CXCL13 activation of c-Myc induces RANK ligand expression in stromal/preosteoblast cells in the oral squamous cell carcinoma tumor-bone microenvironment.

CXC chemokine ligand-13 (CXCL13) has been implicated in oral squamous cell carcinoma (OSCC) tumor progression and osteolysis. The tumor necrosis factor family member RANKL (receptor activator of NF-κB ligand), a critical bone resorbing osteoclastogenic factor, has an important role in cancer invasion of bone/osteolysis. Here, we show high-level expression of CXCL13 in primary human OSCC tumor specimens; however, human bone marrow-derived stromal (SAKA-T) and murine preosteoblast (MC3T3-E1) cells produce at very low level. Recombinant CXCL13 (0-15 ng/ml) dose dependently induced CXCR5 expression in SAKA-T and MC3T3-E1 cells. Conditioned media obtained from OSCC cell lines increased the RANKL expression and an antibody against the CXCL13 specific receptor, CXCR5 markedly decreased RANKL expression in these cells. Furthermore, CXCL13 increased hRANKL-Luc promoter activity. Superarray screening identified c-Myc and NFATc3 transcription factors upregulated in CXCL13-stimulated SAKA-T cells. Immunohistochemical analysis of OSCC tumors that developed in athymic mice demonstrated RANKL and NFATc3 expression in tumor and osteoblast cells, however, showed p-c-Myc expression specific to osteoblastic cells at the tumor-bone interface. We further identified NFATc3 expression, but not c-Myc activation in primary human OSCC tumor specimens compared with adjacent normal tissue. Also, CXCL13 significantly increased p-ERK1/2 in SAKA-T and MC3T3-E1 cells. siRNA suppression of c-Myc expression markedly decreased CXCL13-induced RANKL and NFATc3 expression in preosteoblast cells. Chromatin-immuno precipitation assay confirmed p-c-Myc binding to the hRANKL promoter region. In summary, c-Myc activation through CXCL13-CXCR5 signaling axis stimulates RANKL expression in stromal/preosteoblast cells. Thus, our results implicate CXCL13 as a potential therapeutic target to prevent OSCC invasion of bone/osteolysis.

Transgenic mice with OIP-1/hSca overexpression targeted to the osteoclast lineage develop an osteopetrosis bone phenotype.

Regulatory mechanisms operative in bone-resorbing osteoclasts are complex. We previously defined the Ly-6 gene family member OIP-1/hSca as an inhibitor of osteoclastogenesis in vitro; however, a role in skeletal development is unknown. In this study, we developed transgenic mice with OIP-1/hSca expression targeted to the osteoclast lineage that develop an osteopetrotic bone phenotype. Humeri from OIP-1 mice showed a significant increase in bone mineral density and bone mineral content. microCT analysis showed increased trabecular thickness and bone volume. OIP-1 mice have dense sclerotic cortical bone with absence of spongiosa and inadequate formation of marrow spaces compared to wild-type mice. Moreover, complete inhibition of osteoclasts and marrow cavities in calvaria suggests defective bone resorption in these mice. OIP-1 mouse bone marrow cultures demonstrated a significant decrease (41%) in osteoclast progenitors and inhibition (39%) of osteoclast differentiation/bone resorption. Western blot analysis further demonstrated suppression of TRAF-2, c-Fos, p-c-Jun, and NFATc1 levels in RANKL-stimulated osteoclast precursors derived from OIP-1 mice. Therefore, OIP-1 is an important physiological inhibitor of osteoclastogenesis and may have therapeutic value against bone loss in vivo.

IFN-gamma enhances osteoclast generation in cultures of peripheral blood from osteopetrotic patients and normalizes superoxide production.

Interferon-gamma (IFN-gamma) treatment increases osteoclastic bone resorption in vivo in patients with malignant osteopetrosis (OP). The treatment effect was studied in vitro in osteoclasts generated by culturing peripheral white blood cells (PWBC) from OP patients and normal human control subjects. Osteoclasts were treated with or without IFN-gamma prior to the end of the culture period. Osteoclasts from normal subjects were large in size (161 +/- 18 microm in diameter) with >10 nuclei per osteoclast. These cells showed intense staining for tartrate-resistant acid phosphatase (TRAP), expressed abundant calcitonin receptors (CTR), and formed numerous resorption pits on bovine bone slices, indicative of authentic osteoclasts. In contrast, similarly cultured osteoclasts from OP patients were smaller in size (18 +/- 3 microm in diameter), with 2-3 nuclei per osteoclast, and stained lightly for TRAP. However, IFN-gamma treatment of osteoclasts from OP patients resulted in the formation of larger osteoclasts (171 +/- 33 microm in diameter) with >10 nuclei per cell, similar in appearance to osteoclasts from normal subjects. IFN-gamma stimulation increased the intensity of TRAP staining (p < 0.0001) to levels near that of the normal osteoclasts. Unstimulated osteoclasts from 6 OP patients had a significantly lower baseline level of superoxide production, as measured by nitroblue tetrazolium reduction (p < 0.0001), compared with normal osteoclasts. IFN-gamma markedly increased (p < 0.0001) superoxide production. Whereas there was a 3-fold increase in superoxide generation in OP patients' osteoclasts, osteoclasts from control subjects had only a small and insignificant increase in superoxide production after IFN-gamma treatment.

