Stimulation of osteoclastic bone resorption by hydrogen peroxide.

The molecular mechanisms underlying the pathophysiology of bone destruction still remain poorly understood. We have found that hydrogen peroxide (H2O2), a reactive oxygen species (ROS), is a potent stimulator of osteoclastic bone resorption and cell motility. A marked enhancement of bone resorption was noted when rat osteoclasts, cultured on devitalised bovine cortical bone, were exposed to 10 nM [H2O2]. Apart from exposing osteoclasts to a low extracellular pH, which is known to enhance osteoclastic bone resorption, we provide first evidence for a molecule that stimulates osteoclastic bone resorption in osteoclast cultures that do not respond to parathyroid hormone and 1, 25 dihydroxyvitamin D3. We envisage that both basic biological and practical clinical implications may eventually follow from these studies.     

Clinical usefulness of bisphosphonates in oncology: treatment of bone metastases, antitumoral activity and effect on bone resorption markers

The present article overviews the role of bisphosphonates for the treatment and prevention of bone metastases and their antiangiogenic effects and antitumoral activity. The skeleton is a frequent and clinically relevant site of metastasis in cancer patients. The major events related to bone metastases include bone pain, bone loss, hypercalcemia, spinal cord compression, and fractures. On the basis of their radiographic features, bone metastases are classified as osteoblastic, osteoclastic, or mixed. The primary goals of treatment of bone metastases are reduction of the risk of pathological fractures and other skeletal-related events, and pain control. Bisphosphonates are used to prevent pathological fractures by inhibition of osteoclasts. Recent studies suggest that bisphosphonates have some direct antitumoral activity, mainly mediated through the blockade of angiogenic pathways. Further clinical studies are needed to determine the optimal treatment duration, timing and schedule of bisphosphonates, assess their role as adjuvant therapy for the prevention of bone metastases, and establish their antiangiogenic activity in association with standard cytotoxic and hormonal drugs for treatment of patients with advanced disease.     

Osteoclast-independent bone resorption by fibroblast-like cells

To date, mesenchymal cells have only been associated with bone resorption indirectly, and it has been hypothesized that the degradation of bone is associated exclusively with specific functions of osteoclasts. Here we show, in aseptic prosthesis loosening, that aggressive fibroblasts at the bone surface actively contribute to bone resorption and that this is independent of osteoclasts. In two separate models (a severe combined immunodeficient mouse coimplantation model and a dentin pit formation assay), these cells produce signs of bone resorption that are similar to those in early osteoclastic resorption. In an animal model of aseptic prosthesis loosening (i.e. intracranially self-stimulated rats), it is shown that these fibroblasts acquire their ability to degrade bone early on in their differentiation. Upon stimulation, such fibroblasts readily release acidic components that lower the pH of their pericellular milieu. Through the use of specific inhibitors, pericellular acidification is shown to involve the action of vacuolar type ATPases. Although fibroblasts, as mesenchymal derived cells, are thought to be incapable of resorbing bone, the present study provides the first evidence to challenge this widely held belief. It is demonstrated that fibroblast-like cells, under pathological conditions, may not only enhance but also actively contribute to bone resorption. These cells should therefore be considered novel therapeutic targets in the treatment of bone destructive disorders.     

Stimulation of bone resorption by various prostaglandins in organ culture.

The ability of E, F, A and B prostaglandins to stimulate bone resorption was demonstrated in organ culture. All of the compounds tested were able to increase the release of previously incorporated 45Ca from fetal rat bone by 60 to 135 per cent at maximally effective doses, but prostaglandins of the E series were 10- to 100- fold more potent than F, A or B prostaglandins. Compounds with two double bonds in the side chain were usually more potent than those with one double bond. PGE2 stimulation of bone resorption increased linearly with the logarithm of the medium concentration over the range of 10(-9)M to 10(-5)M, then decreased at higher concentrations. PGE2 stimulated bone resorption more slowly than did parathyroid hormone but caused complete resorption after six days in the culture system. Equilibrium dialysis studies showed no significant binding of F, and 16-34% binding of E and A prostaglandins to bovine serum albumin, which was present in the medium at 1 mg/ml. These differences in albumin binding could not account for differences in potency.     

Lipopolysaccharide-induced bone resorption is mediated by prostaglandins

In organ cultures of new-born mouse calvaria both bovine parathyroid hormone and a lipopolysaccharide preparation from E. coli promoted bone resorption. Dead bone cultures did not respond to either of these agents. When bone prostaglandin synthesis was inhibited by indomethacin the lipopolysaccharide no longer promoted bone resorption. Similar treatment did not affect the ability of parathyroid hormone to cause bone dissolution.     

