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. 2004 Jun 7;199(11):1513-22.
doi: 10.1084/jem.20040474.

Nuclear factor of activated T cells balances angiogenesis activation and inhibition

Tetiana A Zaichuk  1 , Emelyn H Shroff, Rebekah Emmanuel, Stephanie Filleur, Thomas Nelius, Olga V Volpert
Affiliations

Nuclear factor of activated T cells balances angiogenesis activation and inhibition

Tetiana A Zaichuk et al. J Exp Med. .

Abstract

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 1beta-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.

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Figures

Figure 1.
Figure 1.
NFAT deactivation by PEDF. (a) NFAT dephosphorylation by bFGF. Cell extracts of bFGF-induced human microvascular ECs (5 ng/ml, 15 min) were precipitated with phosphoserine antibody and analyzed by immunoblotting with NFATc2 antibody. Note the decreased NFATc2 phosphorylation in the presence of bFGF. (b) PEDF restored NFATc2 phosphorylation in activated ECs. VEGF- or bFGF-stimulated ECs were treated with PEDF (10 nM, 15 min). Note the decrease in phospho-NFATc2 by angiogenic stimuli and higher phosphorylation levels in the activated cells exposed to inhibitory PEDF. (c and d) Inhibition of NFATc2 nuclear localization by PEDF. HUVECs grown on gelatinized coverslips were treated with 200 pg/ml VEGF or 5 ng/ml bFGF and 10 nM PEDF and stained for NFATc2. Note the predominance of the cells with nuclear NFATc2 in the presence of bFGF or VEGF compared with untreated control and cytoplasmic NFATc2 localization in the presence of PEDF. The data were quantified using MetaView software package (d).
Figure 2.
Figure 2.
The role of JNK kinases in NFATc2 deactivation by PEDF. (a) PEDF failed to activate p38MAPK or Erk1/2 kinases. Confluent HUVECs were stimulated with 200 pg/ml VEGF or 5 ng/ml bFGF. 10 nM PEDF was added where indicated. Cell extracts were analyzed by Western blotting with antibodies for active phosphorylated forms of Erk1/2 (p-Erk) and for dually phosphorylated p38 (pp38). The blots were reprobed for total Erk and p38 to ensure equal loading. The representative result of three independent experiments is shown. (b) PEDF enhanced JNK activation in stimulated ECs. HUVECs were treated as indicated with VEGF or bFGF + PEDF for 15 min. Cell extracts were resolved by SDS-PAGE and analyzed by Western blotting with antibodies for active, dually phosphorylated JNK (top, ppJNK) or total JNK (bottom) as a loading control. Three independent experiments were performed with similar results. (c) PEDF-dependent NFATc2 phosphorylation required JNK kinases. VEGF-stimulated HUVECs were treated with PEDF alone or in combination with 100 nM of SP600125, a generic JNK inhibitor. Cell lysates were immunoprecipitated with phosphoserine antibody and analyzed by Western blotting with NFATc2 antibody. Note the increase in NFATc2 phosphorylation by PEDF in stimulated ECs and its attenuation in the presence of JNK inhibitor. (d) Increased JNK activity in PEDF-treated cells. HUVECs were treated with bFGF (15 min, 5 ng/ml) and/or PEDF, and JNK activity was assessed by immunocomplex kinase assay with exogenous recombinant GST-NFATc2 substrate. Note the decrease in JNK activity in bFGF-treated ECs and the capacity for NFATc2 phosphorylation in cells treated with bFGF + PEDF. Western blot with JNK antibodies was performed to ensure equal loading. The result is representative of three independent experiments. (e) NFATc2 redistribution by PEDF was JNK dependent. HUVECs were plated on gelatinized coverslips; treated with indicated combinations of VEGF, PEDF, and JNK inhibitor SP600125; and stained for NFATc2. JNK blockade caused persistent NFATc2 nuclear localization (active state) despite PEDF treatment. (f and g) PEDF caused physical interaction between JNK and NFATc2. Quiescent or activated HUVECs (induced with VEGF or bFGF, as indicated) were treated for 15 min with PEDF. Nuclear or cytosolic extracts were precipitated with JNK-1– or JNK-2–specific antibody and analyzed by Western blotting with NFATc2 pAb. Purity of the fractions was determined by blotting with antibodies against GM130 protein. (f) PEDF increased interaction between JNK-2 and NFATc2 in the cytosol of stimulated ECs. (g) NFATc2 interacted with both JNK-1 and JNK-2 in the nuclei; note the increased complex formation by PEDF in stimulated but not in quiescent ECs.
Figure 3.
Figure 3.
JNK inhibitor attenuated EC apoptosis by PEDF and interfered with its antiangiogenic activity. (a) PEDF-induced apoptosis was JNK dependent. HUVECs were treated with bFGF to maintain survival and with PEDF and/or JNK inhibitor SP600125 for 18 h. Cells positive for DNA fragmentation (apoptosis) were detected by in situ TUNEL assay and quantified using MetaView software. The data from two independent experiments are presented. (white bars) bFGF alone. (black bars) bFGF + PEDF. SEM are shown. *, Significantly different from background (P < 0.005). **, No significant difference from background (P < 0.25). (b) PEDF inhibited EC migration via JNK kinases. JNK inhibitor was used in the EC chemotaxis assay. Note that PEDF ability to block migration up the VEGF gradient was abolished by SP500125. (gray bars) VEGF alone. (black bars) VEGF + PEDF. *, Significant difference from VEGF-induced migration (P < 0.05). (c and d) JNK kinases were essential to PEDF antiangiogenic activity in vivo. Mice received corneal implants containing indicated combinations of bFGF, PEDF, and SP600125. Vascularization was examined by slit-lamp microscopy. Multiple capillaries reaching implants were scored as a positive response, and the data were presented as the number of positive corneas out of the total implanted (c). SP600125 was neutral alone and had no effect on bFGF-induced angiogenesis, but reversed PEDF inhibitory activity. Photographs of representative corneas are shown (d).
Figure 4.
Figure 4.
PEDF decreased NFAT binding to DNA consensus sequences and to c-FLIP promoter. (a) PEDF treatment lowered NFATc2 DNA-binding activity in stimulated ECs. HUVECs were treated for 1 h with indicated combinations of bFGF and PEDF, and nuclear extracts were examined by EMSA with NFAT consensus oligonucleotide (ODN). The same ODN (×10 excess, unlabeled) was used as a specific competitor and SP1 consensus ODN was used as a nonspecific competitor. Note the strong decrease in specific band intensity in the presence of PEDF, the supershift in the presence of NFATc2 antibody, and the decreased intensity of the supershifted band due to PEDF. (b–e) c-FLIP regulation by PEDF. VEGF- or bFGF-induced HUVECs were treated for 4 h (b and c) or 2 h (d and e) with PEDF alone or in combination with SP600125 and used for Western blotting for c-FLIP protein (b), for semi-quantitative RT-PCR for c-FLIP mRNA (c), or in chromatin ChIP assay (d and e). (b) Note the decreased c-FLIP protein due to PEDF, and the reversal by SP600125. (c) PEDF decreased c-FLIP mRNA in stimulated ECs. Three independent experiments were performed with similar results. (d and e) PEDF decreased NFAT binding to c-FLIP promoter in vivo. ChIP with NFATc2 antibody was performed on ECs activated with VEGF (d) or bFGF (e) treated with PEDF. Note the dramatic decrease in the PCR-amplified DNA fragment in the presence of PEDF.
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