Biomedical subjects

T Morinaga

Publications and source records attributed to T Morinaga.

At least 19 recordsLinked to original sources

PubMed2000

Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction.

Osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF, also called CSF-1). When mouse bone marrow cells were cultured with M-CSF, M-CSF-dependent bone marrow macrophages (M-BMM phi) appeared within 3 d. Tartrate-resistant acid phosphatase-positive osteoclasts were also formed when M-BMM phi were further cultured for 3 d with mouse tumor necrosis factor alpha (TNF-alpha) in the presence of M-CSF. Osteoclast formation induced by TNF-alpha was inhibited by the addition of respective antibodies against TNF receptor 1 (TNFR1) or TNFR2, but not by osteoclastogenesis inhibitory factor (OCIF, also called OPG, a decoy receptor of ODF/RANKL), nor the Fab fragment of anti-RANK (ODF/RANKL receptor) antibody. Experiments using M-BMM phi prepared from TNFR1- or TNFR2-deficient mice showed that both TNFR1- and TNFR2-induced signals were important for osteoclast formation induced by TNF-alpha. Osteoclasts induced by TNF-alpha formed resorption pits on dentine slices only in the presence of IL-1alpha. These results demonstrate that TNF-alpha stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL-RANK system. TNF-alpha together with IL-1alpha may play an important role in bone resorption of inflammatory bone diseases.

Acid Phosphatase↗

PubMed1999

Nucleotide sequences of genes for ribosomal protein L41 and tRNAThr(AGU) from Coprinus cinereus.

The nucleotide sequences of genes for the homolog in Coprinus cinereus of the eukaryotic ribosomal protein L41 and for tRNAThr(AGU) are reported. The gene for tRNAThr(AGU) was located upstream of the gene for the L41 ribosomal protein, and these genes were adjacent to each other but in opposite orientations. The deduced amino acid sequence of ribosomal protein L41 exhibited strong homology to those of L41 proteins of several yeasts. The 56th amino acid of the deduced protein was proline, as it is in the L41 protein of a cycloheximide-sensitive strain of yeast. The putative secondary structure of the tRNA gene resembled the characteristic cloverleaf structure of tRNAs. Elements resembling an A-box and a B-box were found in the gene for tRNAThr(AGU). These boxes are known as internal promoter elements in genes for eukaryotic tRNAs.

Amino Acid Sequence↗

PubMed1999

A mutant of deleted variant of hepatocyte growth factor (dHGF) with alanine substitution in the N-terminal basic region has higher activity in vivo.

In a previous study, we generated a mutant of dHGF (deleted variant of hepatocyte growth factor), termed #2, with higher specific activity than dHGF in assays of mitogenic activity on rat hepatocytes and America opossum kidney epithelial cells (OK). In the present study, we examine in vivo hepatotropic and renotropic activities of #2 and its distribution to target tissues, liver and kidney. Administration of #2 to normal rats significantly increased serum levels of total protein, albumin, free-cholesterol, and HDL-cholesterol and liver weight in a dose-dependent manner. Analysis of these parameters suggests that #2 is more potent than dHGF as a hepatotropic factor in vivo. In addition, #2 reduced mortality of mercuric chloride-administered mice and the effect was stronger than that of dHGF. When injected to mice, a larger amount of #2 than dHGF was rapidly distributed to the liver. Sixty minutes after injection, the concentrations of #2 in plasma, liver, and kidney were higher than those of dHGF. These distribution properties and the higher mitogenic activity in vitro may explain why #2 exerts more potent in vivo biological activity than dHGF.

Acute Kidney Injury↗

PubMed1999

Basic fibroblast growth factor induces osteoclast formation by reciprocally regulating the production of osteoclast differentiation factor and osteoclastogenesis inhibitory factor in mouse osteoblastic cells.

Basic fibroblast growth factor (bFGF) induced osteoclast formation in co-cultures of mouse spleen cells and osteoblasts. Osteoclastogenesis inhibitory factor (OCIF) and a selective cyclooxygenase-2 (COX-2) inhibitor, NS-398, abolished bFGF-induced osteoclast formation. bFGF did not affect spleen cells, but it did affect osteoblasts, to stimulate osteoclast formation. Northern blot analysis revealed that bFGF up-regulated the expression of osteoclast differentiation factor (ODF) and COX-2 and down-regulated the expression of OCIF in primary osteoblastic cells. NS-398 abolished the increase of ODF mRNA, but it had no effect on the decrease of OCIF mRNA. NS-398 suppressed the binding of (125)I-labeled OCIF to osteoblastic cells treated with bFGF. Enzyme-linked immunosorbent assay showed that bFGF inhibited OCIF production by osteoblastic cells, and the inhibition was not affected by NS-398. We conclude that bFGF induces osteoclast formation by stimulating ODF production through COX-2-mediated prostaglandin synthesis and by suppressing OCIF production through a mechanism independent of prostaglandin synthesis.

