Biomedical subjects

D M Findlay

Publications and source records attributed to D M Findlay.

At least 19 recordsLinked to original sources

PubMed2005

Osteoprotegerin (OPG) is localized to the Weibel-Palade bodies of human vascular endothelial cells and is physically associated with von Willebrand factor.

Recent studies demonstrate roles for osteoprotegerin (OPG) in both skeletal and extra-skeletal tissues. Although its role in preventing osteoclast (OC) formation and activity is well documented, emerging evidence suggests a role of OPG in endothelial cell survival and the prevention of arterial calcification. In this communication, we show that vascular endothelial cells in situ, and human umbilical vein endothelial cells (HUVEC) in vitro, express abundant OPG. In HUVEC, OPG co-localizes with P-selectin and von Willebrand factor (vWF), within the Weibel-Palade bodies (WPB). Treatment of HUVEC with the pro-inflammatory cytokines, tumor necrosis factor (TNF)-alpha and IL-1beta, resulted in mobilization from the WPBs and subsequent secretion of OPG protein into the culture supernatant. Furthermore, TNF-alpha treatment of HUVEC resulted in a sustained increase in OPG mRNA levels and protein secretion over the 24-h treatment period. Reciprocal immunoprecipitation experiments revealed that while not associated with P-Selectin, OPG is physically complexed with vWF both within the WPB and following secretion from endothelial cells. Interestingly, this association was also identified in human peripheral blood plasma. In addition to its interaction with vWF, we show that OPG also binds with high avidity to the vWF reductase, thrombospondin (TSP-1), raising the intriguing possibility that OPG may provide a link between TSP-1 and vWF. In summary, the intracellular localization of OPG in HUVEC, in association with vWF, together with its rapid and sustained secretory response to inflammatory stimuli, strongly support a modulatory role in vascular injury, inflammation and hemostasis.

Carrier Proteins↗

PubMed2005

Death to the bad guys: targeting cancer via Apo2L/TRAIL.

All higher organisms consist of an ordered society of individual cells that must communicate to maintain and regulate their functions. This is achieved through a complex but highly regulated network of hormones, chemical mediators, chemokines and other cytokines, acting as ligands for intra or extra-cellular receptors. Ligands and receptors of the tumor necrosis factor (TNF) superfamilies are examples of signal transducers, whose integrated actions influence the development, homeostasis and adaptive responses of many cells and tissue types. Apo2L/TRAIL is one of several members of the tumour necrosis factor superfamily that induce apoptosis through the engagement of death receptors. Apo2L/TRAIL interacts with an unusually complex receptor system, which in humans comprises two death receptors and three decoy receptors. This molecule has received considerable attention recently because of the finding that many cancer cell types are sensitive to Apo2L/TRAIL-induced apoptosis, while most normal cells appear to be resistant to this action of Apo2L/TRAIL. In this review, we specifically emphasise on the actions of Apo2L/TRAIL with respect to its apoptotic signaling pathways and summarise what is known about its physiological role. The potential therapeutic usefulness of Apo2L/TRAIL, especially in combination with chemotherapeutic agents, is also discussed in some detail.

Apoptosis↗

PubMed2005

Identification of differentially expressed genes between osteoarthritic and normal trabecular bone from the intertrochanteric region of the proximal femur using cDNA microarray analysis.

