Stavroulaki Melanthia, Kardassis Dimitris, Chatzaki Ekaterini, Sakellaris George, Lindschau Carsten, Haller Hermann, Tosca Androniki, Krasagakis Konstantin
Department of Dermatology, Faculty of Medicine, University of Crete, Heraklion, Greece.
J Cell Physiol. 2008 Feb;214(2):363-70. doi: 10.1002/jcp.21207.
Transforming growth factor-beta (TGF-beta), a potent inhibitor of normal melanocyte growth, does not significantly suppress growth of melanoma cells. The mechanism of melanocyte desensitization to TGF-beta in the transformation process remains largerly unknown. We investigated whether the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) may induce melanocyte resistance to TGF-beta. Cell proliferation and DNA synthesis of normal human melanocytes were strongly inhibited by TGF-beta, whereas in the presence of TPA remained largerly unaffected. The inactive phorbol ester 4alpha-phorbol 12,13 didecanoate did not modify the TGF-beta antiproliferative effect, whereas the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol counteracted TGF-beta effects. Protein kinase C (PKC) is the major cellular receptor of tumor promoting phorbol esters. PKC-alpha expression and phosphorylation were almost completely downregulated under combined treatment with TGF-beta + TPA at 24 and 72 h, as shown by immunoblots. Confocal microscopy demonstrated that TGF-beta-induced nuclear accumulation of PKC-alpha was abolished in the presence of TPA at the same time points. The selective PKC inhibitor Ro-31-8220 weakened the TGF-beta antiproliferative effect. Smads are central mediators for TGF-beta signal transduction. Smad-dependent transcriptional activity was suppressed in TGF-beta-treated melanocytes in the presence of TPA, as well as in ALK5 (constitutively active type I TGF-beta receptor)- or Smad3 + Smad4-transfected melanocytes in the presence of Ro-31-8220. In addition, an antisense oligodeoxynucleotide against PKC-alpha abolished TGF-beta-driven Smad-mediated transcription. These findings show that tumor promoting phorbol esters induce melanocyte resistance to TGF-beta, associated with downregulation of PKC-alpha and suppression of Smad-dependent transcription. This may represent an important mechanism for expansion of melanocytes exposed to PKC-targeting tumor promoters.
转化生长因子-β（TGF-β）是正常黑素细胞生长的强效抑制剂，但对黑素瘤细胞的生长并无显著抑制作用。在转化过程中黑素细胞对TGF-β脱敏的机制仍大多未知。我们研究了促肿瘤佛波酯12-O-十四烷酰佛波醇-13-乙酸酯（TPA）是否可诱导黑素细胞对TGF-β产生抗性。TGF-β可强烈抑制正常人黑素细胞的细胞增殖和DNA合成，而在TPA存在的情况下，细胞增殖和DNA合成大多不受影响。无活性的佛波酯4α-佛波醇12,13-二癸酸酯不会改变TGF-β的抗增殖作用，而二酰基甘油类似物1-油酰基-2-乙酰基-sn-甘油可抵消TGF-β的作用。蛋白激酶C（PKC）是促肿瘤佛波酯的主要细胞受体。免疫印迹显示，在TGF-β + TPA联合处理24小时和72小时后，PKC-α的表达和磷酸化几乎完全下调。共聚焦显微镜显示，在相同时间点TPA存在的情况下，TGF-β诱导的PKC-α核积累被消除。选择性PKC抑制剂Ro-31-8220减弱了TGF-β的抗增殖作用。Smads是TGF-β信号转导的核心介质。在TPA存在的情况下，TGF-β处理的黑素细胞中，以及在Ro-31-8220存在的情况下，ALK5（组成型活性I型TGF-β受体）或Smad3 + Smad4转染黑素细胞中，Smad依赖的转录活性均受到抑制。此外，针对PKC-α的反义寡脱氧核苷酸消除了TGF-β驱动的Smad介导的转录。这些发现表明，促肿瘤佛波酯可诱导黑素细胞对TGF-β产生抗性，这与PKC-α的下调和Smad依赖的转录抑制有关。这可能是暴露于靶向PKC的肿瘤启动子的黑素细胞扩增的重要机制。
