Biomedical subjects

Y Hakeda

Publications and source records attributed to Y Hakeda.

At least 37 records · Page 2Linked to original sources

PubMed1997

Concanavalin A directly stimulates bone-resorbing activity of osteoclasts and their gene expression of cathepsin K/OC-2.

Concanavalin A (Con A), a plant lectin that recognizes cell-surface glycoproteins, up-regulates osteoclastic bone-resorbing activity of cultured isolated rabbit pure osteoclasts as well as unfractionated bone cells. The effect of Con A was blocked by alpha-methyl mannopyranoside. Several Con A-binding proteins were detected in the plasma membranes from osteoclasts. Furthermore, Con A increased the levels of transcripts of cathepsin K/OC-2, one of the proteases responsible for osteoclastic bone-resorbing activity. These results indicate that Con A directly enhances the function of osteoclasts by the association with surface glycoproteins of osteoclasts.

Animals↗

PubMed1997

Stimulation of osteoblast proliferation by the cartilage-derived growth promoting factors chondromodulin-I and -II.

We previously reported the isolation of the cartilage-derived growth promoting factors chondromodulin-I (ChM-I) and chondromodulin-II (ChM-II) from fetal bovine epiphyseal cartilage. Both of these factors stimulate the growth and matrix formation of chondrocytes in vitro. In the present study, we found that ChM-I and ChM-II stimulated the proliferation of clonal mouse osteoblastic MC3T3-E1 cells as well as primary mouse osteoblasts in culture. Unlike other known growth factors, these factors did not support the proliferation of fibroblasts. Concomitantly with growth stimulation of osteoblasts, there was a reduction of alkaline phosphatase (ALP) activity in the cells, the expression of the differentiated phenotype. These results suggest that epiphyseal cartilage may play a functional role in longitudinal bone growth by production of these unique growth-promoting factors.

Alkaline Phosphatase↗

PubMed1997

Prostaglandin F2alpha stimulates tyrosine phosphorylation and mitogen-activated protein kinase in osteoblastic MC3T3-E1 cells via protein kinase C activation.

PGF2alpha stimulates the proliferation of clonal osteoblastic MC3T3-E1 cells via PGF2alpha receptor linked to phospholipase C activation. To elucidate intracellular events elicited by this receptor, we examined the effects of PGF2alpha on tyrosine phosphorylation and mitogen-activated protein kinase (MAPK) activity in MC3T3-E1 cells. PGF2alpha rapidly raised the level of phosphotyrosine of cellular proteins with Mr values of 62, 68, 72, 76, 82, 125, and 150 kDa. This PGF2alpha-induced tyrosine phosphorylation of proteins (except for pp62) was blocked by down-regulating protein kinase C (PKC) by 12-O-tetradecanoylphorbol 13-acetate pretreatment and by GF 109203X, a potent specific PKC inhibitor. The addition of PGF2alpha also transiently activated MAPK in the same range of concentrations that stimulated tyrosine phosphorylation. In addition, PGF2alpha augmented the MAPK kinase kinase activity of Raf-1, whereas basal activity of MAPK/extracellular signal-regulated protein kinase kinase was less than that of Raf-1 and was little affected by PGF2alpha. Like the tyrosine phosphorylation, these activations of Raf-1 and MAPK activities were reduced by inhibition and down-regulation of PKC. Genistein, a potent inhibitor of tyrosine kinases, did not block the Raf-1 induced by PGF2alpha, indicating a tyrosine kinase-independent pathway for Raf-1 activation. However, the tyrosine kinase inhibitor partially inhibited the MAPK activity, suggesting an involvement of another Raf-1-independent kinase cascade for activation of MAPK by PGF2alpha. Fluprostenol, a specific agonist of PGF2alpha receptor, mimicked the actions of PGF2alpha consistent with a PGF2alpha receptor pathway. Thus, the action of PGF2alpha on osteoblastic MC3T3-E1 cells appears to involve a single receptor that uses diverse interacting signal transduction systems.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1997

Down-regulation of gap junctional intercellular communication between osteoblastic MC3T3-E1 cells by basic fibroblast growth factor and a phorbol ester (12-O-tetradecanoylphorbol-13-acetate).

