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Mechanisms of the inhibitory effects of epigallocatechin-3 gallate on platelet-derived growth factor-BB-induced cell signaling and mitogenesis¹

ARTUR-ARON WEBER^*, THOMAS NEUHAUS, ROMANITA ADRIANA SKACH, JÜRGEN HESCHELER, HEE-YUL AHN, KARSTEN SCHRÖR^*, YON KO and AGAPIOS SACHINIDIS^,2

^* Institute of Pharmacology and Clinical Pharmacology, University Clinic of Düsseldorf, Germany;
Medical Policlinic, University of Bonn, Germany,
Department of Pharmacology, College of Medicine, Chungbuk National University, South Korea, and
Center of Physiology and Pathophysiology, University of Cologne, Germany

²Correspondence: University of Cologne Center of Physiology and Pathophysiology, Robert-Koch-Str. 39 50931 Cologne, Germany. E-mail: a.sachinidis{at}uni-koeln.de

SPECIFIC AIMS

An enhanced activity of the platelet-derived growth factor (PDGF) -receptor (PDGF-R) and the ß-receptor (PDGF-Rß) contributes to the development of proliferative diseases such as tumor and atherosclerosis. We have previously demonstrated that green tea catechins containing a galloyl group in the third position of the catechin structure interfere with PDGF-BB-induced mitogenic signaling pathways by inhibiting tyrosine phosphorylation of the PDGF-Rß. In this study, the molecular mechanisms of the inhibitory effects of epigallocatechin-3 gallate (EGCG) on PDGF-induced cell signaling and mitogenesis were investigated.

PRINCIPAL FINDINGS

1. Effects of epigallocatechin-3 gallate on platelet-derived growth factor-BB-induced cell signaling and mitogenesis
In human vascular smooth muscle cells (VSMC), EGCG (50 µM) almost completely (85±6%, n=3) inhibited PDGF-BB-induced phosphorylation of PDGF-R. Only a partial (25±5%, n=3) inhibition of receptor phosphorylation was observed upon stimulation with PDGF-AA (Fig. 1 A). As described previously for the PDGF-Rß phosphorylation, ECG also inhibited phosphorylation of PDGF-R while EC was not effective (not shown). In agreement with these findings, EGCG completely inhibited PDGF-BB-induced cell proliferation. PDGF-AA-induced cell proliferation was only inhibited by 50% (Fig. 1B ).

View larger version (36K): [in this window] [in a new window]   Figure 1. Effects of EGCG on PDGF-induced phosphorylation of the PDGF-R and proliferation of human VSMC. A) Human VSMC were stimulated with 50 ng/ml PDGF for 5 min. Tyrosine-phosphorylated PDGF-R (180 kDa) was detected by immonoprecipitation/Western blotting. B) Human VSMC were stimulated with 50 ng/ml PDGF-AA or -BB for 24 h. Cell counts were measured by automated counting (control=100%). Dara are means ± SD; n = 3, *P < 0.05 vs. PDGF.

The experiments presented thus far indicate some specificity of EGCG for the effects of PDGF-BB as compared with the PDGF-AA isoform. Thus, experiments were carried out with porcine aortic endothelial cells (AEC) stably transfected with either PDGF-R or PDGF-Rß. In porcine AEC expressing PDGF-R, EGCG had no effects on the PDGF-AA- or PDGF-AB-induced tyrosine-phosphorylation of the PDGF-R. Similarly, EGCG did not inhibit phosphorylation of PDGF-AA- or PDGF-AB-induced phosphorylation of PLC-1 or of ERK-1/2. However, PDGF-BB-induced phosphorylation of PDGF-R, PLC-1, or ERK-1/2 was concentration-dependently inhibited by EGCG. In addition, EGCG did not affect PDGF-AA-induced increase in [Ca²⁺]_i, but markedly (60%) inhibited PDGF-BB-induced [Ca²⁺]_i signals. Interestingly, EGCG inhibited the mitogenic effects of PDGF-AA and -AB by 50%. However, PDGF-BB-induced mitogenesis was almost completely blocked by EGCG.

