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Nuclear factor of activated T cells balances angiogenesis activation and inhibition.

Zaichuk TA, Shroff EH, Emmanuel R, Filleur S, Nelius T, Volpert OV - J. Exp. Med. (2004)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.

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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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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fig4: 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.

Mentions: To evaluate PEDF effect on NFAT binding to the cognate DNA sequences, we used EMSA with the dual NFAT consensus binding site from the IL-2 promoter (36, 42). bFGF induced NFATc2 binding to a specific oligonucleotide, which was reduced in the presence of unlabeled specific oligonucleotide, but not in the presence of the nonspecific oligonucleotide containing a binding site for SP-1. NFAT antibody induced supershift in mobility of the DNA–NFATc2 complexes. Predictably, PEDF reversed increased NFAT DNA binding in activated ECs (Fig. 4 a).


Nuclear factor of activated T cells balances angiogenesis activation and inhibition.

Zaichuk TA, Shroff EH, Emmanuel R, Filleur S, Nelius T, Volpert OV - J. Exp. Med. (2004)

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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Related In: Results  -  Collection

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fig4: 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.
Mentions: To evaluate PEDF effect on NFAT binding to the cognate DNA sequences, we used EMSA with the dual NFAT consensus binding site from the IL-2 promoter (36, 42). bFGF induced NFATc2 binding to a specific oligonucleotide, which was reduced in the presence of unlabeled specific oligonucleotide, but not in the presence of the nonspecific oligonucleotide containing a binding site for SP-1. NFAT antibody induced supershift in mobility of the DNA–NFATc2 complexes. Predictably, PEDF reversed increased NFAT DNA binding in activated ECs (Fig. 4 a).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.

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.

Show MeSH
Related in: MedlinePlus