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G D Roodman

Publications and source records attributed to G D Roodman.

At least 37 records · Page 2Linked to original sources
PubMed2000

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

Paget's 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 Paget's 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 Paget's 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 Paget's disease.

Antibodies↗
PubMed2000

Expansile skeletal hyperphosphatasia: a new familial metabolic bone disease.

We describe a new familial metabolic bone disease characterized by expanding hyperostotic long bones, early onset deafness, premature tooth loss, and episodic hypercalcemia. The condition affects a mother and daughter studied at the age of 36 years and 11 years, respectively. Both individuals lost all hearing in early childhood and suffered premature shedding of teeth. Skeletal pains began just before puberty. Swelling and aching of most middle phalanges in the hands is an especially troublesome manifestation. The mother also had episodes of symptomatic hypercalcemia first documented in late childhood and subsequently during intercurrent illness and postpartum lactation. Radiographs show hyperostosis and/or osteosclerosis predominantly in the skull and appendicular skeleton. Long bones also are expanded considerably, especially the middle phalanges in the fingers. The mother's skeletal abnormalities are more severe. Biochemical parameters of bone turnover, including serum alkaline phosphatase (ALP) activity, are elevated substantially. In the proposita, dynamic histomorphometry of nondecalcified sections of iliac crest revealed rapid skeletal remodeling. In the mother, who had been treated with bisphosphonates, electron microscopy (EM) showed disorganized collagen bundles as well as necrotic and apoptotic bone cells but no osteocytic osteolysis. Measles virus gene transcripts were not detected in peripheral blood monocytes. Karyotyping was normal, 46,XX. Hyperphosphatasia with bone disease previously has been reported as either a sporadic or autosomal recessive condition. Expansile skeletal hyperphosphatasia (ESH) is probably inherited as an autosomal dominant trait with a high degree of penetrance.

Adult↗
PubMed2000

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 Paget's 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 Paget's 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 Paget's 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 Paget's 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 Paget's 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 Paget's disease although the Paget's 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 Paget's 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 Paget's disease compared with normal subjects.

Adult↗
PubMed2000

Effect of telomere and telomerase interactive agents on human tumor and normal cell lines.

Shortening of telomeres along with an up-regulation of telomerase is implicated in the immortality of tumor cells. Targeting either telomeres or telomerase with specific compounds has been proposed as an anticancer strategy. Because telomerase activity and telomeres are found in normal cells, telomere or telomerase targeting agents could induce side effects in normal tissues. We evaluated the effects of telomere and telomerase interactive agents in human tumor and normal cell lines to try to determine the potential side effects those agents might induce in patients. Toxicity of the G-quadruplex interactive porphyrins (TMPyP4, TMPyP2) and azidothymidine (AZT) were tested using a cell-counting technique against normal human cell lines (CRL-2115 and CRL-2120, fibroblasts; NHEK-Ad, adult keratinocytes; CCL-241, small intestinal cells; NCM 460, colonic mucosal epithelial cells) and human tumor cell lines (MDA-MB 231 and Hs 578T, breast cancer; SK-N-FI, neuroblastoma; HeLa, cervix cancer; MIA PaCa-2, pancreatic cancer; HT-29 and HCT-116, colon cancer; DU 145, prostatic cancer cell line). Telomerase activity of these cell lines was measured by a non-PCR-based conventional assay. The effects of TMPgammaP2, TMPyP4, and AZT were also evaluated against normal human bone marrow specimens, using a granulocyte-macrophage colony-forming assay (CFU-GM). AZT showed very low cytotoxic effects against normal and tumor cell lines, with the IC50 values above 200 microM. The IC50 values for TMPyP2 and TMPyP4 in normal human cell lines were in the range of 2.9-48.3 microM and 1.7-15.5 microM, respectively, whereas in tumor cell lines the IC50 values were 11.4-53 microM and 9.0-28.2 microM, respectively. Within the tissue types, keratinocytes were more sensitive to TMPyP4 than fibroblasts, and small intestinal cells were more sensitive than colonic mucosal epithelial cells. The IC50 for TMPyP2 and TMPyP4 in the normal marrow colony-forming assays were 19.3 +/- 5.1 microM and 47.9 +/-1.0 microM, respectively. In conclusion, the in vitro cytotoxicity of the telomere interactive agent TMPyP4 is comparable in human tumor and normal cell lines, which indicates that TMPyP4 could have effects on normal tissues.

