Biomedical subjects

Noriyoshi Kurihara

Publications and source records attributed to Noriyoshi Kurihara.

15 recordsLinked to original sources

PubMed2006

Mutation of the sequestosome 1 (p62) gene increases osteoclastogenesis but does not induce Paget disease.

Paget disease is the most exaggerated example of abnormal bone remodeling, with the primary cellular abnormality in the osteoclast. Mutations in the p62 (sequestosome 1) gene occur in one-third of patients with familial Paget disease and in a minority of patients with sporadic Paget disease, with the P392L amino acid substitution being the most commonly observed mutation. However, it is unknown how p62(P392L) mutation contributes to the development of this disease. To determine the effects of p62(P392L) expression on osteoclasts in vitro and in vivo, we introduced either the p62(P392L) or WT p62 gene into normal osteoclast precursors and targeted p62(P392L) expression to the osteoclast lineage in transgenic mice. p62(P392L)-transduced osteoclast precursors were hyperresponsive to receptor activator of NF-kappaB ligand (RANKL) and TNF-alpha and showed increased NF-kappaB signaling but did not demonstrate increased 1,25-(OH)(2)D(3) responsivity, TAF(II)-17 expression, or nuclear number per osteoclast. Mice expressing p62(P392L) developed increased osteoclast numbers and progressive bone loss, but osteoblast numbers were not coordinately increased, as is seen in Paget disease. These results indicate that p62(P392L) expression on osteoclasts is not sufficient to induce the full pagetic phenotype but suggest that p62 mutations cause a predisposition to the development of Paget disease by increasing the sensitivity of osteoclast precursors to osteoclastogenic cytokines.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Eosinophil chemotactic factor-L (ECF-L) enhances osteoclast formation by increasing in osteoclast precursors expression of LFA-1 and ICAM-1.

ECF-L is a novel autocrine stimulator of osteoclast (OCL) formation that enhances the effects of 1,25-(OH)2D3 and RANK ligand (RANKL) and is increased in inflammatory conditions such as rheumatoid arthritis. ECF-L acts at the later stages of OCL formation and does not increase RANKL expression. Thus, its mechanism of action is unclear. Therefore, RAW 264.7 cells and M-CSF-dependent murine bone marrow macrophage (MDBM) cells were treated with RANKL and/or with recombinant ECF-L expressed as a Fc fusion protein (ECF-L-Fc) to determine their effects on NF-kappaB, AP-1 and JNK activity, and on the expression of the adhesion molecules that have been implicated in OCL formation. These parameters were measured by semiquantitative and PCR and Western blot analysis. In addition, the role of ICAM-1 was further assessed by treating normal mouse marrow cultures with ECF-L-Fc and 10(-10) M 1,25-(OH)2D3 in the presence or absence of a blocking ICAM-1 antibody or treating marrow cultures from ICAM-1 knockout mice with ECF-L and 1,25-(OH)2D3. ECF-L-Fc by itself only modestly increased NF-kappaB binding and JNK activity in RAW 264.7 cells, which was further enhanced by RANKL. In contrast, ECF-L-Fc increased LFA-1alpha and ICAM-1 mRNA levels 1.8-fold in mouse marrow cultures, and anti-ICAM-1 almost completely inhibited OCL formation induced by 10(-10) M 1,25-(OH)2D3 and ECF-L. Furthermore, ECF-L did not increase OCL formation in marrow cultures from ICAM-1 knockout mice. Taken together, these results demonstrate that ECF-L enhances RANKL and 1,25-(OH)2D3-induced OCL formation by increasing adhesive interactions between OCL precursors through increased expression of ICAM-1 and LFA-1.

Animals↗

PubMed2006

Cloning and characterization of the annexin II receptor on human marrow stromal cells.

