Sakamuri V. Reddy

Intracellular Fragmentation of Bone Resorption Products by Reactive Oxygen Species Generated by Osteoclastic Tartrate-resistant Acid Phosphatase

Tartrate-resistant acid phosphatase (TRAP) is highly expressed in bone-resorbing osteoclasts and activated macrophages. It has been suggested that a redox-active iron in the binuclear iron center of TRAP could have the capacity to react with hydrogen peroxide to produce highly destructive reactive oxygen species (ROS). Here we show that TRAP can generate ROS in vitro and that cells over-expressing TRAP produce higher amounts of intracellular ROS than their parent cells. We further demonstrate that these ROS can be targeted to destroy collagen and other proteins. In resorbing osteoclasts, TRAP was found in transcytotic vesicles transporting matrix degradation products through the cell, suggesting that TRAP-facilitated fragmentation of endocytosed material takes place in a specific cellular compartment. These results suggest that bone matrix degradation occurs not only extracellularly in the resorption lacunae but also intracellularly in the transcytotic vesicles. We propose that proteins containing redox-active iron could represent a novel mechanism of physiological fragmentation of organic molecules. This mechanism could be important in tissue remodeling and as a defense mechanism of phagocytosing cells.

Enhanced RANK ligand expression and responsivity of bone marrow cells in Paget’s disease of bone

Pagets disease is characterized by highly localized areas of increased osteoclast (OCL) activity. This suggests that the microenvironment in pagetic lesions is highly osteoclastogenic, or that OCL precursors in these lesions are hyperresponsive to osteoclastogenic factors (or both). To examine these possibilities, we compared RANK ligand (RANKL) mRNA expression in a marrow stromal cell line developed from a pagetic lesion (PSV10) with that in a normal stromal cell line (Saka), and expression in marrow samples from affected bones of Pagets patients with that in normal marrow. RANKL mRNA was increased in PSV10 cells and pagetic marrow compared with Saka cells and normal marrow, and was also increased in marrow from affected bones compared with uninvolved bones from Pagets patients. Furthermore, pagetic marrow cells formed OCLs at much lower RANKL concentrations than did normal marrow. Anti-IL-6 decreased the RANKL responsivity of pagetic marrow to normal levels, whereas addition of IL-6 to normal marrow enhanced RANKL responsivity. Thus, RANKL expression and responsivity is increased in pagetic lesions, in part mediated by IL-6. These data suggest that the combination of enhanced expression of RANKL in affected bones and increased RANKL sensitivity of pagetic OCL precursors may contribute to the elevated numbers of OCLs in Pagets disease.

Osteoclasts expressing the measles virus nucleocapsid gene display a pagetic phenotype

Osteoclasts (OCLs) in Pagets disease are markedly increased in number and size, have increased numbers of nuclei per multinucleated cell, and demonstrate increased resorption capacity and increased sensitivity to 1,25-(OH)(2)D(3), the active form of vitamin D. These cells also contain nuclear inclusions, reminiscent of those seen in paramyxovirus-infected cells, which cross-react with antibodies to measles virus nucleocapsid (MVNP) antigen. To elucidate the role of MV in the abnormal OCL phenotype of Pagets disease, we transduced normal OCL precursors with retroviral vectors expressing MVNP and the MV matrix (MVM) genes. The transduced cells were then cultured with 1,25-(OH)(2)D(3) for14 or 21 days to induce formation of OCL-like multinucleated cells. The MVNP-transduced cells formed increased numbers of multinucleated cells, which contained many more nuclei and had increased resorption capacity compared with multinucleated cells derived from empty vector-transduced (EV-transduced) and MVM-transduced or normal bone marrow cells. Furthermore, MVNP-transduced cells showed increased sensitivity to 1, 25-(OH)(2)D(3), and formed OCLs at concentrations of 1, 25-(OH)(2)D(3) that were 1 log lower than that required for normal, EV-transduced, or MVM-transduced cells. These results demonstrate that expression of the MVNP gene in normal OCL precursors stimulates OCL formation and induces OCLs that express a phenotype similar to that of pagetic OCLs. These results support a potential pathophysiologic role for MV infection in the abnormal OCL activity and morphology that are characteristic of pagetic OCLs.

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.

IL-6 mediates the effects of IL-1 or TNF, but not PTHrP or 1,25(OH)2D3, on osteoclast-like cell formation in normal human bone marrow cultures.

