IRFs Regulate Cell Cycle and Growth Inhibitory Genes in

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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.