G. David Roodman

Advances in Bone Biology: The Osteoclast

Much has been learned about the cell biology and molecular biology of the osteoclast in the last 5 yr. The osteoclast appears to be derived from CFU-GM, the committed monocyte-granulocyte precursor cell. This cell then differentiates into more committed precursors for the osteoclast. The role of the marrow microenvironment appears to be critical for murine osteoclast formation, although in human systems it appears to be nonessential but acts to enhance osteoclast formation and resorption. The osteoclast has been shown to be a secretory cell capable of producing both stimulators and inhibitors of osteoclast formation and resorption. The identification of the role of protooncogenes, such as c-fos and pp60c-src, in osteoclast differentiation and bone resorption has provided important insights into the regulation of normal osteoclast activity. Studies such as these should help us to dissect the pathophysiology of abnormal osteoclastic activity, such as seen in hypercalcemia of malignancy, osteopetrosis, and Pagets disease of bone. Future research is needed to further delineate the signaling pathways involved in osteoclastic bone resorption in response to cytokines and hormones, as well as to identify the molecular events required for commitment of multipotent precursors to the osteoclast lineage. Development of osteoclast cell lines may be possible and would greatly enhance our understanding of the biology of the osteoclast. Utilization of current model systems to examine the effects of cytokines and hormones on osteoclast precursors in vitro and in vivo and the ability to obtain large numbers of highly purified osteoclasts for production of osteoclast cDNA libraries should lead to important new discoveries in osteoclast biology.

Cell biology of the osteoclast.

The osteoclast is a hematopoietic cell derived from CFU-GM and branches from the monocyte-macrophage lineage early during the differentiation process. The marrow microenvironment appears critical for osteoclast formation due to production of RANK ligand, a recently described osteoclast differentiation factor, by marrow stromal cells in response to a variety of osteotropic factors. In addition, factors such as osteoprotegerin, a newly described inhibitor of osteoclast formation, as well as secretory products produced by the osteoclast itself and other cells in the marrow enhance or inhibit osteoclast formation. The identification of the role of oncogenes such as c-fos and pp60 c-src in osteoclast differentiation and bone resorption have provided important insights in the regulation of normal osteoclast activity. Current research is beginning to delineate the signaling pathways involved in osteoclastic bone resorption and osteoclast formation in response to cytokines and hormones. The recent development of osteoclast cell lines may make it possible for major advances to our understanding of the biology of the osteoclast to be realized in the near future.

Role of cytokines in the regulation of bone resorption

SummaryThe process of bone remodeling involves complex interactions between the osteoclast, the primary bone-resorbing cell, and other cells in its microenvironment. These interactions can regulate bone resorption through two processes: (1) effects on the number of osteoclasts present at a given site and (2) effects on the bone-resorbing capacity of individual osteoclasts. Cells present in the osteoclast microenvironment include marrow stromal cells, osteoblasts, macrophages, T-lymphocytes, and marrow cells. These cells, as well as the osteoclast itself, produce cytokines that can affect osteoclast formation and osteoclast activity.In vitro model systems using rodent organ cultures or long-term marrow culture systems, andin vivo models have demonstrated that cytokines such as interleukin-1, M-CSF, tumor necrosis factor, and interleukin-6 can stimulate the formation and bone-resorbing capacity of osteoclasts. In contrast, cytokines such as interleukin-4, γ-interferon, and transforming factor-β inhibit both osteoclast formation and osteoclast activity. The relative proportions of these cytokines in the marrow microenvironment may play a critical role in regulating osteoclast activity. Knowledge of cytokines that affect osteoclast formation and activity and their capacity to modulate the bone-resorbing process should provide critical insights into normal calcium homeostasis and disorders of bone turnover such as osteoporosis and Pagets disease of bone.

Evidence for a Novel Osteosarcoma Tumor-Suppressor Gene in the Chromosome 18 Region Genetically Linked with Paget Disease of Bone

Paget disease of bone, or osteitis deformans, is a bone disorder characterized by rapid bone remodeling resulting in abnormal bone formation. It is the second most common metabolic bone disease after osteoporosis, affecting 3%-5% of subjects aged >40 years. Recent evidence suggests that predisposition to Paget disease may have a genetic component. Genetic linkage analysis of families with multigenerational Paget disease shows linkage to a region of chromosome 18q near the polymorphic locus D18S42. Approximately 1% of Paget patients develop osteosarcoma, which represents an increase in risk that is several thousandfold over that of the general population. Osteosarcoma in Paget patients is the underlying basis for a significant fraction of osteosarcomas occurring after age 60 years. Our analysis of tumor-specific loss of constitutional heterozygosity (LOH) in 96 sporadic osteosarcomas has identified a putative tumor-suppressor locus that maps to chromosome 18q. We have localized this tumor-suppressor locus between D18S60 and D18S42, a region tightly linked to familial Paget disease. Analysis of osteosarcomas from patients with Paget disease revealed that these tumors also undergo LOH in this region. These findings suggest that the association between Paget disease and osteosarcoma is the result of a single gene or two tightly linked genes on chromosome 18.