Superoxide generation and tyrosine kinase.

NADPH oxidase is a multi-subunit enzyme complex responsible for superoxide generation in many cells, for example, B-lymphocytes and osteoclasts. NADPH oxidase is localized on the cell surface and generates superoxide extracellularly. After synthesis, components of this oxidase are transported to the cell membrane where the functional NADPH oxidase complex is assembled. The mechanism by which the membrane-bound components are transported to the cell surface of osteoclasts remains unclear. In this study, we examined the role of tyrosine kinase activity in the transport of NADPH oxidase components. When B-lymphocytes and osteoclasts were treated with herbimycin A, a specific inhibitor of tyrosine kinase, superoxide production was significantly decreased. The amount of p91, the catalytic subunit of NADPH oxidase, was decreased in the cellular membrane of herbimycin A treated cells compared to untreated controls. Similar results were obtained for the movement of a regulatory subunit of the NADPH oxidase complex, p47, in B-lymphocytes. Thus, inhibition of tyrosine kinase decreases superoxide production by disrupting the translocation of the NADPH oxidase complex.

The use of a bone block graft from the chin for alveolar ridge augmentation.

Periodontal disease and tooth loss often leave the patient with an alveolar ridge that is less than adequate for endosseous implant placement. Alveolar bone grafting offers a clinically successful treatment option to regain all or some of this lost bone. This article describes the use of a bone block from the chin for the augmentation of the alveolar ridge. It relates the clinical techniques to the healing process in an effort to better understand the events involved.

Superoxide generation in transformed B-lymphocytes from patients with severe, malignant osteopetrosis.

Severe, malignant osteopetrosis is a disease characterized by osteoclasts that fail to resorb bone. Serious defects in the ability of white blood cells to eradicate infectious agents confound the clinical course. Defective superoxide generation by neutrophils, monocytes, and lymphocytes contributes to this inability to fight infection. To elucidate the mechanisms resulting in the defective superoxide generation observed in osteopetrotic leukocytes, gene expression, translocation, and phosphorylation of the major components that form the functional NADPH oxidase complex were studied in transformed B-lymphocytes. The expression of the p47 subunit of NADPH oxidase was reduced in B-lymphocytes collected from osteopetrotic patients compared to those from controls. Phosphorylation and translocation of p47 to the cell membrane after PMA stimulation was similar in B-lymphocytes from both patients and normal controls. However, total amount of p47 phosphorylation and translocation was reduced in patient samples. This was further supported by the experiment using p47 antisense oligonucleotide. The other major components of the oxidase (p91, p22, p67) were found to be present at normal levels. Thus, the reduction in p47 expression results in reduced ability to assemble a functional NADPH oxidase complex at the membrane of lymphocytes from osteopetrotic patients. This defect translates into reduced superoxide generation and an increased propensity for infection.

Nicotinamide adenine dinucleotide phosphate oxidase in the formation of superoxide in osteoclasts.

Osteoclasts use a variety of chemical agents to degrade bone. One important component of this process is the generation of superoxide. It has been reported that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is the enzyme responsible for superoxide production in phagocyte; however, the NADPH oxidase present in osteoclasts has not been studied in detail. One of the membrane-bound subunits of the NADPH oxidase is gp91(phox) which represents the rate-limiting component for the formation of the NADPH oxidase complex. This study was designed to demonstrate the presence of gp91(phox) in individual osteoclasts using the RT-PCR technique developed for limited numbers of cells. Compared with white cells, 1.8 times the amount of gp91(phox) mRNA was found in osteoclasts. This difference may be related to the size of the osteoclast and the multiple nuclei present. The presence of gp91(phox) in osteoclasts was confirmed at protein level by immunocytochemistry. Osteoclastic superoxide generation is inhibited by diphenylene iodonium, a specific inhibitor of the NADPH oxidase. These studies suggest that superoxide generation by osteoclasts correlates with the activity of NADPH oxidase.