Bone Marrow Cells↗
PubMed1999

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 D(3) (1,25-(OH)(2)D(3)) 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)(2)D(3). 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 mm(2) 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.

Animals↗
PubMed1999

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.

Acid Phosphatase↗
PubMed1999

Cell biology of Paget's disease.

Paget's disease is characterized by markedly increased osteoclast formation and bone resorption followed by excessive new bone formation. Osteoclasts in Paget's disease are increased both in number and size, contain paramyxoviral-like nuclear inclusions, and can have up to 100 nuclei per cell. Marrow culture studies have identified several abnormalities in osteoclast formation in Paget's disease. Osteoclast-like multinucleated cells formed more rapidly in marrow cultures from patients with Paget's disease, produced increased levels of interleukin-6 (IL-6), and expressed high levels of IL-6 receptors compared to normals. IL-6 levels were also increased in bone marrow and peripheral blood of patients with Paget's disease. In addition, osteoclast precursors from patients with Paget's disease are hyperresponsive to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and calcitonin. The increased sensitivity of osteoclast precursors to 1,25(OH)2D3 is mediated through the vitamin D receptor (VDR), since 24-hydroxylase activity is also up-regulated at concentrations of 1,25(OH)2D3 that are one log less than that needed to induce 24-hydroxylase activity in osteoclast precursors from normals. However, VDR numbers and affinity for 1,25(OH)2D3 do not differ in osteoclast precursors from Paget's patients compared to those from normals. Synergistic interactions between cytokines such as IL-6 and 1,25(OH)2D3 also cannot explain the enhanced sensitivity of osteoclast precursors from patients with Paget's disease to 1,25(OH)2D3. Interestingly, coculture studies of osteoclast precursors and cells from the marrow microenvironment of patients with Paget's disease and normals have demonstrated that the marrow microenvironment is more osteoclastogenic than normal. Thus, studies of the cell biology of osteoclasts in Paget's disease have demonstrated an increased rate of osteoclast formation and abnormalities in both osteoclast precursors and the marrow microenvironment. Enhanced IL-6 production by osteoclasts in Paget's disease may further amplify the increased osteoclast formation already ongoing in the pagetic lesion, and may explain the increased bone turnover at uninvolved sites distant from the pagetic lesion.

Calcitriol↗
PubMed1999

Variable disease severity associated with a Paget's disease predisposition gene.

We have recently identified a region on the long arm of chromosome 18 that carries a predisposition gene for Paget's disease using linkage analysis. This region was explored because of earlier studies demonstrating the presence of a gene for another bone disorder, familial expansile osteolysis (FEO) within this region. FEO has many similarities to Paget's disease including osteoclast abnormalities and viral-like nuclear inclusions. Therefore, it was proposed that FEO and Paget's disease are disorders resulting from mutations at the same locus. For our linkage study, we utilized a large kindred with a high incidence of Paget's disease. The propositus in this family had polyostotic disease symptoms beginning at age 31. During the process of characterizing this family, four other family members were diagnosed with Paget's disease. One of the proband's siblings was asymptomatic but had high normal serum alkaline phosphatase levels; Paget's disease was identified only after bone scanning (at age 50). This implies that the predisposition gene does not consistently cause severe disease and raises a question concerning the mechanism of predisposition: Does the predisposition gene affect the age of onset and/or the severity of disease? To further explore this question, two individuals from this kindred, under age 30 and carrying the affected haplotype, were evaluated for early onset Paget's disease. No evidence of disease was observed after thorough evaluation. This implies that the age of onset is highly variable for this locus, indicating variable expression of disease in individuals carrying the same mutation.

Adult↗
PubMed1999

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.

Animals↗
PubMed1999

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 Paget's 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.