Osteoarthritis (OA) is a common age-related joint disease resulting in progressive degenerative damage to articular cartilage. The etiology of primary OA has not yet been determined. However, there is evidence supporting the hypothesis that primary OA is a disease affecting bone remodeling in addition to articular cartilage. In this study, we have used cDNA microarray analysis to compare gene expression in bone between normal (CTL) and OA individuals. Trabecular bone was sampled from the intertrochanteric region of the proximal femur, a site distal to the diseased hip joint. Total RNA was extracted from three pairs of age- and sex-matched CTL and OA bone samples, reverse-transcribed and radioactively labeled to generate cDNA probes, before hybridization with the Research Genetics GF211 human gene microarray filter. The CTL and OA samples were found to have similar levels of gene expression for more than 4000 known human genes. However, forty-one genes were identified that were differentially expressed, twofold or more, between all three CTL-OA sample pairs. Using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis, three genes, fms-like tyrosine kinase 1 (FLT1), plexin B1 (PLXNB1), and small inducible cytokine A2 (SCYA2), were confirmed to be consistently expressed at lower levels in OA, in a majority of twenty age- and sex-matched CTL-OA bone sample pairs tested. FLT1, PLXNB1, and SCYA2 have known or potential roles in angiogenesis and bone remodeling. Down-regulation of these genes is consistent with a role for bone in the pathogenesis of OA.

Aged↗

PubMed2004

Factors regulating osteoclast formation in human tissues adjacent to peri-implant bone loss: expression of receptor activator NFkappaB, RANK ligand and osteoprotegerin.

Aseptic bone loss adjacent to orthopedic joint implants is a common cause of joint implant failure in humans. This study investigates the expression of key regulators of osteoclast formation, receptor activator NFkappaB (RANK), Receptor activator of NFkappaB ligand (RANKL) and osteoprotegerin (OPG), in the peri-implant tissues of patients with osteolysis compared with levels in synovial tissues from osteoarthritic and healthy subjects. Immunohistochemical studies demonstrated that significantly higher levels of RANKL protein (p<0.05) were found in the peri-implant tissues of patients with implant failure than in similar tissues from osteoarthritic and healthy subjects. In contrast, OPG protein levels were similar in all tissues. RANKL, expressed as mRNA and protein, was predominantly associated with cells containing wear particles. Dual labeling studies showed that the cells expressing RANKL protein were macrophages. In situ hybridization studies confirmed that mRNA encoding for these proteins is also expressed by cells in the peri-implant tissues. In addition, RANK mRNA was expressed in cells that contained wear particles. These findings show that abnormally high levels of RANKL are expressed in peri-implant tissues of patients with prosthetic loosening and that these abnormal levels of RANKL may significantly contribute to aseptic implant loosening.

Adult↗

PubMed2004

Sensitivity of fresh isolates of soft tissue sarcoma, osteosarcoma and giant cell tumour cells to Apo2L/TRAIL and doxorubicin.

Chemotherapy is an established treatment modality for bone sarcomas such as osteosarcoma (OS). However, the use of chemotherapy in high-grade soft tissue sarcomas remains controversial, with the most active chemotherapeutic agent, doxorubicin (DOX), reported to have a response rate of, at best only 34% and most studies reporting lower response rates. Apo2L/TRAIL is a member of the tumour necrosis factor (TNF) family of cytokines and induces death of tumour cells, but not normal cells. Its potent apoptotic activity is mediated through cell surface death domain-containing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2. We investigated the efficacy of Apo2L/TRAIL as a single agent, and in combination with clinically relevant chemotherapeutic drugs, in fresh isolates of primary malignant cells obtained from biopsy material. The data presented here demonstrate that, in a range of primary bone related tumours, as well as soft tissue sarcomas, chemotherapeutic agents were only moderately effective, in terms of induction of cell death. Apo2L/TRAIL alone had little or no effect on any bone-related tumour or sarcoma in culture. In contrast, the combination of Apo2L/TRAIL and chemotherapeutic drugs produced a significant increase in tumour cell death, with DOX and Apo2L/TRAIL proving to be the most effective combination. These data suggest the potential for Apo2L/TRAIL to increase the effectiveness of chemotherapeutic drugs in bone and soft tissue sarcomas, while perhaps concurrently allowing a reduction in the exposure to drugs such as DOX, and a consequent reduction in toxicity. The synergistic action between these two different classes of agents has yet to be tested in vivo but may prove clinically relevant in the treatment of this refractive class of malignancies.

