Type I interferons (IFN-␣/␤) are essential for immune defense against viruses and induced through the actions of the cytoplasmic helicases, RIG-I and MDA5, and their downstream adaptor molecule IPS-1. TRAF6 and the downstream kinase TAK1 have been shown to be essential for the production of proinflammatory cytokines through the TLR/MyD88/ TRIF pathway. Although binding of TRAF6 with IPS-1 has been demonstrated, the role of the TRAF6 pathway in IFN-␣/␤ production has not been fully understood. Here, we demonstrate that TRAF6 is critical for IFN-␣/␤ induction in response to viral infection and intracellular double-stranded RNA, poly(I:C). Activation of NF-B, JNK, and p38, but not IRF3, was impaired in TRAF6-deficient mouse embryo fibroblasts in response to vesicular stomatitis virus and poly(I:C). However, TAK1 was not required for IFN-␤ induction in this process, since normal IFN-␣/␤ production was observed in TAK1-deficient mouse embryo fibroblasts. Instead, another MAP3K, MEKK1, was important for the activation of the IFN-␤ promoter in response to poly(I:C). Forced expression of MEKK1 in combination with IRF3 was sufficient for the induction of IFN-␤, whereas suppression of MEKK1 expression by small interfering RNA inhibited the induction of IFN-␤ by poly(I:C). These data suggest that IPS-1 requires TRAF6 and MEKK1 to activate NF-B and mitogen-activated protein kinases that are critical for the optimal induction of type I interferons.The innate immune system serves as a first line defense against viral infection. Host antiviral responses are initiated thorough the recognition of viral components by PRRs,2 including TLRs and RIG-I (retinoic acid-inducible gene I)-like helicases (RLHs) (1-3). Upon recognition, the PRRs trigger intracellular signaling pathways that induce the production of antiviral mediators, such as type I interferons (IFN-␣/␤), IFNstimulated genes, inflammatory cytokines, and chemokines, such as IP-10. The expression of type I IFNs and other antiviral proteins suppresses viral replication and facilitates the adaptive immune responses. dsRNA is one of the viral components recognized by TLR3 and RNA helicases, such as RIG-I and MDA5 (melanoma differentiation-associated protein 5). TLR3 recognizes extracellular viral dsRNA internalized into the endosomes in a certain type of cells, such as DCs, whereas RIG-I and MDA5 detect intracellular viral dsRNA in various types of cells, including fibroblasts (4 -7).The viral recognition by TLR3 and RIG-I/MDA5 results in rapid induction of type I IFNs through the activation of their intracellular signaling molecules (1-3). For instance, TLR3 interacts with an adaptor molecule, TRIF (8, 9), which in turn activates two IKK family proteins, TBK1 (TANK-binding kinase-1) and IKK-i (also known as IKK⑀) (10). Both TBK1 and IKK-i subsequently activate a transcription factor, IRF3, resulting in the initial expression of 12). Another IRF (IFN-regulatory factor) family member, IRF7, which is induced by the initial IFN-␤, elicits further induction of type I IFN genes, in...

Sprouty proteins (Sproutys) inhibit receptor tyrosine kinase signaling and control various aspects of branching morphogenesis. In this study, we examined the physiological function of Sproutys in angiogenesis, using gene targeting and short-hairpin RNA (shRNA) knockdown strategies. Sprouty2 and Sprouty4 double knockout (KO) (DKO) mice were embryonic-lethal around E12.5 due to cardiovascular defects. The number of peripheral blood vessels, but not that of lymphatic vessels, was increased in Sprouty4 KO mice compared with wild-type (WT) mice. Sprouty4 KO mice were more resistant to hind limb ischemia and soft tissue ischemia than WT mice were, because Sprouty4 deficiency causes accelerated neovascularization. Moreover, suppression of Sprouty2 and Sprouty4 expression in vivo by shRNA targeting accelerated angiogenesis and has a therapeutic effect in a mouse model of hind limb ischemia. These data suggest that Sproutys are physiologically important negative regulators of angiogenesis in vivo and novel therapeutic targets for treating peripheral ischemic diseases.

Acute liver failure is associated with significant mortality. However, the underlying pathophysiological mechanism is not yet fully understood. Suppressor of cytokine signaling-1 (SOCS1), which is a negative-feedback molecule for cytokine signaling, has been shown to be rapidly induced during liver injury. Here, using liver-specific SOCS1-conditional-knockout mice, we demonstrated that SOCS1 deletion in hepatocytes enhanced concanavalin A (

The protein known as Spred1 (Sprouty-related Ena ⁄ VASP homology-1 domain-containing protein) has been identified as a negative regulator of growth factor-induced ERK ⁄ mitogenactivated protein kinase activation. Spred1 has also been implicated as the target of micro RNA-126 (miR126), a miRNA located within the Egfl7 gene, and is involved in the regulation of vessel development through its role in regulating VEGF signaling. In this study, we examined the role of miR126 and Spred1 in the hematopoietic system, as miR126 has been shown to be overexpressed in leukemic cells. miR126 levels were down-regulated during mast cell differentiation from bone marrow cells, whereas Spred1 expression was inversely up-regulated. Overexpression of miR126 suppressed Spred1 expression and enhanced ERK activity in primary bone marrow cells and MC9 mast cells, which were associated with elevated FceRI-mediated cytokine production. To confirm the effect of Spred1 reduction in vivo, we generated hematopoietic cell-specific Spred1-conditional knockout mice. These mice showed increased numbers of mast cells, and Spred1-deficient bone marrow-derived mast cells were highly activated by cross-linking of Fce-R stimulation as well as by IL-3 and SCF stimulation. These results suggest that Spred1 negatively regulates mast cell activation, which is modulated by miR126.

Phagocytosis by macrophages is essential for host defense, i.e. preventing invasion of pathogens and foreign materials. (6), and the Rho family proteins (Rho (7) and Rac and Cdc42 (8 -10)) are all required for Fc␥Rs-dependent phagocytosis. LTB 4 is a classical lipid chemoattractant derived from arachidonic acid by the actions of 5-lipoxygenase, 5-lipoxygenase-activating protein (FLAP), and LTA 4 hydrolase. LTB 4 attracts neutrophils and eosinophils by the binding to the LTB 4 -specific G-protein-coupled receptor, BLT1. BLT1, originally identified in our laboratory (11), is expressed in a variety of immune cells, including dendritic cells (12), differentiated T cells (13,14), and mast cells (15). Analysis of BLT1-deficient mice revealed the importance in macrophage biology, as atherogenesis was markedly attenuated in BLT1-deficient mice in an apolipoprotein E (apoE)-null background (16). However, although LTB 4 has been shown to activate macrophage phagocytosis, details of the intracellular mechanism of LTB 4 -dependent activation of phagocytosis remain to be elucidated. Recently, Serezani et al. (17) showed that Fc␥RI engagement results in tyrosine phosphorylation of BLT1 by Src and subsequent formation of a molecular complex of Fc␥RI and BLT1 within lipid rafts that drives phagocytic functions in rat alveolar macrophages. However, it remains to be determined whether there is direct cross-talk between LTB 4 -BLT1 and IgG-Fc␥Rs signaling pathways.In this study, we investigated the function of BLT1 in macrophage phagocytosis using BLT1-deficient mice. BLT1 was necessary for Fc␥R-dependent macrophage