Biomedical subjects

M Kumegawa

Publications and source records attributed to M Kumegawa.

At least 37 records · Page 2Linked to original sources

PubMed2000

Direct stimulation of osteoclastic bone resorption by bone morphogenetic protein (BMP)-2 and expression of BMP receptors in mature osteoclasts.

Bone morphogenetic proteins (BMPs) play an important role in various kinds of pattern formation and organogenesis during vertebrate development. In the skeleton, BMPs induce the differentiation of cells of chondrocytic and osteoblastic cell lineage and enhance their function. However, the action of BMPs on osteoclastic bone resorption, a process essential for pathophysiological bone development and regeneration, is still controversial. In this study, we examine the direct effect of BMPs on osteoclastic bone-resorbing activity in a culture of highly purified rabbit mature osteoclasts. BMP-2 caused a dose- and time-dependent increase in bone resorption pits excavated by the isolated osteoclasts. BMP-4 also stimulated osteoclastic bone resorption. The increase in osteoclastic bone resorption induced by BMP-2 was abolished by the simultaneous addition of follistatin, a BMP/activin binding protein that negates their biological activity. Just as it increased bone resorption, BMP-2 also elevated the messenger RNA expressions of cathepsin K and carbonic anhydrase II, which are key enzymes for the degradation of organic and inorganic bone matrices, respectively. Type IA and II BMP receptors (BMPRs), and their downstream signal transduction molecules, Smad1 and Smad5, were expressed in isolated osteoclasts as well as in osteoblastic cells, whereas type IB BMPR was undetectable. BMPs directly stimulate mature osteoclast function probably mediated by BMPR-IA and BMPR-II and their downstream molecules expressed in osteoclasts. The results presented here expand our understanding of the multifunctional roles of BMPs in bone development.

Animals↗

PubMed2000

Direct and indirect actions of fibroblast growth factor 2 on osteoclastic bone resorption in cultures.

Fibroblast growth factor 2 (FGF-2 or basic FGF) is known to show variable actions on bone formation and bone resorption. This study was undertaken to elucidate the mechanisms whereby FGF-2 affects bone metabolism, especially bone resorption, using three different culture systems. FGF-2 at 10(-9) M and higher concentrations induced osteoclastic cell formation in the coculture system of mouse osteoblastic cells and bone marrow cells, and this induction was abrogated by nonsteroidal anti-inflammatory drugs (NSAIDs). 45Ca release from prelabeled cultured mouse calvariae stimulated by FGF-2 (10(-8) M) was also inhibited by NSAIDs, and the inhibition was stronger by NSAIDs, which are more selective for inhibition of cyclooxygenase 2 (COX-2) than COX-1, suggesting the mediation of COX-2 induction. COX-2 was highly expressed and its messenger RNA (mRNA) level was stimulated by FGF-2 in osteoblastic cells whereas it was undetectable or not stimulated by FGF-2 in cells of osteoclast lineage. To further investigate the direct actions of FGF-2 on osteoclasts, resorbed pit formation was compared between cultures of purified osteoclasts and unfractionated bone cells from rabbit long bones. FGF-2 (> or = 10(-12) M) stimulated resorbed pit formation by purified osteoclasts with a maximum effect of 2.0-fold at 10(-11) M, and no further stimulation was observed at higher concentrations. However, FGF-2 at 10(-9) M - 10(-8) M stimulated resorbed pit formation by unfractionated bone cells up to 9.7-fold. NS-398, a specific COX-2 inhibitor, did not affect the FGF-2 stimulation on purified osteoclasts but inhibited that on unfractionated bone cells. We conclude that FGF-2 at low concentrations (> or =10(-12) M) acts directly on mature osteoclasts to resorb bone moderately, whereas at high concentrations (> or = 10(-9) M) it acts on osteoblastic cells to induce COX-2 and stimulates bone resorption potently.

Acid Phosphatase↗

PubMed2000

Apoptosis-inducing activity of vitamin C and vitamin K.

