ADAM8 enhances osteoclast precursor fusion and osteoclast formation in vitro and in vivo.

ADAM8 expression is increased in the interface tissue around a loosened hip prosthesis and in the pannus and synovium of patients with rheumatoid arthritis, but its potential role in these processes is unclear. ADAM8 stimulates osteoclast (OCL) formation, but the effects of overexpression or loss of expression of ADAM8 in vivo and the mechanisms responsible for the effects of ADAM8 on osteoclastogenesis are unknown. Therefore, to determine the effects of modulating ADAM expression, we generated tartrate-resistant acid phosphatase (TRAP)-ADAM8 transgenic mice that overexpress ADAM8 in the OCL lineage and ADAM8 knockout (ADAM8 KO) mice. TRAP-ADAM8 mice developed osteopenia and had increased numbers of OCL precursors that formed hypermultinucleated OCLs with an increased bone-resorbing capacity per OCL. They also had an enhanced differentiation capacity, increased TRAF6 expression, and increased NF-κB, Erk, and Akt signaling compared with wild-type (WT) littermates. This increased bone-resorbing capacity per OCL was associated with increased levels of p-Pyk2 and p-Src activation. In contrast, ADAM8 KO mice did not display a bone phenotype in vivo, but unlike WT littermates, they did not increase RANKL production, OCL formation, or calvarial fibrosis in response to tumor necrosis factor α (TNF-α) in vivo. Since loss of ADAM8 does not inhibit basal bone remodeling but only blocks the enhanced OCL formation in response to TNF-α, these results suggest that ADAM8 may be an attractive therapeutic target for preventing bone destruction associated with inflammatory disease.

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.

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.

Death receptor-3 mediates apoptosis in human osteoblasts under narrowly regulated conditions.

We previously reported that a soluble form of the TNF-family receptor death receptor-3 (DR3) is expressed in osteoblasts. DR3 regulates death or differentiation in other tissues, and DR3 ligands occur in bone, but the function of DR3 in the osteoblast was unknown. We studied the expression of DR3 and the effects crosslinking antibodies to DR3 or of natural DR3 ligands in human osteoblasts. Western analysis showed that nontransformed osteoblasts and the MG63 osteosarcoma cell line produce both soluble decoy receptor and transmembrane isoforms of DR3. Cell surface labeling showed that low and high DR3-expressing osteoblast populations occur. Verification of by cloning showed a point mutation in DR3 from MG63 cells. Activation of DR3 by antibody crosslinking or with DR3 ligands caused apoptosis in osteoblasts and in MG63 cells, but only in low-density cell cultures. In dense cultures apoptosis did not occur, but nuclear factor-kappaB nuclear translocation was observed under some conditions. Crosslinking of DR3 in high-density MG63 cultures blocked expression of bone matrix elements. DR3 activation in high-density nontransformed osteoblasts had only minor effects on cell maturation. We conclude that DR3 activation can mediate apoptosis in osteoblasts. Its activity is, however, highly restricted by its soluble ligand-binding isoform and possibly also by alternate survival signals. In the presence of survival signals, DR3 may affect cell maturation although effects on differentiation were clearly seen only in the MG63 transformed cell line.

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.

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.

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.

NO-dependent osteoclast motility: reliance on cGMP-dependent protein kinase I and VASP.

The osteoclast degrades bone in cycles; between cycles, the cell is motile. Resorption occurs by acid transport into an extracellular compartment defined by an alphavbeta3 integrin ring. NO has been implicated in the regulation of bone turnover due to stretch or via estrogen signals, but a specific mechanism linking NO to osteoclastic activity has not been described. NO stimulates osteoclast motility, and at high concentrations NO causes detachment and terminates resorption. Here we demonstrate that NO regulates attachment through the cGMP-dependent protein kinase I (PKG I) via phosphorylation of the intermediate protein VASP. VASP colocalized with the alphavbeta3 ring in stationary cells, but alternating bands of VASP and alphavbeta3 occurred when motility was induced by NO donors or cGMP. Redistribution of VASP correlated with its phosphorylation. Dependency of NO-induced motility on PKG I and on VASP was shown by siRNA knockdown of each protein. VASP knockdown also altered distribution of alphavbeta3 at the attachment site. We conclude that PKG I and VASP are essential for reorganization of attachment and cytoplasmic proteins in motility induced by NO or by cGMP.

Negative regulation of RANKL-induced osteoclastic differentiation in RAW264.7 Cells by estrogen and phytoestrogens.