Annexin II is a heterotetramer, consisting of two 11-kDa (p11) and two 36-kDa (p36) subunits, that is produced by osteoclasts and stimulates osteoclast formation. However, its receptor is unknown. We showed that annexin II binds to normal primary human marrow stromal cells and the Paget's marrow-derived PSV10 stromal cell line to induce osteoclast formation. 125I-Labeled annexin II binding assays with PSV10 cells demonstrated that there was a single class of annexin II receptors with a Kd of 5.79 nm and Bmax of 2.13 x 10(5) receptors/cell. Annexin III or annexin V did not bind this receptor. Using 125I-labeled annexin II binding to screen NIH3T3 transfected with a human marrow cDNA expression library, we identified a putative annexin II receptor clone, which encoded a novel 26-kDa type I membrane receptor protein when expressed in HEK 293 cells. HEK 293 cells transformed with the cloned annexin II receptor cDNA showed a similar binding affinity to annexin II as that observed in PSV10 cells. Chemical cross-linking experiments with biotinylated annexin II and intact PSV10 cells identified a 55-kDa band on Western blot analysis that reacted with both an anti-p11 antibody and streptavidin but not anti-p36 antibody. A rabbit polyclonal antibody raised against the putative recombinant annexin II receptor also recognized the same 26-kDa protein band detected in PSV10 cells. Importantly, the annexin II receptor antibody dose-dependently blocked the stimulatory effects of annexin II on human osteoclast formation, demonstrating that the receptor mediates the effects of annexin II on osteoclast formation.

Animals↗

PubMed2006

Normalizing the bone marrow microenvironment with p38 inhibitor reduces multiple myeloma cell proliferation and adhesion and suppresses osteoclast formation.

The multiple myeloma (MM) bone marrow (BM) microenvironment plays a critical role in supporting tumor growth and survival as well as in promoting formation of osteolytic lesions. Recent results suggest that the p38 mitogen-activated protein kinase (MAPK) is an important factor in maintaining this activated environment. In this report, we demonstrate that the p38alpha MAPK inhibitor, SCIO-469, suppresses secretion of the tumor-supportive factors IL-6 and VEGF from BM stromal cells (BMSCs) as well as cocultures of BMSCs with MM cells, resulting in reduction in MM cell proliferation. Additionally, we show that SCIO-469 prevents TNFalpha-induced adhesion of MM cells to BMSCs through an ICAM-1- and VCAM-1-independent mechanism. Microarray analysis revealed a novel set of TNFalpha-induced chemokines in BMSCs that is strongly inhibited by SCIO-469. Furthermore, reintroduction of chemokines CXCL10 and CCL8 to BMSCs overcomes the inhibitory effect of SCIO-469 on TNFalpha-induced MM adhesion. Lastly, we show that SCIO-469 inhibits secretion and expression of the osteoclast-activating factors IL-11, RANKL, and MIP-1alpha as well as prevents human osteoclast formation in vitro. Collectively, these results suggest that SCIO-469 treatment can suppress factors in the bone marrow microenvironment to inhibit MM cell proliferation and adhesion and also to alleviate osteolytic activation in MM.

Animals↗

PubMed2006

Eosinophil chemotactic factor-L (ECF-L) enhances osteoclast formation by increasing ICAM-1 expression.

Eosinophil chemotactic factor-L (ECF-L) is a novel stimulator of osteoclast (OCL) formation that acts at the differentiation/fusion stage of OCL formation, and is a cofactor for RANK ligand (RANKL). We examined the effects of ECF-L on the intracellular signaling pathways utilized by RANKL, and on the expression of ICAM-1/LFA-1 to determine its mechanism of action. RAW 264.7 and bone marrow cells were treated with RANKL and/or ECF-L Fc protein to determine their effect on NF-kappaB and AP-1 activity. ECF-L by itself only modestly increased NF-kappaB binding and JNK activity in RAW 264.7 cells, which were further enhanced by RANKL. In contrast, ECF-L Fc increased LFA-1alpha and ICAM-1 mRNA levels 1.8-fold in mouse marrow cultures, and anti-ICAM-1 almost completely inhibited OCL formation induced by 10(-10) M 1,25-(OH)2D3, and ECF-L Fc. Furthermore, ECF-L Fc did not enhance OCL formation by ICAM-1 knockout (KO) cells. Increased expression of ICAM-1 by ECF-L appears to be critical for its effects on OCL formation.

