Shumpei Niida

γ-Glutamyltranspeptidase Stimulates Receptor Activator of Nuclear Factor-κB Ligand Expression Independent of Its Enzymatic Activity and Serves as a Pathological Bone-resorbing Factor

A novel bone-resorbing factor was cloned using an expression cloning technique, which involved a Xenopus oocyte expression system and an assay for osteoclast formation. A candidate clone was isolated from a BW5147 mouse T-lymphoma cell cDNA library. Sequencing analysis identified the factor as γ-glutamyltranspeptidase (GGT), which is an enzyme involved in glutathione metabolism. The addition of purified GGT protein to mouse bone marrow culture effectively induced formation of osteoclasts. An antibody against GGT inhibited osteoclast formation but not the enzymatic activity. We also demonstrated that an inactive form of GGT, the enzymatic activity of which had been blocked by chemical modification with a specific inhibitor, acivicin, supported osteoclast formation. These results indicate that GGT acts on osteoclast formation independent of its own enzymatic activity. Furthermore, both native GGT and inactive GGT stimulated the expression of the receptor activator of nuclear factor-κB ligand (RANKL) mRNA and protein from bone marrow stromal cells. This report is the first demonstration of a novel biological activity of GGT protein in a manner independent of its enzymatic activity.

Estrogen regulates the production of VEGF for osteoclast formation and activity in op/op mice.

op/op mice have a severe deficiency of osteoclasts because of lacking functional M‐CSF that is an essential factor of osteoclast differentiation and function. We now report that OVX induces osteoclast formation and cures osteopetrosis by increasing the VEGF that regulates osteoclast formation in these mice.

Mannose Receptor Ligand-Positive Cells Express the Metalloprotease Decysin in the B Cell Follicle

Decysin, a gene encoding a disintegrin metalloprotease, is transcribed in human dendritic cells (DC) and germinal centers (GC). We have cloned its murine homologue and show that it is processed by the endoprotease furin before secretion of the catalytic domain. We have defined the cell types that express decysin in mouse spleen in the course of an immune response to T cell-dependent Ags. Like in humans, decysin is transcribed by activated CD11c+ DC that enter the T cell zone from the marginal zone (MZ). In the GC, decysin is expressed by follicular DC and tingible body macrophages. In addition, a MZ cell population expresses decysin and appears to migrate into the B cell follicle. The majority of these follicle-homing cells express the mannose receptor ligand, a marker for the macrophage-like MZ metallophils. The follicle-homing cells are M-CSF dependent, as they are absent in op/op mice that lack functional M-CSF. This suggests that mannose receptor ligand+ MZ metallophils differentiate into cells that migrate from the MZ into the B cell follicle. Decysin represents the first marker for this previously unrecognized cell population of the mouse spleen, which may represent a precursor for GCDC and may be specialized in the transport of unprocessed Ag from the MZ into developing GC.

Induction of hydroxyapatite resorptive activity in bone marrow cell populations resistant to bafilomycin A1 by a factor with restricted expression to bone and brain, neurochondrin

Bone, one of the favored sites for tumor metastasis, is a dynamic organ undergoing formation and resorption. We found bone metastasis with osteolytic lesion in the bone marrow of the femur by injecting BW5147 T-lymphoma cells into the tail vein of AKR mice. To understand this bone destruction, we constructed a cDNA library from BW5147 with a cloning vector that allowed in vitro synthesis of mRNAs, and then identified a particular cDNA clone by adding the conditioned medium from Xenopus oocytes following injection of the mRNA synthesized in vitro to primary bone marrow heterogeneous cell populations on hydroxyapatite thin films. By means of this method, we isolated a factor with 16% leucine residues, termed neurochondrin, that induces hydroxyapatite resorptive activity in bone marrow cells resistant to bafilomycin A1, an inhibitor of macrophage- and osteoclast-mediated resorption. Expression of the gene was localized to chondrocyte, osteoblast, and osteocyte in the bone and to the hippocampus and Purkinje cell layer of cerebellum in the brain. This may provide insights into the molecular mechanisms underlying bone resorption with potential implications for the activation of cells other than macrophages and osteoclasts in bone marrow cells.

Osteoporosis following chronic constriction injury of sciatic nerve in rats

Abstract. Osteopathic changes sometimes occur in patients with complex regional pain syndrome (reflex sympathetic dystrophy and causalgia). We aimed to investigate whether such osteopathic changes occurred in rats with chronic constriction injury (CCI) of the sciatic nerve. A CCI of the sciatic nerve was established in a unilateral hind limb in 39 adult Sprague-Dawley rats, which were killed 1, 2, 3, 5, or 7 weeks after the CCI procedure. Bone mineral content (BMC) and bone mineral density (BMD) in extracted tibial bones were measured using a dual-energy X-ray absorptiometer, and the number of osteoclasts in the metaphyseal regions was counted by the use of tartrate-resistant acid phosphate (TRAP) staining. BMC was significantly decreased, compared with that of the contralateral side, 1 to 7 weeks after CCI, and BMD was decreased 2 to 7 weeks after the procedure in the ipsilateral tibial bones, compared with BMD in the contralateral bones. The number of TRAP-positive multinucleated osteoclasts in the ipsilateral bones was significantly increased at 2, 3, and 5 weeks after the CCI, when compared with the number of these osteoclasts in the contralateral bones. The results of the present study demonstrate that osteopathic changes are associated with chronic constrictive injury of the sciatic nerve.

