Pigment epithelial-derived factor (PEDF), an angiogenesis inhibitor with neurotrophic properties, balances angiogenesis in the eye and blocks tumor progression. Its neurotrophic function and the ability to block vascular leakage is replicated by the PEDF 44-mer peptide (residues 58-101). We analyzed PEDFs' three-dimensional structure and identified a potential receptor-binding surface. Seeking PEDF-based antiangiogenic agents we generated and tested peptides representing the middle and lower regions of this surface. We identified previously unknown antiangiogenic epitopes consisting of the 34-mer (residues 24-57) and a shorter proximal peptide (TGA, residues 16-26) with the critical stretch L19VEEED24 and a fragment within the 44-mer (ERT, residues 78-94), which retained neurotrophic activity. The 34-mer and TGA, but not the 44-mer reproduced PEDF angioinhibitory signals hinged on c-jun-NH2-kinase–dependent nuclear factor of activated T cell deactivation and caused apoptosis. Conversely, the ERT, but not the 34-mer/TGA induced neuronal differentiation. For the 44-mer/ERT, we showed a novel ability to cause neuroendocrine differentiation in prostate cancer cells. PEDF and the peptides bound endothelial and PC-3 prostate cancer cells. Bound peptides were displaced by PEDF, but not by each other, suggesting multiple receptors. PEDF and its active fragments blocked tumor formation when conditionally expressed by PC-3 cells. The 34- and 44-mer used distinct mechanisms: the 34-mer acted on endothelial cells, blocked angiogenesis, and induced apoptosis whereas 44-mer prompted neuroendocrine differentiation in cancer cells. Our results map active regions for the two PEDF functions, signaling via distinct receptors, identify candidate peptides, and provide their mechanism of action for future development of PEDF-based tumor therapies.

It has been demonstrated that vascular endothelial cell growth factor (VEGF) induction of angiogenesis requires activation of the nuclear factor of activated T cells (NFAT). We show that NFATc2 is also activated by basic fibroblast growth factor and blocked by the inhibitor of angiogenesis pigment epithelial–derived factor (PEDF). This suggests a pivotal role for this transcription factor as a convergence point between stimulatory and inhibitory signals in the regulation of angiogenesis.We identified c-Jun NH2-terminal kinases (JNKs) as essential upstream regulators of NFAT activity in angiogenesis. We distinguished JNK-2 as responsible for NFATc2 cytoplasmic retention by PEDF and JNK-1 and JNK-2 as mediators of PEDF-driven NFAT nuclear export.We identified a novel NFAT target, caspase-8 inhibitor cellular Fas-associated death domain–like interleukin 1β–converting enzyme inhibitory protein (c-FLIP), whose expression was coregulated by VEGF and PEDF. Chromatin immunoprecipitation showed VEGF-dependent increase of NFATc2 binding to the c-FLIP promoter in vivo, which was attenuated by PEDF. We propose that one possible mechanism of concerted angiogenesis regulation by activators and inhibitors may be modulation of the endothelial cell apoptosis via c-FLIP controlled by NFAT and its upstream regulator JNK.