Nathalie Lokker

PDGF-D, a new protease-activated growth factor.

Platelet-derived growth factor (PDGF) has been directly implicated in developmental and physiological processes, as well as in human cancer, fibrotic diseases and arteriosclerosis. The PDGF family currently consists of at least three gene products, PDGF-A, PDGF-B and PDGF-C, which selectively signal through two PDGF receptors (PDGFRs) to regulate diverse cellular functions. After two decades of searching, PDGF-A and B were the only ligands identified for PDGFRs. Recently, however, database mining has resulted in the discovery of a third member of the PDGF family, PDGF-C, a functional analogue of PDGF-A that requires proteolytic activation. PDGF-A and PDGF-C selectively activate PDGFR-α, whereas PDGF-B activates both PDGFR-α and PDGFR-β. Here we identify and characterize a new member of the PDGF family, PDGF D, which also requires proteolytic activation. Recombinant, purified PDGF-D induces DNA synthesis and growth in cells expressing PDGFRs. In cells expressing individual PDGFRs, PDGF-D binds to and activates PDGFR-β but not PDGFR-α. However, in cells expressing both PDGFRs, PDGF-D activates both receptors. This indicates that PDGFR-α activation may result from PDGFR-α/β heterodimerization.

CT53518, a novel selective FLT3 antagonist for the treatment of acute myelogenous leukemia (AML)

Up to 30% of acute myelogenous leukemia (AML) patients harbor an activating internal tandem duplication (ITD) within the juxtamembrane domain of the FLT3 receptor, suggesting that it may be a target for kinase inhibitor therapy. For this purpose we have developed CT53518, a potent antagonist that inhibits FLT3, platelet-derived growth factor receptor (PDGFR), and c-Kit (IC(50) approximately 200 nM), while other tyrosine or serine/threonine kinases were not significantly inhibited. In Ba/F3 cells expressing different FLT3-ITD mutants, CT53518 inhibited IL-3-independent cell growth and FLT3-ITD autophosphorylation with an IC(50) of 10-100 nM. In human FLT3-ITD-positive AML cell lines, CT53518 induced apoptosis and inhibited FLT3-ITD phosphorylation, cellular proliferation, and signaling through the MAP kinase and PI3 kinase pathways. Therapeutic efficacy of CT53518 was demonstrated both in a nude mouse model and in a murine bone marrow transplant model of FLT3-ITD-induced disease.

Functional Importance of Platelet-derived Growth Factor (PDGF) Receptor Extracellular Immunoglobulin-like Domains IDENTIFICATION OF PDGF BINDING SITE AND NEUTRALIZING MONOCLONAL ANTIBODIES

The biological effects of platelet-derived growth factor (PDGF) are mediated by α- and β-PDGF receptors (PDGFR), which have an intracellular tyrosine kinase domain and an extracellular region comprising five immunoglobulin-like domains (D1–D5). Using deletion mutagenesis we mapped the PDGF binding site in each PDGFR to the D2–D3 region. In the case of α-PDGFR,125I-PDGF AA and 125I-PDGF BB bound to the full-length extracellular domain, D1–D5, and D2–D3 with equal affinity (K d = 0.21–0.42 nm). Identical results were obtained for 125I-PDGF BB binding to β-PDGFR mutants D1–D5 and D2–D3, establishing that D1, D4, and D5 do not contribute to PDGF binding. Monoclonal antibodies (mAb) directed against individual PDGFR Ig-like domains were used to extend these observations. The anti-D1 mAb 1E10E2 and anti-D5 mAb 2D4G10 had no effect on α- or β-PDGFR function, respectively. In contrast, mAb 2H7C5 and 2A1E2 directed against D2 of the α- and β-receptor, respectively, blocked PDGF binding, receptor autophosphorylation and mitogenic signaling with IC50 values of 0.1–3.0 nm. An anti-D4 mAb 1C7D5 blocked β-receptor autophosphorylation and signaling without inhibiting PDGF binding consistent with the observation that D4 is essential for PDGFR dimerization (Omura, T., Heldin, C.-H., and Ostman, A. (1997)J. Biol. Chem. 272, 12676–12682). mAbs identified here act as potent PDGFR antagonists that can be used as research tools and potentially as therapeutic agents for the treatment of diseases involving unwanted PDGFR signaling.

PDGF C Is A Selective α Platelet-Derived Growth Factor Receptor Agonist That Is Highly Expressed in Platelet α Granules and Vascular Smooth Muscle

Objective—The platelet-derived growth factor (PDGF) family consists of four members, PDGF A, PDGF B, and 2 new members, PDGF C and PDGF D, which signal through the &agr; and &bgr; PDGF receptor (PDGFR) tyrosine kinases. This study was performed to determine the receptor specificity and cellular expression profile of PDGF C. Methods and Results—PDGF C growth factor domain (GFD) was shown to preferentially bind and activate &agr; PDGFR and activate &bgr; PDGFR when it is co-expressed with &agr; PDGFR through heterodimer formation. An investigation of PDGF C mRNA and protein expression revealed that during mouse fetal development, PDGF C was expressed in the mesonephric mesenchyme, prefusion skeletal muscle, cardiac myoblasts, and in visceral and vascular smooth muscle, whereas in adult human tissues expression was largely restricted to smooth muscle. Microarray analysis of various cell types showed PDGF C expression in vascular smooth muscle cells, renal mesangial cells, and platelets. PDGF C mRNA expression in platelets was confirmed by real-time polymerase chain reaction, and PDGF C protein was localized in &agr; granules by immuno-gold electron microscopy. Western blot analysis of platelets identified 55-kDa and 80-kDa PDGF C isoforms that were secreted on platelet activation. Conclusions—Taken together, our results demonstrated for the first time to our knowledge that like PDGF A and B, PDGF C is likely to play a role in platelet biology.