Hiromi Nochi

Identification of autotaxin as a neurite retraction-inducing factor of PC12 cells in cerebrospinal fluid and its possible sources

Cerebrospinal fluid (CSF) induced neurite retraction of differentiated PC12 cells; the action was observed in 15 min (a rapid response) and the activity further increased until 6 h (a long‐acting response) during exposure of CSF to the cells. The CSF action was sensitive to monoglyceride lipase and diminished by homologous desensitization with lysophosphatidic acid (LPA) and by pretreatment with an LPA receptor antagonist Ki16425. Although fresh CSF contains LPA to some extent, the LPA content in the medium was increased during culture of PC12 cells with CSF. The rapid response was mimicked by exogenous LPA, and a long‐acting response was duplicated by a recombinant autotaxin, lysophospholipase D (lyso‐PLD). Although the lyso‐PLD substrate lysophosphatidylcholine (LPC) was not detected in CSF, lyso‐PLD activity and an ∼120‐kDa autotaxin protein were detected in CSF. On the other hand, LPC but not lyso‐PLD activity was detected in the conditioned medium of a PC12 cell culture without CSF. Among neural cells examined, leptomeningeal cells expressed the highest lyso‐PLD activity and autotaxin protein. These results suggest that leptomeningeal cells may work as one of the sources for autotaxin, which may play a critical role in LPA production and thereby regulate axonal and neurite morphological change.

Stimulatory Role of Lysophosphatidic Acid in Cyclooxygenase-2 Induction by Synovial Fluid of Patients with Rheumatoid Arthritis in Fibroblast-Like Synovial Cells

While inflammatory cytokines are well-recognized critical factors for the induction of cyclooxygenase-2 (COX-2) in activated fibroblast-like synovial cells, the roles of biologically active components other than inflammatory cytokines in synovial fluid remain unknown. Herein, we assessed the role of lysophosphatidic acid (LPA), a pleiotropic lipid mediator, in COX-2 induction using synovial fluid of patients with rheumatoid arthritis (RA) in fibroblast-like RA synovial cells. Synovial fluid from RA patients stimulated COX-2 induction, which was associated with prostaglandin E2 production, in RA synovial cells. The synovial fluid-induced actions were inhibited by Gi/o protein inhibitor pertussis toxin and LPA receptor antagonist 3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfanyl) propanoic acid (Ki16425). In fact, LPA alone significantly induced COX-2 expression and enhanced IL-1α- or IL-1β-induced enzyme expression in a manner sensitive to pertussis toxin and Ki16425. RA synovial cells abundantly expressed LPA1 receptor compared with other LPA receptor subtypes. Moreover, synovial fluid contains a significant amount of LPA, an LPA-synthesizing enzyme autotaxin, and its substrate lysophosphatidylcholine. In conclusion, LPA existing in synovial fluid plays a critical role in COX-2 induction in collaboration with inflammatory cytokines in RA synovial cells. Ki16425-sensitive LPA receptors may be therapeutic targets for RA.

Involvement of pertussis toxin‐sensitive GTP‐binding proteins in sphingosine 1‐phosphate‐induced activation of phospholipase CCa2+ system in HL60 leukemia cells

Exogenous sphingosine 1‐phosphate (S1P) induced Ca2+ mobilization, in association with an increase in inositol polyphosphate production reflecting activation of phospholipase C in HL60 leukemia cells. The increase in intracellular Ca2+ concentration ([Ca2+]i) induced by S1P was inhibited by an appropriate treatment of the cells with pertussis toxin (PTX), U73122 (a phospholipase C inhibitor) or phorbol 12‐myristate 13‐acetate (PMA). In parallel with the Ca2+ response, these agents also inhibited inositol polyphosphate production. The S1P‐induced Ca2+ response was also attenuated in the dibutyryl cAMP‐induced differentiated cells, where GTP‐binding protein‐induced Ca2+ response is suggested to be enhanced. Lysophosphatidic acid (LPA) also increased [Ca2+]i in the cells, but the maximal response was about half of that of S1P, and furthermore PTX and dibutyryl cAMP treatment hardly affected the LPA‐induced Ca2+ mobilization. We conclude that exogenous S1P mobilizes Ca2+ through phospholipase C activation. The S1P‐induced enzyme activation is at least partly mediated by PTX‐sensitive GTP‐binding protein‐coupled receptors which may be different from LPA receptors.

