Biomedical subjects

K Higashio

Publications and source records attributed to K Higashio.

At least 37 records · Page 2Linked to original sources

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

Osteoclastogenesis inhibitory factor (OCIF) directly inhibits bone-resorbing activity of isolated mature osteoclasts.

Osteoclastogenesis inhibitory factor (OCIF) was previously reported to specifically inhibit osteoclast development by interrupting the action of osteoclast differentiation factor (ODF), which is expressed in stromal cells and plays an important role in osteoclastogenesis. Here we report the direct action of OCIF on isolated rabbit mature osteoclasts to inhibit their functional bone-resorbing activity. The cell population employed in this study consisted of mature osteoclasts with more than 95% of purity. The inhibition by OCIF was dose dependent and observed as early as 6 h after the OCIF addition. An OCIF-binding protein of 140 kDa was detected on the plasma membrane of osteoclasts. ODF with a Mr of 40 kDa was recently isolated as a ligand for OCIF and shows to be identical to TRANCE/RANKL. However, ODF was not detected in osteoclasts. OCIF did not have any impact on the mRNA levels of cathepsin K/OC2 and carbonic anhydrase II responsible for degradation of organic and inorganic bone matrices, respectively, or on osteoclast apoptosis. However, OCIF reduced or disrupted the formation of F-actin ring in isolated osteoclasts, the cytoskeletal structure of which is correlated with bone resorption. These findings demonstrate that OCIF directly inhibits osteoclast function through an ODF-independent mechanism besides blocking the generation of osteoclasts.

Actins↗

PubMed1998

Transforming growth factor-beta stimulates the production of osteoprotegerin/osteoclastogenesis inhibitory factor by bone marrow stromal cells.

Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) is a recently identified cytokine that belongs to the tumor necrosis factor receptor superfamily and regulates bone mass by inhibiting osteoclastic bone resorption. The present study was undertaken to determine whether OPG/OCIF is produced in bone microenvironment and how the expression is regulated. A transcript for OPG/OCIF at 3.1 kilobases was detected in bone marrow stromal cells (ST2 and MC3T3-G2/PA6) as well as in osteoblastic cells (MC3T3-E1). Transforming growth factor-beta1 (TGF-beta1) markedly increased the steady-state level of OPG/OCIF mRNA in a dose-dependent manner, while TGF-beta1 suppressed the mRNA expression of tumor necrosis factor-related activation-induced cytokine (TRANCE)/receptor activator of NF-kappaB ligand (RANKL), a positive regulator of osteoclastogenesis to which OPG/OCIF binds. The effect of TGF-beta1 on the expression of OPG/OCIF mRNA was transient, with a peak level at 3-6 h. The up-regulation of OPG/OCIF mRNA by TGF-beta1 in ST2 cells did not require de novo protein synthesis and involved both a transcriptional and a post-transcriptional mechanism. Western blot analysis and an enzyme-linked immunosorbent assay revealed that TGF-beta1 significantly increased the secretion of OPG/OCIF protein by ST2 cells at 6-24 h. In murine bone marrow cultures, TGF-beta1 markedly inhibited the formation of tartrate-resistant acid phosphatase-positive multinucleated osteoclast-like cells in the presence of 1,25-dihydroxyvitamin D3, whose effect was significantly reversed by a neutralizing antibody against OPG/OCIF. These results suggest that TGF-beta1 negatively regulates osteoclastogenesis, at least in part, through the induction of OPG/OCIF by bone marrow stromal cells and that the balance between OPG/OCIF and TRANCE/RANKL in local environment may be an important determinant of osteoclastic bone resorption.

Animals↗

PubMed1998

Osteoclastogenesis inhibitory factor suppresses osteoclast survival by interfering in the interaction of stromal cells with osteoclast.