Activation of NF-kappaB in human osteoblasts by stimulators of bone resorption.

Several bone resorptive stimuli affect osteoclasts indirectly by modulating the production and release of osteoblastic factors. Using electrophoretic mobility shift assays, we found that not only tumour necrosis factor-alpha (TNF-alpha) but also interleukin-1beta and parathyroid hormone (PTH) caused dose and time-related increases in nuclear factor kappaB (NF-kappaB)-DNA binding in Saos-2 human osteoblastic (hOB) cells. Activation of NF-kappaB by TNF-alpha was reproduced in primary hOBs. In contrast, consistent with their previously reported lack of response to steroid hormones, Saos-2 cells did not respond to 1,25-dihydroxyvitamin D(3). We suggest that NF-kappaB activation in osteoblastic cells constitutes an important pathway in osteoblast-mediated resorptive signalling.     

Inhibition of bone resorption in culture by inhibitors of thiol proteinases.

Leupeptin, antipain, tosyl-lysylchloromethane (Tos-Lys-CH2Cl) and benzyloxy-carbonylphenylalanylalanyldiazomethane (Z-Phe-Ala-CHN2) inhibit reversibly the resorption induced by parathyroid hormone or heparin in cultured mouse bones. Leupeptin and antipain do not affect collagenase production and activity or the enhanced secretion of beta-glucuronidase induced by the bone-resorbing agents. They might thus act by a direct (extracellular?) inhibition of lysosomal thiol proteinases.     

Role of vitamin D in bone resorption.

The idea that vitamin D must function at the bone site to promote bone mineralization has long existed since its discovery as an anti-rachitic agent. However, the definite evidence for this is still lacking. In contrast, much evidence has accumulated that 1 alpha,25(OH)2D3 in involved in bone resorption. 1 alpha,25(OH)2D3 tightly regulates differentiation of osteoclast progenitors into osteoclasts. Osteoclast progenitors have been thought to belong to the monocyte-macrophage lineage. 1 alpha,25(OH)2D3 greatly stimulates differentiation and activation of mononuclear phagocytes. Recent reports have indicated that differentiation of mononuclear phagocytes into osteoclasts is strictly regulated by osteoblastic cells, the process of which is also stimulated by 1 alpha,25(OH)2D3. In the differentiation of mononuclear phagocytes into osteoclasts, the target cells for 1 alpha,25(OH)2D3 appear to be osteoblastic stromal cells. Osteoblastic cells produce several proteins such as BGP, MGP, osteopontin and the third component of complement (C3) in response to the vitamin. They appear to be somehow involved in osteoclast differentiation and functions. Thus, 1 alpha,25(OH)2D3 seems to be involved in the differentiation of osteoclast progenitors into osteoclasts directly and also by an indirect mechanism involving osteoblastic cells. The precise role of osteoblastic cells in osteoclast development has to be elucidated in the future.     

Inhibition of angiogenic factor- and tumour-induced angiogenesis by gamma linolenic acid.

Angiogenesis, the formation of new blood vessels, is an essential feature of malignant tumour development. Gamma linolenic acid (GLA), a n-6 polyunsaturated fatty acid (PUFA), inhibits the growth and metastasis of a variety of tumour cells, including breast, prostate, pancreatic cancer and hepatoma cells and also has anti-metastatic effects on endothelial cells. In the current study, we tested whether GLA inhibited angiogenesis induced by tumour cells. A rat aortic ring assay and in vitro tube formation of human vascular endothelial cells were used to determine angiogenesis (spontaneous, angiogenic factor- and tumour cells-induced). Inclusion of GLA in this 3-D matrix culture system significantly inhibited angiogenesis from aortic rings in a concentration-dependent manner. The results from tube formation of vascular endothelial cell further confirmed that GLA suppressed angiogenesis. Furthermore, in the cell motility assay (phagokinetic assay and endothelial wounding assay), a significant reduction of the motility of vascular endothelial cells by GLA was seen. It is concluded that gamma linolenic acid inhibits angiogenic factor and tumour-induced angiogenesis in vitro at least in part via its inhibitory effect on the motility of vascular endothelial cells.     