To address the relation between osteoblast growth and cell-to-cell communication, we examined the effects of basic fibroblast growth factor (bFGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA), both potent stimulators of osteoblastic proliferation, on gap junctional intercellular communication between osteoblastic MC3T3-E1 cells. The level of intercellular communication was estimated by a photobleaching method. TPA inhibited the degree of intercellular communication in two different time-dependent manners. The early (< 1 h) inhibition by TPA was consistent with an increase in the phosphorylation of connexin 43 (Cx43). The later inhibition was caused by reduction in the total amount of Cx43 on the plasma membrane, due to the decrease in the level of Cx43 transcripts. These qualitative and quantitative modulations by TPA were inhibited by a selective inhibitor of protein kinase C, GF109203X. bFGF also attenuated the gap junctional intercellular communication. However, short exposure (< 5 h) to bFGF did not affect the communication. The fact that the growth factor immediately stimulated the phosphorylation of Cx43 indicates that the phosphorylation site(s) affected by bFGF was not involved in the inhibition of communication. The decrease in the intercellular communication level was detected by the longer exposure (> 8 h) to bFGF and paralleled the decline in the Cx-mRNA level. This inhibitory effect of bFGF was abolished by the addition of a tyrosine kinase inhibitor, herbimycin A. Thus, gap junctional intercellular communication between osteoblasts was down-regulated by osteoblastic mitogens through different mechanisms of the modulation of Cx43.

3T3 Cells↗

PubMed1996

Mammalian mature osteoclasts as estrogen target cells.

The decrease in estrogen levels that follows the onset of menopause causes rapid bone loss, resulting in osteoporosis. However, the mechanism by which this occurs remains unclear, especially concerning the regulation of osteoclasts, i.e. the bone-resorbing cells. Using a pit assay involving isolated mature osteoclasts from rabbit long bones, we found that estrogen inhibited the bone-resorbing activity in a dose- and time-dependent manner. Furthermore, we clarified by Northern analysis that estrogen down-regulated the mRNA levels of cathepsin K/OC-2 and that putative estrogen receptor (ER) mRNA was expressed in these osteoclasts. Moreover, other sizes of mRNAs that hybridized with ER cDNA probe were found in these cells. Our results suggest that osteoclasts may be indeed target cells for estrogen and that estrogen might regulate a part of bone metabolism through osteoclasts.

Animals↗

PubMed1996

Vitamin K2 inhibits osteoclastic bone resorption by inducing osteoclast apoptosis.

In contrast to vitamin K1(VK1), vitamin K2(VK2) inhibited osteoclastic bone resorption by unfractionated bone cells and isolated osteoclasts. To investigate the mechanism of inhibition of osteoclastic bone resorption by VK2, we examined the effect of this vitamin on osteoclast apoptosis using a DNA-binding fluorescent dye, Hoechst 33258. In unfractionated bone cells and isolated osteoclasts on dentin slices, we first demonstrated that VK2 induced osteoclast apoptosis, but VK1 did not. Moreover, cycloheximide inhibited VK2-induced osteoclast apoptosis. These results suggest the possibility that VK2 inhibits osteoclastic bone resorption by targeting osteoclasts to undergo apoptosis, which leads to cell death.

Animals↗

PubMed1996

Intact insulin-like growth factor binding protein-5 (IGFBP-5) associates with bone matrix and the soluble fragments of IGFBP-5 accumulated in culture medium of neonatal mouse calvariae by parathyroid hormone and prostaglandin E2-treatment.

