Takeshi Kameyama

ZAPS is a potent stimulator of signaling mediated by the RNA helicase RIG-I during antiviral responses

The poly(ADP-ribose) polymerases (PARPs) participate in many biological and pathological processes. Here we report that the PARP-13 shorter isoform (ZAPS), rather than the full-length protein (ZAP), was selectively induced by 5′-triphosphate–modified RNA (3pRNA) and functioned as a potent stimulator of interferon responses in human cells mediated by the RNA helicase RIG-I. ZAPS associated with RIG-I to promote the oligomerization and ATPase activity of RIG-I, which led to robust activation of IRF3 and NF-κB transcription factors. Disruption of the gene encoding ZAPS resulted in impaired induction of interferon-α (IFN-α), IFN-β and other cytokines after viral infection. These results indicate that ZAPS is a key regulator of RIG-I signaling during the innate antiviral immune response, which suggests its possible use as a therapeutic target for viral control.

The microRNA miR-485 targets host and influenza virus transcripts to regulate antiviral immunity and restrict viral replication

A single miRNA exhibits both an inhibitory effect on the antiviral response and an antiviral effect on viral replication. Targets foreign and domestic The cytosolic protein RIG-I (retinoic acid–inducible gene I) is a sensor of viral RNA, and its activation induces the host’s antiviral response. Ingle et al. found that infection of various human and mouse cells with RNA viruses, including the H5N1 influenza virus, resulted in the increased production of the microRNA miR-485, which targeted RIG-I mRNA for degradation. As a result, antiviral signaling was inhibited and viral replication was enhanced. However, when cells were exposed to increased amounts of virus, mir-485 was expressed, but viral replication was inhibited. Under these conditions, miR-485 targeted PB1 mRNA, which is a viral transcript required for H5N1 replication. Together, these data suggest that miR-485 exhibits bispecificity, with the extent of infection determining its target. MicroRNAs (miRNAs) are small noncoding RNAs that are responsible for dynamic changes in gene expression, and some regulate innate antiviral responses. Retinoic acid–inducible gene I (RIG-I) is a cytosolic sensor of viral RNA; RIG-I activation induces an antiviral immune response. We found that miR-485 of the host was produced in response to viral infection and targeted RIG-I mRNA for degradation, which led to suppression of the antiviral response and enhanced viral replication. Thus, inhibition of the expression of mir-485 markedly reduced the replication of Newcastle disease virus (NDV) and the H5N1 strain of influenza virus in mammalian cells. Unexpectedly, miR-485 also bound to the H5N1 gene PB1 (which encodes an RNA polymerase required for viral replication) in a sequence-specific manner, thereby inhibiting replication of the H5N1 virus. Furthermore, miR-485 exhibited bispecificity, targeting RIG-I in cells with a low abundance of H5N1 virus and targeting PB1 in cells with increased amounts of the H5N1 virus. These findings highlight the dual role of miR-485 in preventing spurious activation of antiviral signaling and restricting influenza virus infection.

Presence of dominant negative mutation of TP53 is a risk of early recurrence in oral cancer

Genetic alteration of p53 is a significant determining factor in the carcinogenesis. The loss of function, mutant p53 can possess a dominant negative effect on wild-type p53 and may also exert gain-of-function activity. It is, however, not clear how p53 functional status due to various types of mutation results in outcome of patients with oral cancer. A total of 60 oral SCC samples were subjected to yeast functional assay that screens human p53 function in yeast, and sequencing for determination of p53 mutations. The detected mutants were further investigated for their dominant negative activity using a yeast-based transdominance assay that tests dominant negative activity of a mutant p53 over wild-type p53 by coexpressing the mutant and wild-type p53 in a yeast transcriptional reporter system. p53 mutation was found in 42 out of 60 of which 10 (24%) exhibited dominant negative activity and 32 (76%) without dominant activity (recessive mutation). The remaining 18 (30%) were considered to have wild-type p53. The patients with dominant negative mutation had significantly shorter disease-free survival than patients with no mutation (log-rank test, p<0.001) and those with a recessive mutation (p<0.016). There were slight significant differences in disease-free survival were found between the patients with tumours harbouring a recessive p53 mutation and those with tumours harbouring a wild-type p53 (p<0.038). The presence and absence of a dominant negative p53 mutation may thus provide a predictor of early recurrence in oral SCC patients.

