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 -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。我们之前报道过，低剂量PMA使RL细胞对TGF-b1敏感，而DB细胞仍然不敏感。我们最近的研究表明，tgf -b1介导的Smad2和Smad3的磷酸化在DB细胞中不存在，而tgf -b1诱导的Smad3和Smad2的磷酸化在低剂量PMA存在的RL细胞中观察到。对TGF-b受体（TbR）状态的检查显示，RL细胞和DB细胞表面都有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。我们已经证明，转染野生型，而不是c末端截断形式的受体II，使DB细胞对tgf -b1介导的生长抑制有反应。对TRII基因的分析显示，受体II信息缺失，在用去甲基化剂处理后，受体II信息被逆转，这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位点的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。

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.