Osteoclastogenesis inhibitory factor (OCIF) was originally identified as a factor inhibiting osteoclast (OC) formation. The number of OC in rats treated with OCIF decreased, suggesting that OCIF inhibits OC formation in vivo; however, it is also possible that OCIF affects the number of OC by promoting apoptosis of OC. To address this issue, the effects of OCIF on the survival of OC were examined using well established mouse culture systems. OCIF dose-dependently inhibited OC formation in mouse marrow cultures. Addition of OCIF during day 0-3 did not alter the peak levels of OC formation on day 7 and 8. However, the addition of OCIF during day 5 and thereafter resulted in the rapid decrease of the number of OC. OCIF inhibited the survival of OC formed in mouse marrow cultures in dose- and time-dependent manners. The involvement of stromal cells in OC survival was examined using crude and stromal cell-depleted OC populations. OCIF dramatically inhibited the survival of crude OC populations rich with stromal cells. However, in stromal cell-depleted OC populations, OC spontaneously decreased in the absence of OCIF and OCIF did not enhance the decrease further at least up to 48 h. Apoptotic OC were detected in detached cell populations treated with OCIF in mouse marrow cultures and a specific inhibitor for caspase-3 rescued the death of OC. OCIF mutant lacking the death domain homologous regions inhibited OC survival, though the potency was much less than native OCIF. Taken together, OCIF inhibited not only OC recruitment but also OC survival. OCIF inhibited OC survival by interfering the interaction of stromal cells with OC.

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 mediates an essential signal for bone resorption induced by 1 alpha,25-dihydroxyvitamin D3, prostaglandin E2, or parathyroid hormone in the microenvironment of bone.

Osteoclast differentiation factor (ODF), a ligand for osteoprotegerin (OPG)/osteoclastogenesis-inhibitory factor (OCIF), induces osteoclast-like cell formation in vitro. To elucidate the role of ODF in the microenvironment of bone, we examined effects of ODF, OPG/OCIF, and anti-ODF polyclonal antibody on bone resorption using a fetal mouse long bone culture system. A genetically engineered soluble-form ODF (sODF) elicited bone resorption in a concentration-dependent manner and OPG/OCIF blocked the bone resorption. Anti-ODF polyclonal antibody, which neutralizes ODF activity, negated bone resorption induced by 1 alpha,25-dihydroxyvitamin D3, parathyroid hormone, or prostaglandin E2. OPG/OCIF also abolished bone-resorbing activity elicited by these bone-resorbing agents. Interleukin 1 alpha-stimulated bone resorption was also significantly suppressed by anti-ODF polyclonal antibody and OPG/OCIF. Thus, we conclude that ODF plays a critical role in bone resorption in the microenvironment of bone.

Animals↗

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

Preventive effects of the deleted form of hepatocyte growth factor against various liver injuries.

The effects of a naturally occurring deleted form of hepatocyte growth factor (HGF) on hepatic disorder were studied in various models of hepatic failure. The pretreatment of rats and mice with the deleted form of HGF prevented the liver injuries and coagulopathy induced by endotoxin, dimethylnitrosamine and acetaminophen and reduced the mortality due to hepatic dysfunction induced by these hepatotoxins. The concurrent administration of the deleted form of HGF also prevented the liver injury and hepatic fibrosis in mice treated with alpha-naphthylisothiocyanate and in rats treated with dimethylnitrosamine. Moreover, the deleted form of HGF normalized the results of the bromosulphalein-clearance test and ameliorated jaundice in rats with periportal cholangiolitic hepatopathy induced by alpha-naphthylisothiocyanate. The deleted form of HGF also reversed the coagulopathy in rats with hepatic disorder induced by dimethylnitrosamine or by 70% resection of cirrhotic liver (induced by carbon tetrachloride). In Long Evans cinnamon rats receiving vehicle, 20 out of 21 animals died within 4 days after the onset of jaundice. After infusion of the deleted form of HGF for 4 days, 7 out of 20 Long-Evans cinnamon rats survived. These results indicate that the deleted form of HGF could have therapeutic potency in patients with severe hepatic failure.

Animals↗

PubMed1998

Possible involvement of p21/waf1 in the growth inhibition of HepG2 cells induced by hepatocyte growth factor.