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.

Antibodies to Kaposi's Sarcoma—Associated Herpesvirus (Human Herpesvirus 8) in Patients with Multiple Myeloma

Kaposis sarcoma-associated herpesvirus (KSHV) serologic assays were used to detect specific antibodies to KSHV lytic and latent antigens in 27 patients with multiple myeloma, 27 control patients with other cancers, and 50 random blood donors. Antibodies to KSHV recombinant minor capsid antigen orf65 were found in 81% of patients with multiple myeloma, 22% of control patients with other cancers, and 6% of the blood donors. Antibodies to KSHV latent nuclear antigens were found in 52% of patients with multiple myeloma, 26% of control patients with other cancers, and 2% of the blood donors. All of the 11 patients with progressive multiple myeloma were KSHV-seropositive. Antibodies to Epstein-Barr virus nuclear antigen 1 were present in 89% of patients with multiple myeloma, 93% of control patients with other cancers, and 88% of the blood donors. These data support the possible association of KSHV infection with multiple myeloma, particularly with progressive disease.

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.

Further characterization of the murine collagenase (type IVB) gene promoter and analysis of mRNA expression in murine tissues

The collagenase B type IV (Col4B) gene is highly expressed in the osteoclast, the primary bone-resorbing cell. However, factors that regulate expression of the Col4B gene are not well characterized. A murine P1 genomic clone containing a 94 kb sequence insert which contains the Col4B gene was isolated. A 4 kb EcoR1 DNA fragment containing the 5 flanking sequence of the gene was further subcloned and restriction mapped. Putative transcription factors such as SRY, Lyf-1, and GATA1 and 2, binding motifs were identified by sequence analysis in this promoter region. Enhancer and suppressor regions were mapped by transient expression of Col4B gene promoter deletion mutant-luciferase reporter gene constructs in HepG2 cells. Col4B mRNA expression in different murine tissues was analyzed by reverse transcription-polymerase chain reaction and demonstrated high levels of expression in bone, clavaria, spleen and thymus. This promoter provides a valuable tool for targeting gene expression to the osteoclast.

23(S)25(R)-1,25-Dihydroxyvitamin D3-lactone, a naturally occurring metabolite of 1,25-dihydroxyvitamin D3, inhibits osteoclast-like cell formation in human bone marrow cultures

Abstract: We have used a human bone marrow culture system that forms multinucleated cells (MNCs), 50% of which express the osteoclast phenotype, to examine the 23(S)25(R)-1,25-dihydroxyvitamin D3-26,23-lactone (1,25-D3-lactone) on osteoclast-like cell formation. The 1,25-D3-lactone is a vitamin D3 metabolite that has recently been detected in human serum under physiological conditions at concentrations of approximately 131 pg/ml (3 × 10−10 M) and can inhibit bone resorption induced by 1,25-dihydroxyvitamin D3 (1,25-D3) in vivo and in vitro. We examined the effects of the 1,25-D3-lactone on the formation of MNC that cross-reacted with 23C6 monoclonal antibody (23C6-positive MNC), which preferentially binds to osteoclasts. All metabolites of 1,25-D3 except the 1,25-D3-lactone increased both total and 23C6-positive MNC formation in a dose-dependent manner. In contrast, the 1,25-D3-lactone inhibited both total and 23C6-positive MNC formation, whether the cultures were treated with 1,25-D3, parathyroid hormone, or interleukin-1β, all potent stimulators of MNC formation. This inhibitory action of 1,25-D3-lactone on MNC formation was very similar to the inhibitory effects of calcitonin. These data suggest that (1) 1,25-D3-lactone is a potent natural inhibitor of formation of cells with the osteoclast phenotype at physiological concentrations and (2) the inhibition of these cells by 1,25-D3-lactone may not result solely from its competitive binding to the 1,25-D3 receptor.

Epitope mapping of recombinant human procathepsin D.

Procathepsin D is the proform of the aspartic proteinase, cathepsin D. The mature form, cathepsin D, is a low specificity proteinase found in the lysosome where the intracellular protein catabolism carried out by it is well understood. The proform, procathepsin D is also expressed in secretory and membrane-associated forms.1,2 These non-lysosomal forms of procathepsin D are associated with a wide variety of physiological and pathophysiological functions including metastasis of breast cancer,3 tissue remodeling, antigen presentation in the MHC class II pathway,4 and general role in the immune system.5 The structural bases whereby this proteinase can perform these varied and specific functions are unknown. Several possibilities for membrane-association have been proposed, none has been conclusively demonstrated. The propeptide has no hydrophobic regions or C-terminal membrane anchor sequences that may be responsible for membrane association. Membrane association may be due to surface exposed residues of the propeptide. It seems likely that most of the non-lysosomal functions involve interactions of the cellular machinery with the surface features of the folded propeptide region such as propeptide specific receptors.6 Therefore there is interest in the conformation of procathepsin D. A demonstration of a difference in the conformations of the various forms of this proteinase will add to the limited knowledge about the structural bases of the functions of the non-lysosomal forms of this proteinase.