Adolescent↗

PubMed2003

Progressive resistance of BTK-143 osteosarcoma cells to Apo2L/TRAIL-induced apoptosis is mediated by acquisition of DcR2/TRAIL-R4 expression: resensitisation with chemotherapy.

Apo2 ligand (Apo2L, also known as TRAIL) is a member of the tumour necrosis factor (TNF) family of cytokines that selectively induces the death of cancer cells, but not of normal cells. We observed that recombinant Apo2L/TRAIL was proapoptotic in early-passage BTK-143 osteogenic sarcoma cells, inducing 80% cell death during a 24 h treatment period. Apo2L/TRAIL-induced apoptosis was blocked by caspase inhibition. With increasing passage in culture, BTK-143 cells became progressively resistant to the apoptotic effects of Apo2L/TRAIL. RNA and flow cytometric analysis demonstrated that resistance to Apo2L/TRAIL was paralleled by progressive acquisition of the decoy receptor, DcR2. Blocking of DcR2 function with a specific anti-DcR2 antibody restored sensitivity to Apo2L/TRAIL in a dose-dependent manner. Importantly, treatment of resistant cells with the chemotherapeutic agents doxorubicin, cisplatin and etoposide reversed the resistance to Apo2L/TRAIL, which was associated with drug-induced upregulation of mRNA encoding the death receptors DR4 and DR5. BTK-143 cells thus represent a useful model system to investigate both the mechanisms of acquisition of resistance of tumour cells to Apo2L/TRAIL and the use of conventional drugs and novel agents to overcome resistance to Apo2L/TRAIL.

Apoptosis↗

PubMed2003

Induction of cell death of human osteogenic sarcoma cells by zoledronic acid resembles anoikis.

The aim of this study was to investigate the cytotoxic activity of the third-generation nitrogen-containing bisphosphonate zoledronic acid (ZOL) as a single agent, and in combination with clinically relevant anticancer drugs, in a panel of human osteogenic sarcoma cell lines (HOS, BTK-143, MG-63, SJSA-1, G-292, and SAOS2). We found that ZOL, when used alone, reduced cell number in a dose- and time-dependent manner, due either to cell cycle arrest in S-phase or to the induction of apoptosis. In the sensitive HOS, BTK-143, and G-292 cell lines, genomic DNA fragmentation and morphological changes characteristic of apoptosis were evident, and cells became nonadherent. Induction of apoptosis in osteosarcoma cells by ZOL was associated with caspase activation. However, coaddition of the broad-spectrum caspase inhibitors, z-VAD-fmk, Boc-D-fmk, or the caspase-3-specific inhibitor z-DEVD fmk, failed to protect these cells from ZOL-induced apoptosis. Our data support a ZOL-specific induction of cell apoptosis that involves cell detachment (anoikis), and in which caspase activation occurs secondarily to, and is redundant as a mediator of cell death. The addition of geranylgeraniol, an intermediate of the mevalonate pathway, suppressed the ZOL-induced apoptosis, suggesting that the cytotoxic effects of ZOL in osteosarcoma cells were mediated by the mevalonate pathway. While treatment of osteosarcoma cells with the chemotherapeutic agents doxorubicin or etoposide decreased cell viability, combination of these agents with ZOL did not significantly augment apoptosis in any of the cell lines tested. These observations suggest that ZOL has direct effects on the proliferation and survival of osteosarcoma cells in vitro, which has implications for future therapy of osteosarcoma.

Anoikis↗

PubMed2002

Human osteoblasts are resistant to Apo2L/TRAIL-mediated apoptosis.

