The anti-phospholipid syndrome (APS) is characterized by thrombosis and the presence of anti-phospholipid antibodies (aPL). Tissue factor (TF), the major initiator of the coagulation system, is induced on monocytes by aPL in vitro, explaining, in part, the pathophysiology in this syndrome. However, little is known regarding the nature of the aPL-induced signal transduction pathways leading to TF expression. In this study, we investigated aPL-inducible genes in PBMC using cDNA array system and real-time PCR. Our results indicated that the mitogen-activated protein kinase (MAPK) pathway was related to TF expression when PBMCs were treated, in the presence of beta(2)Glycoprotein I (beta(2)GPI), with human monoclonal anti-beta(2)GPI antibodies [beta(2)GPI-dependent anti-cardiolipin antibodies (aCL/beta(2)GPI)]. Western blotting studies using monocyte cell line (RAW264.7) demonstrated that p38 MAPK protein was phosphorylated with nuclear factor kappaB (NF-kappaB) activation by monoclonal aCL/beta(2)GPI treatment, and that SB203580, a specific p38 MAPK inhibitor, decreased the aCL/beta(2)GPI-induced TF mRNA expression. The p38 MAPK phosphorylation, NF-kappaB translocation and TF mRNA expression triggered by aCL/beta(2)GPI were abolished in the absence of beta(2)GPI. These results demonstrated that the p38 MAPK signaling pathway plays an important role in aPL-induced TF expression on monocytes and suggest that the p38 MAPK may be a possible therapeutic target to modify a pro-thrombotic state in patients with APS.

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Defective signaling or repair of DNA double-strand breaks has been associated with developmental defects and human diseases. The E3 ligase RING finger 168 (RNF168), mutated in the human radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties syndrome, was shown to ubiquitylate H2A-type histones, and this ubiquitylation was proposed to facilitate the recruitment of p53-binding protein 1 (53BP1) to the sites of DNA double-strand breaks. In contrast to more upstream proteins signaling DNA double-strand breaks (e.g., RNF8), deficiency of RNF168 fully prevents both the initial recruitment to and retention of 53BP1 at sites of DNA damage; however, the mechanism for this difference has remained unclear. Here, we identify mechanisms that regulate 53BP1 recruitment to the sites of DNA double-strand breaks and provide evidence that RNF168 plays a central role in the regulation of 53BP1 functions. RNF168 mediates K63-linked ubiquitylation of 53BP1 which is required for the initial recruitment of 53BP1 to sites of DNA double-strand breaks and for its function in DNA damage repair, checkpoint activation, and genomic integrity. Our findings highlight the multistep roles of RNF168 in signaling DNA damage.ubiquitin | G2/M checkpoint | HR repair pathways | NHEJ repair pathway T he DNA-damage response (DDR) is critical for genomic integrity (1) and is regulated by posttranslational modifications (PTMs) such as ubiquitylation of histones by the E3 ligases RING finger 8 (RNF8) and RING finger 168 (RNF168). Other PTMs important for DDR include dimethylation of histone H4 (H4K20me2), which allows p53-binding protein 1 (53BP1), a key mediator of DDR, to interact with chromatin.Mutations of RNF168 have been associated with the human radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties (RIDDLE) syndrome, (2-4). RNF168 has an N-terminal RING finger domain, three ubiquitin (Ub)-binding domains (UBDs); two motif interacting with Ub (MIU) domains; and one Ub interacting motif (UIM)-and MIU-related (UMI) UBD (3,5,6). Current data support RNF168 function in DNA double-strand break (DSB) signaling downstream of H2A.X, mediator of DNA damage checkpoint 1 (MDC1), and RNF8 and indicate its requirement for 53BP1 recruitment to DSB sites (3, 5). Through its UBDs, RNF168 recognizes RNF8 ubiquitylated non-nucleosomal protein(s) at DSB-flanking sites, leading to its recruitment at these sites of DNA damage (7). With the Ubconjugating enzyme UBC13, RNF168 initiates ubiquitylation of lysine (K) 13 or 15 of histones H2A and H2A.X, leading to the recruitment of DDR proteins, including 53BP1, to DSBs. Although H2A.X, MDC1, and RNF8 are important for the retention of 53BP1 at these DSBs, its initial and transient recruitment to DNA breaks still occurs in their absence (8-11). In contrast, deficiency of Rnf168 in mouse embryonic fibroblasts (MEFs) completely abolishes 53bp1 recruitment to DSB sites (12). Similar to 53bp1 −/− mice, but in contrast to H2a.x −/− mice (13), young Rnf168 −/− males are ferti...