Cyclooxygenase‐2 Expression and Prostaglandin E2 Production in Response to Acidic pH Through OGR1 in a Human Osteoblastic Cell Line

Acidosis has been shown to induce depletion of bone calcium from the body. This calcium release process is thought to be partially cell mediated. In an organ culture of bone, acidic pH has been shown to induce cyclooxygenase‐2 (COX‐2) induction and prostaglandin E2 (PGE2) production, resulting in stimulation of bone calcium release. However, the molecular mechanisms whereby osteoblasts sense acidic circumstances and thereby induce COX‐2 induction and PGE2 production remain unknown. In this study, we used a human osteoblastic cell line (NHOst) to characterize cellular activities, including inositol phosphate production, intracellular Ca2+ concentration ([Ca2+]i), PGE2 production, and COX‐2 mRNA and protein expression, in response to extracellular acidification. Small interfering RNA (siRNA) specific to the OGR1 receptor and specific inhibitors for intracellular signaling pathways were used to characterize acidification‐induced cellular activities. We found that extracellular acidic pH induced a transient increase in [Ca2+]i and inositol phosphate production in the cells. Acidification also induced COX‐2 induction, resulting in PGE2 production. These proton‐induced actions were markedly inhibited by siRNA targeted for the OGR1 receptor and the inhibitors for Gq/11 protein, phospholipase C, and protein kinase C. We conclude that the OGR1/Gq/11/phospholipase C/protein kinase C pathway regulates osteoblastic COX‐2 induction and subsequent PGE2 production in response to acidic circumstances.

Effects of high-molecular-weight hyaluronates on the functions of guinea pig polymorphonuclear leukocytes

The effect of hyaluronate (HA) with a molecular weight of 1,900,000 (HA190) purified from products of Streptococcus equi on various functions of guinea pig peritoneal polymorphonuclear leukocytes (PMNs) was examined. HA190 effectively inhibited both the chemotactic activity of PMNs for a chemotactic peptide, formyl-Met-Leu-Phe, and the phagocytic activity of the cells for serum-opsonized zymosan (SOZ) in a dose-dependent manner. When the inhibitory activity of the HAs of different molecular weights, including HA190, HA of molecular weight 800,000 (HA80), and HA of molecular weight 300,000 (HA30), was assessed, the HA190 showed the strongest inhibitory activity and HA30 the lowest activity. In contrast, significant inhibition of the superoxide generation by PMNs upon stimulation with SOZ was not observed even in the presence of HA190 and HA80 at the highest concentration used (2.5 mg/mL). This finding indicated that the HAs studied did not prevent the interaction of PMNs with stimuli. From these data it is concluded that the binding of high-molecular-weight HAs to the HA receptors on PMNs may produce intracellular signals that are responsible for suppression of phagocytosis and chemotaxis but not for superoxide generation.

High‐Molecular‐Weight Hyaluronic Acids Inhibit Chemotaxis and Phagocytosis but Not Lysosomal Enzyme Release Induced by Receptor‐Mediated Stimulations in Guinea Pig Phagocytes

The effects of high‐molecular‐weight (HMW) hyaluronic acids (HAs) of 1.9 × 106 Da, 8 × 105 Da and 3 × 105 Da on the receptor‐mediated functions of guinea pig peritoneal phagocytes were studied. HMW‐HAs of 1.9 × 106 Da (HA190) and 8 × 105 Da (HA80) effectively inhibited the chemotactic activity of polymorphonuclear leukocytes (PMNs) for formyl‐Met‐Leu‐Phe (fMLP). The degree of inhibition was dose‐dependent and the concentrations of HA190 and HA80 required for 50% inhibition were 0.5–1.5 mg/ml and 1.5–2.5 mg/ml, respectively. HMW‐HA of 3 × 105 Da (HA30) hardly affected the chemotaxis within a concentration range of 0.5–5.0 mg/ml. The phagocytic activities of PMNs and macrophages (Møs) for serum‐opsonized zymosan (SOZ) and polystyrene latex particles were also inhibited by these HAs in a dose‐ and molecular‐weight‐dependent manner and HA190 was again the most inhibitory. By contrast, the release of lysosomal enzyme from Møs stimulated with SOZ was not significantly affected by HMW‐HAs at any concentration used. Furthermore, the binding of [3H]fMLP with PMNs and the rosette formation of Møs with SOZ were not influenced by the presence of HMW‐HAs. These findings suggested that the binding of HMW‐HAs to the HA receptors on PMNs and Møs might produce certain intracellular signals which would be responsible for the suppression of the chemotaxis and the phagocytosis but not for the release of lysosomal enzyme. For the generation of such signals, higher‐molecular‐weight HMW‐HAs would be more effective than lower one.

Ovarian cancer G protein-coupled receptor 1-dependent and -independent vascular actions to acidic pH in human aortic smooth muscle cells

Atherosclerosis is a chronic inflammation disease characterized by acidic micromilieu and the accumulation of numerous bioactive lipid mediators, such as lysophosphatidic acid (LPA) and prostaglandins, in the atherosclerotic lesion. Chronic acidification induced various effects on vascular smooth muscle cells, but the molecular mechanisms underlying these effects remain unknown. In this study, we examine the role of proton-sensing ovarian cancer G protein-coupled receptor 1 (OGR1) in extracellular acidification-induced regulation of cyclooxygenase (COX)-2 induction, PGI(2) production, MAPK phosphatase (MKP)-1 expression, and plasminogen activator inhibitor (PAI)-1 expression and proliferation in human aortic smooth muscle cells (AoSMCs). Experiments with knockdown with small interfering RNA specific to OGR1 and specific inhibitors for G proteins showed that acidification-induced COX-2 expression, PGI(2) production, and MKP-1 expression, but not PAI-1 expression and inhibition of proliferation, were dependent on OGR1 and mainly mediated by G(q/11) protein. LPA remarkably enhanced, through the LPA(1) receptor/G(i) protein, the OGR1-mediated vascular actions to acidic pH. In conclusion, acidic pH-induced vascular actions of AoSMCs can be dissected to OGR1-dependent and -independent pathways: COX-2 expression, PGI(2) production, and MKP-1 expression are mediated by OGR1, but PAI-1 expression and inhibition of proliferation are not. LPA, which is usually thought to be a proatherogenic lipid mediator, may exert antiatherogenic actions under acidic micromilieu through cross-talk between LPA(1)/G(i) protein and OGR1/G(q/11) protein.