Apoptosis-inducing activity of vitamins C and K and of their analogs are reviewed. Vitamin C shows both reducing and oxidizing activities, depending on the environment in which this vitamin is present. Higher concentrations of vitamin C induce apoptotic cell death in various tumor cell lines including oral squamous cell carcinoma and salivary gland tumor cell lines, possibly via its prooxidant action. The apoptosis-inducing activity of ascorbate is stimulated by Cu2+, lignin and ion chelator, and inhibited by catalase, Fe3+, Co2+ and saliva. On the other hand, at lower concentrations, ascorbic acid displays an antioxidant property, preventing the spontaneous and stress or antitumor agent-induced apoptosis. Sodium 5,6-benzylidene-L-ascorbate, intravenous administration of which induces degeneration of human inoperable tumors and rat hepatocellular carcinoma in vivo, induces apoptotic or non-apoptotic cell death, depending on the types of target cells. On the other hand, elevation of intracellular concentration of ascorbic acid by treatment with ascorbate 2-phosphate or dehydroascorbic acid makes the cells resistant to the oxidative stress-induced apoptosis. Vitamin K2, which has a geranylgeranyl group as a side chain,and vitamin K3 induces apoptosis of various cultured cells including osteoclasts and osteoblasts, by elevating peroxide and superoxide radicals. Synergistic apoptosis-inducing actions have been found between vitamins C and K, and between these vitamins and antiproliferative agents. The possible therapeutic application of these vitamins is discussed.

Animals↗

PubMed2000

[Recent progress in studies on osteocytes--osteocytes and mechanical stress].

Although osteocytes are of the most abundant cells in bone, our knowledge about the role of osteocytes in bone metabolism is still poor compared with that about osteoblasts and osteoclasts, both being on the surface of bone. Osteocytes are terminally differentiated bone-forming cells. During bone formation, some of the osteoblasts lining the surface of bone are incorporated into the newly formed osteoid matrix and become osteocytes, while the other osteoblasts remain on the surface as lining cells. During this transition from osteoblasts to osteocytes, the cells lose numerous osteoblastic phenotypes and acquire osteocytic characteristics such as high expression of osteocalcin and particularly their specific morphology. Osteocytes are connected with each other in bone and with osteoblasts on the bone surface through canaliculi, forming cellular networks; and gap-junctions present at the contact sites mediate their intercellular communication. Several roles of osteocytes in bone have been proposed so far. Of them, based on the morphological characteristics of osteocytes, sensation of mechanical stress loaded onto bone is suspected to be one of their functions. One of the mechanical stresses on bone is fluid shear stress. Between the osteocyte's plasma membrane and the bone matrix is the periosteocytic space. This space exists both in the lacunae and in the canaliculi, and it is filled with extracellular fluid (ECF). Application of mechanical stress to bone locally deforms the tissue. This periodical deformation subsequently causes an increase in the flow of ECF in the periosteocytic space, resulting in shear stress on the surface of the osteocytes. Experimental studies demonstrated that bone cells were equivalently or more sensitive to the fluid shear stress than epithelial cells. Osteocytic cells cultured enhanced expression of prostaglandin (PG) G/H synthase-2 (COX-2) mRNA in response to shear stress. PGE2 is a potent regulator of proliferation and function of osteoblasts and osteoclasts. Therefore, a metabolic response by osteoblasts and osteoclasts lining the bone surface may be caused by PGE2 produced by osteocytes in response to shear stress when the prostanoid reaches the surface through the canaliculi. In conclusion, osteocytes play an important role in sensing extracellular mechanical stress, and the mechanical signals mediated by osteocytes may regulate the overall metabolism of cells in bone tissue.

Animals↗

PubMed1999

CD38/ADP-ribosyl cyclase: A new role in the regulation of osteoclastic bone resorption.

The multifunctional ADP-ribosyl cyclase, CD38, catalyzes the cyclization of NAD(+) to cyclic ADP-ribose (cADPr). The latter gates Ca(2+) release through microsomal membrane-resident ryanodine receptors (RyRs). We first cloned and sequenced full-length CD38 cDNA from a rabbit osteoclast cDNA library. The predicted amino acid sequence displayed 59, 59, and 50% similarity, respectively, to the mouse, rat, and human CD38. In situ RT-PCR revealed intense cytoplasmic staining of osteoclasts, confirming CD38 mRNA expression. Both confocal microscopy and Western blotting confirmed the plasma membrane localization of the CD38 protein. The ADP-ribosyl cyclase activity of osteoclastic CD38 was next demonstrated by its ability to cyclize the NAD(+) surrogate, NGD(+), to its fluorescent derivative cGDP-ribose. We then examined the effects of CD38 on osteoclast function. CD38 activation by an agonist antibody (A10) in the presence of substrate (NAD(+)) triggered a cytosolic Ca(2+) signal. Both ryanodine receptor modulators, ryanodine, and caffeine, markedly attenuated this cytosolic Ca(2+) change. Furthermore, the anti-CD38 agonist antibody expectedly inhibited bone resorption in the pit assay and elevated interleukin-6 (IL-6) secretion. IL-6, in turn, enhanced CD38 mRNA expression. Taken together, the results provide compelling evidence for a new role for CD38/ADP-ribosyl cyclase in the control of bone resorption, most likely exerted via cADPr.

