Characterization Of TGF-b Signaling In a B-cell Lymphoma Cell Line

肿瘤细胞的一个共同特征是逃避TGF-β 1介导的生长抑制作用。我们感兴趣的是两个B细胞淋巴瘤细胞系，DB和RL，是抵抗TGF-β 1介导的生长抑制。 我们以前报道过，低剂量PMA使RL细胞对TGF-β 1敏感，而DB细胞保持不敏感。 我们最近发现，TGF-β 1介导的Smad 2和Smad 3的磷酸化在DB细胞中不存在，而TGF-β 1诱导的Smad 3和Smad 2的磷酸化在低剂量PMA存在下在RL细胞中观察到。TGF-β受体（TbR）的状态的检查显示，RL和DB细胞在其细胞表面上均具有TGF-β受体I（TbRI），而TGF-β受体II（TbRII）仅存在于RL细胞的细胞表面上。我们已经证明，野生型，而不是C-末端截短形式的受体II的转染呈现DB细胞响应TGF-β 1介导的生长抑制。对TRII基因的分析揭示了受体II信息的缺失，这在用去甲基化剂处理后被逆转，表明启动子甲基化可能是基因沉默的原因。启动子分析揭示了与基因沉默相关的-25和-140处的CpG甲基化。我们已经表明，启动子甲基化也参与沉默TbRII基因在另一个B细胞淋巴瘤细胞系，Akata。 关于RL细胞对TGF-β 1介导的生长抑制的无反应性，我们发现瞬时TGF-β 1信号传导是抵抗的原因。 TbRII的分析揭示了配体诱导的受体以时间依赖性方式下调。 在低剂量PMA的情况下，RL细胞通过稳定TbRII和维持TGF-β 1信号传导来恢复对TGF-β 1的敏感性。 PMA效应是由于MEK活化和TbRII通过与活化的MEK 1结合而稳定。 MEK抑制剂U 0126阻断PMA诱导的TbRII上调。 在HaCaT和BJAB细胞中，两种TGF-β敏感的细胞系U 0126诱导TbRII下调并阻断随后的TGF-β信号传导。 在HEK 293 A细胞中，组成型活性的MEK 1，但不是组成型活性的ERK 2，诱导TbRII的上调。 此外，TbRII与组成型活性MEK 1发生物理相互作用，但不与野生型MEK 1发生物理相互作用，表明活性MEK 1参与稳定TbRII。 总的来说，我们的数据表明MEK 1通过稳定TbRII来调节细胞对TGF-β信号传导的敏感性的新机制。 我们目前正在研究MEK稳定TbRII的潜在机制。

One common feature of neoplastic cells is evasion of TGF-b1-mediated growth inhibitory effects. We are interested in two B-cell lymphoma cell lines, DB and RL, that are resistant to TGF-b1-mediated growth suppression. We have reported previously that low dose PMA rendered RL cells sensitive to TGF-b1, whereas DB cells remained insensitive. We have shown recently that the TGF-b1-mediated phosphorylation of Smad2 and Smad3 were absent in DB cells, whereas TGF-b1-induced phosphorylation of both Smad3 and Smad2 were observed in RL cells in presence of low dose PMA. Examination of the status of the TGF-b receptors (TbR) revealed that both RL and DB cells had TGF-b receptors I (TbRI) on their cell surface, whereas TGF-b receptors II (TbRII) were present only on the cell surface of RL cells. We have demonstrated that transfection of wild-type, but not a C-terminal truncated form of receptor II rendered the DB cells responsive to TGF-b1-mediated growth suppression. Analysis of the TRII gene revealed the absence of the receptor II message, which was reversed upon treatment with demethylating agent, indicating that the promoter methylation might be the cause of gene silencing. Promoter analysis revealed CpG methylations at -25 and -140 that correlated with the gene silencing. We have shown that the promoter methylation was also involved in silencing TbRII gene in another B-cell lymphoma cell line, Akata. Regarding the unresponsiveness of RL cells to TGF-b1-mediated growth suppression, we have found that the transient TGF-b1 signaling is responsible for the resistance. Analysis of TbRII revealed ligand-induced receptor down-regulation in a time-dependent manner. With a low dose of PMA, RL cells restored the sensitivity to TGF-b1 by stabilizing TbRII and sustaining TGF- signaling. The PMA effects were due to MEK activation and the stabilization of TbRII through binding to activated MEK1. The MEK inhibitor U0126 blocked PMA-induced up-regulation of TbRII. In HaCaT and BJAB cells, two TGF-b-sensitive cell lines, U0126 induced down-regulation of TbRII and blocked subsequent TGF-b signaling. In HEK293A cells, constitutively active MEK1, but not constitutively active ERK2, induced up-regulation of TbRII. Furthermore, TbRII physically interacted with the constitutively active MEK1, but not with wild type MEK1, indicating involvement of active MEK1 in stabilizing TbRII. Collectively, our data suggest a novel mechanism for MEK1 in regulating the sensitivity of cells to TGF-b signaling by stabilizing TbRII. We are currently investing the mechanism underlying the stabilization of TbRII by MEK.