A potent interleukin‐6 (IL‐6) antagonist (Sant 5), which binds tightly to the IL‐6α receptor but has impaired gp130 heterodimerization, has been developed recently by site‐directed mutagenesis of human IL‐6. We report here that Sant 5 inhibits IL‐6‐stimulated osteoclast‐like multinucleated cell (MNC) formation in human marrow cultures but also inhibits the stimulatory effects of IL‐1 or tumor necrosis factor alpha (TNF‐α in these cultures. We further show that a neutralizing antibody to IL‐6 also inhibits the stimulatory effects of IL‐1 or TNF‐α in these cultures. In contrast, Sant 5 had no effect on parathyroid hormone related protein (PTHrP) or 1,25‐dihydroxyvitamin D3 stimulated MNC formation in human marrow cultures. Transfection of a human marrow stromal cell line, which normally induces osteoclast formation through production of IL‐6, with the Sant 5 cDNA driven by a cytomegalovirus (CMV) promoter blocked the capacity of these cells to stimulate osteoclast‐like cell formation. These Sant 5 transfected cells and conditioned media from these cells also inhibited the stimulatory effects of the parent cell line on MNC formation. These data suggest that IL‐6 mediates the effects of IL‐1 and TNF on human osteoclast formation, but in contrast to murine systems, does not mediate the effects of PTHrP. These data further demonstrate that stromal cells transfected with the Sant 5 cDNA can constitutively produce high levels of the IL‐6 antagonist and inhibit osteoclast formation in vitro.

Sequence analysis of measles virus nucleocapsid transcripts in patients with Paget's disease

It has been debated for almost 30 years whether Pagets disease of bone results from paramyxoviral infection of osteoclasts (OCs). Paramyxoviral‐like nuclear inclusions are found in OCs from patients with Pagets disease, and measles virus (MV) or canine distemper virus (CDV) messenger RNA (mRNA) transcripts have been detected by in situ hybridization in bone cells from pagetic lesions. Furthermore, immunocytochemical studies have shown the presence of several paramyxoviral species in OCs from patients with Pagets disease. However, others have been unable to detect paramyxoviral transcripts in bone samples from patients with Pagets disease or marrow cultures from involved sites of patients with Pagets disease. Furthermore, no one has been able to isolate an infectious virus from pagetic bone samples or marrow cells from patients with Pagets disease, and a full‐length viral gene has not been sequenced from pagetic samples. In this study, we have obtained the full‐length sequence for the MV nucleocapsid (MVNP) gene in bone marrow from an involved site from a patient with Pagets disease and more than 700 base pairs (bps) of MVNP sequence in 3 other patients with Pagets disease. These sequences were undetectable in four normal marrow samples studied simultaneously. The sequences from the patients contained multiple mutations that differed from the Edmonston strain MVNP gene. These findings are consistent with the presence of a chronic MV infection in affected sites from these patients with Pagets disease.

Annexin II increases osteoclast formation by stimulating the proliferation of osteoclast precursors in human marrow cultures

Annexin II (AXII), a calcium-dependent phospholipid-binding protein, has been recently found to be an osteoclast (OCL) stimulatory factor that is also secreted by OCLs. In vitro studies showed that AXII induced OCL formation and bone resorption. However, the mechanism of action by which AXII acts as a soluble extracellular protein to induce OCL formation is unknown. In this paper, we demonstrate that AXII gene expression is upregulated by 1,25-dihydroxyvitamin D3 [1, 25-(OH)2D3] and that addition of AXII significantly increased OCL-like multinucleated cell formation. Time-course studies suggested that AXII acted on the proliferative stage of OCL precursors and that AXII increased thymidine incorporation in OCL precursors. Moreover, AXII enhanced the growth of CFU-GM, the earliest identifiable OCL precursor, when bone marrow cultures were treated with low concentrations of GM-CSF. This capacity of AXII to induce OCL precursor proliferation was due to induction of GM-CSF expression, because the addition of neutralizing antibodies to GM-CSF blocked the stimulatory effect of AXII on OCL formation. RT-PCR analysis using RNA from highly purified subpopulations of marrow cells demonstrated that T cells, especially CD4(+) T cells, produced GM-CSF in response to AXII. Furthermore, FACS(R) analysis of T-cell subpopulations treated with fluorescein-labeled AXII suggested that the CD4(+), but not CD8(+), subpopulation of T cells express an AXII receptor. Taken together, these data suggest that AXII stimulates OCL formation by activating T cells through a putative receptor to secrete GM-CSF. GM-CSF then expands the OCL precursor pool to enhance OCL formation.

