Caspase-8 Inactivation in T Cells Increases Necroptosis and Suppresses Autoimmunity in Bim−/−Bim^{−/−} Mice

Dysregulation of either the extrinsic or intrinsic apoptotic pathway can lead to various diseases including immune disorders and cancer. In addition to its role in the extrinsic apoptotic pathway, caspase-8 plays nonapoptotic functions and is essential for T cell homeostasis. The pro-apoptotic BH3-only Bcl-2 family member Bim is important for the intrinsic apoptotic pathway and its inactivation leads to autoimmunity that is further exacerbated by loss of function of the death receptor Fas. We report that inactivation of caspase-8 in T cells of $Bim^{−/−}$ mice restrained their autoimmunity and extended their life span. We show that, similar to $caspase-8^{−/−}$ T cells, $Bim^{−/−}$ T cells that also lack caspase-8 displayed elevated levels of necroptosis and that inhibition of this cell death process fully rescued the survival and proliferation of these cells. Collectively, our data demonstrate that inactivation of caspase-8 suppresses the survival and proliferative capacity of $Bim^{−/−}$ T cells and restrains autoimmunity in $Bim^{−/−}$ mice

Genomic Instability, Defective Spermatogenesis, Immunodeficiency, and Cancer in a Mouse Model of the RIDDLE Syndrome

Eukaryotic cells have evolved to use complex pathways for DNA damage signaling and repair to maintain genomic integrity. RNF168 is a novel E3 ligase that functions downstream of ATM,γ-H2A.X, MDC1, and RNF8. It has been shown to ubiquitylate histone H2A and to facilitate the recruitment of other DNA damage response proteins, including 53BP1, to sites of DNA break. In addition, RNF168 mutations have been causally linked to the human RIDDLE syndrome. In this study, we report that Rnf168−/− mice are immunodeficient and exhibit increased radiosensitivity. Rnf168−/− males suffer from impaired spermatogenesis in an age-dependent manner. Interestingly, in contrast to H2a.x−/−, Mdc1−/−, and Rnf8−/− cells, transient recruitment of 53bp1 to DNA double-strand breaks was abolished in Rnf168−/− cells. Remarkably, similar to 53bp1 inactivation, but different from H2a.x deficiency, inactivation of Rnf168 impairs long-range V(D)J recombination in thymocytes and results in long insertions at the class-switch junctions of B-cells. Loss of Rnf168 increases genomic instability and synergizes with p53 inactivation in promoting tumorigenesis. Our data reveal the important physiological functions of Rnf168 and support its role in both γ-H2a.x-Mdc1-Rnf8-dependent and -independent signaling pathways of DNA double-strand breaks. These results highlight a central role for RNF168 in the hierarchical network of DNA break signaling that maintains genomic integrity and suppresses cancer development in mammals

Inactivation of Chk2 and Mus81 Leads to Impaired Lymphocytes Development, Reduced Genomic Instability, and Suppression of Cancer

Chk2 is an effector kinase important for the activation of cell cycle checkpoints, p53, and apoptosis in response to DNA damage. Mus81 is required for the restart of stalled replication forks and for genomic integrity. Mus81Δex3-4/Δex3-4 mice have increased cancer susceptibility that is exacerbated by p53 inactivation. In this study, we demonstrate that Chk2 inactivation impairs the development of Mus81Δex3-4/Δex3-4 lymphoid cells in a cell-autonomous manner. Importantly, in contrast to its predicted tumor suppressor function, loss of Chk2 promotes mitotic catastrophe and cell death, and it results in suppressed oncogenic transformation and tumor development in Mus81Δex3-4/Δex3-4 background. Thus, our data indicate that an important role for Chk2 is maintaining lymphocyte development and that dual inactivation of Chk2 and Mus81 remarkably inhibits cancer

Role of Pirh2 in Mediating the Regulation of p53 and c-Myc

Ubiquitylation is fundamental for the regulation of the stability and function of p53 and c-Myc. The E3 ligase Pirh2 has been reported to polyubiquitylate p53 and to mediate its proteasomal degradation. Here, using Pirh2 deficient mice, we report that Pirh2 is important for the in vivo regulation of p53 stability in response to DNA damage. We also demonstrate that c-Myc is a novel interacting protein for Pirh2 and that Pirh2 mediates its polyubiquitylation and proteolysis. Pirh2 mutant mice display elevated levels of c-Myc and are predisposed for plasma cell hyperplasia and tumorigenesis. Consistent with the role p53 plays in suppressing c-Myc-induced oncogenesis, its deficiency exacerbates tumorigenesis of Pirh2−/− mice. We also report that low expression of human PIRH2 in lung, ovarian, and breast cancers correlates with decreased patients' survival. Collectively, our data reveal the in vivo roles of Pirh2 in the regulation of p53 and c-Myc stability and support its role as a tumor suppressor