We examined the distribution of insulin-like growth factor binding proteins (IGFBPs) in cultured neonatal mouse calvariae. IGFBP-3 and -4 were predominantly found in the conditioned medium. IGFBP-2 was partitioned between conditioned medium and bone and extracellular matrix (BECM), while intact (31-kDa) IGFBP-5 was most abundant in BECM extracts. After treatment with parathyroid hormone (PTH, 10(-8) M) or prostaglandin E2 (PGE2, 10(-6) M), immunoreactive IGFBP-5 accumulated in the conditioned medium in a 21-kDa form which did not bind IGF-I on Western ligand blots. PTH and PGE2 did not alter the level of steady-state IGFBP-5 mRNA, nor markedly stimulate IGFBP-5 synthesis in the calvariae, and thus accumulation of 21-kDa IGFBP-5 was largely due to release from BECM. This accumulation of truncated IGFBP-5 in the conditioned medium was not dependent on osteoclastic bone resorption, since it was not blocked by calcitonin or a bisphosphonate which inhibited PTH- and PGE2-stimulated 45Ca-release. The conditioned medium from PTH- or PGE2-treated cultures degraded recombinant human IGFBP-5 into lower molecular weight fragments. Addition of IGF-1 at 10(-8) M into the culture resulted in accumulation of native 31-kDa IGFBP-5. However, even in the presence of IGF-1, the native IGFBP-5 was degraded and the 21-kDa product accumulated in the culture medium. These results suggested a possible proteolytic mechanism for 21-kDa IGFBP-5 accumulation, responsive to PTH and PGE2. Aprotinin, leupeptin, cystatin, and bestatin did not inhibit the effects of PTH and PGE2 in the cultures. The localization of IGFBP-5 in BECM and its release and proteolysis induced by PTH and PGE2 could play a role in the local regulation of bone metabolism.

Animals↗

PubMed1996

Human calcitonin has the same inhibitory effect on osteoclastic bone resorption by human giant cell tumor cells as salmon calcitonin.

Human calcitonin (hCT) has been reported to have a less hypocalcemizing effect on rats and to have a lower binding affinity for the receptor of mouse osteoclasts than salmon CT(sCT). In this study we comparatively examined the effect of hCT and sCT on osteoclastic bone-resorbing activity of unfractionated cells obtained from human giant cell tumor of bone and from rabbit and mouse long bones. We found that hCT had the same inhibitory effect as sCT on the bone-resorbing activity of human and rabbit osteoclastic cells, but a different one on that of mouse cells. These results indicate that the activity of drugs should be assayed using human cells if possible.

Animals↗

PubMed1995

Retinoic acid directly stimulates osteoclastic bone resorption and gene expression of cathepsin K/OC-2.

Vitamin A metabolites such as all-trans-retinoic acid (all-trans-RA) affect several steps of metabolic processes in vertebrates. In the last few years, several studies have shown the effect of RA on bone formation and metabolism. However, mechanisms of its action still remain unclear, especially with respect to the regulation of bone cells. Therefore, this study was carried out to clarify how RA regulates the activity of osteoclasts. Using a pit assay involving unfractionated bone cells, including osteoclasts obtained from rabbits, we found that RA stimulated an increase in the bone-resorbing activity in a dose- and time-dependent manner. Furthermore, this effect occurred more rapidly than that of treatments with 1 alpha,25-dihydroxyvitamin D3. However, this effect of RA may be partly related to cross-talk between osteoclasts and other types of cells. Therefore we studied the effect of RA on isolated osteoclasts. We found that all-trans-RA regulates the gene expression of cathepsin K/OC-2, a dominant cysteine proteinase, at the transcriptional level in mature osteoclasts isolated from rabbits. Moreover, retinoic acid-receptor alpha mRNA and retinoid X-receptor beta mRNA were expressed in these mature osteoclasts. Our results indicate that osteoclasts are target cells for RA and that RA might regulate a part of bone formation and metabolism through osteoclasts.

Animals↗

PubMed1995

Basic fibroblast growth factor regulates IGF-I binding proteins in the clonal osteoblastic cell line MC3T3-E1.

