Abstract 287: Proteolytic regulation of PDGF-D activity by matriptase in human prostate cancer cells
Authors: HUANG WEI , Kim Hyeong-Reh C. 
Published: February 2011

Platelet-derived growth factor (PDGF) proteins are potent inducers of cell proliferation, transformation, and differentiation. PDGF-D, a newly identified PDGF ligand, has a unique two-domain structure with an NH2-terminal CUB domain and a COOH-terminal PDGF/VEGF growth factor domain (GD). Proteolytic removal of the CUB domain is required for PDGF-D to activate its cognate β-PDGF receptor (β-PDGFR). We previously showed that the human prostate cancer cells, LNCaP and PC3, are able to process the latent PDGF-D into an active growth factor. We found that urokinase plasminogen activator (uPA), a secreted serine protease, is the activator of latent PDGF-D in PC3 cells. However, in LNCaP cells, which do not express uPA, latent PDGF-D was shown to be processed pericellularly instead of by a secreted protease. By microarray analysis, matriptase, a type II transmembrane serine protease, was found to be the putative serine protease in LNCaP cells to process latent PDGF-D.

In this study, to test whether latent PDGF-D is a substrate of matriptase, time- and dose-dependent experiments of incubating purified recombinant PDGF-D with purified catalytic domain of matriptase were performed. Full-length PDGF-D dimer (FL-D) was processed into growth factor domain dimer (GD-D) in a two-step manner involving generation of an intermediate hemidimer (HD) composed of one full-length monomer (FL-M) and one growth factor domain monomer (GD-M). Through mutagenesis analyses, we identified YR247GR249↓S in the hinge region as a matriptase cleavage site for the proteolytic activation of PDGF-D. Interestingly, FL PDGF-D was first processed into 18kDa GD and then was further processed into 15kDa GD, indicating there is a matriptase cleavage site within PDGF-D growth factor domain apart from the one in the hinge region. Mutagenesis analyses identified R340RGR343 ↘A within growth factor domain to be a matriptase cleavage site. Through sequence alignment of PDGF-D GD with PDGF-B, the matriptase cleavage site R340RGR343 A is implicated in the interaction of PDGF-D GD with β-PDGFR. The subsequent β-PDGFR activation study confirmed that matriptase-mediated cleavage at R340RGR343A results in deactivation of PDGF-D. Interestingly, although PDGF-D HD is a biological inactive form of PDGF-D, HD is able to be activated by pericellular serine protease. More interestingly, PDGF-D HD can function as a dominant-negative ligand of PDGF-B/β-PDGFR signaling, presumably through its binding to one subunit of β-PDGFR, thereby inhibiting PDGF-B-mediated receptor dimerization. Lastly, we report that while PDGF-D FL-D is easily detected in the conditioned medium, a preferred deposition of PDGF-D HD and GD was detected in the extracellular matrix of LNCaP-PDGF-D cells.

Collectively, this study provides molecular insights into matriptase-mediated complex regulation of PDGF-D activity and extracellular localization in human prostate carcinoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 287.