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

B 细胞淋巴瘤细胞系中 TGF-b 信号转导的表征

• 批准号: 7963874
• 负责人: Dan Longo
• 金额: $ 36.74万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7963874
• 关键词: B-Cell Lymphomas; Binding; C-terminal; Cell Line; Cell surface; Cells; Data; Development; Dose; Down-Regulation; Gene Silencing; Genes; Goals; Growth; Laboratory Study; Ligands; MAP2K1 gene; MAPK1 gene; MEKs; Mediating; Methylation; Phenotype; Phosphorylation; Receptor Down-Regulation; Reporting; Resistance; Signal Transduction; Time; Transfection; U-0126; Up-Regulation; cell type; human TGFB1 protein; inhibitor/antagonist; interest; neoplastic cell; novel; promoter; receptor

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.

肿瘤细胞的一个共同特征是逃避 TGF-b1 介导的生长抑制作用。我们对两种 B 细胞淋巴瘤细胞系 DB 和 RL 感兴趣，它们对 TGF-b1 介导的生长抑制具有抵抗力。 我们之前曾报道过，低剂量的 PMA 使 RL 细胞对 TGF-b1 敏感，而 DB 细胞仍然不敏感。 我们最近发现，DB 细胞中不存在 TGF-b1 介导的 Smad2 和 Smad3 磷酸化，而在低剂量 PMA 存在的 RL 细胞中观察到 TGF-b1 诱导的 Smad3 和 Smad2 磷酸化。对 TGF-b 受体 (TbR) 状态的检查表明，RL 和 DB 细胞的细胞表面都有 TGF-b 受体 I (TbRI)，而 TGF-b 受体 II (TbRII) 仅存在于细胞表面RL 细胞。我们已经证明，转染野生型而非C端截短形式的受体II可使DB细胞对TGF-b1介导的生长抑制产生反应。对 TRII 基因的分析显示，受体 II 信息不存在，而在用去甲基化剂处理后，该信息被逆转，表明启动子甲基化可能是基因沉默的原因。启动子分析显示 -25 和 -140 处的 CpG 甲基化与基因沉默相关。我们已经证明，启动子甲基化也参与了另一种 B 细胞淋巴瘤细胞系 Akata 中 TbRII 基因的沉默。 关于 RL 细胞对 TGF-b1 介导的生长抑制无反应，我们发现短暂的 TGF-b1 信号传导是造成抵抗的原因。 TbRII 分析揭示了配体诱导的受体以时间依赖性方式下调。 使用低剂量的 PMA，RL 细胞通过稳定 TbRII 和维持 TGF-信号传导来恢复对 TGF-b1 的敏感性。 PMA 效应是由于 MEK 激活以及 TbRII 通过与激活的 MEK1 结合而稳定。 MEK 抑制剂 U0126 阻断 PMA 诱导的 TbRII 上调。 在 HaCaT 和 BJAB 细胞（两种 TGF-b 敏感细胞系）中，U0126 诱导 TbRII 下调并阻断随后的 TGF-b 信号传导。 在 HEK293A 细胞中，组成型活性 MEK1（而非组成型活性 ERK2）诱导 TbRII 上调。 此外，TbRII 与组成型活性 MEK1 发生物理相互作用，但不与野生型 MEK1 发生相互作用，表明活性 MEK1 参与稳定 TbRII。 总的来说，我们的数据表明 MEK1 通过稳定 TbRII 来调节细胞对 TGF-b 信号传导的敏感性的新机制。 我们目前正在投资 MEK 稳定 TbRII 的机制。