In previous studies, we reported that basic fibroblast growth factor (bFGF) regulates insulin-like growth factor messenger RNAs and protein levels in the osteoblastic MC3T3-E1 cells. In the present study, we examined the expression of insulin-like growth factor binding proteins (IGFBPs) in MC3T3-E1 cells and determined whether bFGF altered IGFBP mRNAs and protein levels. Since previous studies suggested that IGFBPs can inhibit DNA synthesis stimulated by IGF-I, we wondered whether the mitogenic effect of bFGF was altered by exogenous IGFBP-3. Confluent MC3T3-E1 cells were serum-deprived for 24 h and then treated with bFGF for 6-24 h. In control cultures, MC3T3-E1 cells expressed the mRNAs for IGF-I, IGF-II, and IGFBP-2, 4, 5, and 6 but not IGFBP-1 or 3. A 24 h treatment with bFGF at 10(-8) M decreased IGF-I mRNA by 97%, IGF-II mRNA by 73%, IGFBP-2 by 64%, IGFBP-4 by 73%, IGFBP-5 by 95%, and IGFBP-6 by 65%. The inhibitory effect of bFGF on IGF-I and IGFBP mRNA levels was not altered by aphidicolin, an inhibitor of cell replication. bFGF 10 nM decreased IGF-I levels determined by radioimmunoassay after acidification by 45% and 72% at 24 and 48 h, respectively. Western ligand blot for IGF binding proteins revealed that MC3T3-E1 cells expressed IGFBPs of 24, 30, and 34 kD. Treatment with bFGF 10(-8) M decreased the levels of the 24 and 30 kD band at 24 h but increased the 34 kD band.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗

PubMed1995

Hepatocyte growth factor is involved in formation of osteoclast-like cells mediated by clonal stromal cells (MC3T3-G2/PA6).

Osteoclast formation from hemopoietic precursors has been shown to require the support of stromal cells in bone tissue. In this study, we demonstrated that hepatocyte growth factor (HGF) is one of the stromal cell-derived molecules responsible for osteoclast-like cell formation. For our experiments, we used a coculture system for osteoclastic cell formation and activation in which hemopoietic blast cells are cocultured with calvaria-derived stromal MC3T3-G2/PA6 (PA6) cells on dentine slices in the presence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Addition of anti-HGF neutralizing IgG to the cocultures inhibited the formation of osteoclastic cells and their dentine-resorbing activity. We detected a single 6.0-kb transcript for HGF in PA6 cells, and also recognized immunoreactive M(r) 81,000 and 88,000 forms of HGF in conditioned medium (CM) from PA6 cell cultures, the level of which reached 6 ng/ml. Both the CM and HGF stimulated the proliferation of blast cells synergistically with granulocyte-macrophage colony-stimulating factor, resulting in an increased number of osteoclast precursors that respond to 1,25(OH)2D3 that are tartrate-resistant acid phosphatase-positive multinucleate cells in stromal cell-free blast cell cultures in plastic wells. The effect of the CM was diminished by the addition of anti-HGF IgG. However, neither the CM nor HGF stimulated the formation of osteoclastic cells and pits on dentine slices in the absence of PA6 cells. These results suggest that although HGF cannot completely replace stromal cells, it is one of the paracrine mediators produced by stromal cells that act on proliferation of osteoclastic cell precursors.

Acid Phosphatase↗

PubMed1995

Time-lapse microcinematography of osteocytes.

We succeeded in the isolation of osteocytes from parietal bones of 16-day-old chick embryos. Isolated osteocytes showed a typical stellate morphology. More than 95% of these cells reacted with the osteocyte-specific antibody OB 7.3. In culture osteocytes formed gap junctions with each other, as could be established by ACAS. Sixteen-millimeter time-lapse microcinematography of the cells also demonstrated the formation of intercellular connections and gap junctions, and portrayed the interaction between osteocytes and osteoclasts: osteocytes seemed to inhibit osteoclast activity. This cinematography also showed the ability of osteocytes to proliferate after they had been disconnected from each other. Thereafter these cells redifferentiated into osteoblasts that became embedded in bone matrix produced by themselves. These findings suggest that osteocytes might be involved in bone formation during remodeling.