Animals↗

PubMed2006

Alpha9beta1: a novel osteoclast integrin that regulates osteoclast formation and function.

UNLABELLED: We identified a previously unknown integrin, alpha(9)beta(1), on OCLs and their precursors. Antibody to alpha(9) inhibited OCL formation in human marrow cultures, and OCLs from alpha(9) knockout mice had a defect in actin ring reorganization and an impaired bone resorption capacity. INTRODUCTION: Integrins play important roles in osteoclast (OCL) formation and function. Mature OCLs mainly express alpha(v)beta(3) integrin, a heterodimer adhesion receptor that has been implicated in osteoclastic bone resorption. We identified ADAM8, a disintegrin and metalloproteinase, as a novel stimulator of OCL differentiation and showed that the disintegrin domain of ADAM8 mediated its effects on OCL formation. Because the disintegrin domain of ADAM8 does not bind Arg-Gly-Asp (RGD) sequences, we determined which integrin bound ADAM8 and characterized its role in OCL formation and activity. MATERIALS AND METHODS: Chinese hamster ovary cells (CHO) expressing different integrin subunits were tested for their capacity to bind the disintegrin domain of ADAM8. Mouse or human bone marrow cells and purified OCL precursors were tested for alpha(9)beta(1) integrin expression by Western blot, immunocytochemistry, and real-time RT-PCR. A monoclonal antibody to human alpha(9) was used to block alpha(9)beta(1) on OCL precursors stimulated by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] or RANKL. Vertebrae of 7-day-old alpha(9)(-/-) mice and wildtype (WT) littermates were compared using bone histomorphometry and 3D microCT analysis. RESULTS: Alpha(9) integrin was expressed by mouse and human bone marrow-derived OCLs and their precursors. Importantly, the anti-alpha(9) antibody inhibited human OCL formation stimulated by 1alpha,25(OH)(2)D(3) or RANKL dose-dependently. Furthermore, analysis of OCLs formed in marrow cultures from alpha(9)(-/-) mice showed that the OCLs formed were more contracted and formed significantly less bone resorption pits on dentin slices. Histologic analysis of alpha(9)(-/-) vertebrae showed thickened trabecular regions and retained cartilage within vertebral bodies of alpha(9)(-/-) mice. 3D microCT analysis of alpha(9)(-/-) vertebrae also showed a significant increase in trabecular bone volume/total tissue volume and a tendency for decreased trabecular separation compared with WT mice. CONCLUSIONS: These results support a previously unknown role for alpha(9)beta(1) integrin in OCL formation and function.

ADAM Proteins↗

PubMed2006

Experimental models of Paget's disease.

UNLABELLED: We targeted the MVNP gene to the OCL lineage in transgenic mice. These mice developed abnormal OCLs and bone lesions similar to those found in Paget's patients. These results show that persistent expression of MVNP in OCLs can induce pagetic-like bone lesions in vivo. INTRODUCTION: Paget's disease (PD) is one of the most exaggerated examples of abnormal bone remodeling, with increased bone resorption and excessive new bone formation. However, its etiology is unclear. A viral etiology for PD has been suggested based on the presence of paramyxoviral-like nuclear inclusions, detection of measles virus nucleocapsid (MVNP) mRNA or protein in osteoclasts (OCLs) from PD lesions, and in vitro studies showing that transfection of normal OCL precursors with the MVNP gene results in formation of OCLs that express a pagetic phenotype (increased numbers of OCLs; increased responsivity to 1,25(OH)(2)D(3), RANKL, and TNF-alpha; increased expression of the TAF(II)-17 gene, and increased bone resorption capacity). MATERIALS AND METHODS: We targeted MVNP to cells in the OCL lineage in transgenic mice using the TRACP promoter. RESULTS: Histomorphometric analysis showed that there was a 64% increase in OCL perimeter (p = 6.0002) and 37% increase in osteoblast (OBL) perimeter in MVNP mice. In a mouse that was 14 months of age, there was a 225% increase in OBL perimeter and 149% in OBL perimeter. This was accompanied by increased cancellous bone volume (83%) and trabecular width (47%) and number (25%), with a marked increase in the amount of woven bone. In contrast, cancellous bone volume decreased between 3 and 12 months in wildtype (WT) mice, whereas cancellous bone volume in MVNP mice increased over the same time period. Ex vivo studies showed that the numbers of OCLs formed in marrow cultures from MVNP mice were increased, and the OCLs were hyper-responsive to 1,25(OH)(2)D(3) and had an increased bone resorbing capacity compared with WT cultures. CONCLUSION: These results show that expression of MVNP in OCL in vivo results in a bone phenotype that is characteristic of PD.