Apo2 ligand (Apo2L/TRAIL) is a member of the tumor necrosis factor (TNF) cytokine family. Apo2L/TRAIL can selectively induce programmed cell death in transformed cells, although its wide tissue distribution suggests potential physiological roles. We have investigated the expression, in human osteoblast-like cells (NHBC), of Apo2L/TRAIL and the known Apo2L/TRAIL death receptors, DR4 and DR5, and the Apo2L/TRAIL decoy receptors, DcR-1, DcR-2, and osteoprotegerin (OPG). NHBC expressed abundant mRNA corresponding to each of these molecular species. Immunofluorescence staining demonstrated that Apo2L/TRAIL protein was abundant within the cytoplasm of NHBC and OPG was strongly expressed at the cell surface. DR5 and DcR-2 were present in the cell membrane and cytoplasm and DcR-1 was confined to the nucleus. DR4 staining was weak. Neither Apo2L/TRAIL alone, nor in combination with chemotherapeutic agents of clinical relevance to treatment of osteogenic sarcoma, induced cell death in NHBC, as assessed morphologically and by activation of caspase-3. In contrast, the human osteogenic sarcoma cell lines, BTK-143 and G-292, were sensitive to exogenous Apo2L/TRAIL alone, and to the combined effect of Apo2L/TRAIL/cisplatin and Apo2L/TRAIL/doxorubicin treatments, respectively. In NHBC, we observed strong associations between the levels of mRNA corresponding to the pro-apoptotic molecules, Apo2L/TRAIL, DR4, and DR5, and those corresponding to pro-survival molecules, DcR-1, DcR-2, OPG, and FLIP, suggesting that the balance between pro-survival and pro-apoptotic molecules is a mechanism by which NHBC can resist Apo2L/TRAIL-mediated apoptosis. In contrast, osteogenic sarcoma cells had low or absent levels of DcR-1 and DcR-2. These results provide a foundation to explore the role of Apo2L/TRAIL in osteoblast physiology. In addition, they predict that therapeutic use of recombinant Apo2L/TRAIL, in combination with chemotherapeutic agents to treat skeletal malignancies, would have limited toxic effects on normal osteoblastic cells.

Adult↗

PubMed2002

Receptor activator NF-kappaB ligand (RANKL) expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathy, osteoarthritis, and from normal patients: semiquantitative and quantitative analysis.

OBJECTIVES: To compare receptor activator of NF-kappaB ligand (RANKL) production in the synovial tissue from patients with active rheumatoid arthritis (RA), inactive RA, spondyloarthropathies (SpA), osteoarthritis, and from normal subjects. In addition, to establish the cell lineages expressing RANKL in these tissues. METHODS: Immunohistological analysis of frozen synovial tissue biopsy specimens was performed using a monoclonal antibody (mAb) to detect RANKL. Sections were evaluated by computer assisted image analysis and semiquantitative analysis to compare RANKL expression between groups. Dual and sequential labelling with mAb RANKL and cell lineage specific monoclonal antibodies were used to determine the types of cells expressing RANKL. RESULTS: Higher levels of RANKL were expressed in tissues from patients with active RA and SpA than in tissues from patients with inactive RA, osteoarthritis, and from normal subjects. RANKL protein was associated with CD3 antigen-positive lymphocytes and some macrophages. RANKL was predominantly associated with activated, memory T cells (CD45Ro positive cells) in patients with active RA and spondyloarthropathy (SpA). CONCLUSIONS: The highest levels of RANKL were detected in patients with RA with active synovitis and in some patients with SpA. An increase in RANKL in the inflamed joint of patients with RA, produced by infiltrating activated T cells and macrophages, is likely to be an important cause of joint erosions in RA.

Adult↗

PubMed2002

Calcitonin decreases the adherence and survival of HEK-293 cells by a caspase-independent mechanism.

