of the physiological function of PDGF-CC and tPA, and may provide new therapeutic opportunities for thrombolytic and cardiovascular therapies. Platelet-derived growth factor C (PDGF-C) was discovered a few years ago as the third member of the well-characterized PDGF family of growth factors (1). The classical members of this family, PDGF-A and PDGF-B, have been intensively studied and are known to be important for connective tissue growth and maintenance, and overexpression has been observed in several pathological conditions, including malignancies and atherosclerosis (2). Since its discovery, PDGF-C has been shown to play a role in palate formation (3), fibrotic disease development (4,5), and angiogenesis (6,7). Recently a forth member, PDGF-D, has been added to this family of growth factors (8,9). The four PDGF chains assemble into five dimeric isoforms; PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD, that exert their effects on cells through differential signaling via two known tyrosine kinase receptors, PDGFRa and PDGFR-b (10). Unlike the classical members, PDGF-C and PDGF-D have a unique two-domain structure, with a so called CUB domain N-terminal of the conserved growth factor domain (1,8,9). In order for the novel PDGFs to bind and activate the PDGFRs, the N-terminal CUB domains have to be removed through limited proteolysis by extracellular proteases. The origin of the protease involved in the activation of PDGF-DD still remains elusive, whereas the extracellular fibrinolytic protease tissue plasminogen activator (tPA) has been shown to be a potent activator of PDGF-CC (11). tPA is a highly specific serine protease that consists of five structural domains; a finger domain, an epidermal growth factor-like domain, two kringle domains and a trypsin-like protease domain (12). It is best known for its role in vascular fibrinolysis where it converts the JBC Papers in Press. Published on May 23, 2005 as Manuscript M503388200