LILRA2 selectively modulates LPS-mediated cytokine production and inhibits phagocytosis by monocytes

The activating immunoglobulin-like receptor, subfamily A, member 2 (LILRA2) is primarily expressed on the surface of cells of the innate immunity including monocytes, macrophages, neutrophils, basophils and eosinophils but not on lymphocytes and NK cells. LILRA2 cross-linking on monocytes induces pro-inflammatory cytokines while inhibiting dendritic cell differentiation and antigen presentation. A similar activating receptor, LILRA4, has been shown to modulate functions of TLR7/9 in dendritic cells. These suggest a selective immune regulatory role for LILRAs during innate immune responses. However, whether LILRA2 has functions distinct from other receptors of the innate immunity including Toll-like receptor (TLR) 4 and FcγRI remains unknown. Moreover, the effects of LILRA2 on TLR4 and FcγRI-mediated monocyte functions are not elucidated. Here, we show activation of monocytes via LILRA2 cross-linking selectively increased GM-CSF production but failed to induce IL-12 and MCP-1 production that were strongly up-regulated by LPS, suggesting functions distinct from TLR4. Interestingly, LILRA2 cross-linking on monocytes induced similar amounts of IL-6, IL-8, G-CSF and MIP-1α but lower levels of TNFα, IL-1β, IL-10 and IFNγ compared to those stimulated with LPS. Furthermore, cross-linking of LILRA2 on monocytes significantly decreased phagocytosis of IgG-coated micro-beads and serum opsonized Escherichia coli but had limited effect on phagocytosis of non-opsonized bacteria. Simultaneous co-stimulation of monocytes through LILRA2 and LPS or sequential activation of monocytes through LILRA2 followed by LPS led lower levels of TNFα, IL-1β and IL-12 production compared to LPS alone, but had additive effect on levels of IL-10 and IFNγ but not on IL-6. Interestingly, LILRA2 cross-linking on monocytes caused significant inhibition of TLR4 mRNA and protein, suggesting LILRA2-mediated suppression of LPS responses might be partly via regulation of this receptor. Taken together, we provide evidence that LILRA2-mediated activation of monocytes is significantly different to LPS and that LILRA2 selectively modulates LPS-mediated monocyte activation and FcγRI-dependent phagocytosis.     

Bone-marrow-derived myofibroblasts contribute to the cancer-induced stromal reaction

To confirm whether human cancer-induced stromal cells are derived from bone marrow, bone marrow (BM) cells obtained from beta-galactosidase transgenic and recombination activating gene 1 (RAG-1) deficient double-mutant mice (H-2b) were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice (H-2d). The human pancreatic cancer cell line Capan-1 was subcutaneously xenotransplanted into SCID recipients and stromal formation was analyzed on day 14 and on day 28. Immunohistochemical and immunofluorescence studies revealed that BM-derived endothelial cells (X-gal/CD31 or H-2b/CD31 double-positive cells) and myofibroblasts (X-gal/alpha-smooth muscle actin or H-2b/alpha-smooth muscle actin double-positive cells) were present within and around the cancer nests. On day 14, the frequencies of BM-derived endothelial cells and BM-derived myofibroblasts were 25.3+/-4.4% and 12.7+/-9.6%, respectively. On day 28, the frequency of BM-derived endothelial cells was 26.7+/-9.7%, which was similar to the value on day 14. However, the frequency of BM-derived myofibroblasts was significantly higher (39.8+/-17.1%) on day 28 than on day 14 (P<0.05). The topoisomerase IIalpha-positive ratio was 2.2+/-1.2% for the H-2b-positive myofibroblasts, as opposed to only 0.3+/-0.4% for the H-2b-negative myofibroblasts, significant proliferative activity was observed in the BM-derived myofibroblasts (P<0.05). Our results indicate that BM-derived myofibroblasts become a major component of cancer-induced stromal cells in the later stage of tumor development.     

Essential Role of the ε Subunit for Reversible Chemo-Mechanical Coupling in F1-ATPase

F₁-ATPase is a rotary motor protein driven by ATP hydrolysis. Among molecular motors, F₁ exhibits unique high reversibility in chemo-mechanical coupling, synthesizing ATP from ADP and inorganic phosphate upon forcible rotor reversal. The ε subunit enhances ATP synthesis coupling efficiency to > 70% upon rotation reversal. However, the detailed mechanism has remained elusive. In this study, we performed stall-and-release experiments to elucidate how the ε subunit modulates ATP association/dissociation and hydrolysis/synthesis process kinetics and thermodynamics, key reaction steps for efficient ATP synthesis. The ε subunit significantly accelerated the rates of ATP dissociation and synthesis by two- to fivefold, whereas those of ATP binding and hydrolysis were not enhanced. Numerical analysis based on the determined kinetic parameters quantitatively reproduced previous findings of two- to fivefold coupling efficiency improvement by the ε subunit at the condition exhibiting the maximum ATP synthesis activity, a physiological role of F₁-ATPase. Furthermore, fundamentally similar results were obtained upon ε subunit C-terminal domain truncation, suggesting that the N-terminal domain is responsible for the rate enhancement.     

