Pigment epithelium-derived factor and its phosphomimetic mutant induce JNK-dependent apoptosis and p38-mediated migration arrest

Abstract

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 (ECs), 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. 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 a stronger effect. The stronger activity of EEE-PEDF was correlated with a better binding to laminin receptors. Furthermore, the proapoptotic and antimigratory activities of WT-PEDF and EEE-PEDF were found regulated by differential activation of two distinct MAPK pathways, namely JNK and p38, respectively. We show that JNK and p38 phosphorylation is much higher in cells treated with EEE-PEDF. JNK leads to apoptosis of ECs, whereas p38 leads to anti-migratory 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.

FIGURE 1.

The effect of WT-PEDF and EEE-PEDF on proliferation rate of endothelial and cancer cells. The rate of BAEC (A), MDA-MB-231 (B), HCT116 (C), and U87-MG (D) cell proliferation following treatment for 48 h with the indicated concentrations of WT-PEDF or EEE-PEDF was analyzed using the methylene blue colorimetric assay. Data represent mean ± S.E. (n = 3). *, p < 0.05, treated versus control; #, p < 0.05, EEE-PEDF versus WT-PEDF.

FIGURE 2.

The effect of WT-PEDF and EEE-PEDF on apoptosis of cultured endothelial cells. A, representative TUNEL-labeled ×20 fields of BAEC treated with WT-PEDF or EEE-PEDF as indicated. Left, time course of WT-PEDF- and EEE-PEDF-induced apoptosis (PEDF constructs used at a concentration of 20 nm). Right, dose-dependent effect of WT-PEDF and EEE-PEDF on BAEC apoptosis. Green, TUNEL; blue, DAPI. B, quantification of the time course (top) and dose response (bottom) of the relative apoptotic rate in BAEC treated with WT-PEDF or EEE-PEDF. Data represent mean ± S.E. (n = 4). *, p < 0.05, treated versus control; #, p < 0.05, EEE-PEDF versus WT-PEDF. C, time course (left) and dose response (right) effect of the PEDF constructs on caspase-3 cleavage (cCasp3) as assessed by immunoblotting with anti-cleaved caspase-3 (α-cCasp3).

FIGURE 3.

The effect of WT-PEDF and EEE-PEDF on endothelial and cancer cell migration. A, migration of BAEC and MDA-MB-231 cells in the presence of bFGF (20 ng/ml) and either WT-PEDF or EEE-PEDF as evaluated by Transwell assay. Shown are representative photographs of crystal violet-stained ×20 fields of migrated cells taken from the bottom side of the polycarbonate membranes. B, following visualization, stain was extracted with methanol and migration was quantified by A₅₄₀ measurement. Data shown represent mean ± S.E. (n = 3). *, p < 0.05, treated versus control; #, p < 0.05, EEE-PEDF versus WT-PEDF. C, migration of BAEC and MDA-MB-231 cells in the presence of bFGF (20 ng/ml) and either WT-PEDF or EEE-PEDF (both at 20 nm) as evaluated by wound healing assay. D, quantification of the results in C. Data shown represent mean ± S.E. (n = 3). *, p < 0.01, treated versus control.

FIGURE 4.

Binding of WT-PEDF and EEE-PEDF to recombinant PNPLA2 and LR in in vitro binding assay. COS-7 cells were transfected with either GFP-PNPLA2 (A) or GFP-LR (B). Forty eight hours after transfection, cells were lysed, and recombinant proteins were immunoprecipitated (IP) using anti-GFP antibody. Thereafter, aliquots of washed immunoprecipitate-bound A/G beads were incubated with 4 μg of recombinant GST-WT-PEDF or GST-EEE-PEDF. Following incubation, the beads were boiled, and resolved protein complexes were analyzed by immunoblotting (IB) with anti-PEDF antibody. WCL, whole cell lysate. WT, WT-PEDF protein; EEE, EEE-PEDF protein.

FIGURE 5.

The effect of WT-PEDF and EEE-PEDF on the activity of MAPK cascades in BAEC and MDA-MB-231 cells. A–E, the effect of WT-PEDF and EEE-PEDF (20 nm) on p38α, JNK1/2 (A), ERK1/2 (B), MKK3, MKK6, MKK4, and MKK7 (D) phosphorylation in BAEC was analyzed by immunoblotting with antiphospho-specific antibodies. The time course of WT-PEDF- and EEE-PEDF-induced MAPK (C) and MAP2K (E) phosphorylation in BAEC was quantified from three independent immunoblotting experiments. *, p < 0.05, EEE-PEDF versus WT-PEDF. F–H, the effect of PEDF constructs on p38α, JNK1/2 (F), and ERK1/2 (G) phosphorylation in MDA-MB-231 cells was analyzed by immunoblotting with antiphospho-specific antibodies. Time course of WT-PEDF- and EEE-PEDF-induced MAPKs phosphorylation in MDA-MB-231 cells (H) was quantified from three independent immunoblotting experiments. *, p < 0.05, EEE-PEDF versus WT-PEDF. In all experiments, peroxyvanadate (VOOH; 200 μm H₂O₂, 100 μm vanadate) was used as a positive control of MAPK/MAPKK activation.