ADP-ribosyl Cyclase↗

PubMed1999

High mobility group-like protein in bovine milk stimulates the proliferation of osteoblastic MC3T3-E1 cells.

The active component in bovine milk on the proliferation of osteoblastic MC3T3-E1 cells was purified and identified. Growth-promoting activity was measured by [(3)H]thymidine incorporation on the cell. The molecular weight of the purified protein was 10 kDa. The amino-terminal sequence of this 10-kDa protein was identical to bovine high mobility group protein (HMG) 1. This 10-kDa protein is suggested to be a basic protein and to have an HMG box, a consensus sequence motif among the HMG family. From these results, we named this protein HMG-like protein. HMG is a ubiquitous nonhistone component of chromatin and considered to be implicated in DNA replication. We found this protein in milk, and it showed a growth-promoting activity. We propose the possibility that HMG-like protein existed in milk and plays an important role for neonate in bone formation by activating osteoblasts.

Amino Acid Sequence↗

PubMed1999

Molecular and functional evidence for calcineurin-A alpha and beta isoforms in the osteoclast: novel insights into cyclosporin A action on bone resorption.

We provide the first molecular evidence for the presence of a functional serine/threonine phosphatase, calcineurin-A (CN-A), in the osteoclast. Polymerase chain reaction (PCR) of an osteoclast cDNA library, together with restriction mapping, revealed two isoform sequences, alpha and beta. We then examined the functionality of the detected CN-A by assessing the effect of a classical antagonist, cyclosporin A (CsA), in the osteoclast resorption (pit) assay. CsA (0.1 and 1 microg ml-1) potently inhibited bone resorption. The presence of lymphocytes, with or without prior exposure to CsA in vivo, failed to reverse the CsA-induced resorption-inhibition. Expectedly, CsA had no direct effect on cytosolic Ca2+ levels in fura-2-loaded osteoclasts. These studies are a prelude to further investigations into the possible role of CN-A in osteoclast regulation. Finally, mechanistic studies on the bone effects of CsA, a widely used immunosupressant, should proceed from these observations.

Animals↗

PubMed1999

Etidronate (EHDP) inhibits osteoclastic-bone resorption, promotes apoptosis and disrupts actin rings in isolate-mature osteoclasts.

Bisphosphonates, therapeutic reagents against tumoral bone diseases (Paget's disease or osteoporosis), are potent inhibitors of bone resorption. The mechanisms by which they directly act on mature osteoclasts remain unclear. Using a recently developed technique for isolation of highly purified mammalian mature osteoclasts, we demonstrated that etidronate [ethane-1-hydroxy-1,1-diphosphonate (EHDP), 1-hydroxy-1,1-ethylidenebisphosphonate], inhibited directly osteoclastic bone-resorbing activity by pit assay. In addition, EHDP also directly induced apoptosis and disrupted actin rings in osteoclasts. The data support previous data on non-purified osteoclasts and results in vivo.

Actins↗

PubMed1999

Congenital osteoclast deficiency in osteopetrotic (op/op) mice is improved by ovariectomy and orchiectomy.

We examined the changes in the appearance of osteoclasts in the femora of ovariectomized (OVX) or orchiectomized (ORX) op/op mice. Osteoclasts on the trabecular bone surface of the OVX or ORX op/op mice significantly increased in number seven or eight times in comparison with sham-operated op/op mice. Furthermore, TRAP-positive cells increased about four times in 100-week-old females and males, compared with sham-operated groups. These results have indicated that a sex hormone reduction due to OVX or ORX induces prominent recruitment of osteoclasts in op/op mice.

Animals↗

PubMed1999

Breast cancer cells express cathepsins B and L but not cathepsins K or H.

Lysosomal cysteine proteinases (cathepsins) are considered to play a role in bone degradation mediated by metastatic breast cancers. To evaluate which cathepsin contributes to the osteolysis, we quantitatively determined the expression levels of four cathepsins in two breast cancer cell lines, MCF-7 and MDA-MB-231, by competitive RT-PCR. Cathepsin K, which is the most abundant cathepsin in osteoclasts, was not detected in either cell lines. We also failed to detect cathepsin H mRNA. By contrast, we found significant expression of cathepsins B and L in both cell lines. By Northern blot analysis cathepsin B mRNA was detected in a single form in these cells, whereas osteoclasts contained multiple forms of the mRNA. Cathepsin B protein was also detected by Western blotting as a single immunoreactive band corresponding to its mature enzyme. These findings suggest that osteolysis associated with metastatic breast cancers takes place in a different way from osteoclast-mediated bone resorption.