In porcine AEC expressing PDGF-Rß, PDGF-AA had no effect on tyrosine-phosphorylation of PDGF-Rß. PDGR-Rß phosphorylation induced by PDGF-AB or -BB was inhibited (50-60%) by EGCG. Similar findings were obtained for phosphorylation of PLC-1. In addition, a marked (50%) inhibition of PDGF-BB-induced [Ca²⁺]_i transients by EGCG was observed. Consistently, PDGF-AA did not stimulate mitogenesis of PDGF-Rß–expressing porcine AEC. PDGF-AB- or -BB-induced cell proliferation was completely inhibited by EGCG in this cell system.

2. Molecular and intracellular mechanisms of the inhibitory effects of epigallocatechin-3 gallate
In our experiments, EGCG was incubated with the cells for 24 h and the EGCG-containing medium was removed prior to stimulation with PDGF. Thus, a competitive inhibition of PDGF binding to the receptor is not a likely mechanism for the inhibitory effects of the compound, as described in our study. This is further supported by data, demonstrating that the inhibition of PDGF-BB-induced PDGF-Rß phosphorylation by EGCG could not be overcome by increasing the concentrations of PDGF-BB up to 1000 ng/ml.

To gain insights into the mechanisms of the inhibitory effects of EGCG, possible cellular incorporation of EGCG was determined using [³H]-labeled EGCG. These experiments demonstrated a time-dependent incorporation of EGCG into human VSMC with maximal enrichment after 14 h. In addition the distribution of incorporated EGCG within cellular compartments including plasma and mitochondrial membranes was studied after subcellular fractionation. Interestingly most (85%) of the EGCG was found in the cytoplasmic fraction, while only 2% was found within the cell surface membranes. However, because EGCG inhibits very early events of PDGF signaling (i.e., receptor autophosphorylation) and because morphological analysis revealed that most of the cytoplasmic EGCG probably was stored in inclusion bodies, it was conceivable to study possible mechanisms "located" at the cell surface membrane.

EGCG did not change the fluidity of human VSMC membranes, as determined by the measurement of DPH fluorescence anisotropy (Fig. 2 A). Because the inhibition of PDGF-induced PDGF-Rß phosphorylation was insurmountable, a possible reduction of specific PDGF-BB binding sites was studied in a ligand binding experiments, using rat VSMC. In control cells, the specific binding of [¹²⁵I]-PDGF-BB, as determined by displacement with 50-fold excess of unlabeled PDGF-BB, amounted to 60% (Fig. 2B) . From these data, a K_d value of 300 pM consistent with literature data was calculated. In VSMC treated with EGCG, the total binding of [¹²⁵I]-PDGF-BB was comparable to control cells. Strikingly, EGCG treatment completely abolished specific PDGF-BB binding to its receptors. When [¹²⁵I]-PDGF-BB was incubated with VSMC in the presence of EGCG, a significant reduction of cellular PDGF-BB binding was observed (Fig. 2C ), indicating a direct interaction between EGCG and PDGF-BB.

View larger version (23K): [in this window] [in a new window]   Figure 2. Effects of EGCG membrane fluidity and PDGF-BB binding to VSMC. A) Human VSMC were incubated with 50 µM EGCG for 24 h. Membrane fluidity was assessed by determining the DPH fluorescence polarization. B) Rat VSMC were incubated with 50 µM EGCG for 24 h. PDGF-BB binding was measured by displacement studies, using [125I]-PDGF-BB (means±SD; n=3). C) Rat VSMC were incubated with 50 µM EGCG for 24 h. Then [125I]-PDGF-BB (5 ng) was added to the EGCG-containing starvation medium for 30 min. Subsequently, total [125I]-PDGF-BB binding to rat VSMC was measured (means±SD; n=3, *P<0.05 vs. con).

CONCLUSIONS

This study is the first to propose a mechanism for the antiproliferative effects of EGCG. Evidence is presented for a mechanism, involving an incorporation of EGCG into different cellular compartments, including cell surface membranes, which leads to a non-displaceable binding of PDGF to non-receptor binding sites, most likely resulting in a reduced PDGF-BB binding to the respective receptors.