We recently reported that calcitonin (CT) can profoundly inhibit the growth of HEK-293 cells transfected with the human calcitonin receptor (hCTR). We also obtained preliminary evidence that suggested a role for CT in cell survival, and in the present study we have investigated the pro-apoptotic action of CT, which we observe in conditions of low serum concentration. Under these conditions, we have found that CT treatment of HEK-293 cells stably transfected with the insert-negative form of the human CTR (HR12 cells) caused a time-dependent decrease in cell number associated with loss of cellular attachment. Loss of cellular adherence in CT-treated cultures caused programmed cell death, as shown by Annexin V staining of cells, failure of cells to exclude Trypan Blue dye, condensation and cleavage of nuclear DNA, and appearance of hypodiploid cells in fluorescence-activated cell sorting (FACS) analysis. The accumulation of non-adherent cells and cell death was concomitant with increased intracellular activity of caspase-3. However, inhibition of caspase activation in HR12 cells did not prevent CT-mediated loss of attachment and did not maintain the viability of non-adherent cells, indicating that caspase activation accompanied, but was probably not the cause of, the loss of cell viability. Neither the effects of CT on cell survival nor the activation of caspase-3 were observed in serum-replete conditions, suggesting that serum-derived factors provide protection of cells from CT-induced apoptosis. The inhibitory effects of CT on cell growth were found previously to be related to activation of Erk1/2 MAP kinase. In the present experiments, it was found that the Erk1/2 inhibitor, PD 98059, inhibited the CT-induced loss of cellular adherence and the consequent reduction in cell numbers. These results demonstrate that CT can negatively affect cell survival and they identify roles for cell adherence and MAP kinase activation in this process.

Apoptosis↗

PubMed2001

Osteoprotegerin inhibits osteoclast formation and bone resorbing activity in giant cell tumors of bone.

Osteolysis is a common complication of tumors that arise in, or metastasize to, bone. The recent discovery of key regulators of osteoclast formation and activity, including receptor activator of nuclear factor of kappaB ligand (RANKL), RANK, and osteoprotegerin (OPG), may facilitate new treatment regimes for certain tumors associated with excessive bone loss. We recently showed that the stromal cells of osteolytic giant cell tumors (GCT) of bone express high levels of mRNA encoding RANKL, relative to mRNA for the RANKL antagonist, OPG, compared with the expression patterns of other lytic and nonlytic bone tumors. In this study, we found that expression of RANKL and OPG mRNA continued by the stromal element of these tumors in a constitutive manner for at least 9 days in the absence of giant cells. Immunostaining of unfractionated GCT cultured in vitro revealed punctate cytoplasmic/membranous staining for RANKL and both cytoplasmic and extracellular matrix staining for OPG in stromal cells. Giant cells (osteoclasts) were negative for RANKL staining, but stained brightly for cytoplasmic OPG protein. We also investigated the functional relevance of these molecules for GCT osteolysis by adding recombinant OPG and RANKL to cultured GCT cells. We found that OPG treatment potently and dose-dependently inhibited resorption of bone slices by GCT, and could also inhibit the formation of multinucleated osteoclasts from precursors within the GCT. These effects of OPG were reversed by stoichiometric concentrations of exogenous RANKL. These data indicate that both the processes of osteoclast formation and activation in GCT are promoted by RANKL. Therefore, GCT represent a paradigm for the direct stimulation of osteoclast formation and activity by tumor stromal cells, in contrast to the mechanisms described for osteolytic breast tumors and multiple myeloma. The demonstration of these relationships is important in developing approaches to limit tumor-induced osteolysis.

Bone Neoplasms↗

PubMed2001

Osteoprotegerin and receptor activator of nuclear factor kappaB ligand (RANKL) regulate osteoclast formation by cells in the human rheumatoid arthritic joint.