Constitutive aryl hydrocarbon receptor signaling constrains type I interferon-mediated antiviral innate defense

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxic activity of many environmental xenobiotics. However, its role in innate immune responses during viral infection is not fully understood. Here we demonstrate that constitutive AHR signaling negatively regulates the type I interferon (IFN-I) response during infection with various types of virus. Virus-induced IFN-β production was enhanced in AHR-deficient cells and mice and resulted in restricted viral replication. We found that AHR upregulates expression of the ADP-ribosylase TIPARP, which in turn causes downregulation of the IFN-I response. Mechanistically, TIPARP interacted with the kinase TBK1 and suppressed its activity by ADP-ribosylation. Thus, this study reveals the physiological importance of endogenous activation of AHR signaling in shaping the IFN-I-mediated innate response and, further, suggests that the AHR-TIPARP axis is a potential therapeutic target for enhancing antiviral responses.

Targeted induction of interferon-λ in humanized chimeric mouse liver abrogates hepatotropic virus infection.

Background & Aims The interferon (IFN) system plays a critical role in innate antiviral response. We presume that targeted induction of IFN in human liver shows robust antiviral effects on hepatitis C virus (HCV) and hepatitis B virus (HBV). Methods This study used chimeric mice harboring humanized livers and infected with HCV or HBV. This mouse model permitted simultaneous analysis of immune responses by human and mouse hepatocytes in the same liver and exploration of the mechanism of antiviral effect against these viruses. Targeted expression of IFN was induced by treating the animals with a complex comprising a hepatotropic cationic liposome and a synthetic double-stranded RNA analog, pIC (LIC-pIC). Viral replication, IFN gene expression, IFN protein production, and IFN antiviral activity were analyzed (for type I, II and III IFNs) in the livers and sera of these humanized chimeric mice. Results Following treatment with LIC-pIC, the humanized livers of chimeric mice exhibited increased expression (at the mRNA and protein level) of human IFN-λs, resulting in strong antiviral effect on HBV and HCV. Similar increases were not seen for human IFN-α or IFN-β in these animals. Strong induction of IFN-λs by LIC-pIC occurred only in human hepatocytes, and not in mouse hepatocytes nor in human cell lines derived from other (non-hepatic) tissues. LIC-pIC-induced IFN-λ production was mediated by the immune sensor adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1R domain-containing adaptor molecule-1 (TICAM-1), suggesting dual recognition of LIC-pIC by both sensor adaptor pathways. Conclusions These findings demonstrate that the expression and function of various IFNs differ depending on the animal species and tissues under investigation. Chimeric mice harboring humanized livers demonstrate that IFN-λs play an important role in the defense against human hepatic virus infection.

A role of the sphingosine-1-phosphate (S1P)- S1P receptor 2 pathway in Epithelial Defense Against Cancer (EDAC)

Normal epithelial cells have an ability to sense and actively eliminate neighboring transformed cells, a process called epithelial defense against cancer (EDAC). Exogenous S1P plays a crucial role in EDAC; the S1P–S1PR2 pathway regulates Rho–Rho kinase–filamin in the surrounding normal cells, promoting apical extrusion of RasV12-transformed cells from epithelia.

Helicobacter pylori induces IL-1β protein through the inflammasome activation in differentiated macrophagic cells

More than 50% of people in the world are infected with Helicobacter pylori (H. pylori), which induces various gastric diseases. Especially, epidemiological studies have shown that H. pylori infection is a major risk factor for gastric cancer. It has been reported that the levels of interleukin (IL)-1β are upregulated in gastric tissues of patients with H. pylori infection. In this study, we investigated the induction mechanism of IL-1β during H. pylori infection. We found that IL-1βmRNA and protein were induced in phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells after H. pylori infection. This IL-1β production was inhibited by a caspase-1 inhibitor and a ROS inhibitor. Furthermore, K(+) efflux and Ca(2+) signaling were also involved in this process. These data suggest that NOD-like receptor (NLR) family, pyrin domain containing 3 (NLRP3) and its complex, known as NLRP3 inflammasome, are involved in IL-1β production during H. pylori infection because it is reported that NLRP3 inflammasome is activated by ROS, K(+) efflux and/or Ca(2+) signaling. These findings may provide therapeutic strategy for the control of gastric cancer in H. pylori-infected patients.

