Inhibition of Eph receptor-ephrin ligand interaction by tea polyphenols

Abstract

Tea contains a variety of bioactive chemicals, such as catechins and other polyphenols. These compounds are thought to be responsible for the health benefits of tea consumption by affecting the function of many cellular targets, not all of which have been identified. In a high-throughput screen for small molecule antagonists of the EphA4 receptor tyrosine kinase, we identified five tea polyphenols that substantially inhibit EphA4 binding to a synthetic peptide ligand. Further characterization of theaflavin monogallates from black tea and epigallocatechin-3,5-digallate from green tea revealed that these compounds at low micromolar concentrations also inhibit binding of the natural ephrin ligands to EphA4 and several other Eph receptors in in vitro assays. The compounds behave as competitive EphA4 antagonists, and their inhibitory activity is affected by amino acid mutations within the ephrin binding pocket of EphA4. In contrast, the major green tea catechin, epigallocatechin-3-gallate (EGCG), does not appear to be an effective Eph receptor antagonist. In cell culture assays, theaflavin monogallates and epigallocatechin-3,5-digallate inhibit ephrin-induced tyrosine phosphorylation (activation) of Eph receptors and endothelial capillary-like tube formation. However, the wider spectrum of Eph receptors affected by the tea derivatives in cells suggests additional mechanisms of inhibition besides interfering with ephrin binding. These results show that tea polyphenols derived from both black and green tea can suppress the biological activities of Eph receptors. Thus, the Eph receptor tyrosine kinase family represents an important class of targets for tea-derived phytochemicals.

Fig 1

Theaflavin monogallates and epigallocatechin-3,5-digallate inhibit ephrin binding to a subset of Eph receptors. (A) Curves for inhibition of EphA4 AP binding to immobilized biotinylated KYL peptide (top panels) or ephrin-A5 AP binding to immobilized EphA4 Fc (bottom panels). The calculated IC₅₀ values are indicated. Error bars represent the standard error from 2–3 measurements. (B) IC₅₀ values for inhibition of ephrin-A5 AP binding to immobilized EphA receptor Fc fusion proteins and ephrin-B2 AP binding to immobilized EphB receptor Fc fusion proteins. Error bars represent the standard error for IC₅₀ values calculated from 2–6 experiments.

Fig. 2

Theaflavin monogallates and epigallocatechin-3,5-digallate are competitive inhibitors. (A) Curves showing binding of ephrin-A5 AP to immobilized EphA4 Fc in the presence of the indicated concentrations of theaflavin monogallates and epigallocatechin-3,5-digallate. The curves were fitted according to the Michaelis-Menten equation. Error bars represent the standard errors from duplicate measurements. Dissociation constant (K_D) and maximal binding (B_max) values from two independent experiments were used to determine inhibition constant (K_i) values ± SE. (B) IC₅₀ values for inhibition of wild-type and mutant EphA4 AP binding to immobilized ephrin-A5 Fc. IC₅₀ values obtained with EphA4 mutants were compared to those obtained with wild-type EphA4 by one-way ANOVA and Dunnett’s post-hoc test. *P<0.05, **P<0.01, ***P<0.001. Error bars represent the standard error from 2 experiments for theaflavin monogallates and 3 experiments for epigallocatechin-3,5-digallate. It should be noted that IC₅₀ values are generally slightly higher when using the arrangement with immobilized ephrin-A5 Fc and soluble EphA4 AP.

Fig. 3

Theaflavin monogallates and epigallocatechin-3,5-digallate inhibit ephrin-induced Eph receptor phosphorylation in cells. (A) Cells pretreated for 15 min with the indicated concentrations of theaflavin monogallates or epigallocatechin-3,5-digallate were stimulated with ephrin Fc fusion proteins (+) or Fc as a control (−) for 20 min in the continued presence of the compounds. The histogram shows the average level of phosphorylated receptor normalized to the total amount of receptor in the cell lysates, both measured in ELISA assays. PC3 cells stimulated with 0.25 μg/mL ephrin-A1 Fc were used to measure EphA2 phosphorylation (top panel), while HT22 cells stimulated with 0.5 μg/mL ephrin-A5 Fc were used to measure EphA4 phosphorylation (bottom panel). Values were normalized to those in ephrin-treated cells in the absence of compounds. Error bars represent standard errors from 3–14 measurements in the top panel and from 3–9 measurements in the bottom panel. The levels of EphA2 and EphA4 phosphorylation in cells treated with ephrins Fc fusion proteins in the presence of the compounds were compared with those in cells treated only with ephrins by one-way ANOVA and Dunnett’s post-test. **P<0.01, ***P<0.001. IC₅₀ values were calculated using all normalized measurements, yielding in the case of EphA2 19 μM for theaflavin monogallates and 90 μM for epigallocatechin-3,5-digallate and in the case of EphA4 61 μM for theaflavin monogallates and 190 μM for epigallocatechin-3,5-digallate. (B) Cells pretreated with the indicated concentrations of the compounds for 15 min were stimulated for 20 min with ephrin Fc (+) or Fc (−) as a control in the continued presence of the compounds. PC3 cells stimulated with 0.25 μg/mL ephrin-A1 Fc were used to immunoprecipitate EphA2; HT22 neuronal cells stimulated with 0.5 μg/mL ephrin-A5 Fc were used to immunoprecipitate EphA4; COS cells stimulated with 0.8 μg/mL ephrin-B2 Fc were used to immunoprecipitate EphB2; B16 melanoma cells stimulated with 1.5 μg/mL preclustered ephrin-B2 Fc were used to immunoprecipitate EphB4. Eph receptor immunoprecipitates were probed for phosphotyrosine (PTyr) and reprobed for the Eph receptor immunoprecipitated.

Fig. 4

Tea polyphenols inhibit Eph receptor phosphorylation and capillary-like tube formation in endothelial cells. (A) HUVECs pretreated for 15 min with 50 μM of epigallocatechin-3,5-digallate, theaflavin monogallates or EGCG were stimulated with 0.25 μg/mL ephrin-A1 Fc and 1.5 μg/mL preclustered ephrin-B2 Fc (+) or Fc as a control (−) for 20 min in the continued presence of the compounds. EphA2 and EphB4 immunoprecipitates were probed with an anti-phosphotyrosine antibody (pTyr). Cell lysates were probed for EphA2, EphB4 and β-actin. The lanes are from the same immunoblots, and white vertical lines indicate removal of irrelevant lanes. (B, C) HUVECs plated on Matrigel were treated with the indicated concentrations of tea polyphenols or DMSO control and imaged 18 hours later. (D) The histogram shows average numbers of polygons in 2–4 pictures from each of 3–6 wells and the error bars represent the standard errors. The number of polygons in wells treated with the compounds was compared with that obtained in control wells by one-way ANOVA and Dunnett’s post-hoc test. ***P<0.001 (E) MTT assay to evaluate the number of viable HUVECs after growth in the presence of the indicated compound concentrations for 24 hours. The histogram shows average absorbance at 570 nm in the presence of the compounds normalized to the absorbance in the absence of compounds. Error bars represent standard errors from 3 measurements.