The use of a soft tissue expander in an alveolar bone ridge augmentation for implant placement.

A narrow mandibular posterior alveolar ridge was modified by the use of a soft tissue expander to generate adequate tissue for graft coverage. The principles of osteoperiosteal flaps were combined with guided bone regeneration techniques for an optimum amount of bone at the site.

Osteoclastic superoxide production and bone resorption: stimulation and inhibition by modulators of NADPH oxidase.

Production of superoxide radicals by osteoclasts is necessary for normal bone degradation. White blood cell superoxide, needed for bacterial killing, is produced by activated NADPH oxidase. Since osteoclasts and white blood cells share a common hematopoietic origin, we initiated experiments to test the hypothesis that superoxide radicals at the osteoclast-bone interface are produced by NADPH oxidase. Diphenyl iodonium (IDP), an inhibitor of NADPH oxidase, blocked superoxide generation and decreased osteoclastic bone resorption in cultures of calvarial explants from normal mice. Interferon (IFN) gamma, a stimulant of NADPH oxidase activity, increased superoxide production and bone resorption in cultures of calvarial explants from osteopetrotic (microphthalmic) mice. IDP blocked the stimulatory effects of IFN in this bone resorption model. These data suggest that osteoclastic superoxide is produced by NADPH oxidase.

Functions of the M-CSF receptor on osteoclasts.

Macrophage colony-stimulating factor (M-CSF) receptor has been previously reported to be present in osteoclasts both at mRNA and protein levels. However, the biochemical interactions between M-CSF and its receptor on osteoclasts are less well characterized than in mononuclear phagocytes. In this study, we show that (1) 125I-labeled M-CSF ligand specifically binds to the M-CSF receptor on osteoclasts by autoradiography; (2) binding of M-CSF to the receptor stimulates protein tyrosine phosphorylation in osteoclasts by immunostaining; (3) oxygen-derived free radicals produced by calvarial osteoclasts are increased by M-CSF stimulation (1.37 +/- 0.08, n = 10, P < 0.01); and (4) bone resorption in calvarial explants is enhanced by M-CSF (1.153 +/- 0.09, n = 10, p < 0.001). Thus, our data provide multiple lines of evidences that mouse calvarial osteoclasts are activated by M-CSF. These data suggest that under the conditions present in the calvarial model, M-CSF activates osteoclastic bone resorption.

Long-term treatment of osteopetrosis with recombinant human interferon gamma.

BACKGROUND: Congenital osteopetrosis is a rare osteosclerotic bone disease characterized by both a defect in osteoclastic function and reduced generation of superoxide by leukocytes. The disease is frequently fatal during the first decade of life. A six-month trial of therapy with recombinant human interferon gamma-1b in eight patients with osteopetrosis provided evidence of benefit, prompting this study of more prolonged therapy. METHODS: We studied 14 patients with severe osteopetrosis treated with subcutaneous injections of recombinant human interferon gamma-1b (1.5 micrograms per kilogram of body weight per dose) three times per week for at least 6 months; 11 patients were treated for 18 months. We assessed the effect of therapy by evaluating the patients' clinical status, measuring blood counts and biochemical markers of bone turnover, and performing bone marrow imaging and bone biopsies. RESULTS: After 6 months of therapy, all 14 patients had decreases in trabecular-bone area (determined by histomorphometric analysis of bone-biopsy specimens) and increases in bone marrow space (determined by marrow imaging), and the improvement was sustained in the 11 patients treated for 18 months. The mean (+SD) hemoglobin concentration increased from 7.5 +/- 2.9 to 10.5 +/- 0.3 g per deciliter (P = 0.05), and superoxide generation by granulocyte-macrophage colonies increased (P < 0.001) after 18 months of therapy. In six patients for whom pretreatment data were available, there was a 96 percent decrease in the frequency of infections requiring antibiotic therapy during interferon treatment. There were no side effects necessitating the discontinuation of therapy. CONCLUSIONS: Long-term therapy with interferon gamma in patients with osteopetrosis increases bone resorption and hematopoiesis and improves leukocyte function.

Characterization of M-CSF and its receptor in microphthalmic mice.