Hepatocyte growth factor (HGF) is a potent mitogen for a variety of cell types, but it is also known as an antimitogenic factor for several types of tumor cell lines. The biological processes by which HGF inhibits tumor cell growth remain poorly understood. Here we report a comparative study of HGF-mediated signal transduction events between two opposite responding types of human hepatoblastoma cell lines, HuH6 and HepG2. Following serum starvation, both cell lines were cultured in hepatocyte growth medium (HGM), a chemically defined medium, in the presence or absence of HGF. Under these culture conditions, cell growth in HuH6 was promoted by HGF, while it was inhibited in HepG2. Phosphorylation of p42/mitogen-activated protein (MAP) kinase was observed within 10 min after HGF stimulation in both cell lines. The level of phosphorylated MAP kinase in HuH6 declined to basal levels after 2 hr. However, in HepG2 the phosphorylated form was detectable at 6 hr. p21/waf1 was induced in both cell lines where levels peaked 4-6 hr after HGF stimulation. In HuH6, a marked decrease of p21/waf1 was observed at 8-12 hr, while a high level of p21/waf1 was sustained for at least 24 hr in HepG2. HGF treatment depressed cdk2 activity in a time-dependent manner in HepG2 while the activity increased in HuH6. When serum-starved HepG2 was growth stimulated with serum in the presence or absence of HGF, the cells treated with HGF underwent growth inhibition correlating with a sustained induction of p21/waf1 and a decrease of cdk2 activity. Immunoprecipitation analysis revealed accumulation of cdk2-associated p21/waf1 in the HGF-treated HepG2. Together, the results suggest that sustained induction of p21/waf1 mediates growth inhibition in HepG2 in the presence of HGF.

Blood Proteins↗

PubMed1998

Natural hepatocyte growth factor (HGF) from human serum and a bound form of recombinant HGF with heparan sulfate are indistinguishable in their physicochemical properties.

Natural hepatocyte growth factor (nHGF) purified from human serum showed a difference in molecular mass (Mr) between SDS PAGE (76-90 kDa) and gel filtration chromatography on a Sephadex G-200 column (> 200 kDa), whereas nHGF or recombinant HGF (rHGF) from cell culture medium did almost the same Mr (74-100 kDa). A bound form of rHGF with heparan sulfate (or heparin), and an aggregate form of rHGF itself showed a homogeneous band with a Mr of 76-90 kDa on SDS PAGE, but showed a Mr larger than 200 kDa on a Sephadex G-200 column. Both nHGFs, rHGF and the bound form were basic, but the aggregate form was acidic in ionic nature. No significant difference was found in affinity for heparin among these HGF preparations. The bound form treated by the procedures for purification of nHGF from human serum still showed a larger molecular form. The bound form mimicked physicochemical properties of nHGF purified from human serum. These results suggest that a possible form of nHGF in human serum may be a bound form with heparin-like molecules such as heparan sulfate, which are found in the circulation and on cell surface, and purified as the bound form.

Animals↗

PubMed1998

Osteoclastogenesis inhibitory factor exhibits hypocalcemic effects in normal mice and in hypercalcemic nude mice carrying tumors associated with humoral hypercalcemia of malignancy.

Osteoclastogenesis inhibitory factor (OCIF) is a novel secreted protein that inhibits osteoclastogenesis both in vitro and in vivo. In this study, we examined the effects of OCIF on serum calcium (Ca) concentrations in normal mice and in hypercalcemic nude mice carrying tumors associated with humoral hypercalcemia of malignancy. In normal mice, a single intraperitoneal injection of OCIF reduced serum Ca levels in a dose-dependent manner. Significant decrease in serum Ca (by 1.6 +/- 0.3 mg/dL, n = 5) was observed 2 h after the injection of OCIF at 20 mg/kg and the hypocalcemic effect continued for up to 12 h. Serum phosphate (Pi) concentrations also decreased in response to OCIF. Urinary excretion of Ca, Pi, and creatinine did not change significantly after injection of OCIF or vehicle. In hypercalcemic, tumor-bearing nude mice, a single intraperitoneal injection of OCIF at 20 mg/kg resulted in a dramatic decrease in serum Ca (maximal decrease 2.8 +/- 0.37 mg/dL, n = 11), which continued for up to 24 h. The results suggest that OCIF decreased serum Ca through its inhibitory effect on bone resorption. Furthermore, it is suggested that OCIF has therapeutic potential for the treatment of hypercalcemic conditions such as malignancy-associated hypercalcemia.

Animals↗