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Effects of receptor tyrosine kinase inhibitors on VEGF165 a- and VEGF165 b-stimulated gene transcription in HEK-293 cells expressing human VEGFR2.

Carter JJ, Wheal AJ, Hill SJ, Woolard J - Br. J. Pharmacol. (2015)

Bottom Line: HEK-293 cells expressing the human VEGFR2 and a firefly luciferase reporter gene regulated by an NFAT response element were used for quantitative analysis of the effect of RTKIs on VEGF(165)a- and VEGF(165)b-stimulated luciferase gene expression.VEGF(165)b was a lower-efficacy agonist of the NFAT-luciferase response when compared with VEGF(165)a.Quantitative pharmacological analysis of the interaction of VEGF(165) isoforms and RTKIs with VEGFR2 in intact living cells has provided important insights into the relative affinity and efficacy of VEGF(165)a and VEGF(165)b for activation of the calcineurin- NFAT signalling pathway by this tyrosine kinase receptor.

View Article: PubMed Central - PubMed

Affiliation: Cell Signalling Research Group, School of Life Sciences, University of Nottingham, Nottingham, UK.

No MeSH data available.


Related in: MedlinePlus

The effect VEGF165a on NFAT-mediated gene transcription in VEGFR2 NFAT cells. VEGFR2 NFAT cells were treated with VEGF165a (A and B) or cediranib +1 nM VEGF165a (C). Data are mean ± SEM from quadruplicate determinations in a single representative experiment that was repeated on five separate occasions (A and C). Normalized data from five repeat experiments expressed as a percentage of the response to 10 nM VEGF165a in each experiment (B). Effect of cediranib on basal NFAT-luciferase activity (D). Data are mean ± SEM from quadruplicate determinations in a single representative experiment that was repeated on five separate occasions. The histogram in (A) and (C) show the control response to 1 nM VEGF165a (A and C) and that to VEGF165a in the presence of the vehicle (containing DMSO) for the highest concentration of cediranib used in the competition experiment shown in (C).
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fig01: The effect VEGF165a on NFAT-mediated gene transcription in VEGFR2 NFAT cells. VEGFR2 NFAT cells were treated with VEGF165a (A and B) or cediranib +1 nM VEGF165a (C). Data are mean ± SEM from quadruplicate determinations in a single representative experiment that was repeated on five separate occasions (A and C). Normalized data from five repeat experiments expressed as a percentage of the response to 10 nM VEGF165a in each experiment (B). Effect of cediranib on basal NFAT-luciferase activity (D). Data are mean ± SEM from quadruplicate determinations in a single representative experiment that was repeated on five separate occasions. The histogram in (A) and (C) show the control response to 1 nM VEGF165a (A and C) and that to VEGF165a in the presence of the vehicle (containing DMSO) for the highest concentration of cediranib used in the competition experiment shown in (C).

Mentions: Incubation with VEGF165a produced a concentration-dependent (pEC50 9.66 ± 0.05, n = 10) increase in NFAT-mediated luciferase production in HEK-293 cells expressing VEGFR2 that was 8.30 ± 0.85-fold (n = 10) over basal levels (Table 1; Figure 1A and B). The response to 1 nM VEGF165a was inhibited by the RTKI cediranib in intact HEK-293 cells in a concentration-dependent manner (Figure 1C; Table 2). The pIC50 obtained for cediranib (9.13; Figure 2A, Table 2) was in close agreement with that reported from binding studies with the purified VEGFR2 kinase domain (Davis et al., 2011). It was also noticeable that there was no marked inhibition by cediranib below basal levels at the highest concentration used (Figures 1C and 2A), suggesting that the ability of this RTKI to inhibit other tyrosine kinases (e.g. PDGFR-A, PDGFR-B and EGFR, Davis et al., 2011) did not significantly impact on the response observed. This was confirmed when the effect of cediranib was evaluated for its ability to inhibit basal NFAT-luciferase production (Figure 1D). A significant inhibition (P < 0.05; one way anova) of the small basal NFAT-luciferase response was only observed at concentrations of cediranib above 10 nM (Figure 1D). Analysis of all five repeat experiments indicated that a significant inhibition of basal signalling was only obtained at the two highest concentrations used (P < 0.05; one way anova; n = 5).