Acid Phosphatase↗

PubMed2005

Thalidomide derivative CC-4047 inhibits osteoclast formation by down-regulation of PU.1.

CC-4047, an immunomodulatory analog of thalidomide, inhibits multiple myeloma with unknown effects on the human osteoclast lineage. Early osteoclast progenitors are of hematopoietic origin and differentiate into mature bone resorbing multinucleated osteoclasts. We investigated the effects of CC-4047 and thalidomide on human osteoclastogenesis, using in vitro receptor activator of NFkappa-B ligand/macrophage colony-stimulating factor-stimulated bone marrow cell cultures. Treating bone marrow cultures with CC-4047 for 3 weeks decreased osteoclast formation accompanied by complete inhibition of bone resorption. The inhibitory effect was similar when cultures were treated for 3 weeks or for only the first week (90% inhibition), indicating that CC-4047 inhibits early stages of osteoclast formation. Inhibition of osteoclastogenesis by CC-4047 was mediated by a shift of lineage commitment to granulocyte colony-forming units at the expense of granulocyte-macrophage colony-forming units. Further studies revealed that this shift in lineage commitment was mediated through down-regulation of PU.1. Treatment with thalidomide resulted in significantly less potent inhibition of osteoclast formation and bone resorption. These results provide evidence that CC-4047 blocks osteoclast differentiation during early phases of osteoclastogenesis. Therefore, CC-4047 might be a valuable drug for targeting both tumors and osteoclastic activity in patients with multiple myeloma and other diseases associated with osteolytic lesions.

Angiogenesis Inhibitors↗

PubMed2005

Expression of measles virus nucleocapsid protein in osteoclasts induces Paget's disease-like bone lesions in mice.

UNLABELLED: We targeted the MVNP gene to the OCL lineage in transgenic mice. These mice developed abnormal OCLs and bone lesions similar to those found in Paget's patients. These results show that persistent expression of MVNP in OCLs can induce pagetic-like bone lesions in vivo. INTRODUCTION: Paget's disease (PD) of bone is the second most common bone disease. Both genetic and viral factors have been implicated in its pathogenesis, but their exact roles in vivo are unclear. We previously reported that transfection of normal human osteoclast (OCL) precursors with the measles virus nucleocapsid (MVNP) or measles virus (MV) infection of bone marrow cells from transgenic mice expressing a MV receptor results in formation of pagetic-like OCLs. MATERIALS AND METHODS: Based on these in vitro studies, we determined if the MVNP gene from either an Edmonston-related strain of MV or a MVNP gene sequence derived from a patient with PD (P-MVNP), when targeted to cells in the OCL lineage of transgenic mice with the TRACP promoter (TRACP/MVNP mice), induced changes in bone similar to those found in PD. RESULTS: Bone marrow culture studies and histomorphometric analysis of bones from these mice showed that their OCLs displayed many of the features of pagetic OCLs and that they developed bone lesions that were similar to those in patients with PD. Furthermore, IL-6 seemed to be required for the development of the pagetic phenotype in OCLs from TRACP/MVNP mice. CONCLUSIONS: These results show that persistent expression of the MVNP gene in cells of the OCL lineage can induce pagetic-like bone lesions in vivo.

Animals↗

PubMed2005

Annexin II stimulates RANKL expression through MAPK.