We studied estrogen effects on osteoclastic differentiation using RAW264.7, a murine monocytic cell line. Differentiation, in response to RANKL and colony-stimulating factor 1, was evaluated while varying estrogen receptor (ER) stimulation by estradiol or nonsteroidal ER agonists was performed. The RAW264.7 cells were found to express ERalpha but not ERbeta. In contrast to RANKL, which decreased ERalpha expression and induced osteoclast differentiation, 10 nm estradiol, 3 microm genistein, or 3 microm daidzein all increased ERalpha expression, stimulated cell proliferation, and decreased multinucleation, with the effects of estrogen > or = daidzein > genistein. However, no estrogen agonist reduced RANKL stimulation of osteoclast differentiation markers or its down-regulation of ERalpha expression by more than approximately 50%. Genistein is also an Src kinase antagonist in vitro, but it did not decrease Src phosphorylation in RAW264.7 cells relative to other estrogen agonists. However, both phytoestrogens and estrogen inhibited RANKL-induced IkappaB degradation and NF-kappaB nuclear localization with the same relative potency as seen in proliferation and differentiation assays. This study demonstrates, for the first time, the direct effects of estrogen on osteoclast precursor differentiation and shows that, in addition to effecting osteoblasts, estrogen may protect bone by reducing osteoclast production. Genistein, which activates ERs selectively, inhibited osteoclastogenesis less effectively than the nonselective phytoestrogen daidzein, which effectively reproduced effects of estrogen.

In vitro differentiation of CD14 cells from osteopetrotic subjects: contrasting phenotypes with TCIRG1, CLCN7, and attachment defects.

UNLABELLED: We studied osteoclastic differentiation from normal and osteopetrotic human CD14 cells in vitro. Defects in acid transport, organic matrix removal, and cell fusion with deficient attachment were found. Analysis of genotypes showed that TCIRG1 anomalies correlated with acid transport defects, but surprisingly, organic matrix removal failure correlated with CLCN7 defects; an attachment defect had normal TCIRG1 and CLCN7. INTRODUCTION: Osteopetrotic subjects usually have normal macrophage activity, and despite identification of genetic defects associated with osteopetrosis, the specific developmental and biochemical defects in most cases are unclear. Indeed, patients with identical genotypes often have different clinical courses. We classified defects in osteoclast differentiation in vitro using four osteopetrotic subjects without immune or platelet defects, three of them severe infantile cases, compared with normals. MATERIALS AND METHODS: Osteoclast differentiation used isolated CD14 cells; results were correlated with independent analysis of two key genes, CLCN7 and TCIRG1. CD14 cell attachment and cell surface markers and extent of differentiation in RANKL and colony-stimulating factor (CSF)-1 were studied using acid secretion, bone pitting, enzyme, and attachment proteins assays. RESULTS AND CONCLUSIONS: CD14 cells from all subjects had similar lysosomal and nonspecific esterase activity. With the exception of cells from one osteopetrotic subject, CD14 cells from osteopetrotic and control monocytes attached similarly to bone or tissue culture substrate. Cells from one osteopetrotic subject, with normal CLCN7 and TCIRG1, did not attach to bone, did not multinucleate, and formed no podosomes or actin rings in RANKL and CSF-1. Attachment defects are described in osteopetrosis, most commonly mild osteopetrosis with Glantzman's thrombasthenia. However, this case, with abnormal integrin alphavbeta3 aggregates and no osteoclasts, seems to be unique. Two subjects were compound heterozygotes for TCIRG1 defects; both had CD14 cells that attached to bone but did not acidify attachments; cell fusion and attachment occurred, however, in RANKL and CSF-1. This is consistent with TCIRG1, essential for H+-ATPase assembly at the ruffled border. A compound heterozygote for CLCN7 defects had CD14 cells that fused in vitro, attached to bone, and secreted acid, TRACP, and cathepsin K. However, lacunae were shallow and retained demineralized matrix. This suggests that CLCN7 may not limit H+-ATPase activity as hypothesized, but may be involved in control of organic matrix degradation or removal.

Efectos de la administración prenatal del diazepam en estructuras craneofaciales de ratones Balb/C / Effects of prenatal administration of diazepam on craniofacial structures of Balb/C mice

Una dosis oral de 3 mg/kg diaria de diazepam (DZ) fue administrada a ratones Balb/C durante su gestación. Esta dosis causó una dependencia y sedación hasta de 4 horas. Las crías de estos ratones fueron sacrificadas en los días 1, 3, 7, 14, 21 y 28 postnatales y fueron examinados de anormalidades físicas. Los cráneos y las mandíbulas fueron disecados y pesados para analizarlas estadísticamente. Los ratones del grupo tratado con DZ mostraron un peso menor durante el período de gestación, en comparación con el grupo control. El peso de las mandíbulas y cráneos del grupo DZ también mostró un peso menor en los días 14, 21 y 28 postnatales. La aparición de un tumor en el cráneo fue un hallazgo clínico de esta investigación