Animals↗

PubMed1999

Basic fibroblast growth factor inhibits osteoclast formation induced by 1alpha,25-dihydroxyvitamin D(3) through suppressing the production of osteoclast differentiation factor.

Basic fibroblast growth factor (bFGF) inhibited osteoclast-like cell (OCL) formation in cocultures of mouse spleen cells with either osteoblasts or a stromal cell line, ST2, in the presence of 1alpha, 25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. bFGF directly acted on osteoblasts/stromal cells, but not osteoclast progenitors, to inhibit 1,25(OH)(2)D(3)-induced OCL formation. bFGF suppressed the mRNA expression of osteoclast differentiation factor (ODF) but did not affect that of osteoclastogenesis inhibitory factor (OCIF) in ST2 cells treated with 1,25(OH)(2)D(3) and dexamethasone. Enzyme-linked immunosorbent assay showed that bFGF hardly affected OCIF production in the treated ST2 cells. A genetically engineered soluble form of ODF, but not anti-OCIF neutralizing antibody, abolished bFGF-mediated inhibition of OCL formation. bFGF suppressed the binding of (125)I-labeled OCIF to both ST2 cells and osteoblasts treated with 1,25(OH)(2)D(3). These findings indicate that bFGF inhibits 1,25(OH)(2)D(3)-induced OCL formation via suppression of ODF production by osteoblasts/stromal cells.

Animals↗

PubMed1999

A novel molecular mechanism modulating osteoclast differentiation and function.

Osteoclasts, the multinucleated giant cells that resorb bone, develop from hematopoietic cells of the monocyte/ macrophage lineage. Osteoblasts, as well as bone marrow stromal cells, support osteoclast development through a mechanism of cell-to-cell interaction with osteoclast progenitors. We recently purified and molecularly cloned osteoclastogenesis inhibitory factor (OCIF), which was identical to osteoprotegerin (OPG). OPG/OCIF, a secreted member of the tumor necrosis factor (TNF) receptor family, inhibited differentiation and activation of osteoclasts. A single class of high-affinity binding sites for OPG/OCIF appeared on a mouse bone marrow stromal cell line, ST2, in response to 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] and dexamethasone (Dex). When the binding sites were occupied by OPG/OCIF, ST2 cells failed to support the osteoclast formation from spleen cells. To identify an OPG/OCIF ligand, we screened a cDNA expression library of ST2 cells treated with 1,25(OH)2D3 and Dex using OPG/OCIF as a probe. The cloned molecule was found to be a member of the membrane-associated TNF ligand family, and it induced osteoclast formation from mouse and human osteoclast progenitors in the presence of macrophage colony-stimulating factor (M-CSF) in vitro. Expression of its gene in osteoblasts/stromal cells was up-regulated by osteotropic factors, such as 1,25(OH)2D3, prostaglandin E2 (P(GE2), parathyroid hormone (PTH), and interleukin (IL)-11. A polyclonal antibody against this protein, as well as OPG/OCIF, negated not only the osteoclastogenesis induced by the protein, but also bone resorption elicited by various osteotropic factors in a fetal mouse long bone culture system. These findings led us to conclude that the protein is osteoclast differentiation factor (ODF), a long sought-after ligand that mediates an essential signal to osteoclast progenitors for their differentiation into active osteoclasts. Recent analyses of ODF receptor demonstrated that RANK, a member of the TNF receptor family, is the signaling receptor for ODF in osteoclastogenesis, and that OPG/OCIF acts as a decoy receptor for ODF to compete against RANK. The discovery of ODF, OPG/OCIF, and RANK opens a new era in the investigation of the regulation of osteoclast differentiation and function.

Animals↗

PubMed1998

RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis.

Osteoclast differentiation factor (ODF) is a ligand for osteoclastogenesis-inhibitory factor/osteoprotegerin (OCIF/OPG), and mediates an essential signal for osteoclastogenesis. Soluble-form ODF binds directly to osteoclast progenitors, suggesting the presence of a membrane-bound receptor for ODF (ODFR) on the cells. To understand the ODF-mediated signal transduction mechanism in osteoclastogenesis, we molecularly cloned ODFR from a mouse macrophage-like osteoclast progenitor cell line, C7. Nucleotide sequence analysis revealed that ODFR is identical to RANK, a recently identified member of the tumor necrosis factor receptor (TNFR) family, which is involved in the regulation of dendritic cell function. A polyclonal antibody against the extracellular domain of RANK induced osteoclastogenesis in the presence of macrophage colony-stimulating factor (M-CSF). In contrast, both a genetically engineered soluble RANK and Fab fragment of the antibody blocked the binding of ODF to RANK and ODF-mediated osteoclastogenesis. These results indicate that RANK is the signaling receptor essential for ODF-mediated osteoclastogenesis.