1,25-Dihydroxyvitamin D3 Hypersensitivity of Osteoclast Precursors from Patients with Paget's Disease

Our previous studies suggested that increased osteoclast formation and activity in Pagets disease may be related in part to increased responsiveness of highly purified osteoclast precursors to 1,25‐dihydroxyvitamin D3 [1,25‐(OH)2D3]. However, the basis for this enhanced sensitivity to 1,25‐(OH)2D3 is unclear. To address this question, we examined 24‐hydroxylase and 1,25‐(OH)2D3 receptor (VDR) messenger RNA (mRNA) expression during human osteoclast differentiation from normal subjects and patients with Pagets disease in response to 1,25‐(OH)2D3 as well as VDR content and affinity. Reverse‐transcription polymerase chain reaction (RT‐PCR) analysis of granulocyte–macrophage colony–forming unit (GM‐CFU), the earliest identifiable osteoclast precursor, derived from patients with Pagets disease demonstrated 24‐hydroxylase mRNA expression in response to 1,25‐(OH)2D3 was induced at concentrations of 1,25‐(OH)2D3 that were at least one log less than that required for normal GM‐CFU. VDR mRNA and VDR protein were detected in both immature and more differentiated osteoclast precursors, as well as in osteoclast‐like multinucleated cells (MNCs). However, VDR expression was lower in MNCs than the mononuclear precursor cells. Osteoclast precursors and MNCs from patients with Pagets disease had levels of VDR expression similar to those of normal subjects but showed increased VDR affinity for 1,25‐(OH)2D3. Because the effects of 1,25‐(OH)2D3 are in part mediated by induction of expression of RANK ligand on marrow stromal cells, which in turn stimulates osteoclast formation, we examined expression of RANK ligand mRNA by marrow stromal cell lines derived from patients with Pagets disease and normal subjects in response to 1,25‐(OH)2D3. RT‐PCR analysis showed no difference in sensitivity of marrow stromal cells to 1,25‐(OH)2D3 from normal subjects or patients with Pagets disease although the Pagets stromal cells expressed increased basal levels of RANK ligand mRNA. These results show that VDR protein is expressed in early and more differentiated osteoclast precursors, that expression levels of VDR decline with osteoclast differentiation, and that 1,25‐(OH)2D3 has direct effects on osteoclast precursors. The enhanced sensitivity to 1,25‐(OH)2D3 is an intrinsic property of osteoclast precursors from patients with Pagets disease that distinguishes them from normal osteoclast precursors. Furthermore, our results suggest that an increased affinity of VDR for 1,25‐(OH)2D3 may be responsible for the enhanced 1,25‐(OH)2D3 sensitivity of osteoclast precursors in patients with Pagets disease compared with normal subjects. (J Bone Miner Res 2000;15:228–236)

Expression of Measles Virus Nucleocapsid Protein in Osteoclasts Induces Paget's Disease‐Like Bone Lesions in Mice

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 Pagets patients. These results show that persistent expression of MVNP in OCLs can induce pagetic‐like bone lesions in vivo.

Identification of Human Asparaginyl Endopeptidase (Legumain) as an Inhibitor of Osteoclast Formation and Bone Resorption

We screened a human osteoclast (OCL) cDNA expression library for OCL inhibitory factors and identified a clone that blocked both human and murine OCL formation and bone resorption by more than 60%. This clone was identical to human legumain, a cysteine endopeptidase. Legumain significantly inhibited OCL-like multinucleated cell formation induced by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and parathyroid hormone-related protein (PTHrP) in mouse and human bone marrow cultures, and bone resorption in the fetal rat long bone assay in a dose-dependent manner. Legumain was detected in freshly isolated marrow plasma from normal donors and conditioned media from human marrow cultures. Furthermore, treatment of human marrow cultures with an antibody to legumain induced OCL formation to levels that were as high as those induced by 1,25-(OH)2D3. Implantation in nude mice of 293 cells transfected with the legumain cDNA and constitutively expressing high levels of the protein significantly reduced hypercalcemia induced by PTHrP by about 50%, and significantly inhibited the increase in OCL surface and in OCL number expressed per mm2 bone area and per mm bone surface induced by PTHrP. These results suggest that legumain may be a physiologic local regulator of OCL activity that can negatively modulate OCL formation and activity.