The macrophage colony-stimulating factor (M-CSF) gene expression in osteopetrotic mice (op/op) is defective due to a point-mutation in the M-CSF gene [1]. However, almost all osteopetrotic patients have been shown to have a normal or elevated circulating level of bioactive M-CSF [2]. To investigate the action of the M-CSF in mi/mi mice (microphthalmic; another osteopetrotic mouse mutant), M-CSF levels and its receptors were studied. We found that serum levels of M-CSF in mi/mi mice were not significantly different from normal control mice. The M-CSF receptor binding affinity of spleen-adherent cells was similar to that of control mice. In spleen cells from mi/mi mice, the receptor binding sites per cell (normalized to total spleen cells, M-CSF receptor positive cells, or M-CSF receptor RNA positive cells) were present in a greater number than in spleen cells from phenotypically normal siblings. Northern blot analysis showed that there is no significant difference in transcripts of the M-CSF receptor (c-fms) in the mi/mi and phenotypically normal mice. Unlike the op/op mutant, M-CSF levels, as well as the affinity and number of M-CSF receptors, do not explain the defect in osteoclastic function in the mi/mi mutation.

Superoxide and bone resorption.

The reaction of superoxide with nitroblue tetrazolium produces an electron-dense diformazan precipitate which can be used to localize areas of superoxide production. Transmission electron microscopy was used to demonstrate that diformazan granules formed by the reaction of nitroblue tetrazolium with excess superoxide are electron dense, whereas monoformazan granules generated by hydrogen peroxide were not. On the basis of these observations, superoxide formed along the osteoclast-bone interface was localized by demonstrating the electron-dense diformazan granules between the osteoclastic membrane and the bone surface. The formation of this reaction product was inhibited by a superoxide scavenger, the deferoxamine mesylate-manganese complex (the "green" complex), confirming the specificity of the reaction product. The scavenger also inhibited bone resorption. High concentrations of superoxide generated in vitro at a neutral pH degraded osteocalcin into numerous peptide fragments, demonstrating the ability of superoxide to break peptide bonds. These studies localize superoxide production to the ruffled border space and suggest that superoxide generated at the osteoclast-bone interface is involved in bone matrix degradation.

Osteopetrosis. The pharmaco-physiologic basis of therapy.

Medical treatments of osteopetrosis have attempted to improve hematologic function, reduce the osteosclerotic condition, and/or improve immune function. Prednisone therapy has improved hematologic function in some patients, but has not resulted in a reduction in bone mass. Calcium deficient diets have limited further sclerosis in some patients. High-dose calcitriol and parathormone infusions have stimulated osteoclastic activity. In some patients, high-dose calcitriol has resulted in clinical improvement. Newer treatments, such as interferon gamma and macrophage colony stimulating factor, may alter the osteoclastic and immune defects by stimulating cellular formation and function. These therapies, alone or in combination, ameliorate but do not cure the osteopetrotic condition.

Combination macrophage-colony stimulating factor and interferon-gamma administration ameliorates the osteopetrotic condition in microphthalmic (mi/mi) mice.

Malignant osteopetrosis is a fatal congenital bone disorder characterized by defective osteoclastic function. Death frequently occurs within the first decade of life. The precise molecular defect(s) that causes osteopetrosis is not known. The possibility that osteoclasts, like macrophages, are controlled by interactions with cytokines suggests that these agents may provide a means of increasing osteoclastic function. Macrophage-colony stimulating factor (M-CSF), a cytokine known to enhance macrophage and osteoclast generation, and recombinant human interferon-gamma (rIFN), a cytokine known to stimulate superoxide generation by white cells, were administered to microphthalmic (mi/mi) mice in an attempt to improve the osteopetrotic condition. Each cytokine was administered separately and in combination to neonatal mi/mi mice for 7 consecutive d. Bone turnover, osteoclast numbers, superoxide generation by white cells, and hematocrit were assessed. rIFN, M-CSF, and a combination of the cytokines stimulates oxygen-derived free radical production by white cells and increased bone resorption. rIFN resulted in a reduction in the number of osteoclasts. This reduction in number was ameliorated by M-CSF. M-CSF alone and in combination with rIFN resulted in improved hematopoietic function, increased weight gain, and increased physical activity of the affected mutants.

Nitroblue tetrazolium reduction and bone resorption by osteoclasts in vitro inhibited by a manganese-based superoxide dismutase mimic.

