Cheikh Menaa

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.

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)

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.

Isolation and characterization of a cDNA clone encoding a novel peptide (OSF) that enhances osteoclast formation and bone resorption.

Using an expression cloning approach, we identified and cloned a novel intracellular protein produced by osteoclasts that indirectly induces osteoclast formation and bone resorption, termed OSF. Conditioned media from 293 cells transiently transfected with the 0.9 kb OSF cDNA clone stimulated osteoclast‐like cell formation in both human and murine marrow cultures in the presence or absence 10−9 M 1,25‐dihydroxyvitamin D3. In addition, conditioned media from 293 cells transfected with the OSF cDNA clone enhanced the stimulatory effects of 1,25‐(OH)2D3 on bone resorption in the fetal rat long bone assay. In situ hybridization studies using antisense oligomers showed expression of OSF mRNA in highly purified osteoclast‐like cells from human giant cell tumors of the bone. Northern blot analysis demonstrated ubiquitous expression of a 1.3 kb mRNA that encodes OSF in multiple human tissues. Sequence analysis showed the OSF cDNA encoded a 28 kD peptide that contains a c‐Src homology 3 domain (SH3) and ankyrin repeats, suggesting that it was not a secreted protein, but that it was potentially involved in cell signaling. Consistent with these data, immunoblot analysis using rabbit antisera against recombinant OSF demonstrated OSF expression in cell lysates but not in the culture media. Furthermore, recombinant OSF had a high affinity for c‐Src, an important regulator of osteoclast activity. Taken together, these data suggest that OSF is a novel intracellular protein that indirectly enhances osteoclast formation and osteoclastic bone resorption through the cellular signal transduction cascade, possibly through its interactions with c‐Src or other Src‐related proteins. J Cell Physiol 177:636–645, 1998.

Paget's Disease of Bone: A Disease of the Osteoclast

Pagets disease is a chronic focal disease of the skeleton that affects up to 2-3% of the population over the age of 60 years. There is a genetic predisposition for Pagets disease, with one predisposition locus identified on chromosome 18q-21-22. Osteoclasts and osteoclast precursors from Pagets patients are abnormal and appear hyperresponsive to 1,25(OH)2D3 and RANK ligand and contain paramyxoviral transcripts (Fig. 1). The basis for the abnormalities detected in Pagets disease and the role that the paramyxoviruses may play in this disease are still unclear.

MEASLES VIRUS NUCLEOCAPSID TRANSCRIPT EXPRESSION IS NOT RESTRICTED TO THE OSTEOCLAST LINEAGE IN PATIENTS WITH PAGET'S DISEASE OF BONE

Abundant evidence supports a viral etiology for Pagets disease of bone (PD), however, an infectious virus has not been isolated from PD patients. Thus, it is unclear how the virus is maintained for the many years that the disease persists in patients. We considered if a primitive multipotential hematopoietic stem cell (HSC), which is self-renewing, passes the virus to its differentiated progeny and serves as a reservoir for the pathogen. If a primitive stem cell harbored measles virus (MV), then other hematopoietic lineages derived from this stem cell in PD patients should also express MV transcripts. Therefore, because the human hematopoietic stem cell has not been clearly identified or isolated in large numbers, we isolated RNA from highly purified erythroid and multipotential hematopoietic progenitors that are the precursors for erythroid, granulocyte, megakaryocyte and macrophages (CFU-GEMM), and used RT-PCR to determine if MV nucleocapsid transcripts were present. MV transcripts were detected in PD patients in early erythroid (BFU-E) and more primitive multipotential myeloid progenitors (CFU-GEMM). Nonhematopoietic stromal cells from PD patients did not express MV transcripts. The expression of MV transcripts in erythroid progenitors was further confirmed by in situ hybridization using antisense riboprobes to MV nucleocapsid transcripts. Thus, our findings suggest that the pluripotent HSCs may be a potential reservoir for the virus. We propose that when HSCs, which contain MV, divide they produce a second HSC that serves as a reservoir for the virus and also transmit the virus to their more differentiated progeny in the erythroid and myeloid lineages. This mechanism would permit a defective virus to persist in HSCs of PD patients for many years, since HSCs are usually in G0 phase, and then be transmitted to more differentiated cells. This model further suggests that a mature complete virus that affects cell function could only act pathogenetically in the osteoclast lineage, which offers a permissive milieu.

Cloning and identification of human Sca as a novel inhibitor of osteoclast formation and bone resorption.

Increased osteoclast activity is responsible for the enhanced bone destruction in postmenopausal osteoporosis, Pagets disease, bone metastasis, and hypercalcemia of malignancy. However, the number of known inhibitory factors that block osteoclast formation and bone resorption are limited. Therefore, we used an expression-cloning approach to identify novel factors produced by osteoclasts that inhibit osteoclast activity. A candidate clone was identified and isolated from a human osteoclast-like multinucleated cell (MNC) cDNA library, named osteoclast inhibitory peptide-1 (OIP-1), and the cDNA sequence was determined. This sequence matched that of the recently identified human stem cell antigen, was structurally similar to the mouse Ly-6 gene family, and the sequence predicted it was a glycosyl phosphatidyl inositol (GPI)-anchored protein that had a cleavable COOH-terminal peptide. Western blot analysis of conditioned media from 293 cells transfected with the OIP-1 cDNA clone confirmed that OIP-1 was released into the media as a membrane-bound GPI-linked protein. Interestingly, both recombinant OIP-1 expressed in Escherichia coli (which does not have GPI linker) and OIP-1 expressed by mammalian cells significantly reduced osteoclast-like MNC formation induced by 1,25-dihydroxyvitamin D3 or PTH-related protein in mouse and human bone marrow cultures, and inhibited 45Ca release from prelabeled bone in fetal rat organ cultures. In contrast, recombinant OIP-1 did not inhibit the growth of a variety of other cell types. These data indicate that OIP-1 is a novel, specific inhibitor of osteoclast formation and bone resorption.