FIGURE 6.

The proapoptotic activity of WT-PEDF and EEE-PEDF is mediated by JNK but not by p38α/β or EPK. A, representative TUNEL-labeled ×20 fields of BAEC after pretreatment with either p38α/β (SB203580, 10 μm), JNK1–3 (SP600125, 5 μm), or MEK1/2 (U0126, 5 μm) inhibitors for 1 h followed by incubation with WT-PEDF or EEE-PEDF (20 nm) for 12 h. Green, TUNEL; blue, DAPI. B, quantification of the relative apoptotic rate in TUNEL-labeled BAEC treated as described in A. Data shown are mean ± S.E. (n = 4). *, p < 0.05, treated versus control; #, p < 0.05, EEE-PEDF versus WT-PEDF. C, the effect of p38α/β (SB203580, 10 μm) and JNK1–3 (SP600125, 5 μm) inhibitors on WT-PEDF and EEE-PEDF (20 nm)-induced caspase-3 cleavage (cCasp3) in BAEC, as evaluated by immunoblotting with anticleaved caspase-3 antibody (α-cCasp3).

FIGURE 7.

The antimigratory activity of WT-PEDF and EEE-PEDF is mediated by P38α/β, but not by JNK or ERK. A, migration of BAEC and MDA-MB-231 in the presence of bFGF (20 ng/ml) after pretreatment with p38α/β (SB203580, 10 μm), JNK1–3 (SP600125, 5 μm), or MEK1/2 (U0126, 5 μm) inhibitors for 1 h followed by incubation with either WT-PEDF or EEE-PEDF (20 nm) for 16 h, as evaluated by Transwell assay. Shown are representative photographs of crystal violet-stained ×20 fields of migrated cells taken from the bottom side of the polycarbonate membranes. B and C, following visualization, stain was extracted and migration was quantified for BAEC (B) and MDA-MB-231 (C) by A₅₄₀ measurement. Data represent mean ± S.E. (n = 3). *, p < 0.05, treated versus maximal migration; #, p < 0.05, EEE-PEDF versus WT-PEDF.

FIGURE 8.

The effects of WT-PEDF and EEE-PEDF on MAPK activity, apoptosis, and migration in HUVEC. A, the effect of WT-PEDF and EEE-PEDF (20 nm) on ERK1/2 (right) p38α and JNK1/2 (left) phosphorylation in HUVEC as analyzed by immunoblotting with antiphospho-specific antibodies. For the detection of ERK1/2 phosphorylation, HUVEC were stimulated with PEDF constructs for 15 min, whereas p38α and JNK1/2 phosphorylation was tested after 120 min. B, representative TUNEL-labeled ×20 fields of HUVEC after pretreatment with either p38α/β (SB203580, 10 μm), JNK1–3 (SP600125, 5 μm), or MEK1/2 (U0126, 5 μm) inhibitors for 1 h followed by incubation with WT-PEDF or EEE-PEDF (20 nm) for 24 h. Green, TUNEL; blue, DAPI. C, quantification of the relative apoptotic rate in TUNEL-labeled HUVEC treated as described in B. Data shown are mean ± S.E. (n = 4). *, p < 0.05, treated versus control; #, p < 0.05, EEE-PEDF versus WT-PEDF. D, migration of HUVEC in the presence of bFGF (20 ng/ml) after pretreatment with p38α/β (SB203580, 10 μm), JNK1–3 (SP600125, 5 μm), or MEK1/2 (U0126, 5 μm) inhibitors for 1 h followed by incubation with either WT-PEDF or EEE-PEDF (20 nm) for 24 h, as evaluated by Transwell assay. Shown are representative photographs of crystal violet-stained ×20 fields of migrated cells taken from the bottom side of the polycarbonate membranes. E, following visualization, stain was extracted and migration of HUVEC was quantified by A₅₄₀ measurement. Data represent mean ± S.E. (n = 3). *, p < 0.05, treated versus maximal migration; #, p < 0.05, EEE-PEDF versus WT-PEDF.

FIGURE 9.

Schematic representation of the molecular mechanism of the proapoptotic and antimigratory effect of WT-PEDF and EEE-PEDF towards endothelial and cancer cells.