Although only 2% of the incorporated EGCG was found in cell surface membranes, several lines of evidence favor a surface-membrane-linked mechanism of action of this catechin: Morphological analysis of EGCG-treated human VSMC revealed that most of the cytoplasmic EGCG was stored in vesicle-like inclusion bodies. Thus EGCG incorporated into these structures is not likely to inhibit PDGF-R kinase or other signaling molecules. In addition, the inhibitory actions of EGCG occur at a very early step in the PDGF signal transduction, namely at the site of PDGF-R autophosphorylation. Finally, ligand binding studies demonstrated a non-displaceable biding of PDGF to non-receptor binding sites on EGCG-treated VSMC.

In our studies, treatment with EGCG did not result in a non-specific alteration of physical membrane properties, as examplified by the measurement of membrane fluidity. Thus, a more specific mechanism should explain the antiproliferative effects of EGCG.

In fact, in a separate series of experiments a direct interaction between EGCG and PDGF-BB could be demonstrated. Although a direct binding of soluble EGCG with PDGF-BB does not explain the inhibitory effects in our study because cell stimulation was performed in the absence of EGCG, a "trapping" of PDGF by EGCG, incorporated into the plasma membrane, might have prevented specific binding of PDGF-BB to its respective receptors. This molecular action mode of EGCG provides an attractive model to explain the antiproliferative actions catechins (Fig. 3 ).

View larger version (39K): [in this window] [in a new window]   Figure 3. Proposed mechanism of the inhibitory effects of EGCG on PDGF-BB-induced cell signaling and mitogenesis. EGCG, incorporated into cell surface membranes, leads to a nondisplaceable binding of PDGF-BB to nonreceptor sites resulting in a reduced PDGF binding to the respective receptors.

In this context, it is interesting to note that catechins not only bind to PDGF but are also able to interact with several other proliferation-related proteins. Furthermore, a binding of PDGF to membrane components, such as gangliosides GM1 or GM2, has been reported. These gangliosides have also been shown to bind to other growth factors, such as bFGF. Thus, a "trapping" of growth factors by non-receptor binding sites may represent a general principle in modulation of growth factor activity.

Another interesting aspect of our study is the demonstration of a preferable inhibition of PDGF-BB- as compared with PDGF-AA-induced cell signaling and mitogenesis. Some specificity for PDGF-BB was observed in human VSMC. For example, EGCG only partially inhibited PDGF-AA-induced phosphorylation of PDGF-R, as opposed to an almost complete inhibition of PDGF-BB-induced receptor phosphorylation. This apparent selectivity was supported by experiments with porcine AEC stably transfected with PDGF-R. In these cells, EGCG did not inhibit PDGF-AA-induced phosphorylation of PDGF-R, PLC-1 or ERK-1/2. Similarly, PDGF-AA-induced [Ca²⁺]_i transients were not affected by EGCG. In contrast, EGCG markedly inhibited PDGF-BB-induced receptor phosphorylation and intracellular signaling events. In porcine AEC stably transfected with PDGF-Rß, EGCG inhibited both, PDGF-BB- and PDGF-AB-induced phosphorylation of PDGF-Rß and PLC-1. Thus, the specificity of the inhibitory effects of EGCG for the PDGF-BB-induced cell signaling is not absolute and not necessarily restricted to PDGF. In fact, data from our group demonstrated an inhibition of vascular endothelial cell factor (VEGF)-induced cell signaling by EGCG.

The mechanism of the inhibitory actions of EGCG, as proposed in the present study, may help to better understand the beneficial effects of catechins in human epidemiological studies and in animal atheroscleosis models. The non-competitive inhibition of PDGF-induced mitogenesis by EGCG incorporated into cell membranes, in addition to direct binding of PDGF (and possibly other growth factors), would result in a sustained inhibition of cell proliferation even at low plasma concentrations between doses. Thus, EGCG or other galloyl group-containing plant-derived catechins are attractive candidates to treat proliferative diseases.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj. org/cgi/doi/1096/fj.03-0007fje

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