Animals↗

PubMed1998

Analysis of deleted variant of hepatocyte growth factor by alanine scanning mutagenesis: identification of residues essential for its biological function and generation of mutants with enhanced mitogenic activity on rat hepatocytes.

To understand the structure-function relationship of hepatocyte growth factor (HGF) in more detail, we analyzed one of the other forms of HGF, deleted variant of HGF (dHGF), by alanine scanning mutagenesis. We show here that there are at least four sites important for dHGF to stimulate DNA synthesis in cultured adult rat hepatocytes, and that the residues of HGF essential for exerting its biological activity are not identical to those of dHGF. In addition, two mutants showed a decrease (approximately three-fold) in EC50 compared with wild-type dHGF in an assay of mitogenic activity on rat hepatocytes.

Alanine↗

PubMed1998

Structure of the mouse osteoclastogenesis inhibitory factor (OCIF) gene and its expression in embryogenesis.

Osteoclastogenesis inhibitory factor (OCIF) is a novel soluble-form member of the tumor necrosis factor receptor family and is involved in the regulation of bone mass. Here we isolated genomic and cDNA clones for mouse OCIF and determined their structures. Mouse OCIF gene spans 29 kb and contains five exons of 270, 367, 192, 225 and 1765 bp long. Four cysteine-rich domains and two death domain homologous regions characterized in human OCIF are rigidly conserved in mouse OCIF. The onset of OCIF gene expression in mouse embryogenesis is at day 8.5. In a pregnant female mouse, OCIF gene is expressed in decidua, a maternal tissue surrounding each embryo, immediately after implantation. The isolation of mouse OCIF gene should facilitate studies on OCIF knock-out mice for a better understanding of the role of OCIF in vivo.

Amino Acid Sequence↗

PubMed1998

Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/osteoprotegerin.

Osteoclasts are multinucleated cells that resorb bone. Osteoclastogenesis inhibitory factor (OCIF), also called osteoprotegerin (OPG), acts as a naturally occurring decoy receptor for osteoclast differentiation factor, which mediates an essential signal to osteoclast progenitors for their differentiation into osteoclasts. Here we show that the OCIF/OPG knockout mice exhibited severe osteoporosis due to enhanced osteoclastogenesis when they grew to be adults. These mice were viable and fertile. They exhibited marked bone loss accompanied by destruction of growth plate and lack of trabecular bone in their femurs. The strength of their bones dramatically decreased. These results demonstrate that OCIF/OPG is a key factor acting as a negative regulator against osteoclastogenesis. The OCIF/OPG knockout mice provide the first animal model for osteoporosis without other obvious abnormalities.

Animals↗

PubMed1998

Cloning and characterization of the gene encoding human osteoprotegerin/osteoclastogenesis-inhibitory factor.

The human osteoprotegerin (OPG)-osteoclastogenesis-inhibitory factor (OCIF) gene has been cloned and characterized. The OPG-OCIF gene is a single-copy gene consisting of five exons, and spans 29 kb of the human genome. All the exon/intron boundaries comply with the GT/AG rule. The translation-termination codon is present in exon 5 and a typical poly(A)-addition signal resides 173-nucleotides downstream of the translation-termination codon. A major transcription-initiation site is present 67-nucleotides upstream of the initiation ATG codon. Two minor sites are present further upstream. The 4.2-kb and 6.5-kb transcripts detected in IMR-90 cells were found to contain the 3'-half of intron 2 and the entire intron 2, respectively. In the OPG-OCIF gene, a single intron divides the stretch that encode four Cys-rich motifs, implying diversity from the other members of the tumor necrosis factor receptor (TNFR) family. Two death domain homologous regions (DDHs) present in tandem in OPG-OCIF are encoded separately by exons 4 and 5. The conservation of amino-acid sequences suggests that exon 4 is produced by a duplication of a portion of exon 5.

Alternative Splicing↗

PubMed1998

Osteoclast differentiation factor (ODF) induces osteoclast-like cell formation in human peripheral blood mononuclear cell cultures.