Pyrrolinone derivatives as a new class of P2X3 receptor antagonists Part 2: Discovery of orally bioavailable compounds

Some P2X3 receptor antagonists have been developed as new therapeutic drugs for pain. We discovered a novel chemotype of P2X3 receptor antagonists with a pyrrolinone skeleton. Because of SAR studies to improve bioavailability of lead compound 2, compound (R)-24 was identified, which showed an analgesic effect against neuropathic pain by oral administration. We constructed a human P2X3 homology model as a template for the zebrafish P2X4 receptor, which agreed with SAR studies of pyrrolinone derivatives.     

DEAD-box RNA helicase subunits of the Drosha complex are required for processing of rRNA and a subset of microRNAs

MicroRNAs (miRNAs) control cell proliferation, differentiation and fate through modulation of gene expression by partially base-pairing with target mRNA sequences. Drosha is an RNase III enzyme that is the catalytic subunit of a large complex that cleaves pri-miRNAs with distinct structures into pre-miRNAs. Here, we show that both the p68 and p72 DEAD-box RNA helicase subunits in the mouse Drosha complex are indispensable for survival in mice, and both are required for primary miRNA and rRNA processing. Gene disruption of either p68 or p72 in mice resulted in early lethality, and in both p68(-/-) and p72(-/-) embryos, expression levels of a set of, but not all, miRNAs and 5.8S rRNA were significantly lowered. In p72(-/-) MEF cells, expression of p72, but not a mutant lacking ATPase activity, restored the impaired expression of miRNAs and 5.8S rRNA. Furthermore, we purified the large complex of mouse Drosha and showed it could generate pre-miRNA and 5.8S rRNA in vitro. Thus, we suggest that DEAD-box RNA helicase subunits are required for recognition of a subset of primary miRNAs in mDrosha-mediated processing.     

Presence of supercooling-facilitating (anti-ice nucleation) hydrolyzable tannins in deep supercooling xylem parenchyma cells in <Emphasis Type="Italic">Cercidiphyllum japonicum</Emphasis>

Xylem parenchyma cells (XPCs) in trees adapt to subzero temperatures by deep supercooling. Our previous study indicated the possibility of the presence of diverse kinds of supercooling-facilitating (SCF; anti-ice nucleation) substances in XPCs of katsura tree (Cercidiphyllum japonicum), all of which might have an important role in deep supercooling of XPCs. In the previous study, a few kinds of SCF flavonol glycosides were identified. Thus, in the present study, we tried to identify other kinds of SCF substances in XPCs of katsura tree. SCF substances were purified from xylem extracts by silica gel column chromatography and Sephadex LH-20 column chromatography. Then, four SCF substances isolated were identified by UV, mass and nuclear magnetic resonance analyses. The results showed that the four kinds of hydrolyzable gallotannins, 2,2′,5-tri-O-galloyl-α,β-d-hamamelose (trigalloyl Ham or kurigalin), 1,2,6-tri-O-galloyl-β-d-glucopyranoside (trigalloyl Glc), 1,2,3,6-tetra-O-galloyl-β-d-glucopyranoside (tetragalloyl Glc) and 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranoside (pentagalloyl Glc), in XPCs exhibited supercooling capabilities in the range of 1.5–4.5°C, at a concentration of 1 mg mL⁻¹. These SCF substances, including flavonol glycosides and hydrolyzable gallotannins, may contribute to the supercooling in XPCs of katsura tree.     

RYBP represses endogenous retroviruses and preimplantation- and germ line-specific genes in mouse embryonic stem cells

Polycomb repressive complexes (PRCs) are important chromatin regulators of embryonic stem (ES) cell function. RYBP binds Polycomb H2A monoubiquitin ligases Ring1A and Ring1B and has been suggested to assist PRC localization to their targets. Moreover, constitutive inactivation of RYBP precludes ES cell formation. Using ES cells conditionally deficient in RYBP, we found that RYBP is not required for maintenance of the ES cell state, although mutant cells differentiate abnormally. Genome-wide chromatin association studies showed RYBP binding to promoters of Polycomb targets, although its presence is dispensable for gene repression. We discovered, using Eed-knockout (KO) ES cells, that RYBP binding to promoters was independent of H3K27me3. However, recruiting of PRC1 subunits Ring1B and Mel18 to their targets was not altered in the absence of RYBP. In contrast, we have found that RYBP efficiently represses endogenous retroviruses (murine endogenous retrovirus [MuERV] class) and preimplantation (including zygotic genome activation stage)- and germ line-specific genes. These observations support a selective repressor activity for RYBP that is dispensable for Polycomb function in the ES cell state. Also, they suggest a role for RYBP in epigenetic resetting during preimplantation development through repression of germ line genes and PcG targets before formation of pluripotent epiblast cells.