OBJECTIVE: This study investigated the involvement of the recently identified regulators of osteoclast formation RANKL [receptor activator of nuclear factor kappaB (RANK) ligand, osteoclast differentiation factor, TRANCE, osteoprotegerin ligand] and its natural inhibitor, osteoprotegerin (OPG), in the bone erosion of rheumatoid arthritis (RA). METHODS: mRNA was extracted from cells isolated from the pannus and synovial membrane regions of joints of 11 RA patients. Semiquantitative reverse transcription-polymerase chain reaction was carried out, and the isolated cells were also cultured to determine their ability to form osteoclasts. RESULTS: mRNAs encoding RANKL, RANK, OPG and macrophage-colony stimulating factor were expressed by cells isolated from RA joints. In addition, mRNA encoding for tumour necrosis factor apoptosis-inducing ligand and the osteoclast markers tartrate-resistant acid phosphatase and calcitonin receptor were also often expressed. Osteoclasts capable of forming resorption lacunae were generated from cells in the RA joints. At 50 ng/ml, recombinant OPG completely inhibited the resorptive activity of these cells. There was a significant correlation between the ratio of RANKL mRNA to OPG mRNA and the number of resorption pits produced (P = 0.028). CONCLUSION: These data suggest that RANKL is an essential factor for osteoclast formation by cells in the rheumatic joint and that OPG may prevent the bone erosion seen in RA joints.

Adult↗

PubMed2001

Enhanced apoptosis of soft tissue sarcoma cells with chemotherapy: A potential new approach using TRAIL.

Soft tissue sarcomas are less responsive to conventional chemotherapy when compared to bone sarcomas. We investigated the possibility of enhancing the efficacy of chemotherapy by utilising the recently identified cytokine, tumour necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) in combination with standard chemotherapeutic agents. Fresh human soft tissue sarcomas (rhabdomyosarcoma, fibrosarcoma, malignant fibrous histiocytoma) were obtained at biopsy and dispersed tumour cells were incubated in cell culture with standard cytotoxic agents, either as single agents or in combination with TRAIL. The chemotherapeutic agents were, at best, moderately effective, in terms of induction of cellular apoptosis, although the fibrosarcoma was completely unresponsive to all single agents. TRAIL alone had no effect on any sarcoma cell culture. In contrast, the addition of TRAIL and drug together produced a significant increase in sarcoma cell apoptosis, with TRAIL and doxorubicin the most effective combination.

Journal Article↗

PubMed2001

Regulation of calcitonin receptor by glucocorticoid in human osteoclast-like cells prepared in vitro using receptor activator of nuclear factor-kappaB ligand and macrophage colony-stimulating factor.

Using mouse osteoclast-like cells (OCs), we have shown that treatment with glucocorticoids (GCs) resulted in an increase in calcitonin (CT) binding by enhancing CT receptor (CTR) gene transcription. Additionally, treatment with GCs demonstrated increased sensitivity to CT. There is, however, scant information on the effects of GC or CTR regulation by GCs in human osteoclasts. In this study we examined CTR regulation by GCs and the effects of GCs and CT together in human OCs. OCs were prepared by treatment of peripheral blood mononuclear cells in vitro with soluble receptor activator of nuclear factor-kappaB ligand and macrophage colony-stimulating factor. Treatment of mature OCs with dexamethasone (Dex) resulted in a dose- and time-dependent increase in [(125)I]salmon CT (sCT) binding capacity. Treatment with Dex enhanced CTR messenger RNA (mRNA) expression, suggesting that CTR up-regulation is at least partly due to an increase in de novo CTR synthesis. Triamcinolone and prednisolone reproduced the Dex effect on [(125)I]sCT-specific binding and CTR mRNA expression, but 17beta-estradiol, progesterone, dehydroepiandrosterone, and aldosterone did not. A Scatchard plot analysis showed that Dex enhanced CTR number with a minimal change in the affinity to sCT. Autoradiographic studies using [(125)I]sCT showed that Dex enhanced the CTR density on individual multinuclear OCs. Up-regulation of [(125)I]sCT-specific binding and CTR mRNA expression was seen even in the presence of sCT, but the enhancement diminished subsequently at later times (36-48 h after sCT removal), which was consistent with our previous observation in mouse OCs. This suggests that GCs and CTs act on CTR expression differently, consistent with our previous work using mouse OCs, in which we found that GCs increased transcription of CTR gene expression, whereas CT reduced CTR mRNA stability. The results obtained in this study show that GC increased CTR expression and sensitivity to CT in cells of the human osteoclast lineage and provide the basis for understanding the beneficial effects of combination treatment with GCs and CTs in malignancy-associated hypercalcemia.

