Osteopontin is critically involved in rheumatoid arthritis; however, the molecular cross-talk between osteopontin and joint cell components that leads to the inflammatory joint destruction is largely unknown. We found that not only osteopontin but also tenascin-C and their common receptor, α9 integrin, are expressed at arthritic joints. The local production of osteopontin and tenascin-C is mainly due to synovial fibroblasts and, to a lesser extent, synovial macrophages. Synovial fibroblasts and macrophages express α9 integrin, and autocrine and paracrine interactions of α9 integrin on synovial fibroblasts and macrophages and its ligands contribute differently to the production of proinflammatory cytokines and chemokines. α9 integrin is also involved in the recruitment and accumulation of inflammatory cells. Inhibition of α9 integrin function with an anti-α9 integrin Ab significantly reduces the production of arthrogenic cytokines and chemokines and ameliorates ongoing arthritis. Thus, we identified α9 integrin as a critical intrinsic regulator that controls the development of autoimmune arthritis.

Death associated protein 3 (DAP3) is known to be a highly conserved protein, and is responsible for regulating apoptosis induced by various stimuli. To understand the molecular mechanism of how DAP3 induces apoptosis, we performed yeast two-hybrid screening, and identified a novel DAP3-binding protein termed death ligand signal enhancer (DELE). In this report, we show that DELE actually binds to DAP3 in mammalian cells. We found that the cells stably expressing DELE are susceptible to apoptosis induction by the stimulation of TNF-α and TRAIL. In addition, knockdown of DELE expression rescued the HeLa cells from apoptosis induction by these stimuli. Moreover, activation of caspase-3, caspase-8 and caspase-9 induced by stimulation of TNF-α, anti-Fas or TRAIL was significantly inhibited by the knockdown of DELE expression. These results demonstrated the biological significance of DELE for apoptosis signal mediated by death receptors.

Detachment of adherent epithelial cells from the extracellular matrix induces apoptosis, a process known as anoikis. We have shown that DAP3 is critical for anoikis induction. However, the mechanism for anoikis induction mediated by DAP3 is still unclear. Here, we show that interferon-b promoter stimulator 1 (IPS-1) binds DAP3 and induces anoikis by caspase activation. Recently, IPS-1 has been shown to be critical for antiviral immune responses, although there has been no report of its function in apoptosis induction. We show that overexpression of IPS-1 induces apoptosis by activation of caspase-3, -8, and -9. In addition, IPS-1 knockout mouse embryonic fibroblasts were shown to be resistant to anoikis. Interestingly, IPS-1 expression, recruitment of caspase-8 to IPS-1, and caspase-8 activation were induced after cell detachment. Furthermore, DAP3-mediated anoikis induction was inhibited by knockdown of IPS-1 expression. Therefore, we elucidated a novel function of IPS-1 for anoikis induction by caspase-8 activation.

Tumor necrosis factor-α (TNF-α) has important roles in several immunological events by regulating apoptosis and transcriptional activation of cytokine genes. Intracellular signaling mediated by TNF-receptor-type 1 (TNFR1) is constituted by two sequential protein complexes: Complex-I containing the receptor and Complex-II-containing Caspase-8. Protein modifications, particularly ubiquitination, are associated with the regulation of the formation of these complexes. However, the underlying mechanisms remain poorly defined. Here, we identified CLIP-170-related 59 kDa protein (CLIPR-59) as a novel adaptor protein for TNFR1. Experimental reduction of CLIPR-59 levels prevented induction of apoptosis and activation of caspases in the context of TNF-α signaling. CLIPR-59 binds TNFR1 but dissociates in response to TNF-α stimulation. However, CLIPR-59 is also involved in and needed for the formation of Complex-II. Moreover, CLIPR-59 regulates TNF-α-induced ubiquitination of receptor-interacting protein 1 (RIP1) by its association with CYLD, a de-ubiquitinating enzyme. These findings suggest that CLIPR-59 modulates ubiquitination of RIP1, resulting in the formation of Complex-II and thus promoting Caspase-8 activation to induce apoptosis by TNF-α.

Rat parvovirus (RPV) is nonpathogenic in rats but causes persistent lymphocytotropic infection. We found that RPV was propagated in rat thymic lymphoma cell line C58(NT)D and induced apoptosis. Interestingly, a resistant subclone, C58(NT)D/R, from surviving cells after lytic infection had differentiated phenotypic modifications, such as increased cell adherence, resistance to apoptosis, and suppressed tumorigenicity.Recent molecular studies on parvoviral pathogenicity suggest that the viral nonstructural (NS) protein in which coded genes are highly homologous among parvoviruses correlates with cytotoxicity (2,8,17). The productive and cytotoxic activity of the NS protein is modulated by cellular factors that may vary with the host cell type, particularly in oncogene-transformed cells (2, 13). We have shown that a transcriptional coactivator, CREB binding protein, is required for NS1-mediated viral and cellular transcription in parvovirus-infected cells, resulting in cell proliferation and differentiation to achieve its lytic cycle (16).Newly recognized rodent parvoviruses, also called orphan parvoviruses, are widespread among laboratory mice and rats (21,23,24), and viruses isolated have been classified as mouse parvovirus and rat parvovirus (RPV) (1, 5, 6). Although mouse parvovirus grows well in a murine T-cell clone (L3), causing a cytopathic effect (CPE) (10), effective in vitro RPV propagation has not been established. We found that RPV involved lytic infection in the rat thymic lymphoma cell line C58(NT)D and developed in vitro propagation for RPV. Interestingly, virus-resistant cell clones isolated from subcultures of surviving cells acquired differentiated phenotypes such as reduced tumorigenicity and sensitivity to apoptosis. Viral propagation in cell lines. The RPV strain was isolated from rats infected spontaneously at our facility (23) and passaged three times in specific-pathogen-free newborn SpragueDawley rats. The virus stock was prepared from infected spleens at 7 days postinoculation (p.i.) We initially inoculated a supernatant of 5% infected spleen homogenate into cell lines and primary cell cultures of rats and hamsters to find cells permitting virus propagation. C6 (rat glioma), BRL-3A (Buffalo rat liver), RBL-2H3 (rat basophilic leukemia), BHK-21 (Syrian hamster kidney), and Y3-Ag1.2.3 (rat myeloma) cells were obtained from the Riken Cell Bank, Tsukuba, Japan, and C58(NT)D (rat thymic lymphoma) cells were purchased from the American Type Culture Collection, Manassas, Va. Infected cells were observed for appearance of the viral CPE and examined for presence of the viral antigen by immunofluorescent antibody (IFA) and hemagglutination (HA) ability assays. The isolated virus was propagated only in C58(NT)D cells, not in other cells tested (Table 1). Parvoviral DNA was also detected only in infected C58(NT)D cells (data not shown). In contrast, prototype RV-13 (7) was propagated, with titers in C58(NT)D cells lower than those in other cells. Propagation of the isolate in C58(NT)D cells and a dif...