DNA double-strand break signaling and human disorders

Abstract DNA double-strand breaks are among the most serious types of DNA damage and their signaling and repair is critical for all cells and organisms. The repair of both induced and programmed DNA breaks is fundamental as demonstrated by the many human syndromes, neurodegenerative diseases, immunodeficiency and cancer associated with defective repair of these DNA lesions. Homologous recombination and non-homologous end-joining pathways are the two major DNA repair pathways responsible for mediating the repair of DNA double-strand breaks. The signaling of DNA double-strand breaks is critical for cells to orchestrate the repair pathways and maintain genomic integrity. This signaling network is highly regulated and involves a growing number of proteins and elaborated posttranslational modifications including phosphorylation and ubiquitylation. Here, we highlight the recent progress in the signaling of DNA double-strand breaks, the major proteins and posttranslational modifications involved and the diseases and syndromes associated with impaired signaling of these breaks

Establishment of a newly improved detection system for NF-κB activity

The transcription factor nuclear factor-κB (NF-κB) plays roles in apoptosis, inflammation and oncogenesis. It is important for biological and medical research to understand when proteins of interest are activated in cells, leading to the establishment of a luciferase/EGFP assay to monitor the activation of transcription factors. Here, we describe an improved reporter system for NF-κB, the NF-κB-activated transgene (NAT) system that can detect NF-κB signalling with high sensitivity and specificity. The NAT system consists of large copy numbers of NF-κB consensus sequence and a minimal promoter derived from the mouse interleukin-2 (IL-2) gene. Furthermore, we generated NAT systems with stable or unstable luciferase/EGFP proteins. Stable and unstable types of luciferase/EGFP are suitable for analyzing the accumulation of and the real-time activity of NF-κB signal, respectively. Our findings suggest that the NAT system is effective for in vivo imaging of NF-κB signalling using cells or animals

Ubiquitylation of epsilon-COP by PIRH2 and regulation of the secretion of PSA

Ubiquitylation appears to be involved in the membrane trafficking system including endocytosis, exocytosis, and ER-to-Golgi transport. We found that PIRH2, which was identified as an interacting protein for androgen receptor or p53, interacts with and ubiquitylates the ε-subunit of coatmer complex, ε-COP. PIRH2 promotes the ubiquitylation of ε-COP in vitro and in vivo and consequently promotes the degradation of ε-COP. The interaction between PIRH2 and ε-COP is affected by the presence of androgen, and PIRH2 in the presence of androgen promotes ubiquitylation of ε-COP in vivo. Furthermore, overexpression of the wild type of PIRH2 in prostate cancer cells causes downregulation of the secretion of prostate-specific antigen (PSA), a secretory protein in prostate epithelial cells and one of diagnostic markers for prostate cancer. Our results indicate that PIRH2 functions as a regulator for COP I complex

Ligand-dependent transcription of estrogen receptor α is mediated by the ubiquitin ligase EFP

Estrogen-mediated ubiquitylation and subsequent degradation of the estrogen receptor α (ERα) appears to be involved in the transcriptional activity of ERα. We show that the estrogen-responsive finger protein (EFP) interacts with and ubiquitylates ERα. EFP promoted the ubiquitylation of ERα in vitro and in vivo and consequently promoted the degradation of ERα. The interaction between EFP and ERα was greatly enhanced in the presence of estrogen. The action of EFP on ERα in the presence of estrogen resulted in a robust interaction between ERα and Tip60, one of the transcriptional coactivators, leading to activation of ERα transcriptional activity. However, a dominant negative mutant of EFP lacking the RING domain prolonged the half-life of ERα and inhibited the transcription by ERα. Our results indicate that EFP functions as a cofactor for ERα-mediated transcription, thus suggesting that ERα-mediated transcription is closely linked to the ubiquitylation of ERα