Acid Phosphatase↗

PubMed2001

The osteoclastogenic molecules RANKL and RANK are associated with periprosthetic osteolysis.

Extensive osteolysis adjacent to implants is often associated with wear particles of prosthetic material. We have investigated if RANKL, also known as osteoprotegerin ligand, osteoclast differentiation factor or TRANCE, and its natural inhibitor, osteoprotegerin (OPG), may be important in controlling this bone loss. Cells isolated from periprosthetic tissues containing wear particles expressed mRNA encoding for the pro-osteoclastogenic molecules, RANKL, its receptor RANK, monocyte colony-stimulating factor (M-CSF), interleukin (IL)-1beta, tumour necrosis factor (TNF)alpha, IL-6, and soluble IL-6 receptor, as well as OPG. Osteoclasts formed from cells isolated from periprosthetic tissues in the presence and absence of human osteoblastic cells. When osteoclasts formed in the absence of osteoblastic cells, markedly higher levels of RANKL mRNA relative to OPG mRNA were expressed. Particles of prosthetic materials also stimulated human monocytes to express osteoclastogenic molecules in vitro. Our results suggest that ingestion of prosthetic wear particles by macrophages results in expression of osteoclast-differentiating molecules and the stimulation of macrophage differentiation into osteoclasts.

Carrier Proteins↗

PubMed2001

The ratio of messenger RNA levels of receptor activator of nuclear factor kappaB ligand to osteoprotegerin correlates with bone remodeling indices in normal human cancellous bone but not in osteoarthritis.

The determinants of cancellous bone turnover and trabecular structure are not understood in normal bone or skeletal disease. Bone remodeling is initiated by osteoclastic resorption followed by osteoblastic formation of new bone. Receptor activator of nuclear factor kappaB ligand (RANKL) is a newly described regulator of osteoclast formation and function, the activity of which appears to be a balance between interaction with its receptor RANK and with an antagonist binding protein osteoprotegerin (OPG). Therefore, we have examined the relationship between the expression of RANKL, RANK, and OPG and indices of bone structure and turnover in human cancellous bone from the proximal femur. Bone samples were obtained from individuals with osteoarthritis (OA) at joint replacement surgery and from autopsy controls. Histomorphometric analysis of these samples showed that eroded surface (ES/BS) and osteoid surface (OS/BS) were positively associated in both control (p < 0.001) and OA (p < 0.02), indicating that the processes of bone resorption and bone formation remain coupled in OA, as they are in controls. RANKL, OPG, and RANK messenger RNA (mRNA) were abundant in human cancellous bone, with significant differences between control and OA individuals. In coplotting the molecular and histomorphometric data, strong associations were found between the ratio of RANKL/OPG mRNA and the indices of bone turnover (RANKL/OPG vs. ES/BS: r = 0.93, p < 0.001; RANKL/OPG vs. OS/BS: r = 0.80, p < 0.001). These relationships were not evident in trabecular bone from severe OA, suggesting that bone turnover may be regulated differently in this disease. We propose that the effective concentration of RANKL is related causally to bone turnover.

Adult↗

PubMed2001

Expression of alternatively-spliced MDM2 transcripts in giant cell tumours of bone.