Adenocarcinoma↗

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

Calcium-sensing receptor in mature osteoclasts, which are bone resorbing cells.

Bone metabolism consists of osteoblast-mediated bone formation coupled to osteoclastic resorption of bone. Osteoclastic bone resorption plays an important role in normal skeletal development and the maintenance of its integrity throughout life. Although osteoclastic activity is thought to be under the control of feedback regulation by extracellular cations, the molecular mechanism of detecting extracellular cations within the bone microenvironment remains to be clarified. In the present study we showed by reverse transcription-polymerase chain reaction and Northern blot analysis that cultured mature osteoclasts express the calcium-sensing receptor (CaSR) mRNA. The nucleotide sequence of rabbit osteoclast CaSR was approximately 90% identical to that of CaSR cDNA from human, bovine, and rat parathyroid glands. Moreover, the activity of osteoclastic bone resorption, as determined by pit formation, was regulated by extracellular calcium ion as well as its agonists that are known to act through the CaSR. We conclude that CaSR, homologous to that identified in parathyroid glands, is present in mature osteoclasts and calcium ion released from bone may directly regulate osteoclastic bone resorption.

Animals↗

PubMed1998

Joint fluid from patients with failed total hip arthroplasty stimulates pit formation by mouse osteoclasts on dentin slices.

Periprosthetic bone resorption has been implicated in the failure of total joint arthroplasty. Osteolysis is reported to be associated with bone resorption induced by bone-resorbing cytokines, which are released from macrophages and fibroblasts in periprosthetic tissues after stimulation by wear debris generated in the joint cavity. Recent reports have suggested the concept of the effective joint space, which includes all periprosthetic regions that are accessible to joint fluid and wear debris. In this study, we examined the levels of interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6R), and tartrate-resistant acid phosphatase (TRAP) in joint fluid after failed total hip arthroplasty (THA) with osteolysis and investigated whether the joint fluid could activate osteoclastic bone resorption using unfractionated mouse bone cells cultured on dentin slices. Histochemical analysis showed the presence of more TRAP-positive cells in synovial capsules from failed THA patients when compared with osteoarthritis (OA) patients (controls). The levels of IL-6, sIL-6R, and TRAP in joint fluid from failed THA patients were significantly higher than in OA patients. Mouse osteoclasts cultured on dentin slices with joint fluid from failed THA patients with osteolysis produced a significant increase of pit area, whereas cells cultured with joint fluid from OA patients did not. Interestingly, osteoclastic bone resorption on dentin slices was significantly correlated with TRAP activity in joint fluid (p < 0.0001). These results suggest that joint fluid containing bone-resorbing cytokines is produced by synovial capsules in failed THA patients with osteolysis and may activate osteoclasts around the prosthesis in combination with those produced by interface tissues, thus contributing to periprosthetic bone resorption.

Acid Phosphatase↗

PubMed1998

Tyro 3 receptor tyrosine kinase and its ligand, Gas6, stimulate the function of osteoclasts.

Bone is continuously being formed and resorbed. This process is accomplished by the precise coordination of two cell types: osteoblasts and osteoclasts. Osteoclasts are large, multinucleated cells that are derived from the same hematopoietic precursors as macrophages. However, these bone-resorbing cells are difficult to study directly because of their relative inaccessibility. The purification of primary osteoclasts from rabbit bones by their adherent nature provides an opportunity for investigating the molecules in osteoclasts. We have examined the expression of receptor tyrosine kinase by polymerase chain reaction (PCR) and found that Tyro 3 was frequently identified from primary osteoclasts in PCR cloning. Immunohistochemistry revealed that Tyro 3 was expressed on the multinucleated osteoclasts which were positive for tartrate-resistant acid phosphatase (TRAP), but not on mononuclear TRAP-positive cells. The Tyro 3 ligand, Gas6, induced the phosphorylation of Tyro 3 receptors in osteoclasts in two to five min. Gas6 and protein S directly enhanced the bone resorbing activity of mature osteoclasts. This effect of Gas6 was inhibited by the addition of a tyrosine kinase inhibitor, herbimycin A. However, Gas6 did not affect the differentiation of osteoclasts from bone marrow cells. Gas6 and protein S are dependent on vitamin K, a cofactor for the enzyme responsible for carboxylation of glutamic acid residues. The findings in this study are the first to indicate a new biological activity of Gas6 and protein S as a direct regulator of osteoclastic function; they give an insight into the role of these vitamin K-dependent ligands in bone resorption in vivo.