Osteoblast-osteoclast interaction in bone resorption. Preliminary results

Osteoclastic bone resorption has been evaluated in vitro by release of tritiated collagen fragments from 3H-proline prelabeled bone particles incubated for 48 hours in presence of avian isolated osteoclasts. Cells were co-incubated with periosteum-free chick calvarial fragments by interposition of 0.4 micron millipore membrane transwells, in presence or absence of 10(-8) M 1.34 bovine parathyroid hormone (PTH). Results demonstrated that i) calvaria exert a stimulating effect over osteoclastic bone resorption which was 1.8 fold enhanced with respect to controls (p less than 0.001). ii) the stimulating effect is exerted by calvarium-derived soluble molecules capable of crossing the 0.4 micron millipore membrane interposed between calvarial fragments and osteoclasts, iii) in this experimental system no further enhancement of calvarial stimulating effect is operated by PTH treatment.     

Rac-GTPase, osteoclast cytoskeleton and bone resorption.

The members of the Rho-GTPase subfamily, Rac1 and Rac2, are intimately involved in the organization of the cytoskeleton, and the p21-activated kinases or PAKs are targets of these proteins. Rac1 and Rac2 are also essential components of NADPH oxidase, the enzyme responsible for generating free radicals. The cytoskeleton modulates the adhesion of osteoclasts to bone and its subsequent resorption. These cells contain NADPH diaphorase activity, and free radicals influence bone resorption. The influence of Rac1, Rac2 and PAK1 on the cytoskeleton, resorbing activity and NADPH diaphorase activity of disaggregated rat osteoclasts was investigated by permeabilisation with saponin and introducing specific anti-Rac1, anti-Rac2 or anti-PAK1 antibodies. Rhodamine-phalloidin stain was used to identify actin in osteoclasts cultured on plastic slides, and the bone-slice method was used to measure resorption. Saponin permeabilisation did not affect the cytoskeletal organization or bone resorption. Anti-Rac antibodies caused dose- and time-dependent cytoskeletal changes. The osteoclasts rounded up and developed retraction fibers; actin rings were disrupted and large actin dots were seen at the periphery of the cells. Osteoclast resorptive activity was depressed after incubation with the antibodies. The total area resorbed by treated cells and the mean pit area were smaller than those of controls. Anti-PAK1 antibody caused similar changes. None of the antibodies altered the NADPH diaphorase activity. Thus, Rac-GTPases are present in rat osteoclasts and are involved in the organization of the actin cytoskeleton and in resorptive activity. These effects may be mediated by PAK1 kinase, but do not influence osteoclast NADPH diaphorase activity.     

Inhibition of bone resorption in vitro by serine-esterase inhibitors

The effect of two synthetic serine esterase inhibitors, N-alpha-dansyl(p-guanidino)phenylalaninepiperidine hydrochloride (I 2581) and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (D-Phe-Pro-Arg-CH2Cl), on bone resorption in organ cultured mouse calvaria from neonatal mice has been examined. Mineral mobilization was assessed by analyzing the release of 45Ca, stable calcium (Ca2+) and inorganic phosphate (Pi). Organic matrix degradation was studied by analyzing the release of 3H from [3H]proline-labelled bones, and by quantifying the amounts of hydroxyproline in bone after culture. It was found that I 2581, at and above 30 mumol/l, dose-dependently inhibited 45Ca release induced by thrombin, parathyroid hormone (PTH), prostaglandin E2 and 1-alpha-hydroxyvitamin D-3. I 2581 (50 mumol/l) inhibited PTH-stimulated release of 3H from [3H]proline-labelled bones, and this effect was reversible after withdrawal of I 2581. I 2581 (50 mumol/l) inhibited the release of Ca2+, Pi, beta-glucuronidase and beta-N-acetylglucosaminidase in bones stimulated by PTH and 1-alpha-hydroxyvitamin D-3, without affecting the release of lactate dehydrogenase. In parallel, I 2581 decreased PTH and 1-alpha-hydroxyvitamin D-3 induced reduction of hydroxyproline levels in bones after culture. I 2581 (50 mumol/l) did not affect the basal release of 45Ca, Ca2+, beta-glucuronidase and beta-N-acetylglucosaminidase, nor the basal amounts of hydroxyproline in bones after culture. D-Phe-Pro-Arg-CH2Cl (100 mumol/l) significantly inhibited PTH- and PGE2-induced release of 45Ca without affecting basal release of radioactive calcium. These data indicate that activation of serine proteinase(s) may be a necessary step in the mechanism of action of several stimulators of bone resorption.     