Unique reciprocal changes of hepatocellular membrane transporter expression and fluidity in rats with selective biliary obstruction

BACKGROUND AND AIMS: The localized occlusion of bile ducts by intrahepaticlithiasis and intrahepatic neoplasms dose not cause severe jaundice, although the reason is unclear. The aim of this study was to clarify the molecular change of the liver in partial cholestasis, especially with respect to transporter expression (Mrp2, Mrp3, Bsep, P-gps) and membrane fluidity. METHODS AND RESULTS: After 3 days of selective bile duct ligation in Sprague-Dawley rats, the obstructed lobe (OL) and non-OL were analyzed. Canalicular and sinusoidal membrane fluidity were decreased in the OL compared with the non-OL. Mrp2 protein expression was significantly lower in the OL (by 62.9+/-6.9%) when compared with the non-OL. Mdr1b and Mdr2 mRNA were up-regulated in the OL when compared with the non-OL. Interestingly, Mrp3 protein and mRNA were induced in both the non-OL and OL of rats without hyperbilirubinemia. CONCLUSIONS: It seems that the reduction of membrane fluidity in the OL is a local response to localized cholestasis, while the induction of Mrp3 observed in both the OL and non-OL may be a response of the whole liver to localized impairment of bile secretion, although the regulatory mechanism is yet to be established.

Recruitment of osteoclasts in the mandibular condyle of growing osteopetrotic (op/op) mice after a single injection of macrophage colony-stimulating factor

The purpose was to elucidate histological changes in the mandibular condyle and ramus in growing osteopetrotic (op/op) mice after a single injection of macrophage colony-stimulating factor (M-CSF). M-CSF (5 microg) was injected into 6-, 11-, 26-, 56- and 86-day-old op/op mice, and the mice were killed 4 days after the injection. In normal mice, the condyle was substantially wider than the ramus beneath it, and enlargement and ossification of the condyle occurred after weaning. These changes were not found in the uninjected and injected op/op mice, the condyles of which were occupied by hypertrophic cartilage cells, and the hypertrophic cell layer was thicker and more irregular in the arrangement of epiphyseal cell columns. In spite of the lack of bone resorption in uninjected and injected op/ op mice, ossification of the mandibular ramus occurred, but later than that of normal mouse. The number of tartrate-resistant acid phosphatase-positive cells in the injected op/op and normal mice approached a maximum at 30 days and then gradually decreased up to 90 days of age, although the numbers were substantially different for all ages. The uninjected op/op mice had no visible osteoclasts until 15 days and their number then increased significantly from 60 to 90 days of age. These results were considered due to the difference in biological responses of bony structures to M-CSF injection in the op/op mice. The influences of mechanical stimuli from masticatory functions, which are deficient in op/op mice, might also be responsible for the differences in bony architecture between the op/op and normal mice.

Sexual dimorphism in the trigeminal motor neurons innervating the mouse masseter muscle

The extrafusal and intrafusal muscle fibers in the masseter muscle are innervated by motoneurons in the trigeminal motor nucleus (Mo 5). In the present study, we found that the number of trigeminal motor neurons in the male mouse was significantly larger than that in the female. However, we could not detect any significant difference between male and female mice in the number of sensory neurons in the trigeminal mesencephalic nucleus (Me 5). This is the first report on sexual dimorphism in masticatory motoneurons of mammals.

MicroRNA transcriptome analysis on hypertrophy of ligamentum flavum in patients with lumbar spinal stenosis

Introduction Molecular pathways involved in ligamentum flavum (LF) hypertrophy are still unclarified. The purpose of this study was to characterize LF hypertrophy by microRNA (miRNA) profiling according to the classification of lumbar spinal stenosis (LSS). Methods Classification of patients with LSS into ligamentous and non-ligamentous cases was conducted by clinical observation and the morphometric parameter adopting the LF/spinal canal area ratio (LSAR) from measurements of magnetic resonance imaging (MRI) T2 weighed images. LF from patients with ligamentous stenosis (n=10) were considered as the degenerative hypertrophied samples, and those from patients with non-ligamentous LSS (n=7) and lumbar disc herniation (LDH, n=3) were used as non-hypertrophied controls. Profiling of miRNA from all samples was conducted by Agilent microarray. Microarray data analysis was performed with GeneSpring GX, and pathway analysis was performed using Ingenuity Pathway Analysis. Results The mean LSAR in the ligamentous group was significantly higher than that in the control group (0.662±0.154 vs 0.301±0.068, p=0.0000171). Ten significantly differentially expressed miRNA were identified and taken as a signature of LF hypertrophy: nine miRNA showed down-regulated expression, and one showed up-regulated expression in the ligamentous LF. Among those, miR-423-5p (rs=-0.473, p<0.05), miR-4306 (rs=-0.628, p<0.01), miR-516b-5p (rs=-0.629, p<0.01), and miR-497-5p (rs=0.461, p<0.05) were correlated to the LSAR. Pathway analysis predicted aryl hydrocarbon receptor signaling (p<0.01), Wnt/β-catenin signaling (p<0.01), and insulin receptor signaling (p<0.05) as canonical pathways associated with the miRNA signature. Conclusions Classification based on quantification of the MRI axial image is useful for studying hypertrophy of the LF. Aryl hydrocarbon receptor and Wnt/β-catenin signaling may be involved in LF hypertrophy.

Generation of a transgenic mouse line for conditional expression of human IL-6

IL-6 is a cytokine that is involved in various physiological and pathological conditions, and approaches using gain-of-function transgenic animals have contributed in elucidating IL-6 function. However, studies of the multiple functions of IL-6 in vivo are very time consuming because they require the generation of transgenic mice that harbor the gene encoding IL-6 under the control of specific promoters to mimic different pathologies. Here, we report the establishment of a conditional human IL-6 transgenic mouse, LGL-IL6, which conditionally expresses human IL-6 by taking advantage of the well-characterized Cre recombinase drivers.