Acid Phosphatase↗

PubMed1998

Effect of monoclonal anti-human gp130 antibody (GPX7) on bone turnover in normal and ovariectomized rats.

We examined the effects of a monoclonal antihuman gp130 antibody (GPX7), which is known to inhibit interleukin-6 (IL-6) and leukemia inhibitory factor-mediated responses in human cells on the bone metabolism in normal and ovariectomized (OVX), 7-month-old, Wistar rats for 8 weeks. After confirming the cross-reactivity of the antibody in suppressing the IL-6-mediated proliferation of rat liver cells, GPX7 was injected once a week at doses of 1 (low dose) or 4 (high dose) mg/kg body weight (BW). In the lumbar body, bone mineral density values and the trabecular bone volume (BV/TV) were maintained in the GPX7 groups. The values of the trabecular osteoclast surface and number in the GPX7 high-dose group were significantly smaller than those in the OVX controls. The double-labeled surface and bone formation rates in the GPX7 high-dose group were significantly increased. In the proximal tibia, however, the bone mineral content and BV/ TV values in the GPX7 groups were smaller, but the trabecular thickness value in the GPX7 high-dose group was larger than in the OVX control. The single-labeled surface in the GPX7 high-dose group was significantly larger than that in the OVX control rats. Though the parameter values of trabecular osteoclasts were apparently smaller, the differences were not significant. 17-beta estradiol (0.125 mg/kg BW a week) administration prevented the bone loss by reducing the parameters of bone formation and resorption in both the lumbar and the proximal tibia. The antibody administration to the normal rats did not cause any significant changes in the parameters of bone mass and turnover. These data demonstrate that while GPX7 modulates the bone turnover after ovariectomy in rats, it does not compensate for the action of estrogen after ovariectomy in rats.

Alkaline Phosphatase↗

PubMed1998

Large scale isolation of osteoclast-specific genes by an improved method involving the preparation of a subtracted cDNA library.

BACKGROUND: Osteoclasts play crucial roles in bone resorption, which triggers bone remodeling. Molecular mechanisms underlying these osteoclast-specific biological functions remain elusive because only a limited number of osteoclast-specific genes have been identified. To circumvent this, we isolated a large number of osteoclast-specific genes by preparing a subtracted cDNA library of high quality. RESULTS: We first constructed a plasmid expression vector (pAP3neo) that allowed an efficient subtraction. Then, we improved the standard protocols for preparation of the cDNA library and the subsequent subtraction procedure. Using our protocol, we prepared a rabbit osteoclast cDNA library of high complexity. Subsequently, we prepared an osteoclast-specific cDNA library of high complexity by subtracting it with biotin-labelled mRNA, derived from rabbit spleen through the biotin-avidin method. The resulting library included a high proportion of full-length cDNA inserts. Using DNA dot blot analysis, we found that the osteoclast-specific cDNA clones were highly enriched in this subtracted cDNA library, i.e. nearly 70% of the analysed clones were primarily detected in osteoclasts but not in spleen. Multiple-tissue Northern analysis also showed that many of these clones were expressed almost exclusively in osteoclasts. DNA sequencing of randomly selected clones showed that 424 cDNA species out of 1136 analysed were novel. DNA sequencing also showed that our subtracted cDNA library was almost equalized, suggesting that the library may contain almost all of the osteoclast-specific genes. CONCLUSION: From these data, we conclude that our subtraction protocols, and the subsequent procedure for the analysis of the isolated clones developed here, are useful for the comprehensive isolation and identification of transcriptionally up- or down-regulated genes in general.

Animals↗

PubMed1997

Estrogen inhibits bone resorption by directly inducing apoptosis of the bone-resorbing osteoclasts.

Estrogen deficiency causes bone loss, which can be prevented by estrogen replacement therapy. Using a recently developed technique for isolation of highly purified mammalian osteoclasts, we showed that 17 beta-estradiol (E2) was able to directly inhibit osteoclastic bone resorption. At concentrations effective for inhibiting bone resorption, E2 also directly induced osteoclast apoptosis in a dose- and time-dependent manner. ICI164,384 and tamoxifen, as pure and partial antagonists, respectively, completely or partially blocked the effect of E2 on both inhibition of osteoclastic bone resorption and induction of osteoclast apoptosis. These data suggest that the protective effects of estrogen against postmenopausal osteoporosis are mediated in part by the direct induction of apoptosis of the bone-resorbing osteoclasts by an estrogen receptor- mediated mechanism.

Animals↗

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↗