3428

IFI16

interferon, gamma-inducible protein 16;IFI16;interferon, gamma-inducible protein 16

gamma-interferon-inducible protein 16|interferon-gamma induced protein IFI 16|interferon-inducible myeloid differentiation transcriptional activator

1q22

protein-coding

Recent studies have implicated interferon signaling in the regulation of cellular senescence. However, the role of specific interferon-inducible proteins in cellular senescence remains to be defined. Here we report that IFI 16, an interferon-inducible transcriptional modulator from the p200-protein family, contributes to cellular senescence of prostate epithelial cells. Normal human prostate epithelial cells (PrEC) in culture expressed detectable levels of IFI 16, and the levels increased more than fourfold when cells approached cellular senescence. Consistent with a role of IFI 16 in cellular senescence, human prostate cancer cell lines either did not express IFI 16 or expressed a variant form, which was primarily detected in the cytoplasm of prostate cancer cells and not in the nucleus. Moreover, overexpression of functional IFI 16 in human prostate cancer cell lines inhibited colony formation. Additionally, ectopic expression of IFI 16 in clonal prostate cancer cell lines was associated with a senescence-like phenotype, production of senescence-associated beta-galactosidase (a biochemical marker for cellular senescence), and reduction of S-phase cells in culture. Importantly, upregulation of p21WAF1 and inhibition of E2F-stimulated transcription accompanied inhibition of cell growth by IFI 16 in prostate cancer cell lines. Collectively, our observations support the idea that increased levels of IFI 16 in PrECs contribute to senescence-associated irreversible cell growth arrest.

IFN-inducible IFI16 protein (encoded by IFI16 gene at 1q23.1) is the human member of the IFN-inducible structurally related p200 family proteins. Increased expression of the IFI16 protein, a positive modulator of p53-mediated transcription, in normal old human diploid fibroblasts (HDF) is associated with cellular senescence-mediated cell growth arrest. However, the underlying mechanisms that contribute to transcriptional activation of the IFI16 gene in old HDFs remain to be elucidated. Here, we reported that functional activation of p53 in normal young HDFs and p53-null Saos2 cell line resulted in transcriptional activation of the IFI16 gene. We identified a potential p53 DNA-binding site (indicated as IFI16-p53-BS) in the 5-regulatory region of the IFI16 gene. Importantly, p53 bound to IFI16-p53-BS in a sequence-specific manner in gel-mobility shift assays. Furthermore, p53 associated with the 5-regulatory region of the IFI16 gene in chromatin immunoprecipitation assays. Interestingly, p53 associated with the regulatory region of the IFI16 gene only on treatment of cells with DNA-damaging agents or in the old, but not in the young, HDFs. Importantly, our promoter-reporter assays, which were coupled with site-directed mutagenesis of IFI16-p53-BS, showed that p53 activates transcription of the IFI16 gene in HDFs through the p53 DNA-binding site. Together, our observations provide support for the idea that up-regulation of IFI16 expression by p53 and functional interactions between IFI16 protein and p53 contribute to cellular senescence.

The IFN-inducible IFI16 and AIM2 proteins act as innate immune sensors for cytosolic double-stranded DNA (dsDNA). On sensing dsDNA, the IFI16 protein induces the expression of IFN-beta whereas the AIM2 protein forms an inflammasome, which promotes the secretion of IL-1beta. Given that the knockdown of IFI16 expression in human diploid fibroblasts (HDF) delays the onset of cellular senescence, we investigated the potential roles for the IFI16 and AIM2 proteins in cellular senescence. We found that increased IFI16 protein levels in old (vs. young) HDFs were associated with the induction of IFN-beta. In contrast, increased levels of the AIM2 protein in the senescent (vs. old) HDFs were associated with increased production of IL-1beta. The knockdown of type I IFN-alpha receptor subunit, which reduced the basal levels of the IFI16 but not of the AIM2, protein delayed the onset of cellular senescence. Accordingly, increased constitutive levels of IFI16 and AIM2 proteins in ataxia telangiectasia mutated (ATM) HDFs were associated with the activation of the IFN signaling and increased levels of IL-1beta. The IFN-beta treatment of the young HDFs, which induced the expression of IFI16 and AIM2 proteins, activated a DNA damage response and also increased basal levels of IL-1beta. Interestingly, the knockdown of AIM2 expression in HDFs increased the basal levels of IFI16 protein and activated the IFN signaling. In contrast, the knockdown of the IFI16 expression in HDFs decreased the basal and dsDNA-induced activation of the IFN signaling. Collectively, our observations show differential roles for the IFI16 and AIM2 proteins in cellular senescence and associated secretory phenotype.