We have reported that osteoclast differentiation factor (ODF) expressed on the plasma membrane of osteoblasts/ stromal cells is a ligand for osteoclastogenesis inhibitory factor (OCIF). A genetically engineered soluble form of ODF (sODF) induced osteoclast-like multinucleated cells (OCLs) in the presence of M-CSF in mouse spleen cell cultures. Osteoblasts/stromal cells were not required in this process. To elucidate the mechanism of human osteoclastogenesis, human peripheral blood mononuclear cells (PBMCs) were cultured for 7 days with sODF and human M-CSF in the presence or absence of dexamethasone. Treatment of human PBMCs with sODF together with M-CSF induced OCLs, which expressed tartrate-resistant acid phosphatase and vitronectin receptors, produced cAMP in response to calcitonin, and formed resorption pits on dentine slices. OCLs were also formed from the adherent cell population of human PBMCs. Dexamethasone was required for human OCL formation in culture of whole PBMCs but not in culture of the adherent cell population. OCL formation was strongly inhibited by OCIF simultaneously added. These results clearly indicate that like in mouse osteoclastogenesis, ODF is a critical factor for human osteoclastogenesis. The present study also indicates that OCIF acts as a naturally occurring decoy receptor for ODF in inhibiting signal transduction in human osteoclast formation.

Animals↗

PubMed1998

Identification of heparin-binding stretches of a naturally occurring deleted variant of hepatocyte growth factor (dHGF).

A deleted variant of hepatocyte growth factor (dHGF) is a naturally occurring major variant of HGF, which lacks five consecutive amino acid residues in the first kringle domain. While both HGF and dHGF bind to heparin, the residues involved in the binding to heparin have not been identified in either protein. To identify the residues involved in the binding, we made a series of dHGF mutants in which basic residues in the N-terminal and the first kringle domains were replaced with alanine residue. The analysis of heparin-binding ability revealed that three stretches, 42RCTRNK in the hairpin loop structure, and 2RKRR and 27KIKTKK in the N-terminal basic region, are involved in the binding. Alanine substitution of each basic residue except 3K and 27K in the stretches reduced the heparin-binding ability of dHGF, and the decrease was additive. Conversely, lysine substitution of 37D, 38Q or 64Q in the N-terminal domain increased heparin-binding ability. These results suggest that stretches distant from each other in the primary structure come into close proximity when the polypeptide folds into protein, and form a heparin-binding site with clusters of basic residues.

Amino Acid Sequence↗

PubMed1998

Characterization of monomeric and homodimeric forms of osteoclastogenesis inhibitory factor.

Osteoclastogenesis inhibitory factor (OCIF) is present naturally as two molecular forms, a monomer and a homodimer. The two forms of recombinant human OCIF (rOCIF) produced by Chinese hamster ovary (CHO) cells were purified to homogeneity. Determination of the C-terminal amino-acid sequences of the two forms of rOCIF revealed that the monomeric rOCIF lacked several amino acids including Cys379, which is involved in the intermolecular disulfide bond, in its C-terminal region. The two forms of rOCIF were indistinguishable in stability, sialic acid content, and specific activity in inhibition of osteoclastogenesis. In contrast, the homodimeric rOCIF was stronger in heparin-binding ability than the monomeric rOCIF. The homodimeric rOCIF was significantly shorter in initial half-life and smaller in AUC value in rats than the monomeric rOCIF, but exerted more potent biological activity in reducing the calcium concentration in serum of rats than did the monomeric rOCIF.

Amino Acid Sequence↗

PubMed1998

Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL.

Osteoclasts, the multinucleated cells that resorb bone, develop from hematopoietic cells of monocyte/macrophage lineage. Osteoclast-like cells (OCLs) are formed by coculturing spleen cells with osteoblasts or bone marrow stromal cells in the presence of bone-resorbing factors. The cell-to-cell interaction between osteoblasts/stromal cells and osteoclast progenitors is essential for OCL formation. Recently, we purified and molecularly cloned osteoclastogenesis-inhibitory factor (OCIF), which was identical to osteoprotegerin (OPG). OPG/OCIF is a secreted member of the tumor necrosis factor receptor family and inhibits osteoclastogenesis by interrupting the cell-to-cell interaction. Here we report the expression cloning of a ligand for OPG/OCIF from a complementary DNA library of mouse stromal cells. The protein was found to be a member of the membrane-associated tumor necrosis factor ligand family and induced OCL formation from osteoclast progenitors. A genetically engineered soluble form containing the extracellular domain of the protein induced OCL formation from spleen cells in the absence of osteoblasts/stromal cells. OPG/OCIF abolished the OCL formation induced by the protein. Expression of its gene in osteoblasts/stromal cells was up-regulated by bone-resorbing factors. We conclude that the membrane-bound protein is osteoclast differentiation factor (ODF), a long-sought ligand mediating an essential signal to osteoclast progenitors for their differentiation into osteoclasts. ODF was found to be identical to TRANCE/RANKL, which enhances T-cell growth and dendritic-cell function. ODF seems to be an important regulator in not only osteoclastogenesis but also immune system.