Inhibition of parathormone-stimulated bone resorption by type I interferon

The effect of Type I interferon on bone resorption was studied by measuring its effect on parathormone-stimulated calcium release from neonatal murine calvaria in vitro. A pure human recombinant leukocyte interferon hybrid of the A and D subtypes was used, which has high antiviral activity on mouse cells. Calcium release was inhibited in a dose dependent fashion with 50% inhibition at about 10(-10) M or 600 U/ml, and the inhibition was reversible. The presence of interferon was required before or during the activation phase of the resorptive response, when the formation of osteoclasts from precursor cells would occur. When added to actively resorbing bone it had no effect. The data suggest that Type I interferon can inhibit the parathormone-regulated development of active osteoclasts, possibly by inhibiting osteoclast precursor differentiation.     

Glutamatergic regulation of bone resorption

There has been increasing evidence during the last years that glutamate (Glu), the major neuromediator of the nervous system, contributes to the local regulation of bone cell functions. Several classes of Glu receptors and transporters, as well as molecules involved in glutamate signal transduction in neuronal tissue, were identified in bone. While recent findings suggest that Glu may participate in mechanisms underlying bone formation, several studies indicate that Glu may also control bone resorption. Ionotropic NMDA and metabotropic Glu receptors are expressed by osteoclasts and electrophysiological studies have demonstrated that NMDA receptors (NMDAR) are functional on these cells. In vitro studies have shown that NMDAR are important for osteoclast function since several specific antagonists of NMDAR which block the current induced by Glu in these cells also inhibit bone resorption. Preliminary studies investigating the mechanisms of action of NMDAR antagonists on bone resorption are reviewed in this paper. There is also growing evidence that NMDAR are expressed throughout the osteoclastic differentiation sequence and that antagonists of NMDAR affect osteoclastogenesis. Very few in vivo studies have however investigated the role of Glu in skeletal metabolism and bone resorption and clearly further work is required to demonstrate the relevance of glutamate signaling in the physiology of bone resorption in vivo.     

Stereophotogrammetric observations on bone resorption by isolated rabbit osteoclasts

Although the osteoclast has long been recognized as the major agent of bone resorption, it has previously only been possible to assess resorption by these cells in bone, where their response to environmental factors may be modified by, or even mediated by, the other cell types also present. We used a carborundum saw to prepare slices of human cortical bone which demonstrate a predictable surface appearance. Osteoclasts were disaggregated from neonatal rabbit long bone and settled onto these slices. After incubation for 24 hr we found that osteoclasts had formed distinctive excavations in the bone surface. This technique makes it possible, for the first time, to analyse the direct and indirect effects of hormones and cells on osteoclastic bone resorption.     

Neuropeptidergic regulation of bone resorption and bone formation

Immunohistochemical studies have revealed an extensive network of nerve fibers in the vicinity and within the skeleton, not only in the periosteum of bone but also in cortical and trabecular bone as well as in the bone marrow. Phenotyping of the skeletal nerve fibers have demonstrated the expression of a restrictive panel of different signalling molecules including neuropeptides, neurotransmitters and neurotrophins. In this review, the presence of receptors for the neuropeptides vasoactive intestinal peptide, calcitonin gene-related peptide and substance P on osteoblasts and osteoclasts and the capacity of these receptors to regulate bone formation, osteoclast formation and activity are described. These findings, together with data obtained by chemically and surgically targeted nerve deletion and observations made in paraplegic patients, strongly suggest that neuro-osteogenic interactions play an important role in skeletal function.     

Stimulation of bone resorption by lipoxygenase metabolites of arachidonic acid

We have studied the effect of leukotrienes, (LT): B4, C4, D4 and E4 and the hydroxyeicosatetraenoic acids (HETEs) 5-HETE and 12-HETE on bone resorption in vitro. Resorption was measured by colorimetric assay of calcium released from neonatal mouse calvaria maintained in organ culture for 72h. All the LTs and HETEs stimulated bone resorption, with optimum responses at picomolar or nanomolar concentrations. The responses were biphasic, with a decreasing effect at higher concentrations. In contrast, prostaglandin E2 (PGE2) stimulated resorption only at 10nM and above. Indomethacin partially inhibited resorption by LTB4, LTC4 and LTD4, but did not affect resorption stimulated by LTE4, 5-HETE and 12-HETE. These results indicate that lipoxygenase products of arachidonic acid are highly potent bone resorbing factors and may play an important role in the localised bone loss associated with inflammatory lesions.     

Stimulation of bone resorption in organ culture by salt-free extracts of Solanum glaucophyllum.

Addition of a desalted aqueous extract made from dried leaves of Solanum glaucophyllum produced a marked stimulation of bone resorption in a culture system in vitro. This result indicates that, like the biologically active forms of vitamin D, the active material in the plant not only stimulates intestinal calcium absorption but increases bone mobilization as well.