UNLABELLED: We report that AX-II, in addition to inducing GM-CSF expression, also increases membrane-bound RANKL synthesis by marrow stromal cells and does so through a previously unreported MAPK-dependent pathway. Thus, both GM-CSF and RANKL are required for AX-II stimulation of OCL formation. INTRODUCTION: Annexin II (AX-II) is an autocrine/paracrine factor secreted by osteoclasts (OCLs) that stimulates human OCL formation and bone resorption in vitro by inducing bone marrow stromal cells and activated CD4+ T cells to produce granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF in turn increases OCL precursor proliferation and further enhances OCL formation. However, the induction of GM-CSF by AX-II cannot fully explain its effects on OCL formation. In this study, we tested the capacity of AX-II to induce the expression of RANKL and the corresponding signaling pathways AX-II employs in human marrow stromal cells to induce RANKL. We also showed that both GM-CSF and RANKL are required for OCL formation induced by AX-II. MATERIALS AND METHODS: Real-time RT-PCR and Western blot analysis were used to detect RANKL and osteoprotegerin (OPG) mRNA and protein expression in unfractionated human bone marrow mononuclear cells stimulated with AX-II. Soluble RANKL in the conditioned medium was analyzed by ELISA. Activation of the MAPK pathway by AX-II was tested by Western blot. The effects of OPG and anti-GM-CSF on AX-II-induced OCL formation were also examined. RESULTS AND CONCLUSION: In addition to upregulating GM-CSF mRNA, AX-II increased RANKL mRNA expression dose-dependently in unfractionated human bone marrow mononuclear cells and modestly increased soluble RANKL in unfractionated human bone marrow mononuclear cell conditioned medium. However, AX-II markedly increased membrane-bound RANKL on human bone marrow stromal cells. Treatment of marrow stromal cells with AX-II activated MAP-kinase (ERKs) and PD 98059 abolished the effect but did not block the increase in GM-CSF. Interestingly, OPG, a natural decoy receptor for RANKL, or anti-GM-CSF partially inhibited OCL formation by AX-II in human bone marrow cells, and the combination of OPG and anti-GM-CSF completely blocked AX-II-induced OCL formation. These data show that AX-II stimulates both the proliferation and differentiation of OCL precursors through production of GM-CSF and RANKL respectively.

Annexin A2↗

PubMed2004

(23S)-25-Dehydro-1{alpha}-hydroxyvitamin D3-26,23-lactone, a vitamin D receptor antagonist that inhibits osteoclast formation and bone resorption in bone marrow cultures from patients with Paget's disease.

Osteoclast (OCL) precursors from patients with Paget's disease (PD) and normal OCL precursors transduced with the measles virus nucleocapsid protein gene (MVNP) are hyperresponsive to 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)] and can form OCLs at physiologic concentrations of 1alpha,25-(OH)(2)D(3). This hyperresponsivity to 1alpha,25-(OH)(2)D(3) is due to increased expression of TATA box-associated factor II-17, a potential coactivator of the vitamin D receptor. Hyperresponsivity to 1alpha,25-(OH)(2)D(3) may permit OCL formation in PD patients with low levels of 1alpha,25-(OH)(2)D(3) and play a role in the pathogenesis of PD. Therefore, we tested the effects of a vitamin D antagonist, (23S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647), to determine its potential to inhibit the enhanced OCL formation and bone resorption seen in patients with PD. TEI-9647, by itself, was not a vitamin D receptor agonist and did not induce OCL formation in vitro, even at 10(-6) m. However, it dose-dependently (10(-10) m to 10(-6) m) inhibited osteoclast formation induced by concentrations of 1alpha,25-(OH)(2)D(3) (41 pg/ml, 10(-10) m) detected in PD patients by bone marrow cells of patients with PD and MVNP-transduced colony-forming unit-granulocyte macrophage (CFU-GM) cells, which form pagetic-like OCL. Moreover, bone resorption by OCLs derived from MVNP-transduced CFU-GM treated with 10(-9) m 1alpha,25-(OH)(2)D(3) was dose-dependently inhibited by TEI-9647 (10(-9) m to 10(-6) m). Furthermore, 10(-7) m TEI-9647 by itself did not cause 1alpha,25-(OH)(2)D(3)-dependent gene expression but almost completely suppressed expression of the TATA box-associated factor II-17 and 25-hydroxyvitamin D(3)-24-hydroxylase genes induced by 1alpha,25-(OH)(2)D(3) treatment of MVNP-transduced CFU-GM cells. These results demonstrate that TEI-9647 can suppress the excessive bone resorption and OCL formation seen in marrow cultures from patients with PD.

