Weber Artur-Aron, Neuhaus Thomas, Skach Romanita Adriana, Hescheler Jürgen, Ahn Hee-Yul, Schrör Karsten, Ko Yon, Sachinidis Agapios
Institute of Pharmacology and Clinical Pharmacology, University Clinic of Düsseldorf, Germany.
FASEB J. 2004 Jan;18(1):128-30. doi: 10.1096/fj.03-0007fje. Epub 2003 Nov 20.
An enhanced activity of receptor tyrosine kinases (RTKs), such as the platelet-derived growth factor (PDGF) alpha-receptor (PDGF-Ralpha) or the PDGF beta-receptor (PDGF-Rbeta), is involved in the development of proliferative diseases. 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-Rbeta. However, the underlying cellular and molecular mechanisms are unknown. Using human vascular smooth muscle cells (VSMC) and porcine endothelial cells (AEC) stably transfected with PDGF-Ralpha and -beta, respectively, we demonstrate that EGCG preferably inhibited PDGF-BB isoform-mediated signal transduction pathways and cell proliferation. To elucidate cellular and molecular mechanisms of the inhibitory effects of EGCG, we studied the distribution of incorporated EGCG into cellular compartments after subcellular fractionation. Interestingly, most (85%) of the EGCG was found in the cytoplasmic fraction, whereas only ~2% was found within the cell plasma membranes. However, no alteration of membrane fluidity has been observed after treatment of VSMC with 50 microM EGCG. Binding studies with [125I]-PDGF-BB on EGCG-treated VSMC demonstrated that the specific binding of PDGF-BB was completely abolished. Moreover, when [125I]-PDGF-BB was incubated with VSMC in the presence of EGCG, a 50% reduction of cellular [125I]-PDGF-BB binding was observed. Our findings suggest that plasma membrane incorporated EGCG or soluble EGCG directly interacts with PDGF-BB, thereby preventing specific receptor binding.
受体酪氨酸激酶（RTK）活性增强，如血小板衍生生长因子（PDGF）α受体（PDGF-Rα）或PDGFβ受体（PDGF-Rβ），参与增殖性疾病的发展。我们之前已经证明，在儿茶素结构第三位含有没食子酰基的绿茶儿茶素通过抑制PDGF-Rβ的酪氨酸磷酸化来干扰PDGF-BB诱导的促有丝分裂信号通路。然而，其潜在的细胞和分子机制尚不清楚。分别使用稳定转染了PDGF-Rα和-β的人血管平滑肌细胞（VSMC）和猪内皮细胞（AEC），我们证明表没食子儿茶素没食子酸酯（EGCG）优先抑制PDGF-BB亚型介导的信号转导通路和细胞增殖。为了阐明EGCG抑制作用的细胞和分子机制，我们在亚细胞分级分离后研究了掺入的EGCG在细胞区室中的分布。有趣的是，大部分（85%）的EGCG存在于细胞质部分，而在细胞膜中仅发现约2%。然而，用50μM EGCG处理VSMC后未观察到膜流动性的改变。用[125I]-PDGF-BB对EGCG处理的VSMC进行结合研究表明，PDGF-BB的特异性结合完全被消除。此外，当在EGCG存在下将[125I]-PDGF-BB与VSMC一起孵育时，观察到细胞[125I]-PDGF-BB结合减少了50%。我们的研究结果表明，掺入细胞膜的EGCG或可溶性EGCG直接与PDGF-BB相互作用，从而阻止特异性受体结合。