Short-term mechanical stress inhibits osteoclastogenesis via suppression of DC-STAMP in RAW264.7 cells.

Mechanical stress is an important factor in bone homeostasis, which is maintained by a balance between bone resorption by osteoclasts and bone formation by osteoblasts. However, little is known about the effects of mechanical stress on osteoclast differentiation. In this study, we examined the effects of short-term mechanical stress on osteoclastogenesis by applying tensile force to RAW264.7 cells stimulated with receptor activator of nuclear factor-κB ligand (RANKL) using a Flexercell tension system. We counted the number of osteoclasts that were tartrate-resistant acid phosphatase (TRAP)-positive and multinucleated (two or more nuclei) with or without application of mechanical stress for 24 h. Osteoclast number was lower after mechanical stress compared with no mechanical stress. Furthermore, mechanical stress for up to 24 h caused downregulation of osteoclast-specific gene expression and fusion-related molecule [dendritic cell specific transmembrane protein (DC-STAMP), osteoclast stimulatory transmembrane protein (OC-STAMP), E-cadherin, Integrin αV and Integrin β3] mRNA levels. Protein expression of DC-STAMP decreased with mechanical stress for 24 h compared to the control without mechanical stress, whereas the expression of E-cadherin, Integrin αV and Integrin β3 was slightly decreased. Nuclear factor of activated T cells c1 (NFATc1) mRNA levels were decreased at 6 h and increased at 12 and 24 h compared with the control. The levels of NFATc2, NFATc3 mRNA did not change compared with the control group. By contrast, mechanical stress for 24 h significantly enhanced NFAT transcriptional activity compared with the control, despite a decrease in DC-STAMP mRNA and protein levels. These results suggest that short-term mechanical stress strongly inhibits osteoclastogenesis through the downregulation of DC-STAMP and other fusion-related molecules and that short-term mechanical stress induces a negative regulatory mechanism that cancels the enhancement of NFAT transcriptional activity.

Induction of ICAM-1 and VCAM-1 on the Mouse Lingual Lymphatic Endothelium with TNF-α

This study investigated the TNF-α-induced ICAM-1 and VCAM-1 expression on mouse lingual lymphatic vessels. All podoplanin-positive lymphatic vessels expressed PECAM-1. In the lamina propria mucosae of TNF-α-treated tongue, almost all initial lymphatics expressed ICAM-1. There were initial lymphatics with the VCAM-1 expression and also the vessels without the expression. In the tunica muscularis of TNF-α-treated tongue, collecting lymphatic vessels expressed ICAM-1, but rarely expressed VCAM-1 whereas blood vessels simultaneously expressed ICAM-1 and VCAM-1. The ICAM-1-positive rate increased with TNF-α to 75% from 10% on initial lymphatics, and to 40% from 0% on collecting lymphatic vessels while it increased to 90% from 45% on blood vessels. The VCAM-1-positive rate increased with TNF-α to 30% from 0% on initial lymphatics, and to 5% from 0% on collecting lymphatic vessels while it increased to 75% from 5% on blood vessels. These findings suggest that the lingual lymphatic endothelium has the ability to express ICAM-1, and VCAM-1 to a lesser extent than the ICAM-1 induction with TNF-α, and that the ICAM-1 and VCAM-1 induction predominantly occurs in the initial lymphatics compared with collecting lymphatic vessels.

Characterization of Innate Immune Signalings Stimulated by Ligands for Pattern Recognition Receptors

The innate immunity is an essential step as the front line of host defense, and its aberrant activation particularly in response to nucleic acids is closely related to the pathogenesis of autoimmune and inflammatory diseases. Characterization of the innate immune signalings may provide a pathophysiological insight for better understanding of human diseases. Nucleic acid-mediated activation of pattern recognition receptors triggers the activation of two major intracellular signaling pathways, which are dependent on NF-κB and interferon regulatory factors, transcriptional factors. This leads to the subsequent induction of inflammatory cytokines and type I and III interferons. In this chapter, we first overview the representative families of nucleic acid sensors and their ligands and then show the fundamental techniques for extracellular or intracellular stimulation with these nucleic acid ligands and for detection of innate immune response, that is, IFN and proinflammatory cytokine induction, as assessed by luciferase assay, quantitative RT-PCR (qRT-PCR), and enzyme-linked immunosorbent assay.