Bone Marrow Cells↗

PubMed2004

Role of TAFII-17, a VDR binding protein, in the increased osteoclast formation in Paget's Disease.

UNLABELLED: In contrast to normal OCL precursors, pagetic OCL precursors express MVNP and form OCL at physiologic concentrations of 1,25(OH)2D3, as do normal OCL precursors transfected with the MVNP gene. Using a GST-VDR chimeric protein, we identified TAFII-17 as VDR binding protein expressed by pagetic OCL precursors and MVNP transduced normal OCL precursors. TAF(II)-17 was in part responsible for the increased 1,25(OH)2D3 responsivity of pagetic OCL precursors. INTRODUCTION: Pagetic osteoclasts (OCLs) and their precursors express measles virus nucleocapsid protein (MVNP) and form large numbers of OCLs at low concentrations of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Similarly, normal OCL precursors transfected with MVNP also form OCLs at low concentrations of 1,25(OH)2D3. These results suggest that expression of MVNP in OCL precursors enhances vitamin D receptor (VDR)-mediated gene transcription. MATERIALS AND METHODS: To determine the mechanism for the increased OCL formation capacity of pagetic OCL precursors in response to 1,25(OH)2D3, lysates from pagetic and MVNP-transduced normal OCL precursors were incubated with a GST-VDR chimeric protein. RESULTS: A 17-kDa peptide that bound VDR was detected in MVNP-transduced cells and pagetic OCL precursors treated with 1,25(OH)2D3. This peptide was identified as TAFII-17, a component of the TFIID transcription complex. Expression of increased levels of TAFII-17 in cells allowed TAFII-17 to bind to VDR at low concentrations of 1,25(OH)2D3. An antisense oligonucelotide (AS-ODN) to TAFII-17 significantly decreased OCL formation in response to 1,25(OH)2D3 in pagetic but not normal marrow cultures by approximately 40%. Transfection of TAFII-17 or MVNP into NIH3T3 cells increased VDR transcriptional activity as measured by DR-3 reporter assays. CONCLUSION: These data show that expression of the MVNP gene in OCL precursors results in increased levels of TAFII-17. TAFII-17 can bind VDR at low concentrations of 1,25(OH)2D3. These results suggest that MVNP expression in Paget's OCL precursors increases expression of a component(s) of the VDR transcription complex that can increase OCL formation.

Bone Marrow Cells↗

PubMed2004

Paget's disease-a VDR coactivator disease?

Paget's disease is the most exaggerated example of bone remodeling with increased osteoclastic bone resorption followed by excessive bone formation. One of the earliest findings in our studies of Paget's disease is that pagetic osteoclast (OCL) precursors are hyper-responsive to 1,25-(OH)(2)D(3) and form OCL at concentrations of 1,25-(OH)(2)D(3) that are physiologic rather than pharmacologic. The increased responsivity to 1,25-(OH)(2)D(3) is not due to increased levels of the Vitamin D receptor (VDR) or to increased infinity of 1,25-(OH)(2)D(3) for VDR. We have recently shown using GST-VDR chimeric protein pull-down assays that TAF(II)-17, a member of the TAF(II)-D transcription complex, is increased in OCL precursors from patients with Paget's disease compared to normals. We further showed that TAF(II)-17 can enhance VDR mediated gene transcription and allow formation of the transcription complex at very low levels of 1,25-(OH)(2)D(3). In addition, coactivators of VDR including CPB300 and DRIP205 are also increased in OCL precursors from Paget's patients. These data suggest that the enhanced sensitivity of OCL precursors for 1,25-(OH)(2)D(3) in Paget's disease results from increased expression of coactivators of VDR and suggest that part of the pathophysiology underlying OCL formation in Paget's disease may result from enhanced expression of VDR coactivators.