Antigens, CD34↗
PubMed1999

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.

Annexin A2↗
PubMed1999

Characterization of immortalized osteoclast precursors developed from mice transgenic for both bcl-X(L) and simian virus 40 large T antigen.

We recently developed an immortalized osteoclast (OCL) precursor cell line that forms large numbers of OCLs. This cell line was derived from mice doubly transgenic for bcl-X(L) and large T antigen that was targeted to cells in the OCL lineage (bcl-X(L)/Tag cells). We have now characterized these cells in terms of their surface and enzymatic phenotype, responsiveness to osteotropic factors, and differentiation potential. The bcl-X(L)/Tag cells expressed interleukin-1 receptors 1 and 2, gelatinase B (MMP9), as well as Mac-1, CD16/CD32 (Fcgamma receptors), CD45.2 (common leukocyte marker), CD86 (costimulatory molecule expressed on B cells, follicular dendritic cells, and thymic epithelium), major histocompatibility complex I, and nonspecific esterase when cocultured with MC3T3E1 cells. However, they did not express the antigens for F4/80 (mature macrophage/dendritic cell marker) by immunostaining. Treatment of bcl-X(L)/Tag cells, cocultured with MC3T3E 1 cells, with the combination of 1,25-dihydroxyvitamin D3 and dexamethasone induced high levels of OCL formation. The bcl-X(L)/Tag cells formed large numbers of OCLs when cultured with RANK ligand and macrophage colony-stimulating factor in the absence of feeder cells. In the absence of RANK ligand and a feeder cell layer, 100% of the cells differentiated into F4/80-positive cells. However, neither PTH nor PTH-related protein enhanced OCL formation by bcl-X(L)/Tag cells even when they were cocultured with primary osteoblasts, suggesting that they differ from primary mouse bone marrow cells in their responsiveness to PTH/PTH-related protein. Thus, bcl-X(L)/Tag cells have many of the properties of primary mouse OCL precursors and should be very useful for studies of OCL differentiation and divergence of OCL precursors from the macrophage lineage.

Animals↗
PubMed1998

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, Paget's 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.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1998

Immortalization of osteoclast precursors by targeting Bcl -XL and Simian virus 40 large T antigen to the osteoclast lineage in transgenic mice.

Cellular and molecular characterization of osteoclasts (OCL) has been extremely difficult since OCL are rare cells, and are difficult to isolate in large numbers. We used the tartrate-resistant acid phosphatase promoter to target the bcl-XL and/or Simian Virus 40 large T antigen (Tag) genes to cells in the OCL lineage in transgenic mice as a means of immortalizing OCL precursors. Immunocytochemical studies confirmed that we had targeted Bcl-XL and/or Tag to OCL, and transformed and mitotic OCL were readily apparent in bones from both Tag and bcl-XL/Tag mice. OCL formation in primary bone marrow cultures from bcl-XL, Tag, or bcl-XL/Tag mice was twofold greater compared with that of nontransgenic littermates. Bone marrow cells from bcl-XL/Tag mice, but not from singly transgenic bcl-XL or Tag mice, have survived in continuous culture for more than a year. These cells form high numbers of bone-resorbing OCL when cultured using standard conditions for inducing OCL formation, with approximately 50% of the mononuclear cells incorporated into OCL. The OCL that form express calcitonin receptors and contract in response to calcitonin. Studies examining the proliferative capacity and the resistance of OCL precursors from these transgenic mice to apoptosis demonstrated that the increased numbers of OCL precursors in marrow from bcl-XL/Tag mice was due to their increased survival rather than an increased proliferative capacity compared with Tag, bcl-XL, or normal mice. Histomorphometric studies of bones from bcl-XL/Tag mice also confirmed that there were increased numbers of OCL precursors (TRAP + mononuclear cells) present in vivo. These data demonstrate that by targeting both bcl-XL and Tag to cells in the OCL lineage, we have immortalized OCL precursors that form bone-resorbing OCL with an efficiency that is 300-500 times greater than that of normal marrow.

Acid Phosphatase↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Amino Acid Sequence↗