Animals↗

PubMed1994

Divalent cations elevate cytosolic calcium of chick osteocytes.

Though it is proposed that osteocytes have several functions in bone metabolism, only limited definite information including results of calcium regulation has been forthcoming. We demonstrate here for the first time that the concentration of divalent cations, such as calcium, nickel and cadmium, directly regulates the cytosolic calcium concentration in osteocytes.

Animals↗

PubMed1994

Identification of matrix metalloproteinase 9 in rabbit osteoclasts.

Osteoclasts are multinucleate giant cells that play key roles in bone resorption. To identify genes predominantly expressed in osteoclasts, we screened a cDNA library of osteoclasts with cDNA probes of osteoclasts and alveolar macrophages. Clones specifically hybridizing to the osteoclast probe were isolated and sequenced. The nucleotide sequence of one such clone, F17, was found to share significant similarity with the sequences of human and mouse matrix metalloproteinase 9 (MMP-9) cDNA. By isolation and sequencing of the full-length cDNA, F17 was revealed to encode the rabbit counterpart of MMP-9. By Northern blotting, messenger RNA for MMP-9 was found to be highly and predominantly expressed in isolated osteoclasts when compared with its level in alveolar macrophages and other tissues. By gelatin zymography, gelatinase activity was detected in conditioned medium of isolated osteoclasts, suggesting that MMP-9 is secreted by isolated osteoclasts. Expression of MMP-9 was also observed in in vivo osteoclasts in metacarpal bones of newborn rabbits by in situ hybridization. These facts suggest that MMP-9 is one of the major proteases produced by osteoclasts and possibly plays a role in osteoclastic bone resorption.

Amino Acid Sequence↗

PubMed1994

Molecular cloning of a possible cysteine proteinase predominantly expressed in osteoclasts.

Osteoclasts are multinucleate giant cells playing key roles in bone resorption. These cells solubilize mineralized bone matrix by means of acid and protease action; however, the precise mechanism of this process is not well known. Recently, we succeeded in the isolation of pure osteoclasts from rabbit bones and constructed a cDNA library. Using a differential screening procedure, two genes expressed predominantly in osteoclasts compared with spleen cells were isolated (Tezuka, K., Sato, T., Kamioka, H., Nijweide, P. J., Tanaka, K., Matsuo, T., Ohta, M., Kurihara, N., Hakeda, Y., and Kumegawa, M. (1992) Biochem. Biophys. Res. Commun. 186, 911-917). One of them, OC-2, was found to encode a possible cysteine proteinase structurally related to cathepsins L and S. By in situ hybridization, OC-2 was confirmed to be expressed in osteoclasts in vivo. By Northern blot analysis, OC-2 was highly and preferentially expressed in osteoclasts compared with other tissues such as kidney, liver, spleen, and lung. The predominant expression of OC-2 in osteoclasts may suggest that OC-2 encodes a protein, possibly a cysteine proteinase, that plays an important role in osteoclastic bone resorption.

Amino Acid Sequence↗

PubMed1994

Stimulatory effect of a phorbol ester on expression of insulin-like growth factor (IGF) binding protein-2 and level of IGF-I receptors in mouse osteoblastic MC3T3-E1 cells.

We examined the relationship between signal transduction and the expression of insulin-like growth factor I (IGF-I), IGF-I receptor level, and IGF binding proteins (IGFBPs) in murine clonal osteoblastic MC3T3-E1 cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, decreased the secretion of immunoreactive IGF-I into the medium, whereas dibutyryl cAMP (Bt2cAMP) augmented the secretion. In contrast, TPA increased the level of type I IGF receptor on the cells. Furthermore, MC3T3-E1 cells produced and secreted at least three different IGFBPs with molecular masses of 24, 30, and 34 kDa, and the 24-kDa IGFBP was predominant under normal conditions. However, TPA specifically increased the secretion of the 34-kDa IGFBP. The N-terminal amino acid sequence of the purified 34-kDa IGFBP was nearly identical with that of rat IGFBP-2. Furthermore, the 34-kDa IGFBP was immunoreactive to anti-IGFBP-2 antiserum. The level of IGFBP-2 mRNA in the cells was increased by TPA, indicating that the increase in IGFBP-2 secretion results from the stimulation of IGFBP-2 production. In contrast, Bt2cAMP affected neither IGF-I receptor number nor the IGFBP secretion. These results indicate that the production of IGF-I and the expression of IGF-I receptors and IGFBP-2 are up-regulated by the activation of adenylate cyclase and protein kinase C, respectively, in osteoblastic MC3T3-E1 cells.