Calcitriol↗

PubMed2004

Vitamin D antagonist, TEI-9647, inhibits osteoclast formation induced by 1alpha,25-dihydroxyvitamin D3 from pagetic bone marrow cells.

(23S)-25-Dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) functions an antagonist of the 1alpha,25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)) nuclear receptor (VDR)-mediated differentiation of human leukemia (HL-60) cells [J. Biol. Chem. 274 (1999) 16392]. We examined the effect of vitamin D antagonist, TEI-9647, on osteoclast formation induced by 1alpha,25-(OH)(2)D(3) from bone marrow cells of patients with Paget's disease. TEI-9647 itself never induced osteoclast formation even at 10(-6)M, but dose-dependently (10(-10) to 10(-6)M) inhibited osteoclast formation induced by physiologic concentrations of 1alpha,25-(OH)(2)D(3) (41 pg/ml, 10(-10)M) from bone marrow cells of patients with Paget's disease. At the same time, 10(-8)M of TEI-9647 alone did not cause 1alpha,25-(OH)(2)D(3) dependent gene expression, but almost completely suppressed TAF(II)-17, a potential coactivator of VDR and 25-hydroxyvitamin D(3)-24-hydroxylase (25-OH-D(3)-24-hydroxylase) gene expression induced by 10(-10)M 1alpha,25-(OH)(2)D(3) in bone marrow cells of patients with Paget's disease. Moreover, TEI-9647 dose-dependently inhibited bone resorption induced by 10(-9)M 1alpha,25-(OH)(2)D(3) by osteoclasts produced by RANKL and M-CSF treatment of measles virus nucleocapsid gene transduced bone marrow cells. These results suggest that TEI-9647 acts directly on osteoclast precursors and osteoclasts, and that TEI-9647 may be a novel agent to suppress the excessive bone resorption and osteoclast formation in patients with Paget's disease.

Base Sequence↗

PubMed2002

Identification of the functional domain of osteoclast inhibitory peptide-1/hSca.

Osteoclast (OCL) activity is controlled by local factors produced in the bone microenvironment. We previously identified a novel inhibitor of OCL formation that is produced by OCLs (osteoclast inhibitory peptide-1/human Sca [OIP-1/hSca]). OIP-1/hSca is a glycosylphosphatidylinositol (GPI)-linked membrane protein (16 kDa) that is cleaved from the OCL surface. Immunocytochemical staining further confirmed the expression of OIP-1/hSca in OCL formed in mouse bone marrow cultures. However, the structure/function mechanisms responsible for the inhibitory effects of OIP-1/hSca on OCL formation are unknown. Therefore, we expressed deletion mutants of OIP-1 in 293 cells and tested their effects on OCL formation. These studies indicated that the carboxy-terminal peptide (c-peptide) region is critical for OIP-1/hSca activity. A 33 amino acid OIP-1 c-peptide (10-100 ng/ml) significantly inhibited 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced OCL formation and pit formation capacity of OCL on dentine slices in human bone marrow cultures. Furthermore, the c-peptide (10-100 ng/ml) significantly inhibited early human OCL precursor (granulocyte-macrophage colony-forming unit [GM-CFU]) colony formation in methylcellulose cultures. The polyclonal antibody against the OIP-1 c-peptide neutralized the inhibitory effect of OIP-1 c-peptide on OCL formation in mouse bone marrow cultures in vitro. These results show that the OIP-1 c-peptide is the functional domain of OIP-1 and that availability of neutralizing antibody specific to the OIP-1 c-peptide should provide important mechanistic insights into OIP-1/hSca inhibition of osteoclastogenesis in the bone microenvironment.

ATPases Associated with Diverse Cellular Activitie↗