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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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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).
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fig1: 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).

Mentions: The activation and subcellular distribution of NFAT family members is determined by phosphorylation (36–38); NFAT dephosphorylation/activation was shown previously to mediate VEGF-induced angiogenesis (25). In addition to reproducing results by Hernandez et al. (Fig. 1, b and c; reference 25), we found that another inducer, bFGF, also triggered NFATc2 dephosphorylation and nuclear transfer (Fig. 1, a–d).


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)

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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211785&req=5

fig1: 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).
Mentions: The activation and subcellular distribution of NFAT family members is determined by phosphorylation (36–38); NFAT dephosphorylation/activation was shown previously to mediate VEGF-induced angiogenesis (25). In addition to reproducing results by Hernandez et al. (Fig. 1, b and c; reference 25), we found that another inducer, bFGF, also triggered NFATc2 dephosphorylation and nuclear transfer (Fig. 1, a–d).

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