Giant cell tumours of bone (GCT) are characterized histologically by multinucleated bone resorbing giant cells in a background of ovoid spindle-shaped mesenchymal cells. Current evidence suggests that the latter comprise the tumour element of these lesions, although there are basic questions as to the factors that contribute to the tumourigenesis and progression of GCT. The deregulation of the p53/MDM2 pathway is an important pathogenetic event in many tumour types, prompting us to assess the expression of MDM2 by the stromal cells and giant cells of GCT. Northern blot analysis demonstrated that most of the GCT samples examined expressed increased levels of MDM2 when compared to normal human bone cells. However, Southern analysis failed to show any evidence of MDM2 gene amplification in the same samples, suggesting that increased levels of MDM2 mRNA were not a direct result of gene amplification, but rather due to altered transcriptional regulation of MDM2 gene. By RT-PCR analysis we found that 7/8 giant cell tumours expressed strongly a short alternatively spliced variant of MDM2, whereas other tumours of bone and normal human bone cells expressed predominantly full length MDM2. Sequence analysis confirmed this variant to be MDM2-b, a variant previously reported to confer a transformed phenotype. Cell fractionation of the GCTs has shown that the MDM2-b splice variant was expressed exclusively in the stromal population, whereas the full length MDM2 was expressed in the multinucleated giant cells of these lesions. Overexpression of a green fluorescent protein-tagged MDM2-b in human embryonic kidney cells (HEK-293), demonstrated predominantly nuclear localisation. Immunoprecipitation studies showed that MDM2-b is unable to physically associate with the p53 tumour suppressor protein. These results are consistent with the hypothesis that the stromal cells comprise the tumour element in giant cell tumours of bone and we speculate that expression of the MDM2-b splice variant contributes to their transformed phenotype in a p53 independent manner.

Alternative Splicing↗

PubMed2000

Coordinated cytokine expression by stromal and hematopoietic cells during human osteoclast formation.

An in vitro culture system to generate human osteoclasts (OC) was recently described in which OC precursors in the human peripheral blood mononuclear cell (PBMC) population differentiate in the presence of murine ST-2 stromal cells. We used this culture system to define the cytokine environment in which human OC form and to determine the separate contributions of the stromal and hematopoietic elements. We designed a panel of reverse transcriptase-polymerase chain reaction (RT-PCR) primers that specifically amplify the respective murine or human mRNA species that correspond to cytokines and their cognate receptors previously shown to promote or inhibit OC differentiation. ST-2 cells were cocultured with human PBMC for up to 21 days in the presence of 1alpha,25(OH)(2) vitamin D(3), dexamethasone, and recombinant human macrophage-colony stimulating factor (M-CSF). OC formation was monitored by the appearance of cells that were positive for tartrate-resistant acid phosphatase (TRAP) and able to form resorption lacunae on slices of dentine. We found that the ST-2 cells in these cultures express messenger RNA (mRNA) encoding a repertoire of many of the reported osteoclastogenic factors (interleukins [IL]-1/IL-1R1, IL-11, IL-6/IL-6R, and IL-17 transforming growth factor [TGF]-beta), as well as the recently described OC differentiation factor (ODF/TRANCE/RANKL). The stromal cells also expressed mRNA encoding two molecules shown to be inhibitory to osteoclastogenesis, osteoprotegerin (OPG) and IL-18. OPG, IL-1, IL-1R1, IL-6, IL-6R, IL-11R, IL-17, IL-18, IL-18R, TGF-beta, and M-CSF were expressed by both the stromal cells and the PBMC. Expression of mRNA encoding RANK, IL-1R2, and c-fms, was specific for the PBMC. In addition, PBMC were found to express sIL-6R, granulocyte macrophage (GM)-CSF, GM-CSFRalpha, and tumor necrosis factor (TNF)-alpha. Whereas this indicated that human OC formation occurs in a complex environment of many positive and negative influences, we identified three apparent features of the cytokine environment that may be a characteristic of normal osteoclast formation. First, the ratio of mouse ODF:OPG mRNA was found to increase during the cocultures, consistent with a key role for ODF in the promotion by stromal cells of OC formation. Second, we found that mRNA encoding IL-1 and IL-17, as well as IL-6 and sIL-6R, were coordinately expressed by the PBMC. Third, analysis of the culture medium showed that the PBMC secreted IL-1, IL-6, and TNF-alpha protein only in coculture with ST-2 cells during the first few days of osteoclast development. We conclude that human OC formation occurs in a complex environment of many positive and negative influences; however, these are likely to be strictly regulated by a coordinated cytokine response of both stromal and hematopoietic cells.

Animals↗