Pigment Epithelium-Derived Factor and its Phosphomimetic Mutant Induce JNK-Dependent Apoptosis and P38-Mediated...

Pigment epithelium-derived factor (PEDF) is a potent endogenous inhibitor of angiogenesis, and a promising anticancer agent. We have previously shown that PEDF can be phosphorylated, and that distinct phosphorylations differentially regulate its physiological functions. We also demonstrated that triple phosphomimetic mutant (EEE-PEDF), has significantly increased antiangiogenic activity, and is much more efficient than WT-PEDF in inhibiting neovascularization and tumor growth. The enhanced antiangiogenic effect was associated with a direct ability to facilitate apoptosis of tumor-residing endothelial cells (EC), and subsequently, disruption of intratumoral vascularization. In the present report, we elucidated the molecular mechanism by which EEE-PEDF exerts more profound effects at the cellular level.

Here we used Western blotting, as well as in vitro binding, proliferation, apoptosis and migration assays to follow the signaling components responsible for the PEDF and EEE-PEDF effects.

We found that EEE-PEDF suppresses EC proliferation due to caspase-3-dependent apoptosis, and also inhibits migration of the EC much better than WT-PEDF. Although WT-PEDF and EEE-PEDF did not affect proliferation and did not induce apoptosis of cancer cells, these agents efficiently inhibited cancer cell motility, with EEE-PEDF showing stronger effect. The stronger activity of EEE-PEDF was correlated to a better binding to laminin receptors. Furthermore, the proapoptotic and antimigratory activities of WT-PEDF and EEE-PEDF were found respectively regulated by differential activation of two distinct MAPK pathways, namely JNK and p38. We show that JNK and p38 phosphorylation is much higher in cells treated with EEE-PEDF. JNK leads to apoptosis of ECs, while p38 leads to antimigratory effect in both EC and cancer cells.

These results reveal the molecular signaling mechanism by which the phosphorylated PEDF exerts its stronger antiangiogenic, antitumor activities.