Enhanced expression of Fc receptors on neutrophils from calves with leukocyte adhesion deficiency

The expression of Fc receptors for immunoglobulin G(IgG) and concanavalin A (con A)‐binding receptors, luminol‐dependent chemiluminescent (LDCL) responses, and the effect of anti‐bovine IgG on LDCL responses were evaluated in neutrophils from Holstein calves with leukocyte adhesion deficiency (BLAD). Neutrophils from affected calves showed a 2.1‐ to 2.5‐fold increase in Fc receptor expression compared with those of control calves by flow cytometric analysis. Con A‐binding activities of neutrophils from affected calves were similar to those of control calves. Neutrophils from a calf with BLAD, when stimulated with zymosan opsonized with bovine serum (OPZ), heat‐aggregated bovine IgG (Agg‐bovine IgG), sheep red blood cells (SRBC) sensitized with anti‐SRBC antibody (SRBC‐anti‐SRBC Ab), or con A had LDCL responses of 36 (P<0.05), 77, 126 and 119% of peak LDCL values of controls, respectively. The NBT‐reducing value of neutrophils from a calf with BLAD when stimulated with Agg‐bovine IgG after pretreatment with anti‐bovine IgG was 116.5% of the values of neutrophils from control calves, but the difference was not significant. The LDCL responses of neutrophils from a control calf and a calf with BLAD stimulated with OPZ were inhibited markedly by pre‐incubation with anti‐bovine IgG antiserum at concentrations ranging from 1.25 to 20 or 40 μg/ml. Although an increase in Fc receptor expression on neutrophils from calves with BLAD was observed, the LDCL responses stimulated with SRBC‐anti‐SRBC Ab and NBT‐reducing activity stimulated with Agg‐bovine IgG after pretreatment with anti‐bovine IgG did not correlate significantly with the increased Fc receptor expression. These results support that neutrophil functions mediated by the Fc receptors are associated synergistically with the presence of the complement receptor type 3 (CR3)(CD11b/CD18).

Biosynthesis of B2-integrin, intracellular calcium signalling and functional responses of normal and CD18-deficient bovine neutrophils

1Biosynthesis of CD11/CD18 in bovine leucocytes, intracellular Ca2+ ([Ca2+]i) signalling, chemiluminescent responses and membrane fluidity of neutrophils and the effects of D-mannose on neutrophils from control heifers and a heifer with bovine leucocyte adhesion deficiency (BLAD) were measured. The synthesis of CD11/CD18 complex was clearly detected in leucocytes from a normal heifer, but not in a BLAD-affected heifer. The transient phase of increased [Ca2+]i was clearly detected in neutrophils from a heifer with BLAD stimulated with opsonised zymosan, aggregated bovine immunoglobulin G or concanavalin A, whereas the sustained phase was deficient or significantly decreased compared with control heifers. [Ca2+]i signalling of neutrophils from control heifers and a heifer with BLAD stimulated with phorbol myristate acetate via an 11b/CD18-independent pathway showed no transient phase, and the subsequent increase in [Ca2+]i was almost identical in neutrophils from affected and control heifers. [Ca2+]i concentration and chemiluminescent responses of neutrophils from a control heifer were clearly decreased by treatment with anti-CD18 and anti-IgG antibodies. No differences in membrane fluidity were detected between neutrophils derived from control and CD18-deficient cattle. D-mannose binds mainly to Fc rather than CD18 receptors, and decreased Agg-IgG induced [Ca2+]i and the chemiluminescent response of neutrophils. The [Ca2+]i responses and Agg-IgG induced chemiluminescent responses of neutrophils from control heifers and a BLAD-affected heifer were inhibited by D-mannose. The characteristic changes of [Ca2+]i signalling and functional responses of B2-integrin-deficient neutrophils were demonstrated.

Phosphorylation of NADPH-cytochrome c reductase in guinea pig peritoneal macrophages stimulated with phorbol myristate acetate

The plasma membrane‐bound NADPH‐cytochrome c reductase of guinea pig macrophages (Mφ) was found to be phosphorylated when [32P]phosphate‐labeled cells were stimulated with 12‐phorbol 13‐myristate acetate (PMA). The time course of phosphorylation was parallel to that of O− 2‐generating activity elicited. These results suggest that the reductase participates as a flavoprotein in activation of the respiratory burst NADPH oxidase, when phosphorylated.