Adenylyl Cyclases↗

PubMed1994

Calcitonin inhibits osteopontin mRNA expression in isolated rabbit osteoclasts.

Recent evidence indicates that osteopontin (Opn), one of the bone matrix proteins, plays an important role in the attachment of osteoclasts to bone matrix. Besides being elaborated by osteoblasts, this protein is also produced by osteoclasts. The present study was performed to examine the effect of calcitonin (CT) on Opn mRNA expression of isolated rabbit osteoclasts and to clarify the second messenger signaling of this effect. Eel CT inhibited Opn mRNA expression as well as bone-resorbing activity of isolated rabbit osteoclasts. Eel CT caused a transient increase in intracellular calcium followed by a sustained increase as well as an increase in cAMP production in these cells. Dibutyryl-cAMP (10(-4) M) and Sp-cAMPS (10(-4) M), an activator of cAMP-dependent protein kinase (PKA), as well as A23187 (10(-7) M), a calcium ionophore, and phorbol myristate acetate (10(-7) M), an activator of protein kinase C (PKC), caused a significant inhibition of Opn mRNA expression, and suppressed bone-resorbing activity of isolated osteoclasts. The present study is the first to demonstrate that CT inhibits Opn mRNA expression in isolated rabbit osteoclasts, presumably through the activation of PKA and calcium/PKC pathways, by which the bone-resorbing activity might be attenuated subsequently.

Animals↗

PubMed1994

Regulation of the two prostaglandin G/H synthases by parathyroid hormone, interleukin-1, cortisol, and prostaglandin E2 in cultured neonatal mouse calvariae.

A second prostaglandin G/H synthase (PGHS-2), encoded by a gene separate from that for the original PGHS (PGHS-1), has recently been identified. We have shown that PGHS-2 is expressed in cultured mouse calvariae and have compared regulation of PGHS-2 and PGHS-1 messenger RNA (mRNA) levels. PGHS-2 mRNA was not detectable in freshly isolated bones, but was induced during culture and further stimulated by interleukin-1 (IL-1) and PTH. Both factors also increased PGHS-2 protein levels. Changes in medium prostaglandin E2 (PGE2) production correlated with increases in PGHS-2 mRNA levels. However, with IL-1, PGE2 production was increased more than PGHS-2 mRNA levels (treated/control ratio, 3.4 and 1.5, respectively), whereas with PTH there was a closer correspondence (2.0 and 2.1). Cortisol reduced PTH-stimulated PGE2 production (treated/control ratio decreased from 3.1 to 0.2) more than PGHS-2 mRNA levels (2.8 to 0.8). In the presence of exogenous arachidonic acid, changes in PGHS-2 mRNA levels with IL-1, PTH, and cortisol correlated closely with changes in PGE2 production. PGE2 itself increased PGHS-2 mRNA, and nonsteroidal antiinflammatory drugs decreased PGHS-2 mRNA levels by 80%. In contrast, PGHS-1 mRNA was expressed constitutively and was not affected by IL-1, PTH, or cortisol when measured by competitive reverse transcriptase-polymerase chain reaction. We conclude that regulation of PGE2 production is predominantly through PGHS-2, rather than PGHS-1; that IL-1 and cortisol may also regulate arachidonic acid release; and that PGE2 may amplify its own production through stimulation of PGHS-2.

Animals↗