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: 8335774
• 负责人: Luigi Ferrucci
• 金额: $ 30.19万
• 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/8335774
• 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抗性。 我们先前报道说，低剂量PMA对TGF-B1敏感的RL细胞呈现，而DB细胞仍然不敏感。 我们最近表明，DB细胞中不存在TGF-B1介导的SMAD2和SMAD3的磷酸化，而在低剂量PMA的存在下，在RL细胞中观察到TGF-B1诱导的SMAD3和SMAD2的TGF-B1诱导的磷酸化。对TGF-B受体（TBR）状态的检查表明，RL和DB细胞在其细胞表面上均具有TGF-B受体I（TBRI），而TGF-B受体II（TBRII）仅存在于RL细胞的细胞表面上。我们已经证明了野生型的转染，但没有C末端的受体II截断形式使DB细胞对TGF-B1介导的生长抑制有反应。对TRII基因的分析表明，没有受体II消息，该消息在用脱甲基化剂治疗后反转，表明启动子甲基化可能是造成基因沉默的原因。启动子分析显示，与基因沉默相关的-25和-140的CpG甲基化。我们已经表明，启动子甲基化也参与了另一个B细胞淋巴瘤细胞系Akata中的TBRII基因的沉默。 关于RL细胞对TGF-B1介导的生长抑制的无反应性，我们发现瞬态TGF-B1信号传导是抗电阻的原因。 对TBRII的分析显示，配体诱导的受体下调以时间依赖的方式。 RL细胞在低剂量的PMA中，通过稳定TBRII并维持TGF信号传导来恢复对TGF-B1的敏感性。 PMA效应是由于MEK激活和TBRII与活化的MEK1结合而引起的。 MEK抑制剂U0126阻断了PMA诱导的TBRII上调。 在HACAT和BJAB细胞中，两个TGF-B敏感的细胞系，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，但没有组成性活性ERK2诱导TBRII上调。 此外，TBRII与组成型活性MEK1进行了物理相互作用，但与野生型MEK1相互作用，表明活性MEK1参与稳定TBRII。 总体而言，我们的数据提出了MEK1的一种新型机制，可以通过稳定TBRII来调节细胞对TGF-B信号的敏感性。 我们目前正在投资MEK稳定TBRII的基础机制。