IRFs Regulate Cell Cycle and Growth Inhibitory Genes in

干扰素调节因子(IRF)家族的两个成员IRF-1和ICSBP(干扰素共识序列结合蛋白)是肿瘤抑制因子，对造血细胞有很强的生长抑制作用。我们之前已经证明，IRF-1和ICSBP在小鼠晶状体中有差异表达，并且它们在发育中的晶状体中水平或空间分布的扰动抑制了晶状体的生长。在这项研究中，我们研究了它们抑制晶状体细胞生长的分子机制。将小鼠IRF-1或ICSBP的编码序列克隆到哺乳动物表达载体中，并将其导入α-Tn4-1晶状体上皮细胞系。采用核糖核酸酶保护实验、Western blotting、免疫沉淀、体外增殖实验和凝胶漂移实验检测IRF-1和ICSBP对晶状体上皮细胞生长、细胞周期转录、肿瘤抑制、细胞凋亡和生长抑制基因的影响。我们发现IRF-1和ICSBP都抑制晶状体细胞的生长，并且这种生长抑制作用是通过上调细胞周期调节蛋白(p130，周期蛋白依赖的激酶抑制物p21和p27)、凋亡蛋白(Caspase 1)和肿瘤抑制因子p53来实现的。虽然两种IRF功能重叠，但对晶状体细胞也有不同的影响；IRF-1和ICSBP诱导p21，ICSBP也促进p27和IRF-1的表达。两种蛋白均不影响Rb、Bcl2/w/x、Bak、Bax或Bad基因的表达。我们进一步证明，IRF-1/ICSBP与这些基因启动子中的干扰素反应元件相互作用，是诱导细胞凋亡和生长调节蛋白的途径。我们首次提供了由IRF-1和ICSBP介导的JAK/STAT信号通路调节对晶状体生长和分化至关重要的蛋白质的经验证据。因此，我们的数据提示，这一参与造血细胞生长和分化的重要转录因子家族也可能通过影响晶状体中的细胞周期和生长因子/细胞因子信号而在调控晶状体生长和分化中发挥作用。

Two members of the Interferon regulatory factor (IRF) family, IRF-1 and ICSBP (Interferon consensus sequence binding protein) are tumor suppressors and have potent growth inhibitory effects on hematopoietic cells. We previously showed that that IRF-1 and ICSBP are differentially expressed in the mouse lens and that perturbation of their levels or spatial distribution in the developing lens inhibited lens growth. In this study, we investigated the molecular mechanisms underlying their growth inhibitory effects in lens cells. Coding sequence of murine IRF-1 or ICSBP was cloned into a mammalian expression vector and used to transfect the a-TN4-1 lens epithelial cell line. Stable transfectants were established by selection in hygromycin B. The effects of IRF-1 or ICSBP on lens cell growth, transcription of cell cycle, tumor suppressor, apoptosis, and growth inhibitory genes were examined by RNase protection assay, Western blotting, immunoprecipitation, in vitro proliferation assay and gel-shift assay. We show that both IRF-1 and ICSBP inhibite the growth of the lens cells and that the growth inhibitory effects are mediated by up-regulation of cell cycle regulatory proteins (p130, cyclin-dependent kinase inhibitors p21 & p27), apoptotic-proteins (caspase 1) and the tumor suppressor p53. Although the two IRFs exhibit overlapping functions, they also have distinct effects on the lens cells; whereas IRF-1 and ICSBP induce p21, ICSBP also enhance p27 and IRF-1 expression. Neither protein affected Rb, Bcl-2/w/x, Bak, Bax or Bad gene expression. We further show that induction of the apoptotic and growth regulatory proteins is mediated by IRF-1/ICSBP interactions with IFN responsive elements in promoters of these genes. We provide for the first time empirical evidence that JAK/STAT signaling pathways mediated by IRF-1 and ICSBP regulate proteins that are critical to lens growth and differentiation. Our data therefore suggest that this important family of transcription factors implicated in hematopoietic cell growth and differentiation may also play a role in controling lens growth and differentiation through their effects on the cell cycle and growth factors/cytokine signaling in the lens.