Oxygen-derived free radicals are produced by osteoclasts. Oxygen radical formation occurs at the osteoclast/bone surface interface. This location next to bone implies that oxygen radicals, including but not limited to superoxide, are needed for bone resorption. Compounds that scavenge superoxide are being developed as pharmaceutical agents to inhibit the damaging effects of oxygen radical formation on tissues. One such scavenger is the Desferal-manganese complex (DMnC). DMnC reduced the amount of formazan staining produced by the interaction of oxygen radicals with nitroblue tetrazolium (NBT) in both individual mouse calvarial osteoclasts in tissue explants and isolated osteoclasts. As a result of the reduced concentrations of oxygen radicals, DMnC inhibited bone resorption by calvarial explants and isolated osteoclasts. Superoxide dismutase (SOD) inhibited NBT reduction and bone resorption by isolated osteoclasts but to a lesser degree than DMnC. Inhibition of bone resorption in the isolated osteoclast system increased in parallel to the concentration of DMnC in cultures. Desferal without Mn had no effect on bone resorption by isolated osteoclasts. These results support the hypothesis that osteoclasts produce oxygen radicals as part of the process of bone resorption.

Recombinant human interferon gamma therapy for osteopetrosis.

A defect in leukocytic superoxide formation has been demonstrated in patients with congenital osteopetrosis. This leukocyte defect appears to be related to defective bone resorption. Because recombinant human interferon gamma therapy enhances superoxide production in patients with chronic granulomatous disease, we sought to determine whether a similar strategy could reverse the osteopetrotic condition. Interferon gamma, 1.5 micrograms/kg three times a week, was administered by subcutaneous injection for 6 months to eight patients with osteopetrosis. Urinary hydroxyproline and urinary calcium excretion increased markedly during therapy in parallel with a significant decrease in trabecular bone volume. Bone marrow scans demonstrated increased bone marrow production. The hemoglobin concentration, platelet count, and leukocyte production of superoxide increased significantly. No serious infections were encountered during the therapy. These data suggest that interferon gamma administration enhances bone resorption and leukocyte function in patients with osteopetrosis.

Osteoclastic superoxide generation: taking control of bone resorption using modulators of superoxide concentrations.

We have examined the role of superoxide in bone resorption by stimulating defective superoxide production and bone resorption in patients with osteopetrosis and inhibiting superoxide production and bone resorption in murine calvarial explants. Interferon gamma treatment did stimulate superoxide generation and bone resorption in patients with osteopetrosis as evidenced by a reduction in bone volume and an increase in biochemical markers of bone resorption. Further, lowering the superoxide concentrations within calvarial osteoclasts using a scavenger, desferal manganese, decreased bone resorption. We conclude that superoxide generation by osteoclasts is necessary for normal osteoclastic function.

Oxygen derived free radicals in osteoclasts: the specificity and location of the nitroblue tetrazolium reaction.

Oxygen derived free radicals are generated by osteoclasts. In a novel culture system, isolated rat osteoclasts were stained when nitroblue tetrazolium (NBT) was reduced by cellular oxidants to formazan, an insoluble precipitate. Superoxide dismutase (SOD) inhibited the accumulation of formazan by the isolated osteoclasts. Osteoclasts in mouse calvarial organ cultures also reduced NBT to formazan. The reaction products were localized to the area of the osteoclast-bone interface. At the light microscopic level, the formazan granules appeared to be concentrated within the cytoplasm. Formazan accumulation was significantly inhibited by calcitonin (hCT). The inhibition of NBT reduction by SOD indicates that the isolated osteoclasts were capable of producing superoxide. The localization of the formazan granules between the external osteoclastic membrane and the bone, and the inhibition of this reaction during hCT exposure suggests that oxygen derived free radicals may contribute to bone resorption.

Interleukin-2 stimulates osteoclastic activity: increased acid production and radioactive calcium release.

Recombinant human interleukin-2 (IL-2) was studied to determine effects on acid production by individual osteoclasts in situ on mouse calvarial bones. This analysis was performed using a microspectrofluorimetric technique to quantify acid production in individual cells. Radioactive calcium release was determined using calvarial bones in a standard tissue culture system. This allowed us to correlate changes in acid production with a measure of bone resorption. IL-2 stimulated acid production and bone resorbing activity. Both effects were inhibited by calcitonin. No stimulation of bone resorption occurred when IL-2-containing test media was incubated with a specific anti-IL-2 antibody and ultrafiltered. Our data demonstrated a correlation between acid production and bone resorbing activity in mouse calvaria exposed to parathyroid hormone (PTH). The data obtained from cultured mouse calvaria exposed to IL-2 demonstrated similar stimulatory effects to those seen during PTH exposure. These data suggest that calvaria exposed to IL-2 in vitro have increased osteoclastic acid production corresponding with increased bone resorption.