Animals↗

PubMed1998

Characterization of structural domains of human osteoclastogenesis inhibitory factor.

Osteoclastogenesis inhibitory factor (OCIF) is a heparin-binding secretory glycoprotein that belongs to the tumor necrosis factor receptor (TNFR) family. OCIF is present both as a approximately 60-kDa monomer and a disulfide-linked homodimer. We attempted to characterize the seven structural domains of OCIF by determining the capabilities of various OCIF mutants to inhibit osteoclastogenesis, to interact with heparin, and to form dimers. We also examined a potential of domains 5 and 6, death domain homologous regions (DDHs), for inducing cell death by expressing OCIF/Fas fusion proteins. Our results show that: (i) the N-terminal portion of OCIF containing domains 1-4, which have structural similarity to the extracellular domains of the TNFR family proteins, is sufficient to inhibit osteoclastogenesis; (ii) a heparin-binding site is located in domain 7, and affinity for heparin does not correlate with the inhibitory activity; (iii) Cys-400 in domain 7 is the residue responsible for dimer formation; and (iv) the C-terminal portion containing domains 5 and 6, DDHs, has a high potential for mediating a cytotoxic signal when it is expressed in cells as an OCIF/Fas fusion protein in which the transmembrane region of Fas is inserted in front of DDHs.

Apoptosis↗

PubMed1998

Safety evaluation of Nostoc flagelliforme (nostocales, Cyanophyceae) as a potential food.

The safety of the alga genus Nostoc flagelliforme Born. etFlah. as a human food source was evaluated in an oral acute toxicity study and in a 28-day oral subacute toxicity study using rats. In the acute toxicity study, the dried powder of N. flagelliforme was orally administered to male and female rats at a dose of 1250 mg/kg and 2500 mg/kg. Neither mortality nor changes in general condition were observed in either the study groups or the control group over a 14-day observation period. In the subacute toxicity study, N. flagelliforme powder was administered orally to male and female rats at a dose of 500 mg/kg and 1000 mg/kg for a period of 28 days. Neither mortality nor changes in general condition were observed in either the treatment group or the control group throughout the 28-day administration period. No reduction in food consumption or body weight gain was observed in the experimental animals. Ophthalmological tests performed at the end of the administration period revealed no abnormalities within the ophthalmological parameters. In the haematological tests and serum biochemical tests performed at the time of completion of the administration period, no adverse effects of N. flagelliforme were observed. At autopsy, organ weight at the end of the experimental period and histopathological tests of specimens obtained from the autopsied animals revealed no significant influences of N. flagelliforme. In conclusion, considering the absence of adverse effects of N. flagelliforme in this study, findings in the oral acute toxicity study and the 28-day oral subacute toxicity study may indicate the safety of N. flagelliforme for human consumption. This study is in agreement with the novel nutraceutical idea "Phycophagism".

Animals↗

PubMed1998

Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro.

The morphogenesis and remodeling of bone depends on the integrated activity of osteoblasts that form bone and osteoclasts that resorb bone. We previously reported the isolation of a new cytokine termed osteoclastogenesis inhibitory factor, OCIF, which specifically inhibits osteoclast development. Here we report the cloning of a complementary DNA of human OCIF. OCIF is identical to osteoprotegerin (OPG), a soluble member of the tumor-necrosis factor receptor family that inhibits osteoclastogenesis. Recombinant human OPG/OCIF specifically acts on bone tissues and increases bone mineral density and bone volume associated with a decrease of active osteoclast number in normal rats. Osteoblasts or bone marrow-derived stromal cells support osteoclastogenesis through cell-to-cell interactions. A single class of high affinity binding sites for OPG/OCIF appears on a mouse stromal cell line, ST2, in response to 1,25-dihydroxyvitamin D3. An anti-OPG/OCIF antibody that blocks the binding abolishes the biological activity of OPG/OCIF. When the sites are blocked with OPG/OCIF, ST2 cells fail to support osteoclastogenesis. These results suggest that the sites are involved in cell-to-cell signaling between stromal cells and osteoclast progenitors and that OPG/OCIF inhibits osteoclastogenesis by interrupting the signaling through the sites.

Amino Acid Sequence↗