Effects of receptor tyrosine kinase inhibitors on VEGF165 a- and VEGF165 b-stimulated gene transcription in HEK-293 cells expressing human VEGFR2.

Carter JJ, Wheal AJ, Hill SJ, Woolard J - Br. J. Pharmacol. (2015)

The effect VEGF165a on NFAT-mediated gene transcription in VEGFR2 NFAT cells. VEGFR2 NFAT cells were treated with VEGF165a (A and B) or cediranib +1 nM VEGF165a (C). Data are mean ± SEM from quadruplicate determinations in a single representative experiment that was repeated on five separate occasions (A and C). Normalized data from five repeat experiments expressed as a percentage of the response to 10 nM VEGF165a in each experiment (B). Effect of cediranib on basal NFAT-luciferase activity (D). Data are mean ± SEM from quadruplicate determinations in a single representative experiment that was repeated on five separate occasions. The histogram in (A) and (C) show the control response to 1 nM VEGF165a (A and C) and that to VEGF165a in the presence of the vehicle (containing DMSO) for the highest concentration of cediranib used in the competition experiment shown in (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4459029&req=5

fig01: The effect VEGF165a on NFAT-mediated gene transcription in VEGFR2 NFAT cells. VEGFR2 NFAT cells were treated with VEGF165a (A and B) or cediranib +1 nM VEGF165a (C). Data are mean ± SEM from quadruplicate determinations in a single representative experiment that was repeated on five separate occasions (A and C). Normalized data from five repeat experiments expressed as a percentage of the response to 10 nM VEGF165a in each experiment (B). Effect of cediranib on basal NFAT-luciferase activity (D). Data are mean ± SEM from quadruplicate determinations in a single representative experiment that was repeated on five separate occasions. The histogram in (A) and (C) show the control response to 1 nM VEGF165a (A and C) and that to VEGF165a in the presence of the vehicle (containing DMSO) for the highest concentration of cediranib used in the competition experiment shown in (C).
Mentions: Incubation with VEGF165a produced a concentration-dependent (pEC50 9.66 ± 0.05, n = 10) increase in NFAT-mediated luciferase production in HEK-293 cells expressing VEGFR2 that was 8.30 ± 0.85-fold (n = 10) over basal levels (Table 1; Figure 1A and B). The response to 1 nM VEGF165a was inhibited by the RTKI cediranib in intact HEK-293 cells in a concentration-dependent manner (Figure 1C; Table 2). The pIC50 obtained for cediranib (9.13; Figure 2A, Table 2) was in close agreement with that reported from binding studies with the purified VEGFR2 kinase domain (Davis et al., 2011). It was also noticeable that there was no marked inhibition by cediranib below basal levels at the highest concentration used (Figures 1C and 2A), suggesting that the ability of this RTKI to inhibit other tyrosine kinases (e.g. PDGFR-A, PDGFR-B and EGFR, Davis et al., 2011) did not significantly impact on the response observed. This was confirmed when the effect of cediranib was evaluated for its ability to inhibit basal NFAT-luciferase production (Figure 1D). A significant inhibition (P < 0.05; one way anova) of the small basal NFAT-luciferase response was only observed at concentrations of cediranib above 10 nM (Figure 1D). Analysis of all five repeat experiments indicated that a significant inhibition of basal signalling was only obtained at the two highest concentrations used (P < 0.05; one way anova; n = 5).

Bottom Line: HEK-293 cells expressing the human VEGFR2 and a firefly luciferase reporter gene regulated by an NFAT response element were used for quantitative analysis of the effect of RTKIs on VEGF(165)a- and VEGF(165)b-stimulated luciferase gene expression.VEGF(165)b was a lower-efficacy agonist of the NFAT-luciferase response when compared with VEGF(165)a.Quantitative pharmacological analysis of the interaction of VEGF(165) isoforms and RTKIs with VEGFR2 in intact living cells has provided important insights into the relative affinity and efficacy of VEGF(165)a and VEGF(165)b for activation of the calcineurin- NFAT signalling pathway by this tyrosine kinase receptor.

View Article: PubMed Central - PubMed

Affiliation: Cell Signalling Research Group, School of Life Sciences, University of Nottingham, Nottingham